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1.  Cigarette Smoke Induces Systemic Defects in Cystic Fibrosis Transmembrane Conductance Regulator Function 
Rationale: Several extrapulmonary disorders have been linked to cigarette smoking. Smoking is reported to cause cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the airway, and is also associated with pancreatitis, male infertility, and cachexia, features characteristic of cystic fibrosis and suggestive of an etiological role for CFTR.
Objectives: To study the effect of cigarette smoke on extrapulmonary CFTR function.
Methods: Demographics, spirometry, exercise tolerance, symptom questionnaires, CFTR genetics, and sweat chloride analysis were obtained in smokers with and without chronic obstructive pulmonary disease (COPD). CFTR activity was measured by nasal potential difference in mice and by Ussing chamber electrophysiology in vitro. Serum acrolein levels were estimated with mass spectroscopy.
Measurements and Main Results: Healthy smokers (29.45 ± 13.90 mEq), smokers with COPD (31.89 ± 13.9 mEq), and former smokers with COPD (25.07 ± 10.92 mEq) had elevated sweat chloride levels compared with normal control subjects (14.5 ± 7.77 mEq), indicating reduced CFTR activity in a nonrespiratory organ. Intestinal current measurements also demonstrated a 65% decrease in CFTR function in smokers compared with never smokers. CFTR activity was decreased by 68% in normal human bronchial epithelial cells exposed to plasma from smokers, suggesting that one or more circulating agents could confer CFTR dysfunction. Cigarette smoke–exposed mice had decreased CFTR activity in intestinal epithelium (84.3 and 45%, after 5 and 17 wk, respectively). Acrolein, a component of cigarette smoke, was higher in smokers, blocked CFTR by inhibiting channel gating, and was attenuated by antioxidant N-acetylcysteine, a known scavenger of acrolein.
Conclusions: Smoking causes systemic CFTR dysfunction. Acrolein present in cigarette smoke mediates CFTR defects in extrapulmonary tissues in smokers.
doi:10.1164/rccm.201304-0733OC
PMCID: PMC3919073  PMID: 24040746
cystic fibrosis transmembrane conductance regulator; cigarette smoking; chronic obstructive pulmonary disease; acrolein
2.  Lessons learned from a randomized trial of airway secretion clearance techniques in cystic fibrosis 
Pediatric pulmonology  2010;45(3):291-300.
Rationale
Airway secretion clearance therapies are a cornerstone of cystic fibrosis care, however longitudinal comparative studies are rare. Our objectives were to compare three therapies [postural drainage and percussion: (postural drainage), flutter device, and high frequency chest wall oscillation: (vest)], by studying 1) change in pulmonary function; 2) time to need for IV antibiotics, 3) use of pulmonary therapies, 4) adherence to treatment, 5) treatment satisfaction, and 6) quality of life.
Methods
Participants were randomly assigned to one of three therapies twice daily. Clinical outcomes were assessed quarterly over 3 years.
Results
Enrollment goals were not met, and withdrawal rates were high, especially in postural drainage (51%) and flutter device (26%), compared to vest (9%), resulting in early termination. FEV1 decline, time to need IV antibiotics, and other pulmonary therapies were not different. The annual FEF25–75% predicted rate of decline was greater in those using vest (p=0.02). Adherence was not significantly different (p=0.09). Overall treatment satisfaction was higher in vest and flutter device than in postural drainage (p<0.05). Health-related quality of life was not different. The rate of FEV1 decline was 1.23% predicted/year.
Conclusions
The study was ended early due to dropout and smaller than expected decline in FEV1. Patients were more satisfied with vest and flutter device. The longitudinal decline in FEF25–75% was faster in vest; we found no other difference in lung function decline, taken together this warrants further study. The slow decline in FEV1 illustrates the difficulty with FEV1 decline as a clinical trial outcome.
doi:10.1002/ppul.21179
PMCID: PMC4163837  PMID: 20146387
cystic fibrosis; Chest Wall Oscillation; drainage, postural, Medication Adherence; patient dropouts
3.  Efficacy and safety of a unique enteric-coated bicarbonate-buffered pancreatic enzyme replacement therapy in children and adults with cystic fibrosis 
Clinical investigation  2013;3(8):723-729.
