IgG Anti-beta Research Articles

Pregnancy outcomes in 1869 pregnancies in a large cohort from the Spanish Society of Rheumatology Lupus Register (RELESSER)

IntroductionObstetric complications are more common in women with systemic lupus erythematosus (SLE) than in the general population. ObjectiveTo assess pregnancy outcomes in women with SLE from the RELESSER cohort after 12 years of follow-up. MethodsA multicentre retrospective observational study was conducted. In addition to data from the RELESSER register, data were collected on obstetric/gynaecological variables and treatments received. The number of term pregnancies was compared between women with pregnancies before and after the diagnosis of SLE. Further, clinical and laboratory characteristics were compared between women with pregnancies before and after the diagnosis, on the one hand, and with and without complications during pregnancy, on the other. Bivariate and multivariate analyses were carried out to identify factors potentially associated with complications during pregnancy. ResultsA total of 809 women were included, with 1869 pregnancies, of which 1395 reached term. Women with pregnancies before the diagnosis of SLE had more pregnancies (2.37 vs 1.87) and a higher rate of term pregnancies (76.8% vs 69.8%, p < 0.001) compared to those with pregnancies after the diagnosis. Women with pregnancies before the diagnosis were diagnosed at an older age (43.4 vs 34.1 years) and had more comorbidities. No differences were observed between the groups with pregnancies before and after diagnosis in antibody profile, including anti-dsDNA, anti-Sm, anti-Ro, anti-La, lupus anticoagulant, anticardiolipin or anti-beta-2-glycoprotein. Overall, 114 out of the 809 women included in the study experienced complications during pregnancy, including miscarriage, preeclampsia/eclampsia, foetal death, and/or preterm birth. Women with complications had higher rates of antiphospholipid syndrome (40.5% vs 9.9%, p < 0.001) and higher rates of positivity for IgG anticardiolipin (33.9% vs 21.3%, p = 0.005), IgG anti-beta 2 glycoprotein (26.1% vs 14%, p = 0.007), and IgM anti-beta 2 glycoprotein (26.1% vs 16%, p = 0.032) antibodies, although no differences were found regarding lupus anticoagulant. Among the treatments received, only heparin was more commonly used by women with pregnancy complications. We did not find differences in corticosteroid or hydroxychloroquine use. ConclusionsThe likelihood of term pregnancy is higher before the diagnosis of SLE. In our cohort, positivity for anticardiolipin IgG and anti-beta-2- glycoprotein IgG/IgM, but not lupus anticoagulant, was associated with a higher risk of poorer pregnancy outcomes.

POS0773 AUTOANTIBODIES IN A MULTI-INSTITUTIONAL INDIAN COHORT (INSPIRE) OF SLE PATIENTS: PREVALENCE, CLUSTER ANALYSIS AND PHENOTYPE ASSOCIATION

