E. Johnston

Carotenoids and Co-Antioxidants in Age-Related Maculopathy: Design and Methods

Age-related macular degeneration (AMD), is the leading cause of blind registration in the Western World among individuals 65 years or older. Early AMD, a clinical state without overt functional loss, is said to be present clinically when yellowish deposits known as drusen and/or alterations of fundus pigmentation are seen in the macular retina. Although the etiopathogenesis of AMD remains uncertain, there is a growing body of evidence in support of the view that cumulative oxidative damage plays a causal role. Appropriate dietary antioxidant supplementation is likely to be beneficial in maintaining visual function in patients with AMD, and preventing or delaying the progression of early AMD to late AMD. The Carotenoids in Age-Related Maculopathy (CARMA) Study is a randomized and double-masked clinical trial of antioxidant supplementation versus placebo in 433 participants with either early AMD features of sufficient severity in at least one eye or any level of AMD in one eye with late AMD (neovascular AMD or central geographic atrophy) in the fellow eye. The aim of the CARMA Study is to investigate whether lutein and zeaxanthin, in combination with co-antioxidants (vitamin C, E, and zinc), has a beneficial effect on visual function and/or prevention of progression from early to late stages of disease. The primary outcome is improved or preserved distance visual acuity at 12 months. Secondary outcomes include improved or preserved interferometric acuity, contrast sensitivity, shape discrimination ability, and change in AMD severity as monitored by fundus photography. This article outlines the CARMA Study design and methodology, including its rationale.

Reduced bacterial colony count of anaerobic bacteria is associated with a worsening in lung clearance index and inflammation in cystic fibrosis.

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Proteomic profile of cystic fibrosis sputum cells in adults chronically infected with Pseudomonas aeruginosa

Lung disease is the main cause of morbidity and mortality in cystic fibrosis (CF), and involves chronic infection and perturbed immune responses. Tissue damage is mediated mostly by extracellular proteases, but other cellular proteins may also contribute to damage through their effect on cell activities and/or release into sputum fluid by means of active secretion or cell death. We employed MudPIT (multidimensional protein identification technology) to identify sputum cellular proteins with consistently altered abundance in adults with CF, chronically infected with Pseudomonas aeruginosa, compared with healthy controls. Ingenuity Pathway Analysis, Gene Ontology, protein abundance and correlation with lung function were used to infer their potential clinical significance. Differentially abundant proteins relate to Rho family small GTPase activity, immune cell movement/activation, generation of reactive oxygen species, and dysregulation of cell death and proliferation. Compositional breakdown identified high abundance of proteins previously associated with neutrophil extracellular traps. Furthermore, negative correlations with lung function were detected for 17 proteins, many of which have previously been associated with lung injury. These findings expand our current understanding of the mechanisms driving CF lung disease and identify sputum cellular proteins with potential for use as indicators of disease status/prognosis, stratification determinants for treatment prescription or therapeutic targets. CF sputum proteomics detects high abundance of NET proteins and identifies proteins correlating negatively with FEV1 http://ow.ly/hnX830aEPki

94 Isolation and characterisation of bacteriophage infecting Prevotella spp. recovered from the cystic fibrosis (CF) airways

Objectives Prevotella spp. are the predominant anaerobes detected in CF sputum samples. Whether these organisms contribute to the pathogenicity of chronic lung disease is currently unclear. Bacterial pathogenicity is increasingly being associated with the carriage of temperate bacteriophage. As such, this study aimed to isolate and characterise bacteriophage infecting Prevotella spp. recovered from CF sputum samples. Methods Prevotella spp. (N = 6) were cultivated in basal anaerobic media, under anaerobic conditions, until late stationary/decline phase. Bacteria were pelleted by centrifugation and the supernatant passed through 0.22 µm filters before bacteriophage were concentrated using high speed centrifugation. Phage particles were stained with 2% uranyl acetate and viewed using a transmission electron microscope. Results Bacteriophage like particles with uniformity and icosahedral capsid structures that characterise many bacteriophage species were recovered from all Prevotella cultures. Conclusion CF Prevotella isolates harbour a variety of bacteriophage like particles. Preliminary investigations suggest that the production of these particles may occur in response to increases in bacterial population density. Ongoing genomic analysis may allow further characterisation of these bacteriophage like particles and provide insight into any potential effects they may have on bacterial virulence.

