Discovery and Validation of a Volatile Signature of Eosinophilic Airway Inflammation in Asthma.

Rationale: Volatile organic compounds (VOCs) in asthmatic breath may be associated with sputum eosinophilia. We developed a volatile biomarker signature to predict sputum eosinophilia in asthma. Methods: VOCs emitted into the space above sputum samples (headspace) from patients with severe asthma (n = 36) were collected onto sorbent tubes and analyzed using thermal desorption gas chromatography-mass spectrometry (GC-MS). Elastic net regression identified stable VOCs associated with sputum eosinophilia ⩾ 3% and generated a volatile biomarker signature. This VOC signature was validated in breath samples from: 1) patients with acute asthma according to blood eosinophilia ⩾0.3 × 109cells/L or sputum eosinophilia of ⩾3% in the UK EMBER (East Midlands Breathomics Pathology Node) consortium (n = 65) and 2) U-BIOPRED-IMI (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes Innovative Medicines Initiative) consortium (n = 42). Breath samples were collected onto sorbent tubes (EMBER) or Tedlar bags (U-BIOPRED) and analyzed by GC-MS (GC × GC-MS for EMBER or GC-MS for U-BIOPRED). Measurements and Main Results: The in vitro headspace identified 19 VOCs associated with sputum eosinophilia, and the derived VOC signature yielded good diagnostic accuracy for sputum eosinophilia ⩾3% in headspace (area under the receiver operating characteristic curve [AUROC] 0.90; 95% confidence interval [CI], 0.80-0.99; P < 0.0001), correlated inversely with sputum eosinophil percentage (rs = -0.71; P < 0.0001), and outperformed fractional exhaled nitric oxide (AUROC 0.61; 95% CI, 0.35-0.86). Analysis of exhaled breath in replication cohorts yielded a VOC signature AUROC (95% CI) for acute asthma exacerbations of 0.89 (0.76-1.0) (EMBER cohort) with sputum eosinophilia and 0.90 (0.75-1.0) in U-BIOPRED, again outperforming fractional exhaled nitric oxide in U-BIOPRED (0.62 [0.33-0.90]). Conclusions: We have discovered and provided early-stage clinical validation of a volatile biomarker signature associated with eosinophilic airway inflammation. Further work is needed to translate our discovery using point-of-care clinical sensors.

The causes of a peripheral blood eosinophilia in a secondary care setting.

BACKGROUND: A peripheral blood eosinophilia of greater than 1.0 × 109 /L is relatively unusual and offers a clue to the underlying diagnosis. In 2003, we established a specialist service to diagnose unexplained eosinophilia. OBJECTIVE: To describe the causes of an eosinophilia in our service and the diagnostic algorithm we developed. METHODS: Subjects were referred by physician colleagues across a range of specialties and undertook standard investigations following a semi-structured protocol. Data were extracted from a bespoke database. RESULTS: Three hundred and eighty two subjects were referred over a 17-year period. Average age was 54 years and 183 (48%) of subjects were female, with 21 of 25 (84%) females in the idiopathic eosinophilic pneumonia group (p < 0001), 22 of 30 (73%) females in the gastrointestinal disease group (p < .008), but 11 of 37 (30%) females in the eosinophilic granulomatosis with polyangiitis group (p < .04). A diagnosis was assigned after systematic evaluation using a pre-defined algorithm in 361 (94.5%) of cases. Fungal allergy (82 subjects: 21%), parasitic infection (61 subjects: 16%) and severe eosinophilic asthma (50 subjects: 13%) were the three commonest individual diagnoses. Hypereosinophilic syndrome (HES) disease including eosinophilic granulomatosis with polyangiitis (EGPA) accounted for 85 subjects (20%) of which seven subjects (2%) had myeloproliferative disease (M-HES). A high IgE was common, and 79 (91%) of subjects with complete data who had an IgE of ≥1000 IU/L had fungal allergy or parasite infection. The serum tryptase was raised in 44 of 302 (14.5%) of individuals across all diagnostic groups, though none had mastocytosis. CONCLUSION: A diagnosis of an unexplained eosinophilia can usually be determined using as semi-structured algorithm. Parasitic infection and fungal allergy often with severe eosinophilic asthma were common causes, whereas HES, particularly myeloproliferative, disease was relatively rare.

Utility of the FebriDx point-of-care test for rapid triage and identification of possible coronavirus disease 2019 (COVID-19).

