Acetylsalicylic Acid Reduces the Severity of Dextran Sodium Sulfate-Induced Colitis and Increases the Formation of Anti-Inflammatory Lipid Mediators

The role of non-steroidal anti-inflammatory drugs in inflammatory bowel disease is controversial, as they have been implicated in disease aggravation. Different from other cyclooxygenase inhibitors, acetylsalicylic acid (ASA) enhances the formation of anti-inflammatory and proresolution lipoxins derived from arachidonic acid as well as resolvins from omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA). In this study, we examined the effect of ASA on murine dextran sodium sulfate colitis. A mouse magnetic resonance imaging (MRI) protocol and post mortem assessment were used to assess disease severity, and lipid metabolites were measured using liquid chromatography-coupled tandem mass spectrometry. Decreased colitis activity was demonstrated by phenotype and MRI assessment in mice treated with ASA, and confirmed in postmortem analysis. Analysis of lipid mediators showed sustained formation of lipoxin A4 and an increase of DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA) after treatment with ASA. Furthermore, in vitro experiments in RAW264.7 murine macrophages demonstrated significantly increased phagocytosis activity after incubation with 17-HDHA, supporting its proresolution effect. These results show a protective effect of ASA in a murine colitis model and could give a rationale for a careful reassessment of ASA therapy in patients with inflammatory bowel disease and particularly ulcerative colitis, possibly combined with DHA supplementation

Assessment of mRNA and microRNA Stabilization in Peripheral Human Blood for Multicenter Studies and Biobanks

In this study we evaluate the suitability of two methods of RNA conservation in blood samples, PAXgene and RNAlater, in combination with variable shipping conditions for their application in multicenter studies and biobanking. RNA yield, integrity, and purity as well as levels of selected mRNA and microRNA species were analyzed in peripheral human blood samples stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient temperatures from the study centers to the central analysis laboratory. Both examined systems were clearly appropriate for RNA stabilization in human blood independently of the shipping conditions. The isolated RNA is characterized by good quantity and quality and well suited for downstream applications like quantitative RT-PCR analysis of mRNA and microRNA. Superior yield and integrity values were received using RNAlater. It would be reasonable to consider the production and approval of blood collection tubes prefilled with RNAlater to facilitate the use of this excellent RNA stabilization system in large studies

Genetic modifiers of radon-induced lung cancer risk: a genome-wide interaction study in former uranium miners

PURPOSE: Radon is a risk factor for lung cancer and uranium miners are more exposed than the general population. A genome-wide interaction analysis was carried out to identify genomic loci, genes or gene sets that modify the susceptibility to lung cancer given occupational exposure to the radioactive gas radon. METHODS: Samples from 28 studies provided by the International Lung Cancer Consortium were pooled with samples of former uranium miners collected by the German Federal Office of Radiation Protection. In total, 15,077 cases and 13,522 controls, all of European ancestries, comprising 463 uranium miners were compared. The DNA of all participants was genotyped with the OncoArray. We fitted single-marker and in multi-marker models and performed an exploratory gene-set analysis to detect cumulative enrichment of significance in sets of genes. RESULTS: We discovered a genome-wide significant interaction of the marker rs12440014 within the gene CHRNB4 (OR = 0.26, 95% CI 0.11-0.60, p = 0.0386 corrected for multiple testing). At least suggestive significant interaction of linkage disequilibrium blocks was observed at the chromosomal regions 18q21.23 (p = 1.2 × 10-6), 5q23.2 (p = 2.5 × 10-6), 1q21.3 (p = 3.2 × 10-6), 10p13 (p = 1.3 × 10-5) and 12p12.1 (p = 7.1 × 10-5). Genes belonging to the Gene Ontology term "DNA dealkylation involved in DNA repair" (GO:0006307; p = 0.0139) or the gene family HGNC:476 "microRNAs" (p = 0.0159) were enriched with LD-blockwise significance. CONCLUSION: The well-established association of the genomic region 15q25 to lung cancer might be influenced by exposure to radon among uranium miners. Furthermore, lung cancer susceptibility is related to the functional capability of DNA damage signaling via ubiquitination processes and repair of radiation-induced double-strand breaks by the single-strand annealing mechanism

