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Melatonin vaping products, touted for their faster absorption than oral melatonin supplements, have been gaining popularity among adolescents as sleep aid. Here, we elucidated the response of human bronchial epithelial cells (hBECs) to high levels of melatonin from vaped aerosols, investigated the uptake of melatonin by hBECs in vitro , and characterized the chemical composition of three commercially available melatonin vapes. Melatonin vape exposure decreased the secretion of chemokines and produced an immunosuppressive gene expression signature. The tested devices contained potential contaminants, including pharmaceuticals and industrial chemicals. Further investigation is needed for melatonin vapes to determine their local and systemic toxicity.

The use of in vitro new approach methodologies (NAMs) to assess respiratory irritation depends on several factors, including the specifics of exposure methods and cell/tissue-based test systems. This topic was examined in the context of human health risk assessment for cleaning products at a 1-day public workshop held on 2 March 2023, organized by the American Cleaning Institute ® (ACI). The goals of this workshop were to (1) review in vitro NAMs for evaluation of respiratory irritation, (2) examine different perspectives on current challenges and suggested solutions, and (3) publish a manuscript of the proceedings. Targeted sessions focused on exposure methods, in vitro cell/tissue test systems, and application to human health risk assessment. The importance of characterization of assays and development of reporting standards was noted throughout the workshop. The exposure methods session emphasized that the appropriate exposure system design depends on the purpose of the assessment. This is particularly important given the many dosimetry and technical considerations affecting relevance and translation of results to human exposure scenarios. Discussion in the in vitro cell/tissue test systems session focused on the wide variety of cell systems with varying suitability for evaluating key mechanistic steps, such as molecular initiating events (MIEs) and key events (KEs) likely present in any putative respiratory irritation adverse outcome pathway (AOP). This suggests the opportunity to further develop guidance around in vitro cell/tissue test system endpoint selection, assay design, characterization and validation, and analytics that provide information about a given assay's utility. The session on applications for human health protection emphasized using mechanistic understanding to inform the choice of test systems and integration of NAMs-derived data with other data sources (e.g., physicochemical properties, exposure information, and existing in vivo data) as the basis for in vitro to in vivo extrapolation. In addition, this group noted a need to develop procedures to align NAMs-based points of departure (PODs) and uncertainty factor selection with current human health risk assessment methods, together with consideration of elements unique to in vitro data. Current approaches are described and priorities for future characterization of in vitro NAMs to assess respiratory irritation are noted., Competing Interests: The authors declare that this workshop and the preparation of the workshop proceedings manuscript were funded by the American Cleaning Institute® (ACI). The funder was involved in conceptualization and organization of the workshop, and in a high-level review of the written proceedings. FK was an employee of ACI. KP was an employee of Clorox, a member company of ACI, which manufactures cleaning products and may use the methods/approaches outlined in this manuscript. KG was an employee of Arkema, a member company of ACI, which manufactures chemicals, some of which are used in cleaning products and may use the methods/approaches outlined in this manuscript. CF was an employee of Ecolab, a member company of ACI, which manufactures cleaning products and may use the methods/approaches outlined in this manuscript. NP was an employee of Ecolab at the time of the workshop, and moved to 3M during the preparation of this manuscript. LH, MB, and AB were consultants paid by ACI. LH and MB were employees of the University of Cincinnati. AB was an employee of Stantec ChemRisk at the time of the workshop and moved to ToxStrategies, Inc. during the preparation of this manuscript. HB and VP were employees of the Institute for in vitro Sciences, which uses some of the in vitro experimental models described in this manuscript for commercial purposes. SB was an employee of Invitrolize, which markets in vitro models for the respiratory tract. PC was an employee of the Wake Forest Institute for Regenerative Medicine at the time of the workshop, became an independent consultant during the writing of this manuscript and is now at UNC Chapel Hill. SD was an employee of the Insight Exposure and Risk Sciences Group. AMJ was an employee of the U.S. EPA Office of Research and Development. SDM was an employee of the U.S. EPA Office of Research and Development at the time of the workshop and was an employee of RTI during the writing of the proceedings. CR was the owner of Roper Toxicology Consulting Limited. MS was an advisor to PETA Science Consortium International e.V. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Haber, Bradley, Buerger, Behrsing, Burla, Clapp, Dotson, Fisher, Genco, Kruszewski, McCullough, Page, Patel, Pechacek, Roper, Sharma and Jarabek.)

