SerpinB1 deficiency is not associated with increased susceptibility to pulmonary emphysema in mice

Chronic obstructive pulmonary disease (COPD) is characterized by emphysema and chronic bronchitis and is a leading cause of morbidity and mortality worldwide. Tobacco smoke and deficiency in α1-antitrypsin (AAT) are the most prominent environmental and genetic risk factors, respectively. Yet the pathogenesis of COPD is not completely elucidated. Disease progression appears to include a vicious circle driven by self-perpetuating lung inflammation, endothelial and epithelial cell death, and proteolytic degradation of extracellular matrix proteins. Like AAT, serpinB1 is a potent inhibitor of serine proteases including neutrophil elastase and cathepsin G. Because serpinB1 is expressed in myeloid and lung epithelial cells and is protective during lung infections, we investigated the role of serpinB1 in preventing age-related and cigarette smoke-induced emphysema in mice. Fifteen-month-old mice showed increased lung volume and decreased pulmonary function compared with young adult mice (3 mo old), but no differences were observed between serpinB1-deficient (KO) and wild-type (WT) mice. Chronic exposure to secondhand cigarette smoke resulted in structural emphysematous changes compared with respective control mice, but no difference in lung morphometry was observed between genotypes. Of note, the different pattern of stereological changes induced by age and cigarette smoke suggest distinct mechanisms leading to increased airway volume. Finally, expression of intracellular and extracellular protease inhibitors were differently regulated in lungs of WT and KO mice following smoke exposure; however, activity of proteases was not significantly altered. In conclusion, we showed that, although AAT and serpinB1 are similarly potent inhibitors of neutrophil proteases, serpinB1 deficiency is not associated with more severe emphysema.

alpha1-Antitrypsin inhalation reduces airway inflammation in cystic fibrosis patients

The airways of cystic fibrosis (CF) patients are characterised by neutrophils that release high amounts of elastase overwhelming the local antiprotease shield. Inhalation of alpha(1)-antitrypsin (AAT) may restore the protease-antiprotease balance and attenuate airway inflammation in CF airways. The aims of the present study were: 1) to assess the best deposition region for inhaled AAT by two different inhalation strategies; and 2) to examine the effect of 4 weeks of AAT inhalation on lung function, protease-antiprotease balance and airway inflammation in CF patients. In a prospective, randomised study, 52 CF patients received a daily deposition by inhalation of 25 mg AAT for 4 weeks targeting their peripheral or bronchial compartment. The levels of elastase activity, AAT, pro-inflammatory cytokines, neutrophils, immunoglobulin G fragments and the numbers of Pseudomonas aeruginosa were assessed in induced sputum before and after the inhalation period. Inhalation of AAT increased AAT levels and decreased the levels of elastase activity, neutrophils, pro-inflammatory cytokines and the numbers of P. aeruginosa. However, it had no effect on lung function. No difference was found between the peripheral and bronchial inhalation mode. In conclusion, although no effect on lung function was observed, the clear reduction of airway inflammation after alpha(1)-antitrypsin treatment may precede pulmonary structural changes. The alpha(1)-antitrypsin deposition region may play a minor role for alpha(1)-antitrypsin inhalation in cystic fibrosis patients.

European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function

An increased or disturbed activation and aggregation of platelets plays a major role in the pathophysiology of thrombosis and haemostasis and is related to cardiovascular disease processes. In addition to qualitative disturbances of platelet function, changes in thrombopoiesis or an increased elimination of platelets, (e. g., in autoimmune thrombocytopenia), are also of major clinical relevance. Flow cytometry is increasingly used for the specific characterisation of phenotypic alterations of platelets which are related to cellular activation, haemostatic function and to maturation of precursor cells. These new techniques also allow the study of the in vitro response of platelets to stimuli and the modification thereof under platelet-targeted therapy as well as the characterisation of platelet-specific antibodies. In this protocol, specific flow cytometric techniques for platelet analysis are recommended based on a description of the current state of flow cytometric methodology. These recommendations are an attempt to promote the use of these new techniques which are at present broadly evaluated for diagnostic purposes. Furthermore, the definition of the still open questions primarily related to the technical details of the method should help to promote the multi-center evaluation of procedures with the goal to finally develop standardized operation procedures as the basis of interlaboratory reproducibility when applied to diagnostic testing.

