Störfaktoren ökotoxikologischer Testverfahren mit Daphnia magna: Immobilisationstest, heat shock proteins und Metallothionein

Toxizitätstests mit Daphnia magna sind Grundpfeiler der ökotoxikologischen Bewertung von Chemikalien. Sie sind standardisiert; dennoch weisen die betrachteten Zielparameter, wie der EC50 (OECD Richtlinie 202) und die Reproduktionsrate (OECD Richtlinie 211), teils erhebliche Unterschiede im Vergleich mehrerer Labors auf. Verschiedene Störfaktoren können hierfür verantwortlich sein. (1) Art der Chemikalienapplikation: Zahlreiche Chemikalien sind schwer wasserlöslich und bedürfen Lösungsvermittlern zur Exposition im Toxizitätstest und in Biomarkerstudien. In dieser Arbeit wurden unterschiedliche Methoden und Kombinationen der Lösungsmittelapplikation untersucht. Die exemplarisch ausgewählten Substanzen Diclofenac und DMSO (Lösungsmittel) zeigten eine leicht antagonistische Wirkung im akuten Daphnientest (EC50). Für einen anderen Endpunkt (Hsp70-Level) konnte kein Effekt von DMSO auf die Proteotoxizität von Diclofenac beobachtet werden. (2) Genetische Disposition: In der vorliegenden Arbeit konnten genetische Unterschiede zwischen 7 Klonen von D. magna unterschiedlicher geographischer Herkunft bezüglich der Cd-Toleranz (EC50) und der Hsp70-Stressantwort auf Cd-Exposition festgestellt werden. Auf diese Weise zeigte sich eine Korrelation von Cd-Toleranz mit niedrigen Hsp70-Leveln, welche nicht durch entsprechende Unterschiede in der Akkumulation von Cd bei den untersuchten Klonen zu erklären war. (3) Mikroevolutive Prädisposition: An Zweien dieser Klone konnte gezeigt werden, dass eine 4 Generationen dauernde Akklimation gegenüber einer sublethalen Cd-Konzentration deren Cd-Toleranz geringfügig erhöhen konnte, nicht aber deren Hitzetoleranz. Des Weiteren zeigte sich bei den beiden Klonen ein Trade-off zwischen der Sequestrierung von Cd2+-Ionen und somit der unmittelbaren Bekämpfung des Stressors dienenden Induktion von Metallothionein und der Eindämmung möglicher Proteinschädigung dienender Induktion von Hsp70 im Umgang mit der Cd-Belastung. Die vorliegenden Ergebnisse verdeutlichen, dass sich unterschiedliche mikroevolutive Vorgeschichten einzelner Daphnienklone einerseits in den Resultaten akuter und chronischer ökotoxikologischer Tests niederschlagen können, sowie andererseits auch bei der Interpretation von Hsp70 und Metallothionein als Biomarker beachtet werden müssen

Cathepsin S inhibition suppresses systemic lupus erythematosus and lupus nephritis because cathepsin S is essential for MHC class II-mediated CD4 T cell and B cell priming

Objectives: Major histocompatibility complex (MHC) class II-mediated priming of T and B lymphocytes is a central element of autoimmunity in systemic lupus erythematosus (SLE) and lupus nephritis. The cysteine protease cathepsin S degrades the invariant peptide chain during MHC II assembly with antigenic peptide in antigen-presenting cells; therefore, we hypothesised that cathepsin S inhibition would be therapeutic in SLE. Methods: We developed a highly specific small molecule, orally available, cathepsin S antagonist, RO5461111, with suitable pharmacodynamic and pharmacokinetic properties that efficiently suppressed antigen-specific T cell and B cell priming in vitro and in vivo. Results: When given to MRL-Fas(lpr) mice with SLE and lupus nephritis, RO5461111 significantly reduced the activation of spleen dendritic cells and the subsequent expansion and activation of CD4 T cells and CD4/CD8 double-negative T cells. Cathepsin S inhibition impaired the spatial organisation of germinal centres, suppressed follicular B cell maturation to plasma cells and Ig class switch. This reversed hypergammaglobulinemia and significantly suppressed the plasma levels of numerous IgG (but not IgM) autoantibodies below baseline, including anti-dsDNA. This effect was associated with less glomerular IgG deposits, which protected kidneys from lupus nephritis. Conclusions: Together, cathepsin S promotes SLE by driving MHC class II-mediated T and B cell priming, germinal centre formation and B cell maturation towards plasma cells. These afferent immune pathways can be specifically reversed with the cathepsin S antagonist RO5461111, which prevents lupus nephritis progression even when given after disease onset. This novel therapeutic strategy could correct a common pathomechanism of SLE and other immune complex-related autoimmune diseases

Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3's nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3's subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3's subcellular distribution as determined by High Content Analysis. However, DDX3's nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer

Cathepsin S inhibition combines control of systemic and peripheral pathomechanisms of autoimmune tissue injury

Cathepsin(Cat)-S processing of the invariant chain-MHC-II complex inside antigen presenting cells is a central pathomechanism of autoimmune-diseases. Additionally, Cat-S is released by activated-myeloid cells and was recently described to activate protease-activated-receptor-(PAR)-2 in extracellular compartments. We hypothesized that Cat-S blockade targets both mechanisms and elicits synergistic therapeutic effects on autoimmune tissue injury. MRL-(Fas)lpr mice with spontaneous autoimmune tissue injury were treated with different doses of Cat-S inhibitor RO5459072, mycophenolate mofetil or vehicle. Further, female MRL-(Fas) lpr mice were injected with recombinant Cat-S with/without concomitant Cat-S or PAR-2 blockade. Cat-S blockade dose-dependently reversed aberrant systemic autoimmunity, e.g. plasma cytokines, activation of myeloid cells and hypergammaglobulinemia. Especially IgG autoantibody production was suppressed. Of note (MHC-II-independent) IgM were unaffected by Cat-S blockade while they were suppressed by MMF. Cat-S blockade dose-dependently suppressed immune-complex glomerulonephritis together with a profound and early effect on proteinuria, which was not shared by MMF. In fact, intravenous Cat-S injection induced severe glomerular endothelial injury and albuminuria, which was entirely prevented by Cat-S or PAR-2 blockade. In-vitro studies confirm that Cat-S induces endothelial activation and injury via PAR-2. Therapeutic Cat-S blockade suppresses systemic and peripheral pathomechanisms of autoimmune tissue injury, hence, Cat-S is a promising therapeutic target in lupus nephritis

Functional genetic polymorphisms and female reproductive disorders: Part I: polycystic ovary syndrome and ovarian response

BACKGROUNDThe identification of polymorphisms associated with a disease can help to elucidate its pathogenesis, and this knowledge can be used to improve prognosis for women with a particular disorder, such as polycystic ovary syndrome (PCOS). Since an altered response to ovarian stimulation is also a characteristic of the disease, further knowledge about its aetiology could help in defining the parameters that determine the response of an individual to ovarian stimulation.METHODSPubMed and EMBASE databases were systematically searched for gene association studies published until the end of August 2007, using search criteria relevant to PCOS and ovarian response to stimulation. Data from additional papers identified through hand searches were also included; 139 publications were reviewed.RESULTSSeveral genes involved in ovarian function and metabolism are associated with increased susceptibility to PCOS, but none is strong enough to correlate alone with susceptibility to the disease, or response to therapy. A single-nucleotide polymorphism in exon 10 of the FSH receptor (FSHR) gene, FSHR p.N680S, was consistently identified as having a significant association with ovarian response to FSH.CONCLUSIONSNo consistent association between gene polymorphism and PCOS could be identified. The FSHR gene may play a significant role in the success of ovarian stimulation, and can be used as a marker to predict differences in FSHR function and ovarian response to FSH. Genotyping the FSHR p.N680S polymorphism may provide a means of identifying a population of poor responders before in vitro fertilization procedures are initiated

Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies