Airway Lining Fluid Research Articles

Activation of the Complement and Coagulation Systems in the Small Airways in Asthma

Background: Several studies have shown the importance of the complement and coagulation systems in the pathogenesis of asthma. Objectives: We explored whether we could detect differentially abundant complement and coagulation proteins in the samples obtained from the small airway lining fluid by collection of exhaled particles in patients with asthma and whether these proteins are associated with small airway dysfunction and asthma control. Method: Exhaled particles were obtained from 20 subjects with asthma and 10 healthy controls (HC) with the PExA method and analysed with the SOMAscan proteomics platform. Lung function was assessed by nitrogen multiple breath washout test and spirometry. Results: 53 proteins associated with the complement and coagulation systems were included in the analysis. Nine of those proteins were differentially abundant in subjects with asthma as compared to HC, and C3 was significantly higher in inadequately controlled asthma as compared to well-controlled asthma. Several proteins were associated with physiological tests assessing small airways. Conclusions: The study highlights the role of the local activation of the complement and coagulation systems in the small airway lining fluid in asthma and their association with both asthma control and small airway dysfunction. The findings highlight the potential of complement factors as biomarkers to identify different sub-groups among patients with asthma that could potentially benefit from a therapeutic approach targeting the complement system.

Current smoking alters phospholipid- and surfactant protein A levels in small airway lining fluid: An explorative study on exhaled breath.

Small airways are difficult to access. Exhaled droplets, also referred to as particles, provide a sample of small airway lining fluid and may reflect inflammatory responses. We aimed to explore the effect of smoking on the composition and number of exhaled particles in a smoker-enriched study population. We collected and chemically analyzed exhaled particles from 102 subjects (29 never smokers, 36 former smokers and 37 current smokers) aged 39 to 83 years (median 63). A breathing maneuver maximized the number exhaled particles, which were quantified with a particle counter. The contents of surfactant protein A and albumin in exhaled particles was quantified with immunoassays and the contents of the phospholipids dipalmitoyl- and palmitoyl-oleoyl- phosphatidylcholine with mass spectrometry. Subjects also performed spirometry and nitrogen single breath washout. Associations between smoking status and the distribution of contents in exhaled particles and particle number concentration were tested with quantile regression, after adjusting for potential confounders. Current smokers, compared to never smokers, had higher number exhaled particles and more surfactant protein A in the particles. The magnitude of the effects of current smoking varied along the distribution of each PEx-variable. Among subjects with normal lung function, phospholipid levels were elevated in current smokers, in comparison to no effect of smoking on these lipids at abnormal lung function. Smoking increased exhaled number of particles and the contents of lipids and surfactant protein A in the particles. These findings might reflect early inflammatory responses to smoking in small airway lining fluid, also when lung function is within normal limits.

The seven constitutive respiratory defense barriers against SARS-CoV-2 infection.

Before eliciting an adaptive immune response, SARS-CoV-2 must overcome seven constitutive respiratory defense barriers. The first is the mucus covering the respiratory tract’s luminal surface, which entraps inhaled particles, including infectious agents, and eliminates them by mucociliary clearance. The second barrier comprises various components present in the airway lining fluid, the surfactants. Besides providing low surface tension that allows efficient gas exchange at the alveoli, surfactants inhibit the invasion of epithelial cells by respiratory viruses, enhance pathogen uptake by phagocytes, and regulate immune cells’ functions. The respiratory tract microbiota constitutes the third defense barrier against SARS-CoV-2. It activates the innate and adaptive immune cells and elicits anti-infectious molecules such as secretory IgA antibodies, defensins, and interferons. The fourth defense barrier comprises the antimicrobial peptides defensins, and lactoferrin. They show direct antiviral activity, inhibit viral fusion, and modulate the innate and adaptive immune responses. Secretory IgA antibodies, the fifth defense barrier, besides protecting the local microbiota against noxious agents, also inhibit SARS-CoV-2 cell invasion. If the virus overcomes this barrier, it reaches its target, the respiratory epithelial cells. However, these cells also act as a defense barrier, the sixth one, since they hinder the virus’ access to receptors and produce antiviral and immunomodulatory molecules such as interferons, lactoferrin, and defensins. Finally, the sensing of the virus by the cells of innate immunity, the last constitutive defense barrier, elicits a cascade of signals that activate adaptive immune cells and may inhibit the development of productive infection. The subject of the present essay is discussing these mechanisms.

