Ian Forrest

Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients

Background: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. Methods: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. Results: A median 15% (0–48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-β1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-β1 stimulation. Conclusions: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.

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.

Primary airway epithelial cell culture from lung transplant recipients.

Long-term survival in lung transplantation is limited by the development of obliterative bronchiolitis, a condition characterised by inflammation, epithelial injury, fibroproliferation and obliteration of bronchioles leading to airflow obstruction. To investigate the role of the bronchial epithelium in the pathogenesis of obliterative bronchiolitis the current study aimed to establish primary bronchial epithelial cell cultures (PBEC) from lung allografts. Four to six bronchial brushings were obtained from sub-segmental bronchi of lung allografts. Cells were seeded onto collagen-coated plates and grown to confluence in bronchial epithelial growth medium. Bronchial brushings (nu200a=u200a33) were obtained from 27 patients. PBECs were grown to confluence from 12 out of 33 (39%) brushings. Failure to reach confluence was due to early innate infection. Bacteria were usually isolated from both bronchoalveolar lavage and culture media, but a separate population was identified in culture media only. Primary culture of bronchial epithelial cells from lung transplant recipients is feasible, despite a high rate of early, patient-derived infection. Latent infection of the allograft, identified only by bronchial brushings, may itself be a persistent stimulus for epithelial injury. This technique facilitates future mechanistic studies of airway epithelial responses in the pathogenesis of obliterative bronchiolitis.

Antimicrobial peptides in lung transplant recipients with bronchiolitis obliterans syndrome

Mechanisms other than classical alloimmunity are implicated in the pathogenesis of bronchiolitis obliterans syndrome (BOS). It was hypothesised that antimicrobial peptides (AMPs), elements of the innate immune response, have a role in BOS pathogenesis. Pulmonary expression of the neutrophil-derived AMPs human cathelicidin (hCAP)-18/LL-37 and α-defensins (human neutrophil peptides (HNP) 1–3), and the epithelial cell-derived AMPs human β-defensin (hBD)-2, elafin and secretory leukoprotease inhibitor (SLPI) were measured in stable lung transplant recipients and those with BOS. The relationship between airway pathogens and AMP levels was examined. Bronchoalveolar lavage (BAL) was performed on 44 lung transplant recipients (30 stable, 14 with BOS). BAL was cultured for pathogens and ELISA for AMPs was performed. The presence of airway pathogens was associated with significantly increased levels of neutrophil-derived and epithelial-derived AMPs. When patients without pathogens in BAL fluid were analysed, eight recipients with BOS had elevated hCAP-18/LL-37 and HNP 1–3 compared with 25 stable recipients. hBD-2 and elafin levels were comparable in BOS and stable recipients, but SLPI levels were reduced in BOS. Bronchiolitis obliterans syndrome is associated with elevated airway human cathelicidin 18/LL-37 and human neutrophil peptides 1–3 from activated neutrophils, even in the absence of pathogens. Together with reduced airway secretory leukoprotease inhibitor this may favour nonalloimmune airway injury with reduced antiprotease defence and increased neutrophil degranulation.

Aspiration and allograft injury secondary to gastroesophageal reflux occur in the immediate post-lung transplantation period (prospective clinical trial)

Objectives:To provide novel pilot data to quantify reflux, aspiration, and allograft injury immediately post–lung transplantation. Background:Asymptomatic reflux/aspiration, associated with allograft dysfunction, occurs in lung transplant recipients. Early fundoplication has been advocated. Indications for surgery include elevated biomarkers of aspiration (bile salts) in bronchoalveolar lavage fluid (BALF). Measurements have been mostly documented after the immediate posttransplant period. We report the first prospective study of reflux/aspiration immediately posttransplantation to date. Methods:Lung transplant recipients were recruited over 12 months. At 1 month posttransplantation, patients completed a Reflux Symptom Index questionnaire and underwent objective assessment for reflux (manometry and pH/impedance). Testing was performed on maintenance proton pump inhibitor. BALF was assessed for pepsin, bile salts, interleukin-8 and neutrophils. Results:Eighteen lung transplant recipients, median age of 46 years (range: 22–59 years), were recruited. Eight of 18 patients had abnormal esophageal peristalsis. Five of 17 patients were positive on Reflux Symptom Index questionnaire. Twelve of 17 patients had reflux. Three patients exclusively had weakly acid reflux. Median acid exposure was 4.8% (range: 1%–79.9%) and median esophageal volume exposure was 1.6% (range: 0.7–5.5). There was a median of 72 reflux events (range: 27–147) per 24 hours. A correlation existed between Reflux Symptom Index score and proximal reflux (r = 0.533, P = 0.006). Pepsin was detected in 11 of 15 BALF samples signifying aspiration (median: 18 ng/mL; range: 0–43). Bile salts were undetectable, using spectrophotometry and rarely detectable using dual mass spectrometry (2/15) (levels 0.2 and 1.2 &mgr;mol/L). Lavage interleukin-8 and neutrophil levels were elevated. A correlation existed between proximal reflux events and neutrophilia (r = 0.52, P = 0.03). Conclusions:Lung transplant recipients should be routinely assessed for reflux/aspiration within the first month posttransplant. Reflux/aspiration can be present early postoperatively. Pepsin was detected suggesting aspiration. Bile salts were rarely detected. Proximal reflux events correlated with neutrophilia, linked to allograft dysfunction and mortality. These results support the need for early assessment of reflux/aspiration, which may inform fundoplication.

