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Background: IL-13 is a key T(H)2 cytokine that is implicated in allergic responses., Objective: We evaluated the effects of an anti-IL-13-blocking antibody compared with placebo on repeated nasal allergen challenge responses in hay fever patients out of season., Methods: We performed a parallel group double-blind study of anti-IL-13 (single dose, 6 mg/kg intravenously, n = 16) and placebo (n = 15), with an additional open label group given a topical nasal corticosteroid (n = 5). Subjects received intranasal timothy grass pollen (Phleum pratense P5 allergen), and serial samples of nasal mucosal lining fluid were taken by using synthetic absorptive matrix and by nasal lavage., Results: Administration of anti-IL-13 on day 1 resulted in a significant decrease in IL-13 levels in synthetic absorptive matrix eluates compared with placebo (area under the curve 0-8 hours, change from baseline) during the late phase after nasal allergen challenge on day 5 (P < .05) and day 7 (P < .01). There were no apparent effects of anti-IL-13 treatment on nasal lavage eosinophil numbers or total nasal symptom scores versus placebo. However, in a subgroup with high late-phase IL-13 levels at screening, there was a trend for a decrease in total nasal symptom scores after nasal allergen challenge on day 5, when compared with subjects with low IL-13 levels (P < .10). Nasal fluticasone caused suppression of IL-13 (P < .05 on day 5) as well as IL-5 (P < .01 on day 5) levels in the late phase compared with placebo., Conclusions: Anti-IL-13 had specific pharmacodynamic action in this nasal allergen challenge model, causing profound inhibition of nasal lining fluid IL-13 responses. In addition, there was a possible effect of anti-IL-13 treatment on total nasal symptom scores in a subgroup with high late-phase nasal IL-13 levels at screening., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: The evaluation of exhaled breath profiles by electronic nose (eNose) is considered as a promising non-invasive diagnostic tool, and the discrimination of breathprints between patients with COPD and asthma has been reported. The aim of this study was to assess, whether exhaled breath profile analysis can detect the inflammatory airway response induced by ozone inhalation., Methods: In a randomized double-blind, cross-over study 14 healthy ozone-responsive subjects were exposed to 250 ppb ozone and filtered room air for 3h with intermittent exercise. Blood biomarkers, exhaled NO, exhaled CO, and breathprints (Cyranose 320(®)) were assessed prior and at 3 time points up to 24h post exposure. Induced sputum was collected at baseline and 3h post exposure. Multivariate analysis of eNose data was performed using transformed and normalized datasets., Results: Significantly increased numbers of sputum and blood neutrophils were observed after ozone, whereas the eNose signals showed no differences between exposures and no correlation with neutrophilic airway inflammation. However, independent of ozone exposure, sensor data correlated with serum SP-D levels and to a smaller extent with blood neutrophil numbers., Conclusions: Exhaled breath profiles as measured by the Cyranose 320(®) did not reflect airway responses to ozone. This suggests that exhaled volatiles did not change with ozone challenges or that the changes were below the detection limits. Conversely, the correlation of eNose signals with blood neutrophils and serum SP-D, i.e. markers of systemic inflammation and lung permeability, suggested that the Cyranose 320(®) can detect volatile organic compounds of systemic origin., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Ozone concentrations in ambient air are related to cardiopulmonary perturbations in the aging population. Increased central sympathetic nerve activity induced by local airway inflammation may be one possible mechanism., Methodology/principal Findings: To elucidate this issue further, we performed a randomized, double-blind, cross-over study, including 14 healthy subjects (3 females, age 22-47 years), who underwent a 3 h exposure with intermittent exercise to either ozone (250 ppb) or clean air. Induced sputum was collected 3 h after exposure. Nineteen to 22 hours after exposure, we recorded ECG, finger blood pressure, brachial blood pressure, respiration, cardiac output, and muscle sympathetic nerve activity (MSNA) at rest, during deep breathing, maximum-inspiratory breath hold, and a Valsalva maneuver. While the ozone exposure induced the expected airway inflammation, as indicated by a significant increase in sputum neutrophils, we did not detect a significant estimated treatment effect adjusted for period on cardiovascular measurements. Resting heart rate (clean air: 59±2, ozone 60±2 bpm), blood pressure (clean air: 121±3/71±2 mmHg; ozone: 121±2/71±2 mmHg), cardiac output (clean air: 7.42±0.29 mmHg; ozone: 7.98±0.60 l/min), and plasma norepinephrine levels (clean air: 213±21 pg/ml; ozone: 202±16 pg/ml), were similar on both study days. No difference of resting MSNA was observed between ozone and air exposure (air: 23±2, ozone: 23±2 bursts/min). Maximum MSNA obtained at the end of apnea (air: 44±4, ozone: 48±4 bursts/min) and during the phase II of the Valsalva maneuver (air: 64±5, ozone: 57±6 bursts/min) was similar., Conclusions/significance: Our study suggests that acute ozone-induced airway inflammation does not increase resting sympathetic nerve traffic in healthy subjects, an observation that is relevant for environmental health. However, we can not exclude that chronic airway inflammation may contribute to sympathetic activation.

Background: An environmental challenge chamber (ECC) is a useful tool to expose allergic patients to relevant allergens in a controlled indoor setting and to test anti-allergic treatment. Hitherto, ECC studies with grass pollen are conducted primarily outside of the pollen season to avoid the influence of natural pollen exposure., Objective: To investigate whether an established anti-allergic treatment, a combination of cetirizine (CET) and pseudoephedrine (PSE), shows an equivalent treatment effect within and outside of the grass pollen season when tested in an ECC., Methods: In a randomized, placebo-controlled, double-blind, four-way crossover study, the effect of a combination of 10 mg CET and 120 mg PSE compared with placebo on nasal symptoms, nasal flow, and nasal secretion was investigated in 70 patients with seasonal allergic rhinitis. Subjects underwent four 6-hour pollen challenges in an ECC with administration of the drugs after 2 hours. Two challenges were conducted within the grass pollen season and two out of the grass pollen season., Results: The active treatment significantly improved nasal symptoms and nasal flow and significantly reduced the amount of nasal secretion compared with placebo both within and outside of the pollen season (P < .0001 each). The treatment effect was not different between the seasons (P > .05)., Conclusion: Controlled allergen provocation in an ECC can be used to test anti-allergic treatment both within and outside of the grass pollen season., (Copyright © 2011 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear., Methods: In a cross-sectional study comprising non-smokers (n=10), young--(n=10), elderly smokers (n=20), and smokers with COPD (n=20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis., Results: In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p<0.01) and non-smokers (967(708) ng/ml; p<0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p<0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure., Conclusions: Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker., Trial Registration: no interventional trial.

Background: The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed., Methodology/principal Findings: Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh(-/-) mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh(-/-) mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh(+/+) and Ctsh(-/-) mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh(-/-) mice., Conclusions/significance: We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.

Background: Noninvasive monitoring of airway inflammation is important for diagnosis and treatment intervention of lung disease. Mediators of interest are often nonvolatile molecules that are exhaled as aerosols and captured by breath condensation. Because analysis of exhaled breath condensate has been troublesome in the past, partly due to poor standardization and unknown dilution, we investigated in detail the influence of respiratory variables on exhaled particle number and size distribution during tidal breathing in healthy volunteers., Methods: Particle number was detected by a condensation nuclei counter, and size distribution was determined by a laser spectrometer online with high time resolution while subjects underwent a defined protocol of normal and deep tidal breathing. Intra- and intersubject variability of particle emission was analyzed and physical properties of exhaled aerosols were correlated to pulmonary function variables obtained by body-plethysmography., Results: The particle size distribution was in the submicron range and stable during tidal breathing. Increasing tidal volumes dominantly influenced particle number emission while flow rates had only little effect. Reproducibility within subjects was high, but there was a large variation of particle emission between subjects. The ratio of functional residual capacity to total lung capacity was found to correlate with exhaled particle numbers. This indicates that particle generation is caused by reopening of terminal airways and is dependent on functional residual capacity., Conclusion: We conclude that online determination of exhaled aerosols from the human lungs is a prerequisite to standardize the assessment of nonvolatile mediators by normalization to the aerosol emission rate.

