Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT) studies.

Abstract Inhalation of ambient air particles or engineered nanoparticles (NP) handled as powders, dispersions or sprays in industrial processes and contained in consumer products pose a potential and largely unknown risk for incidental exposure. For efficient, economical and ethically sound evaluation of health hazards by inhaled nanomaterials, animal-free and realistic in vitro test systems are desirable. The new Nano Aerosol Chamber for in-vitro Toxicity studies (NACIVT) has been developed and fully characterized regarding its performance. NACIVT features a computer-controlled temperature and humidity conditioning, preventing cellular stress during exposure and allowing long-term exposures. Airborne NP are deposited out of a continuous air stream simultaneously on up to 24 cell cultures on Transwell® inserts, allowing high-throughput screening. In NACIVT, polystyrene as well as silver particles were deposited uniformly and efficiently on all 24 Transwell® inserts. Particle-cell interaction studies confirmed that deposited particles reach the cell surface and can be taken up by cells. As demonstrated in control experiments, there was no evidence for any adverse effects on human bronchial epithelial cells (BEAS-2B) due to the exposure treatment in NACIVT. The new, fully integrated and transportable deposition chamber NACIVT provides a promising tool for reliable, acute and sub-acute dose-response studies of (nano)particles in air-exposed tissues cultured at the air-liquid interface.

Responses of lung cells to realistic exposure of primary and aged carbonaceous aerosols

Diesel exhaust and wood burning are important sources of ambient atmospheric particles due to increasing numbers of diesel cars and the importance of wood as a source of renewable energy. Inhalation is the predominant route of entry and uptake for fine and ultrafine particles into the body. Health effects of atmospheric particles are still not completely understood. There is consistent evidence from epidemiology that particle exposure contributes to respiratory and cardiovascular diseases. This study aimed at examining acute responses of airway epithelial cells and luminal macrophages after exposure to freshly emitted and photochemically aged carbonaceous aerosols under realistic atmospheric conditions. In addition to a bronchial epithelial cell line advanced cell cultures namely fully differentiated respiratory epithelia and primary surface macrophages were used. Our results demonstrate that a single exposure of the cells to realistic particle doses of 0.3–3 ng diesel or 3–9 ng wood aerosol per cm2 cell surface induces small, particle-specific responses. The release of interleukin-6 and -8 was found to be decreased in differentiated airway epithelia but not in the other cell models studied. Aerosol exposure decreased macrophage phagocytic activity by 45–90%. Cell and tissue integrity remained unaffected. Overall, primary and aged particles from the same combustion induced similar responses in the cell models tested, whereby particles from diesel exhaust affected the cells more than those from wood combustion.

Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

The Pneumococcal Polysaccharide Capsule and Pneumolysin Differentially Affect CXCL8 and IL-6 Release from Cells of the Upper and Lower Respiratory Tract

The polysaccharide capsule and pneumolysin toxin are major virulence factors of the human bacterial pathogen Streptococcus pneumoniae. Colonization of the nasopharynx is asymptomatic but invasion of the lungs can result in invasive pneumonia. Here we show that the capsule suppresses the release of the pro-inflammatory cytokines CXCL8 (IL-8) and IL-6 from the human pharyngeal epithelial cell line Detroit 562. Release of both cytokines was much less from human bronchial epithelial cells (iHBEC) but levels were also affected by capsule. Pneumolysin stimulates CXCL8 release from both cell lines. Suppression of CXCL8 homologue (CXCL2/MIP-2) release by the capsule was also observed in vivo during intranasal colonization of mice but was only discernable in the absence of pneumolysin. When pneumococci were administered intranasally to mice in a model of long term, stable nasopharyngeal carriage, encapsulated S. pneumoniae remained in the nasopharynx whereas the nonencapsulated pneumococci disseminated into the lungs. Pneumococcal capsule plays a role not only in protection from phagocytosis but also in modulation of the pro-inflammatory immune response in the respiratory tract.

The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality.

Evaluation of respiratory function by barometric whole-body plethysmography in healthy dogs.