Macrolide antibiotics and the airway: antibiotic or non-antibiotic effects?

Importance of the field: The macrolides are a class of antibiotics widely prescribed in infectious disease. More recently, there has been considerable interest in potential indications for these agents, in addition to their simple antibacterial indications, in a number of lung pathophysiologies. Areas covered in this review: Demonstrated clinical efficacy of macrolides in diseases such as diffuse panbronchiolitis was difficult to ascribe to a direct antimicrobial action. More recently, positive experiences in dealing with post-transplant bronchiolitis obliterans syndrome suggests that other chronic lung diseases may benefit from macrolide therapy. This is important, as the treatment options for such diseases are often very limited. In this review, potential antibiotic and non-antibiotic beneficial actions of macrolide therapy are discussed and conclusions drawn from a limited but growing literature. What the reader will gain: The reader will gain an overview of lung diseases that may benefit from macrolides, and a consideration of the possible mechanisms underlying such benefit. Take home message: The key message from our review is that this class of agents may prove to be a useful therapeutic option for a range of respiratory diseases, but that further trials and mechanistic studies are required to clarify their role.

Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples.

Background:Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells.Methods:A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells.Results:From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples – three NSCLC and one mesothelioma – were HRR defective and eight samples – one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers – were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1.Conclusions:HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi.

IPF: time for the (ciliary) beat generation?

Idiopathic pulmonary fibrosis (IPF), the most common of the idiopathic interstitial pneumonias remains a disabling, progressive lung disease with extremely poor prognosis, in which no pharmacological intervention significantly alters outcome.1 A relatively poor understanding of the complex pathophysiology of IPF continues to hinder the identification of effective therapies, and of patients most likely to benefit from existing treatments.nnUsual interstitial pneumonia is the histological hallmark of IPF, characterised by temporospatial heterogeneity in which normal lung is interspersed with areas of subpleural interstitial fibrosis, loss of normal alveolar architecture and the presence of fibroblastic foci.2 Histological sections may also show ‘bronchiolisation’ of the distal airway with alveolar structures replaced by enlarged airspaces (forming characteristic honeycomb cysts) lined by epithelial cells more akin to proximal airway epithelium, often ciliated and mucus producing.3 ,4 The potential importance of mucus production in the pathogenesis of IPF has been highlighted by a landmark study demonstrating that a common variant within the putative promoter region of MUC5B (an airway mucin gene) is strongly associated with familial interstitial pneumonia and IPF.5 Moreover, subjects with IPF had significantly higher expression of MUC5B protein levels compared with controls, and this was localised to areas of lung fibrosis. It has been hypothesised that excessive MUC5B may impair the host response to alveolar injury through excess mucus plugging and impaired clearance of inhaled substances and micro-organisms. Alternative theories propose that MUC5B may interfere directly with alveolar repair mechanisms, either through disruption of interactions between type II alveolar epithelial cells and extracellular matrix, or …

Identifying anxiety and depression in interstitial lung disease: use of a simple outpatient screening tool

