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Non-steroidal anti-inflammatory drugs exacerbated respiratory disease (N-ERD) is currently classified as type-2 (T2) immune mediated disease characterized by asthma, chronic rhinosinusitis, and hypersensitivity to cyclooxygenase-1 inhibitors.Our aim was to characterize immunological endotypes of N-ERD based on the gene expression profile in the bronchial epithelium.We analyzed mRNA transcriptome (mRNA-seq) in bronchial brushings from patients with N-ERD (n=22), NSAIDs tolerant asthma (NTA, n=21) and control subjects (n=11). Additionally, lipid and protein mediators were measured in broncho-alveolar lavage fluid (BALF).Initial analysis of the entire asthma group revealed two distinct gene expression signatures: 'T2-high' with increased expression of T2-related genes (e.g., CLCA1, CST1), and 'proinflammatory' characterized by the expression of innate immunity (e.g., FOSB, EGR3) and IL-17A response genes. These endotypes showed similar prevalence in N-ERD and NTA (e.g., T2-high: 33% and 32%, respectively). T2-high asthma was characterized by increased expression of mast cell and eosinophil markers, goblet cell hyperplasia, and elevated LTET2-high signature was found only in one third of N-ERD patients, which was similar to NTA. The proinflammatory endotype, which also occurred in N-ERD, suggests a novel mechanism of severe disease developing on a non-T2 background.

Airway inflammation in asthma is accompanied by reconstruction of the bronchial wall extracellular matrix that most likely occurs with a contribution of matrix metalloproteinases (MMPs). Recently we have reported that omalizumab may decrease reticular basement membrane (RBM) thickness together with fibronectin deposits in asthmatic airways, although mechanisms involved are unknown.In the present study, we have investigated the impact of omalizumab on MMPs concentrations in bronchoalveolar lavage fluid (BAL) of asthmatic subjects in relation to airway remodeling changes in histology.The study group consisted of 13 severe allergic asthmatics treated with omalizumab for at least 12 months. In each subject, clinical and laboratory parameters, bronchoscopy with BAL, and endobronchial biopsy were evaluated before and after the biologic therapy. RBM thickness, fibronectin, and collagen deposits in bronchial mucosa specimens were analyzed in histology. The investigations also included BAL cytology and BAL concentrations of MMP-2, -3, and -9.Omalizumab was related to a decrease in all measured MMPs in BAL (Our data suggest that omalizumab therapy is associated with decreased BAL MMPs concentration in the subgroup of asthma patients. The decline was linked with a reduction in the RBM thickness what might play a beneficial role in airway remodeling.

Background Non-steroidal anti-inflammatory drugs exacerbated respiratory disease (N-ERD) asthma is characterized by chronic rhinosinusitis and intolerance to aspirin and other cyclooxygenase-1 inhibitors. Clinical data point to a heterogeneity within N-ERD phenotype. Objectives To investigate immune mediator profiles in lower airways of patients with N-ERD. Methods Levels of cytokines (luminex) and eicosanoids (mass spectrometry) were measured in bronchoalveolar lavage fluid (BALF) from patients with N-ERD (n=22), NSAIDs-tolerant asthma (NTA, n=21) and control subjects (n=11). mRNA expression was quantified in BALF cells using TaqMan low-density arrays. Results Lower airway eosinophilia was more frequent in N-ERD (54.5%) than in NTA (9.5%, P=0.009). Type-2 (T2) immune signature of BALF cells was more pronounced in eosinophilic subphenotype of N-ERD. Similarly, BALF concentrations of Periostin and CCL26 were significantly increased in eosinophilic N-ERD and correlated with T2-signature in BALF cells. Multiparameter analysis of BALF mediators of all asthma patients revealed the presence of two immune endotypes: ‘T2-like’ with elevated Periostin in BALF, and ‘non-T2/pro-inflammatory’ with higher levels of MMPs and inflammatory cytokines. N-ERD patients were classified mostly as T2 endotype (68%). Changes in eicosanoid profile, e.g. increased LTE4, were limited to N-ERD patients with airway eosinophilia. Blood eosinophilia appeared to be useful predictor of airway T2-signature (AUC=0.83), however, surrogate biomarkers had moderate performance in distinguishing eosinophilic N-ERD (blood eosinophils: AUC=0.72, Periostin: AUC=0.75). Conclusions Lower airway immune profiles show considerable heterogeneity of N-ERD, with skewing toward T2-response and eosinophilic inflammation. Increased production of LTE4 was restricted to a subgroup of patients with lower airway eosinophilia. Clinical implications N-ERD patients are heterogeneous in terms of lower airway inflammation and eicosanoid profiles. Better understanding of inflammatory diversity of N-ERD might help in selection of personalized therapies.

