Resected Lung Tissue Research Articles

Pulmonary and Systemic Immune Profiles Following Lung Volume Reduction Surgery and Allogeneic Mesenchymal Stromal Cell Treatment in Emphysema.

Emphysema in patients with chronic obstructive pulmonary disease (COPD) is characterized by progressive inflammation. Preclinical studies suggest that lung volume reduction surgery (LVRS) and mesenchymal stromal cell (MSC) treatment dampen inflammation. We investigated the effects of bone marrow-derived MSC (BM-MSC) and LVRS on circulating and pulmonary immune cell profiles in emphysema patients using mass cytometry. Blood and resected lung tissue were collected at the first LVRS (L1). Following 6-10 weeks of recovery, patients received a placebo or intravenous administration of 2 × 106 cells/kg bodyweight BM-MSC (n = 5 and n = 9, resp.) in week 3 and 4 before the second LVRS (L2), where blood and lung tissue were collected. Irrespective of BM-MSC or placebo treatment, proportions of circulating lymphocytes including central memory CD4 regulatory, effector memory CD8 and γδ T cells were higher, whereas myeloid cell percentages were lower in L2 compared to L1. In resected lung tissue, proportions of Treg (p = 0.0067) and anti-inflammatory CD163- macrophages (p = 0.0001) were increased in L2 compared to L1, while proportions of pro-inflammatory CD163+ macrophages were decreased (p = 0.0004). There were no effects of BM-MSC treatment on immune profiles in emphysema patients. However, we observed alterations in the circulating and pulmonary immune cells upon LVRS, suggesting the induction of anti-inflammatory responses potentially needed for repair processes.

Expression of Tenascin-C Is Upregulated in the Early Stages of Radiation Pneumonitis/Fibrosis in a Novel Mouse Model.

The lung is a major dose-limiting organ for radiation therapy (RT) for cancer in the thoracic region, and the clarification of radiation-induced lung damage (RILD) is important. However, there have been few reports containing a detailed comparison of radiographic images with the pathological findings of radiation pneumonitis (RP)/radiation fibrosis (RF). We recently reported the upregulated expression of tenascin-C (TNC), an inflammation-associated extracellular matrix molecule, in surgically resected lung tissue, and elevated serum levels were elevated in a RILD patient. Therefore, we have developed a novel mouse model of partial lung irradiation and studied it with special attention paid to the computed tomography (CT) images and immunohistological findings. The right lungs of mice (BALB/c) were irradiated locally at 30 Gy/1fr, and the following two groups were created. In Group 1, sequential CT was performed to confirm the time-dependent changes in RILD. In Group 2, the CT images and histopathological findings of the lung were compared. RP findings were detected histologically at 16 weeks after irradiation; they were also observed on the CT images from 20 weeks. The immunostaining of TNC was observed before the appearance of RP on the CT images. The findings suggest that TNC could be an inflammatory marker preceding lung fibrosis.

Rapid examination of lung tissues by nonlinear microscopy.

Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

Epigenome-Wide Association Studies of COPD and Lung Function: A Systematic Review.

Chronic Obstructive Pulmonary Disease (COPD) results from gene-environment interactions over the lifetime. These interactions are captured by epigenetic changes, such as DNA methylation. This systematic review synthesizes evidence from epigenome-wide association studies (EWAS) related to COPD and lung function. Systematic literature search on PubMed, Embase and CINAHL databases, identified 1947 articles that investigated epigenetic changes associated with COPD/lung function; 17 of them met our eligibility criteria from which data was manually extracted. Differentially methylated positions (DMPs) and/or annotated genes, were considered replicated if identified by ≥2 studies with a p<1 x 10-4. Ten studies profiled DNA methylation changes in blood and 7 in respiratory samples, including surgically resected lung tissue (n=3), small airways epithelial brushings (n=2), bronchoalveolar lavage (n=1) and sputum (n=1). Main results showed: (1) high variability in study design, covariates and effect sizes, which prevented a formal meta-analysis; (2) in blood samples, 51 DMPs were replicated in relation to lung function and 12 related to COPD; (3) in respiratory samples, 42 DMPs were replicated in relation to COPD but none in relation to lung function; and, (4) in COPD vs. control studies, 123 genes (2.6% of total) were shared between ≥1 blood and ≥1 respiratory sample and associated with chronic inflammation, ion transport and coagulation. There is high heterogeneity across published COPD/lung function EWAS studies. A few genes (n=123; 2.6%) were replicated in blood and respiratory samples, suggesting that blood can recapitulate some changes in respiratory tissues. These findings have implications for future research.

