THU0366 SYSTEMATIC CORONARY RISK EVALUATION (SCORE) MISCLASSIFIES CARDIOVASCULAR RISK IN ANTISYNTHETASE SYNDROME: RESULTS OF THE PILOT MULTICENTRIC STUDY RI.CAR.D.A.

Background:Antisynthetase Syndrom (ASyS) is an autoimmune overlap disease characterized by antiaminoacyl-tRNA-synthetase (anti-ARS) antibodies and the classic triad of arthritis, myositis and interstitial lung disease (ILD) (1). Markers of cardiovascular (CV) or cerebrovascular (CVB) risk have never been examined in ASyS.Objectives:Aim of this study (RIsk of CARdiovascular Disease in ASyS: RI.CAR.D.A.) was to test the ability of an established traditional CV risk prediction score (Systematic Coronary Risk Evaluation-SCORE) and its EULAR modified version (mSCORE) to identify ASyS patients at high CV risk. Moreover, we sought to examine for the first time associations of CV surrogate markers with clinical and immunological ASyS parameters.Methods:SCORE/mSCORE and the gold standard marker of aortic stiffness (carotid-femoral pulse wave velocity-cfPWV) were examined in patients with ASyS and healthy controls in a multicenter setting (6 Rheumatology Centers). Moreover, sonography of the common- (CCA), internal- (ICA) and external- (ECA) carotid arteries was performed in subsets of both groups, evaluating carotid intima-media-thickness (cIMT), plaques and duplex-sonographic indices of CBV risk such as the resistance- (RI) and pulsatility-index (PI).Figure 1.Carotid Doppler surrogate markers of cardiovascular and cerebrovascular risk in controls and ASyS (case).cIMTCarotid intima media thickness;CAA(common-),ICA(internal),ECA(external) carotid artery;RIresistance index;PIpulsatility index. (all;p0.9 mm) (SCA) in85.7%of the patients respectively. ROC analyses showed similarly poor diagnostic performances of SCORE/mSCORE in comparison to cfPWV(>10 m/s) and SAC by areas under the curve (AUC) of0.56 (95%CI=0.39-0.73) and0.63 (95%CI=0.3-0.96),respectively. cfPWV and SCA were higher in ASyS patients compared to controls (padj=0.021andp=0.003, respectively). cfPWV and cIMT correlated in the patient group significantly with age (r=0.679; p<0.001 and r=0.664; p<0.001,respectively).Moreover, cfPWV correlated with BMI (padj=0.001) and diabetes(padj=0.043). ACC-RI and ACC-PI showed significant associations with a marker of myositis activity [creatine phosphokinase (CPK):r=0.629;p=0.012andr=0.574;p=0.032, respectively]. Finally, ACI-RI and ACI-PI values were higher in patients with ILD (both;p=0.039).Conclusion:This is the first report of higher aortic stiffness and SCA in ASyS patients compared to controls. Active myositis and presence of ILD were associated with higher CVB risk parameters. Furthermore, SCORE/mSCORE performed poorly in identifying patients at high CV risk and carotid arteriosclerosis compared to cfPWV and CS respectively. Thus, cfPWV and CS could improve CV and CBV screening in ASyS patients.References:[1]Cavagna L, et al. Clinical Spectrum Time Course in Anti Jo-1 Positive Antisynthetase Syndrome.Medicine2015;94:1144.Disclosure of Interests:None declare

Antibodies to the endoplasmic reticulum-resident chaperones calnexin, BiP and Grp94 in patients with rheumatoid arthritis and systemic lupus erythematosus

Objectives. To investigate the presence of autoantibodies against mammalian chaperones of the endoplasmic reticulum (ER) in patients with RA and other immune-mediated diseases. Methods. Sera from healthy donors, from early RA patients with two follow-up samples, patients with SLE, SSc and IBD were collected and analysed for anti-ER chaperone antibodies. Detection of serum IgG antibodies against immunoglobulin heavy chain binding protein (BiP), glucose-regulated protein 94 (Grp94) and calnexin was carried out using ELISA. The specificity of sera positive for individual ER chaperones was confirmed by immunoblotting. Statistical analysis was performed using Welch's t-test, Mann-Whitney U-test, partial correlation and Pearson's correlation. Results. In patients with RA and SLE, autoantibody titres against BiP, Grp94 and calnexin were significantly higher than those in healthy controls. These autoantibodies were detectable in patients with early RA and titres remained stable for at least 6-12 months. Also several SSc and IBD patients exhibited autoantibodies against these ER chaperones; however, titres and frequencies were lower than in RA or SLE patients. Furthermore, anti-calnexin antibodies correlated significantly with the presence of BiP and Grp94 autoantibodies in patients with RA and SLE. Conclusion. Calnexin and Grp94 were identified as novel autoantigens in RA and calnexin in SLE. Since calnexin, Grp94 and BiP are ER-resident proteins of eukaryotic cells, our data suggest that autoantibody generation against ER chaperones is independent of initial exposure to the corresponding bacterial chaperones; rather, ER chaperones may represent genuine autoantigen

CVID-Associated B Cell Activating Factor Receptor Variants Change Receptor Oligomerization, Ligand Binding, and Signaling Responses.

