Exhaled breath particles as a novel tool to study lipid composition of epithelial lining fluid from the distal lung.

BACKGROUND: Surfactant phospholipid (PL) composition plays an important role in lung diseases. We compared the PL composition of non-invasively collected exhaled breath particles (PEx) with bronchoalveolar lavage (BAL) and induced sputum (ISP) at baseline and following endotoxin (LPS) challenges. METHODS: PEx and BAL were collected from ten healthy nonsmoking participants before and after segmental LPS challenge. Four weeks later, PEx and ISP were sampled in the week before and after a whole lung LPS inhalation challenge. PL composition was analysed using mass spectrometry. RESULTS: The overall PL composition of BAL, ISP and PEx was similar, with PC(32:0) and PC(34:1) representing the largest fractions in all three sample types (baseline PC(32:0) geometric mean mol%: 52.1, 56.9, and 51.7, PC(34:1) mol%: 11.7, 11.9 and 11.4, respectively). Despite this similarity, PEx PL composition was more closely related to BAL than to ISP. For most lipids comparable inter-individual differences in BAL, ISP, and PEx were found. PL composition of PEx was repeatable. The most pronounced increase following segmental LPS challenge was detected for SM(d34:1) in BAL (0.24 to 0.52 mol%) and following inhalation LPS challenge in ISP (0.45 to 0.68 mol%). An increase of SM(d34:1) following segmental LPS challenge was also detectable in PEx (0.099 to 0.103 mol%). The inhalation challenge did not change PL composition of PEx. CONCLUSION: Our data supports the peripheral origin of PEx. The lack of PL changes in PEx after inhalation challenge might to be due to the overall weaker response of inhaled LPS which primarily affects the larger airways. Compared with BAL, which always contains lining fluid from both peripheral lung and central airways, PEx analysis might add value as a selective and non-invasive method to investigate peripheral airway PL composition. TRIAL REGISTRATION: NCT03044327, first posted 07/02/2017.

Quantitative analysis of endotoxin-induced inflammation in human lung cells by Chipcytometry.

Chipcytometry is a tool that uses iterative staining cycles with multiple antibodies for a detailed characterization of cells. Cell recognition is based on morphological features. Cells fixed on microfluidic chips can be stored and shipped enabling a centralized analysis, which is important for assessments in multi-center clinical trials. The method was initially implemented for the analysis of cells from peripheral blood. We adapted it to more heterogeneous human lung cells from bronchoalveolar lavage (BAL) fluid and induced sputum (IS). We aimed to assess the performance of Chipcytometry to detect and quantify the endotoxin induced inflammatory response in healthy subjects. BAL and IS samples of 10 healthy subjects were collected prior to and following segmental and inhaled endotoxin challenge. Samples were analyzed by Chipcytometry and were compared with flow cytometry, and differential cell count (DCC). Chipcytometry clearly detected the endotoxin induced inflammatory response which was characterized by a massive increase of neutrophils (BAL: 2.5% to 54.7%; IS: 40.5% to 71.1%) and monocytes (BAL: 7.7% to 24.7%; IS: 8.0% to 14.5%). While some differences between detection methods exist, the overall results were comparable. The ability of Chipcytometry to verify fluorescent signals with morphological features improved the precision of rare cell analysis such as of induced sputum lymphocytes. In conclusion, Chipcytometry enables the quantitative analysis of cells from BAL fluid and IS. Advantages over DCC and flow cytometry include the storage of cells on chips, the ability for re-analysis and the mapping of surface marker binding to morphological information. It therefore appears to be a promising method for use in clinical respiratory drug development.

Raised sputum extracellular DNA confers lung function impairment and poor symptom control in an exacerbation-susceptible phenotype of neutrophilic asthma.

