Demonstration of neutrophil chemotactic activity in the sputum of cystic fibrosis patients with Pseudomonas aeruginosa infection

The pathogenesis of tissue damage in the lungs of cystic fibrosis patients with chronic P. aeruginosa infection is quite complex and not well understood. Inflammatory cells, particularly neutrophils, are accumulated in the damaged tissues and in the sputum. This study demonstrates the presence of a heat-stable neutrophil chemotactic factor(s) in the sputum of CF patients. The chemotactic activity seems to be associated with the endotoxin present in the sputum. Chemotaxis of sol phase sputum was determined in a modified Boyden chamber assay. It was found that the sputum obtained from the patients showed strong chemotactic activity for peripheral blood neutrophils of normal individuals. The activity was greater than twice that of casein, a strong neutrophil chemo-attractant. Sputum at about dilution of 1:50 exhibited chemotactic activity equal to that of casein. Heat treatment of the sputum at 56 degrees C for 30 min, to inactivate complement, and at 100 degrees C for 15 min, to denature other proteins, somewhat reduced the chemotactic activity, but there was still strong chemotactic activity. The presence of endotoxin was demonstrated in both the non-heated and the heated samples by LAL and rocket immunoelectrophoresis. It is concluded that the sputum of CF patients contain chemotactic activity of heat-stable nature and most likely of bacterial origin endotoxin. These factors are involved in attraction of neutrophils to the lungs and sputum of these patients and contribute to the tissue damage of cystic fibrosis.     

Fenofibrate Attenuates Neutrophilic Inflammation in Airway Epithelia: Potential Drug Repurposing for Cystic Fibrosis

A hallmark of cystic fibrosis (CF) lung disease is neutrophilic airway inflammation. Elevated neutrophil counts have been associated with decreased forced expiratory volume in 1 second and poor clinical measures in patients with CF. Interleukin 8 (IL‐8), epithelial neutrophil activating protein 78 (ENA‐78), tumor necrosis factor alpha (TNF‐α), granulocyte macrophage colony‐stimulating factor (GM‐CSF), and granulocyte colony‐stimulating factor (G‐CSF) contribute to neutrophil activation and disease pathogenesis in the airways of patients with CF. Drugs that modify the production of these chemokines in the airways could potentially benefit CF patients. Thus, we determined the effects of fenofibrate on their production in cell populations obtained from the airways. Human small airway epithelial cells and CF bronchial epithelial cells were treated with IL‐1β to induce inflammation. We cotreated the cells with fenofibrate at concentrations ranging from 10 to 50 μM to determine if this drug could attenuate the inflammation. IL‐8, ENA‐78, TNF‐α, GM‐CSF, and G‐CSF production were measured from the cell culture supernates by ELISA. ANOVA statistical testing was conducted using SPSS 17.0. IL‐1β increased the production of each of the chemokines by several fold. Fenofibrate reduced IL‐1β induced production of each of these neutrophilic chemokines at the concentrations used. IL‐1β increases the production of neutrophilic chemokines in airway epithelial cells. Cotreatment with fenofibrate blunts these processes. Fenofibrate should be explored as a therapeutic option to modulate the abundant neutrophilic inflammation observed in CF.     

Purification and cytotoxic potential of myeloperoxidase in cystic fibrosis sputum

The neutrophil myeloperoxidase-H2O2-halide enzyme system produces hypochlorous acid and chlorinated amine compounds capable of killing a variety of target cells. In the present study we hypothesized that the myeloperoxidase enzyme system is one mechanism for airway epithelial damage in patients with cystic fibrosis (CF). Enzyme linked immunosorbent assay detected high antigenic levels of myeloperoxidase in sputum samples of seven patients with CF. Myeloperoxidase was purified to homogeneity from CF sputum and from blood neutrophils by a three-step technique involving dialysis, gel filtration, and ion-exchange chromatography. CF sputum myeloperoxidase and neutrophil myeloperoxidase appeared identical by acid gel electrophoresis and Ouchterlony experiments. CF sputum myeloperoxidase also contained approximately the same enzymatic activity as neutrophil myeloperoxidase. The myeloperoxidase enzyme system was tested for its cytotoxic potential in a tracheal ring culture system. Myeloperoxidase-induced cytotoxicity for airway epithelium was confirmed by light microscopy and radiolabelling experiments. These findings suggest a possible role for neutrophil myeloperoxidase in CF lung disease.     