Binding of the B cell activating factor (BAFF) to its receptor (BAFFR) activates in mature B cells many essential pro-survival functions. Null mutations in the BAFFR gene result in complete BAFFR deficiency and cause a block in B cell development at the transition from immature to mature B cells leading therefore to B lymphopenia and hypogammaglobulinemia. In addition to complete BAFFR deficiency, single nucleotide variants encoding BAFFR missense mutations were found in patients suffering from common variable immunodeficiency (CVID), autoimmunity, or B cell lymphomas. As it remained unclear to which extent such variants disturb the activity of BAFFR, we performed genetic association studies and developed a cellular system that allows the unbiased analysis of BAFFR variants regarding oligomerization, signaling, and ectodomain shedding. In addition to genetic association studies, the BAFFR variants P21R, A52T, G64V, DUP92-95, P146S, and H159Y were expressed by lentiviral gene transfer in DG-75 Burkitt's lymphoma cells and analyzed for their impacts on BAFFR function. Binding of BAFF to BAFFR was affected by P21R and A52T. Spontaneous oligomerization of BAFFR was disturbed by P21R, A52T, G64V, and P146S. BAFF-dependent activation of NF-κB2 was reduced by P21R and P146S, while interactions between BAFFR and the B cell antigen receptor component CD79B and AKT phosphorylation were impaired by P21R, A52T, G64V, and DUP92-95. P21R, G64V, and DUP92-95 interfered with phosphorylation of ERK1/2, while BAFF-induced shedding of the BAFFR ectodomain was only impaired by P21R. Although all variants change BAFFR function and have the potential to contribute as modifiers to the development of primary antibody deficiencies, autoimmunity, and lymphoma, P21R is the only variant that was found to correlate positively with CVID

Triad3a induces the degradation of early necrosome to limit RipK1-dependent cytokine production and necroptosis.

Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, cIAPs, A20, or CYLD. Finally, we show that Triad3a-dependent necrosomal degradation limits necroptosis and production of inflammatory cytokines. These results reveal a new mechanism of shutting off necrosome signaling and may pave the way to new strategies for therapeutic manipulation of inflammatory responses

Polymorphisms in the Hsp70 gene locus are genetically associated with systemic lupus erythematosus

Background Heat shock proteins (Hsps) play a role in the delivery and presentation of antigenic peptides and are thought to be involved in the pathogenesis of multifactorial diseases. Objective To investigate genes encoding cytosolic Hsp70 proteins for associations of allelic variants with systemic lupus erythematosus (SLE). Methods Case-control studies of two independent Caucasian SLE cohorts were performed. In a haplotype-tagging single-nucleotide polymorphism approach, common variants of HspA1L, HspA1A and HspA1B were genotyped and principal component analyses were performed for the cohort from the Oklahoma Medical Research Foundation (OMRF). Relative quantification of mRNA was carried out for each Hsp70 gene in healthy controls. Conditional regression analysis was performed to determine if allelic variants in Hsp70 act independently of HLA-DR3. Results On analysis of common genetic variants of HspA1L, HspA1A and HspA1B, a haplotype significantly associated with SLE in the Erlangen-SLE cohort was identified, which was confirmed in the OMRF cohort. Depending on the cohorts, OR ranging from 1.43 to 1.88 and 2.64 to 3.16 was observed for individuals heterozygous and homozygous for the associated haplotype, respectively. Patients carrying the risk haplotype or the risk allele more often displayed autoantibodies to Ro and La in both cohorts. In healthy controls bearing this haplotype, the amount of HspA1A mRNA was significantly increased, whereas total Hsp70 protein concentration was not altered. Conclusions Allelic variants of the Hsp70 genes are significantly associated with SLE in Caucasians, independently of HLA-DR3, and correlate with the presence of autoantibodies to Ro and La. Hence, the Hsp70 gene locus appears to be involved in SLE pathogenesis

A barcoded flow cytometric assay to explore the antibody responses against SARS-CoV-2 spike and its variants

The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants

IgG autoantibodies bound to surfaces of necrotic cells and complement C4 comprise the phagocytosis promoting activity for necrotic cells of systemic lupus erythaematosus sera

Objective: Accumulation of dying and dead cells is thought to be involved in the etiopathogenesis of systemic lupus erythaematosus (SLE). Clearance has been described mainly for apoptotic cells; however, the knowledge of serum factors participating in the phagocytosis of necrotic cells is limited. Patients and methods: Sera from 18 patients with SLE and 10 normal healthy donors (NHD), and macrophages from 3 NHD were included. Autoantibodies and complement were measured by ELISA and phagocytosis by flow cytometry. Binding of serum IgG to necrotic cells was assessed by flow cytometry and confocal microscopy. Results: Sera from patients with SLE and NHD generally promoted the phagocytosis of necrotic cells by macrophages isolated from NHD. Five independent experiments with macrophages from three different NHD led to similar results. The sera from healthy controls displayed a homogeneous activity, whereas sera from patients with SLE showed a dichotomic behaviour. Only sera containing autoantibodies binding to the surfaces of necrotic cells and sufficient complement showed increased phagocytosis promoting activities. In SLE sera, C4 turned out to be the critical complement component in this process. Sera de-complemented by heat treatment strongly reduced phagocytosis of necrotic cells. Conclusions: Serum components influence the uptake of necrotic cells by phagocytosis competent macrophages from NHD. Complement is required for this process and autoantibodies binding to the surfaces of necrotic cells additionally promote their phagocytosis