95 Susceptibility of CF Prevotella isolates to neutrophil and complement mediated immune clearance

Objectives Prevotella spp. are the predominant anaerobes detected in CF sputum samples. To contribute to disease development bacteria, such as Prevotella, must first survive the hosts immune response. Therefore, this study aimed to determine the susceptibility of CF Prevotella isolates to neutrophil and complement mediated immune clearance. Methods Prevotella spp. (N = 8) isolated from CF patients (N = 8) were incubated with neutrophils from healthy controls (T = 20 mins) and with serum complement (T = 60 mins). Decreases in bacterial viability were calculated relative to controls. Prevotella and P. aeruginosa co-culture neutrophil killing assays were also performed. Table . Survival of isolates Species % Survival in 10% serum (N = 3), mean±SD %Survival in neutrophils (N = 3), mean±SD % Survival of P. aeruginosa, co-cultured with Prevotella, in neutrophils (N = 3), mean±SD P. denticola 100.5±6.4 * 66.13±4.16 44.74±10.56 P. salivae 42.64±4.24 114.3±15.04 * 43.05±2.28 P. nigrescens 70.9±6.99 67.8 48.19±3.17 P. veroralis 61.72±20.1 30.69±8.31 − P. melaninogenica 3.84±1.09 61.43±6.0 66.80±3.26 P. histicola 0 8.19±2.68 − P. pallens 113.3±9.43 * 23.33±5.77 − P. oulorum 101.6±5.4 * 46.03±8.56 − * Resistant. Results See the table. Prevotella isolates displayed resistance to serum killing (N = 3) and phagocytosis (N = 1). Four Prevotella isolates were more resistant to phagocytosis (>60% survival) than P. aeruginosa and S. aureus (∼42 and 54% survival, respectively). When co-cultured with P. melaninogenica, the resistance of P. aeruginosa to phagocytosis increased (P = 0.001). Conclusion Complement resistance & neutrophil evasion/inhibition strategies utilised by Prevotella spp. may enable persistence within the CF lung.

S10 Lung Clearance Index (LCI) and Pseudomonous aeruginosa in adults and children with Cystic Fibrosis (CF)

Introduction LCI obtained from multiple breath washout (MBW) is a sensitive measure of ventilation inhomogeneity in CF. Persistent colonisation with P. aeruginosa is associated with a decline in LCI in children (Kraemer et al. 2006). Further research is required to investigate the relationship between airways infection and LCI in adults. Objective To investigate the sensitivity of LCI to P. aeruginosa in adults and children compared with FEV1%pred and FEF25–75%pred. Methods Stable CF patients from adult & paediatric Northern Ireland CF centres were recruited. LCI was derived from MBW, using 0.2% SF6 and a modified InnocorTMdevice. P. aeruginosa status was determined from routine diagnostic culture of a sputum sample or deep throat swab. Patients categorised as having P. aeruginosa infection met the criteria of chronic infection as defined by the Leeds criteria (Lee et al. 2003). Analysis Receiver-operator characteristic (ROC) curves and area under the receiver operating curves (AUCROC) indicate the level of sensitivity and specificity where 1.0 = perfect discrimination considering sensitivity and specificity. Results Sixty-seven adults were recruited (39M), median (IQR) age 27 (16) years. Mean (SD) FEV1%pred 71.8 (20.3), median (IQR) FEF25–75%pred 40.0 (46.7) and mean (SD) LCI 10.3 (3.0) lung volume turnovers. 49% had P. aeruginosa infection. Forty-three children were recruited (24M), mean (SD) age 11.7 (3.4) years. Mean (SD) FEV1%pred 85.2 (16.6), mean (SD) FEF25–75%pred 66.0 (27.6) and mean (SD) LCI was 7.8 (1.8) lung volume turnovers. 16% had P. aeruginosa infection Compared to FEV1%pred and FEF25–75%pred, LCI had the greatest sensitivity and specificity to discriminate between CF patients with and without P. aeruginosa in both adults and children. Adult AUCROC (SE) for LCI = 0.82 (0.05), p < 0.0001, compared with FEV1%pred = 0.66 (0.07), p = 0.021 and FEF25–75%pred = 0.64 (0.07), p = 0.044 (Figure 1). Child AUCROC (SE) for LCI = 0.85 (0.10), p = 0.004, compared with FEV1%pred = 0.80 (0.12), p = 0.014 and FEF25–75%pred = 0.67 (0.13), p = 0.152. Abstract S10 Figure 1. Adult ROC curve Conclusion LCI is more sensitive and specific to the presence of P. aeruginosa airways infection across the age groups in CF compared with spirometry. This project was funded by a US-Ireland Project Partnership Grant. References Kraemer, R et al 2006, Respiratory Research. Lee, T W R et al 2003, Journal of Cystic Fibrosis.