Altered sphingolipid pathway in SARS-CoV-2 infected human lung tissue.

The SARS-CoV-2 mediated COVID-19 pandemic has impacted millions worldwide. Hyper-inflammatory processes, including cytokine storm, contribute to long-standing tissue injury and damage in COVID-19. The metabolism of sphingolipids as regulators of cell survival, differentiation, and proliferation has been implicated in inflammatory signaling and cytokine responses. Sphingosine-kinase-1 (SK1) and ceramide-synthase-2 (CERS2) generate metabolites that regulate the anti- and pro-apoptotic processes, respectively. Alterations in SK1 and CERS2 expression may contribute to the inflammation and tissue damage during COVID-19. The central objective of this study is to evaluate structural changes in the lung post-SARS-CoV-2 infection and to investigate whether the sphingolipid rheostat is altered in response to SARS-CoV-2 infection. Central and peripheral lung tissues from COVID-19+ or control autopsies and resected lung tissue from COVID-19 convalescents were subjected to histologic evaluation of airspace and collagen deposisiton, and immunohistochemical evaluation of SK1 and CERS2. Here, we report significant reduction in air space and increase in collagen deposition in lung autopsy tissues from patients who died from COVID-19 (COVID-19+) and COVID-19 convalescent individuals. SK1 expression increased in the lungs of COVID-19+ autopsies and COVID-19 convalescent lung tissue compared to controls and was mostly associated with Type II pneumocytes and alveolar macrophages. No significant difference in CERS2 expression was noted. SARS-CoV-2 infection upregulates SK1 and increases the ratio of SK1 to CERS2 expression in lung tissues of COVID-19 autopsies and COVID-19 convalescents. These data suggest an alteration in the sphingolipid rheostat in lung tissue during COVID-19, suggesting a potential contribution to the inflammation and tissue damage associated with viral infection.

Treprostinil Reconstitutes Mitochondrial Organisation and Structure in Idiopathic Pulmonary Fibrosis Cells.

Idiopathic pulmonary fibrosis (IPF) presents as an incurable change in the lung tissue and mitochondrial dysfunction of unknown origin. Treprostinil, a prostacyclin analogue, has been suggested for IPF therapy. This study assessed the effect of treprostinil on the cAMP signalling and mitochondrial activity in healthy lung fibroblasts and fibroblast-like cells from IPF patients. Six control fibroblast strains and six fibroblast-like IPF cell strains were isolated and expanded from freshly resected lung tissue. The cells were grown to confluence before being treated with either transforming growth factor (TGF)-β1, treprostinil, their combination, or a vehicle for up to 2 days. Mitochondria-regulating proteins were analysed using Western blotting and immunofluorescence, and the mitochondria were analysed using cytochrome C, mitochondrial cytochrome C oxidase II (MTCO2), and MTCO4. The IPF cells showed an increased rate of damaged mitochondria, which were significantly reduced when the cells were treated with treprostinil over 24 h. In the control cells, treprostinil prevented TGF-β-induced mitochondrial damage. Treatment with treprostinil modified the expression of several mitochondria-regulating proteins. In both cell types, treprostinil upregulated the expression of PTEN, p21(Waf1/Cip1), beclin1, LC3 II, parkin, PINK1, MTCO2, and MTCO4. In contrast, treprostinil downregulated the phosphorylation of mTOR and the expression of p62, mitofusin1, and mtiofusin2 in IPF cells. This might explain the reduced mitochondrial damage observed in treprostinil-treated IPF cells and suggest an improvement in the mitochondrial function in IPF. In this study, treprostinil improved mitochondrial impairment in vitro, which might, in part, explain the beneficial clinical effects documented in patients.

Spontaneous Pneumothorax Secondary to Bullous Lung Emphysema Positive for Cannabinoids upon Toxicological Examination.

