Search

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+CD138+plasma cells. Interestingly, high levels of CD138+cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.

Background Several animal studies, and one inoculation study in adult asthmatics have shown that interleukin-33 (IL-33) is a major contributor to type-2 inflammation in acute asthma. However, the link between IL-33 and type-2 inflammation has not been shown in naturally occurring asthma exacerbations. Objectives To determine if airway IL-33 is associated with type-2 inflammation measured by type-2 cytokines, FeNO and sputum eosinophils in patients presenting to the Emergency Department with an asthma exacerbations. Methods Adult patients hospitalized due to acute asthma were enrolled. Upper airways were sampled with nasal swabs and lower airways with induced sputum. Cytokines were measured at protein level using a Luminex® assay and mRNA expression level using droplet-digital-PCR. Airway sampling was repeated four weeks after exacerbation. Results At the time of exacerbation, upper airway IL-33 correlated with upper airway IL-5 and IL-13 (R = 0.84, p, Highlights • IL-33 correlates to type 2 cytokines in hospitalized patients with acute asthma. • Airway IL-13 and IL-33 positively correlate with fractional exhaled nitric oxide. • Airway IL-5 correlates to blood eosinophils in naturally occurring acute asthma.

Polymorphisms in P2X4R and CAMKK2 associate with susceptibility to HIV-associated sensory neuropathy (HIV-SN) - a condition likely mediated by TNFα. As single nucleotide polymorphisms (SNPs) and haplotypes of CAMKK2, and a neighbouring gene P2X4R, mark susceptibility to HIV-SN in South Africans living with HIV, we examined the relationship between P2X4R and CAMKK2 genotypes and TNFα production. Peripheral blood mononuclear cells from 129 healthy donors were stimulated with killed Escherichia coli, and concentrations of soluble TNFα were assessed. Their DNA was genotyped for 22 SNPs in P2X4R and CAMKK2. Three SNPs within P2X4R and two SNPs within CAMKK2 influenced concentrations of TNFα, but these SNP did not associate with risk for HIV-SN. This incongruence may reflect differences in P2X4R haplotypes present in Africans and Europeans. However some CAMKK2 haplotypes were found in both populations, so CAMKK2 polymorphisms may impact upon HIV-SN via effects of the protein on pathways other than TNFα.

Background: Hundreds of genetic variants are thought to contribute to variation in asthma risk by modulating gene expression. Methods that increase the power of genome-wide association studies (GWASs) to identify risk-associated variants are needed. Objective: We sought to develop a method that aggregates the evidence for association with disease risk across expression quantitative trait loci (eQTLs) of a gene and use this approach to identify asthma risk genes. Methods: We developed a gene-based test and software package called EUGENE that (1) is applicable to GWAS summary statistics; (2) considers both . cis- and . trans-eQTLs; (3) incorporates eQTLs identified in different tissues; and (4) uses simulations to account for multiple testing. We applied this approach to 2 published asthma GWASs (combined n = 46,044) and used mouse studies to provide initial functional insights into 2 genes with novel genetic associations. Results: We tested the association between asthma and 17,190 genes that were found to have . cis- and/or . trans-eQTLs across 16 published eQTL studies. At an empirical FDR of 5%, 48 genes were associated with asthma risk. Of these, for 37, the association was driven by eQTLs located in established risk loci for allergic disease, including 6 genes not previously implicated in disease cause (eg, . LIMS1, . TINF2, and . SAFB). The remaining 11 significant genes represent potential novel genetic associations with asthma. The association with 4 of these replicated in an independent GWAS: . B4GALT3, . USMG5, . P2RY13, and . P2RY14, which are genes involved in nucleotide synthesis or nucleotide-dependent cell activation. In mouse studies, P2ry13 and P2ry14-purinergic receptors activated by adenosine 5-diphosphate and UDP-sugars, respectively-were upregulated after allergen challenge, notably in airway epithelial cells, eosinophils, and neutrophils. Intranasal exposure with receptor agonists induced the release of IL-33 and subsequent eosinophil infiltration into the lungs. Conclusion: We identified novel associations between asthma and eQTLs for 4 genes related to nucleotide synthesis/signaling and demonstrated the power of gene-based analyses of GWASs.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by progressive decline in pulmonary function. The rate of decline can vary, with some patients remaining stable over longer periods of time and others rapidly progressing [1]. tThe variable progression of this disease makes it difficult to elucidate pathogenic pathways involved in the initiation and progression of IPF. Advances in high-throughput gene-expression analyses have led to improvements in our understanding of disease biology and prognostic gene signatures. We hypothesise that IPF has a unique circulatory transcriptional profile compared to healthy controls, with additional differences between stable and progressive disease likely related to disease pathogenesis. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Miss Clynick has nothing to disclose. Conflict of interest: Dr. Jo has nothing to disclose. Conflict of interest: Dr. Corte has nothing to disclose. Conflict of interest: Dr. Glaspole has nothing to disclose. Conflict of interest: Dr. Grainge has nothing to disclose. Conflict of interest: Dr. Hopkins has nothing to disclose. Conflict of interest: Dr. Reynolds has nothing to disclose. Conflict of interest: Dr. Chapman has nothing to disclose. Conflict of interest: Dr. Walters has nothing to disclose. Conflict of interest: Dr. Zappala has nothing to disclose. Conflict of interest: Dr. Keir has nothing to disclose. Conflict of interest: Dr. Cooper has nothing to disclose. Conflict of interest: Dr. Mahar has nothing to disclose. Conflict of interest: Dr. Ellis has nothing to disclose. Conflict of interest: Dr. Goh has nothing to disclose. Conflict of interest: Dr. Baltic has nothing to disclose. Conflict of interest: Miss Ryan has nothing to disclose. Conflict of interest: Dr. Tan has nothing to disclose. Conflict of interest: Dr. Moodley has nothing to disclose.