100 Comparison of airway microbiota composition in healthy children and children with CF

Introduction and Aims: Although a number of studies have characterized airway microbiota composition in healthy and CF adult populations, limited data is available for the paediatric population. The aim of this study was to define, using culture based methods, the composition of the airway microbiota in age-matched healthy and CF paediatric groups. Methods: Induced sputum samples (CF) and cough swabs (CF, age-matched healthy volunteers) were collected and processed using both aerobic and strict anaerobic bacteriological culture techniques. Bacteria within the samples were detected by plating on selective agars and identified by PCR and sequencing of 16S ribosomal RNA genes. Results: Samples were collected from 21 healthy volunteers (mean (SD) age 11.0 (3.5) yrs; 13M:8F) and from 12 children with CF (mean (SD) age 12.8 (3.2) yrs; 6M:6F) when clinically stable. Bacteria were detected in all samples from CF patients and in 18/21 (86%) healthy volunteers. Aerobic bacteria from genera including Streptococcus, Staphylococcus, Pseudomonas and Haemophilus were detected in both cohorts with Pseudomonas [CF, n = 2/12 (17%); control, n = 1/21 (5%)] and Haemophilus [CF, n = 7/12 (58%); control n = 4/21 (19%)] present in a greater number of CF patients compared to controls. Anaerobic bacteria from genera including Prevotella, Veillonella, and Actinomyces were detected in both cohorts. Conclusion: Aerobic and anaerobic bacteria have been detected by culture in the lungs of children with CF and healthy children. Pseudomonas and Haemophilus, recognised as pathogens in CF patients, were more prevalent in CF patients compared to controls. Funded by US-Ireland Project Partnership Grant.

56 Antibiotic treatment of exacerbation of chronic airways infection

Objectives: Repeated cycles of pulmonary exacerbation of chronic infection causetissue damage and progressive drop in lung function for CF patients so makingeffective treatment and prevention strategies imperative. Our study aims to betterunderstand the response to antibiotics and identify biomarker targets suitable forimproved therapeutic or preventative treatments.Methods: Multidimensional LC-MS/MS and relative quantitation was employed toinvestigate the changes in sputum cellular proteome following antibiotic treatmentin 12 CF patients chronically infected with Pseudomonas aeruginosa.Conclusions: 1989 human proteins were identified in total. 37 proteins out ofthe 318 detected in >80% samples were differentially expressed (p<0.05) afterantibiotic treatment. Of these, 22 exhibited a strong correlation (r = 0.969) betweentheir pre-antibiotic/post-antibiotic ratios and ratios for the same proteinscomparing CF/healthy control cohorts, thus suggesting that successful treatmentpromotes a proteome shift towards a non-CF profile. Ingenuity Pathway Analysissoftware showed antibiotic treatment to most affect the molecular and cellularfunction categories of Cellular Movement (particularly Immune Cell Trafficking),Organismal Injury and Abnormalities, Cell Death and Free Radical Scavenging.67 P. aeruginosa proteins and 17 proteins from other bacterial species werealso detected. Bacterial proteins accounted for 0.5−35% of CF sputum cellularprotein. Although total cellular protein (per g sputum) decreased following antibiotictreatment, the percentage attributable to bacteria did not alter and no bacterialproteins were detected as differentially expressed.

WS11.10 Proteomic analysis of the chronically infected CF airways

Objectives: Progressive lung disease, driven by inflammation secondary to chronicbacterial infection, constitutes the chief burden of CF.We describe a systems biologyinvestigation of the pathogenesis of Pseudomonas aeruginosa infected CF airwaysaiming to identify biomarkers/pathways as potential targets for improved therapyor for use as prognostic indicators.Methods: We employed multi-dimensional LC-MS/MS to semi-quantitatively contrastthe cellular protein profiles of sputum from CF and control cohorts. Importantly,our approach effectively assesses the activity of CF cells directly in theirin vivo environment, so taking into account the interactions between host and thehighly complex CF microbiome.Results: 119 of the 2309 human proteins detected were common to all samples(36 CF + 12 control). Of these, 49 were down-regulated and 29 up-regulatedin CF (p<0.05). Additionally, 21 proteins were detected exclusively in all CFsamples and 17 proteins exclusively in all controls. Analysis for biological relevanceusing Ingenuity Pathway Analysis (IPA) software identified molecular and cellularfunctions belonging to the categories of Cell Death; Cellular Movement; ProteinSynthesis, Degradation, Trafficking & Post-Translational Modification; Cell-to-CellSignalling and Interaction; Free Radical Scavenging; and Cellular Assembly andOrganisation as divergent between the study cohorts.Conclusions: Comparison of the infected CF cellular proteome with IPA diseaseprofiles showed up-regulation of proteins previously identified in Respiratory, Immunologicaland Inflammatory Diseases, and down-regulation of proteins associatedwith Cancer and Neurological Disease.