Atelectasis and survival after bronchoscopic lung volume reduction for COPD.

Bronchoscopic therapies to reduce lung volumes in chronic obstructive pulmonary disease are intended to avoid the risks associated with lung volume reduction surgery (LVRS) or to be used in patient groups in whom LVRS is not appropriate. Bronchoscopic lung volume reduction (BLVR) using endobronchial valves to target unilateral lobar occlusion can improve lung function and exercise capacity in patients with emphysema. The benefit is most pronounced in, though not confined to, patients where lobar atelectasis has occurred. Few data exist on their long-term outcome. 19 patients (16 males; mean±sd forced expiratory volume in 1 s 28.4±11.9% predicted) underwent BLVR between July 2002 and February 2004. Radiological atelectasis was observed in five patients. Survival data was available for all patients up to February 2010. None of the patients in whom atelectasis occurred died during follow-up, whereas eight out of 14 in the nonatelectasis group died (Chi-squared p=0.026). There was no significant difference between the groups at baseline in lung function, quality of life, exacerbation rate, exercise capacity (shuttle walk test or cycle ergometry) or computed tomography appearances, although body mass index was significantly higher in the atelectasis group (21.6±2.9 versus 28.4±2.9 kg·m(-2); p<0.001). The data in the present study suggest that atelectasis following BLVR is associated with a survival benefit that is not explained by baseline differences.

Mannose-binding lectin is present in the infected airway: a possible pulmonary defence mechanism.

BACKGROUND: Mannose-binding lectin (MBL) deficiency has been associated with infections of the respiratory tract and with increased disease severity in cystic fibrosis (CF). The mechanism is uncertain, and could relate either to systemic or local effects. The aim of this study was to determine, in a large cohort of children, whether MBL is present on the airway surface in health or disease. METHODS: Bronchoalveolar lavage (BAL) fluid from children with and without respiratory infection (some with underlying disease) was analysed for MBL and neutrophil elastase (NE). Levels were compared between groups, and correlations were examined with local and systemic inflammatory markers, infective organisms and load. RESULTS: 85 children were recruited to the study. MBL was absent in the lavage of all 7 children without lung infection but present in 62% (8/13) of those with acute pneumonia/pneumonitis, 23% (5/22) with recurrent respiratory tract infections, 17% (1/6) with primary ciliary dyskinesia and 8% (3/37) with CF (p<0.01). Children with acute pneumonia/pneumonitis had significantly higher levels than those in the other groups. There was no relationship with organisms cultured or systemic markers of inflammation, although in the group with detectable MBL in the BAL fluid, the levels correlated positively with levels of NE. CONCLUSIONS: MBL is undetectable in the non-infected airway but is present in a significant number of samples from children with lung infection. The levels found in the BAL fluid could be physiologically active and the protein may therefore be playing a role in host defence.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Reproducibility of dynamically represented acoustic lung images from healthy individuals.

BACKGROUND AND AIM: Acoustic lung imaging offers a unique method for visualising the lung. This study was designed to demonstrate reproducibility of acoustic lung images recorded from healthy individuals at different time points and to assess intra- and inter-rater agreement in the assessment of dynamically represented acoustic lung images. METHODS: Recordings from 29 healthy volunteers were made on three separate occasions using vibration response imaging. Reproducibility was measured using quantitative, computerised assessment of vibration energy. Dynamically represented acoustic lung images were scored by six blinded raters. RESULTS: Quantitative measurement of acoustic recordings was highly reproducible with an intraclass correlation score of 0.86 (very good agreement). Intraclass correlations for inter-rater agreement and reproducibility were 0.61 (good agreement) and 0.86 (very good agreement), respectively. There was no significant difference found between the six raters at any time point. Raters ranged from 88% to 95% in their ability to identically evaluate the different features of the same image presented to them blinded on two separate occasions. CONCLUSION: Acoustic lung imaging is reproducible in healthy individuals. Graphic representation of lung images can be interpreted with a high degree of accuracy by the same and by different reviewers.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Use of ultrasound to enhance nonviral lung gene transfer in vivo.

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.

Computed tomography and cystic fibrosis: promises and problems.