EgeUn###, Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 3 102295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence. Copyright © 2019 by the American Thoracic Society, Háskóli Íslands, HI University of Chicago University of Ulsan, UOU Monash University, MU University of Nottingham University of Edinburgh University of Colorado Denver Universitat de Barcelona University of Pittsburgh, Pitt Harvard School of Dental Medicine Massachusetts General Hospital, MGH University of Pennsylvania, Penn University College Cork, UCC City, University of London, City University of California, San Francisco, UCSF School of Medicine, Vanderbilt University Ege Üniversitesi University of Virginia, UV Aarhus Universitetshospital Central Manchester University Hospitals NHS Foundation Trust Columbia University Novartis Sunovion COPD Foundation Pfizer National Heart, Lung, and Blood Institute, NHLBI: R01-HL097163, UH3-HL123442, P01-HL092870 U.S. Department of Defense, DOD: W81XWH-17-1-0597, U01 HL089897, U01 HL089856 AstraZeneca Siemens Foundation North Carolina GlaxoSmithKline Foundation, 1National Jewish Health, Denver, Colorado; 2School of Public Health; 3Department of Medicine, and 54Department of Immunology, University of Colorado Denver, Denver, Colorado; 4Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; 5Brigham and Women’s Hospital, Harvard School of Medicine, Boston, Massachusetts; 6Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma;7Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 8MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; 9Respiratory Medicine Unit, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; 10Biomedical Research Centre, University of Nottingham, Nottingham, United Kingdom; 11Royal Brompton Hospital and Imperial College, London, United Kingdom; 12Simmons Center for Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania; 13Department of Medicine, University of California, San Francisco, San Francisco, California; 14Gilead Sciences, Foster City, California; 15Department of Medicine, University of Chicago, Chicago, Illinois; 16Department of Medicine, University of Virginia, Charlottesville, Virginia; 17Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; 18National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan; 19National Hospital Organization Tokyo National Hospital, Tokyo, Japan; 20Helmholtz Zentrum München, Neuherberg, Germany; 21University Hospital Munich, Munich, Germany; 22Department of Pulmonology, Ege University Hospital, Bornova, Izmir, Turkey; 23Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia; 24Alfred Hospital and Monash University, Melbourne, Australia; 25Pulmonary Medicine, GB Morgagni Hospital, Forlì, Italy; 26Department of Diseases of the Thorax, Ospedale GB Morgagni, Forlì, Italy; 27Université Paris Diderot and Hôpital Bichat, Paris, France; 28Royal Papworth Hospital and Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom; 29Respiratory Department, University Hospital of Bellvitge, University of Barcelona, Barcelona, Spain; 30National University Hospital of Iceland, University of Iceland, Reykjavik, Iceland; 31Department of Medicine, Columbia University Irving Medical Center, New York, New York; 32Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, Massachusetts; 33Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Aarhus, Denmark; 34Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas,” México City, México; 35Universidad Nacional Autónoma de México, México City, México; 36Cork University Hospital and University College Cork, Cork, Ireland; 37St. Vincent’s University Hospital, Dublin, Ireland; 38School of Medicine, University College Dublin, Dublin, Ireland; 39Department of Respiratory Medicine, First Faculty of Medicine Charles University and Thomayer Hospital, Prague, Czech Republic; 40University of British Columbia, Vancouver, Canada; 41Tokyo Medical and Dental University, Tokyo, Japan; 42Institute for Respiratory Health and; 43Centre for Cell Therapy and Regenerative Medicine, School of Biomedical Sciences, The University of Western Australia, Perth, Australia; 44Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island; 45Advanced Lung Disease and Transplant Program, Inova Fairfax Hospital, Falls Church, Virginia; 46Department of Pulmonary Medicine, Gazi University School of Medicine, Ankara, Turkey; 47Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama; 48Ruhrlandklinik, University Hospital, University of Duisburg-Essen, Essen, Germany; 49Department of Medicine, Tallaght University Hospital, Trinity College Dublin, Dublin, Ireland; 50CardioPulmonary Reserach Center, Alliance Pulmonary Group, Guaynabo, Puerto Rico; 51Department of Internal Medicine, University of Genoa, Genoa, Italy; 52Biomedical Genomics Center, University of Minnesota; Minneapolis, Minnesota; and 53Northwest Genomics Center, University of Washington, Seattle, Washington -- This research was supported by grants from the NHLBI (R01-HL097163, P01-HL092870, and UH3-HL123442) and the Department of Defense (W81XWH-17-1-0597). The COPDGene project was supported by Award Number U01 HL089897 and Award Number U01 HL089856 from the NHLBI. The COPDGene project was also supported by the COPD Foundation through contributions made to an industry advisory board comprised of AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novartis, Pfizer, Siemens, and Sunovion.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by an overall poor prognosis. The rate of disease decline and progression to death can vary, with some patients remaining stable over longer periods of time and others displaying rapid progression [1, 2]. The highly heterogeneous nature of this disease makes it difficult to elucidate the pathways involved in the initiation and progression of IPF and what determines the clinical trajectory of these patients. Advances in high-throughput gene expression analyses have led to improvements in our understanding of disease biology with insights into prognostic and predictive gene signatures [3]. This study sought to characterise differences in gene expression profiles found in the circulation between “stable” and “progressive” IPF patients, to help identify relevant biomarkers that may provide insights into the pathogenesis of progressive IPF. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Miss Clynick has nothing to disclose. Conflict of interest: Dr. Jo has nothing to disclose. Conflict of interest: Dr. Corte has nothing to disclose. Conflict of interest: Dr. Glaspole has nothing to disclose. Conflict of interest: Dr. Grainge has nothing to disclose. Conflict of interest: Dr. Hopkins has nothing to disclose. Conflict of interest: Dr. Reynolds has nothing to disclose. Conflict of interest: Dr. Chapman has nothing to disclose. Conflict of interest: Dr. Walters has nothing to disclose. Conflict of interest: Dr. Zappala has nothing to disclose. Conflict of interest: Dr. Keir has nothing to disclose. Conflict of interest: Dr. Cooper has nothing to disclose. Conflict of interest: Dr. Mahar has nothing to disclose. Conflict of interest: Dr. Ellis has nothing to disclose. Conflict of interest: Dr. Goh has nothing to disclose. Conflict of interest: Dr. Baltic has nothing to disclose. Conflict of interest: Miss Ryan has nothing to disclose. Conflict of interest: Dr. Tan has nothing to disclose. Conflict of interest: Dr. Moodley has nothing to disclose.

