Extracellular purines are biomarkers of neutrophilic airway inflammation.
ABSTRACT: 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.
Project description:Adenosine and related purines have established roles in inflammation, and elevated airway concentrations are predicted in patients with COPD. However, accurate airway surface purine measurements can be confounded by stimulation of purine release during collection of typical respiratory samples.Airway samples were collected noninvasively as exhaled breath condensate (EBC) from 36 healthy nonsmokers (NS group), 28 healthy smokers (S group), and 89 subjects with COPD (29 with GOLD [Global Initiative for Chronic Obstructive Lung Disease] stage II, 29 with GOLD stage III, and 31 with GOLD stage IV) and analyzed with mass spectrometry for adenosine, adenosine monophosphate (AMP), and phenylalanine, plus urea as a dilution marker. Variable dilution of airway secretions in EBC was controlled using ratios to urea, and airway surface concentrations were calculated using EBC to serum urea-based dilution factors.EBC adenosine to urea ratios were similar in NS (0.20 ± 0.21) and S (0.22 ± 0.20) groups but elevated in those with COPD (0.32 ± 0.30, P < .01 vs NS). Adenosine to urea ratios were highest in the most severely affected cohort (GOLD IV, 0.35 ± 0.34, P < .01 vs NS) and negatively correlated with FEV(1) (r = -0.27, P < .01). Elevated AMP to urea ratios were also observed in the COPD group (0.58 ± 0.97 COPD, 0.29 ± 0.35 NS, P < .02), but phenylalanine to urea ratios were similar in all groups. Airway surface adenosine concentrations calculated in a subset of subjects were 3.2 ± 2.7 ?M in those with COPD (n = 28) relative to 1.7 ± 1.5 ?M in the NS group (n = 16, P < .05).Airway purines are present on airway surfaces at physiologically significant concentrations, are elevated in COPD, and correlate with markers of COPD severity. Purinergic signaling pathways are potential therapeutic targets in COPD, and EBC purines are potential noninvasive biomarkers.
Project description:Thiocyanate is a heme peroxidase substrate that scavenges oxidants produced during inflammation and regulates host defense. In cystic fibrosis (CF) patients, increased airway thiocyanate levels are associated with improved lung function. Research on airway thiocyanate is limited, however, because convenient non-invasive airway sampling methods, such as exhaled breath condensate (EBC), yield low concentrations that are difficult to detect with available assays. In the present study, we developed a method for the determination of thiocyanate in dilute samples using isotope dilution headspace gas chromatography-coupled high-resolution, accurate-mass mass spectrometry (GC-HRMS). The method reliably quantified as little as 4 pmol thiocyanate in EBC and could detect even lower amounts. We successfully measured thiocyanate in EBC from seven healthy donors, with a mean ±?SD of 27?±?16?nM and a median inter-assay coefficient of variation of 10.4% over six months. The method was applied to other biological fluids (plasma from the same visit as EBC donation; bronchoalveolar lavage fluid [BALF] from infants with CF; and healthy adult mouse BALF), giving reliable quantification of samples ranging from 10?nM to 100?µM. Thiocyanate concentrations in fluids besides EBC were (from lowest to highest): 0.73?±?0.39?µM in BALF of healthy adult mice (n?=?6); 1.4?±?1.4?µM in BALF from infants with CF (n?=?24); 46?±?22?µM in the plasma of adult volunteers (n?=?7). These results demonstrate the utility of this new method for clinical determination of thiocyanate in EBC and other biological fluids.
Project description:Calgranulins are a family of powerful chemoattractants, which have been implicated as biomarkers in inflammatory diseases. To determine how different respiratory diseases affect the expression of calgranulins, we measured the expression of S100A8/A9 and S100A12 in bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients and healthy volunteers by ELISA. Analysis of calgranulin expression revealed a high level of S100A12 in the lavages of patients suffering from ARDS compared to controls (p<0.001). Based on the hypothesis that the increased expression of S100A12 relative to the S100A8/A9 heterodimer was a characteristic of respiratory diseases with neutrophilic inflammation, we measured calgranulin expression in BALF of cystic fibrosis (CF) patients. Despite similarly elevated levels of S100A8/A9, S100A12 was significantly higher in ARDS compared to CF BALF (p<0.001). The differential expression of calgranulins was unique for inflammatory markers, as an array of cytokines did not differ between CF and ARDS patients. Since ARDS is an acute event and CF a chronic inflammation with acute exacerbations, we compared calgranulin expression in sputum obtained from CF and patients with chronic obstructive lung disease (COPD). Levels of S100A12 and S100A8/9 were elevated in CF sputum compared to COPD sputum, but the ratio of S100A12 to S100A8/A9 was similar in COPD and CF and reflected more closely than seen in healthy controls. The results indicate that the regulation of human calgranulin expression and the ratio of S100A8/A9 to S100A12 may provide important insights in the mechanism of respiratory inflammation.
