Cedric Gunaratnam

Sputum Candida albicans Presages FEV1 Decline and Hospital-Treated Exacerbations in Cystic Fibrosis

BACKGROUND The role of Candida albicans in the cystic fibrosis (CF) airway is underexplored. Considered a colonizer, few question its pathogenic potential despite high isolation frequencies from sputum culture. We evaluated the frequency and identified the strongest predictors of C albicans colonization in CF. Independent associations of colonization with clinical outcomes were determined, and the longitudinal effects of C albicans acquisition on BMI and FEV₁ were evaluated. METHODS A prospective observational study of 89 patients with CF was performed (3,916 sputum samples over 11 years). Frequency of C albicans growth in sputum allowed classification of the cohort into colonizers and noncolonizers. BMI, FEV₁, hospital-treated exacerbations, and other clinical parameters were followed throughout the study to determine association with colonization status. Multivariate regression determined the strongest predictors of colonization and for clinical effects after adjustment for confounders. Repeated-measures analysis of variance assessed the longitudinal effect of colonization on BMI and FEV₁. RESULTS Colonization with C albicans was frequent (49.4%) and best predicted by pancreatic insufficiency (P = .014), osteopenia (P = .03), and cocolonization with Pseudomonas species (P = .002). C albicans colonization significantly predicted hospital-treated exacerbations (P = .004) after adjustment for confounders. Exacerbation rate significantly increased in patients with chronic or intermittent colonizations following first acquisition of C albicans. Colonization accelerated rates of decline for BMI (P < .0001) and FEV₁ (P < .001). CONCLUSION Airway colonization with C albicans presaged a greater rate of FEV₁ decline and hospital-treated exacerbations in CF.

Effect of Estrogen on Pseudomonas Mucoidy and Exacerbations in Cystic Fibrosis

BACKGROUND Women with cystic fibrosis are at increased risk for mucoid conversion of Pseudomonas aeruginosa, which contributes to a sexual dichotomy in disease severity. METHODS We evaluated the effects of estradiol and its metabolite estriol on P. aeruginosa in vitro and in vivo and determined the effect of estradiol on disease exacerbations in women with cystic fibrosis. RESULTS Estradiol and estriol induced alginate production in P. aeruginosa strain 01 and in clinical isolates obtained from patients with and those without cystic fibrosis. After prolonged exposure to estradiol, P. aeruginosa adopted early mucoid morphology, whereas short-term exposure inhibited bacterial catalase activity and increased levels of hydrogen peroxide, which is potentially damaging to DNA. Consequently, a frameshift mutation was identified in mucA, a key regulator of alginate biosynthesis in P. aeruginosa. In vivo levels of estradiol correlated with infective exacerbations in women with cystic fibrosis, with the majority occurring during the follicular phase (P<0.05). A review of the Cystic Fibrosis Registry of Ireland revealed that the use of oral contraceptives was associated with a decreased need for antibiotics. Predominantly nonmucoid P. aeruginosa was isolated from sputum during exacerbations in the luteal phase (low estradiol). Increased proportions of mucoid bacteria were isolated during exacerbations occurring in the follicular phase (high estradiol), with a variable P. aeruginosa phenotype evident in vivo during the course of the menstrual cycle corresponding to fluctuating estradiol levels. CONCLUSIONS Estradiol and estriol induced mucoid conversion of P. aeruginosa in women with cystic fibrosis through a mutation of mucA in vitro and were associated with selectivity for mucoid isolation, increased exacerbations, and mucoid conversion in vivo. (Funded by the Molecular Medicine Ireland Clinician-Scientist Fellowship Programme.).

Effects of Ivacaftor in Patients With Cystic Fibrosis Who Carry the G551D Mutation and Have Severe Lung Disease