Cannabis can be related to respiratory diseases, but the relationship between smoking marijuana and the development of a pneumothorax has scarcely been investigated. We aimed to analyze, in patients with a history of cannabis smoking abuse submitted to lung apicectomy for a primary spontaneous pneumothorax (PSP), the correlation between the presence of cannabinoids in the resected lung and the detection of bullous emphysema within the same tissue. Patients undergoing lung apicectomy for a PSP were prospectively enrolled, and the correlation between the presence of cannabinoids in the resected lung tissue and histological finding of bullous emphysema was investigated with Fisher's exact test. There were 21 male patients, with a median age of 27 years. The cannabinoids found by the toxicological examination in surgical specimens were mainly delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD). In 14/21 patients, cannabinoids were detected in the resected lung tissue, and bullous emphysema was present in 13/14 of these (93%), while bullous emphysema was found in only 1/7 (14%) of the remaining patients who were negative for cannabinoids in the lung tissue, and the difference was found to be statistically significant (p < 0.0009). Our study demonstrated the presence of bullous emphysema in most cannabinoid-positive patients and its absence in most of those who were cannabinoid-negative, supporting the correlation between cannabinoids in the lung tissue and bullous emphysema with the development of a "secondary" spontaneous pneumothorax.

T cell immunity in interstitial lung disease with non-small cell lung cancer patients

ObjectivesLimited treatment options are available for non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD). The rationale for immunotherapy and its adverse events for NSCLC with ILD remains unclear. In this study, we examined T cell profiles and functions in the lung tissues of NSCLC patients with or without ILD to provide evidence for the potential mechanism of immune checkpoint inhibitor (ICI)-related pneumonitis in NSCLC patients with ILD. Material and MethodsWe investigated T cell immunity in the lung tissues of NSCLC patients with ILD to support the application of immunotherapy for these patients. We analyzed T cell profiles and functions in surgically resected lung tissues from NSCLC patients with and without ILD. The T cell profiles of infiltrating cells in lung tissues were analyzed by flow cytometry. T cell functions were measured based on cytokine production by T cells stimulated with phorbol 12-myristate 13-acetate and ionomycin. ResultsThe percentages of CD4+ T cells expressing immune checkpoint molecules (Tim-3, ICOS, and 4-1BB), CD103+CD8+ T cells, and regulatory T (Treg) cells were higher in NSCLC patients with than in those without ILD. A functional analysis of T cells in lung tissues indicated that CD103+CD8+ T cells positively correlated with IFNγ production, whereas Treg cells negatively correlated with IFNγ and TNFα production. Cytokine production by CD4+ and CD8+ T cells did not significantly differ between NSCLC patients with and without ILD, except for TNFα production by CD4+ T cells being lower in the former than in the latter. ConclusionIn NSCLC patients with ILD stable for surgery, T cells were active participants and balanced in part by Treg cells in lung tissues, suggesting the potential development of ICI-related pneumonitis in NSCLC patients with ILD.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines. Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Peribronchiolar Metaplasia: A Marker of Cigarette Smoke-Induced Small Airway Injury in a Rural Cohort.

Peribronchiolar metaplasia (PBM) is considered a reaction to injury characterized by the proliferation of bronchiolar epithelium into immediately adjacent alveolar walls. While an association of PBM with diffuse interstitial lung diseases has been recognized, the clinical significance of PBM remains uncertain. A cohort (n = 352) undergoing surgical resection of a lung nodule/mass in a rural area was retrospectively reviewed. Multivariate logistic regression analysis was performed to determine the association of PBM with clinical, physiological, radiographic, and histologic endpoints. In the total study cohort, 9.1% were observed to have PBM as a histologic finding in resected lung tissue (n = 32). All but one of these patients with PBM were ever-smokers with a median of 42 pack years. Clinical COPD was diagnosed in two-thirds of patients with PBM. Comorbid gastroesophageal reflux disease (GERD) was significantly associated with PBM. All patients with PBM demonstrated radiologic and histologic evidence of emphysema. Measures of pulmonary function were not impacted by PBM. Mortality was not associated with the histologic observation of PBM. In a logistic regression model, centrilobular-ground glass opacity interstitial lung abnormality and traction bronchiectasis on the CT scan of the chest and histologic evidence of fibrosis, desquamative interstitial pneumonia and anthracosis all strongly predicted PBM in the cohort. A constellation of radiologic and histologic smoking-related abnormalities predicted PBM in study cohort. This confirms a co-existence of lung tissue responses to smoking including PBM, emphysema, and fibrosis. Acknowledging the physiologically "silent" nature of small airway dysfunction on pulmonary function testing, our findings support PBM as a histologic marker of small-airway injury associated with cigarette smoking.

