Association of airway cathepsin B and S with inflammation in cystic fibrosis.

Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-a), urine TNF receptor 1 (TNFr1), plasma IL-6, and serum C-reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5)?µg/ml and 1.6 (0.88)?µg/ml, respectively and were found to correlate not only with each other but with NE, TNF-a and IL-8 (in all cases .?<?0.05). Airway cathepsin B further correlated with circulatory IL-6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies. Pediatr. Pulmonol. 2010; 45:860–868.

Antiproteases as therapeutics to target inflammation in cystic fibrosis.

Cystic Fibrosis (CF) is the most common fatal inherited disease of Caucasians, affecting about 1 in 3000 births. Patients with CF have a recessive mutation in the gene encoding the CF transmembrane conductance regulator (CFTR). CFTR is expressed in the epithelium of many organs throughout the exocrine system, however, inflammation and damage of the airways as a result of persistent progressive endobronchial infection is a central feature of CF. The inflammatory response to infection brings about a sustained recruitment of neutrophils to the site of infection. These neutrophils release various pro-inflammatory compounds including proteases, which when expressed at aberrant levels can overcome the endogenous antiprotease defence mechanisms of the lung. Unregulated, these proteases can exacerbate inflammation and result in the degradation of structural proteins and tissue damage leading to bronchiectasis and loss of respiratory function. Other host-derived and bacterial proteases may also contribute to the inflammation and lung destruction observed in the CF lung. Antiprotease strategies to dampen the excessive inflammatory response and concomitant damage to the airways remains an attractive therapeutic option for CF patients.

High concentrations of pepsin in bronchoalveolar lavage from children with cystic fibrosis are associated with high interleukin-8

Background Gastro-oesophageal reflux is common in children with cystic fibrosis (CF) and is thought to be associated with pulmonary aspiration of gastric contents. The measurement of pepsin in bronchoalveolar lavage (BAL) fluid has recently been suggested to be a reliable indicator of aspiration. The prevalence of pulmonary aspiration in a group of children with CF was assessed and its association with lung inflammation investigated. Methods This was a cross-sectional case–control study. BAL fluid was collected from individuals with CF (n=31) and healthy controls (n=7). Interleukin-8 (IL-8), pepsin, neutrophil numbers and neutrophil elastase activity levels were measured in all samples. Clinical, microbiological and lung function data were collected from medical notes. Results The pepsin concentration in BAL fluid was higher in the CF group than in controls (mean (SD) 24.4 (27.4) ng/ml vs 4.3 (4.0) ng/ml, p=0.03). Those with CF who had raised pepsin concentrations had higher levels of IL-8 in the BAL fluid than those with a concentration comparable to controls (3.7 (2.7) ng/ml vs 1.4 (0.9) ng/ml, p=0.004). Within the CF group there was a moderate positive correlation between pepsin concentration and IL-8 in BAL fluid (r=0.48, p=0.04). There was no association between BAL fluid pepsin concentrations and age, sex, body mass index z score, forced expiratory volume in 1 s or Pseudomonas aeruginosa colonisation status. Conclusions Many children with CF have increased levels of pepsin in the BAL fluid compared with normal controls. Increased pepsin levels were associated with higher IL-8 concentrations in BAL fluid. These data suggest that aspiration of gastric contents occurs in a subset of patients with CF and is associated with more pronounced lung inflammation.

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis

Background: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF.

Methods: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods.

Results: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (kappa=0.74) and B cepacia complex (kappa=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3 x 10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3 x 10(6) cfu/g, p=0.046).

Conclusion: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Incidence and risk factors for pulmonary exacerbation treatment failures in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa

Background: Pulmonary exacerbations (PEx) are responsible for much of the morbidity and mortality associated with cystic fibrosis (CF). However, there is a paucity of data on outcomes in CF PEx and factors influencing outcomes.

Methods: We reviewed all PEx in patients infected with Pseudomonas aeruginosa treated with parenteral antibiotics over 4 years at our center. Treatment failures were categorized a priori as those PEx requiring antibiotic regimen change, prolongation of therapy > 20 days because of failure to respond, an early recurrent event within < 45 days, or failure to recover lung function to > 90% of baseline FEV1.