BACKGROUND The development of ivacaftor represents a significant advance in therapeutics for patients with cystic fibrosis (CF) who carry the G551D mutation. Patients with an FEV1 < 40% predicted represent a considerable proportion of eligible patients but were excluded from phase 3 clinical trials, and the effectiveness of the drug in this population is, therefore, unknown. METHODS Data were collected from adult CF centers in the United Kingdom and Ireland with patients enrolled in an ivacaftor compassionate use program (FEV1 < 40% or on lung transplant waiting list). Clinically recorded data were collated from patient records for 1 year prior and for a period of 90 to 270 days following ivacaftor commencement. Each patient was matched to two control subjects who would have met the requirements for the compassionate use program with the exception of genotype. RESULTS Twenty-one patients received ivacaftor for a median of 237 days. Mean FEV1 improved from 26.5% to 30.7% predicted (P = .01), representing a 16.7% relative improvement. Median weight improved from 49.8 to 51.6 kg (P = .006). Median inpatient IV antibiotic days declined from 23 to 0 d/y (P = .001) and median total IV treatment days decreased from 74 to 38 d/y (P = .002) following ivacaftor. Changes in pulmonary function and IV antibiotic requirements were significant compared with control subjects. CONCLUSIONS Ivacaftor was clinically effective in patients with CF who carry the G551D mutation and have severe pulmonary disease. The reductions in treatment requirements were clinically and statistically significant and have not been described in less severe populations.

The Effect of Aspergillus fumigatus Infection on Vitamin D Receptor Expression in Cystic Fibrosis

RATIONALE Aspergillus fumigatus (A. fumigatus) in cystic fibrosis (CF) is increasingly recognized. Although allergic bronchopulmonary aspergillosis (ABPA) leads to deterioration of pulmonary function, the effect of A. fumigatus colonization in the absence of ABPA remains unclear. OBJECTIVES To address this, we examined individuals with CF with A. fumigatus who were ABPA negative to identify the effects of itraconazole therapy on Aspergillus-induced lung inflammation. METHODS The effect of A. fumigatus on nuclear vitamin D receptor (VDR) expression was investigated using qRT-PCR and Western blotting. IL-5 and IL-13 levels were quantified by ELISA. The effect of itraconazole was assessed by a combination of high-resolution computed tomography, lung function test, and microbiological analysis. MEASUREMENTS AND MAIN RESULTS We demonstrate that A. fumigatus down-regulates VDR in macrophages and airway epithelial cells and that the fungal metabolite gliotoxin (Gt) is the main causative agent. Gt overcame the positive effect of 1,25-OH vitamin D(3) on VDR expression in vitro, resulting in increased IL-5 and IL-13 production. In vivo, A. fumigatus positivity was associated with increased Gt in CF bronchoalveolar lavage fluid and increased bronchoalveolar lavage fluid levels of IL-5 and IL-13. After airway eradication of A. fumigatus with itraconazole, we observed decreased Gt, IL-5 and IL-13, improved respiratory symptoms, and diminished high-resolution computed tomography mosaic pattern consistent with sustained pulmonary function. CONCLUSIONS This study provides a rationale for the therapeutic effect of itraconazole and implied that the therapeutic potential of vitamin D supplementation in preventing ABPA is only feasible with concurrent elimination of A. fumigatus to permit VDR expression and its positive functional consequences.

The Role of Short-Chain Fatty Acids, Produced by Anaerobic Bacteria, in the Cystic Fibrosis Airway

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 and inflammatory processes. OBJECTIVES To investigate the capacity of anaerobes to contribute to cystic fibrosis (CF) airway pathogenesis via SCFAs. METHODS Samples of 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFA levels in anaerobic supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of two SCFA receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings and 16HBE14o(-) and CFBE41o(-) cells were evaluated using reverse transcription polymerase chain reaction, Western blot analysis, laser scanning cytometry, and confocal microscopy. SCFA-induced IL-8 secretion was monitored by ELISA. MEASUREMENTS AND MAIN RESULTS Fifty-seven (52.3%) of 109 PWCF were anaerobe positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF younger (n = 24) and older (n = 85) than 6 years of age. All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic, and butyric acids. SCFA levels were higher in BAL samples of adults than in those of children. GPR41 levels were elevated in CFBE41o(-) versus 16HBE14o(-) cells; CF versus non-CF bronchial brushings; and 16HBE14o(-) cells after treatment with cystic fibrosis transmembrane conductance regulator 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 in 16HBE14o(-) cells. CONCLUSIONS This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via up-regulated GPR41.

The basophil surface marker CD203c identifies Aspergillus species sensitization in patients with cystic fibrosis

BACKGROUND Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.

