Neuromedin-U Mediates Rapid Activation of Airway Group 2 Innate Lymphoid Cells in Mild Asthma.

Rationale: In asthma, sputum group 2 innate lymphoid cells (ILC2s) are activated within 7 hours after allergen challenge. Neuroimmune interactions mediate rapid host responses at mucosal interfaces. In murine models of asthma, lung ILC2s colocalize to sensory neuronal termini expressing the neuropeptide neuromedin U (NMU), which stimulates type 2 (T2) cytokine secretion by ILC2s, with additive effects to alarmins in vitro. Objectives: To investigate the effect of the NMU/NMUR1 (NMU receptor 1) axis on early activation of ILC2s in asthma. Methods: Subjects with mild asthma (n = 8) were enrolled in a diluent-controlled allergen inhalation challenge study. Sputum ILC2 expression of NMUR1 and T2 cytokines was enumerated by flow cytometry, and airway NMU levels were assessed by ELISA. This was compared with samples from subjects with moderate to severe asthma (n = 9). Flow sort-purified and ex vivo-expanded ILC2s were used for functional assays and transcriptomic analyses. Measurements and Main Results: Significant increases in sputum ILC2s expressing NMUR1 were detected 7 hours after allergen versus diluent challenge whereby the majority of NMUR1+ ILC2s expressed IL-5/IL-13. Sputum NMUR1+ ILC2 counts were significantly greater in mild versus moderate to severe asthma, and NMUR1+ ILC2s correlated inversely with the dose of inhaled corticosteroid in the latter group. Coculturing with alarmins upregulated NMUR1 in ILC2s, which was attenuated by dexamethasone. NMU-stimulated T2 cytokine expression by ILC2s, maximal at 6 hours, was abrogated by dexamethasone or specific signaling inhibitors for mitogen-activated protein kinase 1/2 and phosphoinositol 3-kinase but not the IL-33 signaling moiety MyD88 in vitro. Conclusions: The NMU/NMUR1 axis stimulates rapid effects on ILC2s and may be an important early activator of these cells in eosinophilic inflammatory responses in asthma.

Benralizumab for allergic asthma: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Benralizumab induces rapid and near-complete depletion of eosinophils from blood and lung tissue. We investigated whether benralizumab could attenuate the allergen-induced late asthmatic response (LAR) in participants with allergic asthma. METHODS: Participants with allergic asthma who demonstrated increased sputum eosinophils and LAR at screening were randomised to benralizumab 30 mg or matched placebo given every 4 weeks for 8 weeks (3 doses). Allergen challenges were performed at weeks 9 and 12 when blood, sputum, bone marrow and bronchial tissue eosinophils and LAR were assessed. RESULTS: 46 participants (mean age 30.9 years) were randomised to benralizumab (n=23) or placebo (n=23). Eosinophils were significantly reduced in the benralizumab group compared with placebo in blood at 4 weeks and sputum and bone marrow at 9 weeks after treatment initiation. At 7 h after an allergen challenge at week 9, sputum eosinophilia was significantly attenuated in the benralizumab group compared to placebo (least squares mean difference -5.81%, 95% CI -10.69- -0.94%; p=0.021); however, the LAR was not significantly different (least squares mean difference 2.54%, 95% CI 3.05-8.12%; p=0.363). Adverse events were reported for seven (30.4%) and 14 (60.9%) participants in the benralizumab and placebo groups, respectively. CONCLUSION: Benralizumab administration over 8 weeks resulted in a significant attenuation of blood, bone marrow and sputum eosinophilia in participants with mild allergic asthma; however, there was no change in the LAR, suggesting that eosinophils alone are not a key component of allergen-induced bronchoconstriction.

Immune Response Dynamics and Biomarkers in COVID-19 Patients.