EBC metabolomics for asthma severity

Asthma is a heterogeneous disease with diverse severity and represents a considerable socio-economic burden. Exhaled Breath Condensate (EBC) is a biofluid directly obtained from the airway lining fluid non-invasively. We attempted to discriminate severe from mild-to-moderate asthma using EBC metabolomics based on both NMR and UHPLC-MS techniques. 36 patients were included in this study (15 patients with severe and 21 with mild-to-moderate asthma). EBC was collected and analyzed using both NMR and UHPLC-MS techniques for possible metabolites. Using PLS and oPLS analysis for the UHPLC-MS data, no metabolite was found to be sufficient for the discrimination of asthma severity. However, when another PLS-regression model was applied five metabolites were found to discriminate severe from mild-to-moderate asthma. Amino-acid lysine was the only metabolite that discriminated the two study groups using NMR data (p= 0.04, t-test with Welch’s correction, AUC 0.66). EBC is an easily available biofluid which directly represents the lower airways but difficult-to-use for metabolomic analysis. Our study presents some encouraging findings for the discrimination of asthma severity subgroups using EBC metabolomics but more well-designed studies with a higher number of patients need to be conducted.

Acute nicotine administration stimulates ciliary activity via α3β4 nAChR in the mouse trachea

Mucociliary clearance, the continuous removal of mucus-trapped particles by cilia-driven directed transport of the airway lining fluid, is the primary innate defense mechanism of the airways. It is potently activated by acetylcholine (ACh) addressing muscarinic receptors with a currently less defined role of nicotinic ACh receptors (nAChR). We here set out to determine their contribution in driving ciliary activity in an explanted mouse trachea preparation utilizing selected agonists and antagonists and nAChR-subunit deficient mice. Nicotine (100 µM) induced an increase in ciliary beat frequency, accompanied by a sharp, but not long lasting increase in particle transport speed (PTS) on the mucosal surface showing marked desensitization within the next 30 min. Nicotine-induced PTS acceleration was sensitive to the general nAChR inhibitors mecamylamine and d-tubocurarine as well as to the α3β4-nAChR antagonist α-conotoxin AulB, but not to other antagonists primarily addressing α3β2-nAChR or α4-, α7- and α9-containing nAChR. Agonists at α3β*-nAChR (epibatidine, cytisine), but not cotinine mimicked the effect. Tracheas from mice with genetic deletion of nAChR subunits α5, α7, α9, α10, α9/10, and β2 retained full PTS response to nicotine, whereas this was entirely lost in tracheas from mice lacking the β4-subunit. Collectively, our data show that nicotinic stimulation of α3β4-nAChR acutely increases PTS to the same extent as the established strong activator ATP. In view of the marked desensitization observed in the present setting, the physiological relevance of these receptors in adapting mucociliary clearance to rapidly changing endogenous or environmental stimuli remains open.

Update on exhaled breath condensate analyses in occupational disease.

The present work represents an update of the review published in this journal by Corradi et al., regarding the use of exhaled breath condensate (EBC) to investigate occupational lung diseases. The relevant literature was searched in the Medline database, assessed through PubMed using key terms such as 'breath AND condensate AND occupational'. Eleven pertinent publications were retrieved between January 2018 and October 2019. One article only was related to occupational allergy, and the conclusion is that EBC hydrogen peroxide is not an useful marker in laboratory animal allergy. The biomarkers of exposure most often assessed with EBC are metals. However, it is controversial whether this approach has any advantage over the conventional environmental monitoring. The biomarkers of effect studied by the majority of investigations were those related to oxidative stress. They appear consistently elevated upon occupational exposures to various agents, including welding fumes, crystalline silica, nanomaterials and chemicals. Although EBC represent a suitable tool to sample airway lining fluid in a noninvasive manner, it remains a niche approach to the investigation of occupational diseases. The confounding influence of EBC dilution should be better addressed in the expression of the results.