BACKGROUND: Extracellular DNA (e-DNA) and neutrophil extracellular traps (NETs) are linked to asthmatics airway inflammation. However, data demonstrating the characterization of airway inflammation associated with excessive e-DNA production and its impact on asthma outcomes are limited. OBJECTIVE: To characterize the airway inflammation associated with excessive e-DNA production and its association with asthma control, severe exacerbations and pulmonary function, particularly, air trapping and small airway dysfunction. METHODS: We measured e-DNA concentrations in induced sputum from 134 asthma patients and 28 healthy controls. We studied the correlation of e-DNA concentrations with sputum neutrophils, eosinophils and macrophages and the fractional exhaled nitric oxide (FeNO). Lung function was evaluated using spirometry, body plethysmography, impulse oscillometry and inert gas multiple breath washout. We stratified patients with asthma into low-DNA and high-DNA to compare lung function impairments and asthma outcomes. RESULTS: Patients with severe asthma had higher e-DNA concentration (54.2 ± 42.4 ng/µl) than patients with mild-moderate asthma (41.0 ± 44.1 ng/µl) or healthy controls (26.1 ± 16.5 ng/µl), (all p values < 0.05). E-DNA concentrations correlated directly with sputum neutrophils (R = 0.49, p < 0.0001) and negatively with sputum macrophages (R = - 0.36, p < 0.0001), but neither with sputum eosinophils (R = 0.10, p = 0.26), nor with FeNO (R = - 0.10, p = 0.22). We found that 29% of asthma patients (n = 39) had high e-DNA concentrations above the upper 95th percentile value in healthy controls (55.6 ng /µl). High-DNA was associated with broad lung function impairments including: airflow obstruction of the large (FEV1) and small airways (FEF50%, FEF25-75), increased air trapping (RV, RV/TLC), increased small airway resistance (R5-20, sReff), decreased lung elasticity (X5Hz) and increased ventilation heterogeneity (LCI), (all P values < 0.05). We also found that high e-DNA was associated with nearly three-fold greater risk of severe exacerbations (OR 2·93 [95% CI 1.2-7.5]; p = 0·012), worse asthma control test (p = 0.03), worse asthma control questionnaire scores (p = 0.01) and higher doses of inhaled corticosteroids (p = 0.026). CONCLUSION: Increased production of extracellular DNA in the airway characterizes a subset of neutrophilic asthma patients who have broad lung function impairments, poor symptom control and increased risk of severe exacerbations.

Noninvasive Monitoring of the Response of Human Lungs to Low-Dose Lipopolysaccharide Inhalation Challenge Using MRI: A Feasibility Study.

BACKGROUND: Development of antiinflammatory drugs for lung diseases demands novel methods for noninvasive assessment of inflammatory processes in the lung. PURPOSE: To investigate the feasibility of hyperpolarized 129 Xe MRI, 1 H T1 time mapping, and dynamic contrast-enhanced (DCE) perfusion MRI for monitoring the response of human lungs to low-dose inhaled lipopolysaccharide (LPS) challenge compared to inflammatory cell counts from induced-sputum analysis. STUDY TYPE: Prospective feasibility study. POPULATION: Ten healthy volunteers underwent MRI before and 6 hours after inhaled LPS challenge with subsequent induced-sputum collection. FIELD STRENGTH/SEQUENCES: 1.5T/hyperpolarized 129 Xe MRI: Interleaved multiecho imaging of dissolved and gas phase, ventilation imaging, dissolved-phase spectroscopy, and chemical shift saturation recovery spectroscopy. 1 H MRI: Inversion recovery fast low-angle shot imaging for T1 mapping, time-resolved angiography with stochastic trajectories for DCE MRI. ASSESSMENT: Dissolved-phase ratios of 129 Xe in red blood cells (RBC), tissue/plasma (TP) and gas phase (GP), ventilation defect percentage, septal wall thickness, surface-to-volume ratio, capillary transit time, lineshape parameters in dissolved-phase spectroscopy, 1 H T1 time, blood volume, flow, and mean transit time were determined and compared to cell counts. STATISTICAL TESTS: Wilcoxon signed-rank test, Pearson correlation. RESULTS: The percentage of neutrophils in sputum was markedly increased after LPS inhalation compared to baseline, P = 0.002. The group median RBC-TP ratio was significantly reduced from 0.40 to 0.31, P = 0.004, and 1 H T1 was significantly elevated from 1157.6 msec to 1187.8 msec after LPS challenge, P = 0.027. DCE MRI exhibited no significant changes in blood volume, P = 0.64, flow, P = 0.17, and mean transit time, P = 0.11. DATA CONCLUSION: Hyperpolarized 129 Xe dissolved-phase MRI and 1 H T1 mapping may provide biomarkers for noninvasive assessment of the response of human lungs to LPS inhalation. By its specificity to the alveolar region, hyperpolarized 129 Xe MRI together with 1 H T1 mapping adds value to sputum analysis. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;51:1669-1676.

Analysis of Human Breath by Millimeter-Wave/Terahertz Spectroscopy.