BACKGROUND: The immune response dynamics in COVID-19 patients remain a subject of intense investigation due to their implications for disease severity and treatment outcomes. We examined changes in leukocyte levels, eosinophil activity, and cytokine profiles in patients hospitalized with COVID-19. METHODS: Serum samples were collected within the first 10 days of hospitalization/confirmed infection and analyzed for eosinophil granule proteins (EGP) and cytokines. Information from medical records including comorbidities, clinical symptoms, medications, and complete blood counts were collected at the time of admission, during hospitalization and at follow up approximately 3 months later. RESULTS: Serum levels of eotaxin, type 1 and type 2 cytokines, and alarmin cytokines were elevated in COVID-19 patients, highlighting the heightened immune response (p < 0.05). However, COVID-19 patients exhibited lower levels of eosinophils and eosinophil degranulation products compared to hospitalized controls (p < 0.05). Leukocyte counts increased consistently from admission to follow-up, indicative of recovery. CONCLUSION: Attenuated eosinophil activity alongside elevated chemokine and cytokine levels during active infection, highlights the complex interplay of immune mediators in the pathogenesis COVID-19 and underscores the need for further investigation into immune biomarkers and treatment strategies.

Progenitor cell-derived basophils: A novel barcoded passive degranulation assay in allergic diseases.

BACKGROUND: Effector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable and high-throughput assays limits the clinical utility. We aimed to develop a high-throughput basophil activation test based on human progenitor cell-derived basophils (PCB) and investigate the role of PCB activation test (PCBAT) in allergic diseases. METHODS: Progenitor cell-derived basophils were differentiated from CD34+ progenitor cells and sensitized with sera from subjects sensitized to cat, peanut or atopic controls. Sensitized PCBs were stimulated with increasing concentrations of the corresponding allergens in vitro. Degranulation was assessed by measuring CD63 expression using flow cytometry. The correlations between PCBAT and clinical allergy were assessed. RESULTS: Following passive sensitization of the mature PCBs with serum and allergen stimulation, an allergen specific dose-dependent increase in CD63 expression was observed. Sera from subjects sensitized to cat (n = 35, of which 17 subjects had clinical reactivity quantified using inhaled allergen challenge), peanut allergic (n = 30, of which 15 subjects had clinical reactivity validated using double blind, placebo controlled food challenges [DBPCFC]), peanut-sensitized but tolerant subjects (n = 5) were used to sensitize PCBs. PCBAT area under the curve (AUC) correlated with sIgE (r2  = .49, p = .001) in subjects sensitized to cat (sIgE ≥ 0.35KU/L). The provocation concentration of inhaled cat allergen (PC20 ) correlated with PCBAT AUC (r2  = .33, p = .016). In subjects sensitized to peanut, PCBAT AUC was highly correlated with sIgE to Ara h 2 (r2  = .59, p < .0001). Peanut threshold cumulative dose during DBPCFC was negatively correlated with PCBAT AUC (r2  = .57, p = .001) and IgE to Ara h1 (r2  = .55, p = .007), but not with sIgE to whole peanut or Ara h2. All peanut-sensitized but tolerant subjects showed no reaction to peanut on PCBAT. CONCLUSION: Progenitor cell-derived basophils activation test is a high-throughput assay, which correlates with clinical allergy and may confer a powerful alternative tool in allergy testing.

Evaluation of the reproducibility of responses to nasal allergen challenge and effects of inhaled nasal corticosteroids.

