Ewfw Alton

Lung clearance index is a sensitive, repeatable and practical measure of airways disease in adults with cystic fibrosis

Background: Lung clearance index (LCI) is a sensitive marker of early lung disease in children but has not been assessed in adults. Measurement is hindered by the complexity of the equipment required. The aims of this study were to assess performance of a novel gas analyser (Innocor) and to use it as a clinical tool for the measurement of LCI in cystic fibrosis (CF). Methods: LCI was measured in 48 healthy adults, 12 healthy school-age children and 33 adults with CF by performing an inert gas washout from 0.2% sulfur hexafluoride (SF6). SF6 signal:noise ratio and 10–90% rise time of Innocor were compared with a mass spectrometer used in similar studies in children. Results: Compared with the mass spectrometer, Innocor had a superior signal:noise ratio but a slower rise time (150 ms vs 60 ms) which may limit its use in very young children. Mean (SD) LCI in healthy adults was significantly different from that in patients with CF: 6.7 (0.4) vs 13.1 (3.8), p<0.001. Ten of the patients with CF had forced expiratory volume in 1 s ⩾80% predicted but only one had a normal LCI. LCI repeats were reproducible in all three groups of subjects (mean intra-visit coefficient of variation ranged from 3.6% to 5.4%). Conclusions: Innocor can be adapted to measure LCI and affords a simpler alternative to a mass spectrometer. LCI is raised in adults with CF with normal spirometry, and may prove to be a more sensitive marker of the effects of treatment in this group.

Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal. Gene Therapy (2001) 8, 1380–1386.

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/ΔF). Here we show that SeV/ΔF is able to mediate β-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/ΔF 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.

A mild variant of cystic fibrosis.

Background. Cystic fibrosis is the most common lethal autosomal recessive disorder among whites. Among Dutch patients with cystic fibrosis, JF508 is the most common mutation and A455E the second most common mutation of the cystic fibrosis transmembrane conductance regulator gene on chromosome 7. A455E is associated with preserved pancreatic function and residual secretion of chloride across membranes. We investigated whether it is also associated with less severe pulmonary disease in patients with cystic fibrosis. Methods. A total of 33 patients with compound heterozygosity for the A455E mutation were matched according to age and sex with patients who were homozygous for the JF508 mutation. The pairs were analyzed with respect to the following outcome variables: age at diagnosis, pulmonary-function values, and the frequency of pseudomonas colonization, pancreatic sufficiency, and diabetes mellitus. Results. Cystic fibrosis was diagnosed at a later age in the patients with the A455E mutation than in the JF508 homozygotes (mean age at diagnosis, 15-0 vs. 3*1 years; P<0.001). Fewer patients with the A455E mutation had pancreatic insufficiency (21 2 percent vs. 93*9 percent, P<0.001), and none had diabetes mellitus (0 percent vs. 27.3 percent, P=0.004). Forced expiratory volume in one second (FEV,) and forced vital capacity (FVC) were significantly higher in the patients with the A455E mutation (mean FEV,, 73.9 percent of the predicted value vs. 54*3 percent of the predicted value; P= 0 002; mean FVC, 88 7 percent of the predicted value vs. 76.3 percent of the predicted value; P= 0.04). Fewer patients with the A455E mutation were colonized with Pseudomonas aeruginosa (33.3 percent vs. 60 6 percent, P= 0.02). Condusions. A455E is a common mutation causing cystic fibrosis in The Netherlands. Although several mutations are known to be associated with less severe pancreatic disease, our findings demonstrate a correlation between the A455E mutation and mild pulmonary disease. Because mortality in this disease depends primarily on the progression of pulmonary disease, patients with the A455E mutation have a better prognosis than patients who are homozygous for the JF508 mutation. (N Engl J Med 1995;333:95-9)

S82 Airway Inflammation is Present by 4 Months in CF Infants Diagnosed on Newborn Screening

