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.

Applying the Transtheoretical Model to Physical Activity Behavior in Individuals With Non-Cystic Fibrosis Bronchiectasis

BACKGROUND: The transtheoretical model has been successful in promoting health behavior change in general and clinical populations. However, there is little knowledge about the application of the transtheoretical model to explain physical activity behavior in individuals with non-cystic fibrosis bronchiectasis. The aim was to examine patterns of (1) physical activity and (2) mediators of behavior change (self-efficacy, decisional balance, and processes of change) across stages of change in individuals with non-cystic fibrosis bronchiectasis.

METHODS: Fifty-five subjects with non-cystic fibrosis bronchiectasis (mean age ± SD = 63 ± 10 y) had physical activity assessed over 7 d using an accelerometer. Each component of the transtheoretical model was assessed using validated questionnaires. Subjects were divided into groups depending on stage of change: Group 1 (pre-contemplation and contemplation; n = 10), Group 2 (preparation; n = 20), and Group 3 (action and maintenance; n = 25). Statistical analyses included one-way analysis of variance and Tukey-Kramer post hoc tests.

RESULTS: Physical activity variables were significantly (P < .05) higher in Group 3 (action and maintenance) compared with Group 2 (preparation) and Group 1 (pre-contemplation and contemplation). For self-efficacy, there were no significant differences between groups for mean scores (P = .14). Decisional balance cons (barriers to being physically active) were significantly lower in Group 3 versus Group 2 (P = .032). For processes of change, substituting alternatives (substituting inactive options for active options) was significantly higher in Group 3 versus Group 1 (P = .01), and enlisting social support (seeking out social support to increase and maintain physical activity) was significantly lower in Group 3 versus Group 2 (P = .038).

CONCLUSIONS: The pattern of physical activity across stages of change is consistent with the theoretical predictions of the transtheoretical model. Constructs of the transtheoretical model that appear to be important at different stages of change include decisional balance cons, substituting alternatives, and enlisting social support. This study provides support to explore transtheoretical model-based physical activity interventions in individuals with non-cystic fibrosis bronchiectasis.

HDAC1 and HDAC2 independently predict mortality in hepatocellular carcinoma by a competing risk regression model in a Southeast Asian population

Histone deacetylases (HDACs) are enzymes involved in transcriptional repression. We aimed to examine the significance of HDAC1 and HDAC2 gene expression in the prediction of recurrence and survival in 156 patients with hepatocellular carcinoma (HCC) among a South East Asian population who underwent curative surgical resection in Singapore. We found that HDAC1 and HDAC2 were upregulated in the majority of HCC tissues. The presence of HDAC1 in tumor tissues was correlated with poor tumor differentiation. Notably, HDAC1 expression in adjacent non-tumor hepatic tissues was correlated with the presence of satellite nodules and multiple lesions, suggesting that HDAC1 upregulation within the field of HCC may contribute to tumor spread. Using competing risk regression analysis, we found that increased cancer-specific mortality was significantly associated with HDAC2 expression. Mortality was also increased with high HDAC1 expression. In the liver cancer cell lines, HEP3B, HEPG2, PLC5, and a colorectal cancer cell line, HCT116, the combined knockdown of HDAC1 and HDAC2 increased cell death and reduced cell proliferation as well as colony formation. In contrast, knockdown of either HDAC1 or HDAC2 alone had minimal effects on cell death and proliferation. Taken together, our study suggests that both HDAC1 and HDAC2 exert pro-survival effects in HCC cells, and the combination of isoform-specific HDAC inhibitors against both HDACs may be effective in targeting HCC to reduce mortality.

Expression of proteins associated with hypoxia and Wnt pathway activation is of prognostic significance in hepatocellular carcinoma

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p < 0.05). We identified a subgroup of hepatocellular carcinoma patients with high expression of both hypoxia and Wnt pathway proteins and found this predictive of poor survival. The therapeutic options for this group might need to be revisited.

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. 

Non-invasive ventilation for cystic fibrosis.

Non-invasive ventilation may be a means to temporarily reverse or slow the progression of respiratory failure in cystic fibrosis. To compare the effect of non-invasive ventilation versus no non-invasive ventilation in people with cystic fibrosis. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings. We searched the reference lists of each trial for additional publications possibly containing other trials.Most recent search: 22 February 2013. Randomised controlled trials comparing a form of pressure preset or volume preset non-invasive ventilation to no non-invasive ventilation in people with acute or chronic respiratory failure in cystic fibrosis. Three reviewers independently assessed trials for inclusion criteria and methodological quality, and extracted data. Fifteen trials were identified; seven trials met the inclusion criteria with a total of 106 participants. Six trials evaluated single treatment sessions and one evaluated a six-week intervention.Four trials (79 participants) evaluated non-invasive ventilation for airway clearance compared with an alternative chest physiotherapy method and showed that airway clearance may be easier with non-invasive ventilation and people with cystic fibrosis may prefer it. We were unable to find any evidence that NIV increases sputum expectoration, but it did improve some lung function parameters.Three trials (27 participants) evaluated non-invasive ventilation for overnight ventilatory support, measuring lung function, validated quality of life scores and nocturnal transcutaneous carbon dioxide. Due to the small numbers of participants and statistical issues, there were discrepancies in the results between the RevMan and the original trial analyses. No clear differences were found between non-invasive ventilation compared with oxygen or room air except for exercise performance, which significantly improved with non-invasive ventilation compared to room air over six weeks. Non-invasive ventilation may be a useful adjunct to other airway clearance techniques, particularly in people with cystic fibrosis who have difficulty expectorating sputum. Non-invasive ventilation, used in addition to oxygen, may improve gas exchange during sleep to a greater extent than oxygen therapy alone in moderate to severe disease. These benefits of non-invasive ventilation have largely been demonstrated in single treatment sessions with small numbers of participants. The impact of this therapy on pulmonary exacerbations and disease progression remain unclear. There is a need for long-term randomised controlled trials which are adequately powered to determine the clinical effects of non-invasive ventilation in cystic fibrosis airway clearance and exercise.

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.