Airway remodelling and its relationship to inflammation in cystic fibrosis

Pulmonary disease is the most important cause of morbidity and mortality in cystic fibrosis (CF). Most patients with CF die from respiratory failure with extensive airway destruction. Airway remodelling, defined as structural airway wall changes, begins early in life in CF but the sequence of remodelling events in the disease process is poorly understood. Airway remodelling in CF has traditionally been thought to be solely the consequence of repeated cycles of inflammation and infection. However, new evidence obtained from developmental, physiological and histopathological studies suggests that there might instead be multiple mechanisms leading to airway remodelling in CF including (1) changes related to infection and inflammation; (2) changes specific to CF as a result of CF transmembrane conductance regulator (CFTR) dysfunction in the airway wall, independent of infection and inflammation; and (3) protective responses to (1) and/or (2). Recent advances in bronchoscopic techniques have allowed airway mucosal (endobronchial) biopsies to be taken in children and even infants. Endobronchial biopsy studies may provide insight into the role and relative contribution of the different mechanisms of airway remodelling in CF, with the main limitation that they assess only changes in proximal large airways and not in peripheral small airways from where CF disease is thought to originate. Findings from biopsy studies could encourage the development of novel therapeutic strategies targeting structural changes in addition to infection and inflammation.

Enhanced antigenicity leads to altered immunogenicity in sulfamethoxazole-hypersensitive patients with cystic fibrosis

Exposure of patients with cystic fibrosis to sulfonamides is associated with a high incidence of hypersensitivity reactions.

Favourable prognostic value of antibodies anti-HSP20 in patients with cystic echinococcosis: a differential immunoproteomic approach

Seeking biomarkers reflecting disease development in cystic echinococcosis (CE), we used a proteomic approach linked to immunological characterisation for the identification of respective antigens. Two-dimensional gel electrophoresis (2-DE) of sheep hydatid fluid, followed by immunoblot analysis (IB) with sera from patients with distinct phases of disease, enabled us to identify by mass spectrometry heat shock protein 20 (HSP20) as a potential marker of active CE. Using IB, antibodies specific to the 34 kDa band of HSP20 were detected in sera from 61/95 (64%) patients with CE, but not in sera from healthy subjects. IB revealed anti-HSP20 antibodies in a higher percentage of sera from patients with active disease than in sera from patients with inactive disease (81 vs. 24%; P = 10(-4)). These primary results were confirmed in a long-term follow-up study after pharmacological and surgical treatment. Herewith anti-HSP20 antibody levels significantly decreased over the course of treatment in sera from patients with cured disease, relative to sera from patients with progressive disease (P = 0.017). Thus, during CE, a comprehensive strategy of proteomic identification combined with immunological validation represents a promising approach for the identification of biomarkers useful for the prognostic assessment of treatment of CE patients.

Emergence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients in Cleveland, Ohio

Linezolid (LZD)-resistant Staphylococcus aureus (LRSA) isolates were monitored from 2000 to 2009 in Cleveland, OH. LRSA first emerged in 2004 only in cystic fibrosis (CF) patients, with 11 LRSA-infected CF patients being identified by 2009. LRSA was isolated from 8 of 77 CF patients with S. aureus respiratory tract infection treated with LZD from 2000 to 2006. Analysis of clinical data showed that the 8 CF patients with LRSA received more LZD courses (18.8 versus 5.9; P = 0.001) for a longer duration (546.5 versus 211.9 days; P < 0.001) and had extended periods of exposure to LZD (83.1 versus 30.1 days/year; P < 0.001) than the 69 with LZD-susceptible isolates. Five LRSA isolates included in the clinical analysis (2000 to 2006) and three collected in 2009 were available for molecular studies. Genotyping by repetitive extrapalindromic PCR and pulsed-field gel electrophoresis revealed that seven of these eight LRSA strains from unique patients were genetically similar. By multilocus sequence typing, all LRSA isolates were included in clonal complex 5 (seven of sequence type 5 [ST5] and one of ST1788, a new single-locus variant of ST5). However, seven different variants were identified by spa typing. According to the Escherichia coli numbering system, seven LRSA isolates contained a G2576T mutation (G2603T, S. aureus numbering) in one to four of the five copies of domain V of the 23S rRNA genes. One strain also contained a mutation (C2461T, E. coli numbering) not previously reported. Two strains, including one without domain V mutations, possessed single amino acid substitutions (Gly152Asp or Gly139Arg) in the ribosomal protein L3 of the peptidyltransferase center, substitutions not previously reported in clinical isolates. Emergence of LRSA is a serious concern for CF patients who undergo prolonged courses of LZD therapy.