The objective of the present study was to assess the validity of barometric whole-body plethysmography (BWBP), to establish reference values, and to standardise a bronchoprovocative test to investigate airway responsiveness using BWBP in healthy dogs. BWBP measurements were obtained from six healthy beagle dogs using different protocols: (1) during three consecutive periods (3.5min each) in two morning and two evening sessions; (2) before and after administration of two protocols of sedation; (3) before and after nebulisation of saline and increasing concentrations of carbachol and histamine both in conscious dogs and in dogs under both protocols of sedation. Enhanced pause (PENH) was used as index of bronchoconstriction. Basal BWBP measurements were also obtained in 22 healthy dogs of different breeds, age and weight. No significant influence of either time spent in the chamber or daytime was found for most respiratory variables but a significant dog effect was detected for most variables. A significant body weight effect was found on tidal volume and peak flow values (P<0.05). Response to carbachol was not reproducible and always associated with side effects. Nebulisation of histamine induced a significant increase in respiratory rate, peak expiratory flow, peak expiratory flow/peak inspiratory flow ratio and PENH (P<0.05). The response was reproduced in each dog at different concentrations of histamine. Sedation with acepromazine+buprenorphine had little influence on basal measurements and did not change the results of histamine challenge. It was concluded that BWBP is a safe, non invasive and reliable technique of investigation of lung function in dogs which provides new opportunities to characterise respiratory status, to evaluate airway hyperresponsiveness and to assess therapeutic interventions.

Owner-reported coughing and nasal discharge are associated with clinical findings, arterial oxygen tension, mucus score and bronchoprovocation in horses with recurrent airway obstruction in a field setting.

REASONS FOR PERFORMING STUDY In clinical practice, veterinarians often depend on owner-reported signs to assess the clinical course of horses with recurrent airway obstruction (RAO). OBJECTIVES To test whether owner-reported information on frequency of coughing and observation of nasal discharge are associated with clinical, cytological and bronchoprovocation findings in RAO-affected horses in nonstandardised field conditions. STUDY DESIGN Cross-sectional study comparing healthy and RAO-affected horses. METHODS Twenty-eight healthy and 34 RAO-affected Swiss Warmblood horses were grouped according to owner-reported 'coughing frequency' and 'nasal discharge'. Differences between these groups were examined using clinical examination, blood gas analyses, endoscopic mucus scores, cytology of tracheobronchial secretion and bronchoalveolar lavage fluid, and airway hyperresponsiveness determined by plethysmography with histamine bronchoprovocation. RESULTS Frequently coughing horses differed most markedly from healthy control animals. Histamine bronchoprovocation-derived parameters were significantly different between the healthy control group and all RAO groups. Mucus grades and tracheobronchial secretion and bronchoalveolar lavage fluid neutrophil percentages had particularly high variability, with overlap of findings between groups. Owner satisfaction with the clinical status of the horse was high, even in severely affected horses. CONCLUSIONS Owner-reported coughing and nasal discharge are associated with specific clinical and diagnostic findings in RAO-affected horses in field settings. While airway hyperresponsiveness differentiates best between healthy horses and asymptomatic RAO-affected horses, the absence of coughing and nasal discharge does not rule out significant neutrophilic airway inflammation. Owner satisfaction with the clinical status of the horse was uninformative.

Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells.

Virus-associated pulmonary exacerbations, often associated with rhinoviruses (RVs), contribute to cystic fibrosis (CF) morbidity. Currently, there are only a few therapeutic options to treat virus-induced CF pulmonary exacerbations. The macrolide antibiotic azithromycin has antiviral properties in human bronchial epithelial cells. We investigated the potential of azithromycin to induce antiviral mechanisms in CF bronchial epithelial cells. Primary bronchial epithelial cells from CF and control children were infected with RV after azithromycin pre-treatment. Viral RNA, interferon (IFN), IFN-stimulated gene and pattern recognition receptor expression were measured by real-time quantitative PCR. Live virus shedding was assessed by assaying the 50% tissue culture infective dose. Pro-inflammatory cytokine and IFN-β production were evaluated by ELISA. Cell death was investigated by flow cytometry. RV replication was increased in CF compared with control cells. Azithromycin reduced RV replication seven-fold in CF cells without inducing cell death. Furthermore, azithromycin increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels. While stimulating antiviral responses, azithromycin did not prevent virus-induced pro-inflammatory responses. Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway. Clinical studies are needed to elucidate the potential of azithromycin in the management and prevention of RV-induced CF pulmonary exacerbations.

Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects.