Complex lung diseases, such as asthma, are influenced by both genetic predisposition and environmental stimuli. The epigenetic landscape of such diseases is attracting increasing interest and research. Epigenetics broadly covers the transient and the inheritable changes to gene expression that are not directly due to changes in nucleotide sequences. Epigenetic mechanisms could have significant impact on asthma-related allergic, immune, and regulatory pathways, as well as on the generation of biomarkers and the heritable transmission of asthma phenotypes. Recent technological advances have allowed mapping of the epigenome and analysis of genome-wide epigenetic contributors to disease. As a result, ground-breaking observations regarding histone post-translational modifications in a number of immunological diseases have emerged. In this review, we look beyond the biological information coded by DNA and review the epigenetic modifications made to histones, with evidence suggesting a role for their modification in asthma.

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

Background To date, no genome-wide association study (GWAS) has considered the combined phenotype of asthma with hay fever. Previous analyses of family data from the Tasmanian Longitudinal Health Study provide evidence that this phenotype has a stronger genetic cause than asthma without hay fever. Objective We sought to perform a GWAS of asthma with hay fever to identify variants associated with having both diseases. Methods We performed a meta-analysis of GWASs comparing persons with both physician-diagnosed asthma and hay fever (n = 6,685) with persons with neither disease (n = 14,091). Results At genome-wide significance, we identified 11 independent variants associated with the risk of having asthma with hay fever, including 2 associations reaching this level of significance with allergic disease for the first time: ZBTB10 (rs7009110; odds ratio [OR], 1.14; P = 4 × 10 −9 ) and CLEC16A (rs62026376; OR, 1.17; P = 1 × 10 −8 ). The rs62026376:C allele associated with increased asthma with hay fever risk has been found to be associated also with decreased expression of the nearby DEXI gene in monocytes. The 11 variants were associated with the risk of asthma and hay fever separately, but the estimated associations with the individual phenotypes were weaker than with the combined asthma with hay fever phenotype. A variant near LRRC32 was a stronger risk factor for hay fever than for asthma, whereas the reverse was observed for variants in/near GSDMA and TSLP . Single nucleotide polymorphisms with suggestive evidence for association with asthma with hay fever risk included rs41295115 near IL2RA (OR, 1.28; P = 5 × 10 −7 ) and rs76043829 in TNS1 (OR, 1.23; P = 2 × 10 −6 ). Conclusion By focusing on the combined phenotype of asthma with hay fever, variants associated with the risk of allergic disease can be identified with greater efficiency.