The anti-inflammatory potential of 17-hydroxydocosahexaenoic acid

Essentielle mehrfach ungesättigte Omega-3 (n-3) und Omega-6 (n-6) Fettsäuren (polyunsaturated fatty acids, PUFA) sind die Vorläufer einer großen Anzahl von bioaktiven Lipidmetaboliten und -mediatoren. Sie greifen in physiologische und pathologische Stoffwechselprozesse ein, können in ihrer Bildung pharmakologisch beeinflusst werden und grundsätzlich pro- und anti- inflammatorische Effekte hervorrufen. Die gleichzeitige Analyse von n-3 und n-6 Lipidmetaboliten eröffnet neue Einsichten, wie diese auf die Regulation inflammatorischer Prozesse einwirken können. In den dieser Arbeit zugrunde liegenden Publikationen wurde die Rolle insbesondere des Lipidmetaboliten 17-Hydroxydocosahexaensäure (17-HDHA) untersucht. In der ersten Studie wurde zunächst eine LC(ESI)-MS/MS-Methode zur simultanen Bestimmung von insgesamt 29 Lipidmetaboliten entwickelt, welche aus der n-6 PUFA Arachidonsäure (AA) sowie den n-3 PUFA Docosahexaensäure (DHA) und Eicosapentaensäure (EPA) entstehen. Diese wurden in nativen und Calcium-Ionophor A23187 (A23187) aktivierten humanen und murinen Blutproben gemessen. Dabei führte die in vitro Aktivierung mit A23187 zu einem signifikanten Anstieg der Lipidmetabolitenbildung. Die Messung im Blut von Wildtyp (WT)-Mäusen und Fat-1-Mäusen, die endogen hohe n-3 PUFA-Konzentrationen aufweisen, zeigte in Fat-1-Tieren im Vergleich zu den WT- Tieren signifikant erhöht 17-HDHA. Die zweite Studie untersuchte chemisch induzierte Lebertumoren in der Maus. Diese zeigte eine geringere Tumor-Bildung in Bezug auf Größe und Anzahl in Fat-1-Mäusen im Vergleich zu ihren WT- Geschwistertieren und signifikant erhöhte 17-HDHA-Konzentrationen im Lebergewebe der Fat-1-Mäuse. In vitro Experimente zeigten außerdem, dass 17-HDHA effektiv die Lipopolysaccharid (LPS)-getriggerte Tumornekrosefaktor alpha (TNF-α)-Bildung in der murinen Makrophagen-Zelllinie RAW 264.7 unterdrücken konnte. Der anti-inflammatorische Effekt von 17-HDHA wurde dann in der dritten Studie in einem Dextran Natriumsulfat (Dextran sulfate sodium, DSS)-induzierten Kolitis-Modell in vivo in WT-Mäusen bestätigt. Es konnte gezeigt werden, dass die intraperitoneale Behandlung mit 17-HDHA die DSS- Kolitis abmildert und signifikant den Gewichtsverlust, den epithelialen Schaden des Kolongewebes und die Makrophageninfiltration lindert. Weitere in vitro Experimente konnten zeigen, dass 17-HDHA die Phagozytoseaktivität in der Makrophagen-Zelllinie RAW 264.7 erhöht. Zusammengefasst etablieren die Ergebnisse dieser Arbeiten 17-HDHA als anti-inflammatorischen Lipidmediator.The essential polyunsaturated omega-3 (n-3) and omega-6 (n-6) fatty acids (PUFA) are precursors of a diverse group of bioactive lipid metabolites and lipid mediators. They influence a multitude of physiological and pathological processes, their synthesis can be modulated by pharmacological means and they can exert both pro-inflammatory and anti-inflammatory actions. The simultaneous analysis of n-3 and n-6 derived lipid metabolites gives insight into their regulatory potential within inflammatory processes. In the publications, which this dissertation is based on, the role of the n-3 PUFA derived lipid mediator 17-hydroxydocosahexaenoic acid (17-HDHA) has been examined. In the first study, a method was established which is based on LC(ESI)-MS/MS analysis and allows the simultaneous measurement of 29 lipid metabolites, that are synthesized from the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Analysis of these 29 lipid metabolites was performed in native and calcium- ionophor A23187 (A23187)-stimulated human and murine blood samples respectively. Here, in vitro activation by A23187 led to a significant increase in lipid mediator synthesis. Analysis of blood samples of wild-type (WT) and Fat-1-mice, that feature endogenously enriched concentrations of n-3 PUFA in all tissues, showed significantly higher levels of 17-HDHA in the Fat-1-mice compared to the WT-mice. The second study examined a model of chemically-induced liver tumorigenesis in mice. A decrease in tumor formation, in terms of size and number was found in Fat-1-mice compared with WT- littermates. Furthermore, concentrations of 17-HDHA in liver tissue of Fat-1-mice were significantly higher than in liver samples of WT-mice. In vitro experiments showed that 17-HDHA effectively inhibits the lipopolysaccharide (LPS)-triggered synthesis of tumor-necrosis-factor alpha (TNF-α) in the murine macrophage cell line RAW 264.7. The anti-inflammatory effect of 17-HDHA was then confirmed in a third study employing a model of dextrane-sodium sulfate (DSS) induced colitis in WT-mice. The intraperitoneal treatment with 17-HDHA led to an ameliorated disease course and reduced body weight loss, decreased epithelial damage of the intestinal mucosa as well as macrophage infiltration in the lamina propria significantly. Further in vitro experiments showed that 17-HDHA increases phagocytic activity in the macrophage cell line RAW 264.7 Taken together the results of the presented work establish 17-HDHA as an anti-inflammatory lipid mediator