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Biomarker für Oxidativen Stress bei Entzündungsreaktionen: Bedeutung von Genpolymorphismen und Genexpression der NADPH-Oxidase unter pro- und anti-inflammatorischen Bedingungen

dc.contributor.advisorBrockmöller, Jürgen Prof. Dr.
dc.contributor.authorGoetze, Robert Günther
dc.titleBiomarker für Oxidativen Stress bei Entzündungsreaktionen: Bedeutung von Genpolymorphismen und Genexpression der NADPH-Oxidase unter pro- und anti-inflammatorischen Bedingungende
dc.title.translatedgenetic polymorphisms and subunit-expression of NADPH-oxidase as biomarkers for oxidative stress including simulation of pro- and anti-inflammatory conditionsde
dc.contributor.refereeOppermann, Martin Prof. Dr.
dc.description.abstractengBackground NADPH oxidases (NOX) comprise a multimeric enzyme system expressed in various types of cells and tissues. The most popular isoform is that in leucocytes termed NOX2 according to the big catalytic subunit and is activatable by assembling of the cytosolic to the membrane-asscociated subunits. By transferring electrons from NADPH to oxygen NOX generates superoxid radicals, which are further metabolized to other reactive oxygen spezies (ROS). First attributed to anti-pathogen activity it was later observed that ROS are implicated in a variety of cellular processes as inflammation, proliferation, differentiation, and apoptosis. Consequently, ROS are regarded to be involved in a plethora of pathological conditions. In the recent years, the potential impact of genetic polymorphisms on the modulation of ROS formation gained increased interest. The aim of this study was to identify biomarkers for NOX2 activity as a surrogate for oxidative stress. Methods As functional trait, the NOX2 activity was quantified in a comprehensive and comparative manner in four different types of peripheral blood mononuclear cells (PBMCs). These cells were isolated of 120 healthy volunteers by gradient centrifugation and cell sorting by flow cytometry. Immediately upon isolation, the phorbol 12-myristate 13-acetate (PMA)-stimulated NOX2 activity was measured by L-012-enhanced chemoluminescence. In the quantitatively predominant PBMC fraction, the T lymphocytes, pro- and anti-inflammatory stimuli were assessed for modulation of NOX2 activity and if the latter is linked to cell viability. NOX2 subunit mRNA expression was ascertained both in the isolated PBMC fractions as well as in 48 commercially available lymphoblastoid cell lines (LCLs). Overall 48 genotypes covering the common genetic diversity in the NOX2 subunits were determined by primer extension method. Results In comparison with T and B lymphocytes, the PMA-stimulated NOX2 activity was 100 to 1000-fold higher in monocytes without concomitant discrepancies in mRNA subunit expression. For this activity, NCF2 subunit expression seemed to be the strongest predictor in these three cell types. With respect to a panel of reference genes, NOX2 subunit expression was ten times lower in LCLs than in the considered PBMC subfractions. The evidence for the regulatory role of NCF2 was further supported in T lymphocytes specifically treated prior to PMA stimulation. Upon treatment with the pro-inflammatory cytokine TGFβ1, transcript levels of NCF2 increased and those of NCF4 decreased coincident with enhanced ROS generation. Vice versa effects were observed upon stimulation with the anti-inflammatory cytokine IL10. NCF4 suppression accompanied by increased amounts of ROS was also seen after radiation of T lymphocytes and LCLs. Furthermore, expression of the big catalytic subunit CYBB was positively correlated with NOX2 activity both without pre-treatment and upon incubation with the pro-inflammatory cytokines TGFβ1 and IL1β. Modulation of NOX2 activity and subunit expression by genetic polymorphisms was rather moderate. With respect to CYBB and NCF4, one polymorphism each showed slight alteration of expression and activity. Conclusion Expression of NCF2 and CYBB appeared as positive and that of NCF4 as negative regulators of NOX2 activity which might become particularly relevant in inflammatory conditions. Genetic polymorphisms in cis-acting elements have not turned out as major contributors to NOX2 expression and
dc.contributor.coRefereeVirsik-Köpp, Patricia Prof. Dr.
dc.subject.gerNADPH-oxidase, NOX-Aktivität, Entzündungsreaktionen, oxidativer Stress, T-Lymphozytende
dc.subject.engNOX-Activity; oxidative stress, T-lymphocytes, inflammatory prcesses, NADPH-oxidasede
dc.affiliation.instituteMedizinische Fakultätde
dc.subject.gokfullPharmakologie (PPN619875518)de

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