Clinical and transcriptomic features of persistent exacerbation-prone severe asthma in U-BIOPRED cohort

Background: Exacerbation-prone asthma is a feature of severe disease. Yet, the basis for its persistency remains unclear. Objectives: To determine the clinical and transcriptomic features of the frequent-exacerbator (FE) and of persistent FEs (PFE) in U-BIOPRED cohort. Methods: We compared features of FE (≥2 exacerbations in past year) to infrequent exacerbators (IE, <2 exacerbations) and of PFE with repeat ≥2 exacerbations during the following year to persistent IE (PIE). Transcriptomic data in blood, bronchial and nasal epithelial brushings, bronchial biopsies and sputum cells were analysed by gene set variation analysis for 103 gene signatures. Results: Of 317 patients, 62.4 % were FE of whom 63.6% were PFE, while 37.6% were IE of whom 61.3% were PIE. Using multivariate analysis, FE was associated with short-acting beta-agonist use, sinusitis and daily oral corticosteroid use, while PFE with eczema, short-acting beta-agonist use and asthma control index. CEA Cell Adhesion Molecule 5 (CEACAM5) was the only differentially-expressed transcript in bronchial biopsies between PE and IE. There were no differentially-expressed genes in the other 4 compartments. There were higher expression scores for Type 2 , T-helper type-17 and Type 1 pathway signatures together with those associated with viral infections in bronchial biopsies from FE compared to IE, while higher expression scores of Type 2, Type 1 and steroid insensitivity pathway signatures in bronchial biopsies of PFE compared to PIE. Conclusion: FE group and its PFE subgroup are associated with poor asthma control while expressing higher Type 1 and Type 2 activation pathways compared to IE and PIE, respectively

Sputum proteomics and airway cell transcripts of current and ex-smokers with severe asthma in U-BIOPRED: an exploratory analysis

Background: Severe asthma patients with a significant smoking history have airflow obstruction with reported neutrophilia. We hypothesise that multi-omic analysis will enable the definition of smoking and ex-smoking severe asthma molecular phenotypes. Methods: The U-BIOPRED severe asthma patients containing current-smokers (CSA), exsmokers (ESA), non-smokers (NSA) and healthy non-smokers (NH) was examined. Blood and sputum cell counts, fractional exhaled nitric oxide and spirometry were obtained. Exploratory proteomic analysis of sputum supernatants and transcriptomic analysis of bronchial brushings, biopsies and sputum cells was performed. Results: Colony stimulating factor (CSF)2 protein levels were increased in CSA sputum supernatants with azurocidin 1, neutrophil elastase and CXCL8 upregulated in ESA. Phagocytosis and innate immune pathways were associated with neutrophilic inflammation in ESA. Gene Set Variation Analysis of bronchial epithelial cell transcriptome from CSA showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to other groups. CXCL5 and matrix metallopeptidase 12 genes were upregulated in ESA and the epithelial protective genes, mucin 2 and cystatin SN, were downregulated. Conclusion: Despite little difference in clinical characteristics, CSA were distinguishable from ESA subjects at the sputum proteomic level with CSA having increased CSF2 expression and ESA patients showed sustained loss of epithelial barrier processes

The effect of BMP-2 and its inhibitors on fracture repair

The aims of this thesis were to investigate certain aspects of the regulation of osteogenesis by BMPs and their physiological inhibitors; to determine whether the balance between the two could have an effect on fracture repair; and to suggest strategies for using this information to provide orthopaedic surgeons with better ways to treat recalcitrant fractures . BMP-2 and its inhibitor chordin were produced endogenously during the in vitro osteogenic differentiation of human mesenchymal stem cells (MSCs) in response to dexamethasone. The addition of exogenous BMP-2 increased the rate of osteogenic differentiation of the MSCs. Knockdown of the BMP inhibitor chordin led to an increase in the rate of osteogenic differentiation of human MSCs, secondary to an increase in the bioavailability of BMP-2. These results raise the possibility that the impaired healing of delayed- and non-unions reflects alterations in the ratios of BMPs to their inhibitors. They further suggest that it may be possible to increase the rate of osteogenesis, and thus improve fracture repair in vivo, by down-regulating the endogenous production of BMP inhibitors. To evaluate these possibilities further, the presence of BMP-2 and -14, as well as their inhibitors chordin and noggin, were investigated by semi-quantitative immunohistochemistry in human fracture biopsies. The expression of BMP-2, BMP-14, chordin and noggin during fracture repair was demonstrated in areas of cartilage and bone formation. Levels of expression of these proteins were compared between healing and non-healing human fractures. There was a decreased relative expression of BMP-2 and BMP-14, compared to the BMP inhibitors, in the non-healing fractures v/s the healing fractures. This suggests an imbalance between BMPs and their inhibitors in fractures that do not heal. These data indicate novel ways to reduce rates of non-union by the local application of BMP-2 or BMP-14, or the blocking of BMP antagonists. Fracture healing may also be impaired by drugs used clinically. The antibiotic tobramycin, commonly used to treat or prevent infections of bone, was shown to reduce the osteogenic potential of human MSCs in vitro. An in vivo study was carried out to determine whether a clinically relevant dose of tobramycin would impair fracture repair induced by BMP-2. The biomechanical and radiological properties of the repair tissue, induced by BMP-2 in a rat femoral defect model, were not affected by the presence of tobramycin. Overall, this thesis demonstrates that the balance between BMPs and their inhibitors has a major influence on the rate of fracture repair and that this balance is altered in non-healing fractures. The data suggest novel, clinically relevant, biological strategies for enhancing bone healing.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The effect of BMP-2 and its inhibitors on fracture repair

