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BackgroundThe presence of antiphospholipid antibodies (aPL) has been observed in patients with COVID-19 (1,2), suggesting that they may be associated with deep vein thrombosis, pulmonary embolism, or stroke in severe cases (3). Antiphospholipid syndrome (APS) is a systemic autoimmune disorder and the most common form of acquired thrombophilia globally. At least one clinical criterion, vascular thrombosis (arterial, venous or microthrombosis) or pregnancy morbidity and at least one laboratory criterion- positive aPL two times at least 12 weeks apart: lupus anticoagulant (LA), anticardiolipin (aCL), anti-β2-glycoprotein 1 (anti-β2GPI) antibody, have to be met for international APS classification criteria(4). Several reports also associate anti-phosphatidylserine/prothrombin antibodies (aPS/PT) with APS.ObjectivesTo combine clinical data on arterial/venous thrombosis and pregnancy complications before and during hospitalisation with aPL laboratory findings at 4 time points (hospital admission, worsening of COVID-19, hospital discharge, and follow-up) in patients with the most severe forms of COVID-19 infection.MethodsPatients with COVID-19 pneumonia were consequetively enrolled, as they were admitted to the General hospital Pancevo. Exclusion criteria were previous diagnosis of inflammatory rheumatic disease and diagnosis of APS. Clinical data were obtained from the medical records. Laboratory results, including LA, aCL, anti-β2GPI, and aPS/PT antibodies were taken at hospital admission, worsening (defined as cytokine storm, connection of the patient to the respirator, use of the anti-IL-6 drug- Tocilizumab), at hospital discharge and at 3-months follow-up and sent to University Medical Centre Ljubljana, Slovenia for analysis. Statistics was performed by using SPSS 21.Results111 patients with COVID-19 pneumonia were recruited; 7 patients died during hospitalisation (none were aPL-positive on admission and at the time of worsening), 3 due to pulmonary artery embolism. All patients were treated according to a predefined protocol which included antibiotics, corticosteroids, anticoagulation therapy and specific comorbidity drugs; patients with hypoxia were supported with oxygen. During hospitalisation, pulmonary artery thrombosis occurred in 5 patients, one was aPL-positive at all time points (was diagnosed with APS), others were negative. In addition, 9/101 patients had a history of thrombosis (5 arterial thrombosis (coronary and cerebral arteries), none of whom was aPL-positive on admission and at follow-up, and 4 venous thrombosis, one of which was aPL-positive at all time points and received an APS diagnosis). Among 9/101 patients with a history of thrombosis, 55.6% were transiently positive at the time of discharge, compared to patients without prior thrombosis, in whom 26.1% were transiently positive at the hospital release (p=0.074). Two patients had a history of pregnancy complications (both had miscarriage after 10th week of gestation), but did not have aPL positivity at any time point.ConclusionAlthough aPL was expected to be associated with vascular disease in the most severe forms of COVID-19, all patients that have died in our cohort were aPL negative. At hospital discharge, 56% of patients with a history of arterial or venous thrombosis had positive aPL that became negative at the 3-months follow-up (were transienlty positive), which should be considered when prescribing therapy after hospitalisation.References[1]Trahtemberg U, Rottapel R, Dos Santos CC, et al. Anticardiolipin and other antiphospholipid antibodies in critically ill COVID-19 positive and negative patients. Annals of the Rheumatic Diseases 2021;80:1236-1240.[2]Stelzer M, Henes J, Saur S. The Role of Antiphospholipid Antibodies in COVID-19. Curr Rheumatol Rep. 2021;23(9):72-4.[3]Xie Y, Wang X, Yang P, Zhang S. COVID-19 complicated by acute pulmonary embolism. Radiology: Cardiothoracic Imaging 2020: 2: e200067.[4]Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, et al. J.Thromb.Haemost. 2006; 4: 295-306.