BACKGROUND: Similar immune responses in the nasal and bronchial mucosa implies that nasal allergen challenge (NAC) is a suitable early phase experimental model for drug development targeting allergic rhinitis (AR) and asthma. We assessed NAC reproducibility and the effects of intranasal corticosteroids (INCS) on symptoms, physiology, and inflammatory mediators. METHODS: 20 participants with mild atopic asthma and AR underwent three single blinded nasal challenges each separated by three weeks (NCT03431961). Cohort A (n = 10) underwent a control saline challenge, followed by two allergen challenges. Cohort B (n = 10) underwent a NAC with no treatment intervention, followed by NAC with 14 days pre-treatment with saline nasal spray (placebo), then NAC with 14 days pre-treatment with INCS (220 µg triamcinolone acetonide twice daily). Nasosorption, nasal lavage, blood samples, forced expiratory volume 1 (FEV1), total nasal symptom score (TNSS), peak nasal inspiratory flow (PNIF) were collected up to 24 h after NAC. Total and active tryptase were measured as early-phase allergy biomarkers (≤30 min) and IL-13 and eosinophil cell counts as late-phase allergy biomarkers (3-7 h) in serum and nasal samples. Period-period reproducibility was assessed by intraclass correlation coefficients (ICC), and sample size estimates were performed using effect sizes measured after INCS. RESULTS: NAC significantly induced acute increases in nasosorption tryptase and TNSS and reduced PNIF, and induced late increases in nasosorption IL-13 with sustained reductions in PNIF. Reproducibility across NACs varied for symptoms and biomarkers, with total tryptase 5 min post NAC having the highest reproducibility (ICC = 0.91). Treatment with INCS inhibited NAC-induced IL-13 while blunting changes in TNSS and PNIF. For a similar crossover study, 7 participants per treatment arm are needed to detect treatment effects comparable to INCS for TNSS. CONCLUSION: NAC-induced biomarkers and symptoms are reproducible and responsive to INCS. NAC is suitable for assessing pharmacodynamic activity and proof of mechanism for drugs targeting allergic inflammation.

Limosilactobacillus reuteri DSM-17938 for preventing cough in adults with mild allergic asthma: A double-blind randomized placebo-controlled cross-over study.

BACKGROUND: Cough is a common troublesome symptom in asthma which is neuronally mediated. Limosilactobacillus reuteri DSM-17938 (L. reuteri DSM-17938) is a probiotic shown to be effective in pre-clinical models at suppressing neuronal responses to capsaicin, a transient receptor potential vanilloid agonist (TRPV1). OBJECTIVE: Investigate the effects of DSM-17938 versus matched placebo on capsaicin-evoked coughs in mild allergic asthmatics. METHODS: We performed a 4-visit, randomized, double-blind, placebo-controlled, two-way cross-over study comparing full dose cough responses with inhaled capsaicin in mild allergic asthmatics after 1 month of treatment with DSM-17938 compared with matched placebo. Randomization and allocation to trial group were carried out by a central computer system. Histamine skin prick testing, airway hyper-responsiveness and inflammatory cells in induced sputum were measured at every visit. Blood was collected to extract PBMCs and stimulated with CD3/CD28 to ascertain the effects of DSM-17938 /placebo on T-cell cytokine responses. RESULTS: Seventeen subjects were recruited and 15 completed the study (8 females, mean age 27.3 years). There was no difference in the change in maximum capsaicin-evoked coughs (Emax) after treatment with L. reuteri DSM-17938 compared with placebo [mean difference 2.07 coughs (95% CI -2.77 to 6.91, p = .38) or relative changes in geometric mean ratios for the dose evoking at least half the Emax (ED50) [1.05 (95% CI 0.31-3.58, p = .94)], concentration evoking 2 coughs (C2) [0.63 (0.26-1.53), p = .28] and 5 coughs (C5) [0.79 (0.25-2.50), p = .67]. There was no effect on histamine skin prick wheal size, intensity of itch sensation, methacholine PC20, airway inflammation or T-cell responses after stimulation with CD3/CD28. There were no serious adverse events. One subject developed a mild upper respiratory tract infection and another mild transient nausea whilst on DSM-17938. CONCLUSION: In this small study in adults with mild allergic asthma, we found no evidence that L. reuteri DSM-17938 has any systemic effects on airway nerves, smooth muscle, sputum inflammatory cells, skin responses or T-cell responses after oral consumption. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT03603522.

Effect of intranasal corticosteroid treatment on allergen-induced changes in group 2 innate lymphoid cells in allergic rhinitis with mild asthma.

