Charles R. Esther

Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis.

BACKGROUND Although nontuberculous mycobacteria (NTM) are recognized pathogens in cystic fibrosis (CF), associations with clinical outcomes remain unclear. METHODS Microbiological data was obtained from 1216 CF patients over 8years (481+/-55patients/year). Relationships to clinical outcomes were examined in the subset (n=271, 203+/-23 patients/year) with longitudinal data. RESULTS Five hundred thirty-six of 4862 (11%) acid-fast bacilli (AFB) cultures grew NTM, with Mycobacterium abscessus (n=298, 55.6%) and Mycobacterium avium complex (n=190, 35.4%) most common. Associated bacterial cultures grew Stenotrophomonas or Aspergillus species more often when NTM were isolated (18.2% vs. 8.4% and 13.9% vs. 7.2%, respectively, p<0.01). After controlling for confounders, patients with chronic M. abscessus infection had greater rates of lung function decline than those with no NTM infection (-2.52 vs. -1.64% predicted FEV(1)/year, p<0.05). CONCLUSIONS NTM infection is common in CF and associated with particular pathogens. Chronic M. abscessus infection is associated with increased lung function decline.

Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis

Ivacaftor, a CFTR potentiator drug used for cystic fibrosis, destabilizes rescued ΔF508 CFTR and interferes with the action of drugs that correct CFTR function. Potentiating Trouble Cystic fibrosis (CF) is a genetic disease caused by mutations of the CF transmembrane conductance regulator (CFTR) ion channel, resulting in pulmonary and other complications. Ivacaftor is the only targeted drug approved for CF, but it is not effective enough to treat the severest and most common form of this disease. Ivacaftor is a “potentiator,” which means that it improves the activity of mutant CFTR but cannot work if there is no CFTR on the cell surface. Other drugs, called “correctors,” help bring mutant CFTR to the cell surface, but two manuscripts by Cholon and Veit and coauthors now show that combining the two types of drugs does not work effectively, because potentiators make CFTR less stable, accelerating the removal of this channel from the cell membrane. Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed “correctors” such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and “potentiators” such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ΔF508. Thus, patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809–mediated CFTR correction in ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 CFTR by VX-770, markedly increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining these drugs to maximize rescue of ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.

Mass spectrometric analysis of biomarkers and dilution markers in exhaled breath condensate reveals elevated purines in asthma and cystic fibrosis.

Exhaled breath condensate (EBC) analyses promise simple and noninvasive methods to measure airway biomarkers but pose considerable methodological challenges. We utilized mass spectrometry to measure EBC purine biomarkers adenosine and AMP plus urea to control for dilutional variability in two studies: 1) a cross-sectional analysis of 28 healthy, 40 cystic fibrosis (CF), and 11 asthmatic children; and 2) a longitudinal analysis of 26 CF children before and after treatment of a pulmonary exacerbation. EBC adenosine, AMP, and urea were readily detected and quantified by mass spectrometry, and analysis suggested significant dilutional variability. Using biomarker-to-urea ratios to control for dilution, the EBC AMP-to-urea ratio was elevated in CF [median 1.3, interquartile range (IQR) 0.7-2.3] vs. control (median 0.75, IQR 0.3-1.4; P < 0.05), and the adenosine-to-urea ratio was elevated in asthma (median 1.5, IQR 0.9-2.9) vs. control (median 0.4, IQR 0.2-1.6; P < 0.05). Changes in EBC purine-to-urea ratios correlated with changes in percent predicted forced expiratory volume in 1 s (FEV(1)) (r = -0.53 AMP/urea, r = -0.55 adenosine/urea; P < 0.01 for both) after CF exacerbation treatment. Similar results were observed using dilution factors calculated from serum-to-EBC urea ratios or EBC electrolytes, and the comparable ratios of EBC electrolytes to urea in CF and control (median 3.2, IQR 1.6-6.0 CF; median 5.5, IQR 1.4-7.7 control) validated use of airway urea as an EBC dilution marker. These results show that mass spectrometric analyses can be applied to measurement of purines in EBC and demonstrate that EBC adenosine-to-urea and AMP-to-urea ratios are potential noninvasive biomarkers of airways disease.

Metabolomic analysis of bronchoalveolar lavage fluid from cystic fibrosis patients

Metabolite profiles of bronchoalveolar lavage fluid (BALF) from pediatric patients with cystic fibrosis (CF) were correlated to the degree of airway inflammation using nuclear magnetic resonance spectroscopy-based metabolomics. BALF was collected from 11 children with CF during clinically indicated bronchoscopy. The spectra from BALF with high levels of neutrophilic airway inflammation displayed signals from numerous metabolites, whereas the spectra from subjects with low levels of inflammation were very sparse. The metabolites identified in samples taken from subjects with high inflammation include known markers of inflammation such as amino acids and lactate, as well as many novel signals. Statistical analysis highlighted the most important metabolites that distinguished the high- from the low-inflammation groups. This first demonstration of metabolomics of human BALF shows that clear distinctions in the metabolic profiles can be observed between subjects experiencing high versus low inflammation and is a first step toward the goal of discovering novel biomarkers of airway inflammation.

Extracellular purines are biomarkers of neutrophilic airway inflammation.

Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF). The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation. Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation. The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.