Background: Recent studies show e-cigarette (EC) users have increased rates of chronic bronchitic symptoms that may be associated with depressed mucociliary clearance (MCC). Little is known about the acute or chronic effects of EC inhalation on in vivo MCC. Methods: In vivo MCC was measured in young adult vapers ( n  = 5 males, mean age = 21) after controlled inhalation of a radiolabeled (Tc99m sulfur colloid) aerosol. Whole-lung clearance of radiolabeled deposited particles was measured over a 90-minute period for baseline MCC and associated with controlled periodic vaping over the first 60 minutes of MCC measurements. The vaping challenge was administered from a fourth generation box mod EC containing unflavored e-liquid (65% propylene glycol/35% vegetable glycerin, 3 mg/mL freebase nicotine). The challenge was administered at the start of each 10-minute interval of MCC measurements and consisted of 1 puff every 30 seconds for 5 minutes (i.e., 10 puffs for each 10-minute period for a total of 60 puffs during the initial 60 minutes of MCC measurements). Results: Compared with baseline, peripheral lung average clearance (%) over the 90 minutes of MCC measures was enhanced, associated with EC challenge, 12 (±6) versus 24 (±6), respectively ( p  < 0.05 by Wilcoxon signed-rank test). Conclusions: Acute enhancement of in vivo MCC during EC challenge is contrary to recent studies showing nicotine-associated slowing of ciliary beat and mucus transport at higher nicotine levels than those used here. However, our findings are consistent with an acute increase in fluid volume and mucin secretion to the bronchial airway surface that is likely short lived. Research reported in this publication was supported by the National Institutes of Health R01HL139369 and registered with ClinicalTrials.gov (NCT03700892).

Objective: Due to recent increases in the use of vaping devices, there is a high demand for research addressing the respiratory health effects of vaping products. Given the constantly changing nature of the vaping market with new devices, flavors, metals, and other chemicals rapidly emerging, there is a need for inexpensive and highly adaptable vaping device exposure systems. Here, we describe the design and validation of a novel in vitro aerosol exposure system for toxicity testing of vaping devices., Materials and Methods: We developed an inexpensive, open-source in vitro vaping device exposure system that produces even deposition, can be adapted for different vaping devices, and allows for experiments to be performed under physiological conditions. The system was then validated with deposition testing and a representative exposure with human bronchial epithelial cells (hBECs)., Results: The Vaping Product Exposure System (VaPES) produced sufficient and uniform deposition for dose-response studies and was precise enough to observe biological responses to vaping exposures. VaPES was adapted to work with both pod and cartridge-based vaping devices., Conclusion: We have designed and validated a novel vaping device exposure system that will eliminate the need to use high-cost commercial exposure systems, lowering the barrier to entry of physiologically relevant vaping studies.

E-cigarette, or vaping product use-associated lung injury (EVALI), is a severe respiratory disorder that caused a sudden outbreak of hospitalized young people in 2019. Using cannabis oil containing vaping products, including vitamin E acetate contaminants, was found to be strongly associated with EVALI. However, the underlying tissue impacts of the condition are still largely unknown. Here, we focused on the vehicle cannabinoid oil (CBD oil) and contaminant vitamin E acetate (VEA) effects on airway epithelial cells. Primary human bronchial epithelial (HBE) cultures were exposed to e-liquid aerosols that contained CBD oil and VEA in combination or the common e-liquid components PG/VG with and without nicotine. Cell viability analysis indicated dramatically increased cell death counts after 3 days of CBD exposure, and this effect was even higher after CBD + VEA exposure. Microscopic examination of the cultures revealed cannabinoid and VEA depositions on the epithelial surfaces and cannabinoid accumulation in exposed cells, followed by cell death. These observations were supported by proteomic analysis of the cell secretions that exhibited increases in known markers of airway epithelial toxicity, such as xenobiotic enzymes, factors related to oxidative stress response, and cell death indicators. Overall, our study provides insights into the association between cannabinoid oil and vitamin E acetate vaping and lung injury. Collectively, our results suggest that the adherent accumulation of CBD oil on airway surfaces and the cellular uptake of both CBD oil- and VEA-containing condensates cause elevated metabolic stress, leading to increased cell death rates in human airway epithelial cultures., (© 2022 John Wiley & Sons Ltd.)