Transport and fate of inhaled particles after deposition onto the airway surface liquid: A 3D numerical study

In this paper, a numerical investigation is carried out to provide insights into the fate of inhaled aerosols after their deposition on the lung lining fluid in both healthy and diseased states. Pulmonary drug delivery is a well-known non-invasive route of administration compared to intravenous delivery. Aerosol particles are formulated and used as drug carriers, which are then sent to the airways using aerosol drug delivery devices. This approach is useful for site-specific treatment of lung diseases, treatment of central nervous system (CNS) disorders and a variety of other diseases. Bioavailability of the inhaled therapeutic particles after landing on the airway lining fluid can be significantly altered by the lung muco-ciliary clearance, a process through which hairlike structures known as cilia beat in a harmonised manner and induce the mucus in the proximal direction, leading to an effective clearance of the foreign inhaled particles entrapped by this sticky layer from the airways. Here, we set up a 3D computational model of ciliary arrays interacting with periciliary liquid film (i.e. confined between the epithelium and mucus layer) and a detailed analysis is conducted to better understand the fate of drug nanoparticles that are able to penetrate the mucus. Consistent with clinical findings, we find that the actions of cilia result in a low rate of drug retention and absorption by the pulmonary tissues in healthy lungs. However, under conditions associated with abnormal ciliary beats, the retention time of particles is notably increased at the site of release. Nonetheless, the results associated with some of the ciliary impairments reveal that deposition of drug aerosols on the ciliated cells may still be a significant challenge. These findings have potentially important implications on the modification of therapeutic drug particles to achieve a higher absorption rate.

P1.11-11 Initial Discovery of Exhaled Small Polar Energetics-Related Metabolites by GC-MS for Lung Cancer Risk Assessment

There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both risk assessment, and earlier diagnosis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including small molecules of polar and non-polar lipid origin, presumably in part from epithelial cellular origins. Here we pilot a GC-MS strategy for measurement of small, polar molecules in exhaled breath condensate in EBC from lung cancer patients and controls. Exhaled breath condensate (EBC) was collected non-invasively, using a handheld device (RTube) in ambulatory subjects engaged/recruited/consented through our pulmonary and thoracic surgery practices, under IRB protocol. A volume of 50 ul of EBC samples were combined with 200uL of methanol, containing 2 internal standards, 1nmol U13_succinate, 5nmol U13_citrate. Then, the samples were vortexed and 240 μl of supernatant was transferred to a sampling vial. The samples were dried under gentle nitrogen flow and derivatized with a two-step derivatization procedure, including a methoxyamine step for 90 minutes, and a silylation step for 60 minutes. QC sample was run multiple times during the analysis. The samples were analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOFMS premier, Waters, USA). A number of 282 variables were detected after alignment and excluding any known artificial peaks, 49 of them were annotated. The data was normalized to the intensity of the sum of all the metabolites. The data set was then imported into SIMCA-p software (Umeå, Sweden) for multivariate analysis. A multivariate case-control ROC discriminant analysis compared the clinical model (AUC 0.72) to clinical plus exhaled small polar discriminant metabolites combined (AUC 0.92), showed incremental discrimination attributable to these metabolites (p=2.22e-62). ROC comparing clinical model (AUC 0.72) to clinical model plus metabolites (AUC 0.92); the difference attributable to metabolites was significant (p=2.22e-62). This exhaled biomarker platform can yield case-control discriminant small polar molecule sets related to known metabolic pathways, some of which are known to be deranged in cancers. Once further distilled and validated, our goal is to apply this non-invasive biomarker approach to prospective cohorts for non-invasive lung cancer risk assessment of the at-risk epithelium, in order to better select higher risk individuals to undergo effective CT screening. Supported by NIH-R21 CA192168-01; DoD-CDMRP- LC150738, NIH-NCI P60DK020541.

SERUM CLARA CELL PROTEIN (CC16) IN RELATION TO PULMONARY FUNCTIONS AMONG WORKERS EXPOSED TO NAPHTHALENE IN PVC MANUFACTURE

AbstractIntroduction: Naphthalene is a harmful environmental tox reduced in serum and/or airway lining fluid of patients with COPD, asthma, bronchopulmonary dysplasia, silicosis, and post-transplant rejection. Aim of work: This study was done to evaluate the use of serum CC16 level as a biological marker for occupational respiratory diseases in relation to pulmonary function tests among workers exposed to naphthalene in polyvinyl chloride (PVC) manufacture. Material and methods: The study included 33 exposed workers and 33 control subjects. The study groups were subjected to full history taking, clinical examination and pulmonary function testing. Serum levels of CC16 and naphthalene levels measured for both groups. Results: Pulmonary functions parameters (FVC, FEV1, FEV1/FVC, FEF 25%, FEF 50%, FEF 75%), were significantly decreased in the exposed group compared to the control and indicated obstructive ventilatory impairment among the exposed workers. Serum naphthalene was significantly higher, while serum CC16 was significantly lower among the exposed group compared to the control. Exposed smokers exhibited the highest serum naphthalene and the lowest serum CC16 among all study groups. The duration of naphthalene exposure positively correlated with the serum level of naphthalene and negatively correlated with pulmonary functions and the serum level of CC16. Also serum level of CC16 correlated negatively with the serum naphthalene level and smoking index.Conclusion: Occupational exposure to naphthalene in PVC manufacture is associated with obstructive ventilatory impairment, with corresponding decrease in the serumlevel of Clara cell protein (CC16), which can be used as a biomarker for respiratory system affection among workers exposed to naphthalene.

