The EvA study: aims and strategy.

The EvA study is a European Union-funded project under the Seventh Framework Programme (FP7), which aims at defining new markers for chronic obstructive pulmonary disease (COPD) and its subtypes. The acronym is derived from emphysema versus airway disease, indicating that the project targets these two main phenotypes of the disease. The EvA study is based on the concept that emphysema and airway disease are governed by different pathophysiological processes, are driven by different genes and have differential gene expression in the lung. To define these genes, patients and non-COPD controls are recruited for clinical examination, lung function analysis and computed tomography (CT) of the lung. CT scans are used to define the phenotypes based on lung density and airway wall thickness. This is followed by bronchoscopy in order to obtain samples from the airways and the alveoli. These tissue samples, along with blood samples, are then subjected to genome-wide expression and association analysis and markers linked to the phenotypes are identified. The population of the EvA study is different from other COPD study populations, since patients with current oral glucocorticoids, antibiotics and exacerbations or current smokers are excluded, such that the signals detected in the molecular analysis are due to the distinct inflammatory process of emphysema and airway disease in COPD.

Deposition, retention, and translocation of ultrafine particles from the central airways and lung periphery.

RATIONALE: Little is known about clearance of ultrafine carbon particles from the different regions of the human lung. These particles may accumulate and present a health hazard because of their high surface area. OBJECTIVES: Technetium Tc 99m ((99m)Tc)-radiolabeled 100-nm-diameter carbon particles were inhaled by healthy nonsmokers, asymptomatic smokers, and by patients with chronic obstructive pulmonary disease (COPD). METHODS: Using a bolus inhalation technique, particle deposition was targeted either to the airways or to the lung periphery, and retention, clearance, and translocation were measured using retained radiotracer imaging. MEASUREMENTS AND MAIN RESULTS: In vitro studies revealed that mean leaching of soluble (99m)Tc-radiotracer from the carbon particles was 4.1 (2.6 [SD]) % after 24 hours. Cumulative (99m)Tc activity in urine at 24 hours was 1.1 (1.3) % of activity deposited in the lungs. In the lung periphery, particle retention was not affected by smoking or pulmonary disease; retention was 96 (3) % after 24 hours. The small amount of clearance could be attributed to leaching of the (99m)Tc label, suggesting negligible particle clearance. In healthy nonsmokers, retention of particles targeted to the airways was 89 (6) and 75 (10) % after 1.5 and 24 hours, respectively. Radiolabel activity did not accumulate in the liver. CONCLUSIONS: Within the limits of detection of our experimental system, most inhaled ultrafine carbon particles are retained in the lung periphery and in the conducting airways without substantial systemic translocation or accumulation in the liver at 48 hours. Repeated exposure may result in significant pulmonary accumulation of ultrafine particles.

Ventilation and aerosolized drug delivery to the paranasal sinuses using pulsating airflow - a preliminary study.

BACKGROUND: Although there is a high incidence of nasal disorders including chronic sinusitis, there is limited success in the topical drug delivery to the nose and the paranasal sinuses. This is caused by the nose being an efficient filter for inhaled aerosol particles and the paranasal sinuses being virtually non-ventilated. METHOD: The objective of this study was to visualize the efficiency of sinus ventilation in healthy volunteers using dynamic 81mKr-gas imaging in combination with pulsating airflows. Furthermore, the deposition and retention of 99mTc-DTPA aerosol particles was assessed. RESULTS: The ventilation of the maxillary and frontal sinuses could be visualized by gamma camera imaging during pulsating airflow. In addition, using pulsating airflow, between 3% and 5% of nasally deposited aerosols penetrated into the paranasal sinuses while during application without pulsation aerosol deposition was below 1%. Furthermore pulsation increased aerosol deposition in the nasal airways by a factor of three. CONCLUSIONS: The study demonstrates the high efficiency of a pulsating airflow in paranasal sinus ventilation and aerosolized drug delivery. This proves that topical drug delivery to the paranasal sinuses in relevant quantities is possible and indicates further clinical studies are necessary.