BACKGROUND: Allergic rhinitis is characterized by rhinorrhea, nasal congestion, sneezing and nasal pruritus. Group 2 innate lymphoid cells (ILC2s), CD4+ T cells and eosinophils in nasal mucosa are increased significantly after nasal allergen challenge (NAC). Effects of intranasal corticosteroids (INCS) on ILC2s remain to be investigated. METHODS: Subjects (n = 10) with allergic rhinitis and mild asthma were enrolled in a single-blind, placebo-controlled, sequential treatment study and treated twice daily with intranasal triamcinolone acetonide (220 µg) or placebo for 14 days, separated by a 7-day washout period. Following treatment, subjects underwent NAC and upper airway function was assessed. Cells from the nasal mucosa and blood, sampled 24 h post-NAC, underwent flow cytometric enumeration for ILC2s, CD4+ T and eosinophil progenitor (EoPs) levels. Cell differentials and cytokine levels were assessed in nasal lavage. RESULTS: Treatment with INCS significantly attenuated ILC2s, IL-5+ /IL-13+ ILC2s, HLA-DR+ ILC2s and CD4+ T cells in the nasal mucosa, 24 h post-NAC. EoP in nasal mucosa was significantly increased, while mature eosinophils were significantly decreased, 24 h post-NAC in INCS versus placebo treatment arm. Following INCS treatment, IL-2, IL-4, IL-5 and IL-13 were significantly attenuated 24 h post-NAC accompanied by significant improvement in upper airway function. CONCLUSION: Pre-treatment with INCS attenuates allergen-induced increases in ILC2s, CD4+ T cells and terminal differentiation of EoPs in the nasal mucosa of allergic rhinitis patients with mild asthma, with little systemic effect. Attenuation of HLA-DR expression by ILC2s may be an additional mechanism by which steroids modulate adaptive immune responses in the upper airways.

Asthma maintenance and reliever therapy: Should this be the standard of care?

OBJECTIVE: The goal of asthma management is to achieve optimal asthma control, defined by the absence of daytime symptoms, nighttime waking, reliever use, functional limitation, and lung function stability, and to also reduce the future risks of asthma exacerbations, deterioration in lung function, and the medication's adverse effects. The most widely used maintenance therapy is inhaled corticosteroids (ICSs). This review considered the evidence in which the combination of the ICS budesonide and the rapid-onset long-acting ß-agonist (LABA) formoterol can be used as a standard of care for maintenance and reliever therapy in moderate to severe asthma. DATA SOURCES: The archival literature of peer-reviewed studies on the efficacy and safety of budesonide-formoterol as maintenance and reliever therapy in moderate to severe asthma. RESULTS: The ICS-LABA combination containing budesonide-formoterol reduces future risk of severe asthma exacerbations and provides similar levels of day-to-day asthma control when compared with using high-dose ICS alone, or combination ICS-LABA therapy and short-acting ß2-agonist as a reliever. CONCLUSION: Budesonide-formoterol as a single combination maintenance and reliever inhaler is effective in reducing asthma exacerbation risk, requires a lower maintenance dose of ICS, and results in a simplified approach to asthma management.

Effect of benralizumab on inflammation in skin after intradermal allergen challenge in patients with moderate-to-severe atopic dermatitis.