Pollen starch granules (PSGs) are allergen particles that get into contact with pulmonary surfactant and phagocytes in the terminal airways. In this study, the effects of surfactant protein D (SP-D) on the interaction of PSGs with phagocytes and on the pulmonary clearance of PSGs were determined. Fluorescently labeled PSGs were incubated in vitro with murine lung macrophages or dendritic cells (DCs) ± recombinant rat SP-D (rrSP-D). In addition, the effect of SP-D on uptake of PSGs by lung macrophages and DCs was studied in vivo. Furthermore, PSGs were instilled in Balb/c mice and the effects of SP-D on total lung clearance were assessed by optical imaging. SP-D treatment increased the number of PSG-positive macrophages and DCs in vitro. Furthermore, SP-D accelerated uptake/binding by alveolar macrophages and reduced the number of PSG-positive tissue macrophages and DCs at 24 hours. However, SP-D did not affect total lung clearance of PSGs and it did not enhance the T-cell proliferation induced by PSG-positive DCs. In conclusion, SP-D increased PSG-positive cells in vitro and accelerated PSG binding/uptake in vivo. The observed effects were limited to cellular clearance mechanisms and did not affect the total clearance of PSGs from the lung.

Prediction of lung innate immune responses is critical for developing new drugs. Well-established immune modulators like lipopolysaccharides (LPS) can elicit a wide range of immunological effects. They are involved in acute lung diseases such as infections or chronic airway diseases such as COPD. LPS has a strong adjuvant activity, but its pyrogenicity has precluded therapeutic use. The bacterial lipopeptide MALP-2 and its synthetic derivative BPPcysMPEG are better tolerated. We have compared the effects of LPS and BPPcysMPEG on the innate immune response in human precision-cut lung slices. Cytokine responses were quantified by ELISA, Luminex, and Meso Scale Discovery technology. The initial response to LPS and BPPcysMPEG was marked by coordinated and significant release of the mediators IL-1β, MIP-1β, and IL-10 in viable PCLS. Stimulation of lung tissue with BPPcysMPEG, however, induced a differential response. While LPS upregulated IFN-γ, BPPcysMPEG did not. This traces back to their signaling pathways via TLR4 and TLR2/6. The calculated exposure doses selected for LPS covered ranges occurring in clinical studies with human beings. Correlation of obtained data with data from human BAL fluid after segmental provocation with endotoxin showed highly comparable effects, resulting in a coefficient of correlation >0.9. Furthermore, we were interested in modulating the response to LPS. Using dexamethasone as an immunosuppressive drug for anti-inflammatory therapy, we found a significant reduction of GM-CSF, IL-1β, and IFN-γ. The PCLS-model offers the unique opportunity to test the efficacy and toxicity of biological agents intended for use by inhalation in a complex setting in humans., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Efficacy testing of drugs in seasonal allergic rhinitis (SAR) is often disturbed by seasonal variations of environmental allergens, and assessment of onset and duration of action is hardly possible under natural conditions. Allergen provocation in an environmental challenge chamber (ECC) can be of added value in this respect. However, the specificity, sensitivity, and reproducibility of outcome measures under both settings are unclear., Objective: The aim of this study was to investigate and compare the diagnostic value (specificity, sensitivity, and reproducibility) of clinical end-points and biomarkers both following allergen provocation in an ECC and under natural conditions., Methods: Sixty adult patients with SAR to grass and 60 healthy subjects were exposed twice to grass pollen in an ECC and observed twice during the pollen season. Symptoms, nasal flow, as well as exhaled and nasal nitric oxide (NO) were investigated., Results: The total nasal symptom score (TNSS) in the ECC had the best reproducibility (intraclass correlation coefficient ICC=0.86) and sensitivity/specificity [area under receiver operating characteristic curve (AUC)=0.99] of all measures. Symptoms in season also had good sensitivity/specificity but were far less reproducible. Nasal flow in the ECC had good sensitivity/specificity but reproducibility was limited. NO measurements showed good reproducibility but sensitivity/specificity were limited, except for exhaled NO in season (AUC=0.75)., Conclusion: The high reproducibility and sensitivity/specificity in the ECC suggests that TNSS is a valuable outcome measure. While exhaled NO can be considered to monitor airway inflammation, nasal NO appears to be unspecific.

Background: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. Our previous work demonstrated that SP-D increases the uptake of SPP by alveolar macrophages. In the present study, we investigated the uptake of SPP in human primary epithelial cells and the potential modulation by SP-D. The patho-physiological consequence was evaluated by measurement of pro-inflammatory mediators., Methods: SPP were isolated from timothy grass and subsequently fluorescently labelled. Human primary bronchial epithelial cells were incubated with SPP or polystyrene particles (PP) in the presence and absence of surfactant protein D. In addition, different sizes and surface charges of the PP were studied. Particle uptake was evaluated by flow cytometry and confocal microscopy. Soluble mediators were measured by enzyme linked immunosorbent assay or bead array., Results: SPP were taken up by primary epithelial cells in a dose dependent manner. This uptake was coincided with secretion of Interleukin (IL)-8. SP-D increased the fraction of bronchial epithelial cells that bound SPP but not the fraction of cells that internalized SPP. SPP-induced secretion of IL-8 was further increased by SP-D. PP were bound and internalized by epithelial cells but this was not modulated by SP-D., Conclusions: Epithelial cells bind and internalize SPP and PP which leads to increased IL-8 secretion. SP-D promotes attachment of SPP to epithelial cells and may thus be involved in the inflammatory response to inhaled allergen.

Human polymorphonuclear PMN constitutively express the enzyme arginase I, which hydrolyzes arginine to ornithine and urea. This arginine consumption has been recognized as a key pathway of myeloid cell-mediated suppression of the adaptive immune system during inflammation, infection, and tumor growth. Eos granulocytes are crucial immunoregulatory and effector cells of allergic inflammation and infections with parasites and helminths and in a variety of tumors. Here, we analyzed if human Eos also express arginase with its potential immunosuppressive consequences. We show that human peripheral blood Eos do not express arginase I or II protein or arginase enzymatic activity. Correspondingly, no metabolism of arginine to ornithine can be detected in Eos-S. Neither Eos apoptosis nor cytokine-mediated cellular activation induces arginase in human Eos in vitro. Finally, we show that arginase activity and protein are also undetectable in Eos of allergic patients from peripheral blood or from BALF activated in vivo during allergic pulmonary inflammation. This work demonstrates a fundamental difference between neutrophil and Eos granulocytes. As Eos are not equipped with the immunosuppressive enzyme arginase, they cannot participate, via arginine limitation, in the suppression of the evolving adaptive immune response in allergy, infections, or tumor immunity.

