Search

Background: Bronchial Thermoplasty (BT) is a bronchoscopic treatment for severe asthma which reduces airway smooth muscle (ASM). The optimal patient responder profile is still under debate. In asthma, mast cell density is increased within the ASM and relates to bronchial hyper responsiveness. Aim: To determine the effect of BT on mast cells in airway wall biopsies and ASM specific area and relate this to treatment response. Methods: 14 severe asthma patients included in the TASMA trial were evaluated. Airway wall biopsies were obtained before and after BT and stained with ASM specific desmin and mast cell specific tryptase antibodies. Mast cells were manually counted and automatic digital analysis was used to calculate ASM area (Fig 1A-B). Results: Before BT, mast cells within ASM had a median of 35.5/mm2 (range 10-83) and 26.5/mm2 (range 12-73) for the total biopsy area. After BT, ASM (%) reduced with 58% (median 12.0% to 4.98% after BT; p Conclusions: Mast cells within the total airway wall seem to increase following BT. Due to BT induced ASM reduction, mast cells might shift from the ASM to the (sub)mucosal area.

Volatile organic compounds (VOCs) in breath can reflect host and pathogen metabolism and might be used to diagnose pneumonia (Oort J Breath Res 2018). We hypothesized that rats with Streptococcus pneumoniae (SP) or Pseudomonas aeruginosa (PA) pneumonia can be discriminated from uninfected controls by gas-chromatography mass-spectrometry (GC-MS) of exhaled breath. Male adult rats (N = 50) received an intra-tracheal inoculation of 1) 200µL saline, 2) 1x107 colony forming units (cfu) of SP or 3) 1x107 cfu of PA. 24 hours later the rats were anaesthetized, tracheotomized and mechanically ventilated. Exhaled breath was analyzed via GC-MS. Area under the receiver operating characteristic curve (AUROCC) was calculated after leave-one-out cross-validation of sparse partial least squares discriminant analysis (sPLS-DA). The AUROCC was 0.85 (95%-CI: 0.73-0.96) for infected vs. non-infected animals. The AUROCC was 0.98 (95%-CI: 0.94-1) for SP vs. PA, 0.92 (95%-CI: 0.83-1) for SP vs. controls and 0.97 (95%-CI: 0.92-1) for PA vs. controls. Exhaled breath analysis discriminated with good accuracy between respiratory infection and no infection, but with much better accuracy between the specific pathogens. Clinical studies should focus more on the discrimination between specific pathogens, instead of the general presence of respiratory infection.

Exhaled volatile organic compounds (VOCs) could potentially serve as non-invasive biomarkers for the diagnosis of respiratory infections. This study aims to discriminate rats infected with Pseudomonas aeruginosa (PA) or Streptococcus pneumoniae (SP) from non-infected rats. Exhaled breath was collected from 50 male rats 24h after intra-tracheal inoculation of 200µL saline, or 1x107 colony forming units of SP or PA. VOCs in exhaled breath trapped on sorbent tubes were analysed with Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) after thermal desorption. The pattern of reaction ions with mass/charge ratio 15 to 200 was analysed using sparse partial least squares discriminant analysis. Area under the receiver operating characteristic curve (AUROCC) calculated after leave-one-out cross-validation of the multivariate discriminant analysis was 0.73 (95%CI: 0.57-0.89) for infected vs. non-infected animals. For SP vs. PA the AUROCC was 0.97 (95%CI: 0.93-1) while for SP vs. controls and PA vs. controls it was 0.67 (95%CI: 0.47-0.86) and 0.72 (95%CI: 0.53-0.91), respectively. Exhaled breath analysis discriminated with higher accuracy between animals infected with specific respiratory pathogens compared to infected vs. non-infected animals. Similar findings were observed for VOCs analysed with Gas Chromatography Mass Spectrometry (see submitted ERS abstract from P. M. van Oort et al.). Our results suggest that breath analysis in infected patients may be helpful in discriminating different pathogens. Furthermore, SIFT-MS has potential as a point-of-care tool to assess respiratory infections in clinical practice.