Mass cytometry to characterize the immune lung cancer microenvironment.

The human tumor microenvironment requires use of high-dimensional single-cell tools to uncover its cellular complexity and functional variety. For decades, flow cytometry has been the technology of choice to explore immune cell diversity in different pathological contexts. Recently, a new format for flow cytometry - termed mass cytometry - has been developed. It allows for simultaneous interrogation of more than 40 different molecular markers, without the need for spectral compensation, which significantly augments the ability of cytometry to evaluate complex cellular systems and processes. Currently, different multiparametric single-cell analysis approaches are being widely adopted to interrogate the human tumor microenvironment. However, important challenges must be addressed when solid tissues are analyzed by single-cell technologies. This protocol describes the challenge and better use of single-cell mass cytometry to dissect tumor infiltrating leukocytes from surgically resected tumoral lung tissues.

Integrated spatial analysis of gene mutation and gene expression for understanding tumor diversity in formalin-fixed paraffin-embedded lung adenocarcinoma.

A deeper understanding of intratumoral heterogeneity is essential for prognosis prediction or accurate treatment plan decisions in clinical practice. However, due to the cross-links and degradation of biomolecules within formalin-fixed paraffin-embedded (FFPE) specimens, it is challenging to analyze them. In this study, we aimed to optimize the simultaneous extraction of mRNA and DNA from microdissected FFPE tissues (φ = 100 µm) and apply the method to analyze tumor diversity in lung adenocarcinoma before and after erlotinib administration. Two magnetic beads were used for the simultaneous extraction of mRNA and DNA. The decross-linking conditions were evaluated for gene mutation and gene expression analyses of microdissected FFPE tissues. Lung lymph nodes before treatment and lung adenocarcinoma after erlotinib administration were collected from the same patient and were preserved as FFPE specimens for 4 years. Gene expression and gene mutations between histologically classified regions of lung adenocarcinoma (pre-treatment tumor in lung lymph node biopsies and post-treatment tumor, normal lung, tumor stroma, and remission stroma, in resected lung tissue) were compared in a microdissection-based approach. Using the optimized simultaneous extraction of DNA and mRNA and whole-genome amplification, we detected approximately 4,000-10,000 expressed genes and the epidermal growth factor receptor (EGFR) driver gene mutations from microdissected FFPE tissues. We found the differences in the highly expressed cancer-associated genes and the positive rate of EGFR exon 19 deletions among the tumor before and after treatment and tumor stroma, even though they were collected from tumors of the same patient or close regions of the same specimen. Our integrated spatial analysis method would be applied to various FFPE pathology specimens providing area-specific gene expression and gene mutation information.

New and effective EGFR-targeted fluorescence imaging technology for intraoperative rapid determination of lung cancer in freshly isolated tissue.