Project description:Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2?years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ?50% of the microbiota, whereas in CF patients aged ?6?years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2?years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.
Project description:Changes in 5'-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5'-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5'-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5'-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5'-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5'-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.
Project description:Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models.
Project description:Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography-defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared "permanent" because they did not dissolve in dilute BALF matrix, they could be solubilized by a previously unidentified reducing agent (P2062), but not N-acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.
Project description:BACKGROUND:There are urgent needs for clinically relevant biomarkers to identify children with cystic fibrosis (CF) at risk for more progressive lung disease and to serve as outcome measures for clinical trials. Our objective was to investigate three targeted biomarkers in a population of asymptomatic CF infants. METHODS:Urine, blood and lung function data were collected for 2 years from clinically stable infants diagnosed with CF by newborn screening. A subset of CF infants had bronchoscopy with lavage performed at 6 months and 1 year. Urine was collected quarterly from healthy control infants. Expectorated sputum and urine were collected quarterly for 2 years from clinically stable CF adults. Desmosine, club cell secretory protein (CCSP) and cathepsin B concentrations were measured and compared. Mixed effects models were used to identify associations between biomarker concentrations and clinical characteristics. Receiver operator characteristic curves were generated to investigate the sensitivity and specificity of the biomarkers. RESULTS:Urinary cathepsin B was significantly higher in CF infants compared to healthy infants (p?=?0.005). CF infant airway and urinary cathepsin B concentrations were significantly lower compared to adult CF subjects (p?=?0.002 & p?=?0.022, respectively). CF infant airway CCSP was significantly higher than adult CF subjects (p?<?0.001). There was a significant correlation between CF infant plasma CCSP and BALF CCSP (p?=?0.046). BALF CCSP was negatively associated with IL-8 (p?=?0.017). There was no correlation between biomarker concentration and FEV0.5. CONCLUSIONS:Cathepsin B and CCSP show promise as biomarkers of inflammation in CF infants. Further study is needed.
Project description:Upper airway cultures guide the identification and treatment of lung pathogens in infants with cystic fibrosis (CF); however, this may not fully reflect the spectrum of bacteria present in the lower airway. Our objectives were to characterize the airway microbiota using bronchoalveolar lavage fluid (BALF) from asymptomatic CF infants during the first year of life and to investigate the relationship between BALF microbiota, standard culture and clinical characteristics.BALF, nasopharyngeal (NP) culture and infant pulmonary function testing data were collected at 6 months and one year of age during periods of clinical stability from infants diagnosed with CF by newborn screening. BALF was analyzed for total bacterial load by qPCR and for bacterial community composition by 16S ribosomal RNA sequencing. Clinical characteristics and standard BALF and NP culture results were recorded over five years of longitudinal follow-up.12 BALF samples were collected from 8 infants with CF. Streptococcus, Burkholderia, Prevotella, Haemophilus, Porphyromonas, and Veillonella had the highest median relative abundance in infant CF BALF. Two of the 3 infants with repeat BALF had changes in their microbial communities over six months (Morisita-Horn diversity index 0.36, 0.38). Although there was excellent percent agreement between standard NP and BALF cultures, these techniques did not routinely detect all bacteria identified by sequencing.BALF in asymptomatic CF infants contains complex microbiota, often missed by traditional culture of airway secretions. Anaerobic bacteria are commonly found in the lower airways of CF infants.
Project description:Interleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD.IL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non-smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils >?3%) or without (sputum eosinophils ?3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry.IL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ?3% (p?<?0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p?<?0.01). Similarly, ST2?+?IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p?<?0.001).Our results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients.