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Non-steroidal anti-inflammatory drugs exacerbated respiratory disease (N-ERD) is currently classified as type-2 (T2) immune mediated disease characterized by asthma, chronic rhinosinusitis, and hypersensitivity to cyclooxygenase-1 inhibitors.Our aim was to characterize immunological endotypes of N-ERD based on the gene expression profile in the bronchial epithelium.We analyzed mRNA transcriptome (mRNA-seq) in bronchial brushings from patients with N-ERD (n=22), NSAIDs tolerant asthma (NTA, n=21) and control subjects (n=11). Additionally, lipid and protein mediators were measured in broncho-alveolar lavage fluid (BALF).Initial analysis of the entire asthma group revealed two distinct gene expression signatures: 'T2-high' with increased expression of T2-related genes (e.g., CLCA1, CST1), and 'proinflammatory' characterized by the expression of innate immunity (e.g., FOSB, EGR3) and IL-17A response genes. These endotypes showed similar prevalence in N-ERD and NTA (e.g., T2-high: 33% and 32%, respectively). T2-high asthma was characterized by increased expression of mast cell and eosinophil markers, goblet cell hyperplasia, and elevated LTET2-high signature was found only in one third of N-ERD patients, which was similar to NTA. The proinflammatory endotype, which also occurred in N-ERD, suggests a novel mechanism of severe disease developing on a non-T2 background.

Natalia Celejewska-Wójcik,1 Aleksander Kania,1 Karolina Górka,1 Paweł Nastałek,1 Krzysztof Wójcik,2 Anna Gielicz,3 Lucyna Mastalerz,1 Marek Sanak,3 Krzysztof Sładek1 1Department of Pulmonology, 2nd Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland; 2 2nd Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland; 3Department of Molecular Biology and Clinical Genetics, Faculty of Medicine, Jagiellonian University Medical College, Kraków, PolandCorrespondence: Natalia Celejewska-Wójcik 2nd Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, ul. Jakubowskiego 2, Krakow, 30-688, PolandTel +48 12 400 30 53Fax +48 12 400 30 67Email natalia.celejewska@gmail.comPurpose: Lipid mediators, particularly eicosanoids, are associated with airway inflammation, especially with the eosinophilic influx. This study aimed to measure lipid mediators and cells in induced sputum, that could possibly reflect the inflammatory process in the bronchial tree of COPD subjects.Patients and Methods: Eighty patients diagnosed with COPD and 37 healthy controls participated in the study. Induced sputum samples were ascertained for differential cell count and induced sputum supernatant concentrations of selected eicosanoids by the means of gas chromatography/mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry.Results: Increased sputum eosinophilia was associated with higher concentrations of selected proinflammatory eicosanoids. In COPD subjects prostaglandin D2 and 11-dehydro-thromboxane B2 correlated negatively with airway obstruction measured by FEV1 and FEV1/FVC values. COPD subjects with disease exacerbations during past 12 months had significantly higher concentrations of prostaglandin D2, 12-oxo-eicosatetraenoic acid and 5-oxo-eicosatetraenoic acid.Conclusion: Stable COPD is often associated with eosinophil influx in the lower airways and elevated concentrations of eicosanoids that is reflected by some disease characteristics.Keywords: chronic obstructive pulmonary disease, eosinophils, eicosanoids

Background Non-steroidal anti-inflammatory drugs exacerbated respiratory disease (N-ERD) asthma is characterized by chronic rhinosinusitis and intolerance to aspirin and other cyclooxygenase-1 inhibitors. Clinical data point to a heterogeneity within N-ERD phenotype. Objectives To investigate immune mediator profiles in lower airways of patients with N-ERD. Methods Levels of cytokines (luminex) and eicosanoids (mass spectrometry) were measured in bronchoalveolar lavage fluid (BALF) from patients with N-ERD (n=22), NSAIDs-tolerant asthma (NTA, n=21) and control subjects (n=11). mRNA expression was quantified in BALF cells using TaqMan low-density arrays. Results Lower airway eosinophilia was more frequent in N-ERD (54.5%) than in NTA (9.5%, P=0.009). Type-2 (T2) immune signature of BALF cells was more pronounced in eosinophilic subphenotype of N-ERD. Similarly, BALF concentrations of Periostin and CCL26 were significantly increased in eosinophilic N-ERD and correlated with T2-signature in BALF cells. Multiparameter analysis of BALF mediators of all asthma patients revealed the presence of two immune endotypes: ‘T2-like’ with elevated Periostin in BALF, and ‘non-T2/pro-inflammatory’ with higher levels of MMPs and inflammatory cytokines. N-ERD patients were classified mostly as T2 endotype (68%). Changes in eicosanoid profile, e.g. increased LTE4, were limited to N-ERD patients with airway eosinophilia. Blood eosinophilia appeared to be useful predictor of airway T2-signature (AUC=0.83), however, surrogate biomarkers had moderate performance in distinguishing eosinophilic N-ERD (blood eosinophils: AUC=0.72, Periostin: AUC=0.75). Conclusions Lower airway immune profiles show considerable heterogeneity of N-ERD, with skewing toward T2-response and eosinophilic inflammation. Increased production of LTE4 was restricted to a subgroup of patients with lower airway eosinophilia. Clinical implications N-ERD patients are heterogeneous in terms of lower airway inflammation and eicosanoid profiles. Better understanding of inflammatory diversity of N-ERD might help in selection of personalized therapies.