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Background:COVID-19 is characterized with pulmonary artery thrombosis, brain and myocardial infarction [1]. Antiphospholipid antibodies (aPLA) are found in COVID-19 patients [2], suggesting that they might be associated with antiphospholipid syndrome (APS), for which the latest research replaces lupus anticoagulans with anti-phosphatidylserine/ prothrombin (aPS/PT) antibodies in cases where it cannot be determined (taking anticoagulants) [4,5].Objectives:In individuals with the most severe types of COVID-19 combining: 1. clinical features on arterial/venous thrombosis and pregnancy issues during hospitalization or in the patient’s personal history; and 2. laboratory data for 9 different aPLAs: anticardiolipin (aCLA), anti-β2-glycoprotein 1 (anti-β2GPI) and aPS/PT antibodies of IgG, IgM, and IgA classes, measured at four time points (hospital admission, worsening of COVID-19, hospital discharge, and the three-month follow-up).Methods:Patients with COVID-19 pneumonia were enrolled in a systematic manner. Previous inflammatory rheumatic illness and APS diagnosis were exclusion criteria. Data on thrombosis and pregnancy pathology were captured by medical records. aCLA, anti-2GPI and aPS/PT antibodies of the IgG, IgM and IgA class were collected at four time points and sent to the University Medical Center Ljubljana in Slovenia for analysis. Values above the 99th percentile of the healthy control population were regarded as positive. SPSS 21 was used for statistics.Results:108 people with COVID-19 pneumonia were included; 7 died during hospitalization (not a single was aPL-positive on admission, although 3 had thrombosis of the pulmonary artery). During hospitalization 5 pts had pulmonary artery thromboembolism, but only one was aPL-positive at all time points (newly diagnosed with APS). Two patients developed arterial thrombosis -one cerebral artery thrombosis and was positive for aCLA on admission, but tested negative at discharge and follow-up, the other developed coronary artery thrombosis with myocardial infarction and tested aPLA negative. Five patients developed microthrombosis, one pulmonary microthrombosis and tested aPLA positive at admission but negative at follow-up, the other four tested aPLA negative. Considering 9/101 patients with a history of thrombosis: 5 had arterial thrombosis of coronary and cerebral arteries but none was aPL-positive and 4 had venous thrombosis, 1 was aPL-positive at all time points and was diagnosed with APS. In 9/101 patients with a history of thrombosis, 55.6% were transiently aPLA positive at the time of discharge, compared to 26.1% of patients without history (p=0.042). Two patients had a history of pregnancy obstacles (both miscarried after the 10th week of gestation), but none was aPL positive.Conclusion:In our study, 2/108 patients (1.85%) were newly diagnosed with APS. Although aPLA was expected to be associated with vascular disease in the most severe forms of COVID-19, it was not confirmed by this study. At hospital discharge, 56% of patients with a history of arterial or venous thrombosis had positive aPLA that negativized at the 3-months follow-up checkout (transienlty positive) and therefore could not be diagnosed with APS. This discovery may aid us in recommending anticoagulant therapy following hospitalization. Patients with severe COVID-19 who tested aPLA positive should get the same anticoagulant medication as negatives, since those antibodies are usually not connected to APS.REFERENCES:[1] Cheng NM, Chan YC, Cheng SW. COVID-19 related thrombosis: A mini-review. Phlebology. 2022 Jun;37(5):326-337.[2] Taha, M.; Samavati, L. Antiphospholipid antibodies in COVID-19: A meta-analysis and systematic review. RMD Open 2021, 7: e001580[3] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, et al. 2006. J.Thromb.Haemost. 4: 295-306[4] Pengo, V. Additional laboratory tests to improve on the diagnosis of antiphospholipid syndrome. J. Thromb. Haemost. 2020, 18, 1846–1848.[5] Egri N, Bentow C, Rubio, L. et al. Anti-Phosphatidylserine/Prothrombin Antibodies at Two Points: Correlation with Lupus Anticoagulant and Thrombotic Risk. Front. Immunol. 2021, 12, 754469.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Sjoegren’s syndrome (SjS) is an understudied systemic autoimmune disease with heterogeneous presentation and many unmet clinical needs. The underlying molecular pathomechanisms that drive SjS are not fully elucidated.Objectives:Our aim was to assess the proteome profiles of the minor salivary gland (MSG) tissues and to identify different subgroups of patients with SjS.Methods:Flash frozen MSG from 18 SjS patients, fulfilling the 2016 ACR/EULAR classification criteria, and 6 sicca controls not fulfilling the classification criteria, were analyzed. Clinical, imaging, functional, laboratory, and histological parameters, including age, gender, salivary gland ultrasound score, Schirmer’s test, unstimulated salivary flow (USF) test, presence of autoantibodies, cryoglobulins, levels of complement components, focus score, numbers of germinal centers, and presence of lymphoepithelial lesions, were collected. Proteome analysis of the MSG tissues was performed by tandem mass spectrometry. Proteins were identified and quantified using Spectronaut software. Differentially expressed proteins were identified using Bayes test. Pearson correlation coefficient r was calculated between log2 protein intensities and the clinical features. Unsupervised hierarchical clustering was performed based on the expression of all detected proteins to identify groups of patients with similar protein expression profiles. Mann-Whitney U test was used to statistically analyze the differences in clinical parameters between the identified clusters.Results:We identified 5577 proteins, 7 of which were differentially expressed between SjS and sicca controls (log2FC >2; p-adj <0,05). All 7 differentially expressed proteins, namely PTPRCAP, IKZF1, CD3E, HDAC3, PIK3CD, SH2D1A, and DOCK10, were upregulated in SjS patients compared to sicca controls (Table 1). PTPRCAP (r=0.585, p-adj=0.013), CD3E (r=0.616, p-adj=0.009), HDAC3 (r=0.448, p-adj=0.0565), PIK3CD (r=0.413, p-adj=0.075), SH2D1A (r=0.671, p-adj=0.004) and DOCK10 (r=0.540, p-adj=0.024) correlated with the focus score in MSG tissues. SH2D1A additionally correlated with cryoglobulins (r=0.590, p-adj=0.067). Unsupervised hierarchical clustering, based on all detected proteins, identified two separate clusters of patients with SjS. Patients from cluster 1 (n=8) had significantly lower focus score compared to patients from cluster 2 (n=10) (median focus score 1.21 vs, 2.0, p=0.002). Additionally, patients from cluster 1 had higher levels of USF compared to patients from cluster 2 (median 0.13 ml/min vs. 0,02 ml/min, p=0,400). Notably, only 29% of patients in cluster 1 tested positive in the USF test, while 70% of patients in cluster 2 showed pathological results.Table 1.List of differentially expressed proteins in SjS compared to sicca controls with their respective Log2FC and p-adjusted values.GeneProteinDifferential expression analysisLog2FCp-adjustedPTPRCAPProtein tyrosine phosphatase receptor type C-associated protein3.580.000IKZF1DNA-binding protein Ikaros3.500.000CD3ET-cell surface glycoprotein CD3 epsilon chain2.550.000HDAC3Histone deacetylase 32.230.000PIK3CDPhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta2.920.000SH2D1ASH2 domain-containing protein 1A3.160.000DOCK10Dedicator of cytokinesis protein 102.180.001Conclusion:The total number of proteins identified in our analysis exceeds that of previous studies and allows us to provide the first list of differentially expressed proteins in MSG tissues in SjS and controls that correlate with SjS-relevant measures. Our results indicate that MSG tissues from patients with different levels of lymphocyte infiltration and salivary gland dysfunction differ in the protein expression profiles.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Neža Štucin: None declared, Katja Perdan Pirkmajer: None declared, Alojzija Hočevar: None declared, Britta Maurer Boehringer-Ingelheim, GSK, Novartis, Otsuka, MSD, Novartis, Boehringer Ingelheim, Jannsen-Cilag, GSK, Novartis, Polona Zigon: None declared, Saša Čučnik: None declared, Kerstin Klein: None declared.