Airway remodelling in children with cystic fibrosis

BACKGROUND: The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls. METHODS: Bronchoalveolar lavage and endobronchial biopsy were performed in a cross-sectional study of 43 children with CF (aged 0.3-16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy. RESULTS: Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase-9 (MMP-9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = -0.45, p<0.05; MMP-9:TIMP-1 ratio: r = -0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 microm vs 4.0 microm, p<0.01) and correlated positively with levels of transforming growth factor-beta(1) (TGF-beta(1); r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function. CONCLUSIONS: This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF-beta(1) concentration but independent of other markers of inflammation.

Methylprednisolone Fails to Preserve Pulmonary Surfactant and Blood-Air Barrier Integrity in a Porcine Cardiopulmonary Bypass Model

BACKGROUND: Pulmonary inflammation after cardiac surgery with cardiopulmonary bypass (CPB) has been linked to respiratory dysfunction and ultrastructural injury. Whether pretreatment with methylprednisolone (MP) can preserve pulmonary surfactant and blood-air barrier, thereby improving pulmonary function, was tested in a porcine CPB-model. MATERIALS AND METHODS: After randomizing pigs to placebo (PLA; n = 5) or MP (30 mg/kg, MP; n = 5), animals were subjected to 3 h of CPB with 1 h of cardioplegic cardiac arrest. Hemodynamic data, plasma tumor necrosis factor-alpha (TNF-alpha, ELISA), and pulmonary function parameters were assessed before, 15 min after CPB, and 8 h after CPB. Lung biopsies were analyzed for TNF-alpha (Western blot) or blood-air barrier and surfactant morphology (electron microscopy, stereology). RESULTS: Systemic TNF-alpha increased and cardiac index decreased at 8 h after CPB in PLA (P < 0.05 versus pre-CPB), but not in MP (P < 0.05 versus PLA). In both groups, at 8 h after CPB, PaO(2) and PaO(2)/FiO(2) were decreased and arterio-alveolar oxygen difference and pulmonary vascular resistance were increased (P < 0.05 versus baseline). Postoperative pulmonary TNF-alpha remained unchanged in both groups, but tended to be higher in PLA (P = 0.06 versus MP). The volume fraction of inactivated intra-alveolar surfactant was increased in PLA (58 +/- 17% versus 83 +/- 6%) and MP (55 +/- 18% versus 80 +/- 17%) after CPB (P < 0.05 versus baseline for both groups). Profound blood-air barrier injury was present in both groups at 8 h as indicated by an increased blood-air barrier integrity score (PLA: 1.28 +/- 0.03 versus 1.70 +/- 0.1; MP: 1.27 +/- 0.08 versus 1.81 +/- 0.1; P < 0.05). CONCLUSION: Despite reduction of the systemic inflammatory response and pulmonary TNF-alpha generation, methylprednisolone fails to decrease pulmonary TNF-alpha and to preserve pulmonary surfactant morphology, blood-air barrier integrity, and pulmonary function after CPB.

Air pollution during pregnancy and lung function in newborns: a birth cohort study

Post-natal exposure to air pollution is associated with diminished lung growth during school age. The current authors aimed to determine whether pre-natal exposure to air pollution is associated with lung function changes in the newborn. In a prospective birth cohort of 241 healthy term-born neonates, tidal breathing, lung volume, ventilation inhomogeneity and exhaled nitric oxide (eNO) were measured during unsedated sleep at age 5 weeks. Maternal exposure to particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)), nitrogen dioxide (NO(2)) and ozone (O(3)), and distance to major roads were estimated during pregnancy. The association between these exposures and lung function was assessed using linear regression. Minute ventilation was higher in infants with higher pre-natal PM(10) exposure (24.9 mL x min(-1) per microg x m(-3) PM(10)). The eNO was increased in infants with higher pre-natal NO(2) exposure (0.98 ppb per microg x m(-3) NO(2)). Post-natal exposure to air pollution did not modify these findings. No association was found for pre-natal exposure to O(3) and lung function parameters. The present results suggest that pre-natal exposure to air pollution might be associated with higher respiratory need and airway inflammation in newborns. Such alterations during early lung development may be important regarding long-term respiratory morbidity.

