Einleitung Adipositas ist ein zunehmendes Problem bei Menschen und Haustieren, z.B. in Pferden. Ponyrassen sind dabei besonders prädisponiert, wobei die Gründe bisher nicht abschließend geklärt werden konnten. Humane Adipositas geht mit einer fettigen Infiltration der Leber einher, die sogenannte Non-Alcoholic Fatty Liver Disease, welche zu einer hepatozellulären Entzündung führt. Es ist bisher nicht bekannt, ob Adipositas in Equiden auch zu hepatischen Veränderungen führt. Menschliche Fettleibigkeit ist zusätzlich mit systemischer Entzündung und gesteigertem oxidativen Stress verbunden. Das führte zu intensiven Untersuchungen von anti-inflammatorischen und antioxidativen Faktoren (z.B. Vitamin A - Retinol und Vitamin E - α-Tocopherol) in der humanen Adipositas Forschung. Viele Studien konnten ein Absinken von Vitamin A und Vitamin E in fettleibigen Menschen feststellen. Ziele Die vorliegende Studie sollte den Einfluss von zunehmendem Körpergewicht (KG) in Ponys und Pferden auf mehrere Parameter untersuchen: (1) Serum Leberenzymaktivitäten und Serum Gallensäuren (GS), (2) Leberfettgehalt, (3) hepatische messenger Ribonukleinsäure (mRNA) Level von Entzündungsmarkern und Markern des Lipidmetabolismus und (4) Serum Konzentrationen von Retinol und α-Tocopherol. Zusätzlich sollten Ponys und Pferde im Verlauf dieser Studie verglichen werden, um eventuelle Gründe für die Rasseprädisposition der Ponys für metabolische Störungen zu identifizieren. Material und Methoden Zehn Shetland Ponys und 9 Warmblut Pferde, die initial nicht adipös waren, wurden über 2 Jahre mit 200% des Erhaltungsbedarfes für umsetzbare Energie gefüttert. Die Entwicklung des KG, des Body Condition Scores (BCS) und des Cresty Neck Scores (CNS) wurde wöchentlich erfasst. Während der Fütterungsphase wurde zu 6 Zeitpunkten (ZP) Blut für die Bestimmung von Serum Leberenzymaktivitäten (Alkaline Phosphatase (ALP), Aspartat Aminotransferase (AST), Glutamat Dehydrogenase (GLDH), Gamma-Glutamyl Transferase (GGT)) und Serum GS entnommen und zu 7 ZP wurde Blut für die Analyse von Serum Retinol und α-Tocopherol gewonnen. An 3 ZP wurde durch Laparotomie Lebergewebe in Vollnarkose entnommen. Die Leberbiopsien wurden histologisch auf ihren Fettgehalt untersucht und mittels quantitativer Echtzeit Polymerase-Kettenreaktion (RT-qPCR) wurden die mRNA Level von Entzündungsmarkern (Nuclear Factor-κB (NF-κB), Interleukin-1β (IL-1β), IL-6, Tumor Nekrose Faktor α (TNFα), Differenzierungsgruppe 68 (CD68), Chemerin) und Lipid Metabolismus Markern (Lipoprotein Lipase (LPL), Fettsäuren Bindungsprotein 1 (FABP1) bestimmt. Die Daten wurden mittels statistischem Software Programm ausgewertet (STATISTICA, version 12, StatSoft GmbH, Hamburg, Deutschland). Nach Prüfung auf Normalverteilung der Daten, wurden geeignete statistische Tests angewendet mit einem statistischen Signifikanzniveau bei P < 0,05. Die Tierschutzkommission des Bezirks Leipzig genehmigte das Projekt in Übereinstimmung mit deutschen Rechtsvorschriften (Nr. TVV 32/15). Ergebnisse Ponys und Pferde zeigten einen signifikanten Anstieg von KG (Mittelwert ± SD; Ponys: 29,9 ± 19,4%; Pferde: 17 ± 6,74%), BCS (Median (25./75. Perzentil); Ponys: 157% (115/349); Pferde: 142% (128/192)) und CNS (Median (25./75. Perzentil); Ponys: 165% (123/500); Pferde: 200% (160/225)) induziert durch die hyperkalorische Fütterung über 2 Jahre. Das ansteigende KG hat keine Steatosis in der Mehrheit der Equiden ausgelöst. Die mRNA Level von IL-6, TNFα, CD68 und IL-1β in der Leber wurden nicht beeinflusst. Die Leber mRNA Level von Chemerin sind signifikant angestiegen in Ponys (x-facher Anstieg: 1,89) und Pferden (x-facher Anstieg: 2,04). Signifikante Unterschiede zwischen den Rassen hinsichtlich der Serum GLDH Aktivitäten, Serum GS Konzentrationen und der hepatischen mRNA LPL Level konnten festgestellt werden. Die Serum α-Tocopherol Konzentrationen stiegen in Ponys und Pferden signifikant an und korrelierten positiv mit der Vitamin E Aufnahme. Die Serum Retinol Konzentrationen fluktuierten während der Studie, ohne mit der Aufnahme zu korrelieren. Schlussfolgerungen Frühe Fettleibigkeit in Equiden führt nicht zwangsläufig zu einer Steatose mit hepatozellulärer Entzündung. Gemäß der Hypothese zeigten Ponys und Pferde allerdings unterschiedliche hepatische Reaktionsmuster nach KG Zunahme. Das könnte die höhere Empfänglichkeit von Ponys für metabolische Erkrankungen erklären. Chemerin konnte als interessanter Marker für die equine Adipositas Forschung identifiziert werden. Serum Konzentrationen von Retinol und α-Tocopherol wurden durch die KG Zunahme nicht beeinflusst.