Introduction: Transbronchial lung cryobiopsy (TBLC) is commonly used in diagnosing interstitial lung diseases (ILDs). Ageneral anesthesia with endotracheal intubation, balloon blockers and fluoroscopy control is the most common modality. Simplifying the procedure without decreasing it’s safety could result in wider use. Prospective, observational study was conducted in three Polish pulmonology centers to evaluate safety and diagnostic yield of TBLC under conscious sedation, without intubation and bronchial blockers and with radial-EBUS guidance instead of fluoroscopy. Material and methods: In patients suspected of ILD, in accordance with high resolution computer tomography (HRCT) selected lung segments were examined with radial-EBUS mini probe without aguide sheath. If the lung infiltrations were visible this locations were preferred. If not, specimens were taken from two different segments of the same lobe. Two to five biopsies with freezing time 5–8 seconds were performed. Moreover ultrasound examination was used to avoid injury of lung vessels. Results: From March 2017 to September 2019 — 114 patients (M: 59, F: 55) of mean (SD) age 54 (14) years were included to the study on the basis of medical history and HRCT. Histopathology was conclusive in 90 (79%) patients and included 16 different diagnoses (sarcoidosis, EAA, COP predominantly). 24 inconclusive biopsies of unclassifiable pulmonary fibrosis were followed up. Complications included five cases (4.4%) of pneumothorax requiring achest tube drainage and aminor and moderate bleeding in few cases. There was no need for use of balloon bronchial blockers. Conclusions: TBLC under conscious sedation guided by radial EBUS mini-probe is novel, reasonable and safe technique for histological diagnosis of ILDs.

Background The role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. In this study, we investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma. Methods Bronchial brushing specimens and brochial mucosal biopsy samples were collected from adult patients with asthma and from age- and gender-matched non-asthmatic individuals. The expression of miR-146a in bronchial brushing specimens, bronchial biopsy tissue sections or cultured primary bronchial epithelial cells was analyzed by RT-qPCR or by in situ hybridization. The expression of direct and indirect miR-146a target genes was determined by RT-qPCR or ELISA. The migration of neutrophils was studied by neutrophil chemotaxis assay and flow cytometry. For statistical analysis, unpaired two-way Student’s t test, one-way ANOVA or linear regression analysis were used. Results Reduced expression of miR-146a was found in bronchial brushing specimens from asthma patients as compared to non-asthmatics and irrespective of the phenotype of asthma. In the same samples, the neutrophil attracting chemokines IL-8 and CXCL1 showed increased expression in patients with neutrophilic asthma and increased IL-33 expression was found in patients with eosinophilic asthma. Linear regression analysis revealed a significant negative association between the expression of miR-146a in bronchial brushings and neutrophil cell counts in bronchoalveolar lavage fluid of patients with asthma. In bronchial biopsy specimens, the level of miR-146a was highest in the epithelium as determined with in situ hybridization. In primary conventional HBEC culture, the expression of miR-146a was induced in response to the stimulation with IL-17A, TNF-α, and IL-4. The mRNA expression and secretion of IL-8 and CXCL1 was inhibited in both stimulated and unstimulated HBECs transfected with miR-146a mimics. Supernatants from HBECs transfected with miR-146a had reduced capability of supporting neutrophil migration in neutrophil chemotaxis assay. Conclusion Our results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.

