C. Alessandri

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

AbstactWe previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid–CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.

Anti-tumour necrosis factor (TNF) α treatment of rheumatoid arthritis (infliximab) selectively down regulates the production of interleukin (IL) 18 but not of IL12 and IL13

Objective: To measure interleukin (IL)18 serum concentrations in patients with rheumatoid arthritis (RA) undergoing infliximab treatment (tumour necrosis factor (TNF) α blockade) and to evaluate the concomitant modification of IL12 and IL13 serum concentrations, two cytokines belonging to the Th1 and Th2 profile respectively and biologically related to IL18. Methods: Ten patients with RA not responding to disease modifying antirheumatic drugs (DMARDs) received intravenous infliximab at a dose of 3 mg/kg at baseline and after two and six weeks. Serum samples were collected from all patients before each infusion and assayed for IL18, IL12, and IL13 by enzyme linked immunosorbent assay (ELISA); IL18 was also measured eight weeks after the last infusion. Results: Serum concentrations of IL18 in all patients were already markedly reduced from baseline after two weeks (p<0.005). Serum IL18 was also decreased in a stable manner after six (p<0.01) and 14 weeks (p<0.01) compared with baseline concentrations. No significant modifications were found in serum concentrations of IL12 and IL13 at any time point. Conclusion: There was a rapid and persistent decrease in serum concentrations of IL18 in all the patients studied. This result provides evidence of an in vivo regulation of IL18 by TNFα and suggests that anti-TNFα therapy is likely to interrupt the synergistic effect between these two cytokines.

Beta-2-glycoprotein I expression on monocytes is increased in anti-phospholipid antibody syndrome and correlates with tissue factor expression

It is well known that monocytes may play an active role in thrombogenesis, since they may express on their surface tissue factor, the major initiator of the clotting cascade. The results of this investigation demonstrate beta‐2‐glycoprotein I (β2‐GPI) mRNA expression by human peripheral blood monocytes, indicating that these cells synthesize β2‐GPI. In addition, we show β2‐GPI expression on cell surface of these cells by flow cytometric analysis, and the presence of this protein in cell lysate by Western blot. Interestingly, β2‐GPI expression on monocytes is significantly increased in patients with anti‐phospholipid syndrome (APS) or systemic lupus erythematosus (SLE) as against healthy blood donors and correlates with tissue factor expression on monocytes. These findings support the view that monocytes are able to synthesize β2‐GPI and suggest that patients with APS may have increased β2‐GPI exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to a prothrombotic diathesis.

Autoantibody Production in Anti‐TNF‐α‐Treated Patients

Abstract:u2002 Targeting tumor necrosis factor alpha (TNF‐α) has offered an additional therapeutic strategy against several rheumatic inflammatory disorders. The current use of TNF‐α inhibitors allows physicians who manage these diseases and patients themselves to testify to an extraordinary efficacy, even though caution for possible adverse events must be maintained. Among these, the occurrence of autoimmune phenomena, encompassing new autoantibody formation and triggering of clinical manifestations, continues to be noted in published reports. Here, we review the current knowledge regarding the autoimmune phenomena linked to anti‐TNF‐α therapy in patients with rheumatic inflammatory disorders.

Safety profile and causes of withdrawal due to adverse events in systemic lupus erythematosus patients treated long-term with cyclosporine A.

Several case reports and uncontrolled trials have established the use of cyclosporine A (CsA) in systemic lupus erythematosus (SLE) but some concerns have been raised because of its kidney damaging effects. We here report the results of a retrospective follow-up study designed to assess the safety profile and causes of discontinuation due to adverse events in SLE patients treated with CsA. We treated 56 SLE patients with oral CsA at doses of 3 ± 5 mg/kg for an average of 26 months. Adverse events not leading to the discontinuation of therapy were observed in 62.5% of the patients, the most frequent being hypertrichosis. CsA was stopped because of the occurrence of side effects in 9=56 (16%) of the patients. The most common were nephrotoxicity (3=9) and the occurrence of tremors (3=9). These effects were always reversible within three months of CsA withdrawal. The patients who were older than 40 y had a significant slightly increased risk of stopping CsA therapy for any adverse events (RR 1.08; CI 95% 1.03 ± 1.14). In comparison with previous studies, this study involved a larger cohort of SLE patients who were evaluated for a longer period of follow-up, and confirmed the good tolerability of CsA in these subjects.

TLC immunostaining for detection of "antiphospholipid" antibodies.

