Therese Small

Cross sectional study of exhaled nitric oxide levels following lung transplantation

BACKGROUND The role of nitric oxide (NO) in the pathophysiology of graft dysfunction following lung transplantation remains unclear. To determine whether measurement of NO in the exhaled breath of lung transplant recipients provides useful information about graft pathology, a cross sectional study was performed on a cohort of recipients as they attended for review. METHODS One hundred and four lung transplant recipients and 55 healthy non-smoking controls were included in the study. Each subject performed three consecutive single breath NO manoeuvres. In recipients NO levels were compared according to current clinical status, presence of any graft pathology, type of lung transplant procedure, indication for transplantation, and current level of immunosuppression. RESULTS Mean (SE) exhaled NO levels were 6.5 (0.61) ppb in the control group, 5.3 (0.46) in clinically well recipients, 10.3 (1.4) in those with lymphocytic bronchiolitis, 10.5 (1.0) in recipients with infection, and 2.5 (0.6) in those with acute vascular rejection. There was no significant difference in NO levels between the control group and lung transplant recipients as a whole (mean difference 0.29 (95% CI –1.17 to 1.75), p = 0.7). Levels were increased significantly in the presence of lymphocytic bronchiolitis (4.98 (95% CI 1.6 to 8.36), p = 0.0002) and infection (5.28 (95% CI 2.9 to 7.56), p<0.0001), but not in acute vascular rejection (2.76 (95% CI 0.97 to 4.55), p = 0.1) compared with exhaled NO in clinically well recipients. Recipients with obliterative bronchiolitis were subdivided according to the grade of their bronchiolitis obliterans syndrome (BOS). Exhaled NO levels in those with BOS grade 1 were 10.0 (1.3) ppb and in those with BOS grades 2 or 3 were 5.1 (0.7) ppb. Compared with those who were clinically well, NO levels were increased in those with BOS grade 1 (4.74 (95% CI 1.8 to 7.69), p < 0.0001) but not in those with BOS grades 2 or 3 (0.19 (95% CI –1.55 to 1.93), p = 0.82). CONCLUSIONS Exhaled NO levels are increased in lung transplant recipients with lymphocytic bronchiolitis, early obliterative bronchiolitis, and infection. These conditions are all associated with the presence of airway inflammation within the graft. The findings suggest that exhaled NO measurements may have a role as a marker of pulmonary allograft dysfunction.

Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients

Obliterative bronchiolitis (OB), the major cause of chronic lung allograft dysfunction, is characterized by airway neutrophilia, inflammation, and remodeling, with progressive fibroproliferation and obliteration of small airways that ultimately leads to patient death. Statins have potential anti-inflammatory effects and have been demonstrated to confer a survival advantage in lung transplant patients. We postulated that the beneficial effects of simvastatin in lung transplantation are in part due to inhibition of the epithelial production of key mediators of neutrophil chemotaxis, inflammation, and airway remodeling. Our objective was to assess the effect of simvastatin on a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts, with specific reference to airway neutrophilia and remodeling. PBEC cultures were stimulated with IL-17 or transforming growth factor (TGF)-beta, with and without simvastatin. Supernatant levels of factors critical to driving airway neutrophilia and remodeling were measured. IL-17 upregulated IL-8, IL-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and VEGF, whereas TGF-beta increased IL-6, GM-CSF, matrix metalloproteinase (MMP)-2, and MMP-9. Simvastatin attenuated effects of both IL-17 and TGF-beta. We have demonstrated the ability of simvastatin to attenuate release of airway neutrophilic and remodeling mediators and to inhibit their upregulation by TGF-beta and IL-17. These data illustrate the potential of simvastatin to alleviate neutrophilic airway inflammation and remodeling in the transplanted lung and may have additional relevance to other neutrophilic airway conditions, such as chronic obstructive pulmonary disease.

