Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

A Concise Review of Exhaled Breath Testing for Respiratory Clinicians and Researchers.

Exhaled breath contains an extensive reservoir of biomolecules. The collection of exhaled breath is noninvasive and low risk. Therefore, its testing is an appealing strategy for the discovery of biomarkers of respiratory diseases. In this concise review, we summarize the evidence of exhaled breath tests for airways diseases and respiratory infections. An overview of breath collection methods in both individuals who are spontaneously breathing and those receiving mechanical ventilation is outlined. We also highlight the challenges in exhaled breath testing and areas for future research.

Defining and Promoting Pediatric Pulmonary Health: Developing Biomarkers for Pulmonary Health.

Children with inherited and/or acquired respiratory disorders often arrive in adolescence and adulthood with diminished lung function that might have been detected and prevented had better mechanisms been available to identify and to assess progression of disease. Fortunately, advances in genetic assessments, low-cost diagnostics, and minimally- invasive novel biomarkers are being developed to detect and to treat respiratory diseases before they give rise to loss of life or lung function. This paper summarizes the Developing Biomarkers for Pulmonary Health sessions of the National Heart, Lung, and Blood Institute- sponsored 2021 Defining and Promoting Pediatric Pulmonary Health workshop. These sessions discussed genetic testing, pulse oximetry, exhaled nitric oxide, and novel biomarkers related to childhood lung diseases.

Desalkylgidazepam blood concentrations in 63 forensic investigation cases.

Desalkylgidazepam, also known as bromonordiazepam, is the latest designer benzodiazepine to appear in postmortem blood samples in British Columbia. Our laboratory was first alerted to the presence of desalkylgidazepam in seized drug samples in May 2022, and the analyte was added to an in-house library shortly thereafter. Previously acquired spectra from routine death investigation cases were reprocessed using the updated library with the first presumptive identification of desalkylgidazepam occurring in a sample received in April 2022. A standard addition method for the quantitation of desalkylgidazepam in blood samples (from femoral, iliac, jugular and subclavian veins) was validated and consequently used to confirm presence and concentrations of the drug in 63 cases, with an average concentration of 42.2 ± 44.0 ng/mL (median concentration: 24.5 ng/mL; range: 3.7-220.6 ng/mL). Similar to detections of other novel benzodiazepines, co-occurrence of desalkylgidazepam with opioids and/or stimulants was common. To our knowledge, this paper is the first to report desalkylgidazepam concentrations in postmortem blood samples.

Effects of pH alteration on respiratory syncytial virus in human airway epithelial cells.

Background: Respiratory syncytial virus (RSV) is a leading cause of respiratory distress and hospitalisation in the paediatric population. Low airway surface pH impairs antimicrobial host defence and worsens airway inflammation. Inhaled Optate safely raises airway surface pH in humans and raises intracellular pH in primary human airway epithelial cells (HAECs) in vitro. We aimed to determine whether raising intracellular pH with Optate would decrease infection and replication of RSV in primary HAECs. Methods: We cultured HAECs from healthy subjects in both air-liquid interface and submerged conditions. We infected HAECs with green fluorescent protein-labelled RSV (GFP-RSV; multiplicity of infection=1) and treated them with Optate or PBS control. We collected supernatant after a 4-h incubation and then every 24 h. We used fluorescence intensity, fluorescent particle counts, plaque assays, Western blots and ELISA to quantitate infection. Results: In submerged culture, fluorescence intensity decreased in Optate-treated cells (48 h p=0.0174, 72 h p≤0.001). Similarly, Optate treatment resulted in decreased fluorescent particle count (48 h p=0.0178, 72 h p=0.0019) and plaque-forming units (48 h p=0.0011, 72 h p=0.0148) from cell culture supernatant. In differentiated HAECs cultured at ALI, Optate treatment decreased fluorescence intensity (p≤0.01), GFP via Western blot and ELISA (p<0.0001), and RSV-fusion protein via ELISA (p=0.001). Additionally, RSV infection decreased as Optate concentration increased in a dose-dependent manner (p<0.001). Conclusions: Optate inhibits RSV infection in primary HAECs in a dose-dependent manner. These findings suggest that Optate may have potential as an inhaled therapeutic for patients with RSV.

Methods to Detect Volatile Organic Compounds for Breath Biopsy Using Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry.