Computed tomography (CT) has two potential roles in the evaluation of patients with cystic fibrosis (CF) lung disease: as a diagnostic test primarily for the detection of supervening complications and as a monitoring tool in clinical research. Interest in the latter role has gained momentum in the last 5 years because of two factors: (1) therapeutic options for CF lung disease are developing rapidly, hence the need for an outcome measure that can be applied in clinical intervention trials; and (2) it has become clear that traditional outcome measures such as pulmonary function tests are relatively insensitive to the early structural damage that occurs in CF. Several recent studies have shown that CT can be used as a potential surrogate outcome measure, although its suitability for this specific role is controversial and still under investigation. This review summarises current concepts relating to the research applications of CT in CF, with particular emphasis on the evidence supporting the use of CT as a surrogate outcome measure in clinical trials.

Gene therapy progress and prospects: cystic fibrosis.

Our first review on progress and prospects in cystic fibrosis (CF) gene therapy was published in this series in October 2002. We now summarize the progress made since then and comment on the prospects for CF gene therapy over the next couple of years. Three clinical trials have been carried out, further supporting the proof-of-principle that gene transfer to the airway epithelium is feasible. Developments in viral and non-viral vectors, as well as recent alternative strategies such as gene repair, trans-splicing and stem cell therapy will be reviewed.

Intravenously administered oligonucleotides can be delivered to conducting airway epithelium via the bronchial circulation.

Topical gene transfer to the airways of cystic fibrosis (CF) patients has been inefficient, partly due to extracellular barriers such as sputum. In an attempt to circumvent these, we assessed whether airway epithelial cells can be transfected by intravenous (i.v.) administration of liposome-complexed or "naked" oligonucleotides (ODNs). The conducting airways are the likely target for CF therapy and are supplied by the bronchial circulation. Consequently, we assessed ODN transfer in the mouse trachea and main bronchi as these are supplied by the bronchial circulation. Liposome-protamine-DNA (LPD) complexes were detected in the bronchial circulation but did not transfect conducting airway epithelial cells, even in the presence of microvascular leakage. In contrast, 'naked' ODNs were delivered to 17% (inter-quartile range (IQR) 10-34%) and 35% (IQR 24-59%) of epithelial cells when injected at 500 microg/animal, without and with microvascular leakage, respectively. Two types of nuclear signal were observed; punctate in cells throughout the airways (3%, IQR 2-6%, and 6%, IQR 4-7%, of cells when delivered without and with microvascular leakage, respectively) and diffuse in a small number of epithelial cells in the proximal trachea. ODNs may be relevant to CF in a variety of ways and these data suggest one way towards implementing their use.

Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo.

We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.

Effect of tolerance induction to immunodominant T-cell epitopes of Sendai virus on gene expression following repeat administration to lung.

Sendai virus (SeV) is able to transfect airway epithelial cells efficiently in vivo. However, as with other viral vectors, repeated administration leads to reduced gene expression. We have investigated the impact of inducing immunological tolerance to immunodominant T-cell epitopes on gene expression following repeated administration. Immunodominant CD4 and CD8 T-cell peptide epitopes of SeV were administered to C57BL/6 mice intranasally 10 days before the first virus administration with transmission-incompetent F-protein-deleted DeltaF/SeV-GFP. At 21 days after the first virus administration, mice were again transfected with DeltaF/SeV. To avoid interference of anti-GFP antibodies, the second transfection was carried out with DeltaF/SeV-lacZ. At 2 days after the final transfection lung beta-galactosidase expression, T-cell proliferation and antibody responses were measured. A state of 'split tolerance' was achieved with reduced T-cell proliferation, but no impact on antiviral antibody production. There was no enhancement of expression on repeat administration; instead, T-cell tolerance was, paradoxically, associated with a more profound extinction of viral expression. Multiple immune mechanisms operate to eradicate viruses from the lung, and these findings indicate that impeding the adaptive T-cell response to the immunodominant viral epitope is not sufficient to prevent the process.

Keratinocyte growth factor therapy in murine oleic acid-induced acute lung injury.