Introduction Cystic fibrosis (CF) newborn screening (NBS) allows early introduction of treatment, often before any symptoms arise with the aim of reducing airway infection and inflammation. UK nationwide screening began in 2007. Aims It is usual clinical practise at our centre to perform fibreoptic bronchoscopy (FOB) at the age of 3 months for all children with CF. The aim of this study was to establish the presence of infection and degree of inflammation in airways in the NBS cohort. Bronchoalveolar lavage fluid (BALF) was cultured and examined for cellular inflammation. Results Infants diagnosed by NBS undergoing routine FOB who had either BALF absolute cell count or differential cell count assessed were included in the study. 44 infants (48% female), median age (range) 15 (7–28) weeks met these criteria. The majority of these infants were symptom free however 13 (29%) had bacterial isolates from their BALF. Comparable data are also available for 71 children with established CF (median age 9.5 years; range 1.9–16.7 years) of whom 46 (65%) were BALF culture positive and for 6 healthy controls (median age 12.3 years, range 10.5–15.4 years). Cellular inflammation was present in the airways in infants diagnosed by NBS, both in those who were culture positive and negative in their BALF. Absolute BALF cell count and neutrophil differential were significantly higher in both NBS and established CF patients compared with healthy controls (p<0.02). Median absolute cell counts and neutrophil differentials can be seen in table 1. For both NBS and established CF, in those who had bacteria isolated from their BALF, an increase in neutrophil differential was seen compared with those culture negative at the time of FOB although this did not reach significance in the NBS group. (NBS CF p= 0.05, established CF p<0.01). Abstract S82 Table 1 Conclusion Our results demonstrate that inflammation is already present by 4 months of age in asymptomatic infants diagnosed through NBS, although at a lower level than seen in established CF. The results underscore the importance of early surveillance and lend support to the evolving focus on this age group for interventional trials.

RSIV. F/HN-MEDIATED GENE THERAPY ENABLES LUNGS TO PRODUCE THERAPEUTICALLY RELEVANT LEVELS OF FVIII

We have previously shown that lung when treated with Sendai virus-mediated gene transfer can produce secreted proteins and release them into the circulation (Griesenbach et al., Mol Therapy 2002). Despite the high levels of transduction efficiency the gene expression is transient and repeated administration is not feasible due to induction of immune responses. To overcome these barriers we developed a lentiviral vector specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV. F/HN) to allow efficient transduction of the airways. Stable expression for >20 months after a single dose and efficient transduction after repeated administration despite detection of anti-rSIV. F/HN neutralising antibodies make the vector an attractive candidate for a large range of disease indications. Here, we first transduced mouse lung with rSIV. F/HN carrying the secreted reporter gene Gaussia luciferase (GLux) or a control virus by nasal instillation (1e6 transduction units (TU)/mouse, n = 5 –6/group). Persistent levels of GLux expression were detectable in lung (3 logs above control) and broncho-alveolar lavage fluid (BALF, 4 logs above control) for at least 12 months. Importantly, even this modest dose of virus lead to significant (p < 0.01) levels of GLux in serum (274 ± 72 RLU/ul, control: 41 ± 6 RLU/ul) which persisted for at least 12 months further supporting the hypothesis that the lung is a suitable, non-invasive factory for production of secreted proteins. Gene therapy strategies for haemophilia have focussed on intravenous or intramuscular delivery of the gene transfer agent. Here, we treated the murine lung with rSIV. F/HN carrying the FVIII cDNA (1.6e8–3.4e8 TU/mouse,) or placebo and assessed whether therapeutically relevant levels of FVIII can be produced. Significant (p < 0.05) and dose-related levels of FVIII were detectable in lungs and BALF 10 and 28 days post-transduction. Dose-related levels of FVIII were also detectable in plasma, which reached a therapeutically relevant level of 3% of normal 1 month after gene transfer. These data support the concept that rSIV. F/HN-mediated transduction of lungs can produce therapeutically relevant and persistent levels of recombinant protein in blood. Reference 1 Griesenbach U, Cassady RL, Ferrari S et al. The nasal epithelium as a factory for systemic protein delivery. Mol Ther. 2002;5:98–103

S69 Development of an in vitro assay to detect chemically-induced changes in ciliary beat frequency