Background: Atopic dermatitis (AD) is a skin barrier dysfunction characterized by tissue eosinophilia. Objective: In patients with AD, we evaluated the effect of eosinophil depletion with benralizumab on markers of inflammation in skin after intradermal allergen challenge. Methods: A total of 20 patients with moderate-to-severe AD completed a randomized, double-blind, placebo-controlled parallel-group study comparing 3 doses of benralizumab (30 mg each) administered subcutaneously every 4 weeks (n = 9) with placebo (n = 11). Allergen and saline control intradermal challenges were conducted before and after treatment, with skin biopsy samples collected 24 hours after challenge. Early and late cutaneous responses were measured by skin wheal size. Levels of eosinophils and IL-5 receptor-α-bearing cells, including eosinophil progenitor (EoP) cells, basophils, and mast cells, in papillary dermis were measured by immunofluorescence microscopy, and levels of EoP cells, hematopoietic progenitor cells, and type 2 innate lymphoid cells in the blood were measured by flow cytometry. Outcomes were compared between the placebo and benralizumab treatment groups by using the Mann-Whitney U test. Results: Benralizumab reduced eosinophil counts in the blood (P < .0001) and allergen-challenged skin, as measured by hematoxylin and eosin staining and eosinophil cationic protein antibody concentration (P < .05). Benralizumab lowered the levels of EoP cells, mast cells, and basophils in the skin, as well as the levels of EoP cells, hematopoietic progenitor cells, and type 2 innate lymphoid cells in the blood (all P < .05). There was a trend toward improvement in the early cutaneous response (P = .095) but no effect on the late cutaneous response. Conclusion: In patients with moderate-to-severe AD, benralizumab treatment significantly inhibited accumulation of eosinophils and other IL-5 receptor-α-expressing cells in the papillary dermis after intradermal allergen challenge. Targeting IL-5 receptor-α-positive cells did not modulate the size of the allergen-induced skin wheal (ClincialTrials.gov identifier NCT03563066).

Machine learning enabled detection of COVID-19 pneumonia using exhaled breath analysis: a proof-of-concept study.

Detection of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) relies on real-time-reverse-transcriptase polymerase chain reaction (RT-PCR) on nasopharyngeal swabs. The false-negative rate of RT-PCR can be high when viral burden and infection is localized distally in the lower airways and lung parenchyma. An alternate safe, simple and accessible method for sampling the lower airways is needed to aid in the early and rapid diagnosis of COVID-19 pneumonia. In a prospective unblinded observational study, patients admitted with a positive RT-PCR and symptoms of SARS-CoV-2 infection were enrolled from three hospitals in Ontario, Canada. Healthy individuals or hospitalized patients with negative RT-PCR and without respiratory symptoms were enrolled into the control group. Breath samples were collected and analyzed by laser absorption spectroscopy (LAS) for volatile organic compounds (VOCs) and classified by machine learning (ML) approaches to identify unique LAS-spectra patterns (breathprints) for SARS-CoV-2. Of the 135 patients enrolled, 115 patients provided analyzable breath samples. Using LAS-breathprints to train ML classifier models resulted in an accuracy of 72.2%-81.7% in differentiating between SARS-CoV2 positive and negative groups. The performance was consistent across subgroups of different age, sex, body mass index, SARS-CoV-2 variants, time of disease onset and oxygen requirement. The overall performance was higher than compared to VOC-trained classifier model, which had an accuracy of 63%-74.7%. This study demonstrates that a ML-based breathprint model using LAS analysis of exhaled breath may be a valuable non-invasive method for studying the lower airways and detecting SARS-CoV-2 and other respiratory pathogens. The technology and the ML approach can be easily deployed in any setting with minimal training. This will greatly improve access and scalability to meet surge capacity; allow early and rapid detection to inform therapy; and offers great versatility in developing new classifier models quickly for future outbreaks.

Use of Asthma Medication During Gestation and Risk of Specific Congenital Anomalies.

Poorly controlled asthma can affect neonatal outcomes including congenital anomalies, which can be reduced with appropriate asthma care during pregnancy. Although there is a concern regarding the safety of asthma medication use during pregnancy and congenital anomalies, the risk of uncontrolled asthma outweighs any potential risks of controller and reliever medication use. Patient education before and during pregnancy is critical to ensure good compliance to therapy and reduce the risk of poor asthma control.

Expanded central role of the respiratory physiotherapists in the community setting.

