Early Detection of Cystic Fibrosis Acute Pulmonary Exacerbations by Exhaled Breath Condensate Metabolomics

The most common cause of death in cystic fibrosis (CF) patients is progressive lung function decline, which is punctuated by acute pulmonary exacerbations (APEs). A major challenge is to discover biomarkers for detecting an oncoming APE and allow for pre-emptive clinical interventions. Metabolic profiling of exhaled breath condensate (EBC) samples collected from CF patients before, during, and after APEs and under stable conditions (n = 210) was performed using ultraperformance liquid chromatography (UPLC) coupled to Orbitrap mass spectrometry (MS). Negative ion mode MS data showed that classification between metabolic profiles from "pre-APE" (pending APE before the CF patient had any signs of illness) and stable CF samples was possible with good sensitivities (85.7 and 89.5%), specificities (88.4 and 84.1%), and accuracies (87.7 and 85.7%) for pediatric and adult patients, respectively. Improved classification performance was achieved by combining positive with negative ion mode data. Discriminant metabolites included two potential biomarkers identified in a previous pilot study: Lactic acid and 4-hydroxycyclohexylcarboxylic acid. Some of the discriminant metabolites had microbial origins, indicating a possible role of bacterial metabolism in APE progression. The results show promise for detecting an oncoming APE using EBC metabolites, thus permitting early intervention to abort such an event.Fil: Zang, Xiaoling. Georgia Institute of Techology; Estados UnidosFil: Monge, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Gaul, David A.. Georgia Institute of Techology; Estados UnidosFil: McCarty, Nael A.. University of Emory; Estados UnidosFil: Stecenko, Arlene. University of Emory; Estados UnidosFil: Fernández, Facundo M.. Georgia Institute of Techology; Estados Unido

Altered prostanoid production by fibroblasts cultured from the lungs of human subjects with idiopathic pulmonary fibrosis

BACKGROUND: Prostanoids are known to participate in the process of fibrogenesis. Because lung fibroblasts produce prostanoids and are believed to play a central role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), we hypothesized that fibroblasts (HF) cultured from the lungs of patients with IPF (HF-IPF) have an altered balance between profibrotic (thromboxane [TX]A(2)) and antifibrotic (prostacyclin [PGI(2)]) prostaglandins (PGs) when compared with normal human lung fibroblasts (HF-NL). METHODS: We measured inducible cyclooxygenase (COX)-2 gene and protein expression, and a profile of prostanoids at baseline and after IL-1β stimulation. RESULTS: In both HF-IPF and HF-NL COX-2 expression was undetectable at baseline, but was significantly upregulated by IL-1β. PGE(2) was the predominant COX product in IL-1β-stimulated cells with no significant difference between HF-IPF and HF-NL (28.35 [9.09–89.09] vs. 17.12 [8.58–29.33] ng/10(6) cells/30 min, respectively; P = 0.25). TXB(2) (the stable metabolite of TXA(2)) production was significantly higher in IL-1β-stimulated HF-IPF compared to HF-NL (1.92 [1.27–2.57] vs. 0.61 [0.21–1.64] ng/10(6) cells/30 min, respectively; P = 0.007) and the ratio of PGI(2) (as measured by its stable metabolite 6-keto-PGF(1α)) to TXB(2) was significantly lower at baseline in HF-IPF (0.08 [0.04–0.52] vs. 0.12 [0.11–0.89] in HF-NL; P = 0.028) and with IL-1β stimulation (0.24 [0.05–1.53] vs. 1.08 [0.51–3.79] in HF-NL; P = 0.09). CONCLUSION: An alteration in the balance of profibrotic and antifibrotic PGs in HF-IPF may play a role in the pathogeneses of IPF