The CF-ABLE Score: A Novel Clinical Prediction Rule for Prognosis in Patients With Cystic Fibrosis

BACKGROUND Determining prognosis and predicting outcomes in cystic fibrosis (CF) is a complex issue, and there have been very few clinically applicable models for this. The aim was to create a simple, practical outcome prediction tool for CF. METHODS Forty-nine consecutive patients with CF from a single center were studied over an 84-month period (2004-2010). All baseline clinical parameters were gathered, and FEV₁ measurements were analyzed over the study period. Using patterns of FEV₁ decline, a tipping point of 52.8% predicted was identified. Other clinical variables were analyzed and correlated with outcome. Poor outcome was defined as death or transplantation. Using age, BMI, lung function (ie, FEV₁), and number of exacerbations in the past 3 months, the CF-ABLE score was created. The score was validated for data from 370 patients from the national Cystic Fibrosis Registry of Ireland. RESULTS The ABLE score uses clinical parameters that are measured at every clinic visit and scored on a scale from 0 to 7. If FEV₁ is < 52%, then 3.5 points are added; if the number of exacerbations in the past 3 months is > 1, then 1.5 points are added; if BMI is < 20.1 kg/m² or age < 24 years, each receive 1 point. CONCLUSIONS Patients with a low score have a very low risk of death or lung transplantation within 4 years; however, as the score increases, the risk significantly increases. Patients who score > 5 points have a 26% risk of poor outcome within 4 years. This score is simple and applicable and better predicts outcome than FEV₁ alone.

A novel neutrophil derived inflammatory biomarker of pulmonary exacerbation in cystic fibrosis

BACKGROUND The focus of this study was to characterize a novel biomarker for cystic fibrosis (CF) that could reflect exacerbations of the disease and could be useful for therapeutic stratification of patients, or for testing of potential drug treatments. This study focused exclusively on a protein complex containing alpha-1 antitrypsin and CD16b (AAT:CD16b) which is released into the bloodstream from membranes of pro-inflammatory primed neutrophils. METHODS Neutrophil membrane expression and extracellular levels of AAT and CD16b were quantified by flow cytometry, Western blot analysis and by 2D-PAGE. Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and AAT:CD16b complex were quantified in CF plasma (n=38), samples post antibiotic treatment for 14 days (n=10), chronic obstructive pulmonary disease (n=10), AAT deficient (n=10) and healthy control (n=14) plasma samples by ELISA. RESULTS Cell priming with IL-8 and TNF-alpha caused release of the AAT:CD16b complex from the neutrophil cell membrane. Circulating plasma levels of IL-8, TNF-alpha and AAT:CD16b complex were significantly higher in patients with CF than in the other patient groups or healthy controls (P<0.05). Antibiotic treatment of pulmonary exacerbation in patients with CF led to decreased plasma protein concentrations of AAT:CD16b complex with a significant correlation with improved FEV1 (r=0.81, P=0.003). CONCLUSION The results of this study have shown that levels of AAT:CD16b complex present in plasma correlate to the inflammatory status of patients. The AAT:CD16b biomarker may become a useful addition to the clinical diagnosis of exacerbations in CF.

Neutrophil Membrane Cholesterol Content is a Key Factor in Cystic Fibrosis Lung Disease

Background Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. Methodology PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n = 48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays. Findings Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. Interpretation Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion.

A specialized method of sputum collection and processing for therapeutic interventions in cystic fibrosis

Cystic fibrosis (CF) lung disease is characterized by aggressive neutrophil-dominated inflammation mediated in large part by neutrophil elastase (NE), an omnivorous protease released by activated or disintegrating neutrophils and a key therapeutic target. To date, several short-term studies have shown that anti-NE compounds can inhibit NE and have anti-inflammatory effects. However, progression to large-scale or multicenter clinical trials has been hampered by the fact that the current gold standard methodology of evaluating airway NE inhibition, bronchoalveolar lavage (BAL), is invasive, difficult to standardize across sites and excludes those with severe lung disease. Attempts to utilize sputum that is either spontaneously expectorated (SS) or induced (IS) have been hindered by poor reproducibility, often due to the various processing methods employed. In this study, we evaluate TEmperature-controlled Two-step Rapid Isolation of Sputum (TETRIS), a specialized method for the acquisition and processing of SS and IS. Using TETRIS, we show for the first time that NE activity and cytokine levels are comparable in BAL, SS and IS samples taken from the same people with CF (PWCF) on the same day once this protocol is used. We correlate biomarkers in TETRIS-processed IS and clinical outcome measures including FEV1, and show stability and reproducible inhibition of NE over time in IS processed by TETRIS. The data offer a tremendous opportunity to evaluate prognosis and therapeutic interventions in CF and to study the full spectrum of people with PWCF, many of whom have been excluded from previous studies due to being unfit for BAL or unable to expectorate sputum.