Lack of an exaggerated inflammatory response on virus infection in cystic fibrosis

Respiratory virus infections play an important role in cystic fibrosis (CF) exacerbations, but underlying pathophysiological mechanisms are poorly understood. We aimed to assess whether an exaggerated inflammatory response of the airway epithelium on virus infection could explain the increased susceptibility of CF patients towards respiratory viruses. We used primary bronchial and nasal epithelial cells obtained from 24 healthy control subjects and 18 CF patients. IL-6, IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and GRO-α/CXCL1 levels in supernatants and mRNA expression in cell lysates were measured before and after infection with rhinoviruses (RV-16 and RV-1B) and RSV. Cytotoxicity was assessed by lactate dehydrogenate assay and flow cytometry. All viruses induced strong cytokine release in both control and CF cells. The inflammatory response on virus infection was heterogeneous and depended on cell type and virus used, but was not increased in CF compared with control cells. On the contrary, there was a marked trend towards lower cytokine production associated with increased cell death in CF cells. An exaggerated inflammatory response to virus infection in bronchial epithelial cells does not explain the increased respiratory morbidity after virus infection in CF patients.

Treatment response of airway clearance assessed by single-breath washout in children with cystic fibrosis

We studied the ability of 4 single-breath gas washout (SBW) tests to measure immediate effects of airway clearance in children with CF.

Practicability of nitrogen multiple-breath washout measurements in a pediatric cystic fibrosis outpatient setting

BACKGROUND: Although lung clearance index (LCI) is a sensitive indicator of mild cystic fibrosis (CF) lung disease, it is rarely measured due to lengthy protocols and the commercial unavailability of multiple-breath washout (MBW) setups and tracer gases. We used a newly validated, commercially available nitrogen (N(2) ) MBW setup to assess success rate, duration, and variability of LCI within a 20 min timeframe, during clinical routine. We also evaluated the relationship between LCI and other clinical markers of CF lung disease. METHODS: One hundred thirty six children (83 with CF) between 4 and 16 years were studied in a pediatric CF outpatient setting. One hundred eighteen out of 136 children were naïve to MBW. Within 20 min, each child was trained, N(2) MBW was performed, and LCI was analyzed. We assessed intra- and between-test reproducibility in a subgroup of children. RESULTS: At least one LCI was feasible in 123 (90%) children, with a mean (range) of 3.3 (1.2-6.4) min per test. Two or more measurements were feasible in 56 (41%) children. Comparing LCI in CF versus controls, LCI mean (SD) was 12.0 (3.9) versus 6.1 (0.9), and the intra- and inter-test coefficient of repeatability was 1.00 versus 0.81 and 0.96 versus 0.62, respectively. LCI was correlated with spirometry, blood gases, and Pseudomonas aeruginosa infection. CONCLUSIONS: Using available N(2) MBW equipment, LCI measurements are practical and fast in children. LCI is correlated with markers of CF lung disease. Longer timeframes would be required for triplicate N(2) MBW tests in inexperienced children. Pediatr Pulmonol. © 2012 Wiley Periodicals, Inc.

A new double-tracer gas single-breath washout to assess early cystic fibrosis lung disease

In cystic fibrosis (CF), tests for ventilation inhomogeneity are sensitive but not established for clinical routine. We assessed feasibility of a new double-tracer gas single-breath washout (SBW) in school-aged children with CF and control subjects, and compared SBW between groups and with multiple-breath nitrogen washout (MBNW). Three SBW and MBNW were performed in 118 children (66 with CF) using a side-stream ultrasonic flowmeter setup. The double-tracer gas containing 5% sulfur hexafluoride and 26.3% helium was applied during one tidal breath. Outcomes were SBW phase III slope (SIII(DTG)), MBNW-derived lung clearance index (LCI), and indices of acinar (S(acin)) and conductive (S(cond)) ventilation inhomogeneity. SBW took significantly less time to perform than MBNW. SBW and MBNW were feasible in 109 (92.4%) and 98 (83.0%) children, respectively. SIII(DTG) differed between children with CF and controls, mean±sd was -456.7±492.8 and -88.4±129.1 mg·mol·L(-1), respectively. Abnormal SIII(DTG) was present in 36 (59%) children with CF. SIII(DTG) was associated with LCI (r= -0.58) and S(acin) (r= -0.58), but not with S(cond). In CF, steeply sloping SIII(DTG) potentially reflects ventilation inhomogeneity near the acinus entrance. This tidal SBW is a promising test to assess ventilation inhomogeneity in an easy and fast way.

High rhinovirus burden in lower airways of children with cystic fibrosis

Rhinovirus (RV)-induced pulmonary exacerbations are common in cystic fibrosis (CF) and have been associated with impaired virus clearance by the CF airway epithelium in vitro. Here, we assess in vivo the association of RV prevalence and load with antiviral defense mechanisms, airway inflammation, and lung function parameters in children with CF compared with a control group and children with other chronic respiratory diseases.