[Unilateral vs. bilateral lung transplantation in obstructive pulmonary disease]

BACKGROUND: The question whether patients suffering from end-stage emphysema who are candidates for lung transplantation should be treated with a single lung or with a double lung transplantation is still unanswered. METHODS: We reviewed 24 consecutive lung transplant procedures, comparing the results of 6 patients with an unilateral and 17 with a bilateral transplantation. PATIENTS AND RESULTS: After bilateral transplantation the patients showed a trend towards better blood gas exchange with shorter time on ventilator and intensive care compared patients after unilateral procedure. Three-year-actuarial survival was higher in the group after bilateral transplantation (83% versus 67%). There was a continuous improvement in pulmonary function in both groups during the first months after transplantation. Vital capacity and forced exspiratory ventilation therapies during the first second were significantly higher in the bilateral transplant group. CONCLUSION: Both unilateral and bilateral transplantation are feasible for patients with end-stage emphysema. Bilateral transplantation results in better pulmonary reserve capacity and faster rehabilitation.

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

Approaching atrial septal defects in pulmonary hypertension

Atrial septal defects (ASDs) are one of the most frequent congenital cardiac malformations, accounting for about 8-10% of all congenital heart defects. The prevalence of pulmonary arterial hypertension (PAH) in adults with an ASD is 8-10%. Different clinical PAH scenarios can be encountered. At one end of the spectrum are adults with no or only mild pulmonary vascular disease and a large shunt. These are patients who can safely undergo shunt closure. In the elderly, mild residual pulmonary hypertension after shunt closure is the rule. At the other end of the spectrum are adults with severe, irreversible pulmonary vascular disease, shunt reversal and chronic cyanosis, that is, Eisenmenger syndrome. These are patients who need to be managed medically. The challenge is to properly classify ASD patients with PAH falling in between the two ends of the spectrum as the ones with advanced, but reversible pulmonary vascular disease amenable to repair, versus the ones with progressive pulmonary vascular disease not responding to shunt closure. There are concerns that adults with progressive pulmonary vascular disease have worse outcomes after shunt closure than patients not undergoing shunt closure. Due to the correlation of pulmonary vascular changes and pulmonary hemodynamics, cardiac catheterization is used in the decision-making process. It is important to consider the hemodynamic data in the context of the clinical picture, the defect anatomy and further noninvasive tests when evaluating the option of shunt closure in these patients.

The chitinase-like protein YKL-40 modulates cystic fibrosis lung disease

The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.

Exogenous gonadotropins have little impact on follicular but considerable effect on serum cytokine concentrations – a comparison between Natural Cycle and stimulated IVF using a multiplexed assay platform