CfDNA in exhaled breath condensate (EBC) and contamination by ambient air: toward volatile biopsies

Exhaled breath is a source of volatile and nonvolatile biomarkers in the body that can be accessed non-invasively and used for monitoring. The collection of lung secretions by conventional methods such as bronchoalveolar lavage, induced sputum collection, and core biopsies is limited by the invasive nature of these methods. Non-invasive collection of exhaled breath condensate (EBC) provides fluid samples that are representative of airway lining fluids. Various volatile and nonvolatile biomarkers can be detected in volatile condensates, such as H2O2, nitric oxide, lipid mediators, cytokines, chemokines, DNA, and microRNAs. Studies have examined cell-free DNA (cfDNA) in plasma samples from non-small-cell lung cancer patients, offering to new insights and fostering development of the liquid biopsy. However, few studies have examined cfDNA in EBC samples. This study examined whether EBC is an appropriate source of cfDNA using housekeeping-gene-specific primer probes and quantitative real-time polymerase chain reaction in healthy subjects. Ambient (room) air is contaminated with DNA, so caution is needed. Preliminary studies indicated that volatile biopsies are becoming an important diagnostic tool in lung cancer.

Exhaled breath condensate pepsin and pH: noninvasive tests for gastroesophageal reflux in acute exacerbations of chronic obstructive pulmonary disease

Introduction Gastroesophageal reflux disease (GERD) has been shown to worsen chronic obstructive pulmonary disease (COPD) control through esophagobronchial reflex and to heighten bronchial reactivity and microaspiration. Exhaled breath condensate (EBC) is an exciting technology that allows an insight into biomolecules of airway lining fluid. Furthermore, diagnosis of GERD in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) by EBC pepsin and pH levels is fast and easy. Patients and methods A total of 50 individuals were included in this study. They were divided into three groups: group A included 20 patients diagnosed with AECOPD and GERD, group B included 20 patients with GERD with chronic cough, and group C (control group) included 10 healthy participants to determine the natural level of pepsin in EBC. EBC pepsin concentration as well as pH was measured among all groups. Reflux symptom index (RSI) was also measured as a tool for GERD diagnosis. Results Patients with AECOPD had increased levels of pepsin in their EBC samples and lower pH level than the other two groups. RSI was significantly higher among patients with COPD with GERD than either GERD patients or healthy controls. Significant associations existed between EBC pepsin and RSI and EBC pH among the COPD group. Conclusion Measurement of EBC pepsin and pH in patients with AECOPD may represent a diagnostic and noninvasive test for GERD among those patients.

Role of zinc insufficiency in fetal alveolar macrophage dysfunction and RSV exacerbation associated with fetal ethanol exposure.

We previously reported that maternal alcohol use significantly increases the risk of sepsis in premature and term newborns. In the mouse, fetal ethanol exposure results in an immunosuppressed phenotype for the alveolar macrophage (AM) and decreases bacterial phagocytosis. In pregnant mice, ethanol decreased AM zinc homeostasis, which contributed to immunosuppression and impaired AM phagocytosis. In this study, we explored whether ethanol-induced zinc insufficiency extended to the pup AMs and contributed to immunosuppression and exacerbated viral lung infections. C57BL/6 female mice were fed a liquid diet with 25% ethanol-derived calories or pair-fed a control diet with 25% of calories as maltose-dextrin. Some pup AMs were treated invitro with zinc acetate before measuring zinc pools or transporter expression and bacteria phagocytosis. Some dams were fed additional zinc supplements in the ethanol or control diets, and then we assessed pup AM zinc pools, zinc transporters, and the immunosuppressant TGFβ1. On postnatal day 10, some pups were given intranasal saline or respiratory syncytial virus (RSV), and then AM RSV phagocytosis and the RSV burden in the airway lining fluid were assessed. Fetal ethanol exposure decreased pup AM zinc pools, zinc transporter expression, and bacterial clearance, but invitro zinc treatments reversed these alterations. In addition, the expected ethanol-induced increase in TGFβ1 and immunosuppression were associated with decreased RSV phagocytosis and exacerbated RSV infections. However, additional maternal zinc supplements blocked the ethanol-induced perturbations in the pup AM zinc homeostasis and TGFβ1 immunosuppression, thereby improving RSV phagocytosis and attenuating the RSV burden in the lung. These studies suggest that, despite normal maternal dietary zinc intake, in utero alcohol exposure results in zinc insufficiency, which contributes to compromised neonatal AM immune functions, thereby increasing the risk of bacterial and viral infections.