BackgroundImmunoglobulin A vasculitis (IgAV) is a small vessel, immune complex vasculitis, involving skin, joints, gastrointestinal tract (GIT) and kidney. While different diagnostic/prognostic and inflammatory markers have already been studied in paediatric IgAV, data on adult cases are scarce1.ObjectivesTo examine the inflammatory cell profile in peripheral blood and cytokine profile in sera of newly diagnosed, biopsy-proven and treatment-naïve adult IgAV compared to healthy blood donors (HBD), and determine associations with IgAV clinical signs.MethodsFlow cytometry of stained, lysed and fixed whole blood was performed in IgAV (n=30), and HBD (n=17) (Miltenyi). Cytokines were quantitated by multiplex bead assay (Luminex), ELISA (IL-6) and immunonephelometry (acute phase serum amyloid A (SAA)) in 57 IgAV vs. 53 HBD.ResultsPercentage of CD16+ neutrophils was significantly higher, while percentages of CD3+ T-cells (including CD4+ and CD8+ cells), as well as CD19+ B-cells were significantly lower in peripheral blood of IgAV patients vs. HBD. The expression of l-selectin (CD62L) on CD16+ neutrophils was significantly increased in IgAV vs. HBD, as were the sera levels of TNF-α (2-fold), IL-6 (3-fold), IL-8 (2.2-fold) and SAA (11.7-fold changed levels) (table 1). Association was found between GIT involvement and lower neutrophil expression of integrin αM (CD11b) (median; IQR: 7.2; 4.2–16.0), compared to skin limited (17.8; 9.9–40.5) IgAV cases (p=0.047). There was no association found between different cytokines and IgAV clinical phenotype.Abstract FRI0516 – Table 1Cell profiles, neutrophil surface proteins and cytokines in IgAV patients compared to HBDMEDIAN (Q25-Q75)MEDIAN (Q25-Q75) Cells(% of WBC)HBD(n=15)IgAV(n=15)P valueCytokines (pg/ml)HBD(n=53)IgAV(n=57)P value Neutrophils51.0(47.1–56.9)67.5(63.6–73.3)<0.001IL-1β0.4(0.4–0.4)0.4(0.4–1.5)ns T-cells26.4(23.0–31.8)16.6(10.2–21.4)<0.001IL-62.0(1.0–6.0)6.0(2.8–14.3)0.015 CD4+T cells13.6(11.7–18.4)10.4(7.7.–14.4)0.003IL-853.3(10.9–195.0)117.1(29.3–443.9)0.018 CD8+T cells10.3(6.0–12.6)5.0(1.9–7,9)0.002IL-919.0(19.0–19.0)19.0(19.0–19.0)ns B-cells3.8(2.9–4.7)2.4(1.6–2.9)0.006IL-101.0(0.8–2.8)0.04(0.04–1.3)ns NK cells4.6(3.8–6.3)3.6(2.3–6.1)nsIL-13665.2(354.1–1668.0)23.0(23.0–102.2)ns Neutrophil surface proteins (MFI)HBD(n=17)IgAV(n=30)IL-231.4(1.4–82.9)1.4(1.4–118.6)ns CD62L56.4(44.9–73.7)86.5(45.4–107.5)0.036TNF-α3.9(0.8–15.5)8.1(3.0–20.2)0.003 CD11b8.7(5.5–27.4)15.7(6.4–31.3)nsSAA (μg/ml)2.8(1.9–4.7)32.8(7.2–168.0)<0.001 ConclusionsWe found significant up-regulation of neutrophils and their CD62L expression, as well as sera levels of IL-6, IL-8, TNF-α and SAA in IgAV, implying a pathogenic role of neutrophils in IgAV. CD11b might represent a promising surface marker of GIT involvement in adult IgAV.Reference[1] Nagy GE, Kemény L, Bata-Csörgő Z, et al. Neutrophil-to-lymphocyte ratio: A biomarker for predicting systemic involvement in adult IgA vasculitis patients. J Eur Acad Dermatol Venereol2017;31(6):1033–1037.AcknowledgementsThe authors would like to thank the Rotary club Zgornji Brnik, Slovenia, as well as Prof. Mauro Peretti and Dr. Suchita Nadkarni from WHRI, Queen Mary, University of London for their support. We would also like to thank the Sovenian Research Agency for financial support.Disclosure of InterestNone declared

BackgroundDeep vein thrombosis (DVT) is frequent and potentially life threatening disease with tendency to reoccur. Anticoagulant treatment of the first episode of DVT usually lasts 3 months. Antiphospholipid syndrome (APS) is an important cause of DVT. However, the APS can be confirmed only 24 weeks after DVT according to the current APS classification criteria.1 Thus, undiagnosed APS patients, who cease anticoagulant therapy after 3 months, might be exposed to a greater risk for recurrent venous thromboembolism. Studies evaluating the significance of positive antiphospholipid antibody (aPL) test in the acute phase of DVT are lacking.ObjectivesTo evaluate whether positive aPL test at the time of acute DVT diagnosis is predictive of APS.MethodsPatients with acute DVT were included into a 24 month prospective study. All patients were given anticoagulants. aCL IgG/IgM and anti-β2GPI IgG/IgM/IgA