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Background Osteoporosis is the most common metabolic bone disease. Postmenopausal loss of estrogen is a major risk factor. Objectives To assess if total ethanolic extract of Sideritis scardica could prevent osteoporosis in ovariectomized female rats. Methods Female Wistar rats were divided in three groups: I-sham operated (control), II-ovariectomized-OV and III-ovariectomized, treated orally with S.scardica extract (300 mg/kg) during 20 weeks-OVT. In 18th experimental week oral glucose tolerance test has been performed, as well measurement of bone mineral density-BMD. After killing, femurs, plasma and erytrocytes were isolated. The following parameters were measured: morfometric bone parameters, wet and dry bone mass, biomechanical testig (three point flexural test), Ca and P in urine and plasma, alkaline phosphatase in plasma, malondialdehyde, catalase activity, reduced gluthation (GSH), total SH groups, free fatty acids and fructozamines. Results There was no significant difference in Ca and P concentration in plasma between groups, but urinary Ca and P were significantly higher in OV rats (Ca 0.67±0.24 mM, P 3.29±0.77 mM) in comparison to control (Ca 0.434±0.21 mM, P 2.32±1.00 mM) and OVT group (Ca 0.52±0.19 mM, P 2.89±0.42 mM) (p 2 , for OV group 2726.68±93.4 N/m 2 and for OVT group 3257.2±86.5 N/m 2 (p Conclusions S. scardica extraxct prevents bone loss in ovariectomized rats, increases bone resistance to mechanical load and reduces cardiovascular metabolic risc factors associated with menopause. Disclosure of Interest None declared

Background and Objectives Besides phagocytosis as mechanism to combat pathogens, neutrophils are able to eject DNA decorated with microbicidal proteins (neutrophil extracellular traps-NETs), that can immobilize and kill microorganisms extracellularly. Procoagulative properties of NETs have already been shown, however, a role of NETs formation in atherosclerotic disease has not been described so far. The process of chromatin release is referred to as NETosis. The aim of this study was to assess if cholesterol crystals are able to induce NETosis in vivo and in vitro . Materials and Methods Polymorphonuclear cells-PMNs were isolated from blood obtained from healthy volunteers after informed consent. Isolated PMNs as well as whole blood were co/cultured with cholesterol crystals. After the treatment, cytospins were prepared and stained for DNA and neutrophil elastase. Extracelullar DNA was quantified by fluorometry. For in vivo experiments, cholesterol crystals were injected into murine air pouches. After 24h, air pouches were lavaged with PBS. After centrifugation cells were used for cytospins and supernatants for cytokines determination. Tophus-like aggregates were isolated and characterised by immune histochemistry and immunobloting. In addition, human autopsy specimens obtained from subject died of cardiac diseases were assessed for presence of NET markers. Results Cholesterol induced dose dependent NETosis in isolated PMNs as well as in whole blood samples. The maximal applied concentration of cholesterol crystals induced 236.5% higher DNA release when compared to control. Cholesterol crystal injected in air pouches induced tophus-like aggregates composed of aggregated NETs attached to the cholesterol crystals. These structures were highly positive for citrullinated histones, markers of NET formation. NET markers were also found in atherosclerotic blood vessels in zones surrounding necrotic core of atheromatous plaque, which is rich in cholesterol crystals Discussion This study showed for the first time ability of cholesterol crystals to induce NETosis in vitro , as well as in vivo . These data may explain prompt thrombus formation in atherosclerotic disease after rupture of fibrous cap of an atheromatous plaque containing huge amount of cholesterol crystals, which will be the subject of our future research.