Volatile organic compounds (VOCs) are byproducts from metabolic pathways that can be detected in exhaled breath and have been reported as biomarkers for different diseases. The gold standard for analysis is gas chromatography-mass spectrometry (GC-MS), which can be coupled with various sampling methods. The current study aims to develop and compare different methods for sampling and preconcentrating VOCs using solid-phase microextraction (SPME). An in-house sampling method, direct-breath SPME (DB-SPME), was developed to directly extract VOCs from breath using a SPME fiber. The method was optimized by exploring different SPME types, the overall exhalation volume, and breath fractionation. DB-SPME was quantitatively compared to two alternative methods involving the collection of breath in a Tedlar bag. In one method, VOCs were directly extracted from the Tedlar bag (Tedlar-SPME) and in the other, the VOCs were cryothermally transferred from the Tedlar bag to a headspace vial (cryotransfer). The methods were verified and quantitatively compared using breath samples (n = 15 for each method respectively) analyzed by GC-MS quadrupole time-of-flight (QTOF) for compounds including but not limited to acetone, isoprene, toluene, limonene, and pinene. The cryotransfer method was the most sensitive, demonstrating the strongest signal for the majority of the VOCs detected in the exhaled breath samples. However, VOCs with low molecular weights, including acetone and isoprene, were detected with the highest sensitivity using the Tedlar-SPME. On the other hand, the DB-SPME was less sensitive, although it was rapid and had the lowest background GC-MS signal. Overall, the three breath-sampling methods can detect a wide variety of VOCs in breath. The cryotransfer method may be optimal when collecting a large number of samples using Tedlar bags, as it allows the long-term storage of VOCs at low temperatures (-80 °C), while Tedlar-SPME may be more effective when targeting relatively small VOCs. The DB-SPME method may be the most efficient when more immediate analyses and results are required.

Nocturnal High-Flow Nasal Cannula Therapy and Sinonasal Symptoms During Cystic Fibrosis Exacerbations.

BACKGROUND: Both nasal obstruction and sleep disturbance are common in patients with cystic fibrosis (CF). In patients with obstructive sleep apnea (OSA), studies suggest that these conditions are related and that nasal congestion improves with CPAP therapy. We hypothesized that subjects admitted to hospital for therapy of an exacerbation of CF would have both nasal symptoms and sleep disturbance and that these would improve with the initiation of nocturnal high-flow nasal cannula therapy (HFNC). METHODS: Twenty-five subjects with an exacerbation of CF were enrolled to randomly receive either 5 d of nocturnal HFNC at 20 L/min in the treatment group or 5 L/min of nocturnal nasal cannula air at ambient temperature and humidity in the low-flow group. On the first and last day of the study, the Sino-Nasal Outcome Test (SNOT-20) was administered to evaluate nasal symptoms, and sleep quality was measured using the Actiwatch 2. RESULTS: Fifteen subjects completed the study (6 HFNC, 9 low flow). We confirmed that subjects had significant sleep disturbance that did not improve over the 5 d of the study. Subjects also had disturbing nasal symptoms that significantly improved only in those receiving HFNC (pre 14 [20] vs post 6 [13], P = .027). CONCLUSIONS: Similar to what has been reported in older subjects with OSA, nocturnal HFNC improves sinonasal symptoms in subjects with an exacerbation of CF. There was no measurable effect on sleep quality, which may be due to the short duration of the study, or to subjects being evaluated while being treated in a hospital setting.

In vitro-in vivocorrelation of aerosol deposition before and after metered-dose inhaler coaching in healthy children.

Although pressurized metered dose inhaler (pMDI) education is a routine part of childhood asthma management and encouraging 'optimal breathing patterns' (i.e. slowly, deeply, completely, and with a mouth seal on the mouthpiece) is an integral part of recommended pMDI education, there is currently no quantifiable way to determine if a child is inhaling their medication correctly or optimally through a valved holding chamber (VHC). The TipsHaler™ (tVHC) is a prototype VHC device that measures inspiratory time, flow, and volume without changing the properties of the medication aerosol. The measurementsin vivorecorded by the tVHC can be downloaded and transferred to a spontaneous breathing lung model to simulate the inhalational patternsin vitroand also determine the deposition of inhaled aerosol mass with each pattern. We hypothesized that pediatric patients' inhalational patterns when using a pMDI would improve after active coaching via tVHC. This would increase the pulmonary deposition of inhaled aerosols in anin vitromodel. To test this hypothesis, we conducted a single-site, prospective, pilot, pre-and-post intervention study paired with a bedside-to-bench experiment. Healthy, inhaler-naïve subjects used a placebo inhaler in conjunction with the tVHC before and after coaching and recorded inspiratory parameters. These recordings were then implemented into a spontaneous breathing lung model during albuterol MDI delivery, and pulmonary deposition of albuterol was quantified. In this pilot study, active coaching resulted in a statistically significant increase in inspiratory time (n= 8,p= 0.0344, 95%CI: 0.082 to ∞). tVHC recorded inspiratory parameters obtained from patients were successfully implemented in thein vitromodel, which demonstrated that both inspiratory time (n= 8,r= 0.78,p <0.001, 95%CI: 0.47-0.92) and volume (n= 8,r= 0.58,p =0.0186, 95%CI: 0.15-0.85) strongly correlate with pulmonary deposition of inhaled drugs.

