Christian Schulz

Expression and Release of Interleukin-8 by Human Bronchial Epithelial Cells from Patients with Chronic Obstructive Pulmonary Disease, Smokers, and Never-Smokers

Background: One of the consistently observed features in chronic obstructive pulmonary disease (COPD) are markedly increased neutrophils in the airways which are accompanied by increased levels of interleukin-8 (IL-8) in induced sputum and bronchoalveolar lavage fluid. To some extent, IL-8 may derive from bronchial epithelial cells since airway epithelium plays a crucial role in initiating and augmenting host defense mechanisms. Objectives: We hypothesized that a marked increase in bronchoepithelial IL-8 expression and release may be found in the airway epithelium of COPD patients compared to ‘healthy’ smokers and never-smokers. Methods: Primary bronchoepithelial cell cultures from COPD patients, smokers, and never-smokers were established. The unstimulated and TNFα-induced IL-8 release was measured by enzyme-linked immunosorbent assay. In addition, mRNA expression levels were quantified by means of reverse transcriptase polymerase chain reaction and light cycler measurements. Results: In subjects with COPD the constitutive and stimulated IL-8 release was significantly higher compared to ‘healthy’ smokers and control subjects, whereas no differences were seen between smokers and the control group. Quantitative assessment of transcript levels confirmed these data, displaying significantly higher mRNA levels in primary bronchial epithelial cells from COPD patients compared to controls (p < 0.05) in uninduced and stimulated conditions (p < 0.05). Conclusions: These results suggest that patients with chronic obstructive airflow limitation are characterized by a significant upregulation of bronchial epithelial IL-8 expression levels and secretion, indicating specific differences in epithelial cell activation in COPD patients compared to smokers and control subjects.

Preoperative Bronchoscopic Assessment of Airway Invasion by Esophageal Cancer: A Prospective Study

BACKGROUNDnBronchoscopy is frequently used to assess invasion of esophageal cancer into the tracheobronchial tree. Prospective studies evaluating the role of bronchoscopy in pretherapeutic staging of esophageal cancer are lacking.nnnSTUDY OBJECTIVESnTo evaluate the diagnostic utility of fiberoptic bronchoscopy for the assessment of airway involvement by esophageal carcinoma and its resectability.nnnPATIENTS AND METHODSnIn a prospective study, we analyzed 150 bronchoscopies in 116 consecutive patients with potentially operable esophageal carcinoma, and correlated the findings with other staging modalities, intraoperative evaluation, and histopathologic data.nnnRESULTSnOne unknown additional bronchial cancer was found. In 32% of bronchoscopies performed in patients with esophageal cancer located above the tracheal bifurcation, some macroscopic abnormality was detected in the trachea and main bronchi, with mobile protrusion of the posterior tracheal wall being the most frequent abnormality (20.7%). When compared with histologic results, normal macroscopic appearance of the trachea and main bronchi had a negative predictive value of 98.5%, but the positive predictive value of all macroscopic abnormalities for the diagnosis of airway involvement was low, particularly after radiation therapy. The overall accuracy of bronchoscopy with multiple brush cytology and biopsy sampling in proving or excluding airway invasion in patients with otherwise operable conditions was 95.8% (95% confidence interval, 88.3 to 99.1%). Bronchoscopy was the sole decisive staging procedure, resulting in exclusion from surgery because of airway invasion, in 9.7% of patients with otherwise potentially operable conditions. The results of bronchoscopy and CT were discordant in 40% of the patients; the specificity and positive predictive value were higher for bronchoscopy than for CT.nnnCONCLUSIONSnWhen performed as the last investigation in the staging workup, bronchoscopy with biopsy and brush cytology is a very accurate procedure in evaluating possible airway invasion of esophageal cancer; macroscopic findings alone are not reliable.