Endoplasmic Reticulum/Golgi Nucleotide Sugar Transporters Contribute to the Cellular Release of UDP-sugar Signaling Molecules

Extracellular UDP-sugars promote cellular responses by interacting with widely distributed P2Y14 receptors, but the mechanisms by which these molecules are released from cells are poorly understood. Given the active role of UDP-sugars in glycosylation reactions within the secretory pathway, we hypothesized that UDP-sugar release includes an exocytotic component. This hypothesis was tested by assessing the contribution of endoplasmic reticulum (ER)/Golgi-resident UDP-GlcNAc transporters to the cellular release of their cognate substrates. A sensitive and highly selective assay for UDP-GlcNAc mass was developed using purified AGX2, an isoenzyme of human UDP-GlcNAc pyrophosphorylase. Robust constitutive release of UDP-GlcNAc was observed in yeast as well as in well differentiated human airway epithelial cells. The human UDP-GlcNAc transporter HFRC1 was overexpressed in human bronchial epithelial cells and was shown to localize in the Golgi and to enhance the surface expression of N-acetylglucosamine-rich glycans. HFRC1-overexpressing cells also displayed increased constitutive and hypotonic stress-stimulated release of UDP-GlcNAc. Yeast mutants lacking Yea4 (the ER UDP-GlcNAc transporter endogenously expressed in Saccharomyces cerevisiae) showed reduced UDP-GlcNAc release. Yea4-deficient cells complemented with Yea4 showed UDP-GlcNAc release rates at levels similar to or higher than wild type cells. Our results illustrate that ER/Golgi lumen constitutes a significant source of extracellular UDP-sugars and therefore plays a critical role in nucleotide sugar-promoted cell signaling.

Lung transplant outcomes in cystic fibrosis patients with pre‐operative Mycobacterium abscessus respiratory infections

Mycobacterium abscessus in cystic fibrosis (CF) patients is considered a contraindication to lung transplantation. We examine the post‐transplant outcomes of CF patients with M. abscessus pre‐transplant.

Primary pulmonary lymphangiectasia in infancy and childhood

Primary pulmonary lymphangiectasia (PPL) is a rare disorder of unknown aetiology characterised by dilatation of the pulmonary lymphatics. PPL is widely reported to have a poor prognosis in the neonatal period and little is known about the clinical features of patients who survive the newborn period. The current authors report the outcome in nine patients diagnosed in infancy with PPL over a 15-yr period at a single university-based hospital clinic and followed for a median of 6 yrs. Although all of the patients initially experienced respiratory distress, respiratory symptoms improved in most patients after infancy and were notably better by the age of 6 yrs. Many patients had poor weight gain in the first years of life, which eventually improved. Radiological scans showed progressive resolution of neonatal infiltrates, but were characterised by hyperinflation and increased interstitial markings in older children. Most patients had evidence of bronchitis and grew pathogenic organisms from quantitative bronchoalveolar lavage culture. Pulmonary function tests showed predominantly obstructive disease that did not deteriorate over time. In conclusion, these results suggest that primary pulmonary lymphangiectasia does not have as dismal a prognosis as previously described and symptoms and clinical findings improve after the first year of life.

Atopic asthmatic subjects but not atopic subjects without asthma have enhanced inflammatory response to ozone

BACKGROUND Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of ozone-induced inflammation has not been determined. OBJECTIVE We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. METHODS Fifty volunteers (25 healthy volunteers, 14 atopic nonasthmatic subjects, and 11 atopic asthmatic subjects not requiring maintenance therapy) underwent a 0.4-ppm ozone exposure protocol. Ozone response was determined based on changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell-surface markers, and cytokine and hyaluronic acid concentrations. RESULTS All cohorts experienced similar decreases in lung function after ozone. Atopic and atopic asthmatic subjects had increased sputum neutrophil numbers and IL-8 levels after ozone exposure; values did not significantly change in healthy volunteers. After ozone exposure, atopic asthmatic subjects had significantly increased sputum IL-6 and IL-1beta levels and airway macrophage Toll-like receptor 4, Fc(epsilon)RI, and CD23 expression; values in healthy volunteers and atopic nonasthmatic subjects showed no significant change. Atopic asthmatic subjects had significantly decreased IL-10 levels at baseline compared with healthy volunteers; IL-10 levels did not significantly change in any group with ozone. All groups had similar levels of hyaluronic acid at baseline, with increased levels after ozone exposure in atopic and atopic asthmatic subjects. CONCLUSION Atopic asthmatic subjects have increased airway inflammatory responses to ozone. Increased Toll-like receptor 4 expression suggests a potential pathway through which ozone generates the inflammatory response in allergic asthmatic subjects but not in atopic subjects without asthma.

A mass spectrometric method to simultaneously measure a biomarker and dilution marker in exhaled breath condensate

Exhaled breath condensate (EBC) collection is a simple and non-invasive method to sample airway secretions, but analysis is limited by extensive and variable dilution of airway secretions within the condensate. To overcome this limitation, we developed a sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to simultaneously detect adenyl purines as biomarkers of inflammation and urea as a dilution marker in EBC. Separation prior to mass spectrometry was achieved using a C18 column with methanol and formic acid as the mobile phase, and characteristic precursor to product ion transitions of m/z 268 to 136 (for adenosine), m/z 348 to 136 (for AMP), and m/z 61 to 44 (for urea) were monitored for quantification. To correct for matrix effects, isotopically labeled adenosine, AMP, and urea were used as internal standards. Using these methods, we detected urea and the adenyl purines adenosine and AMP in EBC from seven subjects with cystic fibrosis (CF) and seven healthy controls and found that the AMP/urea ratio was elevated in the CF samples. These results demonstrate that mass spectrometry can be used successfully in EBC analysis to simultaneously detect a biomarker for airway inflammation and control for variable dilution.