Background: Exhaled breath condensate (EBC) has been used for diagnosing and monitoring respiratory disorders. For clinical purposes the assessment of easy-to-obtain nonspecific markers seems particularly interesting., Objectives: As these measures are related to each other, our objective was to extract the independent information in global EBC markers across a range of respiratory disorders., Methods: EBC was collected from patients with asthma (n = 18), chronic obstructive pulmonary disease (n = 17), and cystic fibrosis (n = 46), as well as from lung transplant (LTX) recipients (n = 14) and healthy controls (n = 26). Samples were assessed for electrical conductivity, ammonia, pH, and nitrite/nitrate. pH was measured after both deaeration with argon and CO(2) standardization. Additionally, the fraction of exhaled nitric oxide (FE(NO)) was assessed. Factor analysis was applied to identify major factors concerning these measures., Results: Three independent factors were detected; the first comprised conductivity, ammonia, and pH, especially when standardized using CO(2), the second nitrite/nitrate, and the third FE(NO). Conductivity and ammonia were highly correlated (r = 0.968; p < 0.001). FE(NO) provided independent information mainly in asthma. The nonspecific EBC markers showed considerable overlap between patient groups and healthy subjects. However, conductivity, ammonia, pH standardized for CO(2) and nitrite/nitrate were increased in LTX recipients compared to healthy controls (p < 0.05 each)., Conclusions: A panel of nonspecific easy-to-obtain exhaled breath markers could be reduced to 3 independent factors. The information content of conductivity, ammonia, and pH after CO(2) equilibration appeared to be similar, while FE(NO) was independent. The increased levels of these biomarkers in LTX might indicate a potential for their use in these patients., (Copyright © 2010 S. Karger AG, Basel.)

Background: Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO(2)) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling in vitro. In addition, TiO(2) effects on surfactant ultrastructure were visualized., Methods: A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 microg/ml) of TiO(2) NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope., Results: TiO(2) NSP, but not MSP, induced a surfactant dysfunction. For TiO(2) NSP, adsorption surface tension (gammaads) increased in a dose-dependent manner from 28.2 + or - 2.3 mN/m to 33.2 + or - 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (gammamin) slightly increased from 4.8 + or - 0.5 mN/m up to 8.4 + or - 1.3 mN/m (p < 0.01) at high TiO(2) NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both gammaads (63.6 + or - 0.4 mN/m) and gammamin (21.1 + or - 0.4 mN/m). Interestingly, TiO(2) NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae., Conclusion: TiO(2) nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.

Nano-sized particles (NSPs) have a diameter of less than 100 nm. When inhaled, they preferentially deposit in the deeper lung, where pulmonary surfactant covers the thin aqueous lining layer. Thus, pulmonary surfactant is the initial contact where NSPs impinge. This can lead to various consequences. For example, binding of NSPs to single surfactant components like phospholipids or surfactant proteins can occur, which might modulate toxic particle effects. Moreover, particle clearance can be modulated. Furthermore, the biophysical surfactant function itself can be disturbed by interaction with NSPs. In addition, surfactant displaces particles into the aqueous hypophase of the lining layer, where they can come into contact with type II pneumocytes. This interaction has been suggested to affect pulmonary surfactant metabolism. The potential interactions of nano-sized particles with the pulmonary surfactant system and the effects on biophysical surfactant function, surfactant metabolism, particle clearance, and on particle-induced toxicity are reviewed.

Background: This study was designed to analyze the role of postoperative donor cell chimerism for the induction and maintenance of transplantation tolerance in a porcine lung transplantation model., Methods: Left-sided single lung transplantation from major histocompatibility mismatched male donors was performed in 27 female minipigs. All received a 28-day course of pharmacologic immunosuppression using various agents, some in combination with preoperative irradiation. Groups for eventual analysis were strictly defined by outcome, that is, pigs with acute rejection before postoperative day 178 (n=16) were allocated into one group, long-term surviving animals (n=11) into the other. Peripheral blood chimerism was monitored by flow cytometry and real-time polymerase chain reaction. Intragraft chimerism was detected from bronchoalveolar lavage fluid (BALF) by fluorescent in situ hybridization., Results: Blood chimerism peaked 1 hour after transplantation and was significantly higher in the group of long-term survivors at that time. Thereafter chimerism rapidly decreased, but tended to remain higher in long-term survivors. In case of acute rejection donor cells were lost, but remained detectable for up to 36 postoperative months in tolerant animals. In BALF, the percentage of male nuclei was equally high under immunosuppression in both groups. Rejecting animals showed a rapid decrease of Y-bearing cells in BALF after drug withdrawal and an almost complete loss when acute rejection occurred. In tolerant pigs, intragraft chimerism remained detectable throughout the follow-up., Conclusions: This study demonstrates a clear correlation of donor leukocyte chimerism with long-term allograft survival in a porcine allogeneic lung transplantation model.

Pulmonary surfactant is a complex mixture of lipids and proteins that reduces the surface tension at the air-liquid interface. In addition to its biophysical function, some surfactant components play an important role for the innate and adaptive immunity of the lung. A negative modulation of the surfactant function was observed in allergic asthma leading to the assumption that the therapeutic application of surfactant components might be beneficial in this disease. So far, there are a number of preclinical and already some clinical studies demonstrating various effects of different surfactant components that were administered with preventive or therapeutic aim in allergic asthma. This review summarizes the current knowledge on the possibilities to treat allergic asthma with surfactant components.

Unlabelled: Antihistamines and nasal decongestants are well-established therapeutics in allergic rhinitis. However, no data are available which directly compare the effect size of the single substances with their combination in a single study including placebo (PLA) treatment., Objective: The aim of this study was to evaluate the effect of a combination of cetirizine (CET) and pseudoephedrine (PSE) and to compare it to treatment with CET or PSE alone and to PLA during grass pollen allergen challenge in an environmental challenge chamber (ECC)., Material and Methods: In a randomized, double-blind, placebo-controlled, four-way crossover study the effect of a combination of 10 mg CET with 120 mg PSE (CET + PSE) versus CET or PSE alone or PLA on symptoms, nasal flow, and nasal secretions was investigated in 49 patients with intermittent allergic rhinitis. Subjects underwent four 6-h pollen exposures in an ECC with administration of the drugs after 2 h., Results: The induction of nasal symptoms, nasal secretion and nasal obstruction (measured as nasal flow) during the first 2 h of pollen exposure was highly reproducible at the 4 consecutive exposures. The symptom of nasal obstruction was significantly reduced after treatment with CET + PSE compared to the treatment with CET or PSE alone or PLA (p < 0.0001). Furthermore, the combination treatment significantly reduced the total nasal symptom score (TNSS) and visual analogue scale score (VAS) compared to the single treatments or PLA. Nasal flow was significantly increased after treatment with CET + PSE and PSE and nasal secretions were significantly reduced by CET + PSE and CET without significant additional improvement of the combination therapy., Conclusion: The combination treatment with CET and PSE is more effective than treatment with single substances in subjects with allergic rhinitis.

Background: There is increasing interest in testing surfactant preparations for asthma therapy. Previously, Curosurf was demonstrated to increase inflammation in allergic asthmatics. So far, little is known about the immunomodulatory effects of therapeutic surfactants, in particular concerning the interaction of surfactant components with eosinophils as key effector cells of the allergic airway inflammation. The aim of the present study was to determine the effect of different therapeutic surfactants on cellular functions of eosinophils., Methods: Eosinophils were isolated from peripheral blood of atopic volunteers and incubated with the natural animal-derived surfactants Curosurf or Alveofact or the synthetic recombinant human surfactant Venticute at different concentrations for up to 42 h., Results: Curosurf and Venticute modulated the viability of eosinophils. While incubation with Curosurf increased the number of necrotic eosinophils after 1, 20 and 42 h, Venticute increased the number of apoptotic and necrotic cells after 1 h, but there were no differences compared with control cells at later time points. All surfactant preparations increased the levels of eosinophil cationic protein after 20 h and, in addition, Curosurf enhanced eosinophil cationic protein release after 42 h. The supernatant of eosinophils induced chemotaxis against autologous eosinophils, and the presence of Curosurf, but not Alveofact or Venticute, augmented the chemotactic effect. Chemotaxis was partly blocked by inhibition of eotaxin but not by inhibition of leukotrienes or platelet-activating factor., Conclusions: Therapeutic surfactants differ in their effects on eosinophil viability and the accompanying release of inflammatory mediators and chemotactic signals. Proinflammatory effects were most pronounced for the natural surfactant Curosurf., (Copyright (C) 2009 S. Karger AG, Basel.)

Background and Objective: Chronic obstructive pulmonary disease (COPD) is one of the most common causes of chronic morbidity and mortality. The "Burden of Obstructive Lung Disease" (BOLD) initiative was established as an international study collecting data about the prevalence of COPD. The Medical University of Hanover took part in this study collecting data representative for Germany., Methods: 683 individuals aged = 40 years from the city and region of Hannover were included in the study. On the basis of standardized questionnaires data were collected on general health, physical and mental capability, smoking habits and occupational exposure to dust. All participants performed spirometry before and after inhalation of salbutamol., Results: The prevalence of COPD, GOLD (Global Health Initiative on Obstructive Lung Disease) severity stage = I, was 13.2% (GOLD stage I: 7.4%; GOLD stage II: 5.0%, GOLD stage III or IV: 0.8%). There was a marked increase of the prevalence of COPD depending on age and smoking habits. The percentage of active smokers in the sample was 20.6 %. Among younger participants the percentage of female smokers was noticeable higher than in older subjects. Although clinical symptoms of COPD, GOLD stage = III correlated with disease severity, only persons with COPD reported reduced physical capability., Conclusion: COPD is a highly prevalent disease. With regard to the increasing life expectancy and the change of smoking habits of the population, a further increase of morbidity and mortality due to COPD must be expected, especially in women.

Rationale: Roflumilast, an investigational, targeted phosphodiesterase 4 inhibitor, reduces the in vitro and in vivo inflammatory activity of cells such as neutrophils, eosinophils, macrophages, and monocytes., Objectives: The aim of this study was to explore the anti-inflammatory properties of roflumilast in a human model of segmental bronchial endotoxin challenge., Methods: In a randomized, placebo-controlled, double-blind, single-center parallel-group study, 37 healthy subjects of either sex were treated for 28 days with either oral roflumilast 500 microg once daily or placebo. At day 29, a baseline bronchoalveolar lavage was performed, followed by segmental endotoxin challenge (4 ng/kg) and saline control challenge. After 24h, bronchoalveolar lavage fluid was sampled from the challenged segments and cells were counted and differentiated., Results: After endotoxin challenge, influx of total cells (difference from baseline) in bronchoalveolar lavage of roflumilast-treated subjects was 36% lower than with placebo (p=0.02). Correspondingly, the influx of neutrophils and eosinophils of roflumilast-treated subjects was 39% (p=0.02) and 74% (p=0.01) lower than with placebo, respectively. In contrast, endotoxin-induced influx of monocytes was not different between roflumilast- and placebo-treated subjects. No significant differences existed between the groups pertaining to endotoxin-induced influx of macrophages and lymphocytes. Roflumilast was well tolerated. No unexpected or serious treatment-emergent signs and symptoms were observed., Conclusions: Roflumilast attenuated the endotoxin-induced influx of neutrophils and eosinophils into the airways. This study demonstrates the anti-inflammatory properties of roflumilast on bronchoalveolar granulocytes in endotoxin-induced airway inflammation in healthy subjects.

We hypothesized that formation of pulmonary emphysema could be diminished after previous activation of stem cells. Animals received either a daily dose of the hematopoietic growth factors (GF; recombinant rat stem cell factor plus recombinant granulocyte colony stimulating factor; n=6, Elastase/GF group) or vehicle (n=9, Elastase/Sham group) starting 3 days before intratracheal instillation of elastase or vehicle and continued for another 25 days. Control animals were treated with NaCl (n=9, Sham/Sham group). On day 25, in all animals, a 2-mL pump was implanted subcutaneously that delivered 200 microg/h 5-bromo-2-desoxyuridine (BrdU) until study termination. Compared to controls, the Elastase/Sham group exhibited elevated total lung capacity (TLC) and functional residual capacity (FRC), significantly increased mean free alveolar pathway, alveolar volume, and decreased septal density. The Elastase/GF group showed (1) a significant increase of TLC and FRC, (2) a significant increase in alveolar size and volume, (3) a significant reduction of septal density, volume, and thickness. Proliferation in lung parenchyma and in terminal bronchioles remained significantly decreased in the Elastase/Sham group and the Elastase/GF group. Blood cell number has significantly increased in the Elastase/GF group. The application of GF-enhanced pulmonary emphysema, presumable because of increased inflammatory activity, was a result of a preventive treatment.

Pneumococcal infections (pneumonia, otitis media, sinusitis, meningitis) are common and usually involve toddlers and the elderly. Currently, two pneumococcal vaccines are in clinical use. The older vaccine consists of pure capsular polysaccharides from 23 pneumococcal serotypes and induces only a limited B-cell response because polysaccharides are poor antigens that stimulate mainly B-cells. In 2000, a vaccination program with a novel 7-valent pneumococcal conjugate vaccine was launched in the U.S. The conjugation of capsular polysaccharides with a highly immunogenic diphtheria toxoid protein induces both a T cell and B cell response that results in specific humoral and mucosal immunity. Since children are the main reservoir of pneumococci, the 7-valent conjugate vaccine seems to eradicate the respective pneumococcal serotypes within the population, as demonstrated by recent US data. Pronounced herd immunity resulted in a decrease in invasive pneumococcal diseases in vaccinees and non-vaccinees as well as in a reduction of antibiotic resistance rates. However, recent data suggest a replacement of vaccine-serotypes by non-vaccine serotypes, which conquer the ecological niche created by the vaccine. In order to encounter this problem a 13-valent conjugated vaccine is currently under development.

Primary graft dysfunction, characterised by intra-alveolar oedema, is a major obstacle in pulmonary transplantation. The present study evaluates the potential of keratinocyte growth factor (palmiferin; DeltaN23-KGF) for the prevention of oedema in lung transplants. Intratracheal instillation of 5 mg x kg(-1) DeltaN23-KGF was performed in Lewis rats on days 3 and 2 before explantation. Control animals obtained an equivalent volume of vehicle. Left lungs were isogeneically transplanted and the graft recipients were sacrificed 1 day later for stereological analysis of intra-alveolar oedema and bronchoalveolar lavage. The total protein and phospholipid content, as well as surfactant proteins, were measured. Surfactant activity was analysed with a pulsating bubble surfactometer. In grafts from control treated donors, the fraction of intra-alveolar oedema amounted to 3.4+/-1.1% of the total parenchymal volume. Treatment of donor lungs with DeltaN23-KGF reduced oedema to a fraction of 1.6+/-0.8%. In the lavage fluid of pulmonary grafts from DeltaN23-KGF-treated donors, the total protein content was decreased compared with vehicle-treated lung transplants, whereas phospholipids did not differ. The protein fraction contained increased amounts of surfactant protein-C after DeltaN23-KGF treatment and surfactant function was improved. Treatment of donor lungs with palifermin protects against intra-alveolar oedema formation upon transplantation. This effect appears to be mediated by an improved surfactant homeostasis.