Newborn screening for cystic fibrosis in Switzerland - Consequences after analysis of a 4 months pilot study

BACKGROUND: Switzerland introduced newborn screening (NBS) for CF in 2011, using an IRT/DNA/IRT protocol. This paper describes the results of the first year and compares two versions of the protocol with different IRT cut-offs, particularly effects on recall rate, sensitivity and specificity. METHODS: IRT cut-offs were >45ng/ml (99.0th percentile) in period 1 (months 1-4) and >50ng/ml (99.2nd percentile) in period 2 (months 5-12). In period 2 we abstained from recalls when none of the 7 most common CF mutations were detected and IRT was <60ng/ml. RESULTS: In periods 1 and 2, 26,535 and 56,663 tests were performed. Recall rates were 0.94% and 0.48%, respectively (p<0.001), PPV increased from 23% to 47% (p=0.024) and sensitivity was 90% and 100%. CONCLUSIONS: Raising initial IRT cut-off from the 99.0th to the 99.2nd percentile and abstaining from recalls for children with an IRT<60ng/ml and carrying no major CFTR mutation significantly reduced the recall rate without affecting sensitivity.

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P ≤ 0.01), the specific effective airway resistance (P ≤ 0.005) and the forced expiratory volume in 1 s (P ≤ 0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.

Progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis

BACKGROUND: Functional deterioration in cystic fibrosis (CF) may be reflected by increasing bronchial obstruction and, as recently shown, by ventilation inhomogeneities. This study investigated which physiological factors (airway obstruction, ventilation inhomogeneities, pulmonary hyperinflation, development of trapped gas) best express the decline in lung function, and what role specific CFTR genotypes and different types of bronchial infection may have upon this process. METHODS: Serial annual lung function tests, performed in 152 children (77 males; 75 females) with CF (age range: 6-18 y) provided data pertaining to functional residual capacity (FRCpleth, FRCMBNW), volume of trapped gas (VTG), effective specific airway resistance (sReff), lung clearance index (LCI), and forced expiratory indices (FVC, FEV1, FEF50). RESULTS: All lung function parameters showed progression with age. Pulmonary hyperinflation (FRCpleth > 2SDS) was already present in 39% of patients at age 6-8 yrs, increasing to 67% at age 18 yrs. The proportion of patients with VTG > 2SDS increased from 15% to 54% during this period. Children with severe pulmonary hyperinflation and trapped gas at age 6-8 yrs showed the most pronounced disease progression over time. Age related tracking of lung function parameters commences early in life, and is significantly influenced by specific CFTR genotypes. The group with chronic P. aeruginosa infection demonstrated most rapid progression in all lung function parameters, whilst those with chronic S. aureus infection had the slowest rate of progression. LCI, measured as an index of ventilation inhomogeneities was the most sensitive discriminator between the 3 types of infection examined (p < 0.0001). CONCLUSION: The relationships between lung function indices, CFTR genotypes and infective organisms observed in this study suggest that measurement of other lung function parameters, in addition to spirometry alone, may provide important information about disease progression in CF.

Chemokines indicate allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

RATIONALE: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a Th2 immune response. Mouse models suggest a critical role for the Th2 chemokines thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in ABPA. OBJECTIVES: To determine whether serum levels of TARC and MDC characterize ABPA in patients with cystic fibrosis (CF) and to examine longitudinally if levels of TARC and MDC indicate ABPA exacerbations in patients with CF. METHODS: Levels of TARC and MDC and levels of Th1 (IL-12 and IFN-gamma) and Th2 (IL-4, IL-5, and IL-13) cytokines were analyzed in serum of 16 patients with CF with ABPA, six non-CF patients with asthma with ABPA, 13 patients with CF colonized with Aspergillus fumigatus, six patients with CF sensitized to A. fumigatus, 12 atopic patients with CF, and 13 non-CF atopic control subjects by ELISA. The longitudinal course of TARC, MDC, and IgE levels was assessed during ABPA episodes. RESULTS: Patients with ABPA had significantly higher serum levels of TARC compared with the other patient groups. Cytokine levels did not differ among the patient groups. Longitudinally, levels of TARC indicated ABPA exacerbations in patients with CF more clearly than IgE levels. In patients with CF and ABPA, levels of TARC correlated positively with specific IgE to A. fumigatus and rAsp f4. CONCLUSIONS: Serum levels of TARC differentiate patients with CF or patients with asthma with ABPA from patients with CF colonized with or sensitized to A. fumigatus, atopic patients with CF, and atopic control subjects. Longitudinally, levels of TARC indicate ABPA exacerbations, suggesting TARC as a marker for identification and monitoring of ABPA in patients with CF.