Results: A total of 101 patients were followed for 452 PEx. Treatment failures were observed in 125 (28%) of PEx; antibiotic regimen change was observed in 27 (6%), prolongation of therapy in 29 (6%), early recurrent events in 63 (14%), and failure to recover lung function to > 90% of baseline FEV1 in 66 (15%). Demographic factors associated with one or more treatment failures per year included advanced airways disease, use of enteric feeds, CF-related diabetes, and CF liver disease but did not include female sex or F508del homozygosity. Increased treatment failure risk was associated with lower admission FEV1 and increased markers of inflammation. At therapeutic completion, increased inflammatory markers correlated with treatment failure. Failure rates decreased with increasing number of active antimicrobial agents used based on in vitro susceptibility (zero, 28/65 [43%]; one, 38/140 [27%]; two, 59/245 [24%]; three, 0/2 [0%]; P = .02).

Conclusions: One-fourth of PEx fail to respond adequately to initial management. Patient demographic and episode-specific clinical information can be used to identify individuals at increased risk of initial management failure.

Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis

This study demonstrated key resistance genes to fluroquinilones in Streptococcci isolated from sputum of people with CF. This suggests that other bacteria which are sometimes considered commensal may be a resovoir for resistance. Jse designed the study with Moore.

Population structure and characterization of viridans group streptococci (VGS) including Streptococcus pneumoniae isolated from adult patients with cystic fibrosis (CF)

A study was undertaken to examine the population structure of viridans group streptococci (VGS) in the sputum of adult patients with cystic fibrosis (CF). Freshly expectorated sputa (n=58) from 45 adult CF patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 190 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 12 species of VGS and included S. anginosus, S. australis, S. cristatus, S. gordonii, S. infantis, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. salivarius and S. sanguinis. The most frequently VGS organism isolated was S. salivarius (47/190; 24.7%), followed by S. mitts (36/190; 19%), S. sanguinis (25/190; 13.2%), S. oralis (20/190; 11.0%), S. pneumoniae (19/190; 10.0%), S. parasanguinis (16/190; 8.4%), S. infantis (11/190; 5.8%), S. gordonii (7/190; 3.7%), S. anginosus (4/190; 2.1%), S. cristatus (2/190; 1.1%), S. australis (1/190; 0.5%), S. mutans (1/190; 0.5%) and S. agalactiae (1/190; 0.5%). All, but four, patients harboured at least one VGS species, which ranged from one to five streptococcal species, with a mean of 2.85 species per patient. There was no clonality at the subspecies level employing ERIC RAPD PCR. Antibiotic susceptibility was determined by Minimum Inhibitory Concentration (MIC) testing against penicillin, erythromycin and ciprofloxacin. Overall, resistance to penicillin with all VGS was 73/190 (38.4%) and 167/190 (87.9%) for erythromycin. With regard to ciprofloxacin, 27/190 (14.2%) were fully resistant, whilst a further 21/190 (11.1%) showed intermediate resistance, which equated to approximately three quarters (74.7%) of isolates being fully sensitive to this agent. In addition, as a comparator control population, we examined antibiotic susceptibility, as above, in a non-CF population comprising 12 individuals (50 VGS isolates), who were not receiving chronic antibiotics. In comparison, 8% and 38% of VGS isolates from non-CF individuals were resistant by disk susceptibility testing to penicillin and erythromycin, respectively. None of the non-CF VGS organisms were resistant to ciprofloxacin, but 42% showed intermediate resistance. (C) 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Effect of Aspergillus fumigatus and Candida albicans on pro-inflammatory response in cystic fibrosis epithelium.

Background
The identification of filamentous fungi and/or yeasts in the airway secretions of individuals with cystic fibrosis (CF) is becoming increasingly prevalent; yet the importance of these organisms in relation to underlying inflammation is poorly defined.

Methods
Cystic fibrosis bronchial epithelial cells (CFBE) and human bronchial epithelial cells (HBE) were co-incubated with Candida albicans whole cells or Aspergillus fumigatus conidia for 24 h prior to the measurement of pro-inflammatory cytokines IL-6 and IL-8 by ELISA.

Results
Treatment of HBE or CFBE with C. albicans whole cells did not alter cytokine secretion. However treatment of CFBE with A. fumigatus conidia resulted in a 1.45-fold increase in IL-6 and a 1.65-fold increase in IL-8 secretion in comparison to basal levels; in contrast there was far less secretion from HBE cells.

Conclusion
Our data indicate that A. fumigatus infection modulates a pro-inflammatory response in CF epithelial cells while C. albicans does not.