A randomised controlled trial of azithromycin therapy in bronchiolitis obliterans syndrome (BOS) post lung transplantation

Background We conducted a placebo-controlled trial of azithromycin therapy in bronchiolitis obliterans syndrome (BOS) post lung transplantation. Methods We compared azithromycin (250 mg alternate days, 12 weeks) with placebo. Primary outcome was FEV1 change at 12 weeks. Results 48 patients were randomised; (25 azithromycin, 23 placebo). It was established, post randomisation that two did not have BOS. 46 patients were analysed as intention to treat (ITT) with 33 ‘Completers’. ITT analysis included placebo patients treated with open-label azithromycin after study withdrawal. Outcome The ITT analysis (n=46, 177 observations) estimated mean difference in FEV1 between treatments (azithromycin minus placebo) was 0.035 L, with a 95% CI of −0.112 L to 0.182 L (p=0.6). Five withdrawals, who were identified at the end of the study as having been randomised to placebo (four with rapid loss in FEV1, one withdrawn consent) had received rescue open-label azithromycin, with improvement in subsequent FEV1 at 12 weeks. Study Completers showed an estimated mean difference in FEV1 between treatment groups (azithromycin minus placebo) of 0.278 L, with 95% CI for the mean difference: 0.170 L to 0.386 L (p=<0.001). Nine of 23 ITT patients in the azithromycin group had ≥10% gain in FEV1 from baseline. No patients in the placebo group had ≥10% gain in FEV1 from baseline while on placebo (p=0.002). Seven serious adverse events, three azithromycin, four in the placebo group, were deemed unrelated to study medication. Conclusions Azithromycin therapy improves FEV1 in patients with BOS and appears superior to placebo. This study strengthens evidence for clinical practice of initiating azithromycin therapy in BOS. Trial registration number EU-CTR, 2006-000485-36/GB.

Exhaled single-breath nitric oxide measurements are reproducible, repeatable and reflect levels of nitric oxide found in the lower airways

Measurement of exhaled nitric oxide (NO) may allow noninvasive assessment of inflammatory disease in the lung. We determined immediate and day-to-day reproducibility of single-breath NO measurements at different points on the exhaled test, and whether levels recorded reflect levels of NO in the lower airways. Using a rapid chemiluminescence analyser, 55 healthy control subjects performed three sequential tests on each of two days. NO levels were compared at the level corresponding with: 1) the time the mouth pressure fell below 4 cmH2O (MP); 2) the plateau of end-exhaled CO2 (CO2); and 3) the NO plateau (NOp). NO levels were measured directly from the lower airways of 15 lung transplant recipients and compared with NO levels from a single-breath test performed in the same cohort. For measurements performed at MP, CO2 and NOp, the mean +/- SD differences between the two closest levels performed on the same day were 0.11+/-0.18, 0.095+/-0.16 and 0.094+/-0.13 parts per billion (ppb), respectively, and between days were 0.18+/-0.76, 0.19+/-0.78 and 0.17+/-0.8 ppb, respectively. End-expiratory levels recorded at the mouth from a single-breath test and in the lower airways were highly correlated (mouth versus trachea r2=0.95, p<0.0001, mouth versus bronchus r2=0.92, p<0.0001). Single-breath exhaled nitric oxide levels are a simple, reproducible and valid measure of nitric oxide production from the lower respiratory tract.

Anti-reflux surgery in lung transplant recipients: outcomes and effects on quality of life

Fundoplication may improve survival after lung transplantation. Little is known about the effects of fundoplication on quality of life in these patients. The aim of this study was to assess the safety of fundoplication in lung transplant recipients and its effects on quality of life. Between June 1, 2008 and December 31, 2010, a prospective study of lung transplant recipients undergoing fundoplication was undertaken. Quality of life was assessed before and after surgery. Body mass index (BMI) and pulmonary function were followed up. 16 patients, mean±sd age 38±11.9 yrs, underwent laparoscopic Nissen fundoplication. There was no peri-operative mortality or major complications. Mean±sd hospital stay was 2.6±0.9 days. 15 out of 16 patients were satisfied with the results of surgery post fundoplication. There was a significant improvement in reflux symptom index and DeMeester questionnaires and gastrointestinal quality of life index scores at 6 months. Mean BMI decreased significantly after fundoplication (p=0.01). Patients operated on for deteriorating lung function had a statistically significant decrease in the rate of lung function decline after fundoplication (p=0.008). Laparoscopic fundoplication is safe in selected lung transplant recipients. Patient benefit is suggested by improved symptoms and satisfaction. This procedure is acceptable, improves quality of life and may reduce deterioration of lung function.