Volatile metabolites differentiate air-liquid interface cultures after infection with Staphylococcus aureus.

Early detection of lung infection is critical to clinical diagnosis, treatment, and monitoring. Measuring volatile organic compounds (VOCs) in exhaled breath has shown promise as a rapid and accurate method of evaluating disease metabolism and phenotype. However, further investigations of the role and function of VOCs in bacterial-host-stress response is required and this can only be realised through representative in vitro models. In this study we sampled VOCs from the headspace of A549 cells at an air-liquid interface (ALI). We hypothesised VOC sampling from ALI cultures could be used to profile potential biomarkers of S. aureus lung infection. VOCs were collected using thin film microextraction (TFME) and were analysed by thermal desorption-gas chromatography-mass spectrometry. After optimising ALI cultures, we observed seven VOCs changed between A549 and media control samples. After infecting cells with S. aureus, supervised principal component-discriminant function analysis revealed 22 VOCs were found to be significantly changed in infected cells compared to uninfected cells (p < 0.05), five of which were also found in parallel axenic S. aureus cultures. We have demonstrated VOCs that could be used to identify S. aureus in ALI cultures, supporting further investigation of VOC analysis as a highly sensitive and specific test for S. aureus lung infection.

Bromazolam Blood Concentrations in Postmortem Cases-A British Columbia Perspective.

Bromazolam is a designer benzodiazepine that was first detected in British Columbia in January 2021. Postmortem cases were analyzed using a comprehensive blood drug screening procedure by liquid chromatography-high-resolution mass spectrometry before being retrospectively analyzed using an in-house novel psychoactive substances data processing method. Bromazolam was detected in 41 postmortem cases in 2021 and quantitatively confirmed by standard addition, using liquid chromatography-tandem mass spectrometry. The mean bromazolam concentration observed was 11.4 ± 53.7 ng/mL (median concentration: 1.6 ng/mL), with a range from 0.5 to 319.3 ng/mL and the majority of cases co-occurring with fentanyl. These low concentrations may be indicative of a presumed enhancement of opioid effects, rather than being used as a stand-alone drug. Bromazolam was always detected with opioids (fentanyl and carfentanil), stimulants (methamphetamine) and/or other benzodiazepines (etizolam and flualprazolam). To our knowledge, this is the first report to provide concentrations of bromazolam in postmortem blood samples in Canada.

The physics of human breathing: flow, timing, volume, and pressure parameters for normal, on-demand, and ventilator respiration.

Normal breathing for healthy humans is taken for granted; it occurs without conscious effort using ambient (1-atmosphere) pressure with 21% oxygen (O2) concentration. The body automatically adjusts for stress, exercise, altitude, and mild disease by increasing the volume and frequency of breathing. Longer term adaptations for exercise and altitude include increases in red blood cell counts and higher concentrations of capillaries in muscle tissue. When more challenging external environmental conditions or pulmonary illnesses exceed the capability for these adaptations, the human system requires technology to maintain sufficient ventilation to preserve life. On the environmental side there are two conditions to be addressed: toxicity of the surrounding atmosphere and changes in external pressure and O2concentration. On the medical side, mechanisms for assisting breathing include O2supplementation at ambient pressure, positive pressure/flow without additional O2, or a combination of both. This overview describes the various technologies applied to maintaining a safe breathing environment. Topics for environmental intervention include filter-based and flowing air-supply masks for toxic environments (occupational and laboratory protection), and on-demand gas supply systems for firefighters, self-contained underwater breathing apparatus divers, and altitude (high performance aircraft, spacecraft) applications. The topics for medical intervention include nasal cannula, continuous positive airway pressure, and medical ventilators. The primary purpose of this article is to provide a basic understanding of normal human breathing and the adaptation of breathing in different environments using available technologies.

Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium.

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air-liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbß expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, ß, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbß gene expression were associated with lower FEV1 in asthma. Both Hbß knockdown and overexpression affected cell morphology. Hbß and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbß gene expression were associated with airflow obstruction. Hbß and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

2020 Year in Review: Pharmacologic Treatments for COVID-19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 infection, has led to a pandemic of acute respiratory illness. Pharmacologic treatments for COVID-19 have included treatments that target infection prevention, prevention of viral replication, reduce inflammation, and manage symptoms of respiratory failure caused by the disease. This is a review of key pharmacologic treatments for COVID-19 based on peer-reviewed articles from 2020.

Benefits of Airway Androgen Receptor Expression in Human Asthma.