Do genetic factors protect for early onset lung cancer? A case control study before the age of 50 years.

BACKGROUND: Early onset lung cancer shows some familial aggregation, pointing to a genetic predisposition. This study was set up to investigate the role of candidate genes in the susceptibility to lung cancer patients younger than 51 years at diagnosis. METHODS: 246 patients with a primary, histologically or cytologically confirmed neoplasm, recruited from 2000 to 2003 in major lung clinics across Germany, were matched to 223 unrelated healthy controls. 11 single nucleotide polymorphisms of genes with reported associations to lung cancer have been genotyped. RESULTS: Genetic associations or gene-smoking interactions was found for GPX1(Pro200Leu) and EPHX1(His113Tyr). Carriers of the Leu-allele of GPX1(Pro200Leu) showed a significant risk reduction of OR = 0.6 (95% CI: 0.4-0.8, p = 0.002) in general and of OR = 0.3 (95% CI:0.1-0.8, p = 0.012) within heavy smokers. We could also find a risk decreasing genetic effect for His-carriers of EPHX1(His113Tyr) for moderate smokers (OR = 0.2, 95% CI:0.1-0.7, p = 0.012). Considered both variants together, a monotone decrease of the OR was found for smokers (OR of 0.20; 95% CI: 0.07-0.60) for each protective allele. CONCLUSION: Smoking is the most important risk factor for young lung cancer patients. However, this study provides some support for the T-Allel of GPX1(Pro200Leu) and the C-Allele of EPHX1(His113Tyr) to play a protective role in early onset lung cancer susceptibility.

Mucociliary and long-term particle clearance in airways of patients with immotile cilia.

Spherical monodisperse ferromagnetic iron oxide particles of 1.9 microm geometric and 4.2 microm aerodynamic diameter were inhaled by seven patients with primary ciliary dyskinesia (PCD) using the shallow bolus technique, and compared to 13 healthy non-smokers (NS) from a previous study. The bolus penetration front depth was limiting to the phase1 dead space volume. In PCD patients deposition was 58+/-8 % after 8 s breath holding time. Particle retention was measured by the magnetopneumographic method over a period of nine months. Particle clearance from the airways showed a fast and a slow phase. In PCD patients airway clearance was retarded and prolonged, 42+/-12 % followed the fast phase with a mean half time of 16.8+/-8.6 hours. The remaining fraction was cleared slowly with a half time of 121+/-25 days. In healthy NS 49+/-9 % of particles were cleared in the fast phase with a mean half time of 3.0+/-1.6 hours, characteristic of an intact mucociliary clearance. There was no difference in the slow clearance phase between PCD patients and healthy NS. Despite non-functioning cilia the effectiveness of airway clearance in PCD patients is comparable to healthy NS, with a prolonged kinetics of one week, which may primarily reflect the effectiveness of cough clearance. This prolonged airway clearance allows longer residence times of bacteria and viruses in the airways and may be one reason for increased frequency of infections in PCD patients.

Liposomal methylprednisolone differentially regulates the expression of TNF and IL-10 in human alveolar macrophages.

Glucocorticoids (GC) are frequently used for therapy of various inflammatory lung diseases by either systemic or inhalative application. Because the oral application often has various side effects and because the inhalative application is not as potent, new formulations of GCs are required. We evaluated the effect of a liposomal (Lip) formulation of methylprednisolone (MP) on the expression of lipopolysaccharide (LPS)-induced proinflammatory tumor necrosis factor (TNF) and antiinflammatory interleukin-10 (IL-10) in human alveolar macrophages (AM). AM were obtained from bronchoalveolar lavage fluids of patients with various inflammatory lung diseases and precultured 20 h+/-MP, either liposomal or free, and then stimulated with LPS. Cells were harvested for analysis of mRNA levels by real-time reverse transcriptase polymerase chain reaction (RT-PCR); supernatants were used to measure protein concentrations by ELISA. We confirm the suppression of LPS-induced TNF production by an average of factor 7 at the mRNA level and factor 3 at the protein level. On the other hand, we detected a strong increase of the IL-10 production by MP. At the mRNA level, liposomal MP alone led to an 18-fold increase, and the LPS-induced IL-10 mRNA was enhanced by factor 2. At the protein level, MP alone had no effect, but LPS-induced IL-10 was increased by factor 2.5. Our data show that liposomal MP can consistently induce IL-10 and reduce TNF when macrophages are exposed for a prolonged period of time. In all respects, liposomal MP had similar activities as free MP, but liposomes were selectively taken up by monocytes and macrophages and not by lymphocytes in blood and in the lung. This suggests that liposomal glucocorticoids when applied locally in the lung may act efficiently but with less side effects.

Mucociliary and long-term particle clearance in the airways of healthy nonsmoker subjects.

Spherical monodisperse ferromagnetic iron oxide particles of 1.9-microm geometric and 4.2-microm aerodynamic diameter were inhaled by 13 healthy nonsmoking subjects using the shallow bolus technique. The bolus width was 100 ml, and the penetration front depth was 150 +/- 27 ml. The mean flow rate during inhalation and exhalation was 250 ml/s. The Fowler dead space and the phase 1 dead space of the airways were 282 +/- 49 and 164 +/- 34 ml, respectively. Deposition was below 20% without breath holding and 51 +/- 8% after an 8-s breath-holding time. We attempted to confine the bolus deposition to the bronchial airways by limiting the bolus front depth to the phase 1 dead space volume. Particle retention was measured by the magnetopneumographic method over a period of 9 mo. Particle clearance from the airways showed a fast and a slow phase; 49 +/- 9% followed the fast phase with a mean half-time of 3.0 +/- 1.6 h and characterized the mucociliary clearance. The remaining fraction was cleared slowly with a half-time of 109 +/- 78 days. The slow clearance phase was comparable to clearance measurements from the lung periphery of healthy nonsmokers, which allowed macrophage-dependent clearance mechanisms of the slow cleared fraction to be taken into account. Despite the fact that part of the slowly cleared particles may originate from peripheral deposition, the data demonstrate that mucociliary clearance does not remove all particles deposited in the airways and that a significant fraction undergoes long-term retention mechanisms, the origin of which is still under discussion.

[Pulmonary lymphangioleiomyomatosis as a rare cause of recurrent spontaneous pneumothorax]. / Pulmonale Lymphangioleiomyomatose als seltene Ursache rezidivierender Spontanpneumothoraces.

BACKGROUND: Pulmonary lymphangioleiomyomatosis is a rare disease that occurs mainly in women of reproductive age. The clinical characteristics include recurrent spontaneous pneumothorax and progressive dyspnea. The features of chest computed tomography are nearly pathognomonic with the detection of bilateral thin-walled cysts. CASE REPORT: A 32-year-old female presented with sudden-onset right-sided chest pain, which aggravated during inspiration, dyspnea at exertion, and cough. She had a history of bilateral recurrent spontaneous pneumothorax. Physical examination showed reduced pulmonary sounds on the right side. Chest X-ray confirmed the diagnosis of recurrent right-sided pneumothorax. Computed tomography showed multiple bilateral bullae. Video-assisted pleurectomy, bulla resection, and bulla coagulation were performed. The diagnosis of pulmonary lymphangioleiomyomatosis was confirmed by pulmonary biopsy. After pleurectomy, the patient remained symptom-free without recurrent pneumothorax. CONCLUSION: Pulmonary lymphangioleiomyomatosis is a rare cause of recurrent pneumothorax and should be considered a differential diagnosis, especially in young women with diffuse bilateral bullous emphysema or tuberous sclerosis.

Drug delivery to paranasal sinuses using pulsating aerosols.