Karolina Górka,1,2 Iwona Gross-Sondej,1,2 Jacek Górka,3 Tomasz Stachura,1,2 Kamil Polok,1,3 Natalia Celejewska-Wójcik,1,2 Sławomir Mikrut,4 Anna Andrychiewicz,5 Krzysztof Sładek,1,2,* Jerzy Soja1,2,* 1Department of Pulmonology and Allergology, University Hospital, Kraków, Poland; 2 2nd Department of Internal Medicine, Jagiellonian University Medical College, Kraków, Poland; 3Centre for Intensive Care and Perioperative Medicine, Jagiellonian University Medical College, Kraków, Poland; 4Faculty of Mining Surveying and Environmental Engineering, AGH University of Science and Technology, Kraków, Poland; 5Department of Endoscopy, University Hospital, Kraków, Poland*These authors contributed equally to this workCorrespondence: Jerzy Soja 2nd Department of Internal Medicine, Jagiellonian University Medical College, ul Jakubowskiego 2, Kraków, 30-688, PolandTel +48 12 400 30 53Fax +48 12 400 30 67Email jerzy.soja@uj.edu.plPurpose: The aim of this study was to evaluate the structural changes of the airways using the endobronchial ultrasound (EBUS) in ACO patients compared to severe asthma and COPD patients.Patients and Methods: The study included 17 patients with ACO, 17 patients with COPD and 33 patients with severe asthma. Detailed clinical data were obtained from all participants. Basic laboratory tests were performed, including measurement of eosinophil counts in blood and serum immunoglobulin E (IgE) concentrations. All patients underwent spirometry and bronchoscopy with EBUS (a 20‑MHz ultrasound probe) to measure the total thicknesses of the bronchial walls and their particular layers in segmental bronchi of the right lower lobe. EBUS allows to distinguish five layers of the bronchial wall. Layer 1 (L1) and layer 2 (L2) were analyzed separately, while the outer layers (layers 3– 5 [L3– 5]) that correspond to cartilage were assessed together.Results: In patients with ACO the thicknesses of the L1 and L2 layers, which are mainly responsible for remodeling, were significantly greater than in patients with COPD and significantly smaller than in patients with severe asthma (median L1= 0.17 mm vs 0.16 mm vs 0.18 mm, p< 0.001; median L2= 0.18 mm vs 0.17 mm vs 0.20 mm, p< 0.001, respectively). The thicknesses of the total bronchial walls (L1+L2+L3– 5) and L3– 5 were significantly smaller in ACO and COPD patients compared to asthma patients (median L1+L2+L3– 5= 1.2 mm vs 1.14 mm vs 1.31 mm, p< 0.001; median L3– 5= 0.85 mm vs, 0.81 mm vs 0.92 mm, p=0.001, respectively).Conclusion: The process of structural changes in the airways assessed by EBUS is more advanced in individuals with ACO compared to patients with COPD, and less pronounced compared to patients with severe asthma. It seems that EBUS may provide useful information about differences in airway remodeling between ACO, COPD and severe asthma.Keywords: asthma-COPD overlap, airway remodeling, total bronchial wall, bronchial wall layers, endobronchial ultrasound

Human rhinoviruses (HRV) are frequent cause of asthma exacerbations, however the influence of airway inflammation on the severity of viral infection is poorly understood. Here, we investigated how cytokine-induced remodeling of airway epithelium modulates antiviral response. We analyzed gene expression response in in vitro differentiated bronchial epithelium exposed to cytokines and next infected with HRV16. IL-13-induced mucous cell metaplasia (MCM) was associated with impaired ciliogenesis and induction of antiviral genes, resulting in lower susceptibility to HRV. Epithelial-mesenchymal transition caused by TGF-β was associated with increased virus replication and boosted innate response. Moreover, HRV infection per se caused transient upregulation of MCM markers and growth factors, followed by low-level virus replication and shedding. Our data suggest that the outcome of HRV infection depends on the type of lower airway inflammation and the extent of epithelial damage. Type-2 inflammation (eosinophilic asthma) may induce antiviral state of epithelium and decrease virus sensitivity, while growth factor exposure during epithelial repair may facilitate virus replication and inflammatory response. Additionally, responses to HRV were similar in cells obtained from asthma patients and control subjects, which implicates that antiviral mechanisms are not intrinsically impaired in asthma, but may develop in the presence of uncontrolled airway inflammation.

Increased airway wall thickness and remodeling of bronchial mucosa are characteristic of asthma and may arise from altered integrin signaling on airway cells. Here, we analyzed the expression of β1-subfamily integrins on blood and airway cells (flow cytometry), inflammatory biomarkers in serum and bronchoalveolar lavage, reticular basement membrane (RBM) thickness and collagen deposits in the mucosa (histology), and airway geometry (CT-imaging) in 92 asthma patients (persistent airflow limitation subtype: n = 47) and 36 controls. Persistent airflow limitation was associated with type-2 inflammation, elevated soluble α2 integrin chain, and changes in the bronchial wall geometry. Both subtypes of asthma showed thicker RBM than control, but collagen deposition and epithelial α1 and α2 integrins staining were similar. Type-I collagen accumulation and RBM thickness were inversely related to the epithelial expression of the α2 integrin chain. Expression of α2β1 integrin on T-cells and eosinophils was not altered in asthma. Collagen I deposits were, however, more abundant in patients with lower α2β1 integrin on blood and airway CD8+ T-cells. Thicker airway walls in CT were associated with lower α2 integrin chain on blood CD4+ T-cells and airway eosinophils. Our data suggest that α2β1 integrin on inflammatory and epithelial cells may protect against airway remodeling advancement in asthma.