The aim of the present study was to find out whether adaption to chronic hypoxia affects the expresion of TNF-α and IL-10 in rat myocardium. TNF-α is a proinflammatory cytokine, which amplifies inflammatory reaction, while IL-10 has opposite antiinflammatory effect. We also measured concentration of nitrotyrosine as a marker of nitrosative stress. We used male Wistar rats divided into four groups: 1) normoxic controls; 2) exposed to continous normobaric hypoxia (10% O2) for three days or 3) for three weeks and 4) exposed to intermittent normobaric hypoxia (10% O2) for three weeks with one hour daily reoxygenation. Cytosolic and membrane proteins (cytosolic and particulate fractions) were obtained from the left ventricle, right ventricle and interventricular septum. Concentrations of TNF-α and IL-10 in both fractions were measured by ELISA. Continous hypoxia increased TNF-α production in particulate fractions from all ventricular parts and decreased the ratio of IL-10/TNF-α in particulate and cytosolic fractions. Intermittent hypoxia redistributed TNF-α from cytosol into the particulate fraction and prevented the drop of IL-10/TNF-α ratio in the cytosolic fraction. The highest concentration of nitrotyrosine was found in the particulate fraction from the right ventricle after three days of hypoxia....

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...

Microglia are the resident macrophage in the central nervous system (CNS) that actively survey their environment and participate in shaping neuronal circuits. Among the transcription factors necessary for microglia development, interferon regulatory factor 8 (IRF8) is a known risk gene for multiple sclerosis and lupus and it has recently been shown to be downregulated in schizophrenia. These studies suggest that lack of microglial IRF8 can subsequently impact neuronal function in disease, but the mechanisms underlying these effects remain unknown. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. To interrogate the impact of disrupted microglial IRF8 signaling on brain circuits, I first show by RNAseq that several genes known to regulate neuronal function are dysregulated basally in Irf8-/- brains. I then found that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to chemically-induced lethal seizures in Irf8-/- mice. Importantly, I also show that developmental synaptic pruning, a key function for microglia, proceeds normally in Irf8-/-mice. Finally, I identified that these IRF8-dependent effects on circuits are due to elevated TNF-α in the CNS as genetic or acute pharmacological blockade of TNF-α in the Irf8-/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. The next steps are to use cell-specific genetic approaches to manipulate this signaling, which I have further developed over the course of this project.

No description available.