During lung cancer surgery, it is very important to define tumor boundaries and determine the surgical margin distance. In previous research, systemically application of fluorescent probes can help medical professionals determine the boundaries of tumors and find small tumors and metastases, thereby improving the accuracy of surgical resection. However, there are very few safe and effective probes that can be applied to clinical trials up to now, which limits the clinical application of fluorescence imaging. Here we developed a new technology that can quickly identify the tumor area in the resected lung tissue during the operation and distinguish the tumor boundary and metastatic lymph nodes. For animal studies, a PDX model of lung cancer was established. The tumors, lungs, and peritumoral muscle tissues of tumor-bearing mice were surgically removed and incubated with a probe targeting epidermal growth factor receptor (EGFR) for 20min, and then imaged by a closed-field near-infrared two-zone (NIR-II) fluorescence imaging system. For clinical samples, ten surgically removed lung tissues and 60 lymph nodes from 10 lung cancer patients undergoing radical resection were incubated with the targeting probe immediately after intraoperative resection and imaged to identify the tumor area and distinguish the tumor boundary and metastatic lymph nodes. The accuracy of fluorescence imaging was confirmed by HE staining and immunohistochemistry. The ex vivo animal imaging experiments showed a fluorescence enhancement of tumor tissue. For clinical samples, our results showed that this new technology yielded more than 85.7% sensitivity and 100% specificity in identifying the tumor area in the resected lung tissue. The average fluorescence tumor-to-background ratio was 2.5 ± 1.3. Furthermore, we also used this technique to image metastatic lymph nodes intraoperatively and showed that metastatic lymph nodes have brighter fluorescence than normal lymph nodes, as the average fluorescence tumor-to-background signal ratio was 2.7 ± 1.1. Calculations on the results of the fluorescence signal in relation to the number of metastatic lymph nodes yielded values of 77.8% for sensitivity and 92.1% for specificity. We expect this new technology to be a useful diagnostic tool for rapid intraoperative pathological detection and margin determination. By using fluorescently labeled anti-human EGFR recombinant antibody scFv fragment to incubate freshly isolated tissues during surgery, the probes can quickly accumulate in lung cancer tissues, which can be used to quickly identify tumor areas in the resected lung tissues and distinguish tumor boundaries and find metastases in lymph nodes. This technology is expected to be used for rapid intraoperative pathological detection and margin determination.

The Cole Relaxation Frequency as a Parameter to Identify Cancer in Lung Tissue: Preliminary Animal and Ex Vivo Patient Studies.

Lung cancer is the world's leading cause of cancer deaths, and diagnosis remains challenging. Lung cancer starts as small nodules; early and accurate diagnosis allows timely surgical resection of malignant nodules while avoiding unnecessary surgery in patients with benign nodules. The Cole relaxation frequency (CRF) is a derived electrical bioimpedance signature, which may be utilized to distinguish cancerous tissues from normal tissues. Human testing ex vivo was conducted with NoduleScan in freshly resected lung tissue from 30 volunteer patients undergoing resection for nonsmall cell lung cancer. The CRF of the tumor and the distant normal lung tissue relative to the tumor were compared to histopathology specimens to establish a potential algorithm for point-of-care diagnosis. For animal testing in vivo, 20 mice were implanted with xenograft human lung cancer tumor cells injected subcutaneously into the right flank of each mouse. Spectral impedance measurements were taken on the tumors on live animals transcutaneously and on the tumors after euthanasia. These CRF measurements were compared to healthy mouse lung tissue. For porcine lung testing ex vivo, porcine lungs were received with the trachea. After removal of the vocal box, a ventilator was attached to pressurize the lung and simulate breathing. At different locations of the lobes, the lung's surface was cut to produce a pocket that could accommodate tumors obtained from in vivo animal testing. The tumors were placed in the subsurface of the lung, and the electrode was placed on top of the lung surface directly over the tumor but with lung tissue between the tumor and the electrode. Spectral impedance measurements were taken when the lungs were in the deflated state, inflated state, and also during the inflation-deflation process to simulate breathing. Among 60 specimens evaluated in 30 patients, NoduleScan allowed ready discrimination in patients with clear separation of CRF in tumor and distant normal tissue with a high degree of sensitivity (97%) and specificity (87%). In the 25 xenograft small animal model specimens measured, the CRF aligns with the separation observed in the human in vivo measurements. The CRF was successfully measured of tumors implanted into ex vivo porcine lungs, and CRF measurements aligned with previous tests for pressurized and unpressurized lungs. As previously shown in breast tissue, CRF in the range of 1kHz-10MHz was able to distinguish nonsmall cell lung cancer versus normal tissue. Further, as evidenced by in vivo small animal studies, perfused tumors have the same CRF signature as shown in breast tissue and human ex vivo testing. Inflation and deflation of the lung have no effect on the CRF signature. With additional development, CRF derived from spectral impedance measurements may permit point-of-care diagnosis guiding surgical resection.

Angiotensin-Converting Enzyme 2 (ACE2), Transmembrane Peptidase Serine 2 (TMPRSS2), and Furin Expression Increases in the Lungs of Patients with Idiopathic Pulmonary Fibrosis (IPF) and Lymphangioleiomyomatosis (LAM): Implications for SARS-CoV-2 (COVID-19) Infections.