The development of community hubs through the Slaintecare initiative will rely on respiratory physiotherapists and clinical nurse specialists for the management of chronic respiratory diseases. The role of the respiratory physiotherapist has evolved dramatically over the last decade. We review the increasing scope of practice of the physiotherapist and the evidence base for same. We pay particular attention to the role of the physiotherapist in areas such as pulmonary rehabilitation, sputum clearance, neuromuscular disease, chronic respiratory failure, ambulatory oxygen assessments and dysfunctional breathing. We give an in depth review of sputum clearance techniques. We also address areas of potential future expansion for the role of the physiotherapist such as prescription and initiation of non-invasive ventilation.

Gene Polymorphisms of Epithelial Cell-Derived Alarmins and Their Effects on Protein Levels and Disease Severity in Patients with COVID-19.

BACKGROUND: The immune response in COVID-19 is characterized by the release of alarmin cytokines, which play crucial roles in immune activation and inflammation. The interplay between these cytokines and genetic variations may influence disease severity and outcomes, while sex differences might further contribute to variations in the immune response. METHODS: We measured the levels of alarmin cytokines in a cohort of COVID-19 and non-COVID-19 patients using a sensitive Meso Scale Discovery system. Additionally, we conducted an SNP analysis to identify genetic variations within the IL-33 and TSLP genes. The association between these genetic variations, cytokine production, and COVID-19 severity was examined. RESULTS: Our findings revealed elevated levels of IL-33 and IL-25 in COVID-19-positive patients compared to COVID-19-negative patients (p < 0.05), indicating their potential as therapeutic targets for disease modulation. Moreover, a minor allele within the IL-33 gene (rs3939286) was found to be associated with a protective effect against severe COVID-19 (p < 0.05), and minor alleles of the TSLP gene (rs2289276 and rs13806933) were found to significantly reduce TSLP protein levels in serum (p < 0.05). Sex-specific effects of TSLP and IL-33 SNPs were observed, suggesting a potential influence of sex hormones and genetic variations on the regulation of cytokine production. CONCLUSION: The present study highlights the importance of alarmin cytokines and genetic variations in COVID-19 severity, providing valuable insights into personalized treatment approaches. Our results suggest that targeting alarmin cytokines may offer potential therapeutic benefits in managing COVID-19. Furthermore, the sex-specific effects of genetic variations emphasize the need to consider individual genetic profiles and sex differences when designing targeted interventions.

The Genetic Factors of the Airway Epithelium Associated with the Pathology of Asthma.

Asthma is a chronic disease of the airways characterized by inflammation, tightened muscles, and thickened airway walls leading to symptoms such as shortness of breath, chest tightness, and cough in patients. The increased risk of asthma in children of asthmatics parents supports the existence of genetic factors involved in the pathogenesis of this disease. Genome-wide association studies have discovered several single nucleotide polymorphisms associated with asthma. These polymorphisms occur within several genes and can contribute to different asthma phenotypes, affect disease severity, and clinical response to different therapies. The complexity in the etiology of asthma also results from interactions between environmental and genetic factors. Environmental exposures have been shown to increase the prevalence of asthma in individuals who are genetically susceptible. This review summarizes what is currently known about the genetics of asthma in relation to risk, response to common treatments, and gene-environmental interactions.

The Role of Airway Epithelial Cell Alarmins in Asthma.

The airway epithelium is the first line of defense for the lungs, detecting inhaled environmental threats through pattern recognition receptors expressed transmembrane or intracellularly. Activation of pattern recognition receptors triggers the release of alarmin cytokines IL-25, IL-33, and TSLP. These alarmins are important mediators of inflammation, with receptors widely expressed in structural cells as well as innate and adaptive immune cells. Many of the key effector cells in the allergic cascade also produce alarmins, thereby contributing to the airways disease by driving downstream type 2 inflammatory processes. Randomized controlled clinical trials have demonstrated benefit when blockade of TSLP and IL-33 were added to standard of care medications, suggesting these are important new targets for treatment of asthma. With genome-wide association studies demonstrating associations between single-nucleotide polymorphisms of the TSLP and IL-33 gene and risk of asthma, it will be important to understand which subsets of asthma patients will benefit most from anti-alarmin therapy.