Background
Pancreatic enzyme replacement therapy (PERT) is used to treat exocrine pancreatic insufficiency in cystic fibrosis.
Results/Methods
Efficacy and safety of a unique enteric-coated (EC) bicarbonate-buffered PERT product (PERTZYE®/PANCRECARB®; Digestive Care, Inc., Bethlehem, PA, USA) was studied in a randomized, double-blind, placebo-controlled cross-over design. Subjects were stabilized on EC-bicarbonate-buffered PERT and a high-fat diet. During two treatment periods, subjects were randomized to EC-bicarbonate-buffered PERT or placebo, followed by a 72-h stool collection employing an ingested stool dye marker. Mean coefficient of fat absorption with EC-bicarbonate-buffered PERT was 82.5% compared with 46.3% with the placebo (absolute difference 36.2%; p < 0.001), a 78.2% improvement for active over placebo. Similar improvements in nitrogen absorption were observed. Overall stool frequency and stool weight decreased (p < 0.001). No safety concerns were identified.
Summary
EC-bicarbonate-buffered PERT is effective in treating cystic fibrosis-associated exocrine pancreatic insufficiency.
doi:10.4155/cli.13.62
PMCID: PMC4157687  PMID: 25210613
bicarbonate-buffered pancrelipase; coefficient of fat absorption; cystic fibrosis; exocrine pancreatic insufficiency; pancreatic enzyme replacement therapy
4.  Acquired defects in CFTR-dependent β-adrenergic sweat secretion in chronic obstructive pulmonary disease 
Respiratory Research  2014;15(1):25.
Rationale
Smoking-induced chronic obstructive pulmonary disease (COPD) is associated with acquired systemic cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Recently, sweat evaporimetry has been shown to efficiently measure β-adrenergic sweat rate and specifically quantify CFTR function in the secretory coil of the sweat gland.
Objectives
To evaluate the presence and severity of systemic CFTR dysfunction in smoking-related lung disease using sweat evaporimetry to determine CFTR-dependent sweat rate.
Methods
We recruited a cohort of patients consisting of healthy never smokers (N = 18), healthy smokers (12), COPD smokers (25), and COPD former smokers (12) and measured β-adrenergic sweat secretion rate with evaporative water loss, sweat chloride, and clinical data (spirometry and symptom questionnaires).
Measurements and main results
β-adrenergic sweat rate was reduced in COPD smokers (41.9 ± 3.4, P < 0.05, ± SEM) and COPD former smokers (39.0 ± 5.4, P < 0.05) compared to healthy controls (53.6 ± 3.4). Similarly, sweat chloride was significantly greater in COPD smokers (32.8 ± 3.3, P < 0.01) and COPD former smokers (37.8 ± 6.0, P < 0.01) vs. healthy controls (19.1 ± 2.5). Univariate analysis revealed a significant association between β-adrenergic sweat rate and female gender (β = 0.26), age (−0.28), FEV1% (0.35), dyspnea (−0.3), and history of smoking (−0.27; each P < 0.05). Stepwise multivariate regression included gender (0.39) and COPD (−0.43) in the final model (R2 = 0.266, P < 0.0001).
Conclusions
β-adrenergic sweat rate was significantly reduced in COPD patients, regardless of smoking status, reflecting acquired CFTR dysfunction and abnormal gland secretion in the skin that can persist despite smoking cessation. β-adrenergic sweat rate and sweat chloride are associated with COPD severity and clinical symptoms, supporting the hypothesis that CFTR decrements have a causative role in COPD pathogenesis.
doi:10.1186/1465-9921-15-25
PMCID: PMC4015030  PMID: 24568560
5.  Urinary Desmosine: A Biomarker of Structural Lung Injury during CF Pulmonary Exacerbation 
Pediatric pulmonology  2012;47(9):10.1002/ppul.22525.