P1.11-12 Interrogation of an Exhaled MicroRNA Panel for Lung Cancer Risk Assessment

There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both risk assessment of the ex-smoker, and earlier diagnosis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including nucleic acids, presumably in part from epithelial cellular origins. MicroRNAs play important regulatory roles in many processes, including carcinogenesis. Here we further develop and begin validation of the detection of microRNAs in EBC from lung cancer patients and controls. EBC was collected non-invasively, using a handheld commercial RTube® device in a clinical series of ambulatory subjects. We generated a 40 miR panel based on literature-derived microRNAs, bronchial brushed microRNA discovery by collaboration, and a lung parenchymal tissue discovery effort we have performed using microRNA-seq on lung tumors and surrounding non-tumor tissue. The PCR primers were designed using our previously published RNA-specific RT-PCR technique. All samples were run twice with positive and negative controls. Calls was made of individual miR present or absent in a given EBC sample, by one of two replicates being positive. The data were analyzed by random forests. We applied the panel to EBC from 177 individuals, 89 NSCLC predominantly early stage (I and II) cases and 88 controls of similar smoking history. We used an empirically-derived base clinical model that included age, smoking status, pack-years, quit-years, and underlying lung disease. MicroRNA signatures alone discriminated cases from controls with ROC-AUCs of 0.64-0.76. Both analyses revealed a small set of incrementally informative miRs (e.g., miRs-21**, 33b**, 96*,105*, `130b.3p*, 200a*, 200b**, 205, 212***, 221***, 345***, 767**, 944*, 1269a***, 1293*, 1910**, 3648*, 3662*, where number of ‘*’ implies number of appearances in each of three models). Here some modest incremental case-control discriminant capacity was conferred by the respective microRNA signature over and above the base clinical model. The magnitude of the increment was typically 2-3% [RF AUC 0.84 clinical model=>0.86 clinical+microRNA (all lung cancer histologies, Welch p=1.33e-05); 0.82=>0.84 (adenocarcinomas, Welch p=3.13e-03); 0.84=>0.86 (NSCLC, Welch p=4.10e-05). Quantitative RT-PCR using this platform set-up was not sufficiently robust to further analyze. This new exhaled biomarker platform can yield case-control discriminant microRNA sets. albeit modest in incremental impact as formulated so far. Once quantified, further distilled and validated, our goal is to test this non-invasive biomarker approach to prospective cohorts for non-invasive lung cancer risk assessment, in order to non-invasively better select higher risk individuals to undergo effective CT screening.

Non-volatile compounds in exhaled breath condensate: review of methodological aspects.

In contrast to bronchial and nasal lavages, the analysis of exhaled breath condensate (EBC) is a promising, simple, non-invasive, repeatable, and diagnostic method for studying the composition of airway lining fluid with the potential to assess lung inflammation, exacerbations, and disease severity, and to monitor the effectiveness of treatment regimens. Recent investigations have revealed the potential applications of EBC analysis in systemic diseases. In this review, we highlight the analytical studies conducted on non-volatile compounds/biomarkers in EBC. In contrast to other related articles, this review is classified on the basis of analytical techniques and includes almost all the applied methods and their methodological limitations for quantification of non-volatile compounds in EBC samples, providing a guideline for further researches. The studies were identified by searching the SCOPUS database with the keywords "biomarkers," "non-volatile compounds," "determination method," and "EBC."