Alveolar type II (ATII) cell proliferation and differentiation are important mechanisms in repair following injury to the alveolar epithelium. KGF is a potent ATII cell mitogen, which has been demonstrated to be protective in a number of animal models of lung injury. We have assessed the effect of recombinant human KGF (rhKGF) and liposome-mediated KGF gene delivery in vivo and evaluated the potential of KGF as a therapy for acute lung injury in mice. rhKGF was administered intratracheally in male BALB/c mice to assess dose response and time course of proliferation. SP-B immunohistochemistry demonstrated significant increases in ATII cell numbers at all rhKGF doses compared with control animals and peaked 2 days following administration of 10 mg/kg rhKGF. Protein therapy in general is very expensive, and gene therapy has been suggested as a cheaper alternative for many protein replacement therapies. We evaluated the effect of topical and systemic liposome-mediated KGF-gene delivery on ATII cell proliferation. SP-B immunohistochemistry showed only modest increases in ATII cell numbers following gene delivery, and these approaches were therefore not believed to be capable of reaching therapeutic levels. The effect of rhKGF was evaluated in a murine model of OA-induced lung injury. This model was found to be associated with significant alveolar damage leading to severe impairment of gas exchange and lung compliance. Pretreatment with rhKGF 2 days before intravenous OA challenge resulted in significant improvements in PO2, PCO2, and lung compliance. This study suggests the feasibility of KGF as a therapy for acute lung injury.

Gene therapy for cystic fibrosis: an example for lung gene therapy.

Gene therapy is currently being evaluated for a wide range of acute and chronic lung diseases. The requirement of gene transfer into the individual cell types of the complex lung structure will very much depend on the target disease. Over the last decade, the gene therapy community has recognized that there is not even one vector that is good for all applications, but that the gene transfer agent has to be carefully chosen. Gene therapy is particularly attractive for diseases that currently do not have satisfactory treatment options and probably easier for monogenic disorders than for complex diseases. Cystic fibrosis (CF) fulfills these criteria and is therefore a good candidate for gene therapy-based treatment. This review will focus on CF as an example for lung gene therapy and discuss the progress made in this field over the last couple of years.

A defective nontransmissible recombinant Sendai virus mediates efficient gene transfer to airway epithelium in vivo.

Recombinant Sendai virus (SeV)-mediated gene transfer to differentiated airway epithelial cells has shown to be very efficient, because of its ability to overcome the intra- and extracellular barriers known to limit gene delivery. However, this virus is transmission competent and therefore unlikely to be suitable for use in clinical trials. A nontransmissible, replication-competent recombinant SeV has recently been developed by deleting the envelope Fusion (F) protein gene (SeV/DeltaF). Here we show that SeV/DeltaF is able to mediate beta-galactosidase reporter gene transfer to the respiratory tract of mice in vivo, as well as to human nasal epithelial cells in vitro. Further, in an ex vivo model of differentiated airway epithelium, SeV/DeltaF gene transfer was not importantly inhibited by native mucus. When compared to the transmission-competent SeV in vivo, no difference in gene expression was observed at the time of peak expression. The development of an F-defective nontransmissible SeV, which can still efficiently mediate gene transfer to the airway epithelium, represents the first important step towards the use of a cytoplasmic RNA viral vector in clinical trials of gene therapy.

Effects of intramyocardial pVEGF165 delivery on regional myocardial blood flow: evidence for a spatial 'delivery-efficacy' mismatch.

The aim of this study was to determine the effects of direct intramyocardial pVEGF165 gene transfer on regional myocardial blood flow in a porcine model of chronic myocardial ischaemia. Pigs underwent placement of an ameroid constrictor around the left circumflex coronary artery. After 3 weeks, animals received direct intramyocardial injections of pVEGF165 (20 x 50 microl at 1 microg/microl, n=11) or a plasmid vector encoding chloramphenicol acetyltransferase (20 x 50 microl at 1 microg/microl, n=11) into a specified target area (TA) of the left lateral wall. At 3 weeks after gene transfer, animals underwent final evaluation including a systematic assessment of regional myocardial blood flow (MBF) under resting and stress conditions. In all, 20 animals (10 per group) reached final studies. There was no change in mean arterial blood pressure or Rentrop collateral score from gene delivery to final studies in either group, nor were there differences between study groups. MBF was significantly higher in the areas adjacent to the TA in the VEGF group under resting (P<0.001) and stress conditions (P<0.05). In addition, pVEGF165 gene transfer abolished flow differences between the adjacent areas and the septum. MBF was not different between study groups in the TA, the anterior wall, or the septum. In conclusion, direct intramyocardial pVEGF165 gene transfer significantly improves myocardial blood flow. However, this effect is limited to the myocardial segments adjacent to the area of gene delivery. These data, therefore, demonstrate a spatial 'delivery-efficacy' mismatch with implications for myocardial gene delivery sites and detection of treatment effects in vivo.