Background: The mechanism of action of Bronchial Thermoplasty (BT) and its related responder profile is unclear. Objective: Investigate if BT-induced airway smooth muscle mass (ASM) change correlates with FEV1. Methods: 16 severe asthma patients were analyzed. Before and after BT, pre- and post-bronchodilator FEV1 was measured and bronchial biopsies were taken and stained with ASM-specific desmin and ASM/myofibroblast/myoepithelial α-smooth muscle actin (α-SMA). Results: BT treatment reduced ASM mass significantly as assessed by both desmin (55%) and α-SMA analysis (40%)(Fig1A). Baseline pre-and post-bronchodilator FEV1 correlated negatively with desmin-determined baseline ASM mass (resp. R=-0.61;p=0.01 and R=-0.62;p=0.01). Furthermore desmin-determined ASM mass decrease correlated negatively with baseline pre- and post-bronchodilator FEV1 (resp. R=-0.61;p=0.01 and R=-0.54;p=0.03 Fig1B). Patients with FEV1 80%(n=10): 2.4%(0.4;6.3 IQR (p=0.01)Fig1C). This correlation was not found with α-SMA staining. Conclusions: The correlation between ASM and FEV1 seems to depend on the fully differentiated contractile ASM only. Patients with a FEV1< 80% show the most reduction of ASM mass after BT treatment. Whether this “obstructive” asthma phenotype qualifies as a responder criteria needs to be determined.

Background: Bronchial Thermoplasty (BT) is a bronchoscopic treatment for severe asthma during which airways are treated with radio-frequent energy and that aims to improve asthma control by reducing airway smooth muscle (ASM). Aim: Determine the effect of BT on ASM mass before and after BT and to compare this with the non-treated right middle lobe (RML) and delayed BT control group. Methods: After obtaining airway biopsies, 11 severe asthma patients were randomized to either direct BT or to 6 months delayed BT treatment (in this control group re-biopsies after 6 months of standard care were obtained). Airway biopsies were taken 6 months post-BT in all patients. ASM mass (% of total biopsy area) was measured by automatic digital image analysis on 2 desmin-stained sections per biopsy. Results: 6 months after BT mean (SD) ASM mass significantly decreased from 11.25 (4.8)% to 4.8 (2.2) % for pre-and post-BT respectively (p=0.007) (n=11, 44 sections). In the non-BT-treated RML, 6 months after BT, ASM mass was 7.3 (±6.2) % vs 10.3 (4.9) % pre BT (p=0.13) (n=9, 36 sections). In the control group, after 6 months of standard care, ASM mass was 10 (±6.1) % vs 7.15 (±4.3) % at the first measurement (p=0.63) (n=4, 16 sections). Conclusions: ASM mass significantly decreased 6 months following BT treatment. No significant change in ASM mass was found in controls or the untreated RML.

Background: Airway remodeling is a key feature in obstructive lung diseases. Near infrared-based optical coherence tomography (OCT) is a novel bronchoscopic imaging technique that generates highly-detailed images of the airway wall. Aim: Identify and quantify human airway wall layers both ex-vivo and in-vivo by OCT and correlate these to histology. Methods: Patients with lung cancer, prior to lobectomy, underwent in-vivo OCT imaging. Ex-vivo OCT imaging was performed in the resected lung lobe after needle insertion for matching with histology. Airway wall layer perimeters were assessed by two independent observers. Airway wall layer areas (total airway wall area, mucosal wall layer area and submucosal wall layer area) were calculated. Results: 13 airways of 5 patients were imaged by OCT. Histology was matched with 51 ex-vivo OCT images and 39 in-vivo OCT images. Significant correlation was found between ex-vivo OCT imaging and histology, in-vivo OCT imaging and histology and ex-vivo and in-vivo OCT imaging for all measurements (Fig 1). Minimal bias was seen in Bland-Altman plots. High inter-observer reproducibility with intra-class correlation coefficients all > 0.90 were detected. Conclusions: OCT is an accurate and reproducible imaging technique for identification and quantification of airway wall layers. Implications: OCT imaging might qualify as the imaging technique of choice to assess airway wall remodeling.