Background Heterogeneity of asthma complicates search for targeted treatment against airway hyperresponsiveness and remodeling. We conducted a systems biology approach study to establish differential co-expression of genes (DCG) in eosinophilic and non-eosinophilic asthma patients and infer their role in the disease. Materials and Methods N = 40 Caucasian adult moderate to severe non-smoking asthma patients (half with eosinophilic asthma) undergone bronchial brush biopsy sampling for mRNA expression using hybridization to cDNA microarray. Mechanistic interpretation of DCG was inferred from existing literature. Results Differentially co-expressed genes bear significance in airway viral infection (ATP1B1, EPS15), arachidonic acid metabolism (CLC, FADS6), cell migration (EPS8L1, STOML3, RhoBTB2), surface receptors endocytosis (STRN4, EPS15, ATP1B1) or decreased expression (CCT7), oxidative stress (DIO3, RhoBTB2), decreased adhesion (ATP1B1, RAPH1, STOML3), epithelial-mesenchymal transition (ASB3, RADX, CCT7, MRPL14, PPP2R3B, RPS13, SLC19A1), myofibroblast differentiation (CCT7), smooth muscle proliferation (ASB3, ATP1B1), airway hyperreactivity (RECK, STOML3, ATP1B1, OR52I1), extracellular matrix remodeling (FBN3, RECK), angiogenesis (GPI, RhoBTB2) and neuronal pathogenesis of asthma (OR52I1, STRN4, TTC3P1, GPI, CABP5) and were linked to asthma in genome- (MRPL14, ASB3, RPS13) and epigenome-wide (CLC, EPS15, GPI, SSCRB4, STRN4) association studies. Signaling pathways involved (especially TGF-β/Smad2/3) are inferred from the co-expression pattern. Conclusion Activity of genes and pathways of known or tentative role in asthma pathogenesis was established in regard to a condition cognizable in clinical practice.

Airway remodeling in asthma is characterized by reticular basement membrane (RBM) thickening, likely related to epithelial structural and functional changes. Gene expression profiling of the airway epithelium might identify genes involved in bronchial structural alterations. We analyzed bronchial wall geometry (computed tomography (CT)), RBM thickness (histology), and the bronchial epithelium transcriptome profile (gene expression array) in moderate to severe persistent (n = 21) vs. no persistent (n = 19) airflow limitation asthmatics. RBM thickness was similar in the two studied subgroups. Among the genes associated with increased RBM thickness, the most essential were those engaged in cell activation, proliferation, and growth (e.g., CDK20, TACC2, ORC5, and NEK5) and inhibiting apoptosis (e.g., higher mRNA expression of RFN34, BIRC3, NAA16, and lower of RNF13, MRPL37, CACNA1G). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (LAMA3, USH2A), involved in ECM remodeling (LTBP1), neovascularization (FGD5, HPRT1), nerve functioning (TPH1, PCDHGC4), oxidative stress adaptation (RIT1, HSP90AB1), epigenetic modifications (OLMALINC, DNMT3A), and the innate immune response (STAP1, OAS2). Cluster analysis revealed that genes linked with RBM thickness were also related to thicker bronchial walls in CT. Our study suggests that the pro-fibrotic profile in the airway epithelial cell transcriptome is associated with a thicker RBM, and thus, may contribute to asthma airway remodeling.

The presented research aims to verify whether physicochemical properties of lung fibroblasts, modified by substrate stiffness, can be used to discriminate between normal and fibrotic cells from idiopathic pulmonary fibrosis (IPF). The impact of polydimethylsiloxane (PDMS) substrate stiffness on the physicochemical properties of normal (LL24) and IPF-derived lung fibroblasts (LL97A) was examined in detail. The growth and elasticity of cells were assessed using fluorescence microscopy and atomic force microscopy working in force spectroscopy mode, respectively. The number of fibroblasts, as well as their shape and the arrangement, strongly depends on the mechanical properties of the substrate. Moreover, normal fibroblasts remain more rigid as compared to their fibrotic counterparts, which may indicate the impairments of IPF-derived fibroblasts induced by the fibrosis process. The chemical properties of normal and IPF-derived lung fibroblasts inspected using time-of-flight secondary ion mass spectrometry, and analyzed complexly with principal component analysis (PCA), show a significant difference in the distribution of cholesterol and phospholipids. Based on the observed distinctions between healthy and fibrotic cells, the mechanical properties of cells may serve as prospective diagnostic biomarkers enabling fast and reliable identification of idiopathic pulmonary fibrosis (IPF).

Introduction: Needle biopsy of enlarged lymph nodes is accepted method for the diagnosis of sarcoidosis, but there is still place for investigating the optimal endosonography guided approach. Objectives: The aim of this study was to assess the relative diagnostic yield of the combination (CUS-b-NA) of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and endoscopic ultrasound fine-needle aspiration (EUS-b-FNA) but also a role of cell blocks (CB) and lymph node localization for the diagnosis of sarcoidosis. Methods: This was a prospective, multicenter study including consecutive patients with clinical diagnosis of stage I or II sarcoidosis. CUS-b-NA with smears and CB technique was performed within all group. If sarcoidosis was not confirmed invasive diagnostics was scheduled and 6 months follow-up was continued. Results: Since Nov 2017 to Sep 2019 fifty patients were enrolled for the final analysis. The overall sensitivity of EBUS-TBNA, EUS-b-FNA and CUS-b-NA was 76.6%, 70.2% and 91.7%, respectively. There were no significant differences between EBUS-TBNA and EUS-b-FNA (P=0.52) but CUS-b-NA had significantly higher yield (P=0.005 and P=0.001). No serious complications after all biopsies were observed. Adding CB to smear technique (P=0.008) and biopsy of the subcarinal lymph nodes (P=0.001) significantly increases the diagnostic yield. Conclusions: A diagnostic yield of CUS-b-NA is higher than endosonographic techniques alone in stage I and II of sarcoidosis. The preparation of cytological material including CB technique and the biopsy of subcarinal lymph node station increases diagnostic efficacy.