Background The effect of renal replacement therapy on drug concentrations in patients with sepsis has not been fully elucidated because the pharmacokinetic properties of many antimicrobials are influenced by both pathophysiological and treatment-related factors. The aim of this study was to determine meropenem concentrations in patients with sepsis before and after the initiation of continuous venovenous hemodialysis with regional citrate anticoagulation (RCA-CVVHD). Methods The study included 15 critically ill patients undergoing RCA-CVVHD due to sepsis-induced acute kidney injury. All participants received 2 g of meropenem every 8 h in a prolonged infusion lasting 3 h. Meropenem concentrations were measured in blood plasma using high-performance liquid chromatography coupled with tandem mass spectrometry. Blood samples were obtained at six-time points prior to and at six-time points after introducing RCA-CVVHD. Results The median APACHE IV and SOFA scores on admission were 118 points (interquartile range [IQR] 97–134 points) and 19.5 points (IQR 18–21 points), respectively. There were no significant differences in the plasma concentrations of meropenem measured directly before RCA-CVVHD and during the first 450 min of the procedure. The drug concentration reached its peak 2 h after initiating the infusion and then steadily declined. Conclusions The concentration of high-dose meropenem (2 g every 8 h) administered in a prolonged infusion was similar before and after the introduction of RCA-CVVHD in patients with sepsis who developed acute kidney injury.

Miroslaw Janczura,1 Rafal Rosa,2 Jerzy Dropinski,3 Anna Gielicz,3 Andrzej Stanisz,4 Katarzyna Kotula-Horowitz,5 Teresa Domagala6 1Faculty of Health Sciences, Jagiellonian University School of Medicine, Krakow, Poland; 2Health Care Centre of the Ministry of the Interior and Administration, Department of Anesthesiology and Intensive Care, Krakow, Poland; 3Department of Internal Medicine, Jagiellonian University School of Medicine, Krakow, Poland; 4Department of Bioinformatics and Telemedicine, Jagiellonian University School of Medicine, Krakow, Poland; 5Health Care Centre of the Ministry of the Interior and Administration, Department of Internal Medicine, Krakow, Poland; 6Department of Medical Biochemistry, Jagiellonian University School of Medicine, Krakow, PolandCorrespondence: Teresa DomagalaDepartment of Medical Biochemistry, Jagiellonian University School of Medicine, Krowoderska 68/11, Krakow, 31-158, PolandTel +48 12 422 74 00Fax +48 12 422 32 72Email domagala32@yahoo.comPurpose: Associations between perceived stress and oxidative stress marker and metabolic syndrome (MetS) components were investigated in a cohort of police officers.Methods: Cross-sectional data from a cohort of non-diabetic subjects (n=233; 19F), median [interquartile range] age 50 [37– 44] years, were analysed. MetS was construed in line with International Diabetes Federation (IDF) criteria and perceived stress with Cohen’s 10-item Perceived Stress Scale. Plasma oxidative stress marker (free 8-iso-prostaglandin F2α; 8-iso-PGF2α), presence of coronary plaque, carotid artery intima-media thickness (cIMT), and physical activity level were also determined.Results: Obesity was established in 100 (42.92%), hypertension in 111 (47.64), whereas MetS was identified in 104 (44.63%) of the study subjects. A significant difference (p=0.003) in plasma 8-iso-PGF2α level, depending on the MetS components status, was noted. The associations of perceived stress with plasma 8-iso-PGF2α level and the select study variables were gender-specific. In multivariate analysis (adjusted for age and current smoking), positive associations of plasma 8-iso-PGF2α levels with PSS score (B=0.108, 95% CI [0.008, 0.209], p=0.03) and systolic blood pressure (B=0.029, 95% CI [0.003, 0.057], p=0.02) in men only were established. Both the perceived stress (OR 1.101, 95% CI [1.001– 1.202], p=0.03) and plasma 8-iso-PGF2α levels (OR 1.223, 95% CI [1.046– 1.432], p=0.01) impacted the prevalence of hypertension. Out of the MetS components, the effect of waist circumference (OR=1.138, 95% CI [1.064– 1.218], p=0.0001) and glucose (B=2.696, 95% CI [1.081– 6.725], p=0.03) were also encountered. No such associations were noted in women, though, neither in univariate nor in multivariate analyses. The prevalence of coronary plaque (0.001), obesity (p< 0.001), hypertension (p< 0.001) and median cIMT value (p=0.005), as well as leisure-time (p=0.04) and total walking physical activity (p=0.03), differed significantly between the subgroups stratified by MetS components status.Conclusion: Both the perceived and oxidative stress were found instrumental in promoting hypertension in a cohort of police officers under study, whereas all study outcomes were conclusively gender-related.Keywords: metabolic syndrome, obesity, hypertension, perceived stress, oxidative stress, physical activity

Background The present study investigated the relationships between psychological stress indices and oxidative stress marker, also when combined with emergent insulin resistance (IR), in the non-diabetic, middle-aged subjects, exposed to frequent/chronic psychological stressors. Methods Cross-sectional data from a cohort of non-diabetic police officers (n = 234; 19F), aged 27–56 years, were used. Plasma inflammatory (CRP, TNF-α), oxidative stress (free 8-iso-prostaglandin F2α; 8-iso-PGF2α) markers, and insulin were measured. The value of homeostasis model assessment of IR index (HOMA-IR) was assumed the threshold value of IR, i.e. 2.04. Free cortisol in urine and perceived stress (psychological stress indices) were also measured. Results In the IR subjects, most biochemical variables, inflammatory markers and urine cortisol were significantly higher, as compared to the non-IR ones. Psychological stress indices were associated with plasma 8-iso-PGF2α [B = 0.139, 95% CI (0.048, 0.230), p = 0.002, and B = 0.007, 95% CI (0.0006, 0.014), p = 0.03; for perceived stress level and cortisol, respectively]. Positive associations were established between plasma 8-iso-PGF2α [B = 0.069, 95% CI (0.016–0.120), p = 0.01] and urine cortisol [B = 0.003, 95% CI (0.0003, 0.005), p = 0.02] with HOMA-IR. Metabolic syndrome, as defined by IDF criteria, was established in 110 study subjects, whereas 136 of them were hypertensive. Waist circumference [B = 0.056, 95% CI (0.039, 0.074), p