Effect of nebulized epoprostenol (prostacyclin) on exhaled nitric oxide in patients with pulmonary hypertension due to congenital heart disease and in normal controls.

Inhaled epoprostenol (prostacyclin) may be used in the treatment of severe pulmonary hypertension, improving oxygenation and reducing pulmonary artery pressures. We have observed symptomatic benefits of epoprostenol in patients with congenital heart disease that extend beyond acute haemodynamic effects of the drug, which has a short biological half-life. The aim of this study was to examine the effects of epoprostenol in patients and normal subjects on exhaled nitric oxide (eNO), based on the hypothesis that the drug may alter the resting vasoconstrictor/vasodilator balance. Nine patients with pulmonary hypertension complicating left-to-right cardiac shunts and nine healthy controls received 100 microgram of nebulized epoprostenol. Exhaled eNO was measured, using a chemiluminescence method, before, immediately after and 18 h after nebulization. There was no significant difference between the two groups in baseline eNO or eNO immediately following nebulized epoprostenol. Epoprostenol produced a delayed elevation in eNO 18 h after nebulization in patients, but not in normal controls. This study supports the concept that epoprostenol, while having no effect on the normal pulmonary circulation, acts on the hypertensive circulation via a mechanism that may result in a delayed alteration of vasoconstrictor/vasodilator balance.

Azithromycin attenuates effects of lipopolysaccharide on lung allograft bronchial epithelial cells

BACKGROUND The bronchial epithelium is a source of mediators that may play a role in the airway inflammation and remodeling of post-transplant obliterative bronchiolitis (OB). Traditional strategies have failed to have an impact on OB. Recent studies have suggested a role for azithromycin in managing the condition. In this study we aimed to determine the effect of azithromycin on LPS-mediated epithelial release of factors relevant to airway neutrophilia and remodeling in a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts. METHODS PBECs were established from bronchial brushings of stable lung transplant recipients and treated with lipopolysaccharide (LPS, 0.1, 1 and 10 microg/ml) for 48 hours. Interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF) protein levels were measured by Luminex analyzer. PBECs were then incubated with LPS and azithromycin, and protein levels were again determined. RESULTS LPS caused a significant increase in IL-8 and GM-CSF at concentrations of 1 and 10 microg/ml, with no effect on VEGF release. Azithromycin caused a significant decrease in the LPS-stimulated IL-8 and GM-CSF release. CONCLUSIONS LPS upregulates release of IL-8 and GM-CSF from PBECs derived from stable lung allografts. Sub-microbicidal concentrations of azithromycin attenuate this and may, therefore, alleviate infection-driven neutrophilic airway inflammation and remodeling in the allograft airway.

Low serum mannose-binding lectin level is not associated with disease severity in non-cystic fibrosis bronchiectasis

Deficiency of mannose-binding lectin (MBL), a serum protein involved in killing and promoting phagocytosis of pathogens, is associated with respiratory infection and disease progression in a number of acute and chronic lung diseases, including cystic fibrosis (CF)- associated bronchiectasis. No such association has been studied in non-CF bronchiectasis (nCF-Br). One hundred and thirty-three adult patients with nCF-Br were studied. Serum MBL levels were measured and deficiency defined using two cut-off levels, i.e. MBL ≤100 ng/ml and ≤600 ng/ml. Parameters of severity included lung function impairment, annual exacerbation and hospital admission rates, breathlessness, and Pseudomonas aeruginosa and Haemophilus influenzae infection rates. The incidence of MBL deficiency using cut-off levels of 100 ng/ml and 600 ng/ml was 10% and 26% respectively, similar to rates seen in the general population. There was no significant difference in mean FEV1% predicted between MBL deficient and sufficient patients at both cut-off levels (≤100 ng/ml: 63.8% vs. 64.6%, P = 0.91; ≤ 600 ng/ml: 66.5% vs. 63.9%, P = 0.56). In addition, exacerbation/hospital admission rates, symptoms of breathlessness and isolation/colonisation rates with P. aeruginosa and H. influenzae were similar in both groups at both cut-off levels. In conclusion, MBL deficiency is not associated with markers of disease severity in patients with nCF-Br.