antibodies were determined by our in-house ELISA2 at inclusion and then every 4 weeks for the first 24 weeks. The last aPL measurement was performed 24 months after inclusion into the study. APS was confirmed if a patient tested positive (medium or high positive aCL and/or presence of anti-β2GPI) 12 and 24 weeks after DVT. Lupus anticoagulants (LA) were tested after cessation of anticoagulation.Results196 patients (111 male, 85 female, age 54±2 years) included in the study had aPL titer assessed at least 5 times. Ultimately, 20/196 (10.2%) patients fulfilled APS classification criteria. Among these, 15/20 (75%) patients had medium or high titer aPL at the time of acute DVT (1 of them had double positive aPL and 2 of them had multiple positive aPL at first aPL determination). Two patients (10%) had low positive aCL IgG and one had low titer aCL IgM. Two patients (10%) were negative for aPL, but had later fulfilled APS criteria due to positive LA. APS was not established in 176/196 (89.8%) patients. Among these, 146/176 (83%) patients were negative for aPL at inclusion, while 30/176 (17%) had low titer aCL IgM or aCL IgG. Altogether, diagnostically important aCL IgG/IgM and/or anti- β2GPI titer at the time of acute DVT had 83% specificity and 90.5% sensitivity for APS. Isolated low titer aCL IgG were more frequent in patients with APS than in patients without APS (χ2=125.6; p<0.001). Completely negative aCL IgG/IgM and anti-β2GPI in the acute phase of DVT had a negative predictive value of 98.6%.ConclusionsHere we show that in acute phase of DVT, positive medium or high titer aCL IgG/IgM or anti-β2GPI is suggestive of APS. In these patients continuation of anticoagulation beyond the initial 3 months should be considered. Patients with negative aPL in the acute phase of DVT do not need further aPL testing; however, LA should be determined. Low aPL titre at the time of acute DVT deems further testing imperative.References[1] Miyakis S, et al.Thromb Haemost2006;4:295–306.2.[2] Cucnik S, et al. Clin Chem Lab Med2000;38:777–783.Disclosure of InterestNone declared

IntroductionInfluenza may cause severe complications in patients with autoimmune inflammatory rheumatic disease (AIRD), to whom vaccinations are especially recommended. However, AIRD patients require cautious scrutiny of immunogenicity as they might exhibit poor antibody response to vaccination, especially when taking immunomodulatory medications.AimThe aim was to determine immunogenicity of seasonal and pandemic influenza vaccine in AIRD patients, its timeline/persistence, and influence of medications on immune response.MethodsOne hundred and thirty-seven AIRD and 54 healthy controls were vaccinated with trivalent seasonal influenza. After 3–5 weeks, 15 healthy controls and 93 AIRD were vaccinated with pandemic influenza vaccine, and 63 of patients were vaccinated a second time after 3–5 weeks. Sera were collected before vaccination, 18–90 days after each vaccination, and more than 180 days after the last vaccination. The immune response was measured using hemagglutination inhibition (HI) assay and IgG/IgA antibodies against influenza A/B with ELISA.ResultsOur findings indicate that following vaccination with seasonal influenza vaccine, seroprotection, seroresponse, and change in geometric mean titers (GMT) in AIRD patients was not compromised compared to healthy. Similarly, we report for pandemic influenza vaccination little added benefit of the second dose. We confirm lowest increase in HI titer in rituximab-treated AIRD compared to other medications. Vaccination largely tilts the balance from negative ELISA A IgG and IgA titers to positive titers in seasonal H1N1 seroresponsive AIRD patients and controls. A significant decrease in HI GMT and seroprotection was observed only in AIRD at > 180 days after vaccination highlighting an absent persistence of immunogenic response in AIRD patients. Due to high initial HI titers for influenza vaccine, we foresee their benefit in personalized medicine in the future.ConclusionInfluenza vaccination is immunologically active for AIRD, with little value of the second dose of the pandemic vaccine and further scrutiny on persistence of immune response to vaccine in AIRD is needed.