Summary
Rationale
Cystic fibrosis (CF) lung disease is characterized by structural changes and remodeling in airway architecture and lung parenchyma. Neutrophilic inflammation and infection lead to injury and breakdown of airway matrix constituents, including elastin. The non-invasive measurement of urinary desmosine (UDes), a breakdown product of elastin, may be reflective of ongoing lung injury and may serve as a biomarker of active short-term damage during pulmonary exacerbation. Our objectives were to measure desmosine in the urine of CF patients hospitalized for treatment of a pulmonary exacerbation and to explore the correlation between desmosine concentration and other markers of clinical improvement, including lung function and inflammatory mediators.
Methods
Urine and blood samples plus lung function measurements were collected at up to three points during hospitalization for treatment of a CF pulmonary exacerbation. We used a repeated measures model, adjusted for age and time between measurements, to compare log transformed urine desmosine concentrations across multiple time points and to correlate those concentrations with related clinical variables. Change in UDes concentration was investigated using a statistical model that incorporated normalization factors to account for variations in urinary concentration.
Results
Desmosine was measured by radioimmunoassay in 155 spot urine samples from 53 CF patients hospitalized for 63 pulmonary exacerbations (range of results: 0–235 pmol Des/mL). Specific gravity adjusted UDes concentration decreased significantly during admission for CF pulmonary exacerbation, p<0.01 (average length of stay = 11 days). No correlation was observed between UDes concentration and lung function or inflammatory markers.
Conclusions
Urinary desmosine decreased significantly following treatment for an acute pulmonary exacerbation and may be a useful biomarker of short-term injury to the CF lung. Further investigation is needed to evaluate the utility of UDes concentration in the long-term progression of CF lung disease.
doi:10.1002/ppul.22525
PMCID: PMC3856884  PMID: 22431382
elastin breakdown; matrix; airway; inflammation
6.  Evaluation of Circulating Proteins and Hemodynamics Towards Predicting Mortality in Children with Pulmonary Arterial Hypertension 
PLoS ONE  2013;8(11):e80235.
Background
Although many predictors have been evaluated, a set of strong independent prognostic mortality indicators has not been established in children with pediatric pulmonary arterial hypertension (PAH). The aim of this study was to identify a combination of clinical and molecular predictors of survival in PAH.
Methods
This single-center, retrospective cohort study was performed from children with PAH between 2001 and 2008 at Children's Hospital Colorado. Blood samples from 83 patients (median age of 8.3 years-old) were obtained. We retrospectively analyzed 46 variables, which included 27 circulating proteins, 7 demographic variables and 12 hemodynamic and echocardiographic variables for establishing the best predictors of mortality. A data mining approach was utilized to evaluate predictor variables and to uncover complex data structures while performing variable selection in high dimensional problems.
Results
Thirteen children (16%) died during follow-up (median; 3.1 years) and survival rates from time of sample collection at 1 year, 3 years and 5 years were 95%, 85% and 79%, respectively. A subset of potentially informative predictors were identified, the top four are listed here in order of importance: Tissue inhibitors of metalloproteinases-1 (TIMP-1), apolipoprotein-AI, RV/LV diastolic dimension ratio and age at diagnosis. In univariate analysis, TIMP-1 and apolipoprotein-AI had significant association with survival time (hazard ratio [95% confidence interval]: 1.25 [1.03, 1.51] and 0.70 [0.54–0.90], respectively). Patients grouped by TIMP-1 and apolipoprotein-AI values had significantly different survival risks (p<0.01).