Priming of neutrophils for enhanced oxidative burst by sputum from cystic fibrosis patients with Pseudomonas aeruginosa infection

Neutrophils accumulate in the lungs of cystic fibrosis (CF) patients and inflict tissue damage by release of oxygen radicals and proteases. Here we report on the ability of sputum to prime neutrophils for enhanced release of oxygen radicals. Sol phase was prepared by ultracentrifugation of sputum obtained from CF patients attending the CF Clinic, Rigshospitalet, Copenhagen. The oxidative burst response of neutrophils from healthy individuals was measured by oxygen consumption, superoxide production and chemiluminescence. Neutrophils were preincubated with sputum or buffer and then stimulated with f-Met-Leu-Phe or zymosan. Appropriate controls were included in the experiments. It was shown that neutrophils preincubated with CF sol phase and stimulated with f-Met-Leu-Phe generated a three- to five-fold higher chemiluminescence response than those preincubated with buffer. There was no enhancement of the response when zymosan was used for stimulation of the cells. Neutrophils incubated with sol phase alone exhibited no response. Attempts were made to identify and partially characterize the priming factor(s). It was found that the sputum samples contained bacterial endotoxins. The priming activity was resistant to heating at 100 degrees C for 15 min, and was present only in fractions with molecules larger than 100 KD. It is suggested that the priming factor(s) consist of bacterial endotoxins and/or immune complexes. Activation and enhanced release of oxygen radicals from neutrophils may play an important role in the host defence as well as pathogenesis of tissue damage in the lungs of these patients.     

哮喘患者诱导痰嗜酸细胞趋化因子的改变及其意义

目的探讨嗜酸细胞趋化因子（嗜酸细胞趋化因子）在嗜酸细胞性气道炎症和气流阻塞发生中的作用。方法收集支气管哮喘（A组）急性发作期患者30例和健康对照者（B组）20例，分别给予肺功能检测和痰诱导检查。用酶联免疫吸附法（ELISA）测定诱导痰上清液中嗜酸细胞趋化因子浓度。结果A组诱导痰嗜酸细胞占白细胞百分比（EOS／Leu％）与B组比较，差异有显著性（P〈0．05）；A组嗜酸细胞趋化因子浓度与B组比较，差异有显著性（P〈0．05）。A组EOS／Leu％、嗜酸细胞趋化因子浓度与一秒钟用力呼气容积占预计值百分比（FEV1％）均呈负相关；A组嗜酸细胞趋化因子浓度与EOS／Leu％呈正相关。结论嗜酸细胞趋化因子可能通过对嗜酸细胞（EOS）的选择性趋化作用使其释放一系列炎性介质参与了嗜酸细胞性气道炎症和气流阻塞的发生机制。     

Increased circulating levels of plasma ATP in cystic fibrosis patients

Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP‐binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin‐luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single‐blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0·01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the ΔF508 genotype had a 54% (n=33, P<0·01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0·4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.     

Prolastin aerosol therapy and sputum taurine in cystic fibrosis

BACKGROUND: Neutrophil elastase in the cystic fibrosis airways inhibits opsonophagocytosis and induces the expression of interleukin-8, a neutrophil chemoattractant. Prolastin is a therapeutic preparation of alpha-1 proteinase inhibitor (alpha1,-PI), a neutrophil elastase inhibitor. The objective of this study was to determine the effects of Prolastin aerosol therapy on airway inflammation in cystic fibrosis. METHODS: The primary endpoint of this study was sputum taurine, an amino-acid present in high concentrations in neutrophils. Sputum taurine correlates with respiratory exacerbations of cystic fibrosis. Seventeen patients with cystic fibrosis were each assigned to three sequential 10-day periods including first, aerosol therapy of 5 ml saline solution bid; second, aerosol therapy of 250 mg Prolastin bid; third, no aerosol therapy. On days 8, 9 and 10 of each period, early morning sputum was collected for the quantification of alpha1-PI, neutrophil elastase activity, IL-8 and taurine. RESULTS: During Prolastin therapy, a 3-fold increase in sputum alpha1-PI was observed (P = 0.002). Baseline values of sputum alpha1-PI correlated with the values obtained after Prolastin aerosol (R = 0.77, P < 0.01). Sputum neutrophil elastase activity remained unchanged but taurine decreased after Prolastin therapy (during therapy P = 0.052, after therapy P = 0.026). Prolastin aerosol therapy had no adverse effect on pulmonary function. CONCLUSIONS: Aerosol therapy with Prolastin in patients with cystic fibrosis leads to a progressive decrease in sputum taurine. This suggests that even in the absence of sustained elastase inhibition, Prolastin aerosol therapy may have a beneficial effect on airway inflammation in patients with cystic fibrosis.     