Techniques are well-established to quantify ciliary beat frequency (CBF), which is often reduced in patients with primary ciliary dyskinesia. This project aims to determine the impact of genetic polymorphisms in the T2R38 (bitter taste) receptor in response to chemical ligands, which is predicted to lead to changes in CBF, which are transient and small in magnitude. These receptors are of interest as they have been shown to ‘sense’ quorum sensing molecules produced by Pseudomonas aeruginosa (Pa) and may thus be disease modifiers in patients with cystic fibrosis. This work describes the development of a rapid CBF assay with sufficient sensitivity to provide a read-out of airway epithelial cell responses to stimulation in vitro. Air-liquid interface (ALI) cultures were obtained from surplus clinical diagnostic samples. Cells in ALI were exposed to phosphate buffered saline (PBS; negative control) and adenosine 5’-triphosphate (ATP; positive control). Experiments were conducted in temperature-controlled wells under a 40× light microscope. Cilia were imaged by high-speed video camera and CBF expressed as a ratio of stimulated/basal frequencies. CBF increased in ALI cultures exposed to ATP (n = 4) but not PBS (n = 3), in experiments at 24–25˚C; this effect was not seen at 37 ˚C. Mean ±SEM stimulated/basal CBF ratio was 1.00 ± 0.05 in the PBS-exposed cells, and 1.31 ± 0.08 in the ATP-exposed cells (p = 0.029). Inter-observer variability (n = 2) was lower than within-sample CBF variability (95% limits of agreement from -0.66 to 1.62 Hz). Intra-observer variability was good with 95% limits of agreement between -0.31 to 0.52 Hz. An assay has been developed to detect rapid changes in CBF in ALI cultures using ATP as a positive control. Further work is being undertaken to a) optimise this assay in epithelial cells in suspension, thus increasing throughput, and b) assess more relevant chemicals and culture media. Once optimised, this assay will be used to study the effects of Pa quorum sensing molecules on ciliated epithelial cells in vitro, from patients of varying TAS2R38 genotypes.Abstract S69 Figure 1

S9 SIFT-MS analysis as a non-invasive determinant of pseudomonas aeruginosa infection in patients with cystic fibrosis

Background There is evidence that Pseudomonas aeruginosa (Pa) produces volatile organic compounds (VOCs) such as hydrogen cyanide (HCN) and 2-aminoacetophenone (2-AA). VOCs in exhaled breath are therefore proposed as potential biomarkers of infection. We hypothesised that selective ion-flow mass spectrometry (SIFT-MS) breath analysis might allow discrimination of CF patients with (CF + Pa) and without Pa (CF-Pa). Methods 79 adults (31 CF + Pa, 22 CF-Pa and 26 healthy controls) provided starved, single tidal exhalation breath samples into NalophanTM bags. Quantification of 15 VOCs was performed within two hours on SIFT-MS. All results are presented as (median parts-per-billion by volume [IQR]). Results 2-AA was significantly higher in CF + Pa than CF-Pa (5.0 [3.4 7.1] vs. 1.3 [0.0 3.2], p <0.01). However, there was significant overlap and median co-efficient of variation was 35.41%; clinical utility is therefore questionable. Dimethyl disulphide was also significantly higher in CF + Pa (95.2 [41.3 211.2 vs. 35.5 [22.1 79.8], p < 0.01). When combined with 2-AA, area under ROC curve was 0.867. Counter to our sputum results, there was no difference in HCN between CF + Pa and CF-Pa (8.1 [5.0 11.9] vs. 6.9 [4.4 11.0], n/s) or between all CF patients and healthy controls (7.8 [4.9 11.5] vs. 7.0 [4.6 11.5], n/s). Our early in vitro data showed decreased butanol above Pa cultures, suggesting consumption. This was replicated in breath with lower levels in CF + Pa vs. CF-Pa (37.4 [24.3 87.6] vs. 91.7 [46.9 143.7], p < 0.05). Of VOCs likely to be of host origin, isoprene was increased in CF vs. controls (108.0 [83.4 195.5] vs. 69.6 [46.9 89], p < 0.01) with no difference between CF + Pa vs. CF-Pa. Acetone was reduced in CF (269.9 [161.9 356.4] vs. 324.9 [236.7 598.9], p < 0.01). Conclusions 2-AA is a potential biomarker of Pa infection but clinical applicability is uncertain. Dimethyl disulphide and butanol also show promise. Mouth-exhaled HCN assessed by SIFT-MS does not appear to fulfil its promise as a Pa biomarker. Other VOCs assessed were either similar between Pa groups or different between healthy controls and CF, but unable to differentiate between Pa status. This study provides proof-of-concept for the development of a non-invasive tool with which to screen for lower airway bacterial infection in CF though a clinically applicable test remains some way off.