Conclusion
Important predictors of mortality were identified from a large number of circulating proteins and clinical markers in this cohort. If confirmed in other populations, measurement of a subset of these predictors could aid in management of pediatric PAH by identifying patients at risk for death. These findings also further support a role for the clinical utility of measuring circulating proteins.
doi:10.1371/journal.pone.0080235
PMCID: PMC3835871  PMID: 24278261
7.  Bronchoalveolar lavage fluid cytokine profiles in neuroendocrine cell hyperplasia of infancy and follicular bronchiolitis 
Background
Neuroendocrine Cell Hyperplasia of Infancy (NEHI) and Follicular Bronchiolitis (FB) are rare pediatric diffuse lung diseases with poorly understood pathogenesis and similar clinical presentations. We sought to determine if cellular and cytokine profiles in bronchoalveolar lavage fluid (BALF) from subjects with NEHI and FB would differ from pediatric disease controls.
Methods
BALF was obtained from forty-one subjects classified into four disease groups: NEHI, Cystic Fibrosis (CF), other airway disease controls (DC), and FB during clinically indicated procedures. BALF cellular profiles and ten cytokines were measured and values compared across groups using descriptive and nonparametric statistics.
Results
Significant BALF cellular and cytokine differences were seen across all groups. NEHI subjects exhibited the lowest total absolute white blood cell (WBC) levels with a higher percentage of BALF alveolar macrophages compared to controls. NEHI also had lower levels of IL-1β, MIP-1β and IL-8 and FB had higher levels of IL-1ra, G-CSF and VEGF compared to all groups. IL-6 was elevated in CF and FB.
Conclusions
BALF cytokine and cellular profiles differed between NEHI, FB, CF and DC subjects. This pilot data suggests different and distinguishing inflammatory responses in the airway, with the least inflammatory being NEHI. These data could have diagnostic implications.
doi:10.1186/1750-1172-8-175
PMCID: PMC3892041  PMID: 24216293
Pediatric interstitial lung disease; Pediatric diffuse lung disease; Children’s interstitial lung disease (chILD); Bronchoalveolar lavage cytokine profiles
8.  Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation 
Thorax  2011;67(1):12-18.
Background
VX-809, a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, has been shown to increase the cell surface density of functional F508del-CFTR in vitro.
Methods
A randomised, double-blind, placebo-controlled study evaluated the safety, tolerability and pharmacodynamics of VX-809 in adult patients with cystic fibrosis (n=89) who were homozygous for the F508del-CFTR mutation. Subjects were randomised to one of four VX-809 28 day dose groups (25, 50, 100 and 200 mg) or matching placebo.
Results
The type and incidence of adverse events were similar among VX-809- and placebo-treated subjects. Respiratory events were the most commonly reported and led to discontinuation by one subject in each active treatment arm. Pharmacokinetic data supported a once-daily oral dosing regimen. Pharmacodynamic data suggested that VX-809 improved CFTR function in at least one organ (sweat gland). VX-809 reduced elevated sweat chloride values in a dose-dependent manner (p=0.0013) that was statistically significant in the 100 and 200 mg dose groups. There was no statistically significant improvement in CFTR function in the nasal epithelium as measured by nasal potential difference, nor were there statistically significant changes in lung function or patient-reported outcomes. No maturation of immature F508del-CFTR was detected in the subgroup that provided rectal biopsy specimens.
Conclusions
In this study, VX-809 had a similar adverse event profile to placebo for 28 days in F508del-CFTR homozygous patients, and demonstrated biological activity with positive impact on CFTR function in the sweat gland. Additional data are needed to determine how improvements detected in CFTR function secondary to VX-809 in the sweat gland relate to those measurable in the respiratory tract and to long-term measures of clinical benefit. Clinical trial number NCT00865904
doi:10.1136/thoraxjnl-2011-200393
PMCID: PMC3746507  PMID: 21825083
9.  Optimizing Nasal Potential Difference Analysis for CFTR Modulator Development: Assessment of Ivacaftor in CF Subjects with the G551D-CFTR Mutation 
PLoS ONE  2013;8(7):e66955.
