Endobronchial ultrasound transbronchial needle aspiration: a hybrid method

Conventional transbronchial needle aspiration (cTBNA) was first performed approximately 30 years ago; however TBNA was not widely adopted until the development of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA). Current EBUS-TBNA needle sizes are limited to 21- and 22-gauge. In order to determine whether a 19-gauge (19G) needle in EBUS-TBNA can further improve the diagnostic yield and simplify the methodology of EBUS-TBNA we developed a hybrid method. Here we report our initial experience in assessing the feasibility of performing EBUS-TBNA using a conventional 19G TBNA needle

Change in cytokine profiles released by mast cells mediated by lung cancer-derived exosome activation may contribute to cancer-associated coagulation disorders

Abstract Background Coagulation disorders are a significant cause of lung cancer mortality. Although mast cells are known to play a role in coagulation abnormalities, their specific role in this process has not yet been elucidated. Method We detected mast cells in the tumor microenvironment using single-cell sequencing data and examined their correlation with thrombosis-related genes, neutrophil-related genes, neutrophil extracellular trap-related signature genes, and immune infiltration levels in lung cancer patients through bioinformatics analysis. Bone marrow mast cell uptake of exosomes isolated from the lung adenocarcinoma cell line A549, which were labeled using PKH67, was observed using confocal microscopy. Mast cell degranulation was detected by measuring the β-hexosaminidase release rate. Additionally, cytokine array analysis was performed to identify altered mediators released by bone marrow mast cells after uptake of the exosomes. Results In our study, we found a close correlation between the proportion of mast cells in lung cancer patients and the expression levels of thrombosis-related genes and neutrophil extracellular trap signature genes, both of which play a key role in thrombophilic disorder. Moreover, we discovered that lung cancer cell-derived exosomes can be taken up by mast cells, which in turn become activated to release procoagulant mediators. Conclusion Our study shows that exosomes derived from lung cancer cells can activate mast cells to release procoagulants that may contribute to abnormal blood clotting in lung cancer patients. Video Abstrac

Involvements of p38 MAPK and oxidative stress in the ozone-induced enhancement of AHR and pulmonary inflammation in an allergic asthma model

Abstract Background Exposure to ambient ozone (O3) increases the susceptivity to allergens and triggers exacerbations in patients with asthma. However, the detailed mechanisms of action for O3 to trigger asthma exacerbations are still unclear. Methods An ovalbumin (OVA)-established asthmatic mouse model was selected to expose to filtered air (OVA-model) or 1.0 ppm O3 (OVA-O3 model) during the process of OVA challenge. Next, the possible involvements of p38 MAPK and oxidative stress in the ozone actions on the asthma exacerbations were investigated on the mice of OVA-O3 model by treating them with SB239063 (a p38 MAPK inhibitor), and/or the α-tocopherol (antioxidant). Biological measurements were conducted including airway hyperresponsiveness (AHR), airway resistance (Raw), lung compliance (CL), inflammation in the airway lumen and lung parenchyma, the phosphorylation of p38 MAPK and heat shock protein (HSP) 27 in the tracheal tissues, and the malondialdehyde (MDA) content and the glutathione peroxidase (GSH-Px) activity in lung tissues. Results In OVA-allergic mice, O3 exposure deteriorated airway hyperresponsiveness (AHR), airway resistance (Raw), lung compliance (CL) and pulmonary inflammation, accompanied by the increased oxidative stress in lung tissues and promoted p38 MAPK and HSP27 phosphorylation in tracheal tissues. Administration of SB239063 (a p38 MAPK inhibitor) on OVA-O3 model exclusively mitigated the Raw, the CL, and the BAL IL-13 content, while α-tocopherol (antioxidant) differentially reduced the BAL number of eosinophils and macrophages, the content of BAL hyaluronan, the peribronchial inflammation, as well as the mRNA expression of TNF-α and IL-5 in the lung tissues of OVA-O3 model. Administration of these two chemical inhibitors similarly inhibited the AHR, the BAL IFN-γ and IL-6 production, the perivascular lung inflammation and the lung IL-17 mRNA expression of OVA-O3 model. Interestingly, the combined treatment of both compounds together synergistically inhibited neutrophil counts in the BALF and CXCL-1 gene expression in the lung. Conclusions O3 exposure during the OVA challenge process promoted exacerbation in asthma. Both p38 MAPK and oxidative stress were found to play a critical role in this process and simultaneous inhibition of these two pathways significantly reduced the O3-elicited detrimental effects on the asthma exacerbation