A 62 years old patient, who was diagnosed with high-grade T1 (T1HG) bladder cancer, after transurethral resection (TURB) (February 2018) and re-TURB (March 2018), with a history of diabetes mellitus type 2, hypertension, dyslipidemia, coronary artery disease and three percutaneous coronary interventions was admitted to the Urology Department [...]

Background Emerging data indicates that extracellular traps (ETs), structures formed by various immune cell types, may contribute to the pathology of noninfectious inflammatory diseases. Histone hypercitrullination is an important step in ETs formation and citrullinated histone H3 (H3cit) is considered a novel and specific biomarker of that process. In the present study we have evaluated circulating H3cit in stable asthmatics and investigated its relationship with asthma severity, pulmonary function and selected blood and bronchoalveolar lavage (BAL) biomarkers. Methods In 60 white adult stable asthmatics and 50 well-matched controls we measured serum levels of H3cit. In asthmatics we also performed bronchoscopy with BAL. We analyzed blood and BAL biomarkers, including interleukin (IL)-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A and interferon γ. For statistical analysis, Mann–Whitney U-test, χ2 test, one-way ANCOVA, ROC curve analysis and univariate linear regression were applied. Independent determinants of H3cit were established in a multiple linear regression model. Results Asthma was characterized by elevated circulating H3cit (17.49 [11.25–22.58] vs. 13.66 [8.66–18.87] ng/ml, p = 0.03). In asthmatics positive associations were demonstrated between serum H3cit and lung function variables, including total lung capacity (TLC) (β = 0.37 [95% CI 0.24–0.50]) and residual volume (β = 0.38 [95% CI 0.25–0.51]). H3cit was increased in asthma patients receiving systemic steroids (p = 0.02), as well as in subjects with BAL eosinophilia above 144 cells/ml (p = 0.02). In asthmatics, but not in controls, circulating H3cit correlated well with number of neutrophils (β = 0.31 [95% CI 0.19–0.44]) and monocytes (β = 0.42 [95% CI 0.29–0.55]) in peripheral blood. Furthermore, BAL macrophages, BAL neutrophils, TLC, high-sensitivity C-reactive protein, Il-12p70 and bronchial obstruction degree were independent determinants of H3cit in a multivariate linear regression model. Conclusions Asthma is characterized by increased circulating H3cit likely related to the enhanced lung ETs formation. Inhibition of ETs might be a therapeutic option in selected asthma phenotypes, such as neutrophilic asthma.

Introduction Needle biopsy of enlarged lymph nodes is an accepted method for the diagnostic workup of sarcoidosis, but the optimal endosonography‑guided approach is yet to be determined. Objectives The aim of our study was to assess the relative diagnostic yield of combined ultrasound‑guided needle aspiration (CUS‑b‑NA), which includes endobronchial ultrasound‑guided transbronchial needle aspiration (EBUS‑TBNA) with endoscopic ultrasound fine‑needle aspiration (EUS‑b‑FNA), as well as the role of the cell block (CB) technique and lymph node localization in the diagnostic workup of sarcoidosis. Patients and methods This was a prospective multicenter study including consecutive patients with clinical suspicion of stage I or II sarcoidosis. CUS‑b‑NA with smears and CB technique were performed in the whole study group. If a biopsy result was not conclusive, an invasive diagnostic workup and a 6-month follow‑up were scheduled. Results Out of 77 screened patients, 54 signed written consent and 50 were enrolled for the final analysis. The overall sensitivity of EBUS‑TBNA, EUS‑b‑FNA, and CUS‑b‑NA was 76.6%, 70.2%, and 91.7%, respectively. There were no differences between EBUS‑TBNA and EUS‑b‑FNA (P = 0.52) but CUS‑b‑NA had a higher diagnostic yield (P = 0.005 and P = 0.001, respectively). Adding the CB method to smear technique (P = 0.008) and biopsy of the subcarinal lymph nodes increased the diagnostic yield (P = 0.001). Conclusions: The diagnostic yield of CUS‑b‑NA is higher than that of endosonographic techniques alone in the diagnostic workup of stage I and II sarcoidosis. The preparation of cytological material including CB and the choice of the subcarinal lymph node station for the biopsy increase the diagnostic efficacy.