Cannabidiol (CBD) vaping products have become widely available in the U.S. since their legalization in 2018. However, little is known about their respiratory health effects. Here we show that aerosolization of commercial CBD vaping products generates a reactive CBD quinone (CBDQ) which forms adducts with protein cysteine residues. Using click chemistry and a novel in vitro vaping product exposure system (VaPES), we further demonstrate that CBDQ forms adducts with human bronchial epithelial cell proteins including Keap1 and activates KEAP1-Nrf2 stress response pathway genes. These results suggest that vaping CBD alters protein function and induces cellular stress pathways in the lung.

Certain e-liquids and aromatic aldehyde flavoring agents were previously identified as inhibitors of microsomal recombinant CYP2A6, the primary nicotine-metabolizing enzyme. However, due to their reactive nature, aldehydes may react with cellular components before reaching CYP2A6 in the endoplasmic reticulum. To determine whether e-liquid flavoring agents inhibited CYP2A6 in a cellular system, we investigated their effects on CYP2A6 using BEAS-2B cells transduced to overexpress CYP2A6. We demonstrated that two e-liquids and three aldehyde flavoring agents (cinnamaldehyde, benzaldehyde, and ethyl vanillin) exhibited dose-dependent inhibition of cellular CYP2A6., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Total and regional deposition of inhaled electronic cigarette (E-cig) particles in the respiratory tract (RT) depends on both physical properties of the inhaled particles and biological factors of users, for example, breathing pattern or puff profile, airway anatomy, and regional ventilation. Accurate particle sizing of E-cig aerosols is essential for predicting particle deposition in the RT. Studies using a variety of sizing methods have shown mass median aerodynamic diameters ranging from 0.2 to 1.2 um and secondary count diameters in the ultrafine range (<0.1 μm). Incorporating these particle sizes into a multiple-path particle dosimetry (MPPD) model shows 10% to 45% total lung deposition by mass and 30% to 80% for ultrafine particles depending on the breathing patterns. These predictions are consistent with experimental measures of deposition fraction of submicron and ultrafine particles. While box-mod-type E-cig devices allow for full "direct-lung" inhalations of aerosol, the more recent pod-based, and disposable E-cigs (e.g., JUUL, Puff Bar, Stig) deliver the aerosol as a "mouth-to-lung" puff, or bolus, that is inhaled early in the breath followed to various degrees by further inhalation of ambient air. Measurement of realistic ventilation patterns associated with these various devices may further improve deposition predictions. Finally, while in vivo measures of RT deposition present a challenge, a recent methodology to radiolabel E-cig particles may allow for such measurements by gamma scintigraphy. Supported by NIH/NHLBI R01HL139369. © 2022 American Physiological Society. Compr Physiol 12: 1-10, year., (Copyright © 2022 American Physiological Society. All rights reserved.)