Neutrophils and macrophages related to the pathogenesis and disease development of chronic obstructive pulmonary disease by the inflammatory reaction

Chronic obstructive pulmonary disease (COPD) is a chronic airway disorder characterized by obstructive airflow limitation which is not completely reversible with treatment. Inflammatory changes in the peripheral airways, especially those with the diameter less than 2mm (so-called small airway disease) have been speculated to be initial steps of COPD. And so it must be quite clear that neutrophils and macrophages play an essential role in the pathogenesis of these lesions. Studies with bronchoalveolar lavage demonstrated an increase in neutrophil numbers and the neutrophil chemoattractant interleukin-8. Recent studies demonstrated that neutrophils and macrophages are increased and contain a variety of proteases, which are involved in cell infiltration and activation. Studies with gene-engineered animals and anti-cytokine treatment will facilitate better understanding the role of neutrophils and macrophages, and eventual novel therapy.     

EPI-hNE4, a proteolysis-resistant inhibitor of human neutrophil elastase and potential anti-inflammatory drug for treating cystic fibrosis

EPI-hNE4 (depelstat) is a potent inhibitor of human neutrophil elastase derived from human inter-alpha-trypsin inhibitor and designed to control the excess proteolytic activity in the sputum of cystic fibrosis patients. We analyzed its resistance to the proteolysis it is likely to encounter at inflammatory sites in vivo. EPI-hNE4 resisted hydrolysis by neutrophil matrix metalloproteases (MMPs) and serine proteases that are released from activated neutrophils in inflammatory lung secretions, including MMP-8 and MMP-9, and the elastase-related protease 3 and cathepsin G. It also resisted degradation by epithelial lung cell MMP-7 but was broken down by the Pseudomonas aeruginosa metalloelastase pseudolysin, when used in a purified system, but not when this protease competed with equimolar amounts of neutrophil elastase. We also investigated the inhibitory properties of EPI-hNE4 at the surface of purified blood neutrophils and in the sputum of cystic fibrosis patients where neutrophil elastase is in both a soluble and a gel phase. The elastase at the neutrophil surface was fully inhibited by EPI-hNE4 and formed soluble complexes. The elastase in cystic fibrosis sputum supernatants was inhibited by stoichiometric amounts of EPI-hNE4, allowing titration of the protease. But the percentage of inhibition in whole sputum homogenates varied from 50 to 100%, depending on the sample tested. EPI-hNE4 was rapidly cleaved by the digestive protease pepsin in vitro. Therefore, EPI-hNE4 seems to be an elastase inhibitor suitable for use in aerosols to treat patients with cystic fibrosis.     

Chronic Pseudomonas aeruginosa infection in cystic fibrosis airway disease: metabolic changes that unravel novel drug targets

The cystic fibrosis (CF) airways have an incompletely characterized defect in innate defense that eventually leads to bacterial infection and airway inflammation. Persistent Pseudomonas aerugionsa infection resulting from defective innate immunity and a bacterial phenotypic switch to a more intractable mucoid form inside the airway are now well established as important components of CF pathogenesis. Broad-based factors leading to chronic infection will be discussed with respect to: bacterial virulence in the context of biofilm formation, quorum sensing machinery and alginate overproduction, and failure of innate lung immunity in CF airways. In addition, a controversial question as to whether inflammation or infection comes first during CF airway disease will be addressed. Finally, a new hypothesis, that P. aeruginosa thrives as biofilms within the thickened anaerobic mucus layers, will be developed.     

