2/20/2023 0 Comments Superoxide d splittCultures were incubated at 37☌ for 48 h before the supernatants were harvested and assayed for IFN-γ. For antigen-specific recall assays, BDC-2.5 T-cells were cultured in 96-well flat-bottom plates at a density of 2 × 10 4 cells/well, with 5,000 islet cells as antigen and 2.5 × 10 4 APCs, in the presence or absence of 17 and 34 μmol/l SOD mimic. Cultures were incubated at 37☌ for 24 h before the supernatants were harvested and assayed by sandwich ELISA for IFN-γ production. For ConA stimulation, BDC-2.5 T-cells were plated at 2 × 10 4 cells/well in 96-well flat-bottom plates with or without 5 × 10 5 irradiated syngeneic spleen cells as APC and ConA (2.5 μg/ml final concentration), in the presence or absence of the SOD mimic at concentrations of 17 and 34 μmol/l. The negative control was BDC-2.5 alone without α-CD3 and α-CD28. ![]() After washing the plates with sterile HBSS and blocking with CM at 37☌ for 1 h, the blocking solution was removed, and the BDC-2.5 T-cell clone (2 × 10 4 cells) was added to the wells in the presence or absence of the SOD mimic at concentrations of 17 and 34 μmol/l. For α-CD3/α-CD28 stimulation, 96-well round-bottom plates were precoated with 0.125 μg/ml α-CD3 and 1 μg/ml α-CD28 for 1 h at 37☌. IFN-γ production by BDC-2.5 was assessed by sandwich enzyme-linked immunosorbent assay (ELISA) analysis of responder T-cells stimulated with α-CD3 and α-CD28, concanavalin A (ConA), or islet cell antigen. The ability of the SOD mimics to scavenge a broad range of ROS allows for their use in inflammatory diseases. SOD mimics have recently been found to rescue vascular contractility in endotoxic shock ( 37), protect neuronal cells from excitotoxic cell death ( 38) and apoptosis ( 39), inhibit lipid peroxidation ( 36, 40), block hydrogen peroxide-induced mitochondrial DNA damage ( 41), and partially rescue a lethal phenotype in a Mn-SOD knockout mouse ( 42). ( 35), and lipid peroxyl radicals ( 36).The Mn porphyrins have a broad antioxidant specificity, which includes scavenging O 2 − in a manner similar to the active metal sites of the mammalian Cu-, Zn- or Mn-containing SODs ( 26– 31).The SOD mimics are designed with a redox-active metal center that catalyzes the dismutation of O 2 We used a pharmacological approach to protect β-cells from the T-cell-mediated ROS and cytokine destruction associated with autoimmune diabetes by using a synthetic metalloporphyrin-based SOD mimic, AEOL-10113. Finally, this compound protected NIT-1 insulinoma cells from interleukin-1β and alloxan cytotoxicity in vitro. In addition, pretreatment of lipopolysaccharide (LPS)-stimulated peritoneal macrophages with SOD mimic inhibited the LPS-dependent increase in TNF-α as well as the NADPH oxidase-dependent release of superoxide. We found that the SOD mimic significantly inhibited antigen-presenting cell-dependent T-cell proliferation and IFN-γ production in vitro. To investigate the mechanisms of protection, in vitro assays for T-cell proliferation and γ-interferon (IFN-γ) production were carried out using the T-cell clone BDC-2.5. Disease was significantly delayed or prevented altogether by treatment of recipient mice with an SOD mimic, AEOL-10113, before transfer of the BDC-2.5 clone. To investigate the effect of SOD mimics and the role of oxidative stress in the development of autoimmune diabetes in vivo, we used a diabetogenic T-cell clone, BDC-2.5, to induce rapid onset of diabetes in young nonobese diabetic-severe combined immunodeficient mice (NOD. Metalloporphyrin-based superoxide dismutase (SOD) mimics scavenge ROS and protect cells from oxidative stress and apoptosis. ![]() Because islet β-cells have a reduced capacity to scavenge free radicals, they are very sensitive to ROS action. Type 1 diabetes is an autoimmune process whereby T-cells recognize pancreatic β-cell antigens and initiate a leukocyte infiltrate that produces proinflammatory cytokines and reactive oxygen species (ROS), ultimately leading to β-cell destruction. We present here the first report of a metalloporphyrin-based antioxidant that can prevent or delay the onset of autoimmune diabetes.
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