Introduction Diagnosis of sarcoidosis is based on clinical status and radiologic specific findings. Tissue confirmation of noncaseating granulomas is crucial. Pathological confirmation of pulmonary sarcoidosis is most commonly accomplished by bronchoscopy, which has a diagnostic yield of approximately 60%-70%. Objectives In this prospective study, we analysed potential benefit of EBUS-TBNA and EBB combination, application of cell blocks and smears with puncturing more than one station of lymph nodes in order to determine optimal strategy in diagnosis of sarcoidosis. Methods About 133 patients with suspicion of sarcoidosis (stage I and stage II) were included in this study. Each patient underwent conventional bronchoscopy with endobronchial biopsy (EBB) followed by the EBUS and puncturing at least two different lymph node stations. Results Positive cytopathological verification of sarcoidosis in our study was obtained in 123 patients (92.5%). EBUS-TBNA was diagnostic in 116 patients (87.2%). EBB was positive in 26 patients (19.55%). Combination of EBUS-TBNA and EBB statistically increased diagnostic yield of sarcoidosis to 92.5%. Sensitivity of EBUS-TBNA with EBB was 93.9%, specificity 100%, PPV 100% and NPV 20%. Conclusions Combining EBUS-TBNA from at least two lymph node stations and EBB increased diagnostic yield of sarcoidosis. Such diagnostic strategy had almost 93% of diagnostic yield in stage I and stage II of sarcoidosis. Taking into account the safety of the whole procedure with endobronchial ultrasonography combined with conventional endoscopy with EBB and its cost effectiveness, TBLB can be intended to diagnose stage III or IV of pulmonary sarcoidosis.

Histone H3 is citrullinated in the process of epigenetic chromatin remodelling and neutrophil extracellular trap (NET) formation. In 76 adult asthmatics and 54 well-matched controls we measured circulating level of citrullinated histone H3 (CitH3) using Cayman ELISA kit. In asthma we also determined the airway cross-sectional geometry of two distal and two peripheral bronchi in lung computed tomography (CT) and performed bronchoscopy with bronchoalveolar lavage (BAL). We assessed blood and BAL biomarkers, including cytokines: IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A, IL-23, INFγ, together with periostin and blood level of disintegrin and metalloproteinase domain-containing protein (ADAM33). We found that CitH3 was higher in asthmatics than controls (16.6 [11.5-21.7] vs. 13.9 [7.14-18.8] ng/ml, p=0.02), independently of asthma severity. Weak correlation was demonstrated between CitH3 and FEV1/VC index (β=-0.15 [-0.27- -0.04]), but none of the CT remodelling indices. In asthma, but not in controls, CitH3 was related to the number of leukocytes (β=0.26 [0.15-0.38]), neutrophils (β=0.25 [0.14-0.37]) monocytes (β=0.42 [0.31-0.53]), erythrocytes (β=0.27 [0.15-0.38]) and platelets (β=0.22 [0.1-0.33]) in peripheral blood. In both groups CitH3 remained in positive relationships with cholesterol levels, but not inflammatory markers. CitH3 was associated with circulating ADAM33 (β=0.19 [0.11-0.18]) and BAL biomarkers: INFγ, IL-5, IL-17A and IL-10 (β=0.19 [0.07-0.31], β=0.22 [0.1-0.33], β=0.19 [0.1-0.3], β=-0.14 [-0.26- -0.02], respectively). Conclusions: We have shown that blood NETs formation might be involved in asthma pathology independently of its severity. Funding: National Science Centre, No DEC-2013/09/B/NZ5/00758

Introduction: A majority of advanced lung cancer (LC) patients should be considered for targeted oncological treatment. CT/PET-CT and EBUS/EUS-b-NA with use of 22G cytological needles are standard staging procedures. Little is known about efficacy of histological core needles to obtain a sufficient material for immunohistochemistry (IHC) and molecular testing (MT). Aims: To evaluate efficacy of EBUS/EUS-b-NA using histological 25G core needles in LC patients stage IIIB/IV. Methods: Consecutive patients with confirmed or suspected advanced LC were included into a single center prospective observational trial. Endosonography was performed with use of 25G core needles. If biopsy was positive for LC material was sent for IHC and MT. If biopsy was negative then repeated with use of standard 22G cytological needles. Results: Since January to February 2019 – 20 patients (M:13, F:7) of mean age (SD) 68 (6.4) years underwent endosonography. A histology was conclusive in 18/20 patients: SCLC – 8, Adenocarcinoma – 6, Squamous-Cell Carcinoma (SCC) – 3, Not otherwise specified (NOS) – 1. In all group sensitivity, accuracy and PPV for histology and IHC tests was (90%, 100%, 100%). In 2 FN patients, in whom undifferentiated cancer cells were only found, EBUS-NA with use of 22G needle confirmed SCLC – 1 and SCC – 1. In 9/11 of NSCLC patients sensitivity, accuracy and PPV for endosonography with use of 25G core needles for MT was (81.8%, 100%, 100%). No complications after biopsies were observed. Conclusions: The endosonography with use of histological 25G core needles is feasible and safe and seems to be a reasonable approach for IHC and MT in advanced LC patients

