ALBERT

Description: Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up-regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide-induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for four days (D5-9) with 300 µM hydrogen peroxide resulted in fewer alkaline phosphatase-positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression ( p  〈 0.05). This suppression was partially blocked when cells were pretreated 12h with 20 µM QRC. Hydrogen peroxide also produced sustained up-regulation of heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC), which was partially blocked in hydrogen peroxide-treated cells that first received QRC pretreatment. The alterations in the antioxidant stress response coincided with alterations in phosphorylated ERK1/2, but not Nrf2. These results suggest that QRC suppresses hydrogen peroxide-induced activation of the antioxidant response, and partially preserves osteoblast phenotypic development. This article is protected by copyright. All rights reserved

Description: Oxidative stress contributes to osteoporosis by suppressing differentiation of osteoblasts, suggesting the osteoblast antioxidant response may be a viable strategy for osteoporosis prevention. Quercetin, an antioxidant flavonol, up-regulates the antioxidant response in many cell types, but studies are needed to understand the effects of quercetin plasma metabolites on the osteoblast antioxidant response. The first specific aim was to examine antioxidant response genes and proteins in osteoblasts exposed to plasma quercetin metabolites. The second specific aim was to identify potential signaling pathways in the osteoblast antioxidant response that mediate the effect of quercetin, specifically Nrf2, ERK1/2, and NFκB p65. Osteoblasts isolated from fetal rat calvaria were treated with doses up to 20 µM of three different quercetin metabolites found in blood plasma after consumption of quercetin-rich foods or supplements: quercetin aglycone (QRC), isorhamnetin (ISO), or quercetin 3-O-glucuronide (Q3G). Alternatively, some cells received a 2:1:1 mixture of all 3 metabolites (10 µM Q3G: 5 µM ISO: 5 µM QRC) to evaluate synergistic effects. Antioxidant response genes and proteins known to be up-regulated by quercetin were analyzed along with Nrf2, ERK1/2, and NFκB proteins. Both QRC and ISO, but not Q3G, up-regulated heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC) at the mRNA and protein level. Synergistic effects of metabolites were not observed. Up-regulation of HO-1 and GCLC was associated with suppression of phosphorylated ERK1/2 and NFκB, but no alterations in Nrf2 protein levels were observed. This study shows that the antioxidant response of osteoblasts is differentially stimulated by quercetin metabolites. This article is protected by copyright. All rights reserved

Description: Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.