neu Transgenic Mice

neu Transgenic Mice

the simplicity of the model, our findings provide further support that the antioxidants present in the respiratory tract lining fluid can offer protec...

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the simplicity of the model, our findings provide further support that the antioxidants present in the respiratory tract lining fluid can offer protection against the oxidative actions of O3 and NO2 at environmentally relevant concentrations.

hypoxia in combination with hepatic steatosis was shown to promote augmented oxidative stress leading to NF-kB inactivation and impaired HIF1α induction, and thereby increased susceptibility to hypoxic injury. Keywords: Fatty liver, Hypoxia, HIF1 signaling, ROS

Keywords: Nitrogen Dioxide, Ozone, Particulate Matter, Antioxidants doi:10.1016/j.freeradbiomed.2012.08.516 Antioxidants, Nutrition and Novel Therapies 7: Oxidative stress impaired HIF1α activation: a novel mechanism for increased vulnerability of steatotic hepatocytes to hypoxic stress S. Anavi, N. Budick Harmelin, Z. Madar, O. Tirosh* The Hebrew University of Jerusalem, Israel Steatosis increases the sensitivity of hepatocytes to hypoxic injury. Thus, this study was designed to elucidate the role of hypoxia inducible factor 1alfa (HIF1α) in steatotic hepatocytes during hypoxia. AML12 hepatocytes and isolated rat hepatocytes were treated with a free fatty acid mixture of oleate and palmitate (2:1,1mM) for 18h, which generated intrahepatocytes fat accumulation. The cells were then exposed to hypoxia (1%oxygen, 6-24h). Following hypoxia, further increase in cellular fat accumulation was shown. In steatotic hepatocytes, a decreased HIF1α activation by hypoxia was observed. The capacity of these cells to express HIF1α dependent genes responsible for the utilization of nutrients for energy was also impaired. This resulted with a significantly lower intracellular ATP levels and greater cell death in steatotic hepatocytes compared with control hepatocytes. In contrast, overexpression of constitutively active HIF1α significantly increased cell viability as well as ATP and GLUT1 mRNA levels in steatotic hepatocytes under hypoxia. Hypoxia significantly enhanced HIF1α mRNA levels in control but not in steatotic hepatocytes. Concomitantly, an increase in oxidative stress was found in steatotic hepatocytes under hypoxic conditions compared with control cells. That included: higher reactive oxygen species generation, lower cellular and nuclear GSH levels and higher accumulation of 4-hydroxynonenal protein adducts. Hypoxia mediated oxidative stress was accompanied by inactivation of basal nuclear factorkappaB (NF-kB) DNA binding. Treatment with N-acetylL-cysteine, a reducing agent, improved NF-kB DNAbinding capacity and restored HIF1α induction. Conversely, overexpression of NF-kB super-suppressor in control hepatocytes ((IkBαΔN-transfected cells) resulted with complete inhibition of HIF1α expression, confirming that indeed NF-kB regulates HIF1α expression in hypoxic hepatocytes. In conclusion,

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doi:10.1016/j.freeradbiomed.2012.08.517 Antioxidants, Nutrition and Novel Therapies 8: Effect of annatto-tocotrienols supplementation on the Development of Mammary Tumors in HER-2/neu Transgenic Mice E. Pierpaoli*, V. Viola, A. Barucca, F. Orlando, F. Galli, M. Provinciali University of Perugia, Italy Desmethylated tocotrienols (T3s), have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-tocotrienols (T3) (90% δ-T3 and 10% γ-T3) on the development of mammary tumors which spontaneously develop in HER-2/neu transgenic mice. The mechanisms involved in the antitumor effect of annatto-T3 were evaluated by studying the apoptosis, the senescent-like growth arrest, the immune modulation, the oxidative effect, and the expression of HER-2/neu in tumoral mammary glands from transgenic mice and in tumor cells. The administration of annattoT3 delayed the development of spontaneous mammary tumors, reduced the number and size of mammary tumor masses, and the size of lung metastasis in transgenic mice. In annatto-T3 supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dosedependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest, and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified dtocotrienols in comparison with untreated tumor cells. Both HER-2/neu mRNA and p185HER-2/neu protein were reduced in tumor cells treated with annatto- T3. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis in tumor cells rather than to a modulation of immune responses.