Background Induced sputum (IS) allows to measure mediators of asthmatic inflammation in bronchial secretions. NSAID‐exacerbated respiratory disease (NERD) is recognized as a distinct asthma phenotype, usually with a severe course, eosinophilic airway inflammation, and increased production of pro‐inflammatory eicosanoids. A more insightful analysis of NERD patients has shown this phenotype to be nonhomogeneous. Objective We aimed to identify possible subphenotypes in a cohort of NERD patients with the means of latent class analysis (LCA). Methods A total of 95 asthma patients with aspirin hypersensitivity underwent sputum induction. High‐performance liquid chromatography or gas chromatography coupled with mass spectrometry was used to profile eicosanoids in induced sputum supernatant (ISS). Sixteen variables covering clinical characteristics, IS inflammatory cells, and eicosanoids were considered in the LCA. Results Three classes (subphenotypes) were distinguished within the NERD cohort. Class 1 subjects had mild‐to‐moderate asthma, an almost equal distribution of inflammatory cell patterns, the lowest concentrations of eicosanoids, and logLTE4/logPGE2 ratio. Class 2 represented severe asthma with impaired lung function despite high doses of steroids. High sputum eosinophilia was in line with higher pro‐inflammatory LTE4 in ISS and the highest logLTE4/logPGE2 ratio. Class 3 subjects had mild‐to‐moderate asthma and were also characterized by eosinophilic airway inflammation, yet increased production of pro‐ (LTE4, PGD2 and 11‐dehydro‐TBX2) was balanced by anti‐inflammatory PGE2. The value of logLTE4/logPGE2 was between values calculated for classes 1 and 3, similarly to disease control and severity. Conclusions LCA revealed three distinct NERD subphenotypes. Our results support a more complex pathobiology of aspirin hypersensitivity. Considering NERD heterogeneity, the relationship between inflammatory pathways and clinical manifestations of asthma may lead to more individualized treatment in difficult to treat patients in the future., Heterogeneity of NERD phenotype reflects differences in inflammatory response measured by airway cells and eicosanoids. Identifying subphenotypes provides a more insightful perception and suggests a need for more individualized approach. In aspect of logLTE4/logPGE2 ratio, latent class analysis assigned subject to different groups better than identification by disease severity or control emphasizing the heterogeneity in NERD subgroup. Abbreviations: LTE4, Leukotriene E4; NERD, NSAID‐exacerbated respiratory disease; PGE2, Prostaglandin E2

Activation of neutrophils is an important mechanism in the pathology of granulomatosis with polyangiitis (GPA). In this study, we evaluated whether extracellular vesicles (EVs) circulating in the plasma of GPA patients could contribute to this process. EVs from the plasma of GPA patients in the active stage of the disease (n = 10) and healthy controls (n = 10) were isolated by ultracentrifugation and characterized by flow cytometry (CD63, CD8) and nanoparticle tracking analysis. Targeted oxylipin lipidomics of EVs was performed by HPLC-MS/MS. EV/oxylipin-induced neutrophil extracellular traps (NETs) were analyzed by confocal microscopy, and released double-stranded DNA (dsDNA) was quantified by PicoGreen fluorescent dye. Reactive oxygen species (ROS) production and neutrophils' EV binding/uptake were evaluated by flow cytometry. Brief priming with granulocyte-macrophage colony-stimulating factor was required for EV-mediated ROS production and dsDNA release. It was observed that priming also increased EV binding/uptake by neutrophils only for EVs from GPA patients. EVs from GPA patients had higher concentrations of leukotriene (LT)B4 and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) as compared with EVs from healthy controls. Moreover, neutrophils stimulated with LTB4 or 5-oxo-ETE produced ROS and released dsDNA in a concentration-dependent manner. These results reveal the potential role of EVs containing oxylipin cargo on ROS production and NET formation by activated neutrophils.

The intestinal epithelial cells reside in close proximity to myofibroblasts and microbiota, which are supposed to have an impact on intestinal stem cells fate and to influence processes of tissue maturation and regeneration. Mechanism underlying these phenomena and their diversity among vertebrates can be studied in 3D organoid cultures. We investigated the growth of chicken embryo intestinal epithelial organoids in Matrigel with and without Toll-like receptors (TLRs) stimulation. The organoid cultures contained also some myofibroblasts with potential to promote intestinal stem cell survival. Organoid cells, expressing TLR4, TLR2 type 1 and TLR2 type 2 were incubated with their agonists (lipopolysaccharide - LPS and Pam3CSK4) or co-cultured with Lactobacillus acidophilus bacteria (LA-5). Pam3CSK4 and LA-5 promoted organoid growth, which was demonstrated by comparing the morphological parameters (mean number and area of organoids). The profile of prostaglandins (PG), known to promote intestinal regeneration, in supernatants from organoid and fibroblast cultures were evaluated. Both PGE2 and PGD2 were detected. As compared to unstimulated controls, supernatants from the Pam3CSK4-stimulated organoids contained twice as much of PGE2 and PGD2. The changes in production of prostaglandins and the support of epithelial cell growth by myofibroblasts are factors potentially responsible for stimulatory effect of TLR2 activation.

Activation of neutrophils is an important mechanism in the pathology of granulomatosis with polyangiitis (GPA). In this study, we evaluated whether extracellular vesicles (EVs) circulating in the plasma of GPA patients could contribute to this process. EVs from the plasma of GPA patients in the active stage of the disease (n = 10) and healthy controls (n = 10) were isolated by ultracentrifugation and characterized by flow cytometry (CD63, CD8) and nanoparticle tracking analysis. Targeted oxylipin lipidomics of EVs was performed by HPLC-MS/MS. EV/oxylipin-induced neutrophil extracellular traps (NETs) were analyzed by confocal microscopy, and released double-stranded DNA (dsDNA) was quantified by PicoGreen fluorescent dye. Reactive oxygen species (ROS) production and neutrophils’ EV binding/uptake were evaluated by flow cytometry. Brief priming with granulocyte-macrophage colony-stimulating factor was required for EV-mediated ROS production and dsDNA release. It was observed that priming also increased EV binding/uptake by neutrophils only for EVs from GPA patients. EVs from GPA patients had higher concentrations of leukotriene (LT)B(4) and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) as compared with EVs from healthy controls. Moreover, neutrophils stimulated with LTB(4) or 5-oxo-ETE produced ROS and released dsDNA in a concentration-dependent manner. These results reveal the potential role of EVs containing oxylipin cargo on ROS production and NET formation by activated neutrophils.