Breath gas analysis is a promising tool for medical research and diagnosis. A particularly powerful technological approach is millimeter-wave/terahertz (mmW/THz) spectroscopy, because it is a very sensitive and highly selective technique. In addition, it offers the potential for compact and affordable sensing systems for wide use. In this work, we demonstrate the capability of a mmW/THz spectrometer for breath analysis. Samples from three volunteers and a sample from ambient air were analyzed with respect to 31 different molecular species. High-resolution absorption spectra were measured by scanning two absorption lines from each species. Out of the 31, a total of 21 species were detected. The results demonstrate the potential of mmW/THz spectroscopy for breath analysis.

Airway and systemic inflammatory responses to ultrafine carbon black particles and ozone in older healthy subjects.

Increased adverse health effects in older subjects due to exposure to ambient air pollutants may be related to the inflammatory response induced by these contaminants. The aim of this study was to assess airway and systemic inflammatory responses in older healthy subjects to a controlled experimental exposure with spark-generated elemental carbon black ultrafine particles (cbUFPs) and ozone (O3). Twenty healthy subjects, age 52-75 years, were exposed on three occasions separated by at least 8 weeks. The exposures to filtered air (FA), to cbUFP (50 µg/m3), or to cbUFP in combination with 250 ppb ozone (cbUFP + O3) for 3 h with intermittent exercise were performed double blind, and in random order. Sputum and blood samples were collected 3.5 h after each exposure. Exposure to cbUFP + O3 significantly increased plasma club cell protein 16 (CC16), the number of sputum cells, the number and percent of sputum neutrophils, and sputum interleukin 6 and matrix metalloproteinase 9. Exposure to cbUFP alone exerted no marked effect, except for an elevation in sputum neutrophils in a subgroup of 13 subjects that displayed less than 65% sputum neutrophils after FA exposure. None of the inflammatory markers was correlated with age, and serum cardiovascular risk markers were not markedly affected by cbUFP or cbUFP + O3. Exposure to cbUFP+O3 induced a significant airway and systemic inflammatory response in older healthy volunteer subjects. The effects induced by cbUFP alone suggest that the inflammation was predominantly mediated by O3, although one cannot rule out that the interaction of cbUFP and O3 played a role.

A European Respiratory Society technical standard: exhaled biomarkers in lung disease.

Breath tests cover the fraction of nitric oxide in expired gas (FeNO), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for FeNO, official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and FeNO, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management.Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members.Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised.Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice.

Disease Progression and Changes in Physical Activity in Patients with Chronic Obstructive Pulmonary Disease.

RATIONALE: Little is known about the role of physical activity in the course of chronic obstructive pulmonary disease (COPD). OBJECTIVES: To assess changes in physical activity in COPD in relation to severity stages and changes in other disease components, and to evaluate the longitudinal association between sustained physical inactivity and disease progression. METHODS: In this prospective cohort study, we measured physical activity (multisensory armband), airflow obstruction (FEV1), health status (St. George's Respiratory Questionnaire), exercise capacity (6-min-walk distance [6MWD]), muscle mass (fat-free mass [FFM]), and systemic inflammation (fibrinogen and high-sensitivity C-reactive protein) over a 3-year period in 137 patients with COPD and 26 with chronic bronchitis (normal spirometry). MEASUREMENTS AND MAIN RESULTS: Independent of baseline disease severity, steps per day, total daily energy expenditure, and (daily) physical activity level (PAL) decreased by 393, 76 kcal, and 0.04 per year, respectively. The decline in PAL was significantly associated with a decline in FEV1 and an increase in St. George's Respiratory Questionnaire total score. Changes in 6MWD, FFM, and inflammatory markers were not associated with changes in PAL. Independent of FEV1, sustained physical inactivity (i.e., PAL(T0andT1) < 1.40) was related to a greater decline in 6MWD and FFM compared with that in patients with some level of activity (i.e., PAL(T0and/orT1) ≥ 1.40; difference, 17 m/yr and 0.87 kg/yr, respectively). CONCLUSIONS: Over time, physical activity substantially decreases across all severity stages of COPD, and this decline is paralleled by a worsening of lung function and health status. Sustained physical inactivity is associated with a progression of exercise intolerance and muscle depletion.

Multi-analyte profiling of inflammatory mediators in COPD sputum--the effects of processing.

Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®). Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®. Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1ß, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently. In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT.

Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.

Impact of endobronchial allergen provocation on macrophage phenotype in asthmatics.

