| 1. |
- Bowden, John A., et al.
(författare)
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Harmonizing lipidomics : NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma
- 2017
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Ingår i: Journal of Lipid Research. - 0022-2275 .- 1539-7262. ; 58:12, s. 2275-2288
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Tidskriftsartikel (refereegranskat)abstract
- As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra-and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium.jlr While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.
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| 2. |
- Emma, Rosalia, et al.
(författare)
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Enhanced oxidative stress in smoking and ex-smoking severe asthma in the U-BIOPRED cohort
- 2018
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 13:9
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF(2 alpha) and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF(2 alpha); IS-transcriptomics; BB-transcriptomics; BBrtranscriptomics). Urinary 8-iso-PGF(2 alpha) was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF(2 alpha) was increased in SAs/ex, median (IQR) = 31.7 (24.5 +/- 44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6 +/- 36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4 +/- 47.7), vs. ESA, median (IQR) = 29.4 (22.3 +/- 40.5), and NSA, median (IQR) = 26.5 (19.6 +/- 16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF(2 alpha) in the smokers/ex-smokers group.
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| 3. |
- Kolmert, Johan
(författare)
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Quantification of inflammatory mediators to explore molecular mechanisms and sub-phenotypes of asthma
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis summarizes a series of studies using liquid chromatography coupled to mass spectrometry methodologies to quantify metabolites of fatty acids (i.e., oxylipins) and histamine in different samples from experimental models and clinical studies with the overall aim to define mechanisms and identify biomarkers for improved sub-phenotyping of asthma. Asthma is characterized by variable airflow obstruction, hyperresponsiveness and chronic inflammation in the airways. The substantial overlap among clinical descriptors has resulted in difficulties to establish diagnosis and predict response to treatment. Instead, a shift in focus towards identifying specific cellular and molecular mechanisms has emerged, aiming to define new treatable traits based on specific cellular and molecular pathways (defined as endotypes). Important pathobiological components involve the release of potent inflammatory mediators, such as histamine, prostaglandins (PGs) and leukotrienes (LTs), that cause bronchoconstriction and airway inflammation. A rapid hydrophilic interaction chromatography method failed to quantify the major histamine metabolite 1,4-methyl-5-imidazoleacetic acid (tele-MIAA) due to ion suppression from inorganic salts present in urine. Ion-pairing chromatography was therefore employed and the resulting increase in precision enabled the detection of higher baseline levels of tele-MIAA in females compared to males (3.0 vs. 2.1 μmol/mmol creatinine, respectively) (Paper I). In addition, levels of tele-MIAA reached up to 30 μmol/mmol creatinine in spot urine samples from mastocytosis patients. Three liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods quantified 130 oxylipins and were able to define kinetic release and enzymatic contribution of mast cell-derived mediators to smooth muscle contraction using isolated and intact airways from humans and guinea pigs in vitro. PGD2 levels were elevated 24-hour post anti-IgE stimulation of human bronchus, suggesting a prolonged mast cell activation (Paper II). Furthermore, exposure to house dust mite (HDM) induced strong release of lipoxygenase-derived LTB4, 5,15-DiHETE, 15-HETE and 15-HEDE along with eosinophilic infiltration in a C57BL/6 murine model of asthma. Interestingly, high levels of cysteinyl-leukotrienes (CysLTs) remained unchanged suggesting a different role of CysLTs in mice (Paper III). Urinary profiles of 11 eicosanoid metabolites in 100 healthy control subjects and 497 asthmatics defined normal baseline levels and revealed increased concentration of PGs, LTE4 and isoprostanes with asthma severity. Consensus clustering of 497 asthmatics identified a five-cluster model with distinct clinical characteristics, which included two new phenotypes, U1 and U5, with low levels of thromboxanes and PGs respectively (Paper IV). At the 12 to 18-month longitudinal time point for the 302 subjects with severe asthma, z-scored eicosanoid concentrations retained the five-cluster profile, despite technical and intra-subject variability. In conclusion, the developed bioanalytical methods were applied to define levels of histamine and eicosanoid metabolites in urine from healthy subjects. In addition, release of multiple oxylipins following mast cell-mediated bronchoconstriction and HDM-induced airway inflammation in model systems were explored to relate functions to levels of lipid mediators. For the first time, grouping of asthmatics according to profiles of eicosanoid metabolites in urine was performed and demonstrated sufficient resolution to identify five sub-phenotypes of asthma possessing distinct clinical characteristics. The presented approaches, for both in vitro and in vivo respiratory research, offer an opportunity to progress the development of new treatment options and suggests a panel of PGs, LTE4 and isoprostanes to be further validated as diagnostic markers in patients with asthma.
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