The dynamics describing the vicious cycle characteristic of cystic fibrosis (CF) lung disease, initiated by stagnant mucus and perpetuated by infection and inflammation, remain unclear. Here we determine the effect of the CF airway milieu, with persistent mucoobstruction, resident pathogens, and inflammation, on the mucin quantity and quality that govern lung disease pathogenesis and progression. The concentrations of MUC5AC and MUC5B were measured and characterized in sputum samples from subjects with CF ( N  = 44) and healthy subjects ( N  = 29) with respect to their macromolecular properties, degree of proteolysis, and glycomics diversity. These parameters were related to quantitative microbiome and clinical data. MUC5AC and MUC5B concentrations were elevated, 30- and 8-fold, respectively, in CF as compared with control sputum. Mucin parameters did not correlate with hypertonic saline, inhaled corticosteroids, or antibiotics use. No differences in mucin parameters were detected at baseline versus during exacerbations. Mucin concentrations significantly correlated with the age and sputum human neutrophil elastase activity. Although significantly more proteolytic cleavages were detected in CF mucins, their macromolecular properties (e.g., size and molecular weight) were not significantly different than control mucins, likely reflecting the role of S-S bonds in maintaining multimeric structures. No evidence of giant mucin macromolecule reflecting oxidative stress-induced cross-linking was found. Mucin glycomic analysis revealed significantly more sialylated glycans in CF, and the total abundance of nonsulfated O-glycans correlated with the relative abundance of pathogens. Collectively, the interaction of mucins, pathogens, epithelium, and inflammatory cells promotes proteomic and glycomic changes that reflect a persistent mucoobstructive, infectious, and inflammatory state.

Background: Globally, a large percentage of men keep a beard at least occasionally. Workplace regulations prohibit beards with N95 respirators, but there is little information on the effect of beards with face masks worn by the public for protection against SARS-CoV-2., Methods and Findings: We examined the fitted filtration efficiency (FFE) of five commonly worn protective face masks as a function of beard length following the US Occupational Safety and Health Administration Quantitative Fit Test: N95 (respirator), KF94 and KN95, surgical/procedure, and cloth masks. A comparison using N95 respirators was carried out in shaven and bearded men. A detailed examination was conducted for beard lengths between 0 and 10 mm (0.5 mm increments). The effect of an exercise band covering the beard on FFE was also tested. Although N95 respirators showed considerable variability among bearded men, they had the highest FFE for beard lengths up to 10 mm. KF94 and KN95 masks lost up to 40% of their FFE. Procedure and cotton masks had poor performance even on bare skin (10-30% FFE) that did not change appreciably with beard length. Marked performance improvements were observed with an exercise band worn over the beard., Conclusions: Though variable, N95 respirators offer the best respiratory protection for bearded men. While KF94 and KN95 FFE is compromised considerably by increasing beard length, they proved better options than procedure and cotton face masks. A simple exercise band improves FFE for face masks commonly used by bearded men during the COVID-19 pandemic., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Ozone (O 3 ) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O 3 oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. By using an alkynyl-tagged form of SecoA and shotgun proteomics, we identified 135 proteins as being modified in bronchial epithelial cells. Among them was NLRP2 (NLR family pyrin domain-containing protein 2), which forms an alkynyl-tagged SecoA-protein adduct at lysine residue 1019 (K1019) in the terminal leucine-rich repeat region, a known regulatory region for NLR proteins. NLRP2 expression in airway epithelial cells was characterized, and CRISPR-Cas9 knockout (KO) and shRNA knockdown of NLRP2 were used to determine its function in O 3 -induced inflammation. No evidence for NLPR2 inflammasome formation or an NLRP2-dependent increase in caspase-1 activity in response to O 3 was observed. O 3 -induced proinflammatory gene expression for CXCL2 and CXCL8 / IL8 was further enhanced in NLRP2-KO cells, suggesting a negative regulatory role. Reconstitution of NLRP2-KO cells with the NLRP2 K1019 mutated to arginine partially blocked SecoA adduction and enhanced O 3 -induced IL-8 release as compared with wild-type NLRP2. Together, our findings uncover NLRP2 as a highly abundant, key component of proinflammatory signaling pathways in airway epithelial cells and as a novel mediator of O 3 -induced inflammation.