BACKGROUND: The role of M2 polarized macrophages (MΦ) during the allergic airway inflammation has been discussed in various animal models. However, their presence and relevance during the chronic and acute phase of allergic airway inflammation in humans has not been fully elucidated so far. In the present study we phenotypically characterized macrophages with regard to M2 polarization in mice, a human in vitro and a human ex vivo model with primary lung cells after endobronchial provocation. RESULTS: Macrophages remained polarized beyond clearance of the acute allergic airway inflammation in mice. Alveolar macrophages of asthmatics revealed increased mRNA expression of CCL13, CCL17 and CLEC10A in response to allergen challenge as well as increased surface expression of CD86. Further, mRNA expression of CCL13, CCL17, and CLEC10A was increased in asthmatics at baseline compared to healthy subjects. The mRNA expression of CCL17 and CLEC10A correlated significantly with the degree of eosinophilia (each P < .01). Furthermore, macrophages from asthmatics released significant amounts of CCL17 protein in vitro which was also found increased in BAL fluid after allergen provocation. CONCLUSIONS: This study supports previous findings of M2 macrophage polarization in asthmatic subjects during the acute course of the allergic inflammation and provides evidence for their contribution to the Th2 inflammation.

Potential prognostic value of biomarkers in lavage, sputum and serum in a five year clinical follow-up of smokers with and without COPD.

BACKGROUND: The aim of this study was to test whether repeatable biomarkers collected from serum, bronchoalveolar lavage (BAL) and sputum of healthy smokers and smokers with COPD would have a prognostic value with respect to the decline in lung function over a 5 year period. METHODS: In 2006/2007 we had repeatedly collected serum, BAL and sputum of 23 healthy smokers and 24 smokers with COPD (GOLD II) and analysed a panel of more than 100 different parameters. In 2012 we reinvited these subjects to assess the change in lung function to enable the investigation of the potential prognostic value of the 2006/2007 markers and to determine the long-term repeatability of selected blood and serum markers. In this follow-up study we performed body-plethysmography, a blood gas analysis and collected blood and urine samples. The change in lung function was compared with 67 markers from BAL, sputum, serum and whole blood that were shown in the 2006/2007 assessment to be repeatable over a 6 week period. RESULTS: We were able to recruit 13 (54%) smokers with COPD and 11 (48%) former healthy smokers that participated in the 2006/2007 study. The decline in lung function was larger in COPD smokers; five of them changed to GOLD III, one to GOLD IV. Two healthy smokers changed to GOLD I. Blood cells, serum von Willebrand factor and alpha-1-antitrypsin showed a good repeatability over 5 years. In COPD smokers a weak correlation between 2006/2007 sputum markers of neutrophilic inflammation and the 5 year change in FEV1/FVC was found. CONCLUSIONS: Our data suggests that inter-individual and group differences are maintained over a five year period. Despite the large panel of markers available for this analysis, a potential prognostic value appears to exist only for some sputum inflammatory markers. If these data can be confirmed in larger COPD cohorts, it would emphasize the value of sputum markers in clinical trials and support the assumption that an anti-inflammatory treatment can have long term benefits in COPD.

Oncostatin M modulates fibroblast function via signal transducers and activators of transcription proteins-3.

Oncostatin M (OSM), an inflammatory cytokine of the interleukin-6 (IL-6) superfamily, plays a key role in various biological processes such as modulation of extracellular matrix (ECM), cell proliferation, cell survival, and induction of inflammation. It has been reported that OSM was increased in asthma and pulmonary fibrosis, and thus OSM may play a role in airway remodeling and the development of lung parenchymal fibrosis. Recruitment of lung fibroblasts to the sites of airway injury and subsequent differentiation into myofibroblasts is believed to contribute to excess ECM deposition. In the current study, we assessed the ability of OSM to modulate fibroblast collagen gel contraction, migration toward fibronectin, and expression of α-smooth muscle actin (α-SMA). We demonstrated that OSM augments gel contraction, chemotaxis, and α-SMA expression. OSM-augmented fibroblast chemotaxis was mediated by the signal transducer and activator of transcription (STAT3) and p38 mitogen-activated protein kinase, while augmentation on gel contraction and α-SMA expression was mediated by STAT3. Neither transforming growth factor-ß1 nor PGE2 was involved in mediating OSM effect on the cells. The Th2 cytokines IL-4 and IL-13, which also are believed to play an important role in promoting lung fibrosis and airway remodeling, act through STAT3, and we demonstrated the potential for additive effects of OSM with IL-4 and IL-13. The present study supports the concept that OSM may contribute to tissue remodeling, which may be additive with IL-4 or IL-13. Blockade of OSM or OSM-mediated STAT3 signaling could be a therapeutic target to regulate lung fibrotic mechanisms.