BackgroundNeutrophils with differential surface protein expression were recently implicated in pathogenesis of Giant Cell Arteritis (GCA)1. However, data are lacking with regard to treatment-naïve GCA and their long-term follow-up.ObjectivesTo determine the expression of l-selectin (CD62L) and integrin αM (CD11b) on CD16+ neutrophils in peripheral blood of newly diagnosed, treatment-naïve GCA cases, at the time of diagnosis (time 0) and during follow-up - at week 1, 4, 12, 24 and 48. In parallel, we aimed to measure also sera levels of serum amyloid A (SAA) and interleukin-6 (IL-6).MethodsPeripheral blood from 33 treatment-naïve GCA patients and 16 healthy blood donors (HBD) was stained, lysed, fixed and analysed by flow cytometry (Miltenyi). SAA and IL-6 were measured by nephelometry and ELISA, respectively. 3/33 GCA patients experienced relapse and were analysed separately during follow-up, at the time of relapse and 12 weeks after relapse. At the time of diagnosis, all patients received steroid treatment and therapy tapering started after 4 weeks. At week 12, some of the patients (14/22) received leflunomide, in addition to steroid therapy.ResultsExpression of CD62L, but not CD11b, was significantly higher on CD16+ neutrophils in treatment-naïve GCA patients (median; IQR: 72.5; 56.2–100.7), as compared to HBD (55.9; 44.5–70.6, p=0.017). Longitudinally, the expression of CD62L significantly decreased in GCA patients from day 0 to week 4 (p=0.005). At week 12 there was an elevation in CD62L which escalated also at week 24. Patients receiving steroids only showed a marked increase at week 48, while patients receiving also leflunomide exhibited a decrease (Fig 1). Expression of CD11b declined from day 0 to week 4, but substantially increased throughout weeks 12, 24 and 48, regardless of therapy used. SAA and IL-6 declined sharply from day 0 to week 1 and 4, with gradual elevation of both at week 12. There was a decline in SAA levels observed in all patients at week 24, while IL-6 increased in patients receiving only steroids. These patients exhibited further elevation of both markers at week 48, while patients receiving steroids, in combination with leflunomide, showed a decrease. In 2/3 patients who experienced a relapse, we could observe an increase in the expression of CD62L at the time of relapse, which was found to be decreased again 12 weeks later. A similar trend was observed for IL-6.ConclusionsNeutrophil CD62L could represent a good surface marker for detection of relapse in GCA. A distinct dichotomy was found for CD62L, as well as SAA and IL-6 in GCA during long term follow-up, with the combination of steroids with leflunomide showing more optimal results.Reference[1] Nadkarni S, et al. Investigational analysis reveals a potential role for neutrophils in GCA disease progression. Circ. Res. 2014;114:242–48.AcknowledgementsThe authors would like to thank the Rotary club Zgornji Brnik, Slovenia, as well as Prof. Mauro Peretti and Dr. Suchita Nadkarni from WHRI, Queen Mary, University of London for their support. The research was conducted within the National Research Programme (#P3–0314), financially supported by the Slovenian Research Agency.Disclosure of InterestNone declared

Oxidative stress has been shown to play a role in modifying antibodies in favor of higher auto-immunoreactivity. We studied the immunoreactivity of oxidized IgG (oxIgG) to β2-glycoprotein I (β2GPI), six peptide sequences corresponding to amino acid clusters on its different domains, to determine their effects on human coronary artery endothelial cells (HCAEC). Human IgG was purified from seven donors, electro-oxidized and checked for immunoreactivity and avidity to β2GPI and to peptides by ELISA. Conformational stability and antibody-antigen complex formation of oxIgG was analyzed by fluorescence spectroscopy and dynamic light scattering. Resting and activated sub-confluent HCAEC were stimulated with oxIgG or IgG. Secreted cytokines were measured by ELISA. Immunoreactivity of seven oxIgG samples increased to 7.5-fold against β2GPI and to 3.8-fold against six peptides as compared to IgG. oxIgG showed low avidity “properties.” Conformational changes and exposure of protein hydrophobic regions were confirmed by an elevation in fluorescence (2.4- to 5.0-fold) on bis-ANS dye binding to oxIgG. oxIgG significantly elevated the release of GROα and IL-8 in resting and activated states of HCAEC. Oxidation alters IgG in favor of autoreactivity toward whole β2GPI and corresponding peptides on different domains of β2GPI and could lead to dysfunction of arterial endothelium by upregulation of chemokines.