Twist: A Regulator of Epithelial-Mesenchymal Transition in Lung Fibrosis

BACKGROUND: Several studies have implicated viral infection as an important factor in the pathogenesis of IPF and related fibrotic lung disorders. Viruses are thought to cause epithelial cell injury and promote epithelial-mesenchymal transition (EMT), a process whereby differentiated epithelial cells undergo transition to a mesenchymal phenotype, and considered a source of fibroblasts in the setting of lung injury. We have demonstrated an association between the epithelial injury caused by chronic herpes virus infection with the murine gamma-herpes virus, MHV68, and lung fibrosis. We hypothesize that EMT in this model of virus-induced pulmonary fibrosis is driven by the expression of the transcription factor Twist. METHODS/FINDINGS: In vitro MHV68 infection of murine lung epithelial cells induced expression of Twist, and mesenchymal markers. Stable overexpression of Twist promoted EMT in MLE15 lung epithelial cells. Transient knockdown expression of Twist resulted in preservation of epithelial phenotype after in vitro MHV68 infection. In concordance, high expression of Twist was found in lung epithelial cells of MHV68 infected mice, but not in mock infected mice. Alveolar epithelial cells from lung tissue of idiopathic pulmonary fibrosis (IPF) patients were strongly positive for Twist. These cells demonstrated features of EMT with low expression of E-cadherin and upregulation of the mesenchymal marker N-cadherin. Finally, IPF tissue with high Twist protein levels was also positive for the herpesvirus, EBV. CONCLUSIONS/SIGNIFICANCE: We conclude that Twist contributes to EMT in the model of virus-induced pulmonary fibrosis. We speculate that in some IPF cases, gamma-herpes virus infection with EBV might be a source of injury precipitating EMT through the expression of Twist

Quality of dietary macronutrients is associated with glycemic outcomes in adults with cystic fibrosis

ObjectivePoor diet quality contributes to metabolic dysfunction. This study aimed to gain a greater understanding of the relationship between dietary macronutrient quality and glucose homeostasis in adults with cystic fibrosis (CF).DesignThis was a cross-sectional study of N = 27 adults with CF with glucose tolerance ranging from normal (n = 9) to prediabetes (n = 6) to being classified as having cystic fibrosis-related diabetes (CFRD, n = 12). Fasted blood was collected for analysis of glucose, insulin, and C-peptide. Insulin resistance was assessed by Homeostatic Model Assessment for Insulin Resistance (HOMA2-IR). Subjects without known CFRD also underwent a 2-h oral glucose tolerance test. Three-day food records were used to assess macronutrient sources. Dietary variables were adjusted for energy intake. Statistical analyses included ANOVA, Spearman correlations, and multiple linear regression.ResultsIndividuals with CFRD consumed less total fat and monounsaturated fatty acids (MUFA) compared to those with normal glucose tolerance (p < 0.05). In Spearman correlation analyses, dietary glycemic load was inversely associated with C-peptide (rho = −0.28, p = 0.05). Total dietary fat, MUFA, and polyunsaturated fatty acids (PUFA) were positively associated with C-peptide (rho = 0.39–0.41, all p < 0.05). Plant protein intake was inversely related to HOMA2-IR (rho = −0.28, p = 0.048). Associations remained significant after adjustment for age and sex.DiscussionImprovements in diet quality are needed in people with CF. This study suggests that higher unsaturated dietary fat, higher plant protein, and higher carbohydrate quality were associated with better glucose tolerance indicators in adults with CF. Larger, prospective studies in individuals with CF are needed to determine the impact of diet quality on the development of CFRD

Sputum from People with Cystic Fibrosis Reduces the Killing of Methicillin-Resistant <i>Staphylococcus aureus</i> by Neutrophils and Diminishes Phagosomal Production of Reactive Oxygen Species

Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs). Staphylococcus aureus has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant S. aureus (MRSA) represents a huge clinical burden in CF due to its association with lung disease and increased resistance to antibiotics. In CF, PMNs are unable to kill and clear MRSA. The reason for this remains largely unknown. Our study found that CF PMNs are as equally capable of killing MRSA as healthy PMNs. We show that the CF sputum, however, significantly impairs the ability of human PMNs to kill CF MRSA isolates. In the absence of CF sputum, PMNs kill MRSA via intracellular mechanisms mediated by phagocytosis, rather than extracellular mechanisms via NET formation. CF sputum does not affect the phagocytosis of MRSA via healthy or CF PMNs. Our results demonstrate that CF sputum exposure impairs phagosomal levels of reactive oxygen species (ROS) in MRSA-phagocytosing PMNs. While phagosomal co-localizations of MRSA with primary granule markers, myeloperoxidase and cathepsin D, were significantly reduced upon CF sputum exposure, that of a third azurophilic granule marker, neutrophil elastase, remained unaffected. This suggests that CF sputum does not compromise the fusion of primary granules with phagosomes but diminishes phagosomal ROS levels via another, likely more specific, mechanism. Overall, we identified the airway environment as an important factor that restricts neutrophils’ oxidative microbicidal activities in CF against MRSA. These results deliver new details of the complex host–pathogen interactions present in the CF lung

Serum anti-PAD4 autoantibodies are present in cystic fibrosis children and increase with age and lung disease severity

Cystic fibrosis (CF) lung disease begins early in childhood and is characterized by neutrophilic inflammation of the airways. Neutrophil extracellular traps (NETs) represent one mechanism by which neutrophils contribute to lung damage. The enzyme peptidylarginine deiminase 4 (PAD4) is required for NET formation. Our overall concept is that NET formation delivers PAD4 outside the neutrophil resulting in autoantibody generation, and this autoimmunity may be a novel mechanism contributing to CF lung disease progression. The aim of this study was to investigate clinical predictors of serum anti-PAD4 autoantibody (PAD4 Ab) levels in CF subjects with a wide range of ages from early childhood through middle age. We measured PAD4 Ab levels in sera from 104 CF subjects. PAD4 Abs were detectable among CF children as young as one year of age and elevated compared to paediatric healthy controls. PAD4 Ab levels increased significantly with age (r = 0.584, p <.001) and correlated with lower lung function (r = −0.481, n = 99, p <.001). PAD4 Abs were elevated in subjects with chronic Pseudomonas aeruginosa airways infection (p <.001), but not with other key clinical CF co-variates including sex, CFTR genotype, sweat chloride, pancreatic enzyme use, nutritional status, recent pulmonary exacerbations, Staphylococcus aureus, or CF-related diabetes. PAD4 Ab levels were also correlated with serum anti-double-stranded DNA IgA autoantibodies, which have similarly been shown to be elevated in CF subjects and associated with lung damage. In multivariable analysis, age and lung function remained correlated with PAD4 Ab levels. In summary, we describe novel findings of anti-PAD4 autoantibodies in CF that are present early in childhood, increase over time with age, and correlate with lung disease severity. Autoimmunity to antigens extruded by NETs appears to be an early event in CF lung disease, and airway autoimmunity related to NET formation is a potential mechanism of lung disease progression in CF.Highlights Serum anti-PAD4 autoantibodies are detected in paediatric CF serum and are elevated compared to healthy paediatric controls Anti-PAD4 autoantibodies increase with age Anti-PAD4 autoantibodies correlate with lower lung function, Pseudomonas aeruginosa airway infection and anti-dsDNA IgA autoantibodies, but not with other key clinical CF co-variates Age and lung function remain correlated with anti-PAD4 autoantibodies in multivariable analysi

Endoscopic Sinus Surgery in Adults With Cystic Fibrosis Effect on Lung Function, Intravenous Antibiotic Use, and Hospitalization