Background: CD100, also known as Sema4D, is an immune semaphorin constitutively expressed on natural killer (NK) cells and T cells. As an immune activation molecule, CD100 has important immunoregulatory effects on NK functions by enhancing the interactions between NK cells and target cells. The aim of this study was to investigate whether hepatitis C virus (HCV) infection affects CD100 expression, and whether interferon-α treatment enhances NK killing activity to facilitate HCV clearance via CD100. Methods: Expression of CD100 on NK cells was evaluated by flow cytometry in patients with chronic HCV infection, with or without pegylated interferon-α-based therapy. NK cell cytotoxicity and interferon (IFN)-γ production were measured by flow cytometry upon culturing the NK cells with K562 and Huh7.5 or HCV JFH-1-infected Huh7.5 cells. Results: The frequency of CD100+ NK cells in HCV-infected individuals was slightly suppressed compared to healthy subjects. IFN-α treatment could significantly upregulate CD100 expression, which was confirmed by in vitro studies using peripheral blood mononuclear cells cocultured with HCV-expressing Huh7.5 cells or IFN-α. Importantly, the expression of CD100 on NK cells from HCV patients was inversely associated with the HCV-RNA levels in the early phase of IFN-α therapy, and the IFN-α upregulated CD100 led to an enhanced NK killing activity through ligations with its receptors plexin-B1/B2 on target cells. Conclusion: These results implied a novel mechanism by which IFN-α enhanced CD100/Plexin-B1/B2 interaction plays an important role in promoting NK functions in patients with chronic hepatitis C.

BACKGROUND: α-tocopherol (AT) and γ-tocotrienol (GT3) are vitamin E isoforms considered to have potential chemopreventive properties. AT has been widely studied in vitro and in clinical trials with mixed results. The latest clinical study (SELECT trial) tested AT in prostate cancer patients, determined that AT provided no benefit, and could promote cancer. Conversely, GT3 has shown antineoplastic properties in several in vitro studies, with no clinical studies published to date. GT3 causes apoptosis via upregulation of the JNK pathway; however, inhibition results in a partial block of cell death. We compared side by side the mechanistic differences in these cells in response to AT and GT3. METHODS: The effects of GT3 and AT were studied on androgen sensitive LNCaP and androgen independent PC-3 prostate cancer cells. Their cytotoxic effects were analyzed via MTT and confirmed by metabolic assays measuring ATP. Cellular pathways were studied by immunoblot. Quantitative analysis and the determination of relationships between cell signaling events were analyzed for both agents tested. Non-cancerous prostate RWPE-1 cells were also included as a control. RESULTS: The RAF/RAS/ERK pathway was significantly activated by GT3 in LNCaP and PC-3 cells but not by AT. This activation is essential for the apoptotic affect by GT3 as demonstrated the complete inhibition of apoptosis by MEK1 inhibitor U0126. Phospho-c-JUN was upregulated by GT3 but not AT. No changes were observed on AKT for either agent, and no release of cytochrome c into the cytoplasm was detected. Caspases 9 and 3 were efficiently activated by GT3 on both cell lines irrespective of androgen sensitivity, but not in cells dosed with AT. Cell viability of non-cancerous RWPE-1 cells was affected neither by GT3 nor AT. CONCLUSIONS: c-JUN is a recognized master regulator of apoptosis as shown previously in prostate cancer. However, the mechanism of action of GT3 in these cells also include a significant activation of ERK which is essential for the apoptotic effect of GT3. The activation of both, ERK and c-JUN, is required for apoptosis and may suggest a relevant step in ensuring circumvention of mechanisms of resistance related to the constitutive activation of MEK1.