Quantitative (immuno) transmission electron microscopy using design-based stereology was performed on specimens collected by means of systematic uniform random sampling of rat lungs, which were fixed by vascular perfusion to stabilize intra-alveolar surfactant in situ. This procedure ensures that the data recorded are representative of the whole organ. Ultrathin sections of specimens embedded at low temperature in Lowicryl HM20 were labeled by indirect immuno-gold staining for surfactant protein A. We observed that, 3 days after treatment of lungs in vivo with truncated keratinocyte growth factor (DeltaN23-KGF), a potent mitogen of alveolar epithelial type II cells, surfactant protein A associated with the tubular myelin fraction of intra-alveolar surfactant was increased by 47% in comparison with buffer-treated control lungs. Despite the marked type II cell hyperplasia, the relative amount of ultrastructural surfactant subtypes was not significantly affected. Because surfactant protein A reduces the sensitivity to inhibition of the biophysical activity of surfactant by exudating plasma proteins, we propose that pretreatment of lungs with DeltaN23-KGF ameliorates adverse effects observed in acute lung injury following, for example, ischemia and reperfusion., ((c) 2007 Wiley-Liss, Inc)

Objective: Despite the introduction of low potassium-based preservation strategies for clinical lung transplantation, relevant early graft dysfunction occurs in up to 20% of cases after lung transplantation. This was found to be frequently associated with postreperfusion surfactant dysfunction. We performed a randomized, prospective study investigating the effect of exogenous surfactant instillation into human donor lungs on posttransplant surfactant function and on clinical outcome., Methods: Exogenous surfactant was instilled into 15 donor lungs before retrieval via bronchoscopy. Bronchoalveolar lavage fluids were taken before instillation as well as 24 hours after transplantation. Surfactant function, phospholipids, and protein content in bronchoalveolar lavage fluids were assessed and clinical data prospectively recorded. Pulmonary function testing was performed 4 weeks after lung transplantation. Additionally, the best forced expiratory volume in 1 second was determined within the first year after lung transplantation. The control group consisted of 14 patients receiving donor lungs without surfactant instillation in randomized order. Pulmonary function test results were further compared with those of 154 consecutive recipients of bilateral lung transplants, which were not involved in the study (historical control)., Results: No deaths occurred during the first year after lung transplantation. Surfactant function in donor lungs was within normal ranges before harvest. In the control group, surfactant function was markedly impaired after reperfusion. This was significantly improved by surfactant substitution. Protein content of the bronchoalveolar lavage fluid in the surfactant group was significantly lower, indicating less leakage through the alveolocapillary membrane. Forced expiratory volume in 1 second after 4 weeks was significantly higher in the surfactant group than in either control group (P = .034 and .01, respectively). Interestingly, the best forced expiratory volume in 1 second during the first year after lung transplantation was significantly higher in both control groups compared with forced expiratory volume measured in examinations 4 weeks after lung transplantation (P = .01). The best forced expiratory volumes in 1 second of control patients were comparable with those in surfactant lungs 4 weeks after transplant., Conclusions: This study indicates a protective effect of exogenous surfactant instillation to donor lungs before retrieval on post-lung transplantation surfactant function and on early clinical outcome. This approach may help to improve the outcome after lung transplantation in the future.

Chronic obstructive pulmonary disease (COPD) is one of the most common chronic diseases worldwide, and its morbidity and mortality are still increasing. Thus, it is expected that, globally, COPD will be the third most common cause of death by 2020. Moreover, COPD leads to enormous health-economic costs (i. e., outpatient and inpatient treatment, inability to work). Nevertheless, the prevalence of COPD is underestimated also in Germany. This is partly because often, the disease is symptomatic only in its advanced stages and as a result it is not diagnosed until late. In addition, the therapeutic effects on the long-term course and the progression of the disease are limited. Therefore, a much deeper understanding of the manifold and complex pathomechanisms of this pathomorphologically varied disease is needed before new therapeutic strategies can be developed. The aim of this review is to discuss current concepts of the pathophysiology of COPD and their scientific bases. The authors will concentrate mainly on a differentiated description of the pathomechanisms which cause chronic obstructive bronchiolitis and obstructive pulmonary emphysema.

Background: Cytochrome P450 monooxygenases (CYP) play an important role in the defense against inhaled toxicants, and expression of CYP enzymes may differ among various lung cells and tissue compartments., Methods: We studied the effects of tobacco smoke in volunteers and investigated gene expression of 19 CYPs and 3 flavin-containing monooxygenases, as well as isoforms of glutathione S-transferases (GST) and uridine diphosphate glucuronosyltransferases (UGT) and the microsomal epoxide hydrolase (EPHX1) in bronchoalveolar lavage cells and bronchial biopsies derived from smokers (n = 8) and nonsmokers (n = 10). We also investigated gene expression of nuclear transcription factors known to be involved in the regulation of xenobiotic metabolism enzymes., Results: Gene expression of CYP1A1, CYP1B1, CYP2S1, GSTP1, and EPHX1 was induced in bronchoalveolar lavage cells of smokers, whereas expression of CYP2B6/7, CYP3A5, and UGT2A1 was repressed. In bronchial biopsies of smokers, CYP1A1, CYP1B1, CYP2C9, GSTP1, and GSTA2 were induced, but CYP2J2 and EPHX1 were repressed. Induction of CYP1A1 and CYP1B1 transcript abundance resulted in increased activity of the coded enzyme. Finally, expression of the liver X receptor and the glucocorticoid receptor was significantly up-regulated in bronchoalveolar lavage cells of smokers., Conclusions: We found gene expression of pulmonary xenobiotic metabolizing enzymes and certain key transcription factors to be regulated in bronchoalveolar lavage cells and bronchial biopsies of smokers. The observed changes demonstrate tissue specificity in xenobiotic metabolism, with likely implications for the metabolic activation of procarcinogens to ultimate carcinogens of tobacco smoke.

Aims: To assess the antihistaminic activity of levocetirizine and fexofenadine 2 h and 24 h after drug administration using facial thermography and to compare the results with those using well-established parameters of antihistaminic activity in the nose and skin., Methods: This was a randomized, double-blind, three-treatment, three-period, single-dose, cross-over study in healthy males taking levocetirizine 5 mg, fexofenadine 120 mg or placebo. The primary endpoint was nasal skin temperature after nasal histamine challenge recorded for 20 min at 2 and 24 h after drug intake. The secondary endpoints were nasal symptoms and a histamine skin prick test., Results: Thirty subjects were randomized. At 2 h after drug intake the inhibition of the nasal temperature increase from baseline was not significantly different between levocetirizine and fexofenadine. At 24 h it was significantly more pronounced after levocetirizine than fexofenadine (difference: least-squares mean: -0.13 degrees C; P < or = 0.024, 95% CI -0.24, -0.02). Both drugs significantly reduced (P < or = 0.001) the mean temperature increase from baseline compared with placebo at 2 and 24 h (least-squares mean increase and (95% CI): levocetirizine, -0.28 degrees C (-0.42, -0.14) and -0.32 degrees C (-0.43, -0.21); fexofenadine -0.35 degrees C (-0.49, -0.21) and -0.19 degrees C (-0.30, -0.08), respectively). Results of nasal symptom score and wheal and flare were consistent with the thermography results., Conclusions: Facial thermography is an objective, non-invasive and sensitive method to study antihistaminic activity at the nose level. Levocetirizine and fexofenadine demonstrate the same activity at 2 h after drug intake, but levocetirizine has a more sustained activity at 24 h.

Background: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival., Methods: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays., Results: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors., Conclusion: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.

Background: The surfactant protein SP-D has been reported to reduce bronchial hyper-responsiveness, blood eosinophilia, and T-helper type 2 cytokines in models of allergic asthma. However, little is known about the functional effect of SP-D on the early airway response upon allergen inhalation, which is an important feature of this disease., Objective: We investigated whether SP-D is able to reduce the immediate allergen-induced mediator release and the early bronchial obstruction in addition to its effects on airway inflammation and bronchial hyperresponsiveness in an Aspergillus fumigatus mouse asthma model., Methods: A. fumigatus-sensitized mice were treated with a recombinant fragment of human SP-D or placebo. Lung functions were measured in orotracheally intubated, spontaneously breathing animals using body plethysmography. In addition, passively sensitized precision-cut lung slices (PCLS) were used to determine the effect of SP-D on allergen-induced histamine release., Results: SP-D inhibited the allergen-induced early airway response and reduced airway hyperresponsiveness compared with placebo. Eosinophilia in bronchoalveolar lavage and lung tissue was reduced after SP-D treatment, possibly by reducing eotaxin levels in the lung. Furthermore, SP-D treatment reduced the allergen-induced histamine release from PCLS., Conclusion: These data suggest that SP-D not only reduces allergen-induced eosinophilic inflammation and airway hyperresponsiveness but also provides protection against early airway obstruction by inhibition of early mediator release.