OBJECTIVES: The Coronavirus disease 2019 (COVID-19) pandemic is straining healthcare resources. Molecular testing turnaround time precludes having results at the point-of-care (POC) thereby exposing COVID-19/Non-COVID-19 patients while awaiting diagnosis. We evaluated the utility of a triage strategy including FebriDx, a 10-minute POC finger-stick blood test that differentiates viral from bacterial acute respiratory infection through detection of Myxovirus-resistance protein A (MxA) and C-reactive protein (CRP), to rapidly isolate viral cases requiring confirmatory testing. METHODS: This observational, prospective, single-center study enrolled patients presenting to/within an acute care hospital in England with suspected COVID-19 between March and April 2020. Immunocompetent patients ≥16 years requiring hospitalisation with pneumonia or acute respiratory distress syndrome or influenza-like illness (fever and ≥1 respiratory symptom within 7 days of enrolment, or inpatients with new respiratory symptoms, fever of unknown cause or pre-existing respiratory condition worsening). The primary endpoint was diagnostic performance of FebriDx to identify COVID-19 as a viral infection; secondary endpoint was SARS-CoV-2 molecular test diagnostic performance compared with the reference standard COVID-19 Case Definition (molecular or antibody detection of SARS-CoV-2). RESULTS: Valid results were available for 47 patients. By reference standard, 35 had viral infections (34/35 COVID-19; 1/35 non-COVID-19; overall FebriDx viral sensitivity 97.1% (95%CI 83.3-99.9)). Of the COVID-19 cases, 34/34 were FebriDx viral positive (sensitivity 100%; 95%CI 87.4-100); 29/34 had an initial SARS-CoV-2 positive molecular test (sensitivity 85.3%; 95%CI 68.2-94.5). FebriDx was viral negative when the diagnosis was not COVID-19 and SARS-Cov-2 molecular test was negative (negative predictive value (NPV) 100% (13/13; 95%CI 71.7-100)) exceeding initial SARS-CoV-2 molecular test NPV 72.2% (13/19; 95%CI 46.4-89.3). The diagnostic specificity of FebriDx and initial SARS-CoV-2 molecular test was 100% (13/13; 95%CI 70-100 and 13/13; 95%CI 85.4-100, respectively). CONCLUSIONS: FebriDx could be deployed as part of a reliable triage strategy for identifying symptomatic cases as possible COVID-19 in the pandemic.

Constituents of Aegle marmelos from Myanmar.

Five compounds (1-5), including three coumarins (1-3) and two alkaloids (4,5) were isolated during the first investigation of the stem bark of Aegle marmelos (L.) Corrêa. collected in Myanmar. Their structures were determined by NMR spectra analysis. Among them, 7´-O-ethylmarmin (1) and 2-O-ethyltembamide (5) were identified as new compounds. Skimmianine (4) showed moderate cytotoxicity against a HeLa cell line, and 7´-O-ethylmarmin (1), marmin (2), and (+)-epoxyaurapten (3) displayed weak radical scavenging activity according to a DPPH scavenging assay.

Mackay Heart Failure Study: Examining the Root Causes, Compliance With Guideline-Based Therapy and Prognosis.

INTRODUCTION: Heart failure patients have poor outcomes comparable to some malignancies; however, the modern guideline directed medical therapy (GDMT) has improved its outcomes. The clinical characteristics and prescribers' compliance with GDMT for heart failure patients have not been studied in the Mackay region. METHODS: A retrospective cohort study of 115 consecutive adult heart failure patients was conducted at our institution. RESULTS: The study cohort consisted of 80% (n=92) males. Ischaemia was the leading cause accounting for 54% (n=62) of the cohort, followed by idiopathic cardiomyopathy at 32% (n=37). Drug-induced and Takotsubo cardiomyopathies were responsible for 11% and 1% respectively. Two (2) patients (2%) had valvular heart disease. Hypertension was present in 57% while diabetes and atrial fibrillation were present in 32% and 43% of patients. Fifty-nine per cent (59%) had a smoking history. All, except four patients, had reduced left ventricular ejection fraction (LVEF <50%) at diagnosis. Among patients with coronary ischaemia, 37% and 31% were revascularised with percutaneous coronary interventions and bypass graft surgeries, respectively. Renin-angiotensin-aldosterone system inhibitors and beta blockers were prescribed in 94% and 95% of the patients, respectively. Mineralocorticoid inhibitors were used in 25% while ivabradine was given to 8% of patients. Nine per cent (9%) of patients received cardiac resynchronisation therapy. Most patients had improvement in functional class and LVEF during follow-up. There were very few mortalities at 3% (n=3) at the median follow-up of 403 (IQR 239-896) days. CONCLUSION: Our study has shed light on heart failure epidemiology in the Mackay region. We found excellent compliance with GDMT and good prognosis for most patients in terms of both symptom and survival.