Airway structural changes are important in asthma pathology and require investigations. In 105 adult asthmatics (53 with fixed obstruction) we determined the airway cross-sectional geometry of two distal and two peripheral bronchi and quantified the low-attenuation lung area (LAA%). We also performed bronchoscopy with BAL and endobronchial biopsy with histological examination. We assessed blood and BAL biomarkers, including IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A, IL-23, INFγ and periostin, together with circulating ADAM33, and investigated interplays between analyzed variables with cluster analyses. Results: Patients with fixed airflow limitation had lower lumen area, higher airway area, increased wall area and wall thickness ratios of peripheral airways, accompanied by raised LAA%. They had also increased blood neutrophils and eosinophils, BAL eosinophilia, higher circulating fibrinogen, INFγ, periostin, and ADAM33. Blood neutrophilia, increased serum HDL, TSH, urea, and shorter APTT were determinants of thicker reticular basement membrane (RBM) of bronchial mucosa. BAL eosinophilia was the only positive predictor of collagen I accumulation. We observed a negative correlation between RBM thickness and collagen I deposit. A cluster analysis revealed three well-separated clusters, characterized by different lung function, biomarkers, collagen I deposit and RBM thickness. Conclusions: Unfavourable airway structural changes are related to the Th2 immunity, higher ADAM33, INFγ, and neutrophil count in blood. Thicker RBM might play a protective role against collagen I accumulation and mucosa fibrosis. Funding: National Science Centre, decision No DEC-2013/09/B/NZ5/00758

Introduction: Airway remodeling is an important factor in persistent obstruction in severe asthma. High resolution computed tomography (HRCT) is an effective method of detecting changes in airway structure. Our aim was to use HRCT to assess changes in airway remodeling in patients with severe allergic asthma who are treated with omalizumab. Material and methods: In 12 patients with severe allergic asthma, HRCT was performed before and after treatment with omalizumab. In selected bronchial airways, parameters were calculated: bronchial wall area (BA), also corrected for body surface area (BSA); percentage of wall area (WA%); and the ratio of luminal area to total bronchial area (Ai/Ao). Clinical response to treatment was assessed using an asthma control questionnaire (ACQ), asthma quality of life questionnaire (AQLQ), and number of exacerbations per year. Assessment included spirometry and blood eosinophilia. Results: Treatment resulted in significant improvement in ACQ (p = 0.035) and AQLQ (p = 0.001). We observed significant reduction in exacerbations per year (p = 0.002) and reduction of daily systemic steroid dose (p = 0.032). FEV₁ and peripheral blood eospinophilia did not change (p = 0.846 and p = 0.221). Airway dimensions (Ai/Ao) of particular bronchi were consistent with the mean of the parameters calculated for all bronchi measured. Although we observed a significant decrease in WA (p = 0.002) and WA/BSA (p = 0.002), WA% and Ai/Ao did not improve (p = 0.39 and p = 0.49). We found no correlations between changes in airways and changes in spirometry or clinical parameters. Conclusion: Despite clinical effectiveness of omalizumab, its effect on airway remodeling may be limited.

Introduction: Diagnosis of sarcoidosis is usually made on the basis of clinical outcome and imaging techniques but its configuration requires detection of noncaseating granulomas in biopsy specimen. The role of bronchoscopic sampling techniques in diagnosis of sarcoidosis is still discussed. Aims and Objectives: To determine the potential benefit of EBUS-TBNA and EBB (endobronchial biopsy) combination, application of cell blocks and puncturing more than one station of lymph nodes in diagnosis of sarcoidosis. Methods: Fifty two patients mean age 46.4±12.9 with suspicion of sarcoidosis (stage I, II) were included in this prospective study. Patients underwent diagnostic conventional bronchoscopy with EBB followed by EBUS-TBNA. One (group 7) or two (group 7, and 4R/4L or 10R/10L or 11R/11L) lymph nodes stations were punctured. Smear and cell blocks were performed and examined. Results: EBB was positive in 16 patients (30.8%). EBUS-TBNA was diagnostic in 86.5%. Combination of EBUS-TBNA and EBB increased diagnostic yield of sarcoidosis (86.5% vs 98.1%; p=0.02). Sensitivity of EBUS-TBNA with EBB was 100%, specificity 98.1%, PPV 100% and NPV 100%. Granulomas from one lymph nodes group were obtained in 78.8%, in group of two stations punctured cytology was positive in 86.5% (p=0.2). Application of cell blocks did not increase diagnostic yield of EBUS-TBNA in sarcoidosis. Conclusions: Combining of EBUS-TBNA of at least two lymph nodes stations and EBB increase diagnostic yield of sarcoidosis. Cell block specimens have not further impact on the diagnostic yield of the procedure.