We previously reported higher ACE2 levels in smokers and patients with COPD. The current study investigates if patients with interstitial lung diseases (ILDs) such as IPF and LAM have elevated ACE2, TMPRSS2, and Furin levels, increasing their risk for SARS-CoV-2 infection and development of COVID-19. Surgically resected lung tissue from IPF, LAM patients, and healthy controls (HC) was immunostained for ACE2, TMPRSS2, and Furin. Percentage ACE2, TMPRSS2, and Furin expression was measured in small airway epithelium (SAE) and alveolar areas using computer-assisted Image-Pro Plus 7.0 software. IPF and LAM tissue was also immunostained for myofibroblast marker α-smooth muscle actin (α-SMA) and growth factor transforming growth factor beta1 (TGF-β1). Compared to HC, ACE2, TMPRSS2 and Furin expression were significantly upregulated in the SAE of IPF (p < 0.01) and LAM (p < 0.001) patients, and in the alveolar areas of IPF (p < 0.001) and LAM (p < 0.01). There was a significant positive correlation between smoking history and ACE2 expression in the IPF cohort for SAE (r = 0.812, p < 0.05) and alveolar areas (r = 0.941, p < 0.01). This, to our knowledge, is the first study to compare ACE2, TMPRSS2, and Furin expression in patients with IPF and LAM compared to HC. Descriptive images show that α-SMA and TGF-β1 increase in the IPF and LAM tissue. Our data suggests that patients with ILDs are at a higher risk of developing severe COVID-19 infection and post-COVID-19 interstitial pulmonary fibrosis. Growth factors secreted by the myofibroblasts, and surrounding tissue could further affect COVID-19 adhesion proteins/cofactors and post-COVID-19 interstitial pulmonary fibrosis. Smoking seems to be the major driving factor in patients with IPF.

SARS-CoV-2 (COVID-19) Adhesion Site Protein Upregulation in Small Airways, Type 2 Pneumocytes, and Alveolar Macrophages of Smokers and COPD - Possible Implications for Interstitial Fibrosis.

BackgroundSmokers and patients with COPD are highly susceptible to SARS-CoV-2 infection, leading to severe COVID-19.MethodsThis cross-sectional study involved resected lung tissues from 16 patients with GOLD stage I or II COPD; of which 8 were current smokers COPD (COPD-CS), and 8 ex-smokers COPD (COPD-ES), 7 normal lung function smokers (NLFS), 9 patients with small airways disease (SAD), and 10 were never-smoking normal controls (NC). Immunostaining for ACE2, Furin, and TMPRSS2 was performed and analysed for percent expression in small airway epithelium (SAE) and counts for positively and negatively stained type 2 pneumocytes and alveolar macrophages (AMs) were done using Image ProPlus V7.0. Furthermore, primary small airway epithelial cells (pSAEC) were analysed by immunofluorescence after exposure to cigarette smoke extract (CSE).ResultsACE2, Furin, and TMPRSS2 expression significantly increased in SAE and type 2 pneumocytes in all the subjects (except Furin for NLFS) compared to NC (p < 0.001). Similar significance was observed for ACE2 positive AM (p < 0.002), except COPD-ES, which decreased in ACE2 positive AMs (p < 0.003). Total type 2 pneumocytes and AMs significantly increased in the pathological groups compared to NC (p < 0.01), except SAD (p = 0.08). However, AMs are significantly reduced in COPD-ES (p < 0.003). Significant changes were observed for tissue co-expression of Furin and TMPRSS2 with ACE2 in SAE, type 2 pneumocytes and AMs. These markers also negatively correlated with lung function parameters, such as FEV1/FVC % predicted, FEF25-75%, DLCO% predicted. A strong co-localisation and expression for ACE2 (p < 0.0001), Furin (p < 0.01), and TMPRSS2 (p < 0.0001) was observed in pSAEC treated with 1% CSE than controls.DiscussionThe increased expression of ACE2, TMPRSS2 and Furin, in the SAE, type 2 pneumocytes and AMs of smokers and COPD are detrimental to lung function and proves that these patient groups could be more susceptible to severe COVID-19 infection. Increased type 2 pneumocytes suggest that these patients are vulnerable to developing post-COVID-19 interstitial pulmonary fibrosis or fibrosis in general. There could be a silently developing interstitial pathology in smokers and patients with COPD. This is the first comprehensive study to report such changes.