Low-dose endotoxin inhalation in healthy volunteers--a challenge model for early clinical drug development.

BACKGROUND: Inhalation of endotoxin (LPS) induces a predominantly neutrophilic airway inflammation and has been used as model to test the anti-inflammatory activity of novel drugs. In the past, a dose exceeding 15-50 µg was generally needed to induce a sufficient inflammatory response. For human studies, regulatory authorities in some countries now request the use of GMP-grade LPS, which is of limited availability. It was therefore the aim of this study to test the effect and reproducibility of a low-dose LPS challenge (20,000 E.U.; 2 µg) using a flow- and volume-controlled inhalation technique to increase LPS deposition. METHODS: Two to four weeks after a baseline sputum induction, 12 non-smoking healthy volunteers inhaled LPS on three occasions, separated by at least 4 weeks. To modulate the inflammatory effect of LPS, a 5-day PDE4 inhibitor (Roflumilast) treatment preceded the last challenge. Six hours after each LPS inhalation, sputum induction was performed. RESULTS: The low-dose LPS inhalation was well tolerated and increased the mean percentage of sputum neutrophils from 25% to 72%. After the second LPS challenge, 62% neutrophils and an increased percentage of monocytes were observed. The LPS induced influx of neutrophils and the cumulative inflammatory response compared with baseline were reproducible. Treatment with Roflumilast for 5 days did not have a significant effect on sputum composition. CONCLUSION: The controlled inhalation of 2 µg GMP-grade LPS is sufficient to induce a significant neutrophilic airway inflammation in healthy volunteers. Repeated low-dose LPS challenges potentially result in a small shift of the neutrophil/monocyte ratio; however, the cumulative response is reproducible, enabling the use of this model for "proof-of-concept" studies for anti-inflammatory compounds during early drug development.

Emissions and uptake of volatiles by sampling components in breath analysis.

The first and most crucial step in breath research is adequate sampling, which plays a pivotal role in quality assurance of breath datasets. In particular, the emissions or uptake of volatile organic compounds (VOCs) by sampling interface materials present a risk of disrupting breath gas samples. This study investigated emissions and uptake by three interface components, namely a silicon facemask, a reusable 3D-printed mouthpiece adapter, and a pulmonary function test filter compatible with the commercial Respiration Collector forIn-VitroAnalysis (ReCIVA) breath sampling device. Emissions were examined before and after (hydro-)thermal treatment of the components, and uptake was assessed by exposing each material to 12 representative breath VOCs comprising alcohols, aldehydes, ketones, carboxylic acids, terpenes, sulphurous and nitrogenous compounds at different target concentration ranges (∼10 ppbVand ∼100 ppbV). Chemical analyses of VOCs were performed using proton transfer reaction-time-of-flight-mass spectrometry (PTR-TOFMS) with supporting analyses via thermal desorption comprehensive two-dimensional gas chromatography-TOFMS (TD-GC×GC-TOFMS). The filter exhibited the lowest overall emissions compared to the mask or adapter, which both had equivalently high emissions (albeit for different compounds). Treatment of the materials reduced the total VOC emissions by 62% in the mask, 89% in the filter and 99% in the adapter. Uptakes of compounds were lowest for the adapter and most pronounced in the mask. In particular, 1-butanol, acetone, 2-butanone, 1,8-cineole and dimethyl sulphide showed negligible uptake across all materials, whereas ethanol, nonanal, acetic acid, butanoic acid, limonene and indole exhibited marked losses. Knowledge of emissions and/or uptake by sampling components is key to reducing the likelihood of erroneous data interpretation, ultimately expediting progress in the field of breath test development.

Prostaglandin E2 stimulates the production of vascular endothelial growth factor through the E-prostanoid-2 receptor in cultured human lung fibroblasts.

Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E(2) (PGE(2)) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE(2) of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbecco's modified Eagle's medium, with or without PGE(2), and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE(2) and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE(2) was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase-A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE(2). The increased release of VEGF induced by PGE(2) was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE(2) can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.