Background: Allergic asthma is associated with airway inflammation and dysfunction of pulmonary surfactant. Because surfactant proteins (SP) account for immunomodulatory functions as well as biophysical functions, we hypothesized that the allergic response in asthma might be accompanied by a dysregulation of SPs., Methods: We measured levels of SP-A, SP-B, SP-C and SP-D by enzyme-linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid of 23 asthma patients and 10 healthy control subjects under well-controlled conditions before and 24 h after segmental allergen provocation. These data were related to surfactant function, Th(2) cytokine levels in BAL fluid and to the degree of eosinophilic inflammation., Results: In patients with asthma, allergen challenge increased BAL levels of SP-B, SP-C and SP-D while SP-A was decreased. For SP-B and SP-D, a moderate increase was also observed after saline challenge. In contrast, no alterations were observed in healthy control subjects. Levels of SP-B and SP-C in asthmatics correlated with the ratio of small to large surfactant aggregates (SA/LA ratio) and correlated negatively with BAL surface activity. Furthermore, increased SP-C but not SP-B levels after allergen challenge correlated with eosinophil numbers, interleukin (IL)-5, and IL-13 in BAL while increased SP-D levels only correlated with eosinophil numbers., Conclusions: This study demonstrates significant alterations of all SPs in BAL fluid after allergen challenge of which SP-C was most closely related to surfactant dysfunction and the degree of the allergic inflammation.

Background: GATA-3 (GATA binding protein 3) and the proto-oncogene c-maf are Th2-regulating transcription factors that control the expression of interleukin (IL)-4 and IL-5, while T-bet (T-box expressed in T cells) is a Th1-specific transcription factor that controls the expression of interferon (IFN-gamma). Allergen provocation in asthmatics induces a Th2-dominated cytokine profile, but so far it is unknown whether the skewed cytokine expression is reflected by the expression of the respective transcriptional regulators., Objective: The aim of this study was to determine the regulation of Th1- and Th2-specific transcription factors and cytokines in 10 atopic subjects with mild asthma and 5 nonatopic healthy controls at baseline and after segmental sham and allergen challenge., Methods: The mRNA expression of GATA-3, c-maf and T-bet was determined by real-time polymerase chain reaction in bronchoalveolar lavage (BAL) cells and bronchial biopsies. The percentage of IL-4+, IL-5+ and IFN-gamma+ BAL T cells was determined by flow cytometry, and BAL levels of these cytokines were measured by ELISA., Results: In BAL cells of asthmatics, the mRNA expression of all transcription factors was increased after allergen challenge. In bronchial biopsies, the basal expression of GATA-3 was increased in asthmatics compared to healthy controls but decreased after allergen challenge. Compared to sham challenge, the percentage of IL-5+/CD4+ BAL T cells was increased after allergen challenge in asthmatics while the percentage of IFN-gamma+/CD4+ and IFN-gamma+/CD8+ T cells was decreased. Expression of c-maf mRNA in BAL cells correlated with IL-4+/CD4+ BAL cells and BAL IL-5 levels., Conclusions: Segmental allergen challenge in asthmatics leads to increased GATA-3, c-maf and T-bet expression in BAL cells but not in bronchial biopsies., (Copyright (c) 2006 S. Karger AG, Basel.)

Background: This study seeks to compare the ability of repeatable invasive and noninvasive lung function methods to assess allergen-specific and cholinergic airway responsiveness (AR) in intact, spontaneously breathing BALB/c mice., Methods: Using noninvasive head-out body plethysmography and the decrease in tidal midexpiratory flow (EF50), we determined early AR (EAR) to inhaled Aspergillus fumigatus antigens in conscious mice. These measurements were paralleled by invasive determination of pulmonary conductance (GL), dynamic compliance (Cdyn) and EF50 in another group of anesthetized, orotracheally intubated mice., Results: With both methods, allergic mice, sensitized and boosted with A. fumigatus, elicited allergen-specific EAR to A. fumigatus (p < 0.05 versus controls). Dose-response studies to aerosolized methacholine (MCh) were performed in the same animals 48 h later, showing that allergic mice relative to controls were distinctly more responsive (p < 0.05) and revealed acute airway inflammation as evidenced from increased eosinophils and lymphocytes in bronchoalveolar lavage., Conclusion: We conclude that invasive and noninvasive pulmonary function tests are capable of detecting both allergen-specific and cholinergic AR in intact, allergic mice. The invasive determination of GL and Cdyn is superior in sensitivity, whereas the noninvasive EF50 method is particularly appropriate for quick and repeatable screening of respiratory function in large numbers of conscious mice.

Mast cells play a key role in allergy and asthma. They reside at the host-environment interface and are among the first cells to make contact with inhaled microorganisms and particulate antigens. Pulmonary surfactant proteins A and D (SP-A and SP-D) function in lung host defense by enhancing microbe phagocytosis and mediating other immune cell functions, but little is known about their effects on mast cells. We hypothesized that SP-A and/or SP-D modulate IgE-dependent mast cell functions. Pollen starch granules (PSG) extracted from Dactylis glomerata and coated with trinitrophenol (TNP) were used as a model of an inhaled organic particulate allergen. Our data revealed that SP-D inhibited by 50% the release of beta-hexosaminidase by peritoneal mast cells sensitized with IgE anti-TNP and stimulated with TNP-PSG. In contrast, SP-A had no effect. Furthermore, SP-D aggregated PSG in a dose-dependent manner, and this aggregation was mediated by SP-D's carbohydrate recognition domain. A single arm SP-D mutant (RrSP-Dser15,20) neither aggregated PSG nor inhibited degranulation, suggesting that multimerization of SP-D is required for maximal PSG aggregation and inhibition of PSG-induced mast cell degranulation. This study is the first to demonstrate that SP-D modulates IgE-mediated mast cell functions, which are important in asthma and allergic inflammation.

Background: Flush perfusion with low-potassium dextran is the standard strategy in clinical lung preservation. Despite improved outcome, endothelial cell injury and surfactant dysfunction remain a significant problem after lung transplantation. The radical scavenger glutathione has been shown to be responsible for the efficacy of Celsior solution in lung preservation. We tested the hypothesis that the addition of glutathione to low-potassium dextran might further improve graft function by ameliorating ischemia-reperfusion injury., Methods: In 12 domestic pigs, lungs were flush preserved with either low-potassium dextran (n = 6) or low-potassium dextran supplemented by 5 mmol glutathione (n = 6). Left single lung transplantation was performed after 24-hour storage in low-potassium dextran at 8 degrees C. After 15 minutes of reperfusion the right main bronchus and pulmonary artery were crossclamped. Hemodynamic and respiratory measures were recorded in 30-minute intervals for a total observation period of 7 hours. Bronchoalveolar lavage fluid was obtained from the native lung and 2 hours after reperfusion from the graft. Bronchoalveolar lavage fluid and surfactant composition, and surfactant function analyses were performed. Neutrophil sequestration was assessed by myeloperoxidase activity assay. Tissue water content was calculated from wet/dry weight ratios at the end of the experiment., Results: In the low-potassium dextran group, 2 animals died during reperfusion. After reperfusion, pulmonary vascular resistance (P = .01) and pulmonary artery pressure remained lower in the glutathione/low-potassium dextran group, which was associated with a higher cardiac output (P = .05) in this group. Also, the oxygenation index at the end of the observation period was higher in the glutathione/low-potassium dextran group compared with the low-potassium dextran group (430 +/- 130 vs 338 +/- 184, respectively; P < .05). The graft water content representing postreperfusion lung edema was not different between the 2 study groups. Alteration of surfactant was less in the glutathione/low-potassium dextran group with a significantly decreased small to large aggregate ratio (P = .03) versus low-potassium dextran group. Myeloperoxidase activity was twice as high in the low-potassium dextran group when compared with the glutathione/low-potassium dextran group (glutathione/low-potassium dextran: 134 +/- 110 mU/g vs low-potassium dextran: 274 +/- 168 mU/g, P = .07)., Conclusion: The addition of glutathione to low-potassium dextran preservation solution reveals beneficial effects on vascular function and surfactant composition in transplanted lungs. Therefore, glutathione ameliorates ischemia-reperfusion injury in a preclinical model of lung transplantation. Future studies are needed to evaluate this promising modification in clinical lung transplantation.