Epigenetic imprinting alterations as effective diagnostic biomarkers for early-stage lung cancer and small pulmonary nodules

BackgroundEarly lung cancer detection remains a clinical challenge for standard diagnostic biopsies due to insufficient tumor morphological evidence. As epigenetic alterations precede morphological changes, expression alterations of certain imprinted genes could serve as actionable diagnostic biomarkers for malignant lung lesions.ResultsUsing the previously established quantitative chromogenic imprinted gene in situ hybridization (QCIGISH) method, elevated aberrant allelic expression of imprinted genes GNAS, GRB10, SNRPN and HM13 was observed in lung cancers over benign lesions and normal controls, which were pathologically confirmed among histologically stained normal, paracancerous and malignant tissue sections. Based on the differential imprinting signatures, a diagnostic grading model was built on 246 formalin-fixed and paraffin-embedded (FFPE) surgically resected lung tissue specimens, tested against 30 lung cytology and small biopsy specimens, and blindly validated in an independent cohort of 155 patients. The QCIGISH diagnostic model demonstrated 99.1% sensitivity (95% CI 97.5–100.0%) and 92.1% specificity (95% CI 83.5–100.0%) in the blinded validation set. Of particular importance, QCIGISH achieved 97.1% sensitivity (95% CI 91.6–100.0%) for carcinoma in situ to stage IB cancers with 100% sensitivity and 91.7% specificity (95% CI 76.0–100.0%) noted for pulmonary nodules with diameters ≤ 2 cm.ConclusionsOur findings demonstrated the diagnostic value of epigenetic imprinting alterations as highly accurate translational biomarkers for a more definitive diagnosis of suspicious lung lesions.

STAT3 and IL-6 Contribute to Corticosteroid Resistance in an OVA and Ozone-induced Asthma Model with Neutrophil Infiltration.

Exposure to high levels of ozone contributes to insensitivity to glucocorticoids in asthma treatment, but the underlying mechanisms are not known. We built two asthma models: a “T2-high” asthma model was established by ovalbumin (OVA) sensitization/challenge and OVA sensitization/challenge combined with ozone exposure (OVA + ozone) was used to induce airway inflammation with increased numbers of neutrophils to simulate “T2-low” asthma. The expression of T-helper (Th)1/2/17-related cytokines was measured by cytokine antibody arrays. Bronchial provocation tests were carried out to evaluate the lung resistance of mice. Hematoxylin and eosin staining, periodic acid-Schiff staining, and immunohistochemical (IHC) analyses of alpha-smooth muscle actin were undertaken to observe morphology changes in lungs. The expression of glucocorticoid receptors (GRs) and phosphorylated-GR (p-GR) was measured by western blotting. Nr3c1 mRNA was quantified by RT-qPCR. Protein expression of proinflammatory cytokines, signal transducer and activator of transcription 3 (STAT3), suppressor of cytokine signaling 3 (SOCS3), and CXCL1 was measured through ELISAs, western blotting, or IHC analyses. Resected lung tissue from seven asthma patients and 10 healthy controls undergoing thoracotomy for pulmonary nodules was evaluated by IHC analyses and ELISAs. In both asthma models, mucus hypersecretion, as well as inflammation, hyperresponsiveness, and remodeling of the airways, was present compared with the control group, whereas the OVA + ozone group showed severe neutrophil infiltration. The expression of Th17-related cytokines (interleukin (IL)-6, IL-17A, IL-21), GR protein, and CXCL1 increased in the OVA + ozone group, whereas the expression of p-GR decreased. Dexamethasone (Dex) could not totally reverse the expression of p-GR and histone deacetylase-2 in the OVA + ozone group. STAT3 expression increased in the OVA + ozone group and could not be completely reversed by Dex, and nor could IL-6 expression. A positive correlation between IL-6 or IL-17A and STAT3 and negative correlation between SOCS3 and STAT3 were shown, suggesting that the IL-6/STAT3 pathway may be involved in OVA + ozone–induced corticosteroid-resistant airway inflammation. In clinical samples, IL-17A expression in lung tissue was positively correlated with percent STAT3-positive area and negatively correlated with SOCS3 expression. The IL-6/STAT3 pathway may contribute to corticosteroid insensitivity in OVA + ozone–induced neutrophilic airway inflammation through regulation of Th17 cells and could provide new targets for individual treatment of corticosteroid resistance in asthma.