Background: Eosinophilic airway inflammation has been demonstrated in 20%–40% of induced sputum samples from patients with stable COPD. Lipid mediators, like eicosanoids are associated with airway inflammation especially with the eosinophilic pattern. Objectives: To investigate the inflammatory cell patterns and eicosanoids in induced sputum in stable COPD. Methods: Induced sputum was collected in COPD and healthy control subjects. Sputum differential cell count and sputum supernatant concentrations of: 5-HETE, 12-HETE, 15-HETE, 5-oxo-ETE, 12-oxo-ETE, 15-oxo-ETE, LTD4, LTE4, LTB4, PGE2, 8-izo-PGE2, PGD2, PGA2, tetranor-PGE-M, tetranor-PGD-M, PGF2a, 8-izo-PGF2a, TBX2, 11-dehydro-TXB2 were measured. Results: In COPD subjects increased eosinophilia in induced sputum (>2%) was found in 42% of subjects (including 34% eosinophilic and 8% mixed eosinophilic and neutrophilic patterns). There was a statistically significant correlation between sputum eosinophilia and induced sputum concentrations of LTE4, LTD4, PGE2, 8-izo-PGE2, 15-HETE,15-oxo-ETE, PGD2, TBX2 and 11-dehydro-TBX2. Induced sputum concentrations of LTE4, LTD4, PGD2, PGE2, 8-izo-PGE2 were significantly higher in COPD subjects compared to healthy controls, whereas concentrations of tetranor-PGE-M and tetranor-PGD-M were lower. Frequent exacerbator COPD subjects had lower sputum but not blood eosinophilia. Conclusions: Stable COPD is often associated with eosinophil influx in the lower airways and elevated concentrations of eicosanoids. Increased sputum eosinophilia is not associated with frequent exacerbations of COPD.

Mediators such as cytokines, eicosanoids, nitric oxide and growth factors may regulate adipogenesis as well as inflammation. It is well documented that production of some form of eicosanoids activates lipid synthesis during adipogenesis but also contributes to the formation of factors maintaining low-level systemic inflammation. Developing nutrients for reduction of adipogenesis and inflammation can enhance preventive efficacy of daily diet. This study examined the effects of free fatty acid influence on changes in lipid biosynthesis and corresponding gene expression during differentiation of human subcutaneous adipose tissue stromal vascular fraction (SVF) cells. Proadipogenic conditions promoted SVF cell differentiation and lipid droplet (LD) formation up to 15 days. This correlated with gene expression of adipocyte differentiation markers as well as inflammatory cytokines and their receptors. Addition of free fatty acids to differentiation medium increased their incorporation during the first period of differentiation (48 h). Presence of eicosanoid acid (EPA) during the initial period of differentiation by elevation of Perilipin 3 protein (TIP47), may be responsible for smaller LD formation. Presence of arachidonic acid (AA) tends to deposit lipids in large form of LDs. Prolongation of differentiation up to 15 days decreased AA or EPA in cellular lipids. PUFA through up-regulation of both phospholipase 2 and enzymes related to eicosanoid production influenced type and quantity of eicosanoids which regulated the extent of SVF cell differentiation. Formation of small LDs and reduction of pro-inflammatory mediators in adipose tissue are the consequence of eicosanoid production with anti-inflammatory potential from EPA.

Background Arachidonic acid metabolites regulate several aspects of airway function including inflammation, muscle contraction and mucous secretion. Objective The aim of this study was to evaluate concentration of selected 5-lipoxygenase (LOX) and cyclooxygenase (COX)-derived eicosanoids in exhaled breath condensate (EBC) during allergen induced bronchoconstriction. Methods The study was performed on 24 allergic rhinitis/asthma patients sensitized to a house dust mite (HDM) Dermatophagoides pteronyssinus (Dp) and 13 healthy controls (HCs). Bronchial challenge with Dp extract was performed only in the allergic patients. Exhaled breath condensate (EBC) samples were collected before (T0) and during Dp-induced bronchoconstriction (TEAR). Eicosanoid concentration was measured using HPLC-tandem mass spectrometry. Results Significant bronchoconstriction after Dp challenge was demonstrated in 15 patients (Rs) while in 9 patients (NRs) no asthmatic response could be detected. At T0 the most abundant eicosanoids in EBC of HDM-allergic patients were LTB4 and 5-oxo-ETE while in HCs EBC concentration of LTB4 was significantly greater than that of 5-oxo-ETE. Allergen challenge resulted in significant increase in EBC concentration of 5-oxo-ETE, LTD4 and 8-iso-PGE2 only in Rs. At TEAR, the relative change of 5-oxo-ETE concentration in EBC correlated with decrease of peripheral blood eosinophilia (R= -0.774; p=0.0012). Moreover, the relative increase of 5-oxo-ETE in EBC at TEAR significantly correlated with the severity of the subsequent late asthmatic response (R=0.683 p=0.007). Conclusion Our study demonstrates significant up-regulation of 5-oxo-ETE synthesis in HDM-allergic patients and indicates possible involvement of that mediator in the pathogenesis of allergic asthma. This article is protected by copyright. All rights reserved.