Nitric oxide (NO) levels are increased in the exhaled air of asthmatics. As NO levels correlate with allergic airway inflammation, NO measurement has been suggested for disease monitoring. In patients with asthma, we previously demonstrated that intrabronchial treatment with a natural porcine surfactant enhanced airway inflammation after segmental allergen provocation. We studied whether local levels of NO reflect the degree of allergic airway inflammation following segmental allergen challenge with or without surfactant pretreatment. Segmental NO, as well as nitrite and nitrate in bronchoalveolar lavage (BAL) fluid, was measured before and after segmental challenge with either saline, saline plus allergen, or surfactant plus allergen in 16 patients with asthma and five healthy subjects. The data were compared with inflammatory BAL cells. Segmental NO levels were increased after instillation of saline (p < 0.05), or surfactant plus allergen in asthmatics (p < 0.05), and values were higher after surfactant plus allergen compared to saline challenge. Nitrate BAL levels were not altered after saline challenge but increased after allergen challenge (p < 0.05) and further raised by surfactant (p < 0.05), whereas nitrite levels were not altered by any treatment. Segmental NO and nitrate levels correlated with the degree of eosinophilic airway inflammation, and nitrate levels also correlated with neutrophil and lymphocyte numbers in BAL. In healthy subjects, NO, nitrite, and nitrate were unaffected. Thus, segmental NO and nitrate levels reflect the degree of allergic airway inflammation in patients with asthma. Measurement of both markers can be useful in studies using segmental allergen provocation, to assess local effects of potential immunomodulators.

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.

Background: Loteprednol etabonate (LE) is a novel soft steroid that was designed to improve the benefit/risk ratio of topical corticosteroid therapy. This study assesses the clinical efficacy and safety of three different doses of LE nasal spray in seasonal allergic rhinitis (SAR)., Methods: In this single-center, double-blind, placebo-controlled, parallel-group trial 165 subjects with SAR to grass pollen received daily single doses of either 100, 200, 400 microg LE nasal spray, or placebo for 14 days. The patients underwent three 4-h allergen challenges with grass pollen in an environmental exposure unit at a screening visit (baseline) and on days 7 and 14 of treatment. Standardized nasal symptom scores were obtained every 20 min. Nasal flow, nasal secretions, and FEV(1) were measured every hour during allergen challenges., Results: After 14 days of treatment, patients who received 400 microg LE had significantly lower total nasal symptom scores compared with those receiving placebo (P = 0.007). LE400 reduced rhinorrhea, nasal congestion, nasal itching, the amount of nasal secretions, and improved nasal flow as compared with placebo (P < 0.05). LE100 and LE200 were not significantly different from placebo. All treatments were well tolerated., Conclusions: Loteprednol 400 microg once daily is superior to placebo and the only effective dose tested in improving nasal symptoms and objective parameters in patients with SAR.

The cytokine IL-6 acts via a specific receptor complex that consists of the membrane-bound IL-6 receptor (mIL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). In this study, we investigated the role of IL-6R components in asthma. We observed increased levels of sIL-6R in the airways of patients with allergic asthma as compared to those in controls. In addition, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130-fraction constant led to suppression of Th2 cells in the lung. By contrast, blockade of mIL-6R induced local expansion of Foxp3-positive CD4+CD25+ Tregs with increased immunosuppressive capacities. CD4+CD25+ but not CD4+CD25- lung T cells selectively expressed the IL-6R alpha chain and showed IL-6-dependent STAT-3 phosphorylation. Finally, in an in vivo transfer model of asthma in immunodeficient Rag1 mice, CD4+CD25+ T cells isolated from anti-IL-6R antibody-treated mice exhibited marked immunosuppressive and antiinflammatory functions. IL-6 signaling therefore controls the balance between effector cells and Tregs in the lung by means of different receptor components. Furthermore, inhibition of IL-6 signaling emerges as a novel molecular approach for the treatment of allergic asthma.

Objectives: The optimal strategy for pulmonary graft preservation remains elusive. Experimental work and initial clinical experience support low-potassium dextran solutions as lung perfusates. We have previously shown a protective effect of prostaglandin E 1 on ischemia-reperfusion injury in lung transplantation by a shift from proinflammatory to anti-inflammatory cytokines in a rat lung transplantation model. In this study, we tested the hypothesis that the addition of a prostacyclin analog (iloprost) to low-potassium dextran might lead to improved surfactant and ultimately graft function., Methods: In a randomized, blinded study with a porcine left single-lung transplantation model, donor lungs were flushed with 1 L of either low-potassium dextran solution or low-potassium dextran solution modified by the addition of 250 microg iloprost (n = 6 in each group). Grafts were stored at 4 degrees C for 24 hours. After transplantation, the right bronchus and pulmonary artery were clamped, and the animals remained dependent on the graft. Posttransplantation graft function was assessed throughout a 7-hour observation period by measuring oxygenation (30-minute intervals), different pulmonary and systemic hemodynamic parameters, and wet/dry lung weight ratios. Bronchoalveolar lavage fluid was obtained before and 2 hours after reperfusion. Surfactant function was measured from bronchoalveolar lavage fluid with a pulsating bubble surfactometer. Neutrophil sequestration was assessed by a myeloperoxidase assay performed on lung tissue specimens taken at the end of the observation period., Results: Pulmonary vascular resistance remained lower in the iloprost group than in the control group (P < .05). Tissue water content after 7 hours of reperfusion remained lower in the iloprost group (P < .05). In addition, significantly reduced myeloperoxidase tissue activity was observed in the iloprost group (P < .05). Although there was no difference in degradation of surface active surfactant large aggregates to small aggregates, the surface tension measured at minimal bubble diameter was lower in the iloprost group (P < .05)., Conclusions: Modification of low-potassium dextran solution with the prostacyclin analog iloprost resulted in a significant amelioration of ischemia-reperfusion injury and improved preservation of surfactant function in transplanted lungs. This intriguing approach merits further evaluation with respect to the mechanisms involved and, ultimately, potential introduction into clinical lung transplantation.

Precise and repeatable measurements of pulmonary function in intact mice are becoming increasingly important for experimental investigations on various respiratory disorders including asthma. Here, we present validation of a novel in vivo method that, for the first time, combines direct and repetitive recordings of standard pulmonary mechanics with cholinergic aerosol challenges in anesthetized, orotracheally intubated, spontaneously breathing mice. We demonstrate that, in several groups of nonsensitized BALB/c mice, dose-related increases in pulmonary resistance and dynamic compliance to aerosolized methacholine are reproducible over short and extended intervals without causing detectable cytological alterations in the bronchoalveolar lavage or relevant histological changes in the proximal trachea and larynx regardless of the number of orotracheal intubations. Moreover, as further validation, we confirm that allergic mice, sensitized and challenged with Aspergillus fumigatus, were significantly more responsive to cholinergic challenge (P < 0.01) and exhibited marked eosinophilia and lymphocytosis in bronchoalveolar lavage fluids as well as significant pathological alterations in laryngotracheal histology compared with nonsensitized mice. We suggest that this approach will provide useful and necessary information on pulmonary mechanics in studies of various respiratory disorders in mice, including experimental models of asthma and chronic obstructive pulmonary disorder, investigations of pulmonary pharmacology, or more general investigations of the genetic determinants of lung function.