S4 Figure 1. Spoken sessions A6 Thorax 2009;64(Suppl IV):A5–A74 common in patients aged .65 (p = 0.048). Anxiety appeared more common in females, but this did not reach statistical significance. The presence of depression and anxiety was not related to severity of lung disease as measured by KCO, FVC or oxygen saturations. Conclusions Use of a simple self-administered screening tool can identify high levels of anxiety and depression in outpatients with interstitial lung disease. Anxiety levels are more common in elderly patients. Identifying psychological problems in this patient group may allow development of new therapeutic options so physicians can improve patient’s symptoms and quality of life. Psychopharmacology, cognitive behavioural therapy or pulmonary rehabilitation may be possible treatment options and merit further study. S5 PHYSIOLOGICAL DETERIORATION VS STABILISATION IN MYOSITIS-ASSOCIATED INTERSTITIAL LUNG DISEASE: PHENOTYPIC DIFFERENCES AND INFLUENCE OF IMMUNOSUPPRESSIVE TREATMENT AM Higton, PDW Kiely, K McNulty, S Grubnic, EJ Edwards, F Chua. St George’s Hospital NHS Trust, London, UK doi:10.1136/thx.2009.127050e Background Interstitial lung disease (ILD) may form a prominent component of idiopathic inflammatory myositis (IIM) and even dominate the clinical course of disease. We observed that certain characteristics were associated with longitudinal worsening of lung disease, including temporal differences that suggest that particular patterns of ILD progression may be expected. Methods Patients on the St George’s Hospital Myositis database with polymyositis (PM), dermatomyositis (DM), overlap syndrome or antisynthetase (Jo-1) syndrome with associated pulmonary abnormalities (as confirmed by CT) were identified. Stable lung disease was accepted as a (10% decrease in forced vital capacity (FVC) and/or a (15% decrease in diffusing capacity (DLCO) over the first year of diagnosis. Results 20 patients with IIM-ILD were identified (mean FVC 70¡18% predicted, mean DLCO 50¡17% predicted at diagnosis). Mean duration of myositis follow-up was 10.75¡6.4 years. 17 of 20 (85%) had >2 measurements of pulmonary function tests (PFTs), with an average total PFT follow-up of 4.7 years. DLCO decline was evident in 7 patients, against a stable/improved pattern in 10 others. Deteriorators tended to have a greater decline in DLCO in their first year of follow-up (213¡13 vs +26¡40 for stable patients p = 0.06). Overall, most patients who experienced early PFT deterioration continued to do so thereafter, albeit at a variable rate. For most, an established decline in DLCO at 3 years from diagnosis heralded further loss subsequently. Deteriorators tended to be younger and more commonly reported respiratory symptoms at initial diagnosis. Amongst them, DM was the most common myositis (5/7, 71%), in contrast to 8/10 (80%) of stable patients with PM (p = 0.008). Afro-Caribbean ethnicity was also more frequent in those with IIM-ILD but did not distinguish deteriorators from stabilisers. Patient numbers were inadequate for ascribing clinical significance to specific ILD patterns. Conclusions For most patients with myositis, ILD is a relatively benign complication that is satisfactorily controlled by immunosuppressive therapy. In those with progressive lung disease, loss of DLCO appears to be a crucial consequence. These individuals more commonly have DM, are younger and have a shorter duration of lung disease. PFT stabilisation following early deterioration is occasionally apparent, although the long-term significance of this observation remains unclear. Cellular interactions in the pathogenesis of pulmonary hypertension S6 REGULATION OF ENDOTHELIN-1 PRODUCTION BY THE TRANSFORMING GROWTH FACTOR/BONE MORPHOGENETIC PROTEIN PATHWAY IN HUMAN PULMONARY ARTERY SMOOTH MUSCLE CELLS PM de Souza, NW Morrell, PD Upton, JES Park, SJ Wort. Unit of Critical Care, NHLI, Imperial College, London, UK; University of Cambridge School of Clinical Medicine, Addenbrooke’s/CUHNHSFT and Papworth Hospitals, Cambridge, UK doi:10.1136/thx.2009.127050f Background Pulmonary artery hypertension is a fatal condition associated with remodelling of pulmonary resistance vessels. There is convincing evidence for the involvement of both the transforming growth factor b (TGFb)/bone morphogenetic protein (BMP) and endothelin (ET-1) pathways in this remodelling process. However, it is unknown how these two pathways interact. Aim To investigate the effect of TGFb1, BMP2 and BMP4 on ET-1 release from normal human pulmonary artery smooth muscle cells (HPASMCs). Methods HPASMCs were grown from resected and morphologically normal pulmonary arteries taken from patients with lung cancer at the Royal Brompton Hospital. Cells were treated with TGFb1 and/ or BMP2 and BMP4 (0, 1 and 10 ng/ml). Following 24 h incubation supernatants were collected and ET-1 concentrations determined by ELISA (R&D, Abingdon, UK). Data were analysed using Student t test. Results TGFb1 dose dependently increased ET-1 release from HPASMCs. TGFb1 (1 ng/ml) significantly increased ET-1 generation by 553% compared with cells treated with medium alone (fig 1; n = 6)). BMP2 (10 ng/ml) and BMP4 (1 and 10 ng/ml) also Abstract S5 Table Deteriorators Stabilisers p Value n = 7 n = 10 Age at diagnosis 39¡15 50¡19 0.18 Ethnicity Black 4 (57%) 5 (50%) White 1 (14%) 5 (50%) 0.14 Other 2 (29%) 0 ILD pattern on HRCT

Nontuberculous mycobacteria in gastrostomy fed patients with cystic fibrosis

Multi-drug resistant Mycobacterium abscessus complex (MABSC) is a form of Nontuberculous mycobacteria (NTM) of special, international concern in Cystic Fibrosis (CF). We hypothesised that gastric juice and percutaneous endoscopic gastrostomy (PEG) feeding devices might yield MABSC isolates. Gastric juice and sputa from sixteen adult PEG fed CF patients and five replaced PEG tubes were studied. Bacterial and fungal isolates were cultured. Mycobacterium were identified by rpoB, sodA and hsp65 gene sequencing and strain typed using variable number tandem repeat. Bacteria and/or fungi grew from all gastric juice, sputa and PEG samples. MABSC were detected in 7 patients. Five had MABSC in their sputum. Two had an identical MABSC strain in their sputum and gastric juice and one had the same strain isolated from their PEG tube and sputum. Two patients who were sputum sample negative had MABSC isolated in their gastric juice or PEG tube. MABSC were therefore identified for the first time from a gastric sample in a minority of patients. We conclude that gastric juice and PEG-tubes may be a potential source of MABSC isolates in CF patients, and these findings warrant further study.