Structures and Bioactivities of Steroidal Saponins Isolated from the Genera Dracaena and Sansevieria.

The species Dracaena and Sansevieria, that are well-known for different uses in traditional medicines and as indoor ornamental plants with air purifying property, are rich sources of bioactive secondary metabolites. In fact, a wide variety of phytochemical constituents have been isolated so far from about seventeen species. This paper has reviewed the literature of about 180 steroidal saponins, isolated from Dracaena and Sansevieria species, as a basis for further studies. Saponins are among the most characteristic metabolites isolated from the two genera. They show a great variety in structural motifs and a wide range of biological activities, including anti-inflammatory, anti-microbial, anti-proliferative effects and, in most case, remarkable cytotoxic properties.

Flavonoids and Stilbenoids of the Genera Dracaena and Sansevieria: Structures and Bioactivities.

The genera Dracaena and Sansevieria (Asparagaceae, Nolinoideae) are still poorly resolved phylogenetically. Plants of these genera are commonly distributed in Africa, China, Southeast Asia, and America. Most of them are cultivated for ornamental and medicinal purposes and are used in various traditional medicines due to the wide range of ethnopharmacological properties. Extensive in vivo and in vitro tests have been carried out to prove the ethnopharmacological claims and other bioactivities. These investigations have been accompanied by the isolation and identification of hundreds of phytochemical constituents. The most characteristic metabolites are steroids, flavonoids, stilbenes, and saponins; many of them exhibit potent analgesic, anti-inflammatory, antimicrobial, antioxidant, antiproliferative, and cytotoxic activities. This review highlights the structures and bioactivities of flavonoids and stilbenoids isolated from Dracaena and Sansevieria.

Bioactive Phytochemical Constituents of Wild Edible Mushrooms from Southeast Asia.

Mushrooms have a long history of uses for their medicinal and nutritional properties. They have been consumed by people for thousands of years. Edible mushrooms are collected in the wild or cultivated worldwide. Recently, mushroom extracts and their secondary metabolites have acquired considerable attention due to their biological effects, which include antioxidant, antimicrobial, anti-cancer, anti-inflammatory, anti-obesity, and immunomodulatory activities. Thus, in addition to phytochemists, nutritionists and consumers are now deeply interested in the phytochemical constituents of mushrooms, which provide beneficial effects to humans in terms of health promotion and reduction of disease-related risks. In recent years, scientific reports on the nutritional, phytochemical and pharmacological properties of mushroom have been overwhelming. However, the bioactive compounds and biological properties of wild edible mushrooms growing in Southeast Asian countries have been rarely described. In this review, the bioactive compounds isolated from 25 selected wild edible mushrooms growing in Southeast Asia have been reviewed, together with their biological activities. Phytoconstituents with antioxidant and antimicrobial activities have been highlighted. Several evidences indicate that mushrooms are good sources for natural antioxidants and antimicrobial agents.

A Review on the Phytochemistry, Medicinal Properties and Pharmacological Activities of 15 Selected Myanmar Medicinal Plants.

Medicinal plants are a reservoir of biologically active compounds with therapeutic properties that over time have been reported and used by diverse groups of people for treatment of various diseases. This review covers 15 selected medicinal plants distributed in Myanmar, including Dalbergia cultrata, Eriosema chinense, Erythrina suberosa, Millettia pendula, Sesbania grandiflora, Tadehagi triquetrum, Andrographis echioides, Barleria cristata, Justicia gendarussa, Premna integrifolia, Vitex trifolia, Acacia pennata, Cassia auriculata, Croton oblongifolius and Glycomis pentaphylla. Investigation of the phytochemical constituents, biological and pharmacological activities of the selected medicinal plants is reported. This study aims at providing a collection of publications on the species of selected medicinal plants in Myanmar along with a critical review of the literature data. As a country, Myanmar appears to be a source of traditional drugs that have not yet been scientifically investigated. This review will be support for further investigations on the pharmacological activity of medicinal plant species in Myanmar.