Human rhinoviruses (RVs) are a major cause of exacerbations in asthma and other chronic airway diseases. A characteristic feature of asthmatic epithelium is goblet cell metaplasia and mucus hypersecretion. Bronchial epithelium is also an important source of lipid mediators, including pro- and antiinflammatory eicosanoids. By using air-liquid interface cultures of airway epithelium from patients with asthma and nonasthmatic control subjects, we compared RV16 replication-induced changes in mRNA expression of asthma candidate genes and eicosanoid production in the epithelium with or without IL-13-induced mucus metaplasia. Mucus metaplastic epithelium was characterized by a 20-fold less effective replication of RV16 and blunted changes in gene expression; this effect was seen to the same extent in patients with asthma and control subjects. We identified ciliary cells as the main target for RV16 by immunofluorescence imaging and demonstrated that the numbers of ciliary cells decreased in RV16-infected epithelium. RV16 infection of mucociliary epithelium resulted in overexpression of genes associated with bronchial remodeling (e.g., MUC5AC, FGF2, and HBEGF), induction of cyclooxygenase-2, and increased secretion of prostaglandins. These responses were similar in both studied groups. These data indicate that structural changes associated with mucus metaplasia renders airway epithelium less susceptible to RV infection. Thus, exacerbations of the lung disease caused by RV may result from severe impairment in mucociliary clearance or activation of immune defense rather than from preferential infection of mucus metaplastic epithelium. Repeated rhinoviral infections of compromised epithelium may contribute to the remodeling of the airways.

The n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce low-grade inflammation associated with obesity. The relationship between therapeutic response to n-3 PUFAs and modification of the transcriptome in obesity or metabolic syndrome remains to be explored.Blood samples were obtained from women with obesity before and after three-months supplementation with a moderate dose of n-3 PUFAs (1.8 g EPA + DHA per day) or from controls. n-3 PUFAs (GC) and plasma concentrations of lipoxins, resolvins, protectin X (GC-MS/MS) and inflammatory markers (ELISA) were measured. Whole blood transcriptome was assayed using microarray.Women supplemented with n-3 PUFAs for 3 months had significantly higher levels of EPA and DHA in plasma phosphatidylcholine. n-3 PUFA supplementation, in contrast to placebo, significantly decreased the concentrations of several inflammatory markers (SELE, MCP-1, sVCAM-1, sPECAM-1, and hsCRP), fasting triglycerides and insulin and increased the concentrations of pro-resolving DHA derivatives in plasma. The microarray data demonstrated effects of n-3 PUFAs on PPAR-?, NRF2 and NF-?B target genes.N-3 PUFAs increased DHA-derived pro-resolving mediators in women with obesity. Elevated resolvins and up-regulation of the resolvin receptor occurred in parallel with activation of PPAR-? target genes related to lipid metabolism and of NRF2 up-regulated antioxidant enzymes.

Background Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry. Objective To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals. Methods Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor. Results Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E2 was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D2 products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B4 has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E4. Two main vascular prostanoids: prostacyclin and thromboxane B2 were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF2α isoprostane was increased in smokers. Conclusion Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.

Background: A special regulatory role for prostaglandin E 2 has been postulated in aspirin-induced asthma. A study was undertaken to investigate the effects of aspirin on the systemic production of prostaglandin E 2 and cysteinyl leucotrienes in patients with asthma. Methods: The urinary concentrations were determined of two main prostaglandin E 2 metabolites (13,14-dihydro-15keto-PGE 2 using a commercial enzyme immunoassay and 9,15-dioxo-11α-hydroxy-2,3,4,5-tetranor-prostane-1,20-dioic acid by gas chromatography/mass spectrometry) and leucotriene E 4 using an immunoassay. Determinations were performed at baseline and following oral aspirin and celecoxib challenges in two well-defined asthma phenotypes: aspirin-sensitive and aspirin-tolerant patients. Results: Aspirin precipitated bronchial reactions in all aspirin-sensitive patients but in none of the aspirin-tolerant patients. Celecoxib 400 mg was well tolerated by all patients except for one with aspirin-induced asthma. At baseline, the mean levels of prostaglandin E 2 metabolites did not differ between the groups. Following different aspirin provocation doses, the mean levels of the two main prostaglandin E 2 metabolites were decreased in the aspirin-tolerant group but remained unchanged in the aspirin-sensitive group. The dose of aspirin had no effect on the magnitude of the response on the prostaglandin E 2 metabolites and its duration. In both groups, urinary prostaglandin E 2 metabolites decreased following celecoxib challenge. No correlation was found between prostaglandin E 2 metabolites and leucotriene E 4 . Conclusions: Aspirin-precipitated asthmatic attacks are not associated with changes in the systemic production of prostaglandin E 2 . In contrast, the systemic production of prostaglandin E 2 becomes depressed by aspirin in non-sensitive patients. This different response might indicate COX-1-dependent prostaglandin E 2 control of inflammatory cells in aspirin-induced asthma. Thus, PGE 2 is released during the clinical reactions to aspirin through an alternative COX-2 pathway. The clinical implications of this finding are in line with current observations of good tolerance of the selective COX-2 inhibitors in aspirin-sensitive patients.

Background Induced sputum (IS) allows to measure mediators of asthmatic inflammation in bronchial secretions. The specific role of induced sputum supernatant (ISS) endogenous bioactive lipid mediators in subtypes of asthma is not well understood. Objective To investigate the interactions between airway inflammation and clinical phenotypes of asthma, we integrated induced sputum supernatant (ISS) eicosanoids and quantitative assessment of infiltrating cells into new subtypes with the means of latent class analysis (LCA). Methods One hundred and thirty-nine asthmatics with and without aspirin hypersensitivity underwent sputum induction. High-performance liquid chromatography or gas chromatography coupled with mass spectrometry was used to profile eicosanoids. Nineteen variables covering clinical characteristics, IS inflammatory cells and eicosanoids were considered in the LCA. Results Four phenotypic asthma classes were distinguished. Class 1 with mild-to-moderate asthma, chronic rhinosinusitis (CRS), high PGA2 in ISS and almost equal distribution of inflammation cell patterns. Class 3 subjects also had mild-to-moderate asthma but without upper airway symptoms. Induced sputum was often paucigranulocytic with low levels of lipid mediators. Classes 2 and 4 represented severe asthma with CRS and impaired lung function despite high doses of steroids. High blood and sputum eosinophilia was in line with high cysteinyl leukotrienes and PGD2 in ISS only in class 2. Class 4 subjects tended to have increased sputum neutrophilia and PGE2 in ISS. Aspirin hypersensitivity was most frequent among class 2 subjects. Conclusions & clinical relevance The LCA revealed four distinct asthma classes differing in eicosanoid pathways.