BackgroundSystemic Lupus Erythematosus (SLE) is characterized by an array of autoantibodies. Different autoantibodies have been associated with different clinical features like anti-dsDNA antibodies with nephritis and anti-phospholipid antibodies with pregnancy loss. However, the prevalence of autoantibodies has been variable across different ethnic groups. Data on the Indian population is limited.ObjectivesTo assess the prevalence of different autoantibodies in a multi-institutional cohort (INSPIRE) of Indian SLE patients and to test their association with various clinical features using cluster analysisMethodsThe patients (n=1053) enrolled in a multi-institutional cohort of Indian patients (Indian SLE inception cohort for Research [INSPIRE]) were included.1 Antibodies were assayed using Immunoline (Euroimmune, Germany) 17 antigen kit. Anti-phospholipid antibodies (IgG and IgM anti-cardiolipin antibodies (ACLs), IgG anti-Beta 2 GpI antibodies) were measured using ELISA (Euroimmune). Lupus anticoagulant was available in a subset of patients.The prevalence data for autoantibodies were analyzed using an intensity of only ++ and above on Immunoline assay as significant. Univariate analysis by Chi-square test was done to identify associations between individual autoantibodies and their clusters with clinical manifestations.ResultsThe clinical features were fever in 702, alopecia in 813, oral ulcers in 628, acute cutaneous lupus (ACLE) in 520, proteinuria in 400, pleural effusion in 181, thrombocytopenia in 250 and autoimmune hemolytic anemia in 137 patients.The prevalence of various autoantibodies by ELISA was anti-dsDNA antibodies in 70.2% (551/784), IgG Anti- beta-2 GpI in 4.47% (42/938), IgG ACL in 6.14% (61/992) and IgM ACL in 7.1% (54/760). Lupus anticoagulant was present in 13.9% (112/ 805). By Immunoline assay, the prevalence for anti-Ro 52, anti-Ro 60, anti-La and anti-Ribosomal P was 28.49%, 33.14%, 10.07% and 24.03% respectively (Table 1).Table 1.Prevalence of different autoantibodies in the INSPIRE lupus cohortS. No.AutoantibodyPrevalence (%) (n=1053)1.dsDNA28.112.Nucleosomes27.833.Histones24.884.Ro_52_SSA28.495.Ro_60_SSA33.146.SSB-La10.077.Ribosomal P24.038.nRNP36.759.Sm33.1410.Scl-703.2311.PM-Scl0.3812.Jo-10.0913.CENP-B0.3814.PCNA1.3315.AMA-M22.28Cluster analysis (Figure 1) revealed association (Odds ratio with 95% confidence interval) of Cluster 1 (antibodies against dsDNA, histones and nucleosomes) with arthritis (1.51 [1.18-1.94]), proliferative nephritis (3.05[2.08-4.48]) and pleural effusion (1.49[1.08-2.05]), cluster 2 (antibodies against Sm, nRNP, Ro52, Ro60 and Ribosomal P) with ACLE (1.3[1.02-1.65]) and non-proliferative nephritis (1.64[1.09-2.46]) and cluster 3 (antiphospholipid antibodies) with thrombocytopenia (3.34[1.57-7.11]).Figure 1.Cluster analysis of autoantibodies (Cluster 1: dsDNA, histone and nucleosome; cluster 2: Sm, nRNP, Ro52, Ro60 and Ribosomal P; cluster 3: cardiolipin, β2GP1 and La and lupus anticoagulant; cluster 4: Scl-70, Jo-1, PCNA, AMA-M2, PM-SCL and CENP-B)ConclusionThe prevalence of anti-Sm antibody and Anti-Ribosomal P antibody is higher whereas that of anti-La antibody is lesser in the Indian SLE patients as compared to other cohorts of SLE patients with different ethnicities.2 Cluster analysis reveals co-occurrence of different autoantibodies in our patients and their significant association with various clinical manifestations which suggests a possible pathogenic role of autoantibodies or a common genetic basis for it.

Plasmin immunization preferentially induces potentially prothrombotic IgG anticardiolipin antibodies in MRL/MpJ mice

To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome. BALB/cJ and MRL/MpJ mice were immunized with Freund's complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin-immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro. Plasmin-immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while plasmin-immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG antiplasmin or IgG aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to beta(2)GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III, and 2 of 10 inhibited inactivation of FXa by antithrombin III. Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti-beta(2)GPI in MRL/MpJ mice, but leads to production of only IgG antiplasmin in BALB/cJ mice. IgG mAb generated from plasmin-immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive potentially prothrombotic aCL in genetically susceptible individuals.

Circulating osteoprotegerin levels are elevated and correlated with antiphospholipid antibodies in patients with systemic lupus erythematosus

Patients with antiphospholipid syndrome (APS) have an increased risk for the development of thrombotic complications. Recent studies indicate that osteoprotegerin (OPG) acts as an important molecule in the development of vascular diseases. The aim of the present study was to examine the association between serum OPG levels and APS manifestations in patients with SLE. Seventy-nine patients with SLE and ninety-two healthy controls, matched for age and sex, were included in this study. Serum levels of OPG, monocyte chemoattractant protein(MCP)-1 and soluble E-selectin were determined by ELISA. At the time of serum sampling, various clinical and laboratory parameters were assessed. We found that serum levels of OPG were significantly higher in patients with SLE than in healthy controls (1236 +/- 82 vs 967 +/- 37 pg/mL, P = 0.003). Particularly, serum OPG levels were significantly higher in SLE patients with APS than those without (1615 +/- 191 vs 1171 +/- 91 pg/mL, P = 0.006). Serum OPG levels correlated with titres of IgG anti-cardiolipin antibody (P = 0.026) and anti-beta(2)-glycoprotein I antibody (P < 0.001). Moreover, serum OPG also correlated with serum levels of sE-selectin (P = 0.002), which is an endothelial cell activation marker, and MCP-1 (P = 0.003), a well known chemokine implicated in thrombogenesis. Collectively, serum OPG levels were increased in SLE patients with APS and correlated with titres of antiphospholipid antibodies, suggesting that OPG might be linked to the development of APS.