Background and Objectives In autoimmune conditions, like rheumatoid arthritis (RA), the own immune system attacks the body and causes inflammation. One of the consequences of inflammation during autoimmune diseases is a changed glycosylation profile of IgG antibodies. RA is not only characterised by an altered N-glycan structure bound to the Fc portion of IgG molecules but also by circulating rheumatoid factors (RF) that recognizes the Fc portion of IgG as antigen. The aim of our work was to investigate the glycosylation of immune complexes circulating in the blood of patients with seropositive and seronegative RA. Material and Methods Sera from 70 patients with RA (RF positive n = 43 and RF negative n = 27) and 8 healthy donors (control) have been used in the analysis. (I) Random serum IgG-complexes were captured with F(ab’) 2 fragments of anti-human-IgG and analysed by ELISA for their interaction with anti-human-IgG, anti-human-IgM, AAL lectin (fucose-specific) and LCA lectin (manose-specific). (II) The IgG complexes eluted from the ELISA-plates were analysed by Western Blots for the presence of IgG, IgM and AAL as well as LCA binding. (III) IgG affinity purified with AAL-, SNA- and jacalin-agarose columns was tested by ELISA as target for IgM rheumatoid factor. Results (I) The accessibility of fucose residues for the AAL lectin of circulating IgG complexes was much higher in patients with RA when compared to healthy donors. This reactivity was independent of the presence of IgM-RF. The accessibility of mannose residues for the LCA lectin of circulating IgG complexes was much higher in patients with RA when compared to healthy donors. This reactivity was only to be observed in the presence of IgM-RF. (II) Western Blot confirmed an increased presence of IgM in IgG complexes from seropositive patients with RA. The fucosylation of IgG did not differ much between all groups. (III) No difference was detected in IgM-RF affinity towards positive and negative fractions of AAL- and SNA-reactive IgG. However, IgM-RF displayed a reduced binding to the minor IgG fraction isolated with jacalin-affinity chromatography. Conclusion In not-denatured IgG circulating in patients with RA the glycan shows a lower accessibility for the fucose-specific AAL lectin, independent of the presence of IgM-RF. In contrast, the increased binding of the LCA lectin is due to the presence of IgM complexed to IgG. IgM-RF did not discriminate between IgG reactive with AAL and SNA lectin. AAL – Aleuria aurantia lectin (α-1,6 core fucose) LCA – Lens culimaris agglutinin (α-mannose) SNA - Sambucus nigra agglutinin (α-2,6-sialic acid)

Background Many recent studies have suggested that smoking is an important risk factor for rheumatoid arthritis (RA), particulary IgM rheumatoid factor (IgM-RF) and antibodies to citrullinated peptide (ACPA) positive RA, as well as a predictor of severe disease. However, there is lack of data on relationship between history of smoking and response to tumor necrosis factor antagonists in patients with RA. Objectives To determine whether cigarette smoking influences the response to etanercept treatment in patients with RA. Methods A history of cigarette smoking was obtained from a questionnaire completed by each patient starting therapy with etanercept since 2008 (n=136). A core set of demographic and clinical variables was recorded at baseline and at 3 and 12 months. The extent of smoking was quantified in pack-years (py), with 1 py equivalent to 20 cigarettes per day for 1 year. The influence of cigarette smoking (current or past) on the response to therapy was evaluated by logistic regression, with never smokers as the referent group. Response to therapy was defined according to the European League Against Rheumatism improvement criteria, based on their 3 or 12-month Disease Activity Score (DAS28) and absolute change in DAS28 from baseline. Results A history of smoking was found in 68/136 (50%) patients. Of these, 50/136 (36.8%) were current smokers at the start of etanercept therapy. There was no significant difference in age, age of onset, sex and disease duration between nonsmokers, past smokers and current smokers. However, there was an increase in the frequency of patients with IgM-RF and ACPA who had smoked, although this did not achieve statistical significance. Compared with never smokers, current smokers were less likely to achieve a good response at 3 months following the start of etanercept therapy (27% versus 41%; p Conclusions RA patients with a history of smoking were more likely to show a poor response to etanercept. Response failure was associated with the intensity of smoking. References G. Westhoff, et al. Rheumatoid arthritis patients who smoke have a higher need for DMARDs and feel worse, but they do not have more joint damage than non-smokers of the same serological group. Rheumatology 2008; 47:849-854 Disclosure of Interest None Declared