Despite widespread exposure to potentially pathogenic mycobacteria present in the soil and in domestic water supplies, it is not clear why only a small proportion of individuals contract pulmonary nontuberculous mycobacterial (NTM) infections. Here, we explore the impact of polymorphisms within three genes: P2X ligand gated ion channel 7 (P2X7R), P2X ligand gated ion channel 4 (P2X4R) and calcium/calmodulin-dependent protein kinase kinase 2 beta (CAMKK2) on susceptibility. Thirty single nucleotide polymorphisms (SNPs) were genotyped in NTM patients (n = 124) and healthy controls (n = 229). Weak associations were found between individual alleles in P2X7R and disease but were not significant in multivariate analyses adjusted to account for gender. Haplotypes spanning the three genes were derived using the fastPHASE algorithm. This yielded 27 haplotypes with frequencies >1% and accounting for 63.3% of the combined cohort. In univariate analyses, seven of these haplotypes displayed associations with NTM disease above our preliminary cut-off (p ≤ 0.20). When these were carried forward in a logistic regression model, gender and one haplotype (SH95) were independently associated with the disease (model p < 0.0001; R2 = 0.05). Examination of individual alleles within these haplotypes implicated P2X7R and CAMKK2 in pathways affecting pulmonary NTM disease.

Although exposure to potentially pathogenic nontuberculous mycobacteria (NTM) via soil and domestic water supplies is common, pulmonary infection and disease are confined to a small proportion of older individuals. Previously, alleles of a polymorphism in IL10 (rs1800896) were associated with NTM disease and we demonstrated elevated production of IL-10 by blood leukocytes from patients with pulmonary NTM. Here seven additional polymorphisms in IL10 were investigated in a larger cohort of Caucasian controls and patients with pulmonary NTM disease. This demonstrated a significant association between pulmonary NTM disease and one polymorphism (rs1518111) in strong linkage disequilibrium with rs1800896.

INTRODUCTION Animal studies, and in vivo inoculation with rhinovirus, have shown that the epithelial-derived cytokine interleukin-33 (IL-33) is a major contributor to Th2-inflammation in acute asthma. However the link between IL-33 and Th2-inflammation has never been shown in naturally occurring cases of acute asthma. AIMS AND OBJECTIVES To determine whether IL-33 plays a role in driving Th2-inflammation in hospitalised acute asthma patients, we aimed to investigate whether the level of IL-33 in the nasal epithelium correlated with levels of Th2 cytokines in upper and lower airways. METHODS Patients admitted to hospital with an acute asthma exacerbation were recruited. Nasal swabs were used to sample nasal fluid representing upper airways, and induced sputum were collected, representing lower airways. Sampling was repeated after 4 weeks, and all samples analysed using Droplet Digital PCR for IL-33, IL-5 and IL-13. RESULTS Nineteen patients were recruited. Nasal IL-33 correlated with IL-5 in both upper and lower airways during acute asthma (0.75, p=0.008 and 0.74, p=0.010, respectively). Similar correlations were found with IL-13 (0.64, p=0.018 and 0.65, p=0.017, respectively). These associations were not significant at the 4-week follow-up. Sputum IL-33 was not associated with IL-5 or IL-13 in either sputum or in the nasal epithelium. CONCLUSION Nasal, but not sputum IL-33 is associated with Th2-promoting cytokines IL-5 and IL-13 in naturally occurring acute asthma cases. These findings support the role of IL-33 as an initiator of Th2-inflammation in acute asthma.

