Search

Pregnancy problems are common in patients with rheumatic disease; indeed, autoimmune disorders and autoantibodies can affect pregnancy progress and lead to maternal complications. Recent studies have highlighted a close association between HMGB1, chronic inflammation, and autoimmune diseases. Thus, in this investigation, we analyzed serum levels of HMGB1, an alarmin which plays a pivotal role in inducing and enhancing immune cell function. Sera from 30 patients with antiphospholipid syndrome (11 primary and 19 secondary APS), 35 subjects with pregnancy morbidity, and 30 healthy women were analysed for HMGB1 and its putative receptor RAGE (sRAGE) by Western blot and for TNF-α by ELISA. Results revealed that APS patients showed significantly increased serum levels of HMGB1, sRAGE, and the proinflammatory cytokine TNF-α, as compared to healthy women. However, also, the pregnancy morbidity subjects showed significantly increased levels of HMGB1 and sRAGE as well as TNF-α compared to healthy women. Our findings suggest that in subjects with pregnancy morbidity, including obstetric APS, elevated levels of HMGB1/sRAGE may represent an alarm signal, indicating an increase of proinflammatory triggers. Further studies are needed to evaluate the role of HMGB1/sRAGE as a possible tool to evaluate the risk stratification of adverse pregnancy outcomes.

Objectives Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). Methods For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. Results Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naive early-active RA patients. Conclusion These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.

Antiphospholipid antibodies (aPLs) are a heterogeneous group of antibodies directed against phospholipids or protein/phospholipid complexes. Currently, aPLs are assessed using either “solid-phase” assays that identify anticardiolipin antibodies and anti-β2-glycoprotein I antibodies or “liquid-phase” assay that identifies lupus anticoagulant. However, in the last few years, “new” antigenic targets and methodological approaches have been employed for refining laboratory diagnosis of antiphospholipid syndrome (APS). In this review the potential diagnostic value of antibodies to domains ofβ2-GPI, prothrombin/phosphatidylserine, vimentin/cardiolipin, protein S, protein C, annexin A2, annexin A5, and phospholipid antigens is discussed. Moreover, new technical approaches, including chemiluminescence, multiline dot assay, and thin layer chromatography (TLC) immunostaining, which utilize different supports for detection of aPL, have been developed. A special focus has been dedicated on “seronegative” APS, that is, those patients with a clinical profile suggestive of APS (thromboses, recurrent miscarriages, or foetal loss), who are persistently negative for the routinely used aPL. Recent findings suggest that, in sera from patients with SN-APS, antibodies may be detected using “new” antigenic targets (mainly vimentin/cardiolipin) or methodological approaches different from traditional techniques (TLC immunostaining). Thus, APS represents a mosaic, in which antibodies against different antigenic targets may be detected thanks to the continuously evolving new technologies.

In the clinical practice it is possible to find patients with clinical signs suggestive of antiphospholipid syndrome (APS), who are persistently negative for the laboratory criteria of APS, that is, anti-cardiolipin antibodies (aCL), anti-β2-GPI antibodies and lupus anticoagulant. Therefore, it was proposed for these cases the term of seronegative APS (SN-APS). In order to detect autoantibodies with different methodological approaches, sera from 24 patients with SN-APS were analysed for anti-phospholipid antibodies using TLC immunostaining, for anti-vimentin/cardiolipin antibodies by enzyme-linked immunosorbent assay (ELISA), and for anti-annexin V and anti-prothrombin antibodies by ELISA and dot blot. Control groups of our study were 25 patients with APS, 18 with systemic lupus erythematosus (SLE), and 32 healthy controls. Results revealed that 13/24 (54.2%) SN-APS sera were positive for aCL (9 of whom were also positive for lysobisphosphatidic acid) by TLC immunostaining, 11/24 (45.8%) for anti-vimentin/cardiolipin antibodies, 3/24 (12.5%) for anti-prothrombin antibodies, and 1/24 (4.2%) for anti-annexin V antibodies. These findings suggest that in sera from patients with SN-APS, antibodies may be detected using “new” antigenic targets (mainly vimentin/cardiolipin) or methodological approaches different from traditional techniques (mainly TLC immunostaining). Thus, SN-APS represents a mosaic, in which antibodies against different antigenic targets may be detected.