Serum S100 calcium-binding protein A4 as a novel predictive marker of acute exacerbation of interstitial pneumonia after surgery for lung cancer

BackgroundAcute exacerbation (AE) of interstitial pneumonia (IP) is the most fatal complication after lung resection for lung cancer. To improve the prognosis of lung cancer with IP, the risk factors of AE of IP after lung resection should be assessed. S100 calcium-binding protein A4 (S100A4) is a member of the S100 family of proteins and is a known marker of tissue fibrosis. We examined the usefulness of S100A4 in predicting AE of IP after lung resection for lung cancer.MethodsThis study included 162 patients with IP findings on preoperative high-resolution computed tomography scan who underwent curative-intent lung resection for primary lung cancer between April 2007 and March 2019. Serum samples were collected preoperatively. Resected lung tissue from 76 patients exhibited usual IP (UIP) pattern in resected lung were performed immunohistochemistry (IHC). Relationship between S100A4 and the incidence of AE of IP and short-term mortality was analyzed.ResultsThe receiver operating characteristic area under the curve for serum S100A4 to predict postoperative AE of IP was 0.871 (95% confidence interval [CI], 0.799–0.943; P < 0.001), with a sensitivity of 93.8% and a specificity of 75.3% at the cutoff value of 17.13 ng/mL. Multivariable analysis revealed that a high serum S100A4 level (> 17.13 ng/mL) was a significant risk factor for AE of IP (odds ratio, 42.28; 95% CI, 3.98–449.29; P = 0.002). A 1-year overall survival (OS) was significantly shorter in patients with high serum levels of S100A4 (75.3%) than in those with low serum levels (92.3%; P = 0.003). IHC staining revealed that fibroblasts, lymphocytes, and macrophages expressed S100A4 in the UIP area, and the stroma and fibrosis in the primary tumor expressed S100A4, whereas tumor cells did not.ConclusionsSerum S100A4 had a high predictive value for postoperative AE of IP and short-term mortality after lung resection.

Biomarker Potential of Preoperative Tumor Size in Determination of the Lymphovascular Invasion in Squamous Cell Lung Cancer and Lung Adenocarcinoma.

Introduction:The invasion of blood and lymph vessels with tumor tissue represents a negative prognostic factor of the disease course in patients with non-small cell lung cancer.Aim:The aim of the study was to determine the marker value of a preoperatively determined size of pulmonary squamous cell carcinoma and adenocarcinoma and its impact on lymphovascular invasion (LVI) in resected lung tissue.Materials and Methods:The conducted observational cross-sectional study included 322 patients with a complete resection of confirmed squamous cell lung carcinoma and lung adenocarcinoma. Preoperative size and type of tumor were determined by a preoperative chest computed tomography scan and cytological/histological analysis of obtained samples, while LVI status was determined by pathohistological analysis of resected tumor lung tissue. Receiver operating characteristic (ROC) curve analysis was performed to assess whether tumor size could serve as a reliable marker for LVI. P < 0.05 was considered statically significant.Results:A statistically significant difference in the frequency of tumor size (P = 0.580) along with LVI (P = 0.656) was not established between the patients with squamous cell lung cancer and lung adenocarcinoma. A ratio between the size of lung adenocarcinoma and LVI status (P < 0.001) was determined as statistically significant, while such a difference was not established in squamous cell lung cancer (P = 0.052). The ROC analysis revealed that tumor size >39 mm in patients with lung adenocarcinoma has obtained a sensitivity of 70.8% and a specificity of 60.9% to differentiate patients with a LVI (areas under the curve [AUC] = 0.70; 95% CI 0.60‒0.79; P < 0.001). A tumor size >4.6 cm in patients with squamous cell lung cancer obtained a sensitivity of 56.5% and a specificity of 60.3% to differentiate patients with a LVI (AUC = 0.59; 95% CI 0.50‒0.67; P = 0.043).Conclusion:The preoperative size of lung adenocarcinoma could be an acceptable marker of LVI presence in resected lung tissue, while in the squamous cell lung cancer, a potential biomarker role of the preoperative size of the tumor was inadequate.