Culture and Differentiation of Primary Human Tracheobronchial Epithelial Cells Using STEMCELL Technologies Pneumacult Media

NOTE: A PDF methods document is attached in the supplementary materials.SCOPE OF APPLICATION (LIMITATIONS)This method describes the culture and differentiation of primary human tracheobronchial epithelial cells (pHBEC). Cells used for this method can be obtained by brush biopsy during clinical bronchoscopy or purchased commercially. This method replaces the previous version described in (Dailey and McCullough, 2021a; b). Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Relationship between nitric oxide levels and the activity of fibrogenic cytokine TGF-β1 and their role in diagnostics of the development of irreversible morphofunctional changes in bronchi of smoking adolescents

The aim is to study the relationship between the level of exhaled nitric oxide (FeNO) and the activity of the fibrogenic cytokine TGF-β1 in blood serum and brush biopsy samples of bronchial mucosa in order to determine their role in the development of irreversible morphological and functional changes in smoking adolescents. Materials and methods. 20 adolescent smokers with chronic bronchitis (CB) (average age – 17.5 ± 0.2 years) were exa­mined. The comparison group consisted of 37 adolescent smokers without respiratory symptoms (average age – 15.9 ± 0.2 years) and 15 healthy adolescents, who never smoked (average age – 15.9 ± 0.4 years). In adolescent smokers the tobacco smoking status was assessed. To confirm active smoking, the nicotine metabolite cotinine was determined in urine. Instrumental methods included spirometry, chest X-ray, tracheobronchoscopy. The FeNO level was measured using a Niox Mino. TGF-β1 level was determined in the blood serum and brush biopsy samples of the bronchial mucosa. Results. The FeNO levels were significantly lower in adolescent smokers with CB in comparison with adolescent smokers without respiratory symptoms (6.1 ± 0.3 ppb versus 8.8 ± 0.6 ppb, P < 0.05). The relationship between the FeNO levels and indicators of the tobacco smoking status has been established in patients with CB and in asymptomatic smokers. There was a significant increase in the TGF-β1 levels in the blood serum in patients with CB compared with asymptomatic smokers (478.7 ± 57.9 pg/ml versus 231.5 ± 23.5 pg/ml, P < 0.05). In smoking adolescents a relationship between a FeNO level and an increased activity of the fibrogenic cytokine TGF-β (r = -0.63; P < 0.05) has been established. In one third of patients the TGF-β1 factor was identified in the bronchial endothelium. The presence of this factor in the bronchial endothelium is a serious prognostic criterion for the risk of developing “inadequate” pneumofibrosis, which can lead to irreversible remodeling processes in the bronchi. Conclusions. Determination of FeNO levels and TGF-β1 in the blood serum in smoking adolescents has a reliable diagnostic value for determining the risk group for the development of irreversible morphological and functional changes in the bronchi and can improve the efficiency of early diagnosis of chronic respiratory pathology.

Bronchoscopic biopsies with radial endobronchial ultrasonographic navigation in the diagnosis of tuberculosis and mycobacteriosis in patients with peripheral lung masses