Nasal potential difference (NPD) is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770) in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1) the average of both nostrils; (2) the most-polarized nostril at each visit; and (3) the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity), the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity), and the delta NPD (measuring CFTR and ENaC activity). The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (±2.8 mV). Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.
doi:10.1371/journal.pone.0066955
PMCID: PMC3724869  PMID: 23922647
10.  Future Directions in Early Cystic Fibrosis Lung Disease Research 
Since the 1989 discovery that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), there has been substantial progress toward understanding the molecular basis for CF lung disease, leading to the discovery and development of new therapeutic approaches. However, the earliest impact of the loss of CFTR function on airway physiology and structure and its relationship to initial infection and inflammation are poorly understood. Universal newborn screening for CF in the United States represents an unprecedented opportunity for investigating CF clinical manifestations very early in life. Recently developed animal models with pulmonary phenotypic manifestations also provide a window into the early consequences of this genetic disorder. For these reasons, the National Heart, Lung, and Blood Institute (NHLBI) convened a working group of extramural experts, entitled “Future Research Directions in Early CF Lung Disease” on September 21–22, 2010, to identify future research directions of great promise in CF. The priority areas identified included (1) exploring pathogenic mechanisms of early CF lung disease; (2) leveraging newborn screening to elucidate the natural history of early lung disease; (3) developing a spectrum of biomarkers of early lung disease that reflects CF pathophysiology, clinical outcome, and response to treatment; (4) exploring the role of genetics/genomics (e.g., modifier genes, gene–environmental interactions, and epigenetics) in early CF pathogenesis; (5) defining early microbiological events in CF lung disease; and (6) elucidating the initial airway inflammatory, remodeling, and repair mechanisms in CF lung disease.
doi:10.1164/rccm.201111-2068WS
PMCID: PMC3360572  PMID: 22312017
cystic fibrosis; airway disease; innate immunity; microbiology; genetics
11.  Update in Cystic Fibrosis 2007 
doi:10.1164/rccm.200801-069UP
PMCID: PMC2720148  PMID: 18460460
12.  Update in Cystic Fibrosis 2006 
doi:10.1164/rccm.200701-160UP
PMCID: PMC2720114  PMID: 17405941
13.  Gene modifiers in cystic fibrosis 
Studies of modifier genes in cystic fibrosis (CF) have often been performed in small or narrowly defined populations, leading to conflicting results. In this issue of the JCI, Dorfman et al. demonstrate in a large, population-based study that two previously studied modifier genes, coding for mannose-binding lectin 2 and TGF-β1, influence pulmonary outcome in pediatric CF patients (see the related article beginning on page 1040). They further show gene-gene interaction between the two, underscoring the complexity of CF lung disease. Their findings provide further impetus to study these molecules and associated signaling pathways in CF. In addition, these findings argue strongly for collecting genotypes of known modifiers prospectively in CF clinical trials as well as in longitudinal studies of infants identified through newborn screening, where the full impact of such modifiers can be defined more precisely.
doi:10.1172/JCI35138
PMCID: PMC2248430  PMID: 18292812
14.  Circulating Cytokines and Growth Factors in Pediatric Pulmonary Hypertension 
Mediators of Inflammation  2012;2012:143428.
Background. Management of pediatric pulmonary hypertension (PH) remains challenging. We have assessed a panel of circulating proteins in children with PH to investigate their value as predictive and/or prognostic biomarkers. From these determinations, we aim to develop a practical, noninvasive tool to aid in the management of pediatric PH. Methods. Twelve cytokines and growth factors putatively associated with lung or vascular disease were examined in plasma specimens from 70 children with PH using multiplex protein array technology. Associations between hemodynamics, adverse events, and protein markers were evaluated. Results. Epidermal growth factor (EGF) and IL-6 were associated with important hemodynamics. Of the twelve proteins, VEGF and IL-6 were significantly, univariately associated with the occurrence of an adverse event, with odds ratios (95% confidence intervals) of 0.56 (0.33–0.98) and 1.69 (1.03–2.77), respectively. When hemodynamic predictors were combined with protein markers, the ability to predict adverse outcomes within the following year significantly increased. Conclusions. Specific circulating proteins are associated with hemodynamic variables in pediatric PH. If confirmed in additional cohorts, measurement of these proteins could aid patient care and design of clinical trials by identifying patients at risk for adverse events. These findings also further support a role for inflammation in pediatric PH.