Background Isoprostanes are bioactive compounds formed by non-enzymatic oxidation of polyunsaturated fatty acids, mostly arachidonic, and markers of free radical generation during inflammation. In aspirin exacerbated respiratory disease (AERD), asthmatic symptoms are precipitated by ingestion of non-steroid anti-inflammatory drugs capable for pharmacologic inhibition of cyclooxygenase-1 isoenzyme. We investigated whether aspirin-provoked bronchoconstriction is accompanied by changes of isoprostanes in exhaled breath condensate (EBC). Methods EBC was collected from 28 AERD subjects and 25 aspirin-tolerant asthmatics before and after inhalatory aspirin challenge. Concentrations of 8-iso-PGF 2α , 8-iso-PGE 2 , and prostaglandin E 2 were measured using gas chromatography/mass spectrometry. Leukotriene E 4 was measured by immunoassay in urine samples collected before and after the challenge. Results Before the challenge, exhaled 8-iso-PGF 2α , 8-iso-PGE 2 , and PGE 2 levels did not differ between the study groups. 8-iso-PGE 2 level increased in AERD group only ( p = 0.014) as a result of the aspirin challenge. Urinary LTE 4 was elevated in AERD, both in baseline and post-challenge samples. Post-challenge airways 8-iso-PGE 2 correlated positively with urinary LTE 4 level ( p = 0.046), whereas it correlated negatively with the provocative dose of aspirin ( p = 0.027). Conclusion A significant increase of exhaled 8-iso-PGE 2 after inhalatory challenge with aspirin was selective and not present for the other isoprostane measured. This is a novel finding in AERD, suggesting that inhibition of cyclooxygenase may elicit 8-iso-PGE 2 production in a specific mechanism, contributing to bronchoconstriction and systemic overproduction of cysteinyl leukotrienes.

Background Aspirin-induced asthma/rhinitis (AIAR) is characterized by the altered metabolism of leukotrienes and proinflammatory prostaglandins. The basal and postchallenge levels of eicosanoids might reflect the clinical and biochemical characteristics of patients with distinct types of hypersensitive responses to aspirin. Objective We compared clinical and eicosanoid profiles of patients with AIAR showing both bronchial and nasal versus isolated nasal responses to aspirin challenge. Methods Twenty-three patients with AIAR underwent the single-blind, oral, placebo-controlled aspirin challenge. The bronchial response (BR) was evidenced by dyspnea and spirometry, whereas the nasal response (NR) was evidenced by nasal symptoms and acoustic rhinometry and/or rhinomanometry. Urinary leukotriene E 4 (uLTE 4 ), serum and urinary stable prostaglandin D 2 metabolite, and 9α,11β-prostaglandin F 2 (9α,11β-PGF 2 ), were determined at baseline and after the aspirin challenge. Results Fifteen subjects showed BR and NR (BNR), whereas 8 showed NR only. Basal uLTE 4 in the BNR group was significantly higher than in the NR group. After aspirin challenge, it increased significantly in both groups. Serum 9α,11β-PGF 2 increased after aspirin challenge in the BNR group only. The patients with BNR had more severe AIAR. Conclusions BNR to aspirin in AIAR indicates a more advanced disease and more profound underlying eicosanoid metabolism disturbances.

BACKGROUND Pro- and anti-inflammatory metabolites of arachidonic acid - eicosanoids - participate in skin homeostasis, affecting the growth and differentiation of keratinocytes. Alterations of 12-lipoxygenase (LOX) and 15-LOX and their metabolites have been described in the epidermis of patients with psoriasis, but systemic production of 12-LOX and 15-LOX eicosanoids has not been studied in the disease. OBJECTIVES To ascertain the frequencies of the genetic variants ALOX12 rs1126667 and ALOX15 rs11568070 in cases and controls, and to compare urinary metabolites of 12(S)-hydroxyeicosatetraenoic acid (HETE) between patients with psoriasis and healthy controls. METHODS Patients with psoriasis (n = 200) were stratified depending on the severity of their dermal lesions. Genotyping was performed using a 5'-nuclease real-time assay. The concentrations of 12(S)-HETE, its metabolites and 15(S)-HETE were determined in urine samples using high-performance liquid chromatography-tandem mass spectrometry. RESULTS Tetranor-12(S)-HETE metabolite excretion was significantly higher in urine of patients with psoriasis, while excretion of 12(S)-HETE was decreased. Neither 12(S)-HETE nor tetranor-12(S)-HETE correlated with the type of disease or severity score. No difference in urinary 15(S)-HETE was found between the study groups. Genotype distribution of the ALOX12 rs1126667 or ALOX15 rs11568070 polymorphisms did not discriminate for the disease or its severity. CONCLUSIONS Systemic metabolism of 12(S)-HETE is accelerated in psoriasis because excretion of the tetranor-12(S)-HETE inactivation product is elevated. No correlation with the severity or extent of psoriasis is detectable. We propose that in patients with psoriasis, 12(S)-HETE to tetranor-12(S)-HETE conversion could be at least a marker for this disease, in which inflammation of the skin can induce microsomal beta-oxidation of this eicosanoid.