Quantification of Rare Circulating Tumor Cells in Non-Small Cell Lung Cancer by Ligand-Targeted PCR

<div><p>Background</p><p>Quantification of circulating tumor cells (CTC) is valuable for evaluation of non-small cell lung cancer (NSCLC). The sensitivity of current methods constrains their use to detect rare CTCs in early stage. Here we evaluate a novel method, ligand-targeted polymerase chain reaction (LT-PCR), that can detect rare CTCs in NSCLC patients.</p><p>Methods</p><p>CTCs were enriched by immunomagnetic depletion of leukocytes and then labeled by a conjugate of a tumor-specific ligand and an oligonucleotide. After washing off free conjugates, the bound conjugates were stripped from CTCs and then analyzed by qPCR. To evaluate the clinical utility, blood samples were obtained from 72 NSCLC patients (33 initially diagnosed and 39 on chemotherapy), 20 benign patients, and 24 healthy donors.</p><p>Results</p><p>Experiments with healthy blood spiked with tumor cells indicated the LT-PCR allows specific detection of CTC. The clinical study showed that the initially diagnosed patients have an average of 20.8 CTC units with metastatic diseases, 11.8 CTC units with localized diseases, and 6.0 CTC units with benign diseases. With the threshold of 8.5 CTC units, the assay can detect 80% of stage I/II, 67% of stage III, and 93% of stage IV cancer. With the benign patients and healthy donors as control group, the method can detect cancer with a sensitivity of 81.8% and a specificity of 93.2%.</p><p>Conclusion</p><p>The LT-PCR would allow quantification of CTC in NSCLC patients at a more sensitive level, providing a potential tool for stratifying malignant lung diseases, especially at early stage.</p></div

Immunostaining of the cultured KB cells and enriched CTCs.

<p>KB cells (A–D) and enriched CTCs from two metastatic NSCLC patients (CTC sample1: E–H; CTC sample 2: I–L) were fixed by methanol, and stained for cytokeratin (A,E and I) and folate receptor (FR) (B, F and J). The cell nucleus was labeled by DAPI (C, G and K). The merged images showed cytokeratin and folate receptor are expressed only in CTCs (H and L) and KB cells (D), not in haematologic cells.</p

Correlation of CTC levels between NSCLC patients and chemotherapy treatment.

<p><i>w/o chemo</i>, initially diagnosed NSCLC patients; <i>with chemo</i>, NSCLC patients on chemotherapy.</p

Outline of the ligand-targeted PCR detection for CTC.

<p>After negative enrichment by immunomagnetic depletion of leukocytes, CTCs were labeled with a conjugate of a tumor-specific ligand and an oligonucleotide. The labeled CTCs were washed thoroughly to remove free conjugates. Then the bound conjugates were stripped from CTC and analyzed by qPCR.</p

Patient characteristics.

<p>Abbreviations: SCC, squamous cell carcinoma.</p

CTC prevalence in initially diagnosed patients.

<p>A) CTC levels in localized and metastatic malignant lung disease, benign lung disease and healthy donors. B) Distribution of CTC levels in different histological subtypes. ADC, adenocarcinoma; SCC, squamous cell carcinoma. C) Receiver operating characteristic (ROC) analysis between malignant lung disease group and benign disease and healthy group.</p

Validation of the ligand-targeted PCR method for CTC detection.

<p>A) Recovery ratio of spiked cell assay. B) Linear regression curve of spiked cell assay. C) Specificity test of the assay. Spiked KB cells were treated with (<i>competed</i>) or without (<i>control</i>) 10 µM folic acid before labeling. D) Healthy donors' blood samples spiked with 20 KB cells <i>(20 cells)</i> or without KB tumor cells <i>(control)</i> were labeled by 10 nM folate-oligonucleotide conjugate <i>(conjugate)</i> or unconjugated-oligonucleotide <i>(oligonucleotide)</i> after enrichment. Data are shown with Mean±SD from three independent assays.</p