Upper airflow obstruction and pulmonary function in acromegaly: relationship to disease activity

Pulmonary function and disease activity were assessed in a large series of patients with acromegaly (19 men and 16 women). Large lungs, defined as a vital capacity greater than 120 per cent of predicted normal occurred in six of 19 males and six of 16 females. Ten of the 12 patients with elevated vital capacity had active disease (growth hormone greater than 5 mU/l during a glucose tolerance test). There was no association with duration of disease. Diffusing capacity was normal overall but DLCO greater than 120 per cent occurred in six of 14 females and one of 18 males. Significant intrathoracic airflow obstruction occurred in eight of 35 patients, six of whom were nonsmokers. Upper (extrathoracic) airflow obstruction was the most common pulmonary function abnormality. A maximal expiratory/inspiratory flow ratio greater than 1.0 at 50 per cent vital capacity occurred in 13 of 18 males and four of 16 female patients, and there was an association with disease activity (17 of 25 subjects with active disease had upper airflow obstruction compared to one of nine in remission; p = 0.01). Nocturnal hypoxaemia occurred in three of 13 patients studied: six of this group had upper airways obstruction. They were all male with elevated growth hormone levels and upper airflow obstruction. In summary, in 35 acromegalics (26 with active disease), large lungs occurred in 12 patients (34 per cent) and upper airflow obstruction in 17 patients (50 per cent). The latter may develop nocturnal hypoxaemia--this was seen in three of six patients with upper airflow obstruction. Upper airways obstruction was more common in males (13 of 18 compared to four of 16 females; p = 0.04) and its presence in males should arouse suspicion of nocturnal hypoxaemia.     

Inhaled hypertonic saline for cystic fibrosis: Reviewing the potential evidence for modulation of neutrophil signalling and function

Cystic fibrosis(CF) is a multisystem disorder with significantly shortened life expectancy. The major cause of mortality and morbidity is lung disease with increasing pulmonary exacerbations and decline in lung function predicting significantly poorer outcomes. The pathogenesis of lung disease in CF is characterised in part by decreased airway surface liquid volume and subsequent failure of normal mucociliary clearance. This leads to accumulation of viscous mucus in the CF airway, providing an ideal environment for bacterial pathogens to grow and colonise, propagating airway inflammation in CF. The use of nebulised hypertonic saline(HTS) treatments has been shown to improve mucus clearance in CF and impact positively upon exacerbations, quality of life, and lung function. Several mechanisms of HTS likely improve outcome, resulting in clinically relevant enhancement in disease parameters related to increase in mucociliary clearance. There is increasing evidence to suggest that HTS is also beneficial through its anti-inflammatory properties and its ability to reduce bacterial activity and biofilm formation. This review will first describe the use of HTS in treatment of CF focusing on its efficacy and tolerability. The emphasis will then change to the potential benefits of aerosolized HTS for the attenuation of receptor mediated neutrophil functions, including down-regulation of oxidative burst activity, adhesion molecule expression, and the suppression of neutrophil degranulation of proteolytic enzymes.     

Migrainous Infarction in a Patient With Sporadic Hemiplegic Migraine and Cystic Fibrosis: A 99mTc-HMPAO Brain SPECT Study

Genetic mutations of sporadic hemiplegic migraine (SHM) are mostly unknown. SHM pathophysiology relies on cortical spreading depression (CSD), which might be responsible for ischemic brain infarction. Cystic fibrosis (CF) is caused by a monogenic mutation of the chlorine transmembrane conductance regulator (CFTR), possibly altering brain excitability. We describe the case of a patient with CF, who had a migrainous stroke during an SHM attack. A 32-year-old Caucasian male was diagnosed with CF, with heterozygotic delta F508/unknown CFTR mutation. The patient experiences bouts of coughing sometimes triggering SHM attacks with visual phosphenes, aphasia, right-sided paresthesia, and hemiparesis. He had a 48-hour hemiparesis triggered by a bout of coughing with hemoptysis, loss of consciousness, and severe hypoxia-hypercapnia. MRI demonstrated transient diffusion hyperintensity in the left frontal-parietal-occipital regions resulting in a permanent infarction in the primary motor area. Later, a brain perfusion SPECT showed persistent diffuse hypoperfusion in the territories involved in diffusion-weighted imaging alteration. Migrainous infarction, depending on the co-occurrence of 2 strictly related phenomena, CSD and hypoxia, appears to be the most plausible explanation. Brain SPECT hypoperfusion suggests a more extensive permanent neuronal loss in territories affected by aura. CF may be then a risk factor for hemiplegic migraine and stroke since bouts of coughing can facilitate brain hypoxia, triggering auras.     