The objective of the study: to evaluate the effectiveness of diagnosis of tuberculosis and mycobacteriosis in bronchobiopsy specimens obtained during navigation by radial endobronchial ultrasonography (rEBUS) in patients with peripheral lung lesions without bacterial excretion.Subjects and methods. A retrospective analysis of the diagnostic effectiveness of bronchoscopic examination with biopsies was carried out in 179 patients (75 men and 104 women) suffering from pulmonary tuberculosis or mycobacteriosis without bacterial excretion; peripheral lung lesions had been visualized by computed tomography (CT). The patients were divided into two groups: 93 underwent bronchoscopy with biopsies with rEBUS navigation, 86 underwent bronchoscopy with classical biopsies and preliminary CT navigation. Each patient underwent multiple biopsies, at least one fluid biopsy (bronchoalveolar lavage or bronchial lavage), and one tissue biopsy (transbronchial lung biopsy or brush biopsy). Specimens collected by all types of bronchobiopsy were sent for microbiological and cytological tests, specimens of pulmonary transbronchial biopsy were additionally sent for histological examination.Results. The diagnosis of tuberculosis was verified by bronchobiopsy in 106 (67.5%) of 158 patients with tuberculosis, but statistically significantly more often in the group with rEBUS navigation versus the group without it – 81.9% (68/83) versus 50.7% (38/75), respectively (pχ2 < 0.01). The diagnosis of non-tuberculous mycobacteriosis was verified by bronchobiopsy in 13 (61.9%) of 21 patients, in the group with rEBUS navigation – in 80.0% (8/10) patients, in the group without it – in 45.5% (5/11) (pφ > 0.05). The use of rEBUS navigation while collecting bronchobiopsy specimens made it possible to increase the etiological verification of tuberculosis using the following microbiological methods: microscopy – from 14.7 to 49.4% (pχ2 < 0.01), molecular genetic – from 41.3 to 72.3% ( pχ2 < 0.01), culture (Bactec MGIT960) – from 44.0 to 67.5% (pχ2 < 0.01) The greatest enhancement of diagnostic effectiveness was achieved in the specimens of bronchoalveolar lavage and bronchial lavage – from 33.3 to 71.1% (pχ2 < 0.01) and in brush biopsy specimens – from 25.6 to 57.6% (pχ2 < 0.01).

Comparison between two cell collecting methods for liquid-based brush biopsies: a consecutive and retrospective study

Background This study compares two different cell collectors, the Orcellex Brush (rigid brush) and the Cytobrush GT (nylon brush), using liquid-based cytology. A comparison of their obtainment procedures was also considered. The aim was to determine the diagnostic accuracy for detection of malignancy in oral brush biopsies. PICO-Statement: In this consecutive and retrospective study we had as population of interests, patients with oral lesions, the intervention was the brush biopsy with two different cell collectors and the control was healthy oral mucosa. The outcome of the study was to compare both cell collectors. Methods From 2009 to 2018, 2018 patients with oral lesions were studied using the nylon brush (666 cases) and rigid brush (1352 cases). In the first cohort five smears per patient were taken with the nylon brush, while each patient received one smear with the rigid brush in the second cohort. These were further processed in a liquid-based procedure. Cytological evaluations were categorised into ‘negative’, which were considered as negative, whereas ‘doubtful’, ‘suspicious’ and ‘positive’ cytological results were overall considered as positive for malignancy in comparison to the final histological diagnoses. Additionally, the clinical expenditure for each collector was estimated. Results 2018 clinically and histologically proven diagnoses were established, including 181 cases of squamous cell carcinomas, 524 lichen, 454 leukoplakias, 34 erythroplakias and 825 other benign lesions. The sensitivity and specificity of the nylon brush was 93.8% (95% CI 91.6–95.5%) and 94.2% (95% CI 91.8–95.5%) respectively, whereas it was 95.6% (95% CI 94.4–96.6%) and 84.9% (95% CI 83.8–87.5%) for the rigid brush. The temporal advantage using the plastic brushes was 4× higher in comparison to the nylon brush. The risk suffering from a malignant oral lesion when the result of the brushes was positive, suspicious, or doubtful was significantly high for both tests (nylon brush OR: 246.3; rigid brush OR: 121.5). Conclusions Both systems have a similar sensitivity, although only the rigid brush achieved a satisfactory specificity. Additional methods, such as DNA image cytometry, should also be considered to improve the specificity. Furthermore, the rigid brush proved to be more effective at taking a sufficient number of cells, whilst also being quicker and presenting less stress for the patient.