Shortages of efficient filtering facepiece respirators leave the public vulnerable to transmission of infectious diseases in small particle aerosols. This study demonstrates that a high-filtration-efficiency facepiece capable of filtering out >95% of 0.05μm particles while being worn can be simply produced with available materials., (Copyright © 2021 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Importance: During the coronavirus disease 2019 (COVID-19) pandemic, the general public has been advised to wear masks or improvised face coverings to limit transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there has been considerable confusion and disagreement regarding the degree to which masks protect the wearer from airborne particles., Objectives: To evaluate the fitted filtration efficiency (FFE) of various consumer-grade and improvised face masks, as well as several popular modifications of medical procedure masks that are intended to improve mask fit or comfort., Design, Setting, and Participants: For this study conducted in a research laboratory between June and August 2020, 7 consumer-grade masks and 5 medical procedure mask modifications were fitted on an adult male volunteer, and FFE measurements were collected during a series of repeated movements of the torso, head, and facial muscles as outlined by the US Occupational Safety and Health Administration Quantitative Fit Testing Protocol. The consumer-grade masks tested included (1) a 2-layer nylon mask with ear loops that was tested with an optional aluminum nose bridge and filter insert in place, (2) a cotton bandana folded diagonally once (ie, "bandit" style) or in a (3) multilayer rectangle according to the instructions presented by the US Surgeon General, (4) a single-layer polyester/nylon mask with ties, (5) a polypropylene mask with fixed ear loops, (6) a single-layer polyester gaiter/neck cover balaclava bandana, and (7) a 3-layer cotton mask with ear loops. Medical procedure mask modifications included (1) tying the mask's ear loops and tucking in the side pleats, (2) fastening ear loops behind the head with 3-dimensional-printed ear guards, (3) fastening ear loops behind the head with a claw-type hair clip, (4) enhancing the mask/face seal with rubber bands over the mask, and (5) enhancing the mask/face seal with a band of nylon hosiery over the fitted mask., Main Outcomes and Measures: The primary study outcome was the measured FFE of common consumer-grade and improvised face masks, as well as several popular modifications of medical procedure masks., Results: The mean (SD) FFE of consumer grade masks tested on 1 adult male with no beard ranged from 79.0% (4.3%) to 26.5% (10.5%), with the 2-layer nylon mask having the highest FFE. Unmodified medical procedure masks with ear loops had a mean (SD) FFE of 38.5% (11.2%). All modifications evaluated in this study increased procedure mask FFE (range [SD], 60.3% [11.1%] to 80.2% [3.1%]), with a nylon hosiery sleeve placed over the procedure mask producing the greatest improvement., Conclusions and Relevance: While modifications to improve medical procedure mask fit can enhance the filtering capability and reduce inhalation of airborne particles, this study demonstrates that the FFEs of consumer-grade masks available to the public are, in many cases, nearly equivalent to or better than their non-N95 respirator medical mask counterparts.

Importance: Procuring respiratory protection for clinicians and other health care workers has become a major challenge of the coronavirus disease 2019 (COVID-19) pandemic and has resulted in nonstandard practices such as the use of expired respirators and various decontamination processes to prolong the useful life of respirators in health care settings. In addition, imported, non-National Institute for Occupational Safety and Health (NIOSH)-approved respirators have been donated or acquired by hospitals as a potential replacement for limited NIOSH-approved N95 respirators., Objective: To assess fitted filtration efficiencies (FFEs) for face mask alternatives used during the COVID-19 pandemic., Design, Setting, and Participants: For this quality-improvement study conducted between April and June 2020, we used the Occupational Safety and Health Administration's Quantitative Fit Testing Protocol for Filtering Facepiece Respirators in a laboratory atmosphere supplemented with sodium chloride particles to assess the FFEs of a variety of respirators worn by a male volunteer and female volunteer., Main Outcomes and Measures: The FFEs of respirators commonly worn by clinicians and other health care workers and available respirator alternatives during the COVID-19 pandemic., Results: Of the 29 different fitted face mask alternatives tested on 1 man and 1 woman, expired N95 respirators with intact elastic straps and respirators subjected to ethylene oxide and hydrogen peroxide sterilization had unchanged FFE (>95%). The performance of N95 respirators in the wrong size had slightly decreased performance (90%-95% FFE). All of the respirators not listed as approved in this evaluation (n = 6) failed to achieve 95% FFE. Neither of the 2 imported respirators authorized for use by the Centers for Disease Control and Prevention that were not NIOSH-approved tested in this study achieved 95% FFE, and the more effective of the 2 functioned at approximately 80% FFE. Surgical and procedural face masks had filtering performance that was lower relative to that of N95 respirators (98.5% overall FFE), with procedural face masks secured with elastic ear loops showing the lowest efficiency (38.1% overall FFE)., Conclusions and Relevance: This quality-improvement study evaluating 29 face mask alternatives for use by clinicians interacting with patients during the COVID-19 pandemic found that expired N95 respirators and sterilized, used N95 respirators can be used when new N95 respirators are not available. Other alternatives may provide less effective filtration.