INTRODUCTION Several airway diseases are characterized by airway remodeling. The current imaging techniques to assess airway wall layers/airway remodeling have limitations. Optical coherence tomography (OCT) is a minimal invasive, high-resolution optical imaging technique which generates real-time, high-detailed images of airway segments. How OCT images relates to histology is largely unknown. AIM Ex-vivo identification and quantification of human airway wall layers by OCT and correlate this to histology. METHODS In 5 lobectomy specimen 13 airways were dissected, marked with suture needles and imaged by OCT. Histology sections were stained with hematoxylin and eosin to visualize airway wall layers and desmin to identify the airway smooth muscle layer. Airway wall thickness was measured by tracing the luminal perimeter (LA) and airway smooth muscle outer perimeter (TA). Airway wall area (WA (mm2)) and airway wall area percentage (WA%) for both OCT and histology where calculated by WA=TA-LA and WA%=WA/TA x100% respectively. RESULTS 51 matched OCT- histology airway cross sections were analyzed. The following separate airway wall layers were identified on OCT imaging; epithelium, basal membrane, lamina propria surrounded by smooth muscle, glands, vessels and cartilage. WA and the WA% measured by OCT correlated with histology (N=51; R 0.76 95% CI 0.61-0.86, p CONCLUSIONS Ex-vivo OCT imaging correlates well with histopathology both for identification of airway wall layers and quantification of airway wall thickness. IMPLICATIONS OCT imaging might qualify as the imaging technique of choice to assess airway wall remodelling.

BACKGROUND: Diagnosing subsets of interstitial lung disease (ILD) is challenging. Confocal laser endomicroscopy (CLE) is a novel imaging technique that provides high detailed images of the alveolar compartment . OBJECTIVES: To identify and quantify characteristics of ILD on CLE images METHODS: In ILD- patients (n=4) scheduled for bronchoscopy, CLE measurements (laser 488 nm, resolution 3,5 µm, field of view 600 µm, Mauna Kea Technologies, France) of the alveolar compartment of 9non-suspected9 and for ILD 9suspected9 areas were obtained based onHRCT. Frame by frame analysis of image density and alveolar septum thickness were extracted with FIJI software. RESULTS: With CLE, the architecture of the alveolar compartment was well depicted and visual characteristics such as: overall density, alveolar septum thickness and filling of the alveolar space, were clearly identified. Frame by frame analysis (n=40) showed higher densities in diseased [30,7 (range 15,4-46,3)] compared to normal appearing alveolar areas [18,5 (range 13,0-23,6)]. Alveolar septum thickness (n=24) varied within diseased areas [ 25,3 µm (range 14-40)]and normal areas [19,7 µm (range 8-42)].(figure) CONCLUSIONS: CLE imaging of the alveolar compartment discriminates normal alveolar sections from diseased parts. Quantification of CLE images might qualify as a minimally invasive and safe way to distinct different ILD patterns during bronchoscopy.