Triglyceride-rich lipoprotein lipolysis products increase blood-brain barrier transfer coefficient and induce astrocyte lipid droplets and cell stress.

Elevation of blood triglycerides, primarily as triglyceride-rich lipoproteins (TGRL), has been linked to cerebrovascular inflammation, vascular dementia, and Alzheimer's disease (AD). Brain microvascular endothelial cells and astrocytes, two cell components of the neurovascular unit, participate in controlling blood-brain barrier (BBB) permeability and regulating neurovascular unit homeostasis. Our studies showed that infusion of high physiological concentrations of TGRL lipolysis products (TGRL + lipoprotein lipase) activate and injure brain endothelial cells and transiently increase the BBB transfer coefficient (Ki = permeability × surface area/volume) in vivo. However, little is known about how blood lipids affect astrocyte lipid accumulation and inflammation. To address this, we first demonstrated TGRL lipolysis products increased lipid droplet formation in cultured normal human astrocytes. We then evaluated the transcriptional pathways activated in astrocytes by TGRL lipolysis products and found upregulated stress and inflammatory-related genes including activating transcription factor 3 (ATF3), macrophage inflammatory protein-3α (MIP-3α), growth differentiation factor-15 (GDF15), and prostaglandin-endoperoxide synthase 2 (COX2). TGRL lipolysis products also activated the JNK/cJUN/ATF3 pathway, induced endoplasmic reticulum stress protein C/EBP homologous protein (CHOP), and the NF-κB pathway, while increasing secretion of MIP-3α, GDF15, and IL-8. Thus our results demonstrate TGRL lipolysis products increase the BBB transfer coefficient (Ki), induce astrocyte lipid droplet formation, activate cell stress pathways, and induce secretion of inflammatory cytokines. Our observations are consistent with evidence for lipid-induced neurovascular injury and inflammation, and we, therefore, speculate that lipid-induced astrocyte injury could play a role in cognitive decline.

Two New Ergosterol Derivatives from the Basidiomycete Cortinarius glaucopus.

Two new sterols 1 and 2 and five known ones 3 - 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1- and 2D-NMR spectra and HR-FABS-MS. The relative configuration of 1 was firmly determined by comparison of the observed 1 H-1 H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6-31 + G(d,p) level of theory, in CHCl3 as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A (1) and B (2), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)-5,6-epoxy-3,7,23-trihydroxystrophast-8-en-14-one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)-3,5-dihydroxyergosta-6,8(14),22-trien-15-one, respectively. Moreover, the configuration of known strophasterol C (3) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A (1) and strophasterol C (3) represent the second finding in nature of steroids with the rare strophastane skeleton.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.

Characterizing blood-brain barrier perturbations after exposure to human triglyceride-rich lipoprotein lipolysis products using MRI in a rat model.

PURPOSE: Previous studies indicated hyperlipidemia may be a risk factor for Alzheimer's disease, but the contributions of postprandial triglyceride-rich lipoprotein (TGRL) are not known. In this study, changes in blood-brain barrier diffusional transport following exposure to human TGRL lipolysis products were studied using MRI in a rat model. METHODS: Male Sprague-Dawley rats (∼180-250 g) received an i.v. injection of lipoprotein lipase (LpL)-hydrolyzed TGRL (n = 8, plasma concentration ≈ 150 mg human TGRL/dL). Controls received i.v. injection of either saline (n = 6) or LpL only (n = 6). The (1) H longitudinal relaxation rate R1 = 1/T1 was measured over 18 min using a rapid-acquired refocus-echo (RARE) sequence after each of three injections of the contrast agent Gd-DTPA. Patlak plots were generated for each pixel yielding blood-to-brain transfer coefficients, Ki , chosen for best fit to impermeable, uni-directional influx or bi-directional flux models using the F-test. RESULTS: Analysis from a 2-mm slice, 2-mm rostral to the bregma showed a 275% increase of mean Ki during the first 20 min after infusion of human TGRL lipolysis product that differed significantly compared with saline and LpL controls. This difference disappeared by 40 min mark. CONCLUSION: These results suggest human TGRL lipolysis products can lead to a transient increase in rat BBB permeability. Magn Reson Med 76:1246-1251, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Induction of ATF3 gene network by triglyceride-rich lipoprotein lipolysis products increases vascular apoptosis and inflammation.