S83 Levels of Antimicrobial Peptides in the Airway of Children with Cystic fibrosis are not related to Serum Vitamin D Concentration

Introduction There is convincing evidence of the clinical health benefits of adequate vitamin D in many respiratory diseases but the evidence in cystic fibrosis (CF) is unclear. There are increasing data on the role of vitamin D as an immunomodulatory agent and it is thought, from in-vitro data, that this may be via induction of the antimicrobial peptides, LL37 and HβD-2. We hypothesised that antimicrobial peptide levels would be increased in children with adequate vitamin D and this could account for reported improvements in lung function via improved airway defence. Aims and methods The main aim was to establish if a relationship exists between vitamin D and antimicrobial peptides, LL37 and HβD-2, and whether any clinically beneficial effects of adequate vitamin D exist in children with CF. Bronchoalveolar fluid (BALF) supernatant levels of LL37 and HβD-2 were by measured by ELISA and serum 25(OH)D2 by mass spectrometry coupled with high-performance liquid chromatography. Results Samples were collected from 120 children with CF (58% female); median age (range) 6.9 (0.1–17.6) years. One third of patients were vitamin D insufficient (<50 nmol/L). Median 25(OH)D2 was 57 nmol/L (range 7–191 nmol/L). LL37 ranged from 0.1–21.9 ng/mL with median value of 0.49 ng/mL and HβD-2 from <15.6 - >1000 pg/ml, median 150 pg/ml. LL37 was significantly correlated with serum neutrophils (r= 0.4, P<0.0001), BALF total cell count (r=0.7, p<0.0001), and BALF neutrophil differential (r= 0.5, p<0.0001). These relationships were not seen with HβD-2. Contrary to our hypothesis neither LL37 nor HβD-2 correlated with vitamin D and no differences were seen between vitamin D ‘adequate’ and ‘insufficient’ patients. There was no association seen between vitamin D and FEV1 (r2=0.01, p=0.4). Conclusions Our results demonstrate that there is not a relationship between serum 25(OH)D2 and BALF HβD-2 or LL37. If vitamin D is involved in the induction of such defence peptides in-vivo, the impact of this on protein levels may be limited in the degradative environment of the inflamed airway. In addition, we found no clinical or physiological effects of vitamin D deficiency. If any beneficial effect of vitamin D on respiratory health does exist in CF, it is small and not mediated via the antimicrobial pathway.

S103 Anti-Pseudomonal Bacteriophage Cocktail Reduces Inflammatory Responses in the Murine Lung

Bacteriophages are naturally occurring viruses that specifically target and infect bacteria and, unlike antibiotics, are able to multiply at infection sites and adapt to resistant bacteria. We hypothesise that bacteriophage cocktails may be useful against P. aeruginosa (Pa) in cystic fibrosis and tested this in a murine infection model. Two strains of Pa were assessed: a) a clinical strain from an adult CF patient, b) a laboratory strain. Both strains were shown to be sensitive to a novel anti-Pseudomonal bacteriophage cocktail on a standard plaque assay. Adult BALB/c mice were inoculated intranasally with 50ul of Pa followed by 20ul of bacteriophage cocktail (treated, n=21) or SM buffer (control, n=21). Twelve mice were sacrificed at 24hrs after infection and the others at 48hr. Bronchoalveolar lavage was serially log diluted, cultured at 37oC and the remainder centrifuged and supernatant stored at –80oC for future analysis of soluble inflammatory markers. Total cell counts were determined using a haemocytometer. Non-quantitative splenic cultures were performed. Results All mice treated with bacteriophage (n=6) had cleared infection at 24hrs compared with none of the controls (n=6) (median [range] CFU/ml 0 [0–0] vs. 1305 [190–4700], p<0.01); inflammatory cell counts did not differ. At the 48hr time point most mice had cleared the infection, with no phage-related differences. However, treated mice demonstrated significantly fewer inflammatory cells in BAL compared with controls (median [range] 4.50 [2.84–5.86] × 104/ml vs. 9.12 [6.93–13.86], p<0.01 for the clinical strain; median [range] 6.04 [5.56–10.60] × 104/ml vs. 9.72 [8.56–15.28], p<0.01 for the laboratory strain). Differential cell counts and measurements of soluble inflammatory mediators are underway. BALB/c mice successfully cle ared this dose of Pa by 48hrs, with an accompanying acute cellular inflammatory response. The reduction in cell number following co-administration of a bacteriophage cocktail to which the organisms were sensitive suggests that Pa was cleared earlier and more effectively in the phage-treated animals; this was confirmed by significant differences in bacterial load at the earlier, 24 hour time point. Further work is underway to explore the therapeutic potential of bacteriophage in pulmonary Pa infection.