Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o-; HBE) and CF cell lines (CFBE14o-; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40 mV and outwardly-rectifying I-V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers--NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.

WS02.7 Initial and chronic MRSA infection in cystic fibrosis

Objectives Chronic MRSA infection, which affects approximately 26% of CF patients in the USA, is associated with declining lung function and poor outcomes (Dasenbrook, 2010). Anaerobic niches have been described within the CF lung, potentially influencing the virulence of MRSA. This study aims to compare initial and chronic CF MRSA isolates, following aerobic and anaerobic culture. Methods Isolates, obtained from CF sputum at first isolation [“early” (n = 10)] or up to 5 years later, during chronic infection [“late” (n = 15)] were cultured in aerobic and anaerobic conditions. Differences in virulence were compared using the Galleria mellonella infection model. Biofilm formation of each isolate was assessed following staining with crystal violet. Production of Δ-haemolysin (Δ-hly), a surrogate marker for expression of the virulence regulator agr, was determined by haemolysis assay. Results MRSA grown in anaerobic conditions had significantly increased virulence in the G. mellonella model (p = 0.007), increased biofilm formation (p = 0.006) and increased Δ-hly production (p<0.0001). No significant difference between Δ-hly production or biofilm formation were observed between early and late isolates; however late isolates were found to be more virulent in the G. mellonella model (p = 0.0002). Conclusion These results suggest that an anaerobic environment, as found in the CF lung, may increase virulence of MRSA and aid in the establishment of chronic infection. Further clinical studies are required to determine how these phenotypic changes are associated with transition to chronic infection and patient outcome.

Frequency of deletion 508 among Irish cystic fibrosis patients

We estimate the incidence of cystic fibrosis in Ireland to be at least 1 case per 1838 live births. We have analysed DNA from 44 Irish CF patients for the presence of deletion 508, using the polymerase chain reaction. The deletion was found in 76% of their chromosomes, and approximately 58% of the patients are homozygous for this deletion. Our results are not significantly different from those found in Canadian or UK patient populations, in which frequencies are higher than those found in Southern European countries.

Shortened Lung Clearance Index is a repeatable and sensitive test in children and adults with cystic fibrosis

Background: Lung clearance index (LCI) derived from sulfur hexafluoride (SF6) multiple breath washout (MBW) is a sensitive measure of lung disease in people with cystic fibrosis (CF). However, it can be time-consuming, limiting its use clinically. Aim: To compare the repeatability, sensitivity and test duration of LCI derived from washout to 1/30th (LCI1/30), 1/20th (LCI1/20) and 1/10th (LCI1/10) to ‘standard’ LCI derived from washout to 1/40th initial concentration (LCI1/40). Methods: Triplicate MBW test results from 30 clinically stable people with CF and 30 healthy controls were analysed retrospectively. MBW tests were performed using 0.2% SF6 and a modified Innocor device. All LCI end points were calculated using SimpleWashout software. Repeatability was assessed using coefficient of variation (CV%). The proportion of people with CF with and without abnormal LCI and forced expiratory volume in 1 s (FEV1) % predicted was compared. Receiver operating characteristic (ROC) curve statistics were calculated. Test duration of all LCI end points was compared using paired t tests. Results: In people with CF, LCI1/40 CV% (p=0.16), LCI1/30 CV%, (p=0.53), LCI1/20 CV% (p=0.14) and LCI1/10 CV% (p=0.25) was not significantly different to controls. The sensitivity of LCI1/40, LCI1/30 and LCI1/20 to the presence of CF was equal (67%). The sensitivity of LCI1/10 and FEV1% predicted was lower (53% and 47% respectively). Area under the ROC curve (95% CI) for LCI1/40, LCI1/30, LCI1/20, LCI1/10 and FEV1% predicted was 0.89 (0.80 to 0.97), 0.87 (0.77 to 0.96), 0.87 (0.78 to 0.96), 0.83 (0.72 to 0.94) and 0.73 (0.60 to 0.86), respectively. Test duration of LCI1/30, LCI1/20 and LCI1/10 was significantly shorter compared with the test duration of LCI1/40 in people with CF (p<0.0001) equating to a 5%, 9% and 15% time saving, respectively. Conclusions: In this study, LCI1/20 was a repeatable and sensitive measure with equal diagnostic performance to LCI1/40. LCI1/20 was shorter, potentially offering a more feasible research and clinical measure.

Physical activity assessment in cystic fibrosis: A position statement

Background

The aim of this position statement was to inform the choice of physical activity tools for use within CF research and clinical settings.