β2‐glycoprotein i is a cofactor for tissue plasminogen activator–mediated plasminogen activation

Regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. The purpose of this study was to investigate the effects of beta(2)-glycoprotein I (beta(2)GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation. The effect of beta(2)GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of beta(2)GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-beta(2)GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured. Beta(2)-glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The beta(2)GPI region responsible for stimulating tPA activity was shown to be at least partly contained within beta(2)GPI domain V. In addition, beta(2)GPI bound tPA with high affinity (K(d) approximately 20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (K(cat)/K(m)) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of beta(2)GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following beta(2)GPI repletion. Stimulation of tPA-mediated plasminogen activity by beta(2)GPI was inhibited by monoclonal anti-beta(2)GPI antibodies as well as by anti-beta(2)GPI antibodies from patients with APS. These findings suggest that beta(2)GPI may be an endogenous regulator of fibrinolysis. Impairment of beta(2)GPI-stimulated fibrinolysis by anti-beta(2)GPI antibodies may contribute to the development of thrombosis in patients with APS.

IgG antibodies to plasminogen and their relationship to IgG anti-β2-glycoprotein 1 antibodies and thrombosis

Reduced fibrinolytic activity has been described in primary antiphospholipid syndrome (PAPS) and may be responsible for thrombotic events. Some evidence supports a relationship between anti-plasminogen (PLG) antibodies, anti-beta(2)-glycoprotein 1 (beta(2)GP1) antibodies, and fibrinolysis, but their relationship is still unclear. The aim of study is to evaluate the association between IgG anti-beta(2)GP1 and IgG anti-PLG antibodies and thrombosis. Two groups of consecutive patients with PAPS and systemic lupus erythematosus (SLE): 32 patients with lupus anticoagulant (LAC), 32 patients without LAC, and 40 healthy controls were included. IgG against beta(2)GP1 and PLG antibodies were measured by enzyme-linked immunosorbent assay, and a value above the 99th percentile of the normal healthy control was considered as positive, and their interrelationship with thrombosis was evaluated by Pearson Chi-squared test. Cross-reactive antibodies binding to PLG and beta(2)GP1 were determined in a competitive and cross-inhibition assay. Levels of fibrinolytic activity in the presence of IgG fractions from patients and healthy controls were examined using a plasmin fluorogenic substrate assay. A high frequency of IgG anti-PLG antibodies (35.9%) was found in 64 patients, and its presence was associated with thrombosis (p = 0.001), which may be due to its ability to inhibit exogenous fibrinolysis. Coexistence of IgG anti-PLG and IgG anti-beta(2)GP1 antibodies was found in 11 of 64 patients and was related with thrombosis (p = 0.001). Cross-reactive antibody binding to PLG and beta(2)GP1 was found in IgG fractions from three patients and a monoclonal anti-beta(2)GP1 antibody BD4, and one of these three patients had thrombotic history. However, no significant association was found between IgG anti-PLG and IgG anti-beta(2)GP1 antibodies in patients. In conclusion, the prevalence of IgG anti-PLG was high in patients with PAPS and SLE and might relate with thrombosis. Cross-reactivity of IgG anti-beta(2)GP1 antibodies with PLG may occur in the sera of patients.

IgG and IgM isotypes of anti-cardiolipin and anti-β 2-glycoprotein i antibodies reflect different forms of recent thrombo-embolic events

We correlated the distribution and levels of serum anti-cardiolipin (aCL) and anti-beta(2)-glycoprotein-1 antibodies (anti-beta(2)-GPI) of the IgG and IgM isotypes to the clinical spectrum of recent (<6 months) thrombo-embolic events in a cohort of 162 patients. Clinical information was obtained by questionnaires from the referring physicians. Cerebro-vascular infarction (CVI) had taken place in 82 patients, deep venous thrombosis (DVT) in 34, pulmonary embolism (PE) in 14, myocardial infarction (MI) in four, and other thromboses in 28 patients. SLE was the most commonly associated rheumatic disease and accounted for 20 (12%) patients. In 124 (77%) patients no underlying rheumatic disease was identified. Isolated IgG aCL was found in 31 of 48 patients with DVT/PE (65%), but in only 21 of 82 patients with CVI (26%); p<0.0001. IgG anti-beta(2)-GPI were detected in 23 (48%) DVT/PE patients, but in only 13 (16%) CVI patients; p<0.001. The IgG class anti-beta(2)-GPI positive patients had significantly higher levels of IgG aCL (mean 65 units) compared to IgG anti-beta(2)-GPI negative patients (mean 29 units); p<0.0001. In contrast, isolated IgM aCL was found in nine (19%) patients with DVT/PE, but in 46 (56%) CVI patients; p<0.0001. Only ten patients had IgM anti-beta(2)-GPI. The present study shows that the IgG and IgM aCL isotypes seem to define different clinical subsets of patients with thrombo-embolic events with IgG aCL being most prevalent in the group having DVT/PE, IgM aCL being found primarily among CVI patients.