Tumour necrosis factor‐α induced CD70 and interleukin‐7R mRNA expression in BEAS‐2B cells

Over the past few years, evidence has emerged for the potential role of the human bronchial epithelial cell in the initiation and progress of inflammation of the airway. Thus, the aim of this study was to investigate the expression pattern of cytokines and immunomodulatory factors in the human bronchial epithelial cell. To elucidate this highly complex expression and regulation pattern, the simian virus‐40 transformed human bronchial-epithelial cell line BEAS‐2B was stimulated with human recombinant tumour necrosis factor (hrTNF)‐α (10u2005ng·mL−1 (specific activity, 2.86×107u2005U·mg−1)) and messenger ribonucleic acid (mRNA) expression pattern was analysed by complementary deoxyribonucleic acid (cDNA) array analysis. Among 375 arrayed cDNA clones, 173 (46%) were detected in BEAS‐2B cells. The levels of expression of 17 genes, including those of monocyte chemoattractant protein (MCP)‐1, intercellular adhesion molecule (ICAM)‐1, growth-related oncogene (GRO) α, β, γ, interleukin (IL)‐7 receptor, CD70, IL‐6, IL‐8, granulocyte-macrophage colony-stimulating factor (GM‐CSF) and regulated in activation, normal T‐cell expressed and secreted (RANTES) were elevated after TNF‐α stimulation. The differential character of 12 clones was further characterised and verified by real time polymerase chain reaction (PCR) analysis of total ribonucleic acid (RNA) isolated from BEAS‐2B cells after 4 or 16u2005h incubation with increasing TNF‐α concentrations (1u2005pg–10u2005ng·mL−1). The authors semiquantified concentration-dependent mRNA upregulation of cytokines and immunology factors identified in the array and could determine threshold values of mRNA increases at 10u2005pg·mL−1–1u2005ng·mL−1 TNF‐α by real-time PCR. For CD70 (CD27 ligand) and interleukin‐7 receptor, which to the best of the authors knowledge have not yet been described in the human bronchial epithelial cell, a rapid and continuous messenger ribonucleic acid increase induced by 100u2005pg·mL−1 tumour necrosis factor‐α after only 60–90u2005min was shown. A potential role for these genes in the inflammatory process in the human bronchial epithelial cell is proposed.

Upregulation of MCAM in primary bronchial epithelial cells from patients with COPD.

An increasing body of evidence indicates that the bronchial epithelium plays a crucial role in the pathophysiology of chronic obstructive pulmonary disease (COPD). The aim of this study was to identify new genes whose bronchoepithelial expression is specifically altered in COPD patients. Primary bronchial epithelial cell (PBEC) cultures were established from exsmokers with stable airflow limitation and never smokers. Complementary deoxyribonucleic acid array technology was used to investigate the differential expression of 847 cytokine and cytokine-related genes between the two groups. Statistical analysis was performed by means of significance analysis of microarrays and Bonferroni-corrected analysis of variance on ranks. Discriminant analysis and light cycler measurements as well as flow cytometry and Western blotting were used to confirm the significance of the array results at both the messenger ribonucleic acid (mRNA) and protein expression levels. With respect to array experiments, melanoma cell adhesion molecule (MCAM) was identified as the sole gene showing highly significant upregulation in PBECs from COPD patients compared to never smokers. Light cycler measurements confirmed these results, revealing a 2.9‐fold and 2.0‐fold increase in MCAM mRNA expression in COPD patients compared to nonsmokers and smokers, respectively. In addition, these differences are associated with higher median protein expression levels. These results strongly suggest involvement of melanoma cell adhesion molecule in the pathophysiology of the chronic airway inflammation seen in patients with chronic obstructive pulmonary disease.

NKG2D-dependent effector function of bronchial epithelium activated alloreactive T cells

Allogeneic haematopoietic stem cell transplantation (SCT) has emerged as a curative therapeutic option. However, the role of graft-versus-host disease in lung injury after SCT has yet to be determined. In the present study, primary bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used to investigate immune responses of allogeneic CD8+ T-cells directed against respiratory epithelial cells. Following stimulation with irradiated bronchial epithelial cells, CD8+ T-cells produced significant amounts of interferon-γ, upregulated alloantigen activation markers and proliferated highly compared with T-cells stimulated with interleukin-2 alone. Furthermore, cytotoxicity assays demonstrated that bronchial epithelial cell-specific and granzyme B-mediated cytolytic activity was induced in CD8+ T-cells. Generation of natural killer (NK) T-cells, NK-like T-cells, cytokine-induced killer cells or lymphokine-activated killer cells could be excluded by phenotyping, culture conditions and neglectable lytic activity following stimulation with interleukin-2 alone. Inhibition experiments showed that lysis of bronchial epithelial cells was not major histocompatibility complex-I restricted, but depended on NK group 2 member D signalling; a stimulatory receptor initially shown to be expressed on NK cells. The present data imply that the respiratory epithelium has an antigen presenting function and directly alloactivates cytotoxic CD8+ T-cells that show nonclassical effector function.