Introduction: Throughout follicular growth and subsequent corpus luteum formation the leukocyte number increases and follicular vascularisation changes. These processes are enhanced under exogenous stimulation with gonadotropins. Cytokines released by leukocytes contribute to further recruitment and vascularisation of the follicle, and they play an important role in regulating ovarian steroidogenesis by influencing theca and granulosa–lutein cell function. Changes in cytokine and vascular endothelial growth factor (VEGF) concentrations in the ovary as a consequence of gonadotropin stimulation may negatively influence oocyte quality. In this project we have compared the intrafollicular production of inflammatory cytokines and growth factors between natural IVF cycles (NC) and classical, gonadotropin-stimulated IVF cycles (gsIVF). Material and Methods: Serum on the day of oocyte retrieval and follicular fluid (FF) were collected in 37 NC and 39 gsIVF cycles. Thirteen women within this population underwent one NC and one gsIVF cycle each. A total of 14 cytokines from Bio-Plex panels I and II were determined in matched serum and FF samples using Luminex xMAP technology on the Bio-Plex(R) platform, using the serum protocol. Results: Tumour necrosis factor-alpha, RANTES, eotaxin and interferon-gamma-induced protein-10 levels were lower in FF than in serum, and thus not further investigated. Interleukin (IL)-6, -8, -10, -15, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median concentrations in FF than in serum, indicating possible ovarian production. Moreover, most of these showed higher evels in the gsIVF than in the NC groups in the serum, but not in the follicular fluid. IL-8 was reduced in gsIVF cycles. Conclusion: The fact that serum but not FF levels of the studied cytokines were higher in the stimulated than in the natural cycles can be attributed to the increased number of active follicles present after controlled ovarian stimulation.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Assessment of bronchodilator responsiveness in preschool children using forced oscillations

BACKGROUND: The forced oscillation technique (FOT) requires minimal patient cooperation and is feasible in preschool children. Few data exist on respiratory function changes measured using FOT following inhaled bronchodilators (BD) in healthy young children, limiting the clinical applications of BD testing in this age group. A study was undertaken to determine the most appropriate method of quantifying BD responses using FOT in healthy young children and those with common respiratory conditions including cystic fibrosis, neonatal chronic lung disease and asthma and/or current wheeze. METHODS: A pseudorandom FOT signal (4-48 Hz) was used to examine respiratory resistance and reactance at 6, 8 and 10 Hz; 3-5 acceptable measurements were made before and 15 min after the administration of salbutamol. The post-BD response was expressed in absolute and relative (percentage of baseline) terms. RESULTS: Significant BD responses were seen in all groups. Absolute changes in BD responses were related to baseline lung function within each group. Relative changes in BD responses were less dependent on baseline lung function and were independent of height in healthy children. Those with neonatal chronic lung disease showed a strong baseline dependence in their responses. The BD response in children with cystic fibrosis, asthma or wheeze (based on both group mean data and number of responders) was not greater than in healthy children. CONCLUSIONS: The BD response assessed by the FOT in preschool children should be expressed as a relative change to account for the effect of baseline lung function. The limits for a positive BD response of -40% and 65% for respiratory resistance and reactance, respectively, are recommended.

Oxygen-sensitive 3He-MRI in bronchiolitis obliterans after lung transplantation

Oxygen-sensitive 3He-MRI was studied for the detection of differences in intrapulmonary oxygen partial pressure (pO2) between patients with normal lung transplants and those with bronchiolitis obliterans syndrome (BOS). Using software developed in-house, oxygen-sensitive 3He-MRI datasets from patients with normal lung grafts (n = 8) and with BOS (n = 6) were evaluated quantitatively. Datasets were acqiured on a 1.5-T system using a spoiled gradient echo pulse sequence. Underlying diseases were pulmonary emphysema (n = 10 datasets) and fibrosis (n = 4). BOS status was verified by pulmonary function tests. Additionally, 3He-MRI was assessed blindedly for ventilation defects. Median intrapulmonary pO2 in patients with normal lung grafts was 146 mbar compared with 108 mbar in patients with BOS. Homogeneity of pO2 distribution was greater in normal grafts (standard deviation pO2 34 versus 43 mbar). Median oxygen decrease rate during breath hold was higher in unaffected patients (-1.75 mbar/s versus -0.38 mbar/s). Normal grafts showed fewer ventilation defects (5% versus 28%, medians). Oxygen-sensitive 3He-MRI appears capable of demonstrating differences of intrapulmonary pO2 between normal lung grafts and grafts affected by BOS. Oxygen-sensitive 3He-MRI may add helpful regional information to other diagnostic techniques for the assessment and follow-up of lung transplant recipients.