Methods

A systematic review of physical activity tools to explore evidence for reliability, validity, and responsiveness. Narrative answers to “four key questions” on motion sensors, questionnaires and diaries were drafted by the core writing team and then discussed at the Exercise Working Group in ECFS Lisbon 2013.

Results and summary

Our current position is that activity monitors such as SenseWear or ActiGraph offer informed choices to facilitate a comprehensive assessment of physical activity, and should as a minimum report on dimensions of physical activity including energy expenditure, step count and time spent in different intensities and sedentary time. The DigiWalker pedometer offers an informed choice of a comparatively inexpensive method of obtaining some measurement of physical activity. The HAES represents an informed choice of questionnaire to assess physical activity. There is insufficient data to recommend the use of one diary over another. Future research should focus on providing additional evidence of clinimetric properties of these and new physical activity assessment tools, as well as further exploring the added value of physical activity assessment in CF.

Is the raised volume rapid thoracic compression technique ready for use in clinical trials in infants with cystic fibrosis?

The European Cystic Fibrosis Society Clinical Trial Network (ECFS-CTN) has established a Standardization Committee to undertake a rigorous evaluation of promising outcome measures with regard to use in multicentre clinical trials in cystic fibrosis (CF). The aim of this article is to present a review of literature on clinimetric properties of the infant raised-volume rapid thoracic compression (RVRTC) technique in the context of CF, to summarise the consensus amongst the group on feasibility and answer key questions regarding the promotion of this technique to surrogate endpoint status.

METHODS: A literature search (from 1985 onwards) identified 20 papers that met inclusion criteria of RVRTC use in infants with CF. Data were extracted and tabulated regarding repeatability, validity, correlation with other outcome measures, responsiveness and reference values. A working group discussed the tables and answered 4 key questions.

RESULTS: Overall, RVRTC in particular forced expiratory volume in 0.5s, showed good clinimetric properties despite presence of individual variability. Few studies showed a relationship between RVRTC and inflammation and infection, and to date, data remains limited regarding the responsiveness of RVRTC after an intervention. Concerns were raised regarding feasibility in multi-centre studies and availability of reference values.

CONCLUSION: The ECFS-CTN Working Group considers that RVRTC cannot be used as a primary outcome in clinical trials in infants with CF before universal standardization of this measurement is achieved and implementation of inter-institutional networking is in place. We advise its use currently in phase I/II trials and as a secondary endpoint in phase III studies. We emphasise the need for (1) more short-term variability and longitudinal 'natural history' studies, and (2) robust reference values for commercially available devices.

A novel inhibitor of channel activating proteases: Putting the CAP on ENaC

Background: Excessive activation of epithelial sodium channels (ENaC) contributes to CF lung pathophysiology due to the resultant dehydration of the airway surface liquid (ASL) and impaired mucociliary clearance. Regulated proteolysis of the endogenous α and γ subunits of ENaC by apical membrane-bound Channel Activating Proteases (CAPs) is a fundamental regulatory mechanism for channel activity. In the CF lung a stark imbalance between the levels of CAPs and their natural inhibitors drives the activation of normally inactive ENaC. On this basis inhibition of CAPs-ENaC signalling represents a potential therapeutic intervention. To this end we have developed a novel cell impermeable active-site directed compound (QUB-TL1) designed to inactivate key trypsin-like CAPs highly relevant in this regard. Objectives & Methods: Utilize differentiated non-CF and CF human airway epithelial cells to assess the impact of QUB-TL1 on a range of parameters including surface CAP activities, ENaC subunit processing/channel activity, ASL height and mucociliary clearance. Results: Treatment of airway epithelial cells with QUB-TL1 results in the significant downregulation of key endogenous CAP activities found to be excessively active at the surface of CF cultures. QUB-TL1-mediated CAP inhibition subsequently causes the internalisation of a pool of processed (active) ENaCγ prominent at the apical surface of CF cultures which correlates with a decline in channel activity. This downregulation of ENaC activity results in an increase in ASL height and improved mucociliary clearance in CF cells. We further find QUB-TL1 uniquely inhibits the ENaC activating enzyme furin, which is in contrast to the alternate trypsin-like CAP inhibitors camostat mesylate and aprotinin. QUB-TL1-mediated furin inhibition correlates with a reduction in neutrophil elastase-induced ENaC activation. Moreover we find QUB-TL1 treatment protects CF cultures from Pseudomonas aeruginosa exotoxin A-induced cytotoxicity. Pseudomonas aeruginosa exotoxin A is a major toxic product activated by furin and positively associated with mortality. Conclusion: The novel inhibitor (QUB-TL1) dampens CAPs-ENaC signalling which improves hydration status mucociliary clearance in CF airway epithelial cell cultures. Moreover this compound provides additional benefit by preventing Pseudomonas aeruginosa exotoxin A-induced cytotoxicity.

