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Contains fulltext : 203366.pdf (publisher's version ) (Open Access)

Contains fulltext : 200258.pdf (publisher's version ) (Closed access), A large number of cardiovascular events are not prevented by current therapeutic regimens. In search for additional, innovative strategies, immune cells have been recognized as key players contributing to atherosclerotic plaque progression and destabilization. Particularly the role of innate immune cells is of major interest, following the recent paradigm shift that innate immunity, long considered to be incapable of learning, does exhibit immunological memory mediated via epigenetic reprogramming. Compelling evidence shows that atherosclerotic risk factors promote immune cell migration by pre-activation of circulating innate immune cells. Innate immune cell activation via metabolic and epigenetic reprogramming perpetuates a systemic low-grade inflammatory state in cardiovascular disease (CVD) that is also common in other chronic inflammatory disorders. This opens a new therapeutic area in which metabolic or epigenetic modulation of innate immune cells may result in decreased systemic chronic inflammation, alleviating CVD, and its co-morbidities.

Objectives. To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients. Methods. Plasma levels of the S100A4 protein were analysed in 40 anti-TNF-alpha naive patients with active RA. Of the 40 patients, 25 were treated with adalimumab and monitored over time. The conformational form of S100A4 was analysed using size-exclusion gel chromatography. TNF-alpha mRNA expression and protein synthesis were analysed by RT-PCR and ELISA, respectively. Results. Baseline levels of S100A4 were significantly correlated with disease activity in RA patients (r = 0.41; P < 0.01). After 12 weeks of treatment with adalimumab, there was an obvious shift in the conformations of S100A4 from the multimeric to the dimeric forms, whereas the total levels of the S100A4 protein remained unchanged. This suggests that the bioactive (multimer) S100A4 may decline in response to successful treatment with adalimumab. In addition, we showed significant up-regulation of TNF-alpha mRNA (P < 0.01), and protein release to the cell culture medium of monocytes stimulated with the S100A4 multimer compared with those treated with the dimer and to the unstimulated monocytes (P < 0.001). Conclusions. This is the first study to show that the levels of the S100A4 protein are correlated with RA disease activity. Furthermore, only the bioactive form, but not the total amount of S100A4, decreases after successful TNF-alpha blocking therapy in patients with RA. These data support an important role for the S100A4 multimer in the pathogenesis of RA

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes

In this chapter, we discuss the current understanding of the possible epigenetics changes that occur in rheumatoid arthritis. In particular, we describe that deregulation ofDNA methylation and histone modifications can occur in the immune system and lead to rheumatoid arthritis. In addition, we discuss the role of rheumatoid arthritis synovial fibroblasts in autoimmunity. Examples of changes in DNA methylation and histone modification occurring in synovial fibroblasts during the disease process are reviewed in this chapter. In conclusion, we discuss the possible use of epigenetic therapy and describe future experiments that can elucidate further the epigenetic changes observed in the disease.

AIMS: Inflammation plays a key role in acute coronary syndromes (ACS). Toll-like receptors (TLR) on leucocytes mediate inflammation and immune responses. We characterized leucocytes and TLR expression within coronary thrombi and compared cytokine levels from the site of coronary occlusion with aortic blood (AB) in ACS patients. METHODS AND RESULTS: In 18 ACS patients, thrombi were collected by aspiration during primary percutaneous coronary intervention. Thrombi and AB from these patients as well as AB from 10 age-matched controls without coronary artery disease were assessed by FACS analysis for cellular distribution and TLR expression. For further discrimination of ACS specificity, seven non-coronary intravascular thrombi and eight thrombi generated in vitro were analysed. In 17 additional patients, cytokine levels were determined in blood samples from the site of coronary occlusion under distal occlusion and compared with AB. In coronary thrombi from ACS, the percentage of monocytes related to the total leucocyte count was greater than in AB (47 vs. 20%, P = 0.0002). In thrombi, TLR-4 and TLR-2 were overexpressed on CD14-labelled monocytes, and TLR-2 was increased on CD66b-labelled granulocytes, in comparison with leucocytes in AB. In contrast, in vitro and non-coronary thrombi exhibited no overexpression of TLR-4. Local blood samples taken under distal occlusion revealed elevated concentrations of chemokines (IL-8, MCP-1, eotaxin, MIP-1alpha, and IP-10) and cytokines (IL-1ra, IL-6, IL-7, IL-12, IL-17, IFN-alpha, and granulocyte-macrophage colony-stimulating factor) regulating both innate and adaptive immunity (all P < 0.05). CONCLUSION: In ACS patients, monocytes accumulate within thrombi and specifically overexpress TLR-4. Together with the local expression patterns of chemokines and cytokines, the increase of TLR-4 reflects a concerted activation of this inflammatory pathway at the site of coronary occlusion in ACS.

OBJECTIVE: To search for novel autoantibodies in patients with rheumatoid arthritis (RA) in an effort to better understand the processes of joint destruction in this disease. METHODS: Using a modified SEREX technique and complementary DNA derived from RA synovium, serpin E2 was identified as a novel autoantigen and was analyzed by immunohistochemistry. Levels of anti-serpin E2 autoantibodies in serum and synovial fluid from patients with RA, osteoarthritis (OA), psoriatic arthritis, and ankylosing spondylitis, and/or from healthy individuals were assessed by enzyme-linked immunosorbent assay. Since serpin E2 is an inhibitor of serine proteases, we studied the inhibitory activity of serpin E2 toward its target, urokinase plasminogen activator (uPA), in vitro in the presence of isolated anti-serpin E2 autoantibodies and in vivo using the uPA activity assay. RESULTS: We identified autoantibodies against serpin E2 by the SEREX technique. Serpin E2 was overexpressed in RA synovial tissues as compared with OA synovial tissues. Significantly higher levels of anti-serpin E2 autoantibodies were present in samples of synovial fluid (28%) and serum (22%) from RA patients as compared with OA patients (0 and 6%, respectively) or with healthy individuals (6% of sera). Most importantly, anti-serpin E2 autoantibodies isolated from RA sera reversed the inhibitory activity of serpin E2 by 70%. Furthermore, the levels of anti-serpin E2 autoantibodies correlated with the uPA activity in vivo. CONCLUSION: This study characterizes a functional property of a novel autoantibody in RA. Since anti-serpin E2 autoantibodies interfere with the inhibitory activity of serpin E2 toward serine proteases, they might facilitate the joint destruction in RA.