doi:10.1155/2012/143428
PMCID: PMC3536060  PMID: 23316102
15.  Update in Cystic Fibrosis 2005 
doi:10.1164/rccm.2601006
PMCID: PMC2662914  PMID: 16632633
16.  Effects of Gender and Age at Diagnosis on Disease Progression in Long-term Survivors of Cystic Fibrosis 
Rationale: Long-term survivors of cystic fibrosis (CF) (age > 40 yr) are a growing population comprising both patients diagnosed with classic manifestations in childhood, and nonclassic phenotypes typically diagnosed as adults. Little is known concerning disease progression and outcomes in these cohorts.
Objectives: Examine effects of age at diagnosis and gender on disease progression, setting of care, response to treatment, and mortality in long-term survivors of CF.
Methods: Retrospective analysis of the Colorado CF Database (1992–2008), CF Foundation Registry (1992–2007), and Multiple Cause of Death Index (1992–2005).
Measurements and Main Results: Patients with CF diagnosed in childhood and who survive to age 40 years have more severe CFTR genotypes and phenotypes compared with adult-diagnosed patients. However, past the age of 40 years the rate of FEV1 decline and death from respiratory complications were not different between these cohorts. Compared with males, childhood-diagnosed females were less likely to reach age 40 years, experienced faster FEV1 declines, and no survival advantage. Females comprised the majority of adult-diagnosed patients, and demonstrated equal FEV1 decline and longer survival than males, despite a later age at diagnosis. Most adult-diagnosed patients were not followed at CF centers, and with increasing age a smaller percentage of CF deaths appeared in the Cystic Fibrosis Foundation Registry. However, newly diagnosed adults demonstrated sustained FEV1 improvement in response to CF center care.
Conclusions: For patients with CF older than 40 years, the adult diagnosis correlates with delayed but equally severe pulmonary disease. A gender-associated disadvantage remains for females diagnosed in childhood, but is not present for adult-diagnosed females.
doi:10.1164/rccm.201001-0092OC
PMCID: PMC2937235  PMID: 20448091
cystic fibrosis; middle aged; aged; gender; outcome assessment
17.  Effect of VX-770 in Persons with Cystic Fibrosis and the G551D-CFTR Mutation 
The New England journal of medicine  2010;363(21):1991-2003.
BACKGROUND
A new approach in the treatment of cystic fibrosis involves improving the function of mutant cystic fibrosis transmembrane conductance regulator (CFTR). VX-770, a CFTR potentiator, has been shown to increase the activity of wild-type and defective cell-surface CFTR in vitro.
METHODS
We randomly assigned 39 adults with cystic fibrosis and at least one G551D-CFTR allele to receive oral VX-770 every 12 hours at a dose of 25, 75, or 150 mg or placebo for 14 days (in part 1 of the study) or VX-770 every 12 hours at a dose of 150 or 250 mg or placebo for 28 days (in part 2 of the study).
RESULTS
At day 28, in the group of subjects who received 150 mg of VX-770, the median change in the nasal potential difference (in response to the administration of a chloride-free isoproterenol solution) from baseline was −3.5 mV (range, −8.3 to 0.5; P = 0.02 for the within-subject comparison, P = 0.13 vs. placebo), and the median change in the level of sweat chloride was −59.5 mmol per liter (range, −66.0 to −19.0; P = 0.008 within-subject, P = 0.02 vs. placebo). The median change from baseline in the percent of predicted forced expiratory volume in 1 second was 8.7% (range, 2.3 to 31.3; P = 0.008 for the within-subject comparison, P = 0.56 vs. placebo). None of the subjects withdrew from the study. Six severe adverse events occurred in two subjects (diffuse macular rash in one subject and five incidents of elevated blood and urine glucose levels in one subject with diabetes). All severe adverse events resolved without the discontinuation of VX-770.