Utilisation of a novel ProteaseTag™ activity immunoassay for the specific measurement of neutrophil elastase in BAL from children with cystic fibrosis

Introduction: Neutrophil elastase (NE) is a serine protease implicated in the pathogenesis of several respiratory diseases including cystic fibrosis (CF). The presence of free NE in BAL is a predictor of subsequent bronchiectasis in children with CF (Sly et al, 2013, NEJM 368: 1963-1970). Furthermore, children with higher levels of sputum NE activity (NEa) tend to experience a more rapid decline in FEV1 over time even after adjusting for age, gender and baseline FEV1 (Sagel et al, 2012, AJRCCM 186: 857-865). Its detection and quantification in biological samples is however confounded by a lack of robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex samples containing multiple proteolytic and hydrolytic enzymes. ELISA systems measure total protein levels which can be a mixture of latent, active and protease-inhibitor complexes. We have therefore developed a novel assay (ProteaseTag™ Active NE Immunoassay), which couples an activity dependent NE-Tag with a specific antibody step, resulting in an assay which is both selective and specific for NEa. Aims: To clinically validate ProteaseTag™ Active NE for the detection of free NEa in BAL from children with CF. Methods: A total of 95 paediatric BAL samples [CF (n=76; 44M, 32F) non-CF (n=19; 12M, 7F)] collected through the Study of Host Immunity and Early Lung Disease in CF (SHIELD CF) were analysed for NEa using ProteaseTag™ Active NE (ProAxsis Ltd) and a fluorogenic substrate-based assay utilising Suc-AAPV-AMC (Sigma). IL-8 was measured by ELISA (R&D Systems). Results were analysed to show comparisons in free NEa between CF and non-CF samples alongside correlations with a range of clinical parameters. Results: NEa measured by ProteaseTag™ Active NE correlated significantly with age (r=0.3, p=0.01) and highly significantly with both IL-8 (r=0.4, p=<0.0001) and the absolute neutrophil count (ANC) (r=0.4, p=<0.0001). These correlations were not observed when NEa was measured by the substrate assay even though a significant correlation was found between the two assays (r=0.8, p<0.0001). A trend towards significance was found between NEa in the CF and non-CF groups when measured by ProteaseTag™ Active NE (p=0.07). Highly significant differences were found with the other inflammatory parameters between the 2 groups (IL-8: p=0.0002 and ANC: p=0.006). Conclusion: NEa as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE has been shown to be a specific and superior tool in the measurement of NEa in paediatric CF BAL samples (supporting data from previous studies using adult CF expectorated samples). The technology is currently being transferred to a lateral flow device for use at Point of Care. Acknowledgements: This work was supported by the National Children’s Research Centre, Dublin (SHIELD CF) and grants from the Medical Research Council and Cystic Fibrosis Foundation Therapeutics.

Efficacy and Long-Term Outcomes of Palivizumab Prophylaxis to Prevent Respiratory Syncytial Virus Infection in Infants with Cystic Fibrosis in Northern Ireland

BACKGROUND: RSV causes considerable morbidity and mortality in children. In cystic fibrosis (CF) viral infections are associated with worsening respiratory symptoms and bacterial colonization. Palivizumab is effective in reducing RSV hospitalization in high risk patient groups. Evidence regarding its effectiveness and safety in CF is inconclusive. CF screening in N. Ireland enabled timely palivizumab prophylaxis, becoming routine in 2002.

OBJECTIVES: To determine the effect of palivizumab on RSV-related hospitalization and compare lung function and bacterial colonization at age 6 years for those born pre- and post-introduction of palivizumab prophylaxis.

METHODS: A retrospective audit was conducted for all patients diagnosed with CF during the period from 1997 to 2007 inclusive. RSV-related hospitalization, time to Pseudomonas aeruginosa (PA) 1st isolate, lung function and growth parameters were recorded. Comparisons were made for outcomes pre- and post-introduction of routine palivizumab administration in 2002. A cost evaluation was also performed.

RESULTS: Ninety-two children were included; 47 pre- and 45 post-palivizumab introduction. The overall RSV-positive hospitalization rate was 13%. The relative risk of RSV infection in palivizumab non-recipients versus recipients was 4.78 (95%CI: 1.1-20.7), P = 0.027. Notably, PA 1st isolate was significantly earlier in the palivizumab recipient cohort versus non-recipient cohort (median 57 vs. 96 months, P < 0.025) with a relative risk of 2.5. Chronic PA infection at 6 years remained low in both groups, with similar lung function and growth parameters. Total costs were calculated at £96,127 ($151,880) for the non-recipient cohort versus £137,954 ($217,967) for the recipient cohort.

