Removal of organic content from diesel exhaust particles alters cellular responses of primary human bronchial epithelial cells cultured at an air-liquid interface

Background Exposure to air pollutants, including diesel particulate matter, has been linked to adverse respiratory health effects. Inhaled diesel particulate matter contains adsorbed organic compounds. It is not clear whether the adsorbed organics or the residual components are more deleterious to airway cells. Using a physiologically relevant model, we investigated the role of diesel organic content on mediating cellular responses of primary human bronchial epithelial cells (HBECs) cultured at an air-liquid interface (ALI). Methods Primary HBECs were cultured and differentiated at ALI for at least 28 days. To determine which component is most harmful, we compared primary HBEC responses elicited by residual (with organics removed) diesel emissions (DE) to those elicited by neat (unmodified) DE for 30 and 60 minutes at ALI, with cigarette smoke condensate (CSC) as the positive control, and filtered air as negative control. Cell viability (WST-1 cell proliferation assay), inflammation (TNF-α, IL-6 and IL-8 ELISA) and changes in gene expression (qRT-PCR for HO-1, CYP1A1, TNF-α and IL-8 mRNA) were measured. Results Immunofluorescence and cytological staining confirmed the mucociliary phenotype of primary HBECs differentiated at ALI. Neat DE caused a comparable reduction in cell viability at 30 or 60 min exposures, whereas residual DE caused a greater reduction at 60 min. When corrected for cell viability, cytokine protein secretion for TNF-α, IL-6 and IL-8 were maximal with residual DE at 60 min. mRNA expression for HO-1, CYP1A1, TNF-α and IL-8 was not significantly different between exposures. Conclusion This study provides new insights into epithelial cell responses to diesel emissions using a physiologically relevant aerosol exposure model. Both the organic content and residual components of diesel emissions play an important role in determining bronchial epithelial cell response in vitro. Future studies should be directed at testing potentially useful interventions against the adverse health effects of air pollution exposure.

Promotion of a cancer-like phenotype, through chronic exposure to inflammatory cytokines and hypoxia in a bronchial epithelial cell line model

Globally, lung cancer accounts for approximately 20% of all cancer related deaths. Five-year survival is poor and rates have remained unchanged for the past four decades. There is an urgent need to identify markers of lung carcinogenesis and new targets for therapy. Given the recent successes of immune modulators in cancer therapy and the improved understanding of immune evasion by tumours, we sought to determine the carcinogenic impact of chronic TNF-α and IL-1β exposure in a normal bronchial epithelial cell line model. Following three months of culture in a chronic inflammatory environment under conditions of normoxia and hypoxia (0.5% oxygen), normal cells developed a number of key genotypic and phenotypic alterations. Important cellular features such as the proliferative, adhesive and invasive capacity of the normal cells were significantly amplified. In addition, gene expression profiles were altered in pathways associated with apoptosis, angiogenesis and invasion. The data generated in this study provides support that TNF-α, IL-1β and hypoxia promotes a neoplastic phenotype in normal bronchial epithelial cells. In turn these mediators may be of benefit for biomarker and/or immune-therapy target studies. This project provides an important inflammatory in vitro model for further immuno-oncology studies in the lung cancer setting.

Investigation of the interaction between non-small cell lung cancer cells and immortalised normal bronchial epithelial cells

Non-small cell lung cancer consists of a diverse range of molecular and pathological features. This may be due in part to the critical interaction between normal and lung cancer cells. Consequently resulting in ‘normal’ cells acting in a malignant fashion. This project aims to identify pathways responsible for this altered ‘normal’ behaviour.

A compact mock circulation loop for the in vitro testing of cardiovascular devices

In vitro cardiovascular device performance evaluation in a mock circulation loop (MCL) is a necessary step prior to in vivo testing.A MCL that accurately represents the physiology of the cardiovascular system accelerates the assessment of the device’s ability to treat pathological conditions. To serve this purpose, a compact MCL measuring 600 ¥ 600 ¥ 600 mm (L ¥ W¥ H) was constructed in conjunction with a computer mathematical simulation.This approach allowed the effective selection of physical loop characteristics, such as pneumatic drive parameters, to create pressure and flow, and pipe dimensions to replicate the resistance, compliance, and fluid inertia of the native cardiovascular system. The resulting five-element MCL reproduced the physiological hemodynamics of a healthy and failing heart by altering ventricle contractility, vascular resistance/compliance, heart rate, and vascular volume. The effects of interpatient anatomical variability, such as septal defects and valvular disease, were also assessed. Cardiovascular hemodynamic pressures (arterial, venous, atrial, ventricular), flows (systemic, bronchial, pulmonary), and volumes (ventricular, stroke) were analyzed in real time. The objective of this study is to describe the developmental stages of the compact MCL and demonstrate its value as a research tool for the accelerated development of cardiovascular devices.

Could BFFB mode breath aerosol play a role in H5N1 transmission?

Recent findings concerning exhaled aerosol size distributions and the regions in the respiratory tract in which they are generated could have significant implications for human to human spread of lower respiratory tract-specific infections. Even in healthy people, measurable quantities of aerosol are routinely generated from the Lower Respiratory Tract (LRT) during breathing(1-3). We have found that there at least three modes in the exhaled aerosol size distribution of healthy adults(4) (see Figure 1). These modes each have a characteristic size and arise from different parts of the respiratory tract. The respiratory bronchioles produce aerosol during breathing, the larynx during speech and the oral cavity also during speech. The model of the resulting droplet size distribution is therefore called the Bronchial Laryngeal Oral (B.L.O.) tri-modal model of expired aerosol.