Poly (D, L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged transgene expression in airway epithelium in vitro, ex vivo and in vivo.

Repeat administration of gene therapy for cystic fibrosis is likely to be essential for long-term clinical efficacy. This may be minimized by the use of slow-release gene transfer preparations with more prolonged expression and longer dosing intervals for the patient. Poly(D-L-lactide-co-glycolide) (PLG) is a biodegradable and biocompatible polymer that has been used to encapsulate plasmid DNA. PLG-DNA microspheres were generated and characterized with respect to morphology, size (80% of particles <5.2 microm), and encapsulation efficiency (50.7+/-2.3%, n=6). Gel electrophoresis of DNA re-extracted from the microspheres confirmed that despite a decrease in the proportion of supercoiled conformation, it had not been degraded by the preparation process. Gene transfer efficiency was tested using microspheres encapsulating the reporter gene beta-galactosidase in vitro on Cos 7 cells and a CF airway epithelial line (CFTEo approximately ) and ex vivo in a sheep tracheal (s.t.) model. In both cases, transgene expression was significantly (P<0.01) lower at the first time point tested (24 h in vitro, 48 h ex vivo) compared to lipid-#67-mediated gene transfer. However, PLG-mediated expression in vitro was sustained at 48 h, while lipid #67-mediated expression levels had dropped significantly (P<0.05) to 50.3+/-13.7 and 38.2+/-2.7% (Cos 7 and CFTEo approximately cells, respectively) of the 24-h level. This pattern was also seen in the s.t. model where at 72 h, PLG-mediated expression was 125.4+/-7.2% of the 48-h level demonstrating significantly (P<0.05) better retention of transfection efficiency than lipid #67, where levels had fallen to approximately half the 48 h level. By 96 h, expression was still retained in the PLG-transfected group (87.3+/-12.5% of 48 h expression) but was undetectable in the lipid -#67-transfected s.t. Finally, PLG microspheres, encapsulating the reporter gene chloramphenicol transferase (CAT, 80 microg) were instilled intranasally into Balb/C mice. Compared to lipid-#67-mediated delivery, where whole lung CAT expression was highest at 48 h (13.7 x 10(3)+/-0.05 CAT U/microg protein, n=6) and then not detectable at further time points, CAT expression was not detectable in PLG-transfected mice at 48 h, but was detectable at 7, 14 and 21 days after transfection. These data demonstrate that PLG-mediated gene transfer can produce prolonged gene expression in airway epithelia. However, gene transfer efficiency still requires significant improvement.

Immunological hurdles to lung gene therapy.

Gene delivery has the potential to offer effective treatment to patients with life-threatening lung diseases such as cystic fibrosis, alpha1-antitrypsin deficiency and lung cancer. Phase I/II clinical trials have shown that, in principle, gene transfer to the lung is feasible and safe. However, gene expression from both viral and non-viral gene delivery systems has been inefficient. In addition to extra- and intracellular barriers, the host innate and acquired immune system represents a major barrier to successful gene transfer to the lung. Results from studies in experimental animals and clinical trials have shown that inflammatory, antibody and T cell responses can limit transgene expression duration and readministration of the gene transfer vector. We will review here how the development of pharmacological and/or immunological agents can modulate the host immune system and the limitations of these strategies. A better understanding of the immunological barriers which exist in the lung might allow for a more sustained expression of the transgene and importantly help overcome the problem of readministration of viral vectors.