BACKGROUND Bronchial thermoplasty (BT) is a novel bronchoscopic treatment for severe asthma that is based on radiofrequent energy delivery to ∼3-10mm airways and aims to reduce airway smooth muscle (ASM). The TASMA study is the first randomised controlled trial that investigates the change in ASM after BT. AIM To determine the change in ASM mass in airway biopsies before and after BT and to compare this with a control group and the non-treated right middle lobe (RML). METHODS 6 severe asthma patients were randomized- after obtaining airway biopsies (pre-BT)- to either direct BT treatment (immediate group) or to 6 months waiting followed by re – airway biopsies and BT treatment (delayed group). From all patients, airway biopsies were taken 6 months post-BT. For each biopsy the ASM mass in percentage of the total biopsy area was measured automatically on 2 desmin-stained sections. RESULTS 6 months after BT mean ASM mass percentage significantly decreased (ASM mass % 11.6±5.5 vs 6±1.2 for pre-and post-BT respectively; p=0.029 (n=6, 23 biopsies)). In the delayed group (ASM mass % 7.6±5.2 vs 11±6.6 for pre-BT and delayed group respectively; P=0.473 (n=3, 12 biopsies)) and in the non-treated RML (ASM mass % 10.9±5.9 vs 10±7 for pre-BT and RML respectively; P=0.820 (n=5, 15 biopsies) no significant differences were detected when compared to pre-BT ASM mass %. CONCLUSIONS ASM mass percentage significantly decreases following BT treatment. ASM mass stays the same over time and in the untreated RML. IMPLICATIONS ASM mass might qualify as a biomarker for Bronchial Thermoplasty response.

BACKGROUND: The identification of subsets of interstitial lung disease (ILD) is challenging. An important hallmark and differentiating factor in ILD is the presence of fibrosis, expressed by thickening of alveolar septa. Optical coherence tomography (OCT) is a minimally invasive, bronchoscopic technique that potentially provides high resolution imaging of the alveolar compartment. Its value in ILD is unknown. OBJECTIVES: To test the feasibility of OCT to characterize and quantify alveolar septa and compare this to histology in ILD patients. METHODS: In patients with ILD on HRCT (n=3) the alveolar compartment was imaged by inserting the OCT probe (St Jude Medical) via the airways to peripheral lung areas and obtaining 5.4cm pull-backs (539 images) or stationary measurements (121 images) of the right lower lobe with subsequent cryo-biopsies at the same location. Dedicated software (FIJI, Olyvia) was used for measurements. RESULTS: OCT was capable of visualizing the alveolar structures in all patients to a depth of 0,9-1,2 mm. Alveoli were visible in 35,7 % of pullback-images and 94% of stationary-images. The average number of alveoli visible in a single OCT image was 15. Mean alveolar septum thickness was 0,12 mm (0,05-0,24) in OCT-images and 0,09 mm (0,04-0,16) in histology. CONCLUSIONS: OCT is capable of visualization and quantification of the alveolar compartment and might be helpful in the diagnostic process of ILD.

Background: A growing body of evidence indicates that age plays a critical role in the development and outcome of ARDS. However, the molecular mechanisms linking aging with ARDS have not been elucidated. An age-dependent imbalance in the pulmonary renin-angiotensin system (RAS) might form an explanation. Objectives: We investigated whether there are age-related changes in the pulmonary RAS in an animal model of acute lung injury (ALI). We hypothesized that during ALI, aging is associated with a shift from the lung protective pathway [Angiotensin converting enzyme (ACE) 2] to the lung injurious pathway [ACE], thereby increasing the inflammatory mediator response and lung injury. Methods: Lung injury was induced in rats of four different age groups (infants, juveniles, adults, and elderly) by intratracheal administration of LPS and mechanical ventilation. Results: With increasing age, ACE activity in bronchoalveolar lavage fluid increased (from 0.05 RFU/min in infants to 0.16 RFU/min in elderly; p for trend = 0.02). In contrast, membrane-bound ACE activity in lung-homogenate declined, indicating shedding. No changes in ACE2 activity were found, thereby shifting the balance in the alveolar compartment towards the injurious pathway. This age-dependent imbalance was associated with increased inflammatory mediator response and lung injury (wet-to-dry ratio and histology). Conclusions: Our data shows that increasing age is associated with higher ACE activity in the alveolar compartment which correlates with aggravated inflammation and lung injury. These changes should be taken into account in terms of dosing and effectiveness of RAS modulating agents for treatment of ARDS.