Rationale: Androgens are potentially beneficial in asthma, but AR (androgen receptor) has not been studied in human airways.Objectives: To measure whether AR and its ligands are associated with human asthma outcomes.Methods: We compared the effects of AR expression on lung function, symptom scores, and fractional exhaled nitric oxide (FeNO) in adults enrolled in SARP (Severe Asthma Research Program). The impact of sex and of androgens on asthma outcomes was also evaluated in the SARP with validation studies in the Cleveland Clinic Health System and the NHANES (U.S. National Health and Nutrition Examination Survey).Measurements and Main Results: In SARP (n = 128), AR gene expression from bronchoscopic epithelial brushings was positively associated with both FEV1/FVC ratio (R2 = 0.135, P = 0.0002) and the total Asthma Quality of Life Questionnaire score (R2 = 0.056, P = 0.016) and was negatively associated with FeNO (R2 = 0.178, P = 9.8 × 10-6) and NOS2 (nitric oxide synthase gene) expression (R2 = 0.281, P = 1.2 × 10-10). In SARP (n = 1,659), the Cleveland Clinic Health System (n = 32,527), and the NHANES (n = 2,629), women had more asthma exacerbations and emergency department visits than men. The levels of the AR ligand precursor dehydroepiandrosterone sulfate correlated positively with the FEV1 in both women and men.Conclusions: Higher bronchial AR expression and higher androgen levels are associated with better lung function, fewer symptoms, and a lower FeNO in human asthma. The role of androgens should be considered in asthma management.

Blood Biomarkers for Detection of Brain Injury in COVID-19 Patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus attacks multiple organs of coronavirus disease 2019 (COVID-19) patients, including the brain. There are worldwide descriptions of neurological deficits in COVID-19 patients. Central nervous system (CNS) symptoms can be present early in the course of the disease. As many as 55% of hospitalized COVID-19 patients have been reported to have neurological disturbances three months after infection by SARS-CoV-2. The mutability of the SARS-COV-2 virus and its potential to directly affect the CNS highlight the urgency of developing technology to diagnose, manage, and treat brain injury in COVID-19 patients. The pathobiology of CNS infection by SARS-CoV-2 and the associated neurological sequelae of this infection remain poorly understood. In this review, we outline the rationale for the use of blood biomarkers (BBs) for diagnosis of brain injury in COVID-19 patients, the research needed to incorporate their use into clinical practice, and the improvements in patient management and outcomes that can result. BBs of brain injury could potentially provide tools for detection of brain injury in COVID-19 patients. Elevations of BBs have been reported in cerebrospinal fluid (CSF) and blood of COVID-19 patients. BB proteins have been analyzed in CSF to detect CNS involvement in patients with infectious diseases, including human immunodeficiency virus and tuberculous meningitis. BBs are approved by the U.S. Food and Drug Administration for diagnosis of mild versus moderate traumatic brain injury and have identified brain injury after stroke, cardiac arrest, hypoxia, and epilepsy. BBs, integrated with other diagnostic tools, could enhance understanding of viral mechanisms of brain injury, predict severity of neurological deficits, guide triage of patients and assignment to appropriate medical pathways, and assess efficacy of therapeutic interventions in COVID-19 patients.

A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects.

BACKGROUND: Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense, and worsens airway inflammation. Inhaled Optate is designed to safely raise airway surface pH and is well tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms. METHODS: We grew primary NHAE cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether Optate would prevent viral infection and replication. Cells were pretreated with Optate or placebo prior to infection (multiplicity of infection = 1), and viral replication was determined with plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial. RESULTS: Optate almost completely prevented viral replication at each time point between 24 h and 120 h, relative to placebo, on both plaque assay and N protein expression (P < .001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs, or oxygenation. CONCLUSIONS: Inhaled Optate may be well suited for a clinical trial in patients with pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regard to pH, isotonicity, and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.

The remaining barriers to normalcy in CF: Advances in assessment of CF lung disease.

Despite early diagnosis of cystic fibrosis (CF) through newborn screening, a substantial proportion of infants and young children with CF still demonstrate physiologic and structural evidence of lung disease progression, such as obstructive airway disease and bronchiectasis. The growing availability of highly effective CF transmembrane conductance regulatory modulator therapy to the vast majority of people with CF has led to the potential to alter the natural history of CF lung disease, but to assess the full impact of these therapies on CF lung disease and to help guide treatment, sensitive measures of early and mild disease are needed. Chest imaging using computed tomography or magnetic resonance imaging is one approach, but technologic barriers and/or concern about exposure to ionizing radiation may limit its use. However, advances in physiologic measurement techniques and exhaled breath analysis offer another option for assessment of CF lung disease.