PURPOSE Primary nasal epithelial cells are used for diagnostic purposes in clinical routine and have been shown to be good surrogate models for bronchial epithelial cells in studies of airway inflammation and remodeling. We aimed at comparing different instruments allowing isolation of nasal epithelial cells. METHODS Primary airway epithelial cell cultures were established using cells acquired from the inferior surface of the middle turbinate of both nostrils. Three different instruments to isolate nasal cells were used: homemade cytology brush, nasal swab, and curette. Cell count, viability, time until a confluent cell layer was reached, and success rate in establishing cell cultures were evaluated. A standard numeric pain intensity scale was used to assess the acceptability of each instrument. RESULTS Sixty healthy adults (median with interquartile range [IQR] age of 31 [26-37] years) participated in the study. Higher number of cells (×10(5) cells/ml) was obtained using brushes (9.8 [5.9-33.5]) compared to swabs (2.4 [1.5-3.9], p < 0.0001) and curettes (5.5 [4.4-6.9], p < 0.01). Cell viability was similar between groups. Cells obtained by brushes had the fastest growth rate, and the success rate in establishing primary cell cultures was highest with brushes (90% vs. 65% for swabs and 70% for curettes). Pain was highest with curettes (VAS score 4.0 [3.0-5.0] out of 10). The epithelial phenotype of the cultures was confirmed through cytokeratin and E-cadherin staining. CONCLUSIONS All three types of instruments allow collection and growth of human nasal epithelial cells with good acceptability to study participants. The most efficient instrument is the nasal brush.

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

A numerical model for inert gas transport in the lung based on fractal airway morphology

Patients suffering from cystic fibrosis (CF) show thick secretions, mucus plugging and bronchiectasis in bronchial and alveolar ducts. This results in substantial structural changes of the airway morphology and heterogeneous ventilation. Disease progression and treatment effects are monitored by so-called gas washout tests, where the change in concentration of an inert gas is measured over a single or multiple breaths. The result of the tests based on the profile of the measured concentration is a marker for the severity of the ventilation inhomogeneity strongly affected by the airway morphology. However, it is hard to localize underlying obstructions to specific parts of the airways, especially if occurring in the lung periphery. In order to support the analysis of lung function tests (e.g. multi-breath washout), we developed a numerical model of the entire airway tree, coupling a lumped parameter model for the lung ventilation with a 4th-order accurate finite difference model of a 1D advection-diffusion equation for the transport of an inert gas. The boundary conditions for the flow problem comprise the pressure and flow profile at the mouth, which is typically known from clinical washout tests. The natural asymmetry of the lung morphology is approximated by a generic, fractal, asymmetric branching scheme which we applied for the conducting airways. A conducting airway ends when its dimension falls below a predefined limit. A model acinus is then connected to each terminal airway. The morphology of an acinus unit comprises a network of expandable cells. A regional, linear constitutive law describes the pressure-volume relation between the pleural gap and the acinus. The cyclic expansion (breathing) of each acinus unit depends on the resistance of the feeding airway and on the flow resistance and stiffness of the cells themselves. Special care was taken in the development of a conservative numerical scheme for the gas transport across bifurcations, handling spatially and temporally varying advective and diffusive fluxes over a wide range of scales. Implicit time integration was applied to account for the numerical stiffness resulting from the discretized transport equation. Local or regional modification of the airway dimension, resistance or tissue stiffness are introduced to mimic pathological airway restrictions typical for CF. This leads to a more heterogeneous ventilation of the model lung. As a result the concentration in some distal parts of the lung model remains increased for a longer duration. The inert gas concentration at the mouth towards the end of the expirations is composed of gas from regions with very different washout efficiency. This results in a steeper slope of the corresponding part of the washout profile.

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic Aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment System varying particle number concentration independent of particle chemistry, and an aerosol Deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully differentiated HBE is most appropriate in future toxicity studies.

Pulmonary neuroendocrine (carcinoid) tumors: European Neuroendocrine Tumor Society expert consensus and recommendations for best practice for typical and atypical pulmonary carcinoids.