Analysis of exocrine pancreatic function in cystic fibrosis: one mild CFTR mutation does not exclude pancreatic insufficiency

BACKGROUND: Cystic fibrosis (CF) is the most common cause of exocrine pancreatic insufficiency in childhood. The aim of the present study is to evaluate the correlation between genotype and exocrine pancreatic insufficiency in CF patients. The special emphasis was put on the analysis of mild CFTR mutations. DESIGN: The study comprised 394 CF patients and 105 healthy subjects (HS). Elastase-1 concentrations were measured in all subjects. RESULTS: Severe pancreatic insufficiency was associated with the presence of two CFTR gene mutations (DeltaF508, N1303K, CFTR dele 2,3 (21kb), G542X, 1717-1G-A, R533X, W1282X, 621GT, 2183AAG, R560T, 2184insA and DeltaI507, G551D, 895T) and mild insufficiency with the presence of at least one mutation (R117H, 3171insC, A155P2, 138insL, 296 + 1G-A, E92GK, E217G, 2789 + 5G-A. 3849 + 1kbC-T/3849 + 1kbC-T) genotype resulted in high elastase-1-values. However, in case of patients with genotype DeltaF508/3849 + 10kbC-T, 1717-1GA/3849 + 10kbC-T as well as with DeltaF508/R334W, both high and low elastase-1 concentrations were found. Low E1 values were found in a patient with DeltaF508/R347P genotype. CONCLUSION: Patients who carry two 'severe' mutations develop pancreatic insufficiency, whereas those who carry at least one 'mild' usually remain pancreatic sufficient. However, the presence of one mild mutation does not exclude pancreatic insufficiency.     

Contributions of loss of lung recoil and of enhanced airways collapsibility to the airflow obstruction of chronic bronchitis and emphysema

We investigated the contributions of intrinsic disease of the airways, loss of lung recoil and enhanced airway collapsibility to the airflow obstruction of 17 patients with chronic bronchitis and emphysema. Airways conductance at low flow (G(aw)), maximum expiratory flow (V(E, MAX)) and static lung recoil pressure [P(st) (l)] were measured at different lung volumes, and conductance-static recoil pressure and maximum flow-static recoil pressure curves constructed. Low values of DeltaG(aw)/DeltaP(st) (l) and DeltaV(E), max/DeltaP(st) (l) were attributed to intrinsic airways disease. Airway collapsibility was assessed by comparing G(aw) with upstream conductance on forced expiration and by the intercept of the maximum flow-static recoil curve on the static recoil pressure axis (P(tm)').All patients had reduced G(aw) at all volumes but in seven DeltaG(aw)/DeltaP(st) (l) was normal. On forced expiration, maximum flow in all patients was reduced more than could be accounted for by loss of lung recoil. DeltaV(E, MAX)/DeltaP(st) (l) was reduced in the patients in whom DeltaG(aw)/P(st) (l) was low. In contrast DeltaV(E, MAX)/DeltaP(st) (l) was normal in three and only slightly reduced in another three of the seven patients with normal DeltaG(aw)/DeltaP(st) (l). In these patients G(aw) greatly exceeded upstream conductance and P(tm)' was increased.We conclude that loss of lung recoil could account for the reduction in resting airways dimensions in 7 of the 17 patients. Enhanced airway collapsibility commonly contributed to reduction in maximum flow. In three patients the airflow obstruction could be entirely accounted for by loss of lung recoil and enhanced airway collapsibility.     