OBJECTIVE: Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. APPROACH AND RESULTS: We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB-dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3(-/-) mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes. CONCLUSIONS: This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Single-molecule quantification of lipotoxic expression of activating transcription factor 3.

Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500 h(-1). After three hours we detected 33,090 ± 3491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ∼3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates.

Analytical Validation of a 37-Gene Next-Generation Sequencing Panel for Myeloid Malignancies and Review of Initial Findings Incorporating Updated 2022 Diagnostic and Prognostic Guidelines.

Myeloid neoplasms are clonal disorders that arise via acquisition of genetic mutations leading to excessive proliferation and defective differentiation. Mutational profiling is vital as it has implications for diagnosis, prognosis, and therapeutic decision-making. Next-generation sequencing (NGS) has become a mainstay in the evaluation of myeloid malignancies, as it enables efficient characterization of multiple genetic changes. Herein, the analytical validation of the 37-gene Archer VariantPlex Core Myeloid panel is reported, using 58 DNA specimens with 87 single-nucleotide variants and 23 insertions/deletions. The panel achieved good depth of coverage, 100% analytical sensitivity and specificity for single-nucleotide variants and insertions/deletions ≤21 bp, and 100% reproducibility, with a reportable limit of detection determined as 5%. The Archer NGS panel can accurately and reproducibly detect variants of clinical significance in myeloid neoplasms. A retrospective analysis of 535 clinical specimens tested with the Archer NGS panel showed a frequency and pattern of mutations across myeloid malignancies that were similar to other published studies. A review of the diagnostic classification of patients with acute myeloid leukemia and myelodysplastic syndrome using the World Health Organization 2017/2022 and International Consensus Classification 2022 guidelines, in addition to European LeukemiaNet 2017/2022 risk stratification of patients with acute myeloid leukemia, was also performed to assess the utility of the molecular information provided by the Archer NGS panel.

Post-pandemic seasonal dynamics of hospitalised COPD exacerbations and aetiologies in the COPD population.

This study uncovered a reversal of seasonal variation in hospitalised COPD exacerbation events after the COVID-19 pandemic. Investigation of updated seasonal exacerbation patterns and triggers can help initiate preventive strategies effectively. https://bit.ly/3wN71h0.

Assessing Inhaled Corticosteroid Adherence and Responsiveness in Severe Asthma Using Beclometasone Dipropionate/Formoterol NEXThaler Dose-Counting and Nitric Oxide Monitoring.

BACKGROUND: Sixty-five percent of people with severe asthma and a fractional exhaled nitric oxide (Feno) greater than or equal to 45 parts per billion (ppb) are nonadherent to inhaled corticosteroids (ICSs). Digital devices recording both time of use and inhaler technique identify nonadherence and ICS responsiveness but are not widely available. As the NEXThaler dose counter activates only at an inspiratory flow rate of 35 L/min, this may provide an alternative to identifying ICS responsiveness. OBJECTIVE: To assess ICS adherence and responsiveness in severe asthma using beclometasone/formoterol (200/6 µg) NEXThaler (BFN) dose-counting. METHODS: Patients with severe asthma with a Feno greater than or equal to 45 ppb were invited to use BFN in place of their usual ICS/long-acting ß2-agonist. Feno, 6-item Asthma Control Questionnaire score, lung function, and blood eosinophil count were monitored for 3 months. A log10ΔFeno of greater than or equal to 0.24 was used to define Feno suppression as the primary marker of ICS responsiveness at day 28. RESULTS: Twenty-seven of 48 (56%) patients demonstrated significant Feno suppression at month 1 (median pre-114, post-48 ppb, P < .001). A small but significant reduction occurred in Feno nonsuppressors. The 6-item Asthma Control Questionnaire score fell a median 1.2 units in Feno suppressors (P < .001) and 0.5 units in nonsuppressors (P = .025). These effects were sustained until month 3 in Feno suppressors, with a significant improvement in FEV1 and blood eosinophils. Sixty-seven percent (18 of 27) of those with baseline ICS/long-acting ß2-agonist prescription refills of 80% or more were Feno suppressors, suggesting prior nonadherence despite adequate prescription collection. Seventy-nine percent of Feno suppressors did not require biologics within mean 11.4 months from initial dose counting. CONCLUSIONS: BFN dose-counting identifies ICS responsiveness in severe asthma with the implication that these patients may not need to progress to biological therapies.