Objective: To evaluate avidity of IgG anti-β2-glycoprotein I antibodies (anti-β2-GPI) in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) in relation to thrombosis, and to demonstrate a possible affinity maturation of IgG anti-β2-GPI during the disease course. Methods: 64 sera from 32 patients (18 with primary or secondary APS, 14 with SLE without APS) and their respective IgG fractions or affinity purified anti-β2-GPI were studied by anticardiolipin (aCL) and anti-β2-GPI enzyme linked immunosorbent assay and by chaotropic assay. Results: Six, 12, and 14 patients had high, low, and heterogeneous avidity IgG anti-β2-GPI, respectively. In 12 patients an increase in antibody avidity was observed over a period of between four and 12 years. More patients with APS were in the high avidity than in the low avidity anti-β2-GPI group, while the opposite was observed for SLE alone (both p<0.05). The most common clinical feature among patients with high avidity anti-β2-GPI was thrombosis, mainly venous thrombosis (p<0.05 and p<0.02, respectively, v the low avidity anti-β2-GPI group). Conclusions: Patients with APS with or without SLE may have anti-β2-GPI of high, low, or heterogeneous avidity. High avidity anti-β2-GPI appear to be associated with thrombosis and APS, while in pure SLE low avidity anti-β2-GPI may prevail. Monitoring of avidity may help elucidate the role of anti-β2-GPI affinity maturation in the pathogenesis of APS.

Objective. The objective of this study was to extend the findings of the preliminary study by measuring the avidity of IgG anti-β2-glycoprotein I antibodies (anti-β2-GPI) on a larger group of patients with primary or secondary antiphospholipid syndrome (APS) and anti-β2-GPI positive patients without APS in the frame of the European Forum on antiphospholipid antibodies (aPL). Methods. Serum from 137 patients with primary APS, APS associated with autoimmune diseases, and patients with autoimmune diseases other than APS from five EU rheumatology centres were tested for anti-β2-GPI antibodies. The 109 patients who were sera positive for anti-β2-GPI by the in-house anti-β2-GPI enzyme-linked immunosorbent assay (ELISA) at the Immunology Laboratory, UMC Ljubljana were selected for further testing on avidity with chaotropic anti-β2-GPI ELISA. Results. High, low and heterogeneous avidity IgG anti-β2-GPI was found in 32/109, 17/109 and 60/109 patients respectively. Significantly more patients with APS were in the high avidity than in the low avidity anti-β2-GPI group, while the opposite was observed for non-APS (both p < 0.001). The most common clinical feature among patients with high avidity anti-β2-GPI was thrombosis, mainly due to venous thrombosis (p < 0.01 and p < 0.001, versus low avidity anti-β2-GPI group). Conclusion. Patients with or without APS had anti-β2-GPI of high, low or heterogeneous avidity. High avidity anti-β2-GPI was associated with thrombosis and APS, while in the low avidity anti-β2-GPI group non-APS (predominantly SLE) patients prevailed. Determination of anti-β2-GPI avidity should be considered in the analytical strategies for further differentiation of patients with anti-β2-GPI antibodies.