INTRODUCTION: Pro-inflammatory signalling by the receptor for advanced glycation end products (RAGE), has been described in stable asthma, and a protective role of soluble RAGE (esRAGE) is well established, but the role of esRAGE and RAGE ligands (including enRAGE) is unknown in acute asthma. We aimed to determine whether RAGE signalling is involved in the increased inflammation seen during asthma exacerbations. METHODS: Adult asthmatics were recruited on presentation with acute asthma to Bispebjerg University Hospital during a one-year period. Spirometry and blood samples were collected within 24 hours of admission and repeated after 12 weeks. RESULTS: Patients (n=47) were 16-45 years old and 70% were females. Serum enRAGE was higher (78 ng/ml 95%CI 62-99, p = 0.001) at exacerbation than follow-up (41 ng/ml 95%CI 29-57), while serum esRAGE was lower (96 pg/ml 95%CI 79-115 vs. 158 pg/ml 95%CI 134-187, respectively, p Both enRAGE and the ratio of enRAGE to esRAGE correlated negatively with FEV 1 at exacerbation adjusting for age, gender, ICS usage, blood neutrophil count and detection of respiratory pathogens (p=0.012, R 2 =0.26 and p=0.014, R 2 =0.26, respectively). Patients with a severe or life threatening exacerbation, as defined by BTS guidelines (n=23), had higher enRAGE (83.9 95%CI 56.8-123.8, p=0.046) than patients with a mild to moderate exacerbation (52.1 95%CI 39.1-69.5). CONCLUSION: A systemic, pro-inflammatory response mediated by RAGE, is increased during acute asthma and correlates with the severity of symptoms.

INTRODUCTION: TSLP is released from the airway epithelium in response to damage and is pivotal in orchestrating type 2 immune responses seen in allergic asthma. However, the role of TSLP in exacerbations of asthma has not been evaluated. AIM: We sought to investigate whether the epithelial damage associated with an asthma exacerbation would increase TSLP expression and whether this in turn would promote an increase in sputum eosinophils. METHODS: During 12 months, adult asthmatics, admitted to hospital with an asthma exacerbation, were recruited. Induced sputum was collected within 24 hours of admission. TSLP mRNA was quantified using Droplet Digital PCR. RESULTS: A total of 47 patients were recruited, of which 28 had sputum samples suitable for gene expression analysis (age 19-43, 61% females). Expression of TSLP mRNA in sputum correlated with the total count of non-squamous cells (Spearman9s rho 0.52, p=0.004). There was an inverse correlation between TSLP mRNA and %-eosinophils (rho -0.46, p=0.02), while there was no correlation with the percentage of neutrophils, macrophages, lymphocytes or columnar epithelial cells, respectively. In patients with suitable sputum at both exacerbation and follow-up (n=18), there was not a significant difference in TSLP expression (p=0.174). The same inverse correlation with TSLP and %-eosinophils was seen at follow-up (rho -0.49, p=0.04). CONCLUSIONS: TSLP expression in sputum correlates with total cell count, and, somewhat surprisingly, inversely with %-eosinophils. While TSLP is well described as a master switch of type 2 inflammation in stable asthma, these results indicate that other factors determine the inflammatory phenotype during asthma exacerbations.

Allergen-specific immunoglobulin E (present in allergic sensitization) has a central role in the pathogenesis of allergic disease. We performed the first large-scale genome-wide association study (GWAS) of allergic sensitization in 5,789 affected individuals and 10,056 controls and followed up the top SNP at each of 26 loci in 6,114 affected individuals and 9,920 controls. We increased the number of susceptibility loci with genome-wide significant association with allergic sensitization from three to ten, including SNPs in or near TLR6, C11orf30, STAT6, SLC25A46, HLA-DQB1, IL1RL1, LPP, MYC, IL2 and HLA-B. All the top SNPs were associated with allergic symptoms in an independent study. Risk-associated variants at these ten loci were estimated to account for at least 25% of allergic sensitization and allergic rhinitis. Understanding the molecular mechanisms underlying these associations may provide new insights into the etiology of allergic disease.

Background Functional variants in the interleukin-6 receptor gene (IL6R) are associated with asthma risk. We hypothesized that genes co-expressed with IL6R might also be regulated by genetic polymorphisms that are associated with asthma risk. The aim of this study was to identify such genes. Methods To identify genes whose expression was correlated with that of IL6R, we analyzed gene expression levels generated for 373 human lymphoblastoid cell lines by the Geuvadis consortium and for 38 hematopoietic cell types by the Differentiation Map Portal (DMAP) project. Genes correlated with IL6R were then screened for nearby single nucleotide polymorphisms (SNPs) that were significantly associated with both variation in gene expression levels (eSNPs) and asthma risk. Results We identified 90 genes with expression levels correlated with those of IL6R and that also had a nearby eSNP associated with disease risk in a published asthma GWAS (N = 20 776). For 16 (18%) genes, the association between the eSNP and asthma risk replicated with the same direction of effect in a further independent published asthma GWAS (N = 27 378). Among the top replicated associations (FDR < 0.05) were eSNPs for four known (IL18R1, IL18RAP, BCL6, and STAT6) and one putative novel asthma risk gene, stomatin-like protein 2 (STOML2). The expression of STOML2 was negatively correlated with IL6R, while eSNPs that increased the expression of STOML2 were associated with an increased asthma risk. Conclusion The expression of STOML2, a gene that plays a key role in mitochondrial function and T-cell activation, is associated with both IL-6 signaling and asthma risk.