Objective: To ascertain the effect of endoscopic sinus surgery (ESS) on lung function, intravenous (IV) antibiotic use, and hospitalization in adults with cystic fibrosis (CF). Design: Retrospective analysis. Settings: Tertiary care center. Patients: Fifteen adults with a diagnosis of CF undergoing ESS between March 1, 2006, and June 31, 2008. Main Outcome Measures: Twelve-month preoperative and 12-month postoperative pulmonary function testing ( forced vital capacity [FVC] and forced expired volume in 1 second [FEV1]), number of IV antibiotic courses, total number of days of intravenous antibiotic use, and number of inpatient hospital days (IHDs) were assessed. Results: Twenty-two adults with CF underwent ESS; 15 patients had adequate data for evaluation. No significant differences were found between mean preoperative and postoperative FEV1 (61.3% vs 59.5%; P =. 41) or FVC (76.4% vs 76.1%; P =. 97) or between best preoperative and postoperative FEV1 (67.4% vs 67.0%; P =. 95) or FVC (84.2% vs 83.0%; P =. 86) (paired samples t test). The number of IV antibiotic courses and the total number of days of IV antibiotic use did not differ between the preoperative and postoperative periods (Wilcoxon signed rank test P =. 61 and P =. 10, respectively). However, the number of IHDs was significantly lower in the 1-year postoperative period (36.7 days) vs the 1-year preoperative period (59.1 days) (Wilcoxon signed rank test, z =-2.20, P =. 03). Conclusions: This preliminary study of ESS in adult CF patients indicates significant reduction in the number of IHDs in the postoperative period. However, there is no evidence that ESS improved lung function or the need for IV antibiotics. Arch Otolaryngol Head Neck Surg. 2012;138(12):1167-117

Feasibility of Early Detection of Cystic Fibrosis Acute Pulmonary Exacerbations by Exhaled Breath Condensate Metabolomics: A Pilot Study

Progressive lung function decline and, ultimately, respiratory failure are the most common cause of death in patients with cystic fibrosis (CF). This decline is punctuated by acute pulmonary exacerbations (APEs), and in many cases, there is a failure to return to baseline lung function. Ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry was used to profile metabolites in exhaled breath condensate (EBC) samples from 17 clinically stable CF patients, 9 CF patients with an APE severe enough to require hospitalization (termed APE), 5 CF patients during recovery from a severe APE (termed post-APE), and 4 CF patients who were clinically stable at the time of collection but in the subsequent 1-3 months developed a severe APE (termed pre-APE). A panel containing two metabolic discriminant features, 4-hydroxycyclohexylcarboxylic acid and pyroglutamic acid, differentiated the APE samples from the stable CF samples with 84.6% accuracy. Pre-APE samples were distinguished from stable CF samples by lactic acid and pyroglutamic acid with 90.5% accuracy and in general matched the APE signature when projected onto the APE vs stable CF model. Post-APE samples were on average more similar to stable CF samples in terms of their metabolomic signature. These results show the feasibility of detecting and predicting an oncoming APE or monitoring APE treatment using EBC metabolites.Fil: Zang, Xiaoling. Georgia Institute of Techology; Estados UnidosFil: Monge, Maria Eugenia. Georgia Institute of Techology; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: McCarty, Nael A.. University of Emory; Estados UnidosFil: Stecenko, Arlene A.. University of Emory; Estados UnidosFil: Fernández, Facundo M.. Georgia Institute of Techology; Estados Unido

Role of the SDF-1/CXCR4 Axis in the Pathogenesis of Lung Injury and Fibrosis

Stromal cell–derived factor-1 (SDF-1) participates in mobilizing bone marrow–derived stem cells, via its receptor CXCR4. We studied the role of the SDF-1/CXCR4 axis in a rodent model of bleomycin-induced lung injury in C57BL/6 wild-type and matrix metalloproteinase (MMP)-9 knockout mice. After intratracheal instillation of bleomycin, SDF-1 levels in serum and bronchial alveolar lavage fluid increased. These changes were accompanied by increased numbers of CXCR4+ cells in the lung and a decrease in a population of CXCR4+ cells in the bone marrow that did not occur in MMP-9−/− mice. Both SDF-1 and lung lysates from bleomycin-treated mice induced migration of bone marrow–derived stem cells in vitro that was blocked by a CXCR4 antagonist, TN14003. Treatment of mice with TN14003 with bleomycin-induced lung injury significantly attenuated lung fibrosis. Lung tissue from patients with idiopathic pulmonary fibrosis had higher numbers of cells expressing both SDF-1 and CXCR4 than did normal lungs. Our data suggest that the SDF-1/CXCR4 axis is important in the complex sequence of events triggered by bleomycin exposure that eventuates in lung repair. SDF-1 participates in mobilizing bone marrow–derived stem cells, via its receptor CXCR4