Objective: Pulmonary preservation for transplantation is associated with ischemia reperfusion injury resulting in endothelial cell and surfactant dysfunction. The purpose of the study was to compare two extracellular type solutions, low potassium dextrane (LPD) and Celsior in their ability of ameliorating lung ischemia reperfusion injury., Methods: In 12 donor pigs, the left lung was perfused with either LPD (n = 6) or Celsior (n = 6) solution. After 24 h cold storage, the lungs were transplanted into 12 recipient animals. After reperfusion of the left lung, the right pulmonary artery and bronchus were clamped. Bronchoalveolar lavage fluid (BALF) was obtained before the surgical procedure and 2 h after reperfusion. Surfactant activity was measured from BALF using a pulsating bubble surfactometer. Hemodynamic and respiratory parameters were assessed in 30-min intervals for 7 post-operative hours., Results: In both study groups two of six animals died from severe ischemia reperfusion injury, thus survival did not differ between groups. Rise of pulmonary vascular resistance index (P = 0.01) and sequestration of neutrophiles (P = 0.08) was less pronounced in Celsior group when compared to LPD animals. A difference in surfactant activity between both groups was not evident after 2 h of reperfusion., Conclusions: Both solutions might provide safe pulmonary preservation for 24 h of cold ischemia. While surfactant activity was affected to the same extent in both groups, Celsior solution provided slightly superior endothelial preservation.

Surfactant reduces surface tension at pulmonary air-liquid interfaces. Although its major component is dipalmitoyl-phosphatidylcholine (PC16:0/16:0), other PC species, principally palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC, and palmitoyloleoyl-PC, are integral components of surfactant. The composition and metabolism of PC species depend on pulmonary development, respiratory rate, and pathologic alterations, which have largely been investigated in animals using radiolabeled precursors. Recent advances in mass spectrometry and availability of precursors carrying stable isotopes make metabolic experiments in human subjects ethically feasible. We introduce a technique to quantify surfactant PC synthesis in vivo using deuteriated choline coupled with electrospray ionization tandem mass spectrometry. Endogenous PC from induced sputa of healthy volunteers comprised 54.0 +/- 1.5% PC16:0/16:0, 9.7 +/- 0.7% palmitoylmyristoyl-PC, 10.0 +/- 1.0% palmitoylpalmitoleoyl-PC, and 13.1 +/- 0.3% palmitoyloleoyl-PC. Infusion of deuteriated choline chloride (3.6 mg/kg body weight) over 3 hours resulted in linear incorporation into PC over 30 hours. After a plateau of 0.61 +/- 0.04% labeled PC between 30 and 48 hours, incorporation decreased to 0.30 +/- 0.02% within 7 days. Compared with native PC, fractional label was initially lower for PC16:0/16:0 (31.9 +/- 8.3%) but was higher for palmitoyloleoyl-PC (21.0 +/- 1.2%), and equilibrium was achieved after only 48 hours. We conclude that infusion of deuteriated choline and electrospray ionization tandem mass spectrometry is useful to investigate surfactant metabolism in humans in vivo.

There is increasing evidence for a role of pulmonary surfactant in asthma and allergic inflammation. In murine asthma models, recent studies have demonstrated that surfactant components downregulate the allergic inflammation. Therefore, we tested the hypothesis that in individuals with mild asthma, a natural porcine surfactant preparation (Curosurf) given before segmental allergen challenge can reduce the allergic airway inflammation. Ten patients with asthma and five healthy control subjects were treated in two segments with either Curosurf or vehicle followed by local allergen challenge. Six additional patients with asthma received Curosurf before allergen challenge in one segment as above, but the second segment was instilled with Curosurf without allergen challenge. Unexpectedly, surfactant treatment augmented the eosinophilic inflammation 24 hours after allergen challenge. A direct chemotactic effect of Curosurf was excluded. However, levels of eotaxin and interleukin-5 were increased in bronchoalveolar lavage after Curosurf treatment, whereas IFN-gamma-levels and numbers of IFN-gamma(+) T cells were decreased. Curosurf had no influence on spreading and retention of allergen determined by allergen uptake in mice. These findings demonstrate that treatment with a natural porcine surfactant results in an augmentation of the eosinophilic inflammation after allergen challenge that is more likely due to immunomodulatory effects than to biophysical properties of the surfactant.

Background: Impaired surfactant function has been demonstrated in patients with asthma. Inhibitory proteins originating from plasma or inflammatory mediators are good candidates to contribute to this dysfunction. Eosinophils are potent effector cells in asthma, which, on activation, release inflammatory mediators, especially reactive granula proteins such as eosinophil cationic protein (ECP)., Objective: Because the potential role of ECP in the inhibition of surfactant function is not known, we tested the hypothesis of whether ECP levels in bronchoalveolar lavage fluid (BALF) of patients with asthma after segmental allergen provocation correlate to surfactant dysfunction. Furthermore, we tested the effect of purified ECP on surfactant function and structure in vitro., Methods: Surfactant isolated from BALF of asthmatic patients was assessed for biophysical function with the Pulsating Bubble Surfactometer and the Capillary Surfactometer and correlated to ECP levels. Purified ECP and plasma proteins at various concentrations were incubated with natural surfactant. Surfactant function was studied with the Capillary Surfactometer, and surfactant structure was determined by electron microscopy., Results: ECP is elevated in BALF from patients with asthma after allergen challenge compared with baseline. ECP levels after allergen challenge correlate well to surfactant dysfunction. In vitro, ECP induces a concentration-dependent inhibition of surfactant function that can be inhibited by antibodies against ECP. ECP is more potent compared with albumin or fibrinogen. Finally, ECP induces severe ultrastructural changes to surfactant vesicles that are more pronounced than changes induced by either fibrinogen or albumin., Conclusions: ECP contributes to surfactant dysfunction in asthma, which in turn could lead to airway obstruction.

Keratinocyte growth factor (KGF) is a mitogen for pulmonary epithelial cells. Intratracheal administration of KGF to adult rats results in alveolar epithelial type II and bronchiolar epithelial cell proliferation. While cellular responses to KGF have been intensively studied, functional consequences regarding lung function are unknown. Therefore, in this study, we sought to investigate whether KGF alters pulmonary function variables. Rats received either recombinant human KGF (rHuKGF) (5 mg/kg) or vehicle intratracheally. Before and on days 3 and 7 after treatment, pulmonary function was determined by body plethysmography. Subsequently, lung histological changes were quantified. rHuKGF induced a transient proliferation of alveolar and bronchiolar epithelial cells. The extent of type II cell hyperplasia was significantly correlated with a transient reduction in tidal volume and an increase in breathing frequency. In addition, quasi-static compliance, total lung capacity, and vital capacity were reduced after rHuKGF instillation, suggesting the development of a transitory restrictive lung disorder. Moreover, reduced expiratory flow rates and forced expiratory volumes, as well as increased functional residual capacity after rHuKGF but not vehicle, suggest obstructive lung function changes. In conclusion, the induction of alveolar and bronchiolar epithelial cell proliferation by KGF is paralleled by moderate functional consequences that should be taken into account when the therapeutic potential of KGF is tested.