Human type-2 CD8 T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia-a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8CRTH2 (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D (PGD) and cysteinyl leukotriene E (LTE) are also increased in the airways of the same group of patients. In vitro PGD and LTE function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

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Background Chronic obstructive pulmonary disease (COPD) is predominantly associated with neutrophilic inflammation. Active neutrophil elastase (NE) is a serine proteinase, secreted by neutrophils, in response to inflammation and pathogen invasion. We sought to investigate if NE could be used as a biomarker for bacterial infection in patients with COPD. Methods NE was quantified using ProteaseTag® Active NE Immunoassay (ProAxsis, Belfast) from the sputum of COPD subjects at stable state, exacerbation and 2 weeks post treatment visit. Results NE was measured in 90 samples from 30 COPD subjects (18 males) with a mean (range) age of 65 (45–81) years and mean (SD) FEV 1 of 47% (18). The geometric mean (95%CI) of NE at stable state was 2454 ng/mL (1460 to 4125 ng/mL). There was a significant increase in NE levels at an exacerbation ( p = 0.003), and NE levels were higher in a bacterial-associated exacerbation (NE log difference 3.873, 95% CI of log difference 1.396 to 10.740, p = 0.011). NE was an accurate predictor of a bacteria-associated exacerbation (area (95%CI) under the receiver operator characteristic curve 0.812 (0.657 to 0.968). Conclusion NE is elevated during exacerbations of COPD. NE may be a viable biomarker for distinguishing a bacterial exacerbation in patients with COPD. Trial registration Leicestershire, Northamptonshire and Rutland ethics committee (reference number: 07/H0406/157 ).

Mycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation experiments employed Mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved MTB detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) occurred at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealing initial sample volumes ≥1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

To explore whether fractional exhaled nitric oxide (FeNO) non-suppression identifies corticosteroid resistance, we analysed inflammatory mediator changes during a FeNO suppression test with monitored high-intensity corticosteroid therapy. In linear mixed-effects models analysed over time, the 15 clinically distinct ‘suppressors’ (ie, ≥42% FeNO suppression) normalised Asthma Control Questionnaire scores (mean±SD, start to end of test: 2.8±1.4 to 1.4±0.9, p<0.0001) and sputum eosinophil counts (median (IQR), start to end of test: 29% (6%–41%) to 1% (1%–5%), p=0.0003) while significantly decreasing sputum prostaglandin D2 (254 (89–894) to 93 (49–209) pg/mL, p=0.004) and numerically decreasing other type-2 cytokine, chemokine and alarmin levels. In comparison, the 19 non-suppressors had persistent sputum eosinophilia (10% (1%–67%) despite high-intensity therapy) with raised end-test inflammatory mediator levels (1.9 (0.9–2.8)-fold greater than suppressors). FeNO non-suppression during monitored treatment implies biological corticosteroid resistance.

BackgroundPentraxin 3 (PTX3) is an acute phase protein, involved in antibacterial resistance. Recent studies have shown PTX3 levels to be elevated in the presence of a bacterial infection and in a murine sepsis model.ObjectiveWe aim to investigate if sputum PTX3 can be used as a biomarker for bacterial infection in subjects with COPD.Materials and methodsSputum samples from 142 COPD patients (102 men) with a mean (range) age of 69 years (45–85) and mean (SD) post-bronchodilator percentage predicted forced expiratory volume in 1 second (FEV1) of 50% (19) were analyzed for PTX3, using a commercial assay at stable state and during an exacerbation. Association with bacteria, from culture, quantitative real-time polymerase chain reaction (qPCR) and colony-forming units (CFU) was investigated.ResultsThe geometric mean (95% CI) PTX3 level at stable state was 50.5 ng/mL (41.4–61.7). PTX3 levels correlated with absolute neutrophil count in sputum (r=0.37; P<0.01), but not FEV1 or health status. There was a weak correlation between PTX3 and bacterial load (CFU: r=0.29, P<0.01; 16S qPCR: r=0.18, P=0.05). PTX3 was a poor predictor of bacterial colonization (defined as >105 CFU/mL at stable state) with a receiver-operating characteristic (ROC) area under the curve (AUC) of 0.59 and 95% confidence interval (CI) 0.43–0.76 (P=0.21). During an exacerbation, there was a modest increase in PTX3 (fold difference 0.15, 95% of difference 0.02–0.29; P=0.02), and PTX3 fared better at identifying a bacteria-associated exacerbation (ROC AUC 0.65, 95% CI 0.52–0.78, P=0.03).ConclusionPTX3 is associated with bacterial infection in patients with COPD, but its utility as a biomarker for identifying a bacteria-associated exacerbation warrants further studies.

37Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid 38 diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The 39 approach is especially advantageous for infections caused by species which grow very slowly 40 in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as 41 little as 0.01% of the total DNA, enrichment of the input material for target sequences by 42 specific amplification and, or depletion of non-target DNA (human, other bacteria) is 43 essential for success. Here, we investigated the potential of isothermal multiple displacement 44 amplification by Phi29 polymerase. We directed the amplification reaction towards 45 Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, 46 their high GC content (approximately 65%) relative to human DNA. Amplified DNA was 47 then sequenced using the Oxford Nanopore Technology MinION. In addition, a model 48 system comprising standardised 'mock clinical samples' was designed. Pooled infection 49 negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) 50 Pasteur strain at concentrations spanning the typical range at which Mycobacterium 51 tuberculosis is found in human sputum samples (10 6 -10 1 BCG cells/ml). To assess the 52 amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and 53 after amplification. Reads from amplified samples, which mapped to a BCG reference 54 genome, comprised short repeated sequences -apparently transcribed multiple times from the 55 same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for 56 BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, 57 increasing to 28,617 at 10 6 BCG cells/ml sample), but BCG genome coverage declined 58 markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical 59 samples allowed direct comparison of data from different Mycobacteria enrichment 3 60 experiments and sequencing runs. However, optimal conditions for multiple displacement 61 amplification of minority Mycobacteria DNAs, remain to be identified. 4 62 5 87 concentration of target organisms. Mycobacteria DNA, for example, can represent as little as 88 0.01% of the total DNA extracted from sputum [11]. Small scale studies employing direct 89 from sample sequencing have reported 0.002 -0.7% sequence coverage of the M. 90 tuberculosis genome (using differential lysis and a DNA extraction kit) [12], and up to 90% 91 genome coverage with 20x depth (20/24 samples), (using the SureSelect target enrichment 92 method, Agilent, USA) [13, 14]. The study by Brown et al. [13] predicted both Mycobacteria 93 species and antibiotic susceptibility, but the cost ($350 per sample) and duration of the 94 protocol (2 to 3 days) could prevent its use. The ideal 'direct from sample' methodology 95 would be simple, low cost and portable, to facilitate use in remote, low-income settings 96 ...

Background Chronic obstructive pulmonary disease (COPD) and asthma have heterogeneous inflammation with inhaled corticosteroids (ICS) as a mainstay of treatment. There is increased prevalence of non-typeable Haemophilus influenzae (NTHi) persistence in airways of patients with neutrophilic airway inflammation, potentially due to suppressed host defence after corticosteroid treatment. Antimicrobial peptides (AMPs) have antimicrobial activity against pathogens and immunomodulatory effects. We investigated whether AMPs associate with NTHi presence in COPD and asthma, and whether ICS alter this. Methods Secretory leukocyte protease inhibitor (SLPI), osteopontin, elafin and beta defensin-1 were measured in sputum supernatants from healthy donors (n=9), asthmatics (n=21) and patients with COPD (n=14). Elafin and beta defensin-1 were measured in a primary human bronchial epithelial cells (HBECs) from healthy and COPD donors infected with NTHi and pre-treated with fluticasone propionate (FP) and budesonide (BUD). Internalised NTHi was quantified by qPCR. Results Sputum SLPI was negatively correlated with FEV1 (p<0.001, r=−0.610), FEV1% predicted (p<0.001, r=−0.583) and FEV1/FVC (p=0.001, r=−0.528). Sputum beta defensin-1 was negatively associated with FEV1 (p<0.001***r=−0.594). SLPI and beta defensin-1 levels in sputum were higher in the healthy controls and COPD group compared to the asthma group (p=0.001 and p=0.014) and (p<0.001 and p=0.007, respectively). ICS use was associated with higher sputum osteopontin compared to those with no ICS use. NTHi infection of COPD HBECs produced higher levels of beta defensin-1 compared to healthy donors (mean (SD) release: 45.1pg/mL (7.3) vs 21.2pg/mL (7.3) respectively, p=0.014). Elafin release from HBECs from COPD donors did not change following NTHi infection; however, elafin from healthy donors was significantly reduced (%mean reduction: 23.7%, 95% confidence intervals (CI) of reduction: 5.3–38.4%, p<0.01). Conclusion Sputum SLPI and beta defensin-1 may be markers to identify those patients with declining lung function. ICS use was associated with higher sputum osteopontin compared to those with no ICS use.