Nicotine is the primary psychoactive chemical in both traditional and electronic cigarettes (e-cigarettes). Nicotine levels in both traditional cigarettes and e-cigarettes are an important concern for public health. Nicotine exposure due to e-cigarette use is of importance primarily due to the addictive potential of nicotine, but there is also concern for nicotine poisoning in e-cigarette users. Nicotine concentrations in e-liquids vary widely. Additionally, there is significant genetic variability in the rate of metabolism of nicotine due to polymorphisms of CYP2A6, the enzyme responsible for the metabolism of approximately 80% of nicotine. Recent studies have shown CYP2A6 activity is also reduced by aromatic aldehydes such as those added to e-liquids as flavoring agents, which may increase nicotine serum concentrations. However, the impacts of flavored e-liquids on CYP2A6 activity are unknown. In this study, we investigated the impact of three flavored e-liquids on microsomal recombinant CYP2A6. Microsomal recombinant CYP2A6 was challenged at e-liquid concentrations ranging up to 0.125% (v/v) and monitored for metabolic activity using a probe molecule approach. Two e-liquids exhibited dose-dependent inhibition of CYP2A6 activity. Mass spectrometry was conducted to identify flavoring agents in flavored e-liquids that inhibited CYP2A6. Microsomal recombinant CYP2A6 was subsequently exposed to flavoring agents at concentrations ranging from 0.03 μM to 500 μM. Cinnamaldehyde and benzaldehyde were found to be the most potent inhibitors of microsomal CYP2A6 of the flavoring agents tested, with identified IC 50 values of 1.1 μM and 3.0 μM, respectively. These data indicate certain aromatic aldehyde flavoring agents are potent inhibitors of CYP2A6, which may reduce nicotine metabolism in vivo. These findings indicate an urgent need to evaluate the effects of flavoring agents in e-cigarette liquids on the pharmacokinetics of nicotine in vivo.

Aldehydes in cigarette smoke (CS) impair mitochondrial function and reduce ciliary beat frequency (CBF), leading to diminished mucociliary clearance (MCC). However, the effects of aldehyde e-cigarette flavorings on CBF are unknown. The purpose of this study was to investigate whether cinnamaldehyde, a flavoring agent commonly used in e-cigarettes, disrupts mitochondrial function and impairs CBF on well-differentiated human bronchial epithelial (hBE) cells. To this end, hBE cells were exposed to diluted cinnamon-flavored e-liquids and vaped aerosol and assessed for changes in CBF. hBE cells were subsequently exposed to various concentrations of cinnamaldehyde to establish a dose-response relationship for effects on CBF. Changes in mitochondrial oxidative phosphorylation and glycolysis were evaluated by Seahorse Extracellular Flux Analyzer, and adenine nucleotide levels were quantified by HPLC. Both cinnamaldehyde-containing e-liquid and vaped aerosol rapidly yet transiently suppressed CBF, and exposure to cinnamaldehyde alone recapitulated this effect. Cinnamaldehyde impaired mitochondrial respiration and glycolysis in a dose-dependent manner, and intracellular ATP levels were significantly but temporarily reduced following exposure. Addition of nicotine had no effect on the cinnamaldehyde-induced suppression of CBF or mitochondrial function. These data indicate that cinnamaldehyde rapidly disrupts mitochondrial function, inhibits bioenergetic processes, and reduces ATP levels, which correlates with impaired CBF. Because normal ciliary motility and MCC are essential respiratory defenses, inhalation of cinnamaldehyde may increase the risk of respiratory infections in e-cigarette users.

Rationale: E-cigarettes have become increasingly popular and little is known about their potential adverse health effects., Objectives: To determine the effects of e-cigarette use on the airways., Methods: Induced sputum samples from cigarette smokers, e-cigarette users, and nonsmokers were analyzed by quantitative proteomics, and the total and individual concentrations of mucins MUC5AC and MUC5B were determined by light scattering/refractometry and labeled mass spectrometry, respectively. Neutrophil extracellular trap (NET) formation rates were also determined for the same groups., Measurements and Main Results: E-cigarette users exhibited significant increases in aldehyde-detoxification and oxidative stress-related proteins associated with cigarette smoke compared with nonsmokers. The levels of innate defense proteins associated with chronic obstructive pulmonary disease, such as elastase and matrix metalloproteinase-9, were significantly elevated in e-cigarette users as well. E-cigarette users' sputum also uniquely exhibited significant increases in neutrophil granulocyte-related and NET-related proteins, such as myeloperoxidase, azurocidin, and protein-arginine deiminase 4, despite no significant elevation in neutrophil cell counts. Peripheral neutrophils from e-cigarette users showed increased susceptibility to phorbol 12-myristate 13-acetate-induced NETosis. Finally, a compositional change in the gel-forming building blocks of airway mucus (i.e., an elevated concentration of mucin MUC5AC) was observed in both cigarette smokers and e-cigarette users., Conclusions: Together, our results indicate that e-cigarette use alters the profile of innate defense proteins in airway secretions, inducing similar and unique changes relative to cigarette smoking. These data challenge the concept that e-cigarettes are a healthier alternative to cigarettes.

Purpose of Review: Vaping is gaining popularity in the USA, particularly among teens and young adults. While e-cigs are commonly represented as safer alternatives to tobacco cigarettes, little is known regarding the health effects of their short- or long-term use, especially in individuals with pre-existing respiratory diseases such as asthma. Flavored e-cig liquids (e-liquids) and e-cig aerosols contain airway irritants and toxicants that have been implicated in the pathogenesis and worsening of lung diseases. In this review, we will summarize existing data on potential health effects of components present in e-cig aerosols, such as propylene glycol, vegetable glycerin, nicotine, and flavorings, and discuss their relevance in the context of asthma., Recent Findings: Recent survey data indicate that adolescents with asthma had a higher prevalence of current e-cig use (12.4%) compared to their non-asthmatics peers (10.2%) and conveyed positive beliefs about tobacco products, especially e-cigs. Similarly, a study conducted among high school students from Ontario, Canada, indicated a greater likelihood of e-cig use in asthmatics as compared to their non-asthmatic peers. Availability of different flavorings is often cited as the main reason among youth/adolescents for trying e-cigs or switching from cigarettes to e-cigs. Occupational inhalation of some common food-safe flavoring agents is reported to cause occupational asthma and worsen asthmatic symptoms. Moreover, workplace inhalation exposures to the flavoring agent diacetyl have caused irreversible obstructive airway disease in healthy workers. Additionally, recent studies report that thermal decomposition of propylene glycol (PG) and vegetable glycerin (VG), the base constituents of e-liquids, produces reactive carbonyls, including acrolein, formaldehyde, and acetaldehyde, which have known respiratory toxicities. Furthermore, recent nicotine studies in rodents reveal that prenatal nicotine exposures lead to epigenetic reprogramming in the offspring, abnormal lung development, and multigenerational transmission of asthmatic-like symptoms. Comparisons of the toxicity and health effects of e-cigs and conventional cigarettes often focus on toxicants known to be present in cigarette smoke (CS) (i.e., formaldehyde, nitrosamines, etc.), as well as smoking-associated clinical endpoints, such as cancer, bronchitis, and chronic obstructive pulmonary disease (COPD). However, this approach disregards potential toxicity of components unique to flavored e-cigs, such as PG, VG, and the many different flavoring chemicals, which likely induce respiratory effects not usually observed in cigarette smokers.

Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. End points included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell-mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids, and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures, and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol. We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations., (Copyright © 2017 the American Physiological Society.)