Chronic rhinosinusitis (CRS) is the major disorder of the upper airways, affecting about 10-15% of the total population. Topical treatment regimens show only modest efficacy, because drug delivery to the posterior nose and paranasal sinuses is still a challenge. Therefore, there is a high rate of functional endoscopic sinus surgery in CRS patients. Most nasally administered aerosolized drugs, like nasal pump sprays, are efficiently filtered by the nasal valve and do not reach the posterior nasal cavity and the sinuses, which are poorly ventilated. However, as highlighted in this review, sinus ventilation and paranasal aerosol delivery can be achieved by using pulsating airflow, offering new topical treatment options for nasal disorders. Radioaerosol inhalation and imaging studies in nasal casts and in healthy volunteers have shown 4-6% of the nasally administered dose within the sinuses. In CRS patients, significant aerosol deposition in the sinus cavities was reported before sinus surgery. After surgery, deposition increased to the amount observed in healthy volunteers. In addition, compared with nasal pump sprays, retention kinetics of the radiolabel deposited in the nasal cavity was prolonged, both in healthy volunteers and in CRS patients. These efficiencies may be sufficient for topical aerosol therapies of sinus disorders and, due to the prolonged retention kinetics, may reduce application modes, but have to be proven in future clinical trials. Pulsating aerosols may offer additional new topical treatment options of nasal and sinus disorders before as well as after surgery.

Topical drug delivery in chronic rhinosinusitis patients before and after sinus surgery using pulsating aerosols.

OBJECTIVES: Chronic rhinosinusitis (CRS) is a common chronic disease of the upper airways and has considerable impact on quality of life. Topical delivery of drugs to the paranasal sinuses is challenging, therefore the rate of surgery is high. This study investigates the delivery efficiency of a pulsating aerosol in comparison to a nasal pump spray to the sinuses and the nose in healthy volunteers and in CRS patients before and after sinus surgery. METHODS: (99m)Tc-DTPA pulsating aerosols were applied in eleven CRSsNP patients without nasal polyps before and after sinus surgery. In addition, pulsating aerosols were studied in comparison to nasal pump sprays in eleven healthy volunteers. Total nasal and frontal, maxillary and sphenoidal sinus aerosol deposition and lung penetration were assessed by anterior and lateral planar gamma camera imaging. RESULTS: In healthy volunteers nasal pump sprays resulted in 100% nasal, non-significant sinus and lung deposition, while pulsating aerosols resulted 61.3+/-8.6% nasal deposition and 38.7% exit the other nostril. 9.7+/-2.0 % of the nasal dose penetrated into maxillary and sphenoidal sinuses. In CRS patients, total nasal deposition was 56.7+/-13.3% and 46.7+/-12.7% before and after sinus surgery, respectively (p<0.01). Accordingly, maxillary and sphenoidal sinus deposition was 4.8+/-2.2% and 8.2+/-3.8% of the nasal dose (p<0.01). Neither in healthy volunteers nor in CRS patients there was significant dose in the frontal sinuses. CONCLUSION: In contrast to nasal pump sprays, pulsating aerosols can deliver significant doses into posterior nasal spaces and paranasal sinuses, providing alternative therapy options before and after sinus surgery. Patients with chronic lung diseases based on clearance dysfunction may also benefit from pulsating aerosols, since these diseases also manifest in the upper airways.

A defect of CD16-positive monocytes can occur without disease.

The CD16-positive monocytes have been first described in 1988 but to date no selective defect in the number of these cells in blood has been reported. We now describe a family in which three of four siblings lack both CD16-positive monocyte subsets, i.e. the nonclassical and the intermediate monocytes. All three had CD16-positive monocytes of 2 cells/µl or less as compared to 52±18 cells/µl in healthy controls. The index case was affected by recurrent pleural effusion and infections and had evidence of an auto-inflammatory condition but no mutation of any of the relevant candidate genes. The other two siblings without CD16-positive monocytes were apparently healthy. There was no defect in serum M-CSF levels and no mutation in the M-CSF and M-CSFR genes. The data indicate that the absence of CD16-positive monocytes in blood does not lead to disease.

Pulsating aerosols for drug delivery to the sinuses in healthy volunteers.

