measured by ALT and AST levels. This work was supported by NIH grant AA013868 and P20 AA17069 to LEN and P20 AA17069 (#6017) to AEF. doi: 405 Oxidative and Nitrative Modification of C1q Affects its Structure, Function and Antigenicity in Systemic Lupus Erythematosus Brent Ryan1,2, Paul Eggleton2, Simon Brown3, Nick Viner2, Ahuva Nissim4, David Isenberg5, Richard Haigh2, and Paul G Winyard2 1Universtity of Oxford, UK, 2Peninsula Medical School, Exeter, UK, 3Queen’s Medical Research Institute, University of Edinburgh, UK, 4Barts and The London Medical School, London, UK, 5Centre for Rheumatology, University College London, UK Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, characterised by inflammation, oxidative stress, impaired apoptotic cell clearance and the presence of autoantibodies, which form immune complexes. Deposition of immune complexes in the kidneys results in inflammation and glomerulonephritis. The clearance of both immune complexes and apoptotic cells is mediated by the complement protein, C1q, and is deficient in SLE patients with glomerulonephritis. We tested: (a) the effects of oxidative modifications on the structure of C1q and (b) whether these modifications were responsible for its increased antigenicity and impaired ability to clear immune complexes and apoptotic cells in SLE patients. Oxidative modification of C1q resulted in gross changes to C1q as observed by SDS-PAGE and size exclusion chromatography. Modified amino acids such as methionine sulfoxide and 3-nitrotyrosine were identified by LC-MS/MS and their spatial positions determined using the published crystal structure of the C1q globular heads. A greater proportion of SLE patients (n=77) had antibodies against H2O2-modified C1q than against native C1q (P<0.05). Peptides corresponding to candidate neo-epitopes from C1q were synthesised in both non-oxidized and oxidized forms. Antibody titres to several oxidised C1q peptides demonstrated negative correlations with renal disease and serum C3 levels in SLE patients with active nephritis. Modification of C1q with physiological and pathophysiological (0.1-100 µM) concentrations of H2O2 or ONOO– increased the ability of C1q to bind to immunoglobulin M and activate complement by up to 400%. Although H2O2 or ONOO–

modification did not alter C1q binding to apoptotic neutrophils, ONOO– modification of C1q completely abolished its ability to clear apoptotic cells. These data suggest that oxidative modification of C1q profoundly affects both the antigenicity and function of C1q in both SLE patients and in normal physiology.  doi: 406 Intracellular ROS Modulate the Tumor Cell Invasion Promoting of Tumor­associated Macrophages Through PPARγ Nuclear Translocation Jing Liu1, Xuzhu Lin1, and Pingping Shen1 1State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University Tumor-associated macrophages (TAMs) have an influence on multiple steps of tumor development including tumor growth, invasion and metastasis. To investigate the activation of TAMs, RAW264.7 and ANA-1 were exposed to the culture supernatant of melanoma B16F1and B16F10 cells. Activated primary TAMs were obtained from mice injected subcutaneously with B16F1 or B16F10 by cell sorting on a FASCAria. We analyzed the phenotypic markers of induced macrophages including CD206,

CCR7, arginase-1. The results showed that the macrophages were activated and gained the ability of promoting tumor cell invasion characterized by up-regulating TNF-α and IL-10 release, as compared with those of their controls. Using immunofluorescence labeling, we analyzed the kinetic profile of intracellular ROS in TAMs. The results showed that the ROS levels in RAW264.7, ANA-1 and primary TAMs were obviously increased after activated by the tumor cell culture supernatant. Through tumor invasion assay, we found that the high levels of intracellular ROS were coinciding with tumor cell invasion promoting of TAMs. Further study indicated that the ROS in TAMs mediated critical steps of nuclear translocation of PPARγ in a MEK1-dependent manner and phosphorylation of PPARγ at S112 by ERK that result in up-regulating of TNF-α and IL-10 release, contributing to tumor cell invasiveness promoting of TAMs. doi: 407 Hypochlorous Acid Mediates Heme Degradation, Free Iron Release, and Protein Aggregation in Lactoperoxidase Carlos E. Souza1, Dhiman Maitra1, Ibrahim Abdulhamid2, Samya Abukhatwa1, Michael P. Diamond1, and Husam M. Abu-Soud1 1Wayne State University, 2Children's Hospital of Michigan Lactoperoxidase (LPO) is a heme-containing protein, present mainly in body fluids and mucosal surfaces such as the airways, milk, and tears. LPO is the major consumer of hydrogen peroxide (H2O2) in the airways through its ability to oxidize thiocyanate to produce hypothiocyanous acid, an antimicrobial agent. Studies of nasal inflammatory diseases, such as cystic fibrosis, demonstrated that LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by leukocytes at sites of inflammation, were both mobile and localized to site of inflammation. The present studies were performed to assess the cross reaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Rapid kinetics measurements revealed that low concentrations of HOCl (<50 µM) mediated the subsequent formation of LPO Compound I and Compound II, which then decayed slowly to the LPO ferric form. Higher concentrations of HOCl (>50 µM) induced subsequent formation of Compounds I and II, followed by heme destruction and protein aggregation. The colorimetric ferrozine-based assay for quantitation of free iron showed a corresponding increase in free iron production as a function of HOCl concentration. Moreover, a reducing and denaturing SDS-PAGE showed several degrees of protein aggregation products. A number of porphyrin degradation products resulting from oxidation cleavage of one or more of the carbon-methane bridges of the LPO porphyrin ring were detected by HPLC and identified by LC-MS. Similarly, exposure of LPO Compound III to increasing HOCl concentrations resulted in heme degradation and iron release. These findings may suggest a role for dose dependent HOCl-mediated LPO heme fragmentation and iron release, implicating it in pathogenic conditions associated with MPO activity. doi: 408 The Cigarette Smoke Component Acrolein Suppresses Innate Macrophage Responses by Direct Alkylation of NF­kB and JNK Page C. Spiess1, Milena Hristova1, David I. Kasahara1, Matthew J. Randall1, and Albert van der Vliet1 1University of Vermont The innate immune system of the respiratory tract is critical in defense against inhaled pathogens and is often compromised in

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