Keywords: Desmethylated tocotrienols, Mammary tumors, Cancer therapy, HER-2/neu transgenic mice

Keywords: superoxide dismutase, knockout mouse, autoantibody, red blood cell

doi:10.1016/j.freeradbiomed.2012.08.518

doi:10.1016/j.freeradbiomed.2012.08.519

Inflammation and Immunity Oral Presentations

Inflammation and Immunity 2:

Inflammation and Immunity 1:

Modulation of TH1/TH17 equilibrium in vitro indicaxanthin from Opuntia ficus indica (L. Mill)

Oxidative stress as a cause for autoimmune hemolytic anemia; supporting evidences from genetically modified mice T. Konno, N. Ohtsuki, N. Kibe, S. Tsunoda, Y. Iuchi, J. Fujii* Yamagata University, Japan Autoimmune hemolytic anemia (AIHA) is the symptom in which antibodies against red blood cells (RBC) are produced and trigger the destruction of RBCs, resulting in hemolytic anemia. We recently found elevation of reactive oxygen species (ROS) levels in RBCs, and augmented production of autoantibodies to RBCs in SOD1-knockout C57BL/6 mice. Since both anemia and autoimmune responses in SOD1-deficient mice are totally rescued by local expression of human SOD1 in erythroid cells, these phenotypes are attributable to oxidative stress in RBC. A causal connection between ROS and AIHA was further confirmed by using an AIHAprone NZB mouse. ROS levels in RBC were originally high in young NZB mice and further increased during aging. Now we have strong evidence implying involvement of ROS in the development of AIHA by making genetically-modified congenic NZB mice. We prepared both human SOD1-trangenic (hSOD1-Tg) NZB mice, which express human SOD1 protein in erythroid cells specifically, and SOD1-deficient (SOD1-KO) NZB mice by back-crossing eight times to NZB mice. The severity of AIHA phenotypes, such as autoantibody production and anemia, and levels of intracellular ROS and oxidative stress markers were positively correlated among the mice. While SOD1 deficiency showed elevated oxidative stress and accelerated mortality, transgenic expression of human SOD1 protein in RBC suppressed ROS levels and concomitantly decreased the autoantibody levels and the death rate. Thus, oxidative stress-mediated autoantibody production may be a more general mechanism for AIHA and related autoimmune diseases. Thus we have now firm evidence that imply involvement of oxidative stress in the autoimmune response in NZB mice. The results obtained in our studies support the hypothetical role of ROS in triggering the autoimmune reaction, and propose a novel approach to mitigate the progression of AIHA by antioxidants.

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M. Allegra*1, L. Rattazzi2, A. Attanzio1, L. Tesoriere1, M.A. Livrea1, F. D'Acquisto2 1 Università di Palermo, Italy, 2Queen Mary University of London, UK Indicaxanthin is a betalainic phytochemical from Cactus Pear Fruit (Opuntia Ficus Indica, L. Mill). We have recently showed that this compound is a reducing and amphipathic molecule, able to penetrate cells and membranes and counteract oxidative damage in vitro. Moreover, it behaves as a signalling molecule and modulates specific redox-dependent pathways in vitro (1). Remarkably, indicaxanthin is highly bioavailable: the ingestion of three cactus pear fruits generates, in humans, an indicaxanthin plasma concentration of 7µM after 3 h (2). In the light of the above reported properties of indicaxanthin, we have here investigated the effects of the pigment on T effector function, differentiating splenocytes in Th0, Th1 and Th17 skewing conditions in presence or absence of indicaxanthin 15µM. Differentiated cells were analysed by flow cytometry (FACS) intracellular staining while cell supernatants were screened by multiplex cytometer beads assay (CBA). Our CBA results show that indicaxanthin inhibits both IFN-gamma and IL-17 production in Th17 conditions. Interestingly, these effects were accompanied by a marked increase in IL-6 levels and a significant reduction in IL-10, together suggesting a modulation of T regulatory differentiation and/or function. FACS intracellular staining of gated T cells in Th17 condition indicated a reduced percentage of IL-17 and a parallel increase in IFN-gamma-producing cells in indicaxanthin-treated cells compared to control. Consistent with this, indicaxanthin almost doubled the number of IFN-gamma+ T cells in splenocytes differentiated in Th1 skewing conditions. Together these results indicate that indicaxanthin exerts an immunomodulatory effect on both antigen-presenting cells and T cells in vitro. In addition, our data suggest that indicaxanthin might either induce a robust Th1mediated protective immunity against extracellular pathogens or suppress a wide variety of Th17-driven autoimmune disorders such as multiple sclerosis or rheumatoid arthritis.

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