BACKGROUND Pulmonary carcinoids (PCs) are rare tumors. As there is a paucity of randomized studies, this expert consensus document represents an initiative by the European Neuroendocrine Tumor Society to provide guidance on their management. PATIENTS AND METHODS Bibliographical searches were carried out in PubMed for the terms 'pulmonary neuroendocrine tumors', 'bronchial neuroendocrine tumors', 'bronchial carcinoid tumors', 'pulmonary carcinoid', 'pulmonary typical/atypical carcinoid', and 'pulmonary carcinoid and diagnosis/treatment/epidemiology/prognosis'. A systematic review of the relevant literature was carried out, followed by expert review. RESULTS PCs are well-differentiated neuroendocrine tumors and include low- and intermediate-grade malignant tumors, i.e. typical (TC) and atypical carcinoid (AC), respectively. Contrast CT scan is the diagnostic gold standard for PCs, but pathology examination is mandatory for their correct classification. Somatostatin receptor imaging may visualize nearly 80% of the primary tumors and is most sensitive for metastatic disease. Plasma chromogranin A can be increased in PCs. Surgery is the treatment of choice for PCs with the aim of removing the tumor and preserving as much lung tissue as possible. Resection of metastases should be considered whenever possible with curative intent. Somatostatin analogs are the first-line treatment of carcinoid syndrome and may be considered as first-line systemic antiproliferative treatment in unresectable PCs, particularly of low-grade TC and AC. Locoregional or radiotargeted therapies should be considered for metastatic disease. Systemic chemotherapy is used for progressive PCs, although cytotoxic regimens have demonstrated limited effects with etoposide and platinum combination the most commonly used, however, temozolomide has shown most clinical benefit. CONCLUSIONS PCs are complex tumors which require a multidisciplinary approach and long-term follow-up.

Postmortem CT versus forensic autopsy: frequent discrepancies of tracheobronchial content findings.

In their daily forensic casework, the authors experienced discrepancies of tracheobronchial content findings between postmortem computed tomography (PMCT) and autopsy to an extent previously unnoticed in the literature. The goal of this study was to evaluate such discrepancies in routine forensic cases. A total of 327 cases that underwent PMCT prior to routine forensic autopsy were retrospectively evaluated for tracheal and bronchial contents according to PMCT and autopsy findings. Hounsfield unit (HU) values of tracheobronchial contents, causes of death, and presence of pulmonary edema were assessed in mismatching and matching cases. Comparing contents in PMCT and autopsy in each of the separately evaluated compartments of the respiratory tract low positive predictive values were assessed (trachea, 38.2 %; main bronchi, 40 %; peripheral bronchi, 69.1 %) indicating high discrepancy rates. The majority of tracheobronchial contents were viscous stomach contents in matching cases and low radiodensity materials (i.e., HU < 30) in mismatching cases. The majority of causes of death were cardiac related in the matching cases and skull/brain trauma in the mismatching cases. In mismatching cases, frequency of pulmonary edema was significantly higher than in matching cases. It can be concluded that discrepancies in tracheobronchial contents observed between PMCT and routine forensic autopsy occur in a considerable number of cases. Discrepancies may be explained by the runoff of contents via nose and mouth during external examination and the flow back of tracheal and main bronchial contents into the lungs caused by upright movement of the respiratory tract at autopsy.

Modifying Post-Operative Medical Care after EBV Implant May Reduce Pneumothorax Incidence.

OBJECTIVE Endoscopic lung volume reduction (ELVR) with valves has been shown to improve COPD patients with severe emphysema. However, a major complication is pneumothoraces, occurring typically soon after valve implantation, with severe consequences if not managed promptly. Based on the knowledge that strain activity is related to a higher risk of pneumothoraces, we asked whether modifying post-operative medical care with the inclusion of strict short-term limitation of strain activity is associated with a lower incidence of pneumothorax. METHODS Seventy-two (72) emphysematous patients without collateral ventilation were treated with bronchial valves and included in the study. Thirty-two (32) patients received standard post-implantation medical management (Standard Medical Care (SMC)), and 40 patients received a modified medical care that included an additional bed rest for 48 hours and cough suppression, as needed (Modified Medical Care (MMC)). RESULTS The baseline characteristics were similar for the two groups, except there were more males in the SMC cohort. Overall, ten pneumothoraces occurred up to four days after ELVR, eight pneumothoraces in the SMC, and only two in the MMC cohorts (p=0.02). Complicated pneumothoraces and pneumothoraces after upper lobe treatment were significantly lower in MMC (p=0.02). Major clinical outcomes showed no significant differences between the two cohorts. CONCLUSIONS In conclusion, modifying post-operative medical care to include bed rest for 48 hours after ELVR and cough suppression, if needed, might reduce the incidence of pneumothoraces. Prospective randomized studies with larger numbers of well-matched patients are needed to confirm the data.