Clinical Investigations: Tumors and MassesPreoperative Bronchoscopic Assessment of Airway Invasion by Esophageal Cancer: A Prospective Study

BACKGROUNDnBronchoscopy is frequently used to assess invasion of esophageal cancer into the tracheobronchial tree. Prospective studies evaluating the role of bronchoscopy in pretherapeutic staging of esophageal cancer are lacking.nnnSTUDY OBJECTIVESnTo evaluate the diagnostic utility of fiberoptic bronchoscopy for the assessment of airway involvement by esophageal carcinoma and its resectability.nnnPATIENTS AND METHODSnIn a prospective study, we analyzed 150 bronchoscopies in 116 consecutive patients with potentially operable esophageal carcinoma, and correlated the findings with other staging modalities, intraoperative evaluation, and histopathologic data.nnnRESULTSnOne unknown additional bronchial cancer was found. In 32% of bronchoscopies performed in patients with esophageal cancer located above the tracheal bifurcation, some macroscopic abnormality was detected in the trachea and main bronchi, with mobile protrusion of the posterior tracheal wall being the most frequent abnormality (20.7%). When compared with histologic results, normal macroscopic appearance of the trachea and main bronchi had a negative predictive value of 98.5%, but the positive predictive value of all macroscopic abnormalities for the diagnosis of airway involvement was low, particularly after radiation therapy. The overall accuracy of bronchoscopy with multiple brush cytology and biopsy sampling in proving or excluding airway invasion in patients with otherwise operable conditions was 95.8% (95% confidence interval, 88.3 to 99.1%). Bronchoscopy was the sole decisive staging procedure, resulting in exclusion from surgery because of airway invasion, in 9.7% of patients with otherwise potentially operable conditions. The results of bronchoscopy and CT were discordant in 40% of the patients; the specificity and positive predictive value were higher for bronchoscopy than for CT.nnnCONCLUSIONSnWhen performed as the last investigation in the staging workup, bronchoscopy with biopsy and brush cytology is a very accurate procedure in evaluating possible airway invasion of esophageal cancer; macroscopic findings alone are not reliable.

Microsatellite analysis of pleural supernatants could increase sensitivity of pleural fluid cytology

Pleural effusions may result from various inflammatory, hemodynamic, or neoplastic conditions. A common diagnostic problem lies in distinguishing malignant from benign pleural effusions using routine cytological evaluation. We studied pleural fluid samples obtained from 14 patients with histologically confirmed malignancy and from 6 patients with benign pleural effusions using 12 microsatellite markers from 8 different chromosomal regions. Supernatants and cellular sediments of all 20 pleural fluid samples were analyzed. Routine cytological examination was 100% specific for malignancy but was only 57% sensitive. Microsatellite analyses of pleural fluid supernatants showed genetic alterations in tumor patients only. However, 50% of pleural effusions that were considered negative for malignancy by routine cytological analysis showed either loss of heterozygosity or microsatellite instability. The sensitivity of pleural fluid examination rose to 79% when routine cytological assessment was supplemented by molecular studies. Our data suggest that microsatellite analysis increases the sensitivity of cytological pleural fluid examination in assessing potential malignancy and that combining cytological and molecular methods may improve yield and certainty in diagnostically challenging cases.

Improved asthma control in patients with severe, persistent allergic asthma after 12 months of nightly temperature-controlled laminar airflow: an observational study with retrospective comparisons.