Attenuation of inhibitory prostaglandin E2 signaling in human lung fibroblasts is mediated by phosphodiesterase 4.

The etiology of chronic obstructive pulmonary disease (COPD) is complex and involves an aberrant inflammatory response. Prostaglandin (PG)E2 is elevated in COPD, is a key modulator of lung fibroblast functions, and may influence COPD progression. Most studies evaluating the effects of PGE2 on lung fibroblasts have used acute exposures. The current study evaluated whether longer-term exposure would induce attenuation of PGE2 signaling as part of an autoregulatory pathway. Human fetal lung fibroblasts were pretreated with PGE2 for 24 hours, and migration and cAMP accumulation in response to acute stimulation with PGE2 were assessed. Fibroblasts from adults with and without COPD were pretreated, and migration was assessed. PGE2 pretreatment attenuated subsequent PGE2-mediated inhibition of chemotaxis and cAMP stimulation. This attenuation was predominantly due to an increase in phosphodiesterase (PDE)4-mediated degradation of cAMP rather than to decreased activation of PGE2 receptors (receptor desensitization). Albuterol- and iloprost-mediated signaling were also attenuated after PGE2 pretreatment, suggesting that activation of PDE4 was able to broadly modulate multiple cAMP-coupled pathways. Lung fibroblasts from adult control subjects pretreated with PGE2 also developed attenuation of PGE2-mediated inhibition of chemotaxis. In contrast, fibroblasts obtained from patients with COPD maintained inhibitory PGE2 signaling after PGE2 pretreatment. These data identify a PDE4-mediated attenuation of PGE2 inhibitory signaling in normal fibroblasts that appears to be altered in COPD fibroblasts. These alterations may contribute to COPD pathogenesis and could provide novel therapeutic targets.

3D-printed mouthpiece adapter for sampling exhaled breath in medical applications.

The growing use of 3D printing in the biomedical sciences demonstrates its utility for a wide range of research and healthcare applications, including its potential implementation in the discipline of breath analysis to overcome current limitations and substantial costs of commercial breath sampling interfaces. This technical note reports on the design and construction of a 3D-printed mouthpiece adapter for sampling exhaled breath using the commercial respiration collector for in-vitro analysis (ReCIVA) device. The paper presents the design and digital workflow transition of the adapter and its fabrication from three commercial resins (Surgical Guide, Tough v5, and BioMed Clear) using a Formlabs Form 3B stereolithography (SLA) printer. The use of the mouthpiece adapter in conjunction with a pulmonary function filter is appraised in comparison to the conventional commercial silicon facemask sampling interface. Besides its lower cost - investment cost of the printing equipment notwithstanding - the 3D-printed adapter has several benefits, including ensuring breath sampling via the mouth, reducing the likelihood of direct contact of the patient with the breath sampling tubes, and being autoclaveable to enable the repeated use of a single adapter, thereby reducing waste and associated environmental burden compared to current one-way disposable facemasks. The novel adapter for breath sampling presented in this technical note represents an additional field of application for 3D printing that further demonstrates its widespread applicability in biomedicine.

Millimeter-wave gas spectroscopy for breath analysis of COPD patients in comparison to GC-MS.

The analysis of human breath is a very active area of research, driven by the vision of a fast, easy, and non-invasive tool for medical diagnoses at the point of care. Millimeter-wave gas spectroscopy (MMWGS) is a novel, well-suited technique for this application as it provides high sensitivity, specificity and selectivity. Most of all, it offers the perspective of compact low-cost systems to be used in doctors' offices or hospitals. In this work, we demonstrate the analysis of breath samples acquired in a medical environment using MMWGS and evaluate validity, reliability, as well as limitations and perspectives of the method. To this end, we investigated 28 duplicate samples from chronic obstructive lung disease patients and compared the results to gas chromatography-mass spectrometry (GC-MS). The quantification of the data was conducted using a calibration-free fit model, which describes the data precisely and delivers absolute quantities. For ethanol, acetone, and acetonitrile, the results agree well with the GC-MS measurements and are as reliable as GC-MS. The duplicate samples deviate from the mean values by only 6% to 18%. Detection limits of MMWGS depend strongly on the molecular species. For example, acetonitrile can be traced down to 1.8 × 10-12mol by the MMWGS system, which is comparable to the GC-MS system. We observed correlations of abundances between formaldehyde and acetaldehyde as well as between acetonitrile and acetaldehyde, which demonstrates the potential of MMWGS for breath research.