Secondary organic aerosol (SOA) derived from the photochemical oxidation of isoprene contributes a substantial mass fraction to atmospheric fine particulate matter (PM 2.5 ). The formation of isoprene SOA is influenced largely by anthropogenic emissions through multiphase chemistry of its multigenerational oxidation products. Considering the abundance of isoprene SOA in the troposphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. In this study, we assessed the effects of isoprene SOA on gene expression in human airway epithelial cells (BEAS-2B) through an air-liquid interface exposure. Gene expression profiling of 84 oxidative stress and 249 inflammation-associated human genes was performed. Our results show that the expression levels of 29 genes were significantly altered upon isoprene SOA exposure under noncytotoxic conditions (p < 0.05), with the majority (22/29) of genes passing a false discovery rate threshold of 0.3. The most significantly affected genes belong to the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor network. The Nrf2 function is confirmed through a reporter cell line. Together with detailed characterization of SOA constituents, this study reveals the impact of isoprene SOA exposure on lung responses and highlights the importance of further understanding its potential health outcomes.

Reliable methods for sampling the nasal mucosa provide clinical researchers with key information regarding respiratory biomarkers of exposure and disease. For quick and noninvasive sampling of the nasal mucosa, nasal lavage (NL) collection has been widely used as a clinical tool; however, limitations including volume variability, sample dilution, and storage prevent NL collection from being used in nonlaboratory settings and analysis of low abundance biomarkers. In this study, we optimize and validate a novel methodology using absorbent Leukosorb paper cut to fit the nasal passage to extract epithelial lining fluid (ELF) from the nasal mucosa. The ELF sampling method limits the dilution of soluble mediators, allowing quantification of both high- and low-abundance soluble biomarkers such as IL-1β, IL-8, IL-6, interferon gamma-induced protein 10 (IP-10), and neutrophil elastase. Additionally, we demonstrate that this method can successfully detect the presence of respiratory pathogens such as influenza virus and markers of antibiotic-resistant bacteria in the nasal mucosa. Efficacy of ELF collection by this method is not diminished in consecutive-day sampling, and percent recovery of both recombinant IL-8 and soluble mediators are not changed despite freezing or room temperature storage for 24 h. Our results indicate that ELF collection using Leukosorb paper sampling of ELF provides a sensitive, easy-to-use, and reproducible methodology to collect concentrated amounts of soluble biomarkers from the nasal mucosa. Moreover, the methodology described herein improves upon the standard NL collection method and provides researchers with a novel tool to assess changes in nasal mucosal host defense status., (Copyright © 2017 the American Physiological Society.)

Exposure to cigarette smoke is known to result in impaired host defense responses and immune suppressive effects. However, the effects of new and emerging tobacco products, such as e-cigarettes, on the immune status of the respiratory epithelium are largely unknown. We conducted a clinical study collecting superficial nasal scrape biopsies, nasal lavage, urine, and serum from nonsmokers, cigarette smokers, and e-cigarette users and assessed them for changes in immune gene expression profiles. Smoking status was determined based on a smoking history and a 3- to 4-wk smoking diary and confirmed using serum cotinine and urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels. Total RNA from nasal scrape biopsies was analyzed using the nCounter Human Immunology v2 Expression panel. Smoking cigarettes or vaping e-cigarettes resulted in decreased expression of immune-related genes. All genes with decreased expression in cigarette smokers (n = 53) were also decreased in e-cigarette smokers. Additionally, vaping e-cigarettes was associated with suppression of a large number of unique genes (n = 305). Furthermore, the e-cigarette users showed a greater suppression of genes common with those changed in cigarette smokers. This was particularly apparent for suppressed expression of transcription factors, such as EGR1, which was functionally associated with decreased expression of 5 target genes in cigarette smokers and 18 target genes in e-cigarette users. Taken together, these data indicate that vaping e-cigarettes is associated with decreased expression of a large number of immune-related genes, which are consistent with immune suppression at the level of the nasal mucosa., (Copyright © 2016 the American Physiological Society.)

Purpose: We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG., Methods: Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl- cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays., Results: Expression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels., Conclusions: This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in DeltaF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of DeltaF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on DeltaF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects DeltaF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies--using differing mechanisms of action--may have additive benefits in treating CF.