CONCLUSION: Palivizumab was effective in reducing RSV-related hospitalization infection in CF patients. Surprisingly, we found a significantly earlier time to 1st isolate of PA in palivizumab recipients which we could not explain by altered or improved diagnostic tests. 

Closed circuit rebreathing to achieve inert gas wash-in for multiple breath wash-out

Multiple breath wash-out (MBW) testing requires prior wash-in of inert tracer gas. Wash-in efficiency can be enhanced by a rebreathing tracer in a closed circuit. Previous attempts to deploy this did not account for the impact of CO2 accumulation on patients and were unsuccessful. We hypothesised that an effective rebreathe wash-in could be delivered and it would not alter wash-out parameters. Computer modelling was used to assess the impact of the rebreathe method on wash-in efficiency. Clinical testing of open and closed circuit wash-in–wash-out was performed in healthy controls and adult patients with cystic fibrosis (CF) using a circuit with an effective CO2 scrubber and a refined wash-in protocol. Wash-in efficiency was enhanced by rebreathing. There was no difference in mean lung clearance index between the two wash-in methods for controls (6.5 versus 6.4; p=0.2, n=12) or patients with CF (10.9 versus 10.8; p=0.2, n=19). Test time was reduced by rebreathe wash-in (156 versus 230 s for CF patients, p<0.001) and both methods were well tolerated. End wash-in CO2 was maintained below 2% in most cases. Rebreathe–wash-in is a promising development that, when correctly deployed, reduces wash-in time and facilitates portable MBW testing. For mild CF, wash-out outcomes are equivalent to an open circuit.

Production of extended-spectrum β-lactamases and the potential indirect pathogenic role of Prevotella isolates from the cystic fibrosis respiratory microbiota

Extended-spectrum β-lactamase (ESBL) production and the prevalence of the β-lactamase-encoding gene blaTEM were determined in Prevotella isolates (n=50) cultured from the respiratory tract of adults and young people with cystic fibrosis (CF). Time-kill studies were used to investigate the concept of passive antibiotic resistance and to ascertain whether a β-lactamase-positive Prevotella isolate can protect a recognised CF pathogen from the action of ceftazidime in vitro. The results indicated that approximately three-quarters (38/50; 76%) of Prevotella isolates produced ESBLs. Isolates positive for ESBL production had higher minimum inhibitory concentrations (MICs) of β-lactam antibiotics compared with isolates negative for production of ESBLs (P<0.001). The blaTEM gene was detected more frequently in CF Prevotella isolates from paediatric patients compared with isolates from adults (P=0.002), with sequence analysis demonstrating that 21/22 (95%) partial blaTEM genes detected were identical to blaTEM-116. Furthermore, a β-lactamase-positive Prevotella isolate protected Pseudomonas aeruginosa from the antimicrobial effects of ceftazidime (P=0.03). Prevotella isolated from the CF respiratory microbiota produce ESBLs and may influence the pathogenesis of chronic lung infection via indirect methods, including shielding recognised pathogens from the action of ceftazidime.

Inhibition of protease-ENaC signaling improves mucociliary function in cystic fibrosis airways

Rationale: In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height
compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of ENaC have therapeutic potential in CF airways to reduce the hyperstimulated sodium and fluid absorption to levels which can restore airways hydration.

Objectives: To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function.

Methods: Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy) and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured by LDH assay.

Measurements and Results: QUB-TL1 inhibits extracellularly-located CAPs, including prostasin, matriptase and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells (AECs). QUB-TL1-mediated CAPs inhibition results in diminished ENaC-mediated Na+ absorption in CF AECs due to internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A induced cell death.

Conclusions: QUB-TL1 corrects aberrant CAP activities providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CFTR mutation.

Prevotella denticola Lipopolysaccharide from a Cystic Fibrosis Isolate Possesses a Unique Chemical Structure

We report the first complete structural characterization of the lipopolysaccharide (LPS) from a cystic fibrosis (CF) clinical isolate of Prevotella denticola (B003V1S1X). Chemical, spectroscopic, and spectrometric analyses revealed a unique rough-type LPS (LOS) structure. The structure has a highly negatively charged heptasaccharide core region containing hexoses, with the first two sugars, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and mannose, highly phosphorylated. Furthermore, the lipid A moiety has the typical structure for the genus Prevotella, and was also highly phosphorylated.

O papel do técnico oficial de contas na elaboração dos relatórios de sustentabilidade:um estudo aplicado às maiores empresas portuguesas

Dissertação, Mestrado, Contabilidade e Finanças, Instituto Politécnico de Santarém, Escola Superior de Gestão e Tecnologia, 2014