Respiratory health and disease in a UK population-based cohort of 85 year olds: The Newcastle 85+ Study

Background People aged 85 years and older are the fastest growing age group worldwide. This study assessed respiratory health, prevalence of respiratory disease and use of spirometry in respiratory diagnosis in a population-based cohort of 85 year olds to better understand respiratory health and disease in this sector of society. Methods A single year birth-cohort of 85 year olds participated in a respiratory assessment at their home or residential institution including self-reporting of symptoms and measurement of spirometry. General practice medical records were reviewed for respiratory diagnoses and treatments. Findings In the 845 participants, a substantial burden of respiratory disease was seen with a prevalence of COPD in medical records of 16.6% (n=140). A large proportion of the cohort had environmental exposures through past or current smoking (64.2%, n=539) and occupational risk factors (33.6%, n=269). Spirometry meeting reliability criteria was performed in 87% (n=737) of participants. In the subgroup with a diagnosis of COPD (n=123), only 75.6% (n=93) satisfied Global Initiative in Obstructive Lung Disease (GOLD) criteria for airflow obstruction, and in a healthy subgroup without respiratory symptoms or diagnoses (n=151), 44.4% (n=67) reached GOLD criteria for airflow obstruction and 43.3% (n=29) National Institute of Health and Care Excellence criteria for at least moderate COPD. Interpretation Spirometry can be successfully performed in the very old, aged 85 years, and may help identify respiratory diseases such as COPD. However interpretation in this age group using current definitions of COPD based on spirometry indices may be difficult and lead to overdiagnosis in a healthy group with transient symptoms.

P112 Deeper phenotyping of non CF bronchiectasis through sputum differential counts

Introduction and Objectives Non CF Bronchiectasis has diverse aetiologies. This includes idiopathic, systemic disease related and as a complication of asthma. Such diversity may be important in determining therapeutic strategies (personalised medicines) and may also be an important consideration in clinical trial design. This is increasingly relevant when neutrophil targeting or eosinophil targeted therapies are being developed. We hypothesised that patients could be phenotyped by sputum cytospins irrespective of suspected aetiology or disease severity. Methods Patients underwent a standardised clinical phenotyping protocol including HRCT chest (Anwar et al 2013). Baseline therapy was recorded. Spontaneous sputa were collected in stable state and spirometry was undertaken according to guidelines. Sputum cell counts were calculated using standard methods with data expressed as medians and ranges. Results Fifty three patients’ data are reported. The M:F ratio was 1:1.4. The mean FEV1 predicted was 62%, mean FEV1/VC ratio was 64%. Forty three (83%) were on inhaled corticosteroids and 24.5% had a historical diagnosis of asthma and /or ABPA. The predominant cell in sputa was neutrophils, median 94 (range 23–100%), macrophages were the 2nd most prevalent cell type median 2.6 (range 0–75%). Eosinophils showed a skewed distribution with median of 0.2 with a range of 0–24.8%. Four patients had sputum eosinophilia >3%. Of these, only 2 had a history of asthma and / or ABPA being diagnosed. Despite historical diagnoses of asthma and / or ABPA in 13 patients the eosinophil percentage was not statistically different to “non asthmatics”. (P = 0.59 Chi Sq test) This group included features of ABPA in 2 patients and significant atopy in another. Conclusions Non CF bronchiectasis patients usually have sputum neutrophil dominance. Sputum eosinophilia is rare (<10% of patients); however such patients may need alternative therapeutic strategies. Excluding bronchiectasis patients with a history of asthma from trials targeting neutrophils seems unnecessary. The neutrophilic predominant profiles in asthmatic bronchiectasis patients suggest either asthma misdiagnosis or that neutrophil predominant asthmatics may be more susceptible to developing bronchiectasis. Longitudinal studies are needed to determine if the sputum cell profiles are static in stable patients. These data may help develop a more personalised medicine approach in bronchiectasis. Abstract P112 Table 1. Differential sputa counts. NEUT EOSIN MACRO LYMPH Median 94.80 0.2 2.60 0.40 Max 100.00 24.80 75.20 7.00 Min 23.40 0.00 0.00 0.00 References Anwar GA, et al. 2013 Phenotyping adults with non-cystic fibrosis bronchiectasis: A prospective observational cohort study. Respir Med. Jul;107(7):1001–7. doi: 10.1016/j.rmed.2013.04.013. Epub 2013 May 11.