Proteomic characterization of human exhaled breath condensate

To improve biomedical knowledge and to support biomarker discovery studies, it is essential to establish comprehensive proteome maps for human tissues and biofluids, and to make them publicly accessible. In this study, we performed an in-depth proteomics characterization of exhaled breath condensate (EBC), a sample obtained non-invasively by condensation of exhaled air that contains submicron droplets of airway lining fluid. Two pooled samples of EBC, each obtained from 10 healthy donors, were processed using a straightforward protocol based on sample lyophilization, in-gel digestion and liquid chromatography tandem-mass spectrometry analysis. Two ‘technical’ control samples were processed in parallel to the pooled samples to correct for exogenous protein contamination. A total of 229 unique proteins were identified in EBC among which 153 proteins were detected in both EBC pooled samples. A detailed bioinformatics analysis of these 153 proteins showed that most of the proteins identified corresponded to proteins secreted in the respiratory tract (lung, bronchi). Eight proteins were salivary proteins. Our dataset is described and has been made accessible through the ProteomeXchange database (dataset identifier: PXD007591) and is expected to be useful for future MS-based biomarker studies using EBC as the diagnostic specimen.

The R213G polymorphism in SOD3 protects against allergic airway inflammation.

Oxidative stress is important in the pathogenesis of allergic asthma. Extracellular superoxide dismutase (EC-SOD; SOD3) is the major antioxidant in lungs, but its role in allergic asthma is unknown. Here we report that asthmatics have increased SOD3 transcript levels in sputum and that a single nucleotide polymorphism (SNP) in SOD3 (R213G; rs1799895) changes lung distribution of EC-SOD, and decreases likelihood of asthma-related symptoms. Knockin mice analogous to the human R213G SNP had lower airway hyperresponsiveness, inflammation, and mucus hypersecretion with decreased interleukin-33 (IL-33) in bronchoalveolar lavage fluid and reduced type II innate lymphoid cells (ILC2s) in lungs. SOD mimetic (Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin) attenuated Alternaria-induced expression of IL-33 and IL-8 release in BEAS-2B cells. These results suggest that R213G SNP potentially benefits its carriers by resulting in high EC-SOD in airway-lining fluid, which ameliorates allergic airway inflammation by dampening the innate immune response, including IL-33/ST2-mediated changes in ILC2s.

Variability of breath condensate pH may contribute to the better understanding of non-allergic seasonal respiratory diseases.

The seasonal variability of certain non-allergic respiratory diseases is not clearly understood. Analysis of the breath condensate, the liquid that can be collected by breathing into a cold tube, has been proposed to bring closer to the understanding of airway pathologies. It has been assumed, that (1) airway lining fluid was a stable body liquid and (2) the breath condensate samples were representative of the airway lining fluid. Research was focussed on the identification of biomarkers indicative of respiratory pathologies. Despite 30years of extended investigations breath condensate analysis has not gained any clinical implementation so far. The pH of the condensate is the characteristic that can be determined with the highest reproducibility. The present paper shows, that contrary to the initial assumptions, breath condensate is not a representative of the airway lining fluid, and the airway lining fluid is not a stable body liquid. Condensate pH shows baseline variability and it is influenced by drinking and by the ambient temperature. The changes in condensate pH are linked to changes in airway lining fluid pH. The variability of airway lining fluid pH may explain seasonal incidence of certain non-allergic respiratory diseases such as the catching of a common cold and the increased incidence of COPD exacerbations and exercise-induced bronchoconstriction in cold periods.

Abstract 4429: An exhaled microRNA panel interrogation and validation as risk biomarkers for lung cancer