Background Sarcoidosis is characterized by exaggerated immune response to unknown agent and can affect different organs. One of the main players in the pathology of the disease are regulatory T cells (Tregs), however, up to date the mechanisms of the possible molecular alterations of this particular cell subset are not known. Methods In the current study we looked for the global transcriptomic changes of miRNAs, using predefined array, and mRNAs (RNA seq analysis) of Tregs of patients with the most predominant form of the disease - acute pulmonary sarcoidosis (PS). For this purpose sorted CD4+/CD25+/CD127- Tregs from peripheral blood (PB) and CD4+/CD25 + Tregs from bronchoalveolar lavage (BAL) were used. Results MiRNA analysis revealed that Tregs isolated from PB and BAL display significantly different miRNA profile, suggesting an important role of the pulmonary microenvironment in creating these changes. Among disease-related miRNAs of PB Tregs we identified miR-155 and miR-223. Moreover, looking at the global transcriptome of PB Tregs, we recognized alterations in TLR-2 signaling pathway and in the downstream of NF-κB apoptosis and proliferation signals. However, induction of TLR-2 expression was found not only in Tregs, but also in the heterogeneous population of peripheral blood mononuclear cells (PBMC) as well as two PBMC subpopulations (CD4+/CD25-and CD4-/CD25-) of patients with PS. This indicates that activation of TLR signaling pathway in sarcoidosis does not occur only in Tregs. Conclusion Our findings offer a deeper insight into the molecular mechanisms of Tregs reduced suppression and increased apoptosis in patients with PS. Based on the current results, future studies should focus on possible therapeutic effect of TLR-2 signaling inhibition.

Introduction Bronchofiberoscopy (BF) is a key tool used in the management of patients with respiratory diseases. Describing factors that contribute to patient anxiety surrounding BF has the potential to influence patient management and willingness to undergo the procedure again in the future if needed. Objectives This study sought to understand what factors influence anxiety and satisfaction experienced by patients undergoing BF under conscious sedation. Methods This study had a prospective observational character and evaluated 463 consecutive patients undergoing BF. Participants were divided into two groups, those undergoing BF for the first time and those who had undergone the procedure at least once in the past. Data were collected from three questionnaires prepared by the research team. Results Patients who had undergone multiple bronchoscopies were more satisfied with their physician's explanation of the procedure (P

Introduction Endoscopic biopsy techniques are useful in the diagnosis of sarcoidosis. There is a need for randomized trials to establish where these procedures fit in the diagnosis of sarcoidosis. Objectives The aim of the study was to compare the diagnostic yield of conventional transbronchial needle aspiration (TBNA) with endobronchial ultrasound-guided TBNA (EBUS-TBNA) and endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in stages I and II of pulmonary sarcoidosis. Patients and methods Patients suspected of sarcoidosis were randomized to undergo TBNA or EBUS-TBNA or EUS-FNA. Patients with negative TBNA and EBUS-TBNA results underwent EUS-FNA and those with negative EUS-FNA results—EBUS-TBNA. If both tests were negative, patients in stage I were scheduled for mediastinoscopy (MS) and those in stage II—for transbronchial lung biopsy (TBLB). Results In 100 patients, 34 TBNA, 30 EBUS-TBNA, and 36 EUS-FNA procedures were performed at baseline. TBNA was positive in 20 patients (58.8%); EBUS-TBNA, in 23 (76.7%); and EUS-FNA, in 31 patients (86.1%). In patients with negative biopsy results, the second procedure was performed. The results of EUS-FNA were positive in 9 patients and of EBUS-TBNA—in none. Of 17 patients with negative results of both procedures, MS was performed in 6 patients and was positive in 2. In the remaining 11 patients, sarcoidosis was confirmed by TBLB. Sensitivity and accuracy of TBNA compared with EBUS-TBNA and EUS-FNA were 62.5% and 64.7%, 79.3% and 80%, and 88.6% and 88.9%, respectively. Sensitivity and accuracy of EBUS-TBNA were higher (P = 0.139) and of EUS-FNA were significantly higher compared with TBNA (P = 0.012). Conclusions In stages I and II of pulmonary sarcoidosis, endoscopic ultrasound is a reasonable approach but EUS-FNA seems to be the method of choice.