<p>  </p> <p>Osteoarthritis (OA) is prevalent in equine and can be career-ending for performance horses due to lameness limitations and decreased quality of life. OA is a progressive, multifactorial disease that compromises the synovial joints' normal function, resulting in subchondral bone and articular cartilage deterioration over time. OA is a complex disease that impacts the entire joint, wherein activation of the innate immune system has an essential role in the disease progression and the development of pain. The synovial membrane, or the synovium, is a crucial contributor to the inflammation of diseased joints, regardless of the intra-articular tissue type initially affected. Synoviocytes are a predominant cell type of the synovium and contribute to inflammation by releasing key mediators and degradative enzymes, such as interleukin (IL)-6, IL-1β, a disintegrin, and metalloproteinase (ADAM) domains, and matrix metalloproteinases (MMPs). The production of pro-inflammatory molecules sequentially influences the expression of degradative enzymes and cartilage destruction. Therefore, the pathophysiological processes within synovial joints afflicted by OA can be further understood by studying the characteristics of synoviocytes.</p> <p>We aimed to investigate the inflammatory component of OA in an <em>in vitro</em> model using a primary cell line of equine fibroblast-like synoviocytes (eqFLS) stimulated with tumor necrosis factor-alpha (TNF-α) to represent an initial inflammatory stimulus. Our studies have shown that stimulating eqFLS with TNF-α for 24 hours significantly increased the gene expression of pro-inflammatory biomarkers. Among several pro-inflammatory candidate genes assayed, only pro-inflammatory cytokine IL-6 gene expression could be detected reproducibly following stimulation with the TNF-α gene in eqFLS. We characterized the pro-inflammatory response of eqFLS and utilized this system to examine the impact of novel therapeutic compounds designed <em>in-silico</em> with the goal of reducing the inflammatory response of eqFLS. A piperazine-based compound (C3) and its derivative (02-09) were primarily designed to mimic the interactions of the growth factor pigment epithelium-derived factor (PEDF) with its receptor, the non-integrin laminin receptor 1 (LAMR1). Based on previous <em>in vitro</em> studies in the laboratory, C3 and 02-09 had been proposed to have a strong potential for inhibiting inflammation while reducing angiogenesis and chondrocyte hypertrophy. The efficacy of these two novel compounds on eqFLS was examined in the present work by assessing the gene expression levels of inflammatory biomarkers, including IL-6, IL-1β, IL-8, ADAMs, and MMPs relative to a control housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in various study designs. An <em>in-vitro</em> screen with the IL-1β promoter driving a reporter green fluorescent protein (GFP) was also designed to detect and track the inflammatory response of eqFLS by imaging following stimulation with or without (+/-) TNF-α relative to controls. This screen will be utilized in future studies to potentially identify more effective compounds in the LAMR1-interacting series. The current findings suggest that the novel compounds, especially 02-09, might exhibit an anti-inflammatory effect on eqFLS; therefore, it is a potential therapeutic agent in modulating inflammation during OA development. </p> <p><br></p>

The expression of the α1C-adrenoceptor subtype in human and rabbit blood vessels has been analyzed using the reverse transcriptase/polymerase chain reaction technique (RT/PCR). The 20 bp primers employed were designed from the bovine α1C-adrenoceptor and flank a least conserved region - the putative third cytoplasmic loop. RT/PCR products generated from rabbit and human brain mRNA both had 93% homology to the bovine α1C-adrenoceptor and were used as species and subtype specific probes in Southern blot analysis of vascular RT/PCR products. Poly A+ RNA was purified from the human saphenous vein and rabbit aorta, renal, pulmonary and central ear arteries and amplified by RT/PCR. Size analysis by agarose gel electrophoresis, together with Southern hybridization of the resulting cDNA products confirm the expression of the α1C-adrenoceptor in these vessels.

Nutritional factors play an important role in the prevention and promotion of colorectal cancer. Vitamin E is a generic term that describes a group of lipid-soluble chain-breaking antioxidants that includes tocopherols and tocotrienols. Vitamin E occurs in nature as eight structurally related forms that include four tocopherols and four tocotrienols. Vitamin E is a potent membrane-soluble antioxidant. Antioxidants like vitamin E (tocopherols) may prevent colon cancer through several different cellular and molecular mechanisms. Vitamin E in the American diet is primarily available in plant-oil rich foods such as vegetable oils, seeds and nuts and these foods vary widely in their content of α-tocopherol and γ-tocopherol [1]. Vitamin E may help prevent colon cancer by decreasing the formation of mutagens arising from the oxidation of fecal lipids, by decreasing oxidative stress in the epithelial cells of the colon and by molecular mechanisms that influence cell death, cell cycle and transcriptional events. Most epidemiological, experimental and clinical studies have evaluated the α-isoform and not the γ-isoform of vitamin E. Recent epidemiological, experimental and mechanistic evidence suggests that γ-tocopherol may be a more potent cancer chemopreventive agent than α-tocopherol. The differences in chemical reactivity, metabolism and biological activity may contribute to these differences in the effects of γ-tocopherol when compared with α-tocopherol. The rationale supporting the development of γ-tocopherol as a colorectal cancer preventive agent is reviewed here.