The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2-glycoprotein I IgG or IgM antibodies (anti-β2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI by ELISA, were also positive for IgG aCl by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (p

Background Autophagy is a fundamental self-degradative process in eukaryotic cells via the lysosomal pathway, involved both in basal turnover of cellular components and in the response to nutrient starvation (1). The immune system utilizes autophagy to restrict intracellular pathogens and regulate adaptive immunity, thus autophagy, as well as apoptosis, could be relevant in induction or loss of self tolerance to intracellular molecules and might act as a source of autoantigens in autoimmune diseases (2). Objectives Aim of our study was to study the monocyte autophagy in patients with active early RA naive to steroids and DMARDs before and after 3 months of therapy. Methods 17 early RA patients (mean age 49 yrs, mean disease duration 21 weeks), fulfilling both 1987 and 2010 ACR criteria, naive to treatment, were recruited after written informed consent. Before (T0) and after 3 months of treatment (T1), DAS28 was calculated, CRP was assessed. Autophagy markers (LC3, Ambra, beclin) were evaluated by flow cytometry on isolated monocytes. As a control group, 10 healthy donors were also evaluated. Results As expected, CRP, GH and DAS 28 score were significantly reduced following treatment with steroids and DMARDs (Table 1). Autophagy markers (LC3, Ambra, beclin) showed a similar behaviour, reducing their expression in monocytes after treatment (Tab.1). Interestingly, the expression of autophagy markers was significantly related to DAS28 (p=0.04 for LC3, p=0.03 for Ambra), to GH (p=0.01 for Ambra), and to CRP (p=0.01 for Ambra, p=0.006 for beclin). Conclusions These data indicate an increased expression of autophagy markers in isolated monocytes from patients with active early RA, suggesting a possible role for autophagy in the pathogenesis of this disease. The relationship between autophagy and acute inflammation is supported by the significant reduction of autophagy markers after therapy, together with their relationship with DAS28, GH, and CRP. Our results may contribute to clarify whether endogenous or pharmacological regulation of autophagy might modify the autoimmune response and the progression of the disease. References Lleo A et Al,J.Autoimmun 2007;29:61-68. Deretic V,Curr Opin Immunol 2009;21:53-62. Disclosure of Interest None Declared

Introduction High mobility group box 1 (HMGB1) is a key mediator of inflammation that is actively secreted by macrophages and/or passively released from damaged cells. The proinflammatory role of HMGB1 has been demonstrated in both animal models and humans, since the severity of inflammatory response is strictly related to serum HMGB1 levels in patients suffering from traumatic insult, including operative trauma. This study was undertaken to investigate HMGB1 production kinetics in patients undergoing major elective surgery and to address how circulating mononuclear cells are implicated in this setting. Moreover, we explored the possible relationship between HMGB1 and the proinflammatory cytokine interleukin-6 (IL-6). Methods Forty-seven subjects, American Society of Anesthesiologists physical status I and II, scheduled for major abdominal procedures, were enrolled. After intravenous medication with midazolam (0.025 mg/Kg), all patients received a standard general anesthesia protocol, by thiopentone sodium (5 mg/Kg) and fentanyl (1.4 μg/Kg), plus injected Vecuronium (0.08 mg/Kg). Venous peripheral blood was drawn from patients at three different times, t0: before surgery, t1: immediately after surgical procedure; t2: at 24 hours following intervention. Monocytes were purified by incubation with anti-CD14-coated microbeads, followed by sorting with a magnetic device. Cellular localization of HMGB1 was investigated by flow cytometry assay; HMGB1 release in the serum by Western blot. Serum samples were tested for IL-6 levels by ELISA. A one-way repeated-measures analysis ANOVA was performed to assess differences in HMGB1 concentration over time, in monocytes and serum. Results We show that: a) cellular expression of HMGB1 in monocytes at t1 was significantly higher as compared to t0; b) at t2, a significant increase of HMGB1 levels was found in the sera of patients. Such an increase was concomitant to a significant down-regulation of cellular HMGB1, suggesting that the release of HMGB1 might partially derive from mononuclear cells; c) treatment of monocytes with HMGB1 induced in vitro the release of IL-6; d) at t2, high amounts of circulating IL-6 were detected as compared to t0. Conclusions This study demonstrates for the first time that surgical/anesthesia trauma is able to induce an early intracellular upregulation of HMGB1 in monocytes of surgical patients, suggesting that HMGB1 derives, at least partially, from monocytes.