OBJECTIVE: Approximately 10 to 15 percent of the European and U.S. population have chronic rhinosinusitis, but effective treatment remains a challenge. There has been limited success using topical drug delivery to the nose and the paranasal cavities/sinuses, in part because most nasally administered aerosol drug formulations are efficiently filtered at the nasal valve and fail to reach the osteomeatal area and sinuses. STUDY DESIGN: Feasibility study. SETTING: Nuclear medicine department. SUBJECTS AND METHODS: Pulsating airflows were applied to the nasal cavity and sinus ventilation was studied in five healthy human volunteers using dynamic (81m)Kr-gas gamma camera imaging. Furthermore, deposition and retention of (99m)Tc-DTPA radiolabeled aerosols delivered by nasal pump sprays or by pulsating aerosols was assessed in each volunteer over a 24-hour period. RESULTS: Only the pulsating airflow demonstrated efficient (81m)Kr-gas ventilation of the paranasal sinuses. No drug was deposited into the sinuses using nasal pump sprays, but up to 6.5 percent of the nasally administered drug was deposited into the sinuses using pulsating airflow. Clearance kinetics of the drug was reduced after pulsating aerosol delivery compared to nasal pump sprays. Residence time of the drug at the site of deposition was up to three-fold longer with pulsating aerosol delivery than with nasal pump sprays. CONCLUSION: Our data support the hypothesis that topical drug delivery in relevant quantities to the nose and osteomeatal areas, including the paranasal sinuses, is possible using pulsating airflows. Furthermore, the frequency of drug applications may be reduced due to a delayed clearance and longer residence time.

Motion and twisting of magnetic particles ingested by alveolar macrophages in the human lung: effect of smoking and disease.

BACKGROUND: Magnetic microparticles being ingested by alveolar macrophages can be used as a monitor for intracellular phagosome motions and cytoskeletal mechanical properties. These studies can be performed in the human lung after voluntary inhalation. The influence of cigarette smoking and lung diseases on cytoskeleton dependent functions was studied. METHODS: Spherical 1.3 microm diameter ferrimagnetic iron oxide particles were inhaled by 17 healthy volunteers (40-65 years), 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 18 patients with chronic obstructive bronchitis (COB). The retained particles were magnetized and aligned in an external 100 mT magnetic field. All magnetized particles induce a weak magnetic field of the lung, which was detected by a sensitive SQUID (superconducting quantum interference device) sensor. Cytoskeletal reorganizations within macrophages and intracellular transport cause stochastic magnetic dipole rotations, which are reflected in a decay of the magnetic lung field, called relaxation. Directed phagosome motion was induced in a weak magnetic twisting field. The resistance of the cytoplasm to particle twisting was characterized by the viscosity and the stiffness (ratio between stress to strain) of the cytoskeleton. RESULTS: One week after particle inhalation and later macrophage motility (relaxation) and cytoskeletal stiffness was not influenced by cigarette smoking, neither in healthy subjects, nor in the patients. Patients with IPF showed in tendency a faster relaxation (p = 0.06). Particle twisting revealed a non-Newtonian viscosity with a pure viscous and a viscoelastic compartment. The viscous shear was dominant, and only 27% of the shear recoiled and reflected viscoelastic properties. In patients with IPF, the stiffness was reduced by 60% (p < 0.02). An analysis of the shear rate and stress dependence of particle twisting allows correlating the rheological compartments to cytoskeletal subunits, in which microtubules mediate the pure viscous (non-recoverable) shear and microfilaments mediate the viscoelastic (recoverable) behavior. The missing correlation between relaxation and particle twisting shows that both stochastic and directed phagosome motion reflect different cytoskeletal mechanisms. CONCLUSION: Faster relaxation and a soft cytoskeleton in patients with IPF indicate alterations in cytoskeleton dependent functions of alveolar macrophages, which may cause dysfunction's in the alveolar defense, like a slower migration, a retarded phagocytosis, a disturbed phagosome lysosome fusion and an impaired clearance.