慢性阻塞性肺疾病CT定量参数改变及其与肺功能指标的关系

目的 观察未经治疗COPD患者CT定量参数变化及其与肺功能指标的关系。方法 回顾性分析99例未经治疗COPD患者资料;根据气流受限程度分为A组(GOLD 1级,轻度受限,36例)、B组(GOLD 2级,中度受限,37例)及C组(GOLD 3、4级,明显受限,26例);分析首检及随访胸部CT定量参数、肺功能指标的差异及其相关性。CT定量参数包括全肺容积、低衰减区百分比(LAA%)、肺血管总计数与肺表面积比值(N_total/LSA)、截面积<5 mm²的肺血管计数与肺表面积比值(N_{<5 mm²}/LSA)、理论气道内周长为10 mm的管壁面积平方根(Pi10)、全肺气道壁体积、壁厚及壁面积百分比(WA%)等,而肺功能指标包括吸入支气管舒张剂后第1秒用力呼气容积(FEV₁)、用力肺活量(FVC)及二者比值(FEV₁/FVC)和FEV₁率(FEV₁%)。结果 相比首检各项指标,随访时各组FVC、FEV_...     

Mechanical airway clearance using the frequencer electro-acoustical transducer in cystic fibrosis

PURPOSE: Clearance of mucus from airways is the cornerstone of therapy for lung disease in patients with cystic fibrosis (CF). This paper describes the operation of the Frequencer, a novel respiratory physiotherapy device comprised of an electro-acoustical transducer. We hypothesized that the Frequencer would be a safe and effective therapy to help clear secretions from the airways of subjects with CF. METHODS: To verify this hypothesis, 22 individuals with CF were recruited to this study comparing sputum production during conventional chest physiotherapy (CCPT) and Frequencer therapy using a crossover design. The sputum weight was the main outcome measure. RESULTS: Sputum weight was found to be a reproducible measure of the efficacy of chest physiotherapy in individual patients. The Frequencer induced airway clearance in patients with CF that was equivalent to that of CCPT. Furthermore, treatment of a 4% mucin preparation ex vivo with the Frequencer significantly reduced the viscosity of the mucin solution as determined in a capillary rheometer. CONCLUSIONS: These results indicate the Frequencer is safe and as effective as CCPT in inducing airway clearance in patients with CF.     

Oncogene-mediated propagation of tracheal epithelial cells from two cystic fibrosis fetuses with different mutations. Characterization of CFT-1 and CFT-2 cells in culture

Abstract. Primary tracheal epithelial cells obtained from two fetuses with cystic fibrosis (CF) were successfully transfected with a plasmid vector recombined with the large T oncogene of SV40. The resulting tracheal cells were propagated in culture for up to 25 passages and retained the mutations of the CF genes carried by the two fetuses, one heterozygous for the S549N and N1303K substitutions (CFT-I cells), and the other homozygous for the most common deletion ΔF508 (CFT-2 cells). The transfected cells: (a) expressed the SV40 large T oncogene, as determined by immunofluorescence and Northern blot analysis; (b) retained typical epithelial morphology, as assessed by the presence of microvilli, desmosomes, gap junctions, and cytokeratin expression; (c) were fully responsive to the cAMP-stimulating agents isproterenol, forskolin and vasoactive intestinal peptide for cAMP production and PKA activation; (d) do not produce any tumour in the athymic nude mice; (e) were diploid and tetraploid with a normal chromosomal complement at early passages, and (f) exhibited the abnormal regulation of chloride conductance characteristic of CF.
These results indicate that CFT-1 and CFT-2 cells constitute a suitable model for: (a) comparison of the maturation and function of the CFTR protein mutated in the two nucleotide-binding domains; (2) analysis of the biochemical defect in CF epithelial airway cells, (c) development of new therapeutic agents, and correction of the CF defect by gene replacement therapy in vitro .     

A strategy to deliver genes to cystic fibrosis lungs: A battle with environment

Cystic fibrosis (CF) sputum, a tenacious biopolymer network accumulating in the airways, critically interferes with the effectiveness of pulmonary gene delivery. To overcome this challenge, nanoparticulate ternary gene-polymer complexes were encapsulated in inhalable dry microparticles containing mannitol. When applied on a layer of artificial sputum, which comprised major components of CF sputum such as DNA and mucin, mannitol microparticles rapidly dissolved in it and enhanced transport of nanoparticles across the sputum layer. Despite the improvement of nanoparticle transport in the artificial sputum, the gene-polymer complex passing the sputum did not show gene transfection because of the significant inactivation by DNA and, to a lesser extent, mucin. Particle size measurement suggested that aggregation of the gene transfer agents was mainly responsible for the activity loss. These results indicate that the delivery of gene transfer agent across CF sputum depended not only on the ability to penetrate the sputum but also on preservation of the activity during and/or after the transport.