A Prospective Study of 1038 Pregnancies on the Predictive Value of Anti‐Annexin V Antibodies for Fetal Loss

Retrospective studies have demonstrated that anti-annexin V (anti-AnxV) antibodies are linked to miscarriage. Their predictive value is, however, unknown. We have carried out a prospective study to evaluate the relationship between anti-AnxV antibodies and the pregnancy outcome. A serum sample was taken from 1038 consecutive healthy women at the beginning of pregnancy. IgG and IgM anti-AnxV antibodies were measured by an ELISA method. The cutoff value was set at 5 units for both IgG and IgM. Out of 1038 women, 116 (11.4%) had a miscarriage by the 22nd week; 10 were lost to follow-up, 10 had an induced abortion, 6 had a preterm delivery, and 896 carried their pregnancy through to term. An adverse outcome of the pregnancy proved to be directly related to the number of previous miscarriages (P = .008) and the age of the woman (P = .002). IgG and IgM anti-AnxV were present in 25% and 27% of the women who miscarried, and in 23% and 28% of those who gave birth (mean antibody concentration IgG, 4.2 vs. 4.4 U/mL; IgM, 3.7 vs. 3.5 U/mL). IgG and IgM anticardiolipin and anti-beta(2)GPI, together with antinuclear, antithyroperoxidase, and antithyroglobulin antibodies, were also measured in the 116 sera of the women with miscarriage and in an equal number of women who gave birth. Their positivity or level proved not to be useful in discriminating between the risk of miscarriage and term delivery. This large-scale prospective study demonstrates that the presence of IgG and IgM anti-AnxV antibodies, when measured in healthy women, does not give a positive predictive lead towards the possibility of a miscarriage, and it is not useful in evaluating the risk of miscarriage at the beginning of pregnancy.

Avidity of Anti-β2-Glycoprotein I and Thrombosis or Pregnancy Loss in Patients with Antiphospholipid Syndrome

We aimed to evaluate avidity of anti-beta(2)-glycoprotein I antibodies (anti-beta(2)-GPIs) in patients with antiphospholipid syndrome (APS) at the time of acute thrombotic events or pregnancy loss as compared with clinical event-free periods. To do so, 69 sera samples from 16 patients (6 with primary APS and 10 with APS secondary to systemic lupus erythematosus ) were selected on the basis of anti-beta(2)-GPI positivity. Avidity of IgG anti-beta(2)-GPIs was determined by chaotropic enzyme-linked immunosorbent assay (ELISA), using increased NaCl concentration during antibody binding. High, heterogeneous, and low-avidity anti-beta(2)-GPIs were measured in APS patients, with no clear pattern regarding the time of thrombotic events or pregnancy failure. In general, anti-beta(2)-GPI avidity did not change substantially during disease course. We concluded that avidity of anti-beta(2)-GPIs appears to be a rather stable parameter in an individual APS patient. Considering the previously shown association of high-avidity anti-beta(2)-GPIs with venous thrombosis, avidity of anti-beta(2)-GPIs may be a better predictor of predisposition to thrombosis and unsuccessful pregnancy than levels of antiphospholipid antibodies, which may fluctuate over time owing to several factors.