Introduction Continuous or episodic allergen exposure is a major risk factor of frequent symptoms and exacerbations for patients with allergic asthma. It has been shown that temperature-controlled laminar airflow (TLA) significantly reduced allergen exposure and airway inflammation and improved quality of life of patients with poorly controlled allergic asthma. Objective The objective was to evaluate the effects of nighttime TLA when used during real-life conditions for 12 consecutive months in addition to the patients’ regular medication. Methods This multicenter, pre- and postretrospective observational study included patients with inadequately controlled moderate-to-severe allergic asthma who received add-on treatment with TLA for 12 consecutive months. Data on medication use, asthma control, asthma symptoms, lung function, use of hospital resources, and exacerbations were collected after 4 and 12 months and compared with corresponding data collected retrospectively from medical records during the year prior to inclusion in the study. Results Data from 30 patients (mean age 28; range 8–70) completing 4 months and 27 patients completing 12 months of TLA use are presented. The mean number of exacerbations was reduced from 3.6 to 1.3 (p<0.0001), and the ratio of asthma-related emergency room visits or hospitalizations diminished from 72.4 to 23.3% (p=0.001) or from 44.8 to 20.0% (p<0.05), respectively, after 12 months of TLA use. The Asthma Control Test index increased from 14.1 to 18.5 (p<0.0001). After 4 months of TLA use, clear improvements can be shown for most variables in line with the data collected after 12 months. Conclusions The addition of TLA to the patients’ regular medication significantly reduced exacerbations, asthma symptoms, and the utilization of hospital resources. The data support that TLA may be an important new non-pharmacological approach in the management of poorly controlled allergic asthma.

Bronchoepithelial Expression of CXCR1 and CXCR2 Does Not Facilitate Transepithelial Migration of Neutrophils

Background: Neutrophilic airway inflammation is one of the key features of chronic obstructive pulmonary disease (COPD). The chemokine receptors 1 (CXCR1) and 2 (CXCR2) are expressed in the bronchial mucosa during chronic inflammation and might be of importance for transepithelial migration of neutrophils. Objectives: This study addressed the role of bronchoepithelial CXCR1 and CXCR2 expression with respect to transepithelial migration of neutrophils. Methods: Primary bronchial epithelial cells (PBECs) derived from COPD patients and healthy controls as well as transiently CXCR1- and CXCR2-transfected Calu-6 cells were used for transepithelial migration assays of neutrophils under various conditions. Epithelial CXCR1 and CXCR2 expression was verified by means of flow cytometry. Results: Transepithelial migration of neutrophils was significantly increased following lipopolysaccharide pretreatment of epithelial cells. Transient transfection of CXCR1 and CXCR2 neither augmented the transepithelial migration of neutrophils, nor did the selective blockade of CXCR1 and CXCR2 have any significant effect on neutrophilic transepithelial migration. In addition, no differences were found in PBECs and neutrophils derived from healthy controls and COPD patients. Conclusions: The data of the present study do not support the hypothesis that bronchoepithelial expression of CXCR1 and/or CXCR2 facilitate transepithelial migration of neutrophils.

Updating the EORTC questionnaire for assessing quality of life in lung cancer patients (EORTC QLQ-LC13)

Background: The EORTC QLQ-LC13 was the first module developed to be used in conjunction with the core questionnaire C30. Major advances have been made with regard to diagnostic and therapeutic options in lung cancer. Therefore, research objective was to develop a revised lung cancer module supported by the EORTC. Methods: EORTC employs a four phase methodology of modules development and Phases I to III have been completed. Phase I generates quality of life issues using a mix of sources. Phase II converts issues into questionnaire items. The Phase III study pre-tests a provisional module to evaluate the importance and acceptability of the items. Biometrical methods primarily included descriptive statistics and basic psychometric analyses. The 48 lung cancer items that emerged from Phases I to III were assessed against a set of pre-specified criteria comprised of patient ratings and distribution properties of the items. Results: 12 international centers participated in the Phase III study. 200 patients were enrolled. Mean age 64 years. 59% were male, 82% had NSCLC, 56% were treated with a palliative approach. Patients had to fill in the EORTC QLQ-C30 and a 48-item provisional lung cancer module and rated the lung cancer items. 29 lung cancer items met the pre-specified cut-off levels. Psychometric analyses suggested a five multi-item scale structure. Conclusions: The updated module retained 12 of the 13 original LC13 items. Further items were identified assessing side effects of targeted therapy, radiochemotherapy, and thoracic surgery. Phase IV will be a field study analyzing the proposed scale structure and assessing the psychometric properties of the module.