This study tested the hypothesis that proinflammatory kinin peptides are involved in modulating human dendritic cell (DC) function. Inflammation is accompanied by an increased maturation of DCs and the generation of kinins, particularly Lys-des[Arg(9)]-bradykinin (Lda-BK). We assessed the role of Lda-BK in the activation and migration of human monocyte-derived DCs (hMo-DCs) matured through the use of LPS, TNF-α + IL-1β, or CD40 ligand. Kinin B(1) and B(2) receptor mRNA and protein expression were assessed by confocal microscopy, flow cytometry, and RT-PCR. The effects of Lda-BK on the migration of mature hMo-DCs were assessed in Transwell chambers, whereas the expression of costimulatory molecules and the secretion of IL-12 were assessed by flow cytometry and ELISA, respectively. The expression of the kinin B(1) receptor (B(1)R) was down-regulated during the maturation of hMo-DCs, whereas the expression of B(2)R was unchanged. The B(1)R agonist Lda-BK was not chemotactic for hMo-DCs matured using LPS, TNF-α + IL-1β, or CD40 ligand, but Lda-BK enhanced the secretion of IL-12p70 and inhibited the secretion of IL-12p40 by mature hMo-DCs. However, the exposure of hMo-DCs matured with TNF-α + IL-1β to Lda-BK for 6 hours decreased subsequent migration in response to Lda-BK, the chemokine CCL19, or Lda-BK combined with CCL19. The expression of B(1)R was increased in hMo-DCs from subjects with asthma compared with subjects without asthma, in keeping with a tendency toward increased in vitro migration of asthmatic hMo-DCs in response to Lda-BK. The increased formation of Lda-BK and the enhanced expression of B(1)R as a consequence of inflammation may alter the migration of mature, antigen-laden DCs to regional lymph nodes in response to CCL19, may modulate the secretion of cytokines by these DCs, and may contribute to the accumulation of mature DCs in the lungs of patients with asthma.

Cystic Fibrosis (CF) is often accompanied by diabetes leading to worsening lung function, the reason for which is unclear. The receptor for advanced-glycation-end-products (RAGE) regulates immune responses and inflammation and has been linked to diabetes and possibly CF. We performed a pilot study to determine if CF and CF-related diabetes (CFRD) are associated with enhanced RAGE expression. Full length (fl)RAGE, soluble (s)RAGE, endogenous soluble (es)RAGE, S100A12 (enRAGE) and advanced-glycation-end-products (AGE) expression was assessed in serum, white blood cells and sputum of patients with CF; diabetes; CFRD and healthy subjects. Sputum enRAGE/sRAGE ratios were high in CF but particularly in CFRD which negatively correlated with % predicted FEV1. Serum AGE and AGE/sRAGE ratios were high in diabetics but not in CF. A complex, multifaceted approach was used to assess the role of RAGE and its ligands which is fundamental to determining their impact on airway inflammation. There is a clear association between RAGE activity in the airways of CF and CFRD patients that is not evident in the vascular compartment and correlates with lung function, in contrast to diabetes. This strongly suggests a role for RAGE in contributing to the inflammatory overdrive seen in CF and to a greater extent in CFRD.

Introduction. Premature ovarian failure (POF) is characterized by amenorrhea, hypergonadotropism and hypoestrogenism in women bellow 40 years. Osteoporosis is one of the late complications of POF. Objective. To correlate collagen type I alpha1 (COLIA1) gene polymorphism with bone mineral density (BMD) in women with POF. Methods. We determined the COLIA1 genotypes SS, Ss, ss in 66 women with POF. Single nucleotide polymorphism (G to T substitution) within the Sp 1-binding site in the first intron of the COLIA1 gene was assessed by polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism (SSCP) analysis. Bone mineral density (BMD) was measured at the lumbar spine region by dual X-ray absorptiometry. Statistics: Kruskal-Wallis ANOVA, Chisquare test, Spearman correlation test. Results. The relative distribution of COLIA1 genotype alleles was SS - 54.4%, Ss - 41.0% and ss - 4.5%. No significant differences were found between genotype groups in body mass index, age, duration of amenorrhea or BMD. A significant positive correlation was observed between BMI and parity. Conclusion. The COLIA1 gene is just one of many genes influencing bone characteristics. It may act as a marker for differences in bone quantity and quality, bone fragility and accelerated bone loss in older women. However, in young women with POF, COLIA1 cannot identify those at higher risk for osteoporosis. [Projekat Ministarstva nauke Republike Srbije, br. ON 173056]

Background The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown. Methodology/Principal Findings Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour. Conclusions/Significance We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.