The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies

The importance of testing for anticardiolipin antibodies (aCL) in the diagnosis of antiphospholipid syndrome (APS) in patients with thrombosis has recently been challenged (ISTH SSC meeting, Boston 2002). We have analyzed the antiphospholipid serology of 123 patients with persistent antiphospholipid antibodies (aPL) attending our hematology department. The cohort was tested for anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies and aCL of IgG and IgM class and for lupus anticoagulant (LA). Ninety-six of these patients fulfilled Sapporo clinical criteria for APS and 70 of these patients had venous and/or arterial thrombosis. Patients with LA plus anti-beta(2)-GPI antibodies had significantly higher levels of IgG aCL and anti-beta(2)-GPI antibodies than those exhibiting positivity for only LA or anti-beta(2)-GPI antibodies (P < 0.05). Patients with aCL IgG levels over 60 GPLU were found in all cases to be positive for LA and anti-beta(2)-GPI antibodies; 25.2% (31/123) of all patients and 26.04% (25/96) of patients fulfilling Sapporo clinical criteria for APS were positive for aCL only. The mean IgG aCL level in the Sapporo clinical criteria positive patients who had aCL only was 11.5 GPLU (normal < 5 GPLU). These data indicate that omission of aCL testing from the clinical investigation of APS could lead to a failure to diagnose the syndrome in a proportion of patients.

A two‐step coagulation test to identify antiβ2‐glycoprotein I lupus anticoagulants

Lupus anticoagulants (LA) are immunoglobulins which inhibit phospholipid (PL)-dependent coagulation tests. LA are not specific, as they may reflect the presence of antibodies to human prothrombin, human beta(2)-Glycoprotein I (beta(2)GPI), an association of previous antibodies or other antibodies. Antibodies to human beta(2)GPI act as in vitro anticoagulants by enhancing the binding of beta(2)GPI to PL, and this binding may be influenced by calcium ion concentration. A reduction in final calcium concentration, from 10 mm to 5 mm, increased coagulation times in both dilute Russell Viper Venom Time (dRVVT) and dilute Prothrombin Time (dPT) when plasmas of patients with antibeta(2)GPI antibodies were used. Ten LA patients showed increased dRVVT and dPT ratios from means of 1.5 to 1.7 (P < 0.001) and 2.4 to 4.3 (P = 0.002), respectively. Instead, all LA-positive antibeta(2)GPI antibody-negative patients showed decreased coagulation times from mean ratios of 1.5 to 1.3 (P = 0.004) in dRVVT and from 2.0 to 1.5 (P = 0.01) in dPT. These results are confirmed by running dRVVT of normal plasma spiked with affinity purified IgG antibeta(2)GPI antibodies. Therefore, when a PL-dependent coagulation test is run twice, at different final calcium concentrations, antibeta(2)GPI LA can be identified.

Antibodies to high-density lipoprotein and beta2-glycoprotein I are inversely correlated with paraoxonase activity in systemic lupus erythematosus and primary antiphospholipid syndrome.

To determine the prevalence of anti-high-density lipoprotein (anti-HDL) antibodies and to establish a possible relationship between anti-HDL, anticardiolipin antibodies (aCL), anti-beta(2)-glycoprotein I (anti-beta(2)GPI), and paraoxonase (PON) activity in patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS). Thirty-two patients with SLE and 36 with primary APS were enrolled in a cross-sectional study. Twenty age- and sex-matched healthy subjects were used as controls. Serum levels of IgG and IgM aCL, anti-beta(2)GPI, and antiprothrombin antibodies and IgG anti-HDL were measured by enzyme-linked immunosorbent assay. Total cholesterol, HDL cholesterol, HDL(2), and HDL(3) were determined by standard enzymatic techniques. PON activity was assessed by quantification of nitrophenol formation, and total antioxidant capacity (TAC) by chemiluminescence. Levels of total HDL, HDL(2), and HDL(3) were reduced in patients with SLE compared with controls (mean +/- SD 0.51 +/- 0.3, 0.37 +/- 0.3, and 0.14 +/- 0.1 mmoles/liter, respectively, versus 1.42 +/- 0.9, 1.01 +/- 0.7, and 0.40 +/- 0.2). Patients with SLE and primary APS had higher titers of anti-HDL antibodies and lower PON activity than controls. In the SLE population, PON activity was inversely correlated with IgG anti-HDL titers (r = -0.48, P = 0.005) whereas in the primary APS population, IgG anti-beta(2)GPI was the only independent predictor of PON activity (r = -0.483, P = 0.003). In the SLE group, anti-HDL was inversely correlated with TAC (r = -0.40, P < 0.02), and PON activity was positively correlated with TAC (r = 0.43, P < 0.02). IgG anti-HDL and IgG anti-beta(2)GPI antibodies are associated with reduced PON activity in patients with SLE and primary APS. Since the physiologic role of PON is to prevent low-density lipoprotein oxidation with its attendant atherogenic effects, the reported interactions may be relevant to the development of atherosclerosis in SLE and primary APS.