Drugs and the respiratory system

17.1 Drugs for bronchial asthma and Chronic Obstructive Pulmonary Disease (COPD) 17.1.1 Introduction to asthma 17.1.2 Introduction to COPD 17.1.3 Drug delivery by inhalation 17.1.4 Drugs to treat 17.1.4.1 β2-adrenoceptor agonists 17.1.4.2 Muscarinic receptor antagonists 17.1.4.3 Leukotriene receptor antagonists 17.1.4.4 Theophylline 17.1.4.5 Oxygen for COPD 17.1.5 Drugs to prevent asthma 31.5.1 Glucocorticoids 31.5.2 Cromolyn sodium 17.1.6 Combination to treat and prevent asthma 17.1.7 Drug for allergic asthma – omalizumab 17.1.8 Emergency treatment of asthma 17.2. Expectorants, mucolytics, cough and oxygen 17.2.1 Introduction to expectorants and mucolytics 17.2.2 Expectorants 17.2.3 Mucolytics 17.2.4 Cough 17.2.5 Oxygen 17.3. Drugs for rhinitis and rhinorrea 17.3.1 Introduction 17.3.2 Histamine and H1-receptor antagonists 17.3.3 Sympathomimetic 17.3.4 Muscarinic receptor antagonists 17.3.4 Cromolyn sodium 17.3.5 Glucocorticoids

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Letter : anxiety score as a risk factor for radial artery vasospasm during radial interventions : a pilot study

I read with interest the article in Angiology that determined the role of anxiety level on radial artery spasm during transradial coronary angiography.1 As the importance of conducting more randomised controlled trials using anxiolytics to define the relation between anxiety and vasospasm was noted by the authors, I offer the following insights for investigators to consider when conducting such research. While previous research has already identified that moderate procedural sedation and opioid analgesia reduces the incidence of vasospasm,2 the identification of risk factors in the present study is hypothesis generating as to how outcomes might be even further improved. It is possible that selectively applying either even more intensive sedation and analgesia or complementary non-pharmacological stress-reducing therapies, such as music therapy or visualisation and attentive behaviour, to patients ‘at-risk’ of vasospasm (women and those with high levels of anxiety prior to the procedure) might lead to even better patient outcomes...

IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF

Background IL-20 is a pleiotrophic member of the IL-10 family and plays a role in skin biology and the development of haematopoietic cells. Recently, IL-20 has been demonstrated to have potential anti-angiogenic effects in non-small cell lung cancer (NSCLC) by down regulating COX-2. Methods The expression of IL-20 and its cognate receptors (IL-20RA/B and IL-22R1) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of this family was examined in normal bronchial epithelial and NSCLC cell lines. Furthermore, the effect of IL-20 on VEGF family members was examined. Results The expression of IL-20 and its receptors are frequently dysregulated in NSCLC. IL-20RB mRNA was significantly elevated in NSCLC tumours (p < 0.01). Protein levels of the receptors, IL-20RB and IL-22R1, were significantly increased (p < 0.01) in the tumours of NSCLC patients. IL-20 and its receptors were found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation. In addition, treatment with recombinant IL-20 resulted in decreased expression of the VEGF family members at the mRNA level. Conclusions This family of genes are dysregulated in NSCLC and are subject to epigenetic regulation. Whilst the anti-angiogenic properties of IL-20 require further clarification, targeting this family via epigenetic means may be a viable therapeutic option in lung cancer treatment. © 2011 Elsevier Ltd. All rights reserved.

Epigenetics underpinning the regulation of the CXC (ELR+) chemokines in non-small cell lung cancer

Background: Angiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR+) chemokine family are powerful promoters of the angiogenic response. Methods: The expression of the CXC (ELR+) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines. Results: Overall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line. Conclusions: CXC (ELR+) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment. © 2011 Baird et al.

EUELC project : a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular - pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARβ genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes. Copyright©ERS Journals Ltd 2009.

Epigenetic regulation of glucose transporters in non-small cell lung cancer

Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Somatostatin receptor expression in lung cancer

Experimental evidence suggests that somatostatin analogues may have a role to play in the management of lung tumours. We evaluated membrane preparations of nine small cell lung cancer (SCLC) cell lines and of tumour samples from 3 patients with non-small cell lung cancer (NSCLC), 1 patient with an atypical carcinoid and another with a bronchial carcinoid for the presence of specific binding sites for RC-160, a potent growth inhibitory octapeptide analogue of somatostatin. Specific binding was noted on six of nine SCLC lines. Radio-receptor assay on the cell line NCI H 69 showed evidence of two specific binding sites for RC-160, one with high affinity and the other with low affinity. Binding sites were also found on all five tumour samples. Scatchard analysis indicated the presence of a single class of receptors with high affinity in each case. Histological assessment of the resected specimens before binding assay showed them to be comprised of tumour cells and necrotic tissue, stroma and/or inflammatory cells. Therefore, the specific binding of RC-160 may be to tissues other than the tumour cells. In 3 patients, from whom the tumour samples were obtained, radiolabelled somatostatin analogue scintigraphy using [111In] pentetreotide was performed prior to surgery. In all cases, the radiolabel localised the disease. This study demonstrates the presence of specific binding sites for RC-160 in SCLC. Furthermore, the detection of specific binding in vitro and in vivo in NSCLC and intrapulmonary carcinoids demonstrates that these tumours contain cells which express specific binding sites for somatostatin. These results suggest that RC-160 may have a role toplay as a therapeutic agent in lung cancer.