Background: The genetic basis for developing asthma has been extensively studied. However, association studies to date have mostly focused on mild to moderate disease and genetic risk factors for severe asthma remain unclear. Objective: To identify common genetic variants affecting susceptibility to severe asthma. Methods: A genome-wide association study was undertaken in 933 European ancestry individuals with severe asthma based on Global Initiative for Asthma (GINA) criteria 3 or above and 3346 clean controls. After standard quality control measures, the association of 480?889 genotyped single nucleotide polymorphisms (SNPs) was tested. To improve the resolution of the association signals identified, non-genotyped SNPs were imputed in these regions using a dense reference panel of SNP genotypes from the 1000 Genomes Project. Then replication of SNPs of interest was undertaken in a further 231 cases and 1345 controls and a meta-analysis was performed to combine the results across studies. Results: An association was confirmed in subjects with severe asthma of loci previously identified for association with mild to moderate asthma. The strongest evidence was seen for the ORMDL3/GSDMB locus on chromosome 17q12-21 (rs4794820, p=1.03×10 (-8) following meta-analysis) meeting genome-wide significance. Strong evidence was also found for the IL1RL1/IL18R1 locus on 2q12 (rs9807989, p=5.59×10 (-8) following meta-analysis) just below this threshold. No novel loci for susceptibility to severe asthma met strict criteria for genome-wide significance. Conclusions: The largest genome-wide association study of severe asthma to date was carried out and strong evidence found for the association of two previously identified asthma susceptibility loci in patients with severe disease. A number of novel regions with suggestive evidence were also identified warranting further study. Copyright Article author (or their employer) 2012.

Summary Background We aimed to identify novel genetic variants affecting asthma risk, since these might provide novel insights into molecular mechanisms underlying the disease. Methods We did a genome-wide association study (GWAS) in 2669 physician-diagnosed asthmatics and 4528 controls from Australia. Seven loci were prioritised for replication after combining our results with those from the GABRIEL consortium (n=26 475), and these were tested in an additional 25 358 independent samples from four in-silico cohorts. Quantitative multi-marker scores of genetic load were constructed on the basis of results from the GABRIEL study and tested for association with asthma in our Australian GWAS dataset. Findings Two loci were confirmed to associate with asthma risk in the replication cohorts and reached genome-wide significance in the combined analysis of all available studies (n=57 800): rs4129267 (OR 1·09, combined p=2·4×10 −8 ) in the interleukin-6 receptor ( IL6R ) gene and rs7130588 (OR 1·09, p=1·8×10 −8 ) on chromosome 11q13.5 near the leucine-rich repeat containing 32 gene ( LRRC32 , also known as GARP ). The 11q13.5 locus was significantly associated with atopic status among asthmatics (OR 1·33, p=7×10 −4 ), suggesting that it is a risk factor for allergic but not non-allergic asthma. Multi-marker association results are consistent with a highly polygenic contribution to asthma risk, including loci with weak effects that might be shared with other immune-related diseases, such as NDFIP1 , HLA-B , LPP , and BACH2 . Interpretation The IL6R association further supports the hypothesis that cytokine signalling dysregulation affects asthma risk, and raises the possibility that an IL6R antagonist (tocilizumab) may be effective to treat the disease, perhaps in a genotype-dependent manner. Results for the 11q13.5 locus suggest that it directly increases the risk of allergic sensitisation which, in turn, increases the risk of subsequent development of asthma. Larger or more functionally focused studies are needed to characterise the many loci with modest effects that remain to be identified for asthma. Funding National Health and Medical Research Council of Australia. A full list of funding sources is provided in the webappendix.

The kinins, bradykinin (BK) and Lys-des[Arg9]-BK, are important inflammatory mediators that act via two specific G protein-coupled kinins, B1 and B2 receptors (B2R). Kinins influence the activity of immune cells by stimulating the synthesis of cytokines, eicosanoids, and chemotactic factors. Whether human dendritic cells (DC) express kinin receptors and whether kinins influence DC function are unknown. Fluorescence immunocytochemistry and RT-PCR were used to demonstrate that immature human monocyte-derived DC (hMo-DC) constitutively expressed kinins B1R and B2R. Kinin receptor expression was induced on the 3rd and 4th days of culture during differentiation of hMo-DC from monocytes and was not dependent on the presence of IL-4 or GM-CSF. Although monocytes also expressed B2R mRNA, the protein was not detected. The kinin agonists BK and Lys-des[Arg9]-BK up-regulated the expression of their respective receptors. BK, acting via the B2R, increased intracellular Ca2+, as visualized by confocal microscopy using the fluorescent Ca2+ dye, Fluor-4 AM. Evaluation of migration in Trans-well chambers demonstrated significant enhancement by BK of migration of immature hMo-DC, which was B2R-dependent. However, kinins did not induce maturation of hMo-DC. The novel finding that kinin receptors are constitutively expressed in immature hMo-DC suggests that these receptors may be expressed in the absence of proinflammatory stimuli. BK, which increases the migration of immature hMo-DC in vitro, may play an important role in the migration of immature DC in noninflammatory conditions and may also be involved in the recruitment of immature DC to sites of inflammation.