Thin-layer chromatography (TLC) is a nonquantitative technique, which has been employed in the detection of antiphospholipid (aPL) antibodies. Antiphospholipid syndrome (APS) is the most frequently acquired thrombophilia, characterized by thrombosis and obstetric manifestations associated to an autoimmune trait, represented by the positivity of antiphospholipid (aPL) antibodies. Immunoassays for anticardiolipin (aCL) and anti-β2 glycoprotein I (aβ2GPI) antibodies and clotting tests for lupus anticoagulant (LA) represent the standard tests for the routine detection of aPL. The term seronegative APS has been used to describe patients with clinical manifestation of APS and persistently negative aPL assessed with routine assays. TLC immunostaining is a useful method for the detection of different antigenic targets of antiphospholipid antibodies; it is able to identify the reactivity of serum aPL experimented with purified phospholipid molecules with a different exposure compared to ELISA methods. This method seems to be applicable in patients who repeatedly tested negative for the standard aPL, i.e., aCL, aβ2GPI, and LA. Therefore, this technique may be proposed as a second step test for the diagnosis of APS.

Acute longitudinal myelitis following Cryptococcus laurentii pneumonia in a patient with systemic lupus erythematosus

Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is reported in about 50% of patients. Among the neuropsychiatric features of SLE, myelopathy, including acute transverse myelitis (ATM) or acute longitudinal myelitis (ALM), represents an uncommon event. A possible vascular aetiology of SLE myelopathies has been hypothesized and it seems to be much more associated to SLE-associated antiphospholipid syndrome (APS). Furthermore, a possible infectious cause of ATM or ALM in healthy subjects has been described. SLE patients are susceptible to infection due to the disease itself or to the immunosuppressive therapy. Cryptococci non-neoformans have been rarely associated to infections in humans. Here we describe the case of a 47-year-old woman with SLE and Sjögren Syndrome who developed an ALM concurrently with a Cryptococcus laurentii pneumonia. The patient was treated with antimycotics, high doses of glucocorticoids and intravenous immunoglobulins with a significant clinical and radiological improvement. As far as we know, this is the first case of Cryptococcus laurentii infection and ALM in a patient with SLE who later developed a seronegative APS. Even though myelopathy may be considered primarily associated to SLE, a possible role of the infection in ALM development cannot be excluded.

Anti-carbamylated protein antibodies in systemic lupus erythematosus patients with articular involvement:

Objective Several studies have evaluated the prevalence of rheumatoid factor (RF) and anti-citrullinated proteins antibodies (ACPA) in systemic lupus erythematosus (SLE) patients but no data are available on the anti-carbamylated proteins (anti-CarP), a new biomarker for rheumatoid arthritis (RA). We evaluated the anti-CarP prevalence in SLE patients with joint involvement and the associations with different phenotypes. Methods Seventy-eight SLE patients with joint involvement were enrolled (F/M 73/5; meanu2009±u2009SD age 47.6u2009±u200911.2 years; meanu2009±u2009SD disease duration 214.3u2009±u2009115.6 months). As control groups, we evaluated SLE patients without joint manifestations (Nu2009=u200915), RA (Nu2009=u200978) and healthy individuals (HS, Nu2009=u200998). Anti-CarP were assessed by home-made ELISA in all patients and controls, RF and ACPA in SLE patients with joint involvement (commercial ELISA kit). Results The prevalence of anti-CarP in SLE patients with joint involvement was similar to RA (pu2009=u2009NS) and significantly higher compared with SLE without joint involvement and HS (pu2009<u20090.0001, pu2009<u20090.0001, respectively). Four patients were positive for all three antibodies: seventy-five percent of these showed Jaccoud arthropathy. Fourty-five percent of ACPA-ve/RF-ve patients were anti-CarPu2009+u2009ve. Conclusions The evaluation of anti-CarP in SLE joint involvement demonstrated a prevalence of almost 50%, similar to RA and significantly higher than SLE without joint involvement and HS.

AB0004 Polymorphisms in Genes in The IL-17 Pathway and B Cell Mediated Immune Response Modulate The Development of Specific Autoimmune Manifestations in Systemic Lupus Erythematosus

Background Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease in which a complex interaction between genetic, environmental and immunological factors determines the susceptibility and the phenotype. Dysregulations in IL-17 pathway as well as in B-cell mediated immune response have been observed in SLE. We have recently demonstrated that polymorphisms in TRAF3IP2 gene are associated with susceptibility for SLE and can predispose for the development of pericarditis (1). TRAF3IP2 encodes for Act1, a molecule that acts as a negative regulator of B cell by inhibiting CD40-mediated signaling and as a positive signaling adapter in IL-17-mediated cellular responses. TRAF6 is essential for the IL-17/Act1-mediated activation of NF-kB, while CD40 is the target for the Act1 mediated inhibition of B cell response. Objectives The primary goal of our study was to evaluate the association of polymorphisms in CD40, TRAF6 and TNFSF4 (OX40) genes with susceptibility to SLE. Secondary objectives were to assess the possible association of these polymorphisms with the clinical and laboratory features. Methods We recruited 315 Italian SLE patients and 278 healthy controls. Genotyping of rs4810485 in CD40, rs4755453 and rs5030437 in TRAF6, and rs2205960 and rs10489265 in TNFSF4 (OX40) SNPs was performed by allelic discrimination assay. A case/control association study and a genotype/phenotype correlation analysis were performed. Results Deviations from Hardy–Weinberg equilibrium for the three SNPs were not observed. None of the studies polymorphisms was associated with susceptibility for SLE. Only CD40 rs4810485 was associated at genotypic (P=0.034) but not at the allelic level. Nonetheless, these polymorphisms seem to contribute to define disease phenotype. TRAF6 was associated with the presence of anemia (P=0.019, OR=1.96), rs2205960 of TNFSF4 was associated with the pericarditis (P=0.013, OR=2.14), and rs4810485 of CD40 with the presence of anti-SSB/La (P=0.014, OR=2.26) and lupus nephritis (P=0.024, OR=1.8). Conclusions We were not able to confirm previous association of TRAF6 and CD40 polymorphisms with susceptibility for SLE. However, such SNPs seem to influence the disease phenotype. In particular, it is of interest the association of rs4810485 in CD40 with lupus nephritis and the presence of anti-SSB/La. Indeed, the modulation of CD40-mediated T cell-dependent antibody response has already been associated with the development of anti-SSB/La in association with SLE-like nephritis in a mouse model (2), probably due to an uncontrolled B cell activation signal. References Perricone C, Ciccacci C, Ceccarelli F, et al. TRAF3IP2 gene and systemic lupus erythematosus: association with disease susceptibility and pericarditis development. Immunogenetics. 2013 Oct;65(10):703–9 Qian Y, Giltiay N, Xiao J, et al. Deficiency of Act1, a critical modulator of B cell function, leads to development of Sjögrens syndrome. Eur J Immunol. 2008 Aug;38(8):2219–28. Disclosure of Interest None declared

SAT0378 Autophagy is Up-Regulated in the Salivary Glands of Primary Sjogren's Syndrome Patients and Correlates with the Focus Score and Disease Activity

Background Autophagy is now considered as a major regulator in trafficking events that activates innate and adaptive immunity and consistent evidence supports its role in autoimmunity (1). Primary Sjogrens syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of exocrine glands by T and B cells that, producing chemokines and cytokines, coordinate the chronic inflammatory process. No data on the role of autophagy in pSS are available in humans, although studies in mice demonstrated its involvement in the salivary and lacrimal gland homeostasis (2,3). Objectives We investigated the autophagy process in salivary gland tissue and in peripheral T lymphocytes from pSS patients to evaluate its possible implication in the pathogenesis of the disease. Methods 30 patients with pSS, 20 patients with sicca syndrome or non-specific-chronic-sialoadenitis and 30 healthy donors were studied. Peripheral T lymphocytes were isolated by standard procedures. Salivary gland biopsies were evaluated by i) H&E to assess histological pattern, the severity of inflammatory infiltrate and the presence of germinal centers, ii) RT-PCR for the expression of autophagy-related genes and IL-23p19 and IL-21 mRNA. Autophagy-related proteins (LC3, Atg5, p62/SQSTM1) were detected in peripheral T lymphocytes by western blot and in salivary gland by immunohistochemistry and immunofluorescence. IL-21 and IL-23p19 serum levels were measured by ELISA. Results Autophagy is up-regulated in T cells from the salivary glands, but not from the peripheral blood, of pSS patients and it is correlated with disease activity and damage indexes. Autophagy is also correlated with the local expression of the pro-inflammatory cytokines IL-21 and IL-23p19, but not with serum levels of these cytokines. Conclusions Our data show that, in pSS, T cells present high levels of autophagy, which may up-regulate the expression of pro-inflammatory cytokines, providing evidence for a role of this process in the pathogenesis of pSS and identifying a possible therapeutic target. References Pierdominici M, Vomero M, Barbati C et al. FASEB J. 2012; 26: 1400-1412. Morgan-Bathke M, Lin HH, Chibly AM et al. J Dent Res. 2013; 92: 911-917. Seo Y, Ji YW, Lee SM, et al. Cell Death Dis. 2014; 5: e1309. Disclosure of Interest None declared