Abstract Background: There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both risk assessment of the ex-smoker, and earlier diagnosis. Recent studies have revealed that microRNAs play important regulatory roles in carcinogenesis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including nucleic acids, presumably in part from epithelial cellular origins. Here we further develop and begin validation of the detection of microRNAs in EBC from lung cancer patients and controls. Methods: Exhaled breath condensate (EBC) was collected non-invasively, using a handheld device in ambulatory subjects. MicroRNA expression profiling using RNA-specific RT-qPCR was performed in EBC in an initial batch (E) of 88 individuals (41 lung cancer cases, 47 controls), and a second batch D of 88 individuals (41, cases and 47 controls). We generated a 39 miR panel based on literature-derived microRNAs, combined with a lung tissue-based discovery effort we have performed using microRNA-seq on lung tumors and surrounding non-tumor tissue. The qPCR primers were designed using our previously published RNA-specific realtime RT-PCR technique. All samples were run twice with positive and negative controls. Results: Qualitative analyses of the first donor batch E was performed on 39 microRNAs measured in 41 cases and 47 controls. Chi-square analysis (univariate) within the training set (batch E) revealed that exhaled miR-31, 33, 105, 200b, 944, 1269a were significantly different between the initial set of cases and controls; their absence marked the cases. In the validation batch (D) preliminary analysis, exhaled miRs 200b (this time present in cases, p=0.044) and 944 (again absent in cases, p=0.052) were case-control discriminant. In addition, test set batch D showed case-control discrimination for additional miRs 130b-3p (present in cases, p=0.035), 212 (absent in cases, p=0.009), 1910a (absent in cases, p=0.006) and other microRNAs that were not seen in the qualitative training set (batch E). Importantly, multi-miR discriminant signatures, quantitative analysis, and risk ROC/modelling analyses are pending as of this AACR abstract deadline. Conclusion: This new exhaled biomarker platform can yield case-control discriminant microRNA sets, including a subset of miRs that can validate in test sets. Once further distilled and validated, our goal is to apply this non-invasive biomarker approach to prospective cohorts for non-invasive lung cancer risk assessment, in order to better select higher risk individuals who should undergo CT screening. Note: This abstract was not presented at the meeting. Citation Format: Simon D. Spivack. An exhaled microRNA panel interrogation and validation as risk biomarkers for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4429. doi:10.1158/1538-7445.AM2017-4429

Method validation of nanoparticle tracking analysis to measure pulmonary nanoparticle content: the size distribution in exhaled breath condensate depends on occupational exposure

A particle exposure assessment based on the dose deposited in the lungs would be the gold standard for the evaluation of any resulting health effects. Measuring particles in exhaled breath condensate (EBC)—a matrix containing water and airway lining fluid—could help to evaluate particle retention in the lungs. This study aimed to (1) validate a nanoparticle tracking analysis (NTA) method for determining the particle number concentration and their hydrodynamic size distribution in EBC, and (2) apply this method to EBC collected from workers exposed to soapstone (n = 55) or quartz dust (n = 12) and controls (n = 11). A standard latex bead solution was used to determine the linear range, limit of detection (LOD), repeatability (coefficient of variation, CV), and bias in spiked EBC. An LM10 NanoSight instrument with NTA version 3.1 software was used for measurement. RTubes® were used for field collection of EBC. The repeatability obtained for a D50 size distribution in EBC showed less than 8% variability, with a bias <7%. The particle concentration was linear in the range ≤2.5 × 108 particles ml−1 with a LOD of 4 × 106 particles ml−1. A recovery of 117 ± 20% at 6.2 × 107 particles ml−1 was obtained with a CV <10% and a bias <20%. EBC from workers exposed to quartz, who experienced the largest exposure to silica particles, consistently exhibited a statistically significant (p < 0.01) higher concentration of particles in their EBC, with a size distribution shift towards larger values than the other groups. Results showed that the NTA technique performed well for characterizing the size distribution and concentrations of particles in EBC. The technique needs to be corroborated with a larger population of workers.

Collectins, H-ficolin and LL-37 reduce influence viral replication in human monocytes and modulate virus-induced cytokine production.

Infiltrating activated monocytes are important mediators of damaging inflammation during influenza A virus (IAV) infection. We show that soluble respiratory proteins [collectins, surfactant proteins D (SP-D) and mannose binding lectin (MBL), H-ficolin and LL-37] inhibit replication of seasonal IAV in human monocytes. The collectins and H-ficolin also increased viral uptake by the cells, while LL-37 did not. H-ficolin was able to inhibit replication of the 2009 pandemic H1N1 strain (Cal09) in monocytes, but SP-D and LL-37 had significantly fewer inhibitory effects on this strain than on seasonal IAV. All of these proteins reduced IAV-induced TNF-α production, even in instances when viral replication was not reduced. We used modified recombinant versions of SP-D, MBL and ficolin to elucidate mechanisms through which these proteins alter monocyte interactions with IAV. We demonstrate the importance of the multimeric structure, and of binding properties of the lectin domain, in mediating antiviral and opsonic activity of the proteins. Hence, soluble inhibitors present in airway lining fluid may aid clearance of IAV by promoting monocyte uptake of the virus, while reducing viral replication and virus-induced TNF-α responses in these cells. However, SP-D and LL-37 have reduced ability to inhibit replication of pandemic IAV in monocytes.