Culture of Primary Human Tracheobronchial Epithelial Cells

This protocol is intended for culture of primary human tracheobronchial epithelial cells (pHBEC) obtained by brush biopsy during clinical bronchoscopy, or purchased commercially, using Lonza-based medium.Note: This is a historical protocol. At the time of publication, this protocol has been superseded by a different version in the McCullough lab; however, it is being published to support the transparency and reproducibility of other studies by which it is referenced.Disclaimer: The information presented here has been reviewed and approved for publication by the US Environmental Protection Agency do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

A Comparison between Total Intravenous Anaesthesia Using Propofol plus Remifentanil and Propofol plus Sevoflurane in Children undergoing Fibreoptic Bronchoscopy：A Randomized, Single-Blind Study

BackgroundFibreoptic bronchoscopy is an important used to evaluate and manage paediatric patients with respiratory disease.This study aimed to compare the effect of two different anaesthesia methods, propofol combined with remifentanil and propofol combined with sevoflurane, on children undergoing fibreoptic bronchoscopy (FOB).Method:Sixty children were enrolled and randomly divided into two groups. In the remifentanil group (Group R), the patients were induced with 4 mg•kg− 1 propofol + 4 µg•kg− 1 remifentanil (slow intravenous injection) and maintained with 0.3–0.5 µg•kg− 1•min− 1 remifentanil. In the sevoflurane group (Group S), the patients were induced with 2 mg•kg− 1 propofol and 8% sevoflurane and maintained with 4–6 vol% sevoflurane. Heart rate (HR), mean blood pressure (MBP) and pulse oxygen saturation (SpO2) were monitored and recorded at four time points: baseline (T0), when the bronchoscope reached glottis (T1), the time of lavage (T2), and the time of brush biopsy (T3). Waking time and the satisfaction score of physicians were recorded, and emergence agitation (EA) was evaluated in the postanesthesia care unit (PACU). Finally, any supplementary medicine or adverse events were also recorded.ResultsCompared with group S, the waking time was significantly shorter and the incidence of EA was significantly lower in Group R. During bronchoscopy, significant differences in MAP or HR were not observed between the two groups at T0. Compared with group S, the HR of group R was significantly decreased at T1, T2 and T3, and MAP was significantly decreased at T1, but the fluctuation range was within 20% of the baseline. A significant difference in SpO2 was not observed between the two groups at any time point. No significant differences in the operation time of FOB, the incidence of complications, such as moving, hypoxemia, bronchospasm, and bucking, or the level of satisfaction were observed between the two groups.ConclusionsTotal intravenous anaesthesia using propofol plus remifentanil in children undergoing fibreoptic bronchoscopy potentially reduces the waking time and decreases the incidence of EA to improve the work efficiency and turnover rate of the outpatient operating room.Trial registrationRegistered at the China Clinical Trial Registry (http://www.chictr.org.cn) (ChiCTR1900026098).

Integration of Airway Inflammation and Remodeling Mechanisms Specific to Eosinophilic Asthma Through Differential Co-Expression of Genes in Bronchial Brush Biopsy Samples

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.

A standalone approach to utilize telomere length measurement as a surveillance tool in oral leukoplakia

Oral Squamous Cell Carcinoma (OSCC) is often preceded by white patch, called oral leukoplakia (OL). Assessing relative telomere length (TL) in OL could be a predicting biomarker. Due to high variability and lack of universal reference, there has been a limited translational application of TL. Here, we describe an approach of evaluating TL using paired PBMC as internal reference and demonstrate its translational relevance. Oral brush biopsy and paired venous blood were collected from 50 male OL patients and 44 male healthy controls. Relative TL was measured by qPCR. TL of each OL sample was normalized to paired PBMC sample (TL ratio). Mean TL ratio in healthy controls with high risk oral habits was shorter than those who did not have these habits (1.093±0.411 and 1.253±0.296, respectively; p=0.071). In OL patients, the mean TL ratio was not only significantly shorter in the patch but also in distal normal oral tissue (0.971±0.317, p=0.0002 and 0.896±0.284, p=0.00001, respectively), relative to healthy control without high risk oral habit. Based on the TL ratio, we proposed a classification of OL into four subgroups. Dysplastic pathology was frequently associated with a subgroup having normal TL ratio at the patch while significantly shorter TL ratio at paired normal distal site. The approach of analyzing TL attrition of oral mucosa, eliminating requirement of external reference DNA, will enable the TL data universally comparable and provide a useful marker to define high risk OL group for follow-up program. Larger studies will further validate the approach and its broader application in other pre-malignant conditions.