Background Altered metabolism of eicosanoids is a characteristic finding in aspirin-exacerbated respiratory disease (AERD). Bronchial challenge with lysyl-aspirin can be used as a confirmatory diagnostic test for this clinical condition. Induced sputum allows to measure mediators of asthmatic inflammation in bronchial secretions. Objectives To investigate the influence of inhaled lysyl-aspirin on sputum supernatant concentration of eicosanoids during the bronchial challenge test. Subjects with asthma hypersensitive to nonsteroidal anti-inflammatory drugs were compared with aspirin-tolerant asthmatic controls. Methods Induced sputum was collected before and following bronchial challenge with lysyl-aspirin. Sputum differential cell count and sputum supernatant concentrations of selected lipoxygenases products: 5-,12-,15-hydroxyeicosatetraenoic acid, cysteinyl leukotrienes, leukotriene B4, 11-dehydro-thromboxane B2, and prostaglandins E2, D2, and F2α and their metabolites, were measured using validated methods of chromatography–mass spectrometry. Results Aspirin precipitated bronchoconstriction in all AERD subjects, but in none of the aspirin-tolerant asthmatics. Phenotypes of asthma based on the sputum cytology did not differ between the groups. Baseline sputum eosinophilia correlated with a higher leukotriene D4 (LTD4) and leukotriene E4 (LTE4) concentrations. LTC4, PGE2, and 11-dehydro-TXB2 did not differ between the groups, but levels of LTD4, LTE4, and PGD2 were significantly higher in AERD group. Following the challenge, LTD4 and LTE4 increased, while PGE2 and LTB4 decreased in AERD subjects only. Conclusions During the bronchial challenge, decrease in PGE2 and its metabolite is accompanied by a surge in bronchoconstrictory cysteinyl leukotrienes produced at the expense of LTB4 in AERD subjects. Bronchial PGE2 inhibition in AERD seems specific and sensitive to a low dose of aspirin.

Background Numerous open trials have demonstrated the beneficial clinical effects of aspirin desensitization (AD) in patients with aspirin-induced asthma (AIA). These beneficial effects might be attributable to aspirin's potent anti-inflammatory properties, but that supposition requires further corroboration. Objective We sought to compare the clinical and biochemical responses to chronic oral AD in 20 patients with AIA and 14 patients with aspirin-tolerant asthma (ATA). All of the patients had chronic rhinosinusitis and nasal polyposis, and these responses were investigated in a pilot, double-blind, placebo-controlled study. Methods Twelve patients with AIA and 6 patients with ATA were randomly assigned to receive 624 mg of aspirin, and 8 patients with AIA and 8 patients with ATA received placebo. Both aspirin and placebo were administered once daily for 6 months. Nasal symptoms, Sino-Nasal Outcome Test (SNOT20) scores, peak nasal inspiratory flows, Asthma Control Questionnaire scores, spirometric parameters, peak expiratory flows, blood eosinophilia, and corticosteroid doses were assessed on a monthly basis. Levels of urinary leukotriene E 4 and the stable plasma prostaglandin (PG) D 2 metabolite 9α,11β-PGF2 were evaluated at baseline and after 1, 3, 5, and 6 months. Results Only the patients with AIA subjected to AD reported improvements in smell and reductions in sneezing and nasal blockade. The SNOT20 and Asthma Control Questionnaire scores of these patients decreased, and their peak nasal inspiratory flows increased. The dosages of inhaled corticosteroids were reduced. There were no changes in leukotriene E 4 or 9α,11β-PGF 2 levels after AD. Conclusion The clinically beneficial effects of AD on nasal and bronchial symptoms occurred only in the patients with AIA.

The purpose of this study was to examine the profile of eicosanoids secreted by human bronchial epithelial cells (HBEC) during their in vitro differentiation toward mucociliary or mucous metaplastic phenotype. Eicosanoids were measured in supernatants by mass spectrometry, and corresponding gene expression by real-time PCR. Primary HBEC produced mainly prostaglandins (PGE2, PGD2) and epoxides (e.g. 14,15-EET), but during further mucociliary differentiation we observed a gradual increase in secretion of lipoxygenase derived HETEs. Treatment with IL-13 and IL-4 induced mucous metaplasia and resulted in downregulation of PG pathway, and potent induction of 15-lipoxygenase (marked release of 15-HETE). The deficiency in PG production sustained during long term culture of mucous metaplastic epithelia. In conclusions, Th2-type cytokines induce changes in eicosanoid metabolism of airway epithelial cells, resulting in an immense induction of 15-lipoxygenase pathway, and inhibition of PG pathways. Deficient production of immunomodulatory PGs may promote chronic inflammation and airway remodeling.

Background Eicosanoids, important signaling and inflammatory molecules, are present in exhaled breath condensate (EBC) in very low concentrations, requiring highly sensitive analytic methods for their quantification. Objective We sought to assess a vast platform of eicosanoids in different asthma phenotypes, including aspirin-intolerant asthma, by means of a recently developed analytic approach based on mass spectrometry. Methods EBC from 115 adult asthmatic subjects (62 with aspirin intolerance) and 38 healthy control subjects were assessed quantitatively for 19 eicosanoids by using complementary HPLC, gas chromatography–mass spectrometry, or both. Palmitic acid concentrations were used as a marker for dilution of condensate samples. Results Asthma was characterized by an increase in arachidonate lipoxygenase products and cysteinyl leukotrienes. The COX pathway was also significantly upregulated in asthmatic subjects. Subjects with aspirin-intolerant asthma were distinguished by a sharp increase in the level of prostaglandin D 2 and E 2 metabolites; their 5- and 15-hydroxyeicosateraenoic acid levels were also higher than in aspirin-tolerant subjects. A classical discriminant analysis permitted us to classify correctly 99% of asthmatic subjects within the study population; the specificity of the analysis was 97%. The eicosanoid profiling allowed for 92% correct classification of aspirin-intolerant subjects. Conclusions The highly sensitive eicosanoid profiling in EBC makes it possible to detect alterations in asthma, especially in its distinct phenotype characterized by hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs. This permits us to discriminate asthmatic subjects from healthy subjects, as well as to distinguish the 2 asthma phenotypes based on the presence or absence of aspirin hypersensitivity.