Funded by: UK Medical Research Council, British Heart Foundation, UK National Institute for Health Research, National Institute for Health Research Cambridge Biomedical Research Centre, European Research Council, and European Commission Framework Programme 7. To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation. Methods: We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453 411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746 171 total participants). Findings: For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0·22 SD (95% CI 0·18-0·25; 12·5%; p=9·3 × 10), concentrations of interleukin 6 decreased by 0·02 SD (-0·04 to -0·01; -1·7%; p=3·5 × 10), and concentrations of C-reactive protein decreased by 0·03 SD (-0·04 to -0·02; -3·4%; p=7·7 × 10). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1·15 (1·08-1·22; p=1·8 × 10) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1·03 (1·02-1·04; p=3·9 × 10). Per-allele odds ratios were 0·97 (0·95-0·99; p=9·9 × 10) for rheumatoid arthritis, 0·99 (0·97-1·01; p=0·47) for type 2 diabetes, 1·00 (0·98-1·02; p=0·92) for ischaemic stroke, and 1·08 (1·04-1·12; p=1·8 × 10) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk. Interpretation: Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations. Publisher PDF

The region spanning the tumor necrosis factor (TNF) cluster in the human major histocompatibility complex (MHC) has been implicated in susceptibility to numerous immunopathological diseases, including type 1 diabetes mellitus and rheumatoid arthritis. However, strong linkage disequilibrium across the MHC has hampered the identification of the precise genes involved. In addition, the observation of "blocks" of DNA in the MHC within which recombination is very rare, limits the resolution that may be obtained by genotyping individual SNPs. Hence a greater understanding of the haplotypes of the block spanning the TNF cluster is necessary. To this end, we genotyped 32 human leukocyte antigen (HLA)-homozygous workshop cell lines and 300 healthy control samples for 19 coding and promoter region SNPs spanning 45 kb in the central MHC near the TNF genes. The workshop cell lines defined 11 SNP haplotypes that account for approximately 80% of the haplotypes observed in the 300 control individuals. Using the control individuals, we defined a further six haplotypes that account for an additional 10% of donors. We show that the 17 haplotypes of the "TNF block" can be identified using 15 SNPs.

BAT1 (D6S81E, UAP56) lies in the central MHC between TNF and HLA-B, a region containing genes that affect susceptibility to immunopathologic disorders. BAT1 protein may be directly responsible for the genetic association, as antisense studies show it can down-regulate inflammatory cytokines. Here we investigate polymorphisms at positions -22 and -348 relative to the BAT1 transcription start site. DNA samples from healthy donors were used to confirm haplotypic associations with the type 1 diabetes-susceptible 8.1 ancestral haplotype (AH; HLA-A1,B8,BAT1-22*C,BAT1-348*C,DR3 ) and the diabetes-resistant 7.1 AH (HLA-A3,B7,BAT1-22*G,BAT1-348*T,DR15). Alleles carried at BAT1-22 and -348 were in linkage disequilibrium. Electrophoretic mobility shift assays using nuclear proteins from T-cells (Jurkat and HT2), monocytes (THP1, U937) and epithelial cells (HeLa and MDA468) demonstrated DNA : protein complexes binding oligonucleotides spanning positions -22 and -348 on the 7.1 AH only. Competition assays, supershifts and molecular weight determinations suggest the complexes include the transcription factors YY1 (at -348) and Oct1 (at -22). Promoter activity was demonstrated using 520 bp and 336 bp fragments cloned from immediately upstream of the transcription start site and carrying all combinations of -22 and -348 alleles, suggesting an unidentified non-polymorphic sequence within 336 bp of the start site drives transcription. The 520 bp fragment of the BAT1 promoter cloned from the 8.1 AH was slightly less efficient than the equivalent from the 7.1 AH, whilst the reverse was observed with 336 bp fragments. This suggests BAT1 transcription on the 7.1 AH is modified by interactions involving DNA flanking positions -22 and -348.

The kinin B1 receptor (B1R) is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood. We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV) of human B1R gene with a shorter 5′untranslated region. This novel SV is ≈35% less stable than the wild-type (WT) transcript in lung adenocarcinoma cells (H2126), but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE) −1020 to −766 bp in H2126; positive response element (PRE) −766 to −410 bp in 16HBE; −410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells) were found. These findings reveal complex regulation of B1R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer.

Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose–response titrations.