CONCLUSIONS
This study to evaluate the safety and adverse-event profile of VX-770 showed that VX-770 was associated with within-subject improvements in CFTR and lung function. These findings provide support for further studies of pharmacologic potentiation of CFTR as a means to treat cystic fibrosis.
doi:10.1056/NEJMoa0909825
PMCID: PMC3148255  PMID: 21083385
18.  Pulmonary exacerbations in cystic fibrosis with negative bacterial cultures 
Pediatric pulmonology  2010;45(6):569-577.
Summary
Background
Pulmonary exacerbations are a major cause of morbidity in cystic fibrosis (CF) and likely contribute to lung function decline. Exacerbations are often associated with characteristic airway bacteria [CF related bacteria (CFRB)]. However, some patients do not have CFRB detected by culture during exacerbations.
Objectives
We sought to determine the proportion of airway cultures negative for CFRB during pulmonary exacerbations, and to characterize patients who were CFRB-negative versus CFRB-positive.
Methods
We performed a retrospective study of patients with CF admitted for a pulmonary exacerbation. Patients were classified as CFRB-positive or CFRB-negative based on admission airway cultures. Demographics, clinical presentation, lung function, history of chronic P. aeruginosa infection and improvement in lung function with treatment were compared between groups.
Main Results
There were 672 admissions for exacerbation involving 211 patients over five years. Seventeen percent were classified as CFRB-negative. Forty-one percent of bronchoalveolar lavage (BAL), 32% of throat and 10% of sputum samples were CFRB-negative. Among patients capable of expectorating sputum, the CFRB-negative group was younger, less likely to have chronic P. aeruginosa, had higher lung function and body mass index (BMI), and had a lower systemic inflammatory response on admission compared to those with CFRB-positive cultures. The two groups had similar numbers of patients with three or more signs and symptoms of a pulmonary exacerbation (88% versus 92%). Both groups returned to baseline lung function following treatment.
Conclusions
A significant number of patients with CF and pulmonary exacerbation did not have typical CF related bacteria detected by culture. Patients without CF related bacteria still had characteristic signs and symptoms of pulmonary exacerbation and responded to treatment. Understanding the causes of illness in these patients may improve the diagnosis and treatment of pulmonary exacerbations in CF.
doi:10.1002/ppul.21221
PMCID: PMC2937349  PMID: 20503282
airway infection; sputum culture; bronchoalveolar lavage; lung function
19.  THE APPLICABILITY OF URINARY CREATININE AS A METHOD OF SPECIMEN NORMALIZATION IN THE CYSTIC FIBROSIS POPULATION 
Background
Urine specimens are commonly used in biomarker research. Urinary creatinine (UCr) is often used to adjust for urine analyte concentration. We aim to explore the applicability of UCr as a normalization method in a cystic fibrosis (CF) population during hospitalization.
Methods
Multiple spot urine samples were collected from CF patients hospitalized for a pulmonary exacerbation. Single spot specimens were obtained from asthmatics and healthy children for comparison. The assumptions and implications from the use of UCr as a normalization factor for urinary desmosine measurements were investigated.
Results
UCr differed significantly across disease groups and decreased significantly over time in the CF population. Differing results were obtained when contrasting normalization by UCr with specific gravity.
Conclusions
UCr levels are not completely attributable to simply variations in urine concentration. Analysis of urinary biomarker measurements should be initiated with an understanding of the relative effects of the normalization process on the results.
doi:10.1016/j.jcf.2010.02.004
PMCID: PMC2883692  PMID: 20227353
Urinary biomarkers; Urinary Creatinine; Cystic Fibrosis; Normalization Factor; Specific Gravity; Pulmonary Exacerbation
20.  Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results 
Human genetics  2009;126(6):763-778.
Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.
doi:10.1007/s00439-009-0724-8
PMCID: PMC2888886  PMID: 19662435
21.  MEASURING AND IMPROVING RESPIRATORY OUTCOMES IN CYSTIC FIBROSIS LUNG DISEASE: OPPORTUNITIES AND CHALLENGES TO THERAPY 
Cystic fibrosis (CF) is a life-shortening disease with significant morbidity. Despite overall improvements in survival, patients with CF experience frequent pulmonary exacerbations and declining lung function, which often accelerates during adolescence. New treatments target steps in the pathogenesis of lung disease, such as the basic defect in CF (CF Transmembrane Conductance Regulator [CFTR]), pulmonary infections, inflammation, and mucociliary clearance. These treatments offer hope but also present challenges to patients, clinicians, and researchers. Comprehensive assessment of efficacy is critical to identify potentially beneficial treatments. Lung function and pulmonary exacerbation are the most commonly used outcome measures in CF clinical research. Other outcome measures under investigation include measures of CFTR function; biomarkers of infection, inflammation, lung injury and repair; and patient-reported outcomes. Molecular diagnostics may help elucidate the complex CF airway microbiome. As new treatments are developed for patients with CF, efforts should be made to balance treatment burden with quality of life. This review highlights emerging treatments, obstacles to optimizing outcomes, and key future directions for research.
doi:10.1016/j.jcf.2009.09.003
PMCID: PMC2830746  PMID: 19833563
Cystic fibrosis; Emerging treatments; Improved outcomes
22.  Reliability of Quantitative Real-Time PCR for Bacterial Detection in Cystic Fibrosis Airway Specimens 
PLoS ONE  2010;5(11):e15101.
The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities ≥102 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.
doi:10.1371/journal.pone.0015101
PMCID: PMC2994853  PMID: 21152087
23.  Airway Infection with a Novel Cupriavidus Species in Persons with Cystic Fibrosis▿  
Journal of Clinical Microbiology  2009;47(9):3026-3028.
We describe the recovery and identification of a bacterium that represents a new species of the genus Cupriavidus from cultures of respiratory tract specimens from two patients with cystic fibrosis (CF). The elucidation of the role of this species in CF lung disease will require an evaluation of a greater number of cases.
doi:10.1128/JCM.00846-09
PMCID: PMC2738123  PMID: 19605576
24.  Guidelines for Diagnosis of Cystic Fibrosis in Newborns through Older Adults: Cystic Fibrosis Foundation Consensus Report 
The Journal of pediatrics  2008;153(2):S4-S14.
Newborn screening (NBS) for cystic fibrosis (CF) is increasingly being implemented and is soon likely to be in use throughout the United States, because early detection permits access to specialized medical care and improves outcomes. The diagnosis of CF is not always straightforward, however. The sweat chloride test remains the gold standard for CF diagnosis but does not always give a clear answer. Genotype analysis also does not always provide clarity; more than 1500 mutations have been identified in the CF transmembrane conductance regulator (CFTR) gene, not all of which result in CF. Harmful mutations in the gene can present as a spectrum of pathology ranging from sinusitis in adulthood to severe lung, pancreatic, or liver disease in infancy. Thus, CF identified postnatally must remain a clinical diagnosis. To provide guidance for the diagnosis of both infants with positive NBS results and older patients presenting with an indistinct clinical picture, the Cystic Fibrosis Foundation convened a meeting of experts in the field of CF diagnosis. Their recommendations, presented herein, involve a combination of clinical presentation, laboratory testing, and genetics to confirm a diagnosis of CF.
doi:10.1016/j.jpeds.2008.05.005
PMCID: PMC2810958  PMID: 18639722
25.  Circulating RNA Transcripts Identify Therapeutic Response in Cystic Fibrosis Lung Disease 
Rationale: Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV1, a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics.
Objectives: We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry.
Methods: Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV1% predicted were regressed with transcript changes.
Measurements and Main Results: Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV1 alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV1.
Conclusions: Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV1 may enhance current outcomes measures for treatment response.
doi:10.1164/rccm.200803-387OC
PMCID: PMC2577728  PMID: 18723435
cystic fibrosis; peripheral blood mononuclear cells; biomarkers; pulmonary exacerbation

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