Background: Increased levels of leukotrienes (LTs) in exhaled breath condensate (EBC) are associated with asthma and bronchial hyperresponsiveness (BHR), whereas eicosanoids generated through the 15-lipoxygenase (LO) pathway (15- hydroxyeicosatetraenoic acid [HETE] and eoxins) have been less studied. Objective: We investigated whether metabolites of the 5- and 15-LO pathways in EBC are associated with childhood asthma, asthma severity, and clinical parameters. Methods: The present study included 131 school-aged children (27 children with problematic severe asthma, 80 children with mild-to-moderate asthma, and 24 healthy children) from the Severe Asthma Recognized in Childhood study and 19 children with other nonasthmatic chronic lung diseases. Clinical work-up included spirometry, fractional exhaled nitric oxide measurements, skin prick testing, and methacholine challenge. Eicosanoids were analyzed in EBC by using mass spectrometry and are reported as concentrations (in picograms per milliliter) and eicosanoid/palmitic acid (PA) ratios. Results: Eoxin C4/PA, eoxin D4/PA, eoxin E4/PA, 15-HETE/PA, and LTC4/PA ratios were significantly increased in asthmatic versus healthy children. Eoxin D4/PA and LTE4/PA ratios were also significantly higher in children with BHR. A nonsignificant trend was observed toward higher eoxin/PA ratios with increasing asthma severity. In contrast to asthma, children with chronic lung disease had the highest 15-HETE/PA, LTC4/PA, LTE4/PA, and LTB4/PA ratios. Conclusion: The results point to increased activity of the 15-LO inflammatory pathway in childhood asthma. Mass spectrometric analyses of EBC demonstrate that increased eoxin levels not only accompany the increased 5-LO product LTC4 but are also associated with BHR. These markers might represent a new therapeutic target for asthma treatment. Background: Increased levels of leukotrienes (LTs) in exhaled breath condensate (EBC) are associated with asthma and bronchial hyperresponsiveness (BHR), whereas eicosanoids generated through the 15-lipoxygenase (LO) pathway (15- hydroxyeicosatetraenoic acid [HETE] and eoxins) have been less studied. Objective: We investigated whether metabolites of the 5- and 15-LO pathways in EBC are associated with childhood asthma, asthma severity, and clinical parameters. Methods: The present study included 131 school-aged children (27 children with problematic severe asthma, 80 children with mild-to-moderate asthma, and 24 healthy children) from the Severe Asthma Recognized in Childhood study and 19 children with other nonasthmatic chronic lung diseases. Clinical work-up included spirometry, fractional exhaled nitric oxide measurements, skin prick testing, and methacholine challenge. Eicosanoids were analyzed in EBC by using mass spectrometry and are reported as concentrations (in picograms per milliliter) and eicosanoid/palmitic acid (PA) ratios. Results: Eoxin C4/PA, eoxin D4/PA, eoxin E4/PA, 15-HETE/PA, and LTC4/PA ratios were significantly increased in asthmatic versus healthy children. Eoxin D4/PA and LTE4/PA ratios were also significantly higher in children with BHR. A nonsignificant trend was observed toward higher eoxin/PA ratios with increasing asthma severity. In contrast to asthma, children with chronic lung disease had the highest 15-HETE/PA, LTC4/PA, LTE4/PA, and LTB4/PA ratios. Conclusion: The results point to increased activity of the 15-LO inflammatory pathway in childhood asthma. Mass spectrometric analyses of EBC demonstrate that increased eoxin levels not only accompany the increased 5-LO product LTC4 but are also associated with BHR. These markers might represent a new therapeutic target for asthma treatment.

Background: Prostaglandin D2 (PGD2) is a major cyclooxygenase product generated by activated mast cells during an allergic response. Assessment of PGD2 and its metabolites in patients with asthma has mostly been performed in urine, bronchoalveolar lavage fluid and induced sputum, whereas human plasma determinations have been performed only sporadically. Methods: In 32 patients with allergic asthma and 50 healthy non-allergic controls, baseline plasma and urinary levels of 9α,11s-PGF2, a primary PGD2 metabolite, were assessed by gas chromatography/mass spectrometry. Serum tryptase levels were measured by fluoroenzyme immunoassay and urinary leukotriene E4 (LTE4) by ELISA. In a subgroup of 10 asthmatics (randomly selected from the 32 study patients) in whom bronchial allergen challenges with specific allergens (Dermatophagoides pteronyssinus, n = 4, mixed grass pollens, n = 6) were carried out, measurements were taken both before and after provocation. Results: At baseline no significant differences between mean plasma and urinary levels of the PGD2 metabolite and serum tryptase levels were found in asthmatics or controls. Asthmatic patients had significantly higher urinary LTE4 levels. Allergen challenge resulted in a significant early increase in the mean plasma 9α,11s-PGF2 level and only a borderline but significant increase in the urinary 9α,11s-PGF2 level within 2 hours after provocation. The challenge did not produce statistically significant changes in serum tryptase levels. Urinary LTE4 levels remained significantly increased 4 hours after provocation. Conclusions: PGD2 is actively involved in the early asthmatic response to allergens. Measurement of 9α,11s-PGF2 release into plasma rather than urine following allergen challenge is a sensitive marker of enhanced PGD2 synthesis, most probably due to mast cell activation.

A total of 7.5% of the males and 2.2% of the females had persistent wheezing complaints. Wheezing symptoms were found to be significantly high among males and smokers. The prevalence of allergic rhinitis and dermatitis were 24.1% and 6.8% respectively. Rhinitis rates were 16.5% among males and 7.6% among females. The eczema rate was 5.2% among males and 1.6% among females. Prevalence of having a pet at home was 11.7% and family history of atopy was 11.9%. Pet ownership [OR: 1.25 (0.94–1.65)] and family history of atopy [(OR: 3.77(2.88–4.94)] were significantly associated with rhinitis. Smoking rate was 23.3% in the whole group, 20.3% in males and 3.0% in females. Wheezing, asthma-like symptoms, cumulative asthma, current asthma and rhinitis were less common related in students who did not smoke. The rates of asthma, and asthma-like symptoms were found to be higher among smoking students than in non-smoking students, statistically. The low prevalence of asthma in our low-income population may depend on geographical and climatic variation, as well as to the low socioeconomic status of our population. The higher prevalence of rhinitis may depend on that some students may have confused the question about rhinitis and some with sinusitis or other nonallergic rhinitis complaints may have answered the same question with yes , because nonallergic rhinitis patients may have the same complaints like allergic rhinitis patients (5). In conclusion, asthma and/or asthmalike symptoms among university students in the eastern region of Turkey are lower than the developed countries, and habitual smoking is an important risk factor for allergic diseases.