The aims of this thesis were to investigate certain aspects of the regulation of osteogenesis by BMPs and their physiological inhibitors; to determine whether the balance between the two could have an effect on fracture repair; and to suggest strategies for using this information to provide orthopaedic surgeons with better ways to treat recalcitrant fractures . BMP-2 and its inhibitor chordin were produced endogenously during the in vitro osteogenic differentiation of human mesenchymal stem cells (MSCs) in response to dexamethasone. The addition of exogenous BMP-2 increased the rate of osteogenic differentiation of the MSCs. Knockdown of the BMP inhibitor chordin led to an increase in the rate of osteogenic differentiation of human MSCs, secondary to an increase in the bioavailability of BMP-2. These results raise the possibility that the impaired healing of delayed- and non-unions reflects alterations in the ratios of BMPs to their inhibitors. They further suggest that it may be possible to increase the rate of osteogenesis, and thus improve fracture repair in vivo, by down-regulating the endogenous production of BMP inhibitors. To evaluate these possibilities further, the presence of BMP-2 and -14, as well as their inhibitors chordin and noggin, were investigated by semi-quantitative immunohistochemistry in human fracture biopsies. The expression of BMP-2, BMP-14, chordin and noggin during fracture repair was demonstrated in areas of cartilage and bone formation. Levels of expression of these proteins were compared between healing and non-healing human fractures. There was a decreased relative expression of BMP-2 and BMP-14, compared to the BMP inhibitors, in the non-healing fractures v/s the healing fractures. This suggests an imbalance between BMPs and their inhibitors in fractures that do not heal. These data indicate novel ways to reduce rates of non-union by the local application of BMP-2 or BMP-14, or the blocking of BMP antagonists. Fracture healing may also be impaired by drugs used clinically. The antibiotic tobramycin, commonly used to treat or prevent infections of bone, was shown to reduce the osteogenic potential of human MSCs in vitro. An in vivo study was carried out to determine whether a clinically relevant dose of tobramycin would impair fracture repair induced by BMP-2. The biomechanical and radiological properties of the repair tissue, induced by BMP-2 in a rat femoral defect model, were not affected by the presence of tobramycin. Overall, this thesis demonstrates that the balance between BMPs and their inhibitors has a major influence on the rate of fracture repair and that this balance is altered in non-healing fractures. The data suggest novel, clinically relevant, biological strategies for enhancing bone healing

“T2-high” in severe asthma related to blood eosinophil, exhaled nitric oxide and serum periostin

Copyright ©ERS 2019. Type-2 (T2) immune responses in airway epithelial cells (AECs) classifies mild–moderate asthma into a T2-high phenotype. We examined whether currently available clinical biomarkers can predict AEC-defined T2-high phenotype within the U-BIOPRED cohort. The transcriptomic profile of AECs obtained from brushings of 103 patients with asthma and 44 healthy controls was obtained and gene set variation analysis used to determine the relative expression score of T2 asthma using a signature from interleukin (IL)-13-exposed AECs. 37% of asthmatics (45% nonsmoking severe asthma, n=49; 33% of smoking or ex-smoking severe asthma, n=18; and 28% mild–moderate asthma, n=36) were T2-high using AEC gene expression. They were more symptomatic with higher exhaled nitric oxide fraction (FeNO) and blood and sputum eosinophils, but not serum IgE or periostin. Sputum eosinophilia correlated best with the T2-high signature. FeNO (30 ppb) and blood eosinophils (300 cells·µL −1 ) gave a moderate prediction of T2-high asthma. Sputum IL-4, IL-5 and IL-13 protein levels did not correlate with gene expression. T2-high severe asthma can be predicted to some extent from raised levels of FeNO, blood and sputum eosinophil counts, but serum IgE or serum periostin were poor predictors. Better bedside biomarkers are needed to detect T2-high