[High expression of anti-ß2 glycoprotein I (ß2GPI) autoantibody exacerbates inflammation and immune dysfunction in connective tissue disease].

Objective To observe the expression of anti-ß2 glycoprotein I (ß2GPI) autoantibody in connective tissue diseases and its relationship with the degree of inflammation and immune function. Methods Patients with broad connective tissue diseases including connective tissue disease (CTD), rheumatoid arthritis (RA), Sjogren's syndrome (SS), and systemic lupus erythematosus (SLE) were observed. ß2GPI was quantified by chemiluminescence, ESR was measured by Weil's method, and C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated polypeptide (CCP) antibody were measured by automatic biochemical analyzer. Results ß2GPI and their subtypes were significantly higher in RA patients compared with CTD, SS, and SLE patients. CRP was positively associated with anti-ß2GPI antibody and anti-ß2GPI antibody IgM in patients with connective tissue disease. ESR was positively associated with anti-ß2GPI antibody. Anti-ß2GPI antibody and anti-ß2GPI antibody IgM were elevated in the abnormal CRP group compared with the normal CRP group. Compared with the ESR normal group, anti-ß2GPI antibody and anti-ß2GPI antibody IgG were elevated in the ESR abnormal group. Anti-ß2GPI antibody was positively correlated with ESR and anti-CCP antibody in RA patients. Anti-ß2GPI antibody IgG was positively correlated with RF. Conclusion ß2GPI can be used as a predictor of the degree of inflammation and assessment of immune disorders in CTD.

An allosteric redox switch in domain V of ß2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition.

ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding.

Beta-2-glycoprotein I (ß2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). ß2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of ß2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of ß2GPI. Atomic force microscopy images suggested that reduced ß2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced ß2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of ß2GPI reduction on the protein structure and its implications for antibody binding in APS patients.

Apolipoprotein H drives hepatitis B surface antigen retention and endoplasmic reticulum stress during hepatitis B virus infection.

BACKGROUND: Apolipoprotein H (APOH), also known as beta2-glycoprotein I (beta2-GPI), is an acute phase protein in hepatitis B virus (HBV) infection and binds to hepatitis B surface antigen (HBsAg) with high-affinity. APOH expression is upregulated by HBV and the large surface protein (LHBs), but also elevated in HBV-related hepatoma cells. Previous studies show that intracellular retention of HBsAg induces endoplasmic reticulum (ER) stress, a key driver of hepatocyte damage during chronic liver injury, but the mechanisms are unclear. We hypothesize that APOH mediates HBV-induced ER stress through increased retention of HBsAg. METHODS: VR-APOH-myc and VR-LHBs-flag plasmids were constructed by PCR using pcDNA3.1(-)-APOH or an HBV expression vector, respectively. APOH and ER stress markers were examined at protein and mRNA levels by Western Blot or RT-qPCR. HBsAg titer was assayed by ELISA. RNA-seq was performed to elucidate the transcriptional impact of APOH manipulation in HBV-producing cells (HepG2.2.15 cells). RESULTS: We found that HBV upregulates APOH expression in 293 T cells, and APOH overexpression subsequently inhibits secretion of HBsAg. Next, we show that LHBs overexpression in conjunction with APOH leads to ER stress in 293 T cells, as evidenced by production of the binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), as well as increased splicing of X-box binding protein 1 (XBP1). We further observed that loss of beta2-GPI reduced CHOP expression in HepG2.2.15 cells, while beta2-GPI overexpression enhanced CHOP production. CONCLUSION: The interaction of beta2-GPI and HBV initiates ER stress through driving intracellular retention of HBsAg and activates the UPR.

Complement Activation and Thrombin Generation by MBL Bound to ß2-Glycoprotein I.

ß2-Glycoprotein I (ß2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to ß2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of ß2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and ß2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to ß2-GPI was detected on the surface of HUVECs, and colocalization of MBL with ß2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because ß2-GPI-mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the ß2-GPI/MBL complex may contribute to amplify similar activities of anti-ß2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.

Anti-beta-2-glycoprotein I domain 1 identifies antiphospholipid antibodies-related injuries in patients with concomitant lupus nephritis.

BACKGROUND: In this study we aimed to evaluate the usefulness of domain profiling of Beta-2-glycoprotein I(ß2GPI)-Domain-1 (D1) antibodies in relation to antiphospholipid antibodies (aPL)-related nephropathy (aPL-N) in patients with biopsy-proven lupus nephritis (LN). METHODS: Of 124 consecutive patients (96 women, mean age 45.5 ± 12.3 years, mean disease duration 14.7 ± 9.6 years) fulfilling the 1982 criteria for systemic lupus erythematosus (SLE), we identified 39 patients (mean age 39.84 ± 8.6 years, mean disease duration 11.3 ± 7.7 years) with the following characteristics: (a) biopsy-proven LN; (b) no previous diagnosis of antiphospholipid syndrome (APS) according to the current classification criteria. RESULTS: Patients with both LN and aPL-N had higher median aß2GPI-D1 antibody titres (220.1 CU, 25-75th IQ 29.1-334.2) as compared those with LN alone (46.5 CU, 25-75th IQ 12.5-75.1) (p = 0.0087). Median aß2GPI-D1 antibody titres were higher in patients with acute thrombotic microangiopathy (aTMA) (N = 7) (250.1 CU, 25-75th IQ 61.2-334.2) vs. with LN alone (46.5 CU, 25-75th IQ 12.5-75.1 CU) (p = 0.0009). Having a Global Antiphospholipid Syndrome Score > 10 confers an increased probability of having acute features of aTMA (OR 6.25, 95%CI 1.2-31.8). As compared to other aPL, aß2GPI-D1 antibodies have the best diagnostic accuracy for aTMA as evaluated by performances in Area Under the Curves in a ROC analysis. CONCLUSIONS: aß2GPI-D1 antibodies detection might provide a second-line assay to be performed in aß2GPI positive patients with LN, allowing more accurate stratification of the renal vascular involvement risk, thus potentially leading to a more tailored management.

B cell-derived anti-beta 2 glycoprotein I antibody contributes to hyperhomocysteinaemia-aggravated abdominal aortic aneurysm.

AIMS: Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation. METHODS AND RESULTS: ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-ß2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-ß2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in µMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into µMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with µMT mice that received plasma IgG from control mice, µMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-ß2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining. CONCLUSION: Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-ß2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

Atherosclerosis: Beyond the lipid storage hypothesis. The role of autoimmunity.

Atherosclerosis has long been considered as a lipid storage disease. Recent data suggest that autoimmune mechanisms seem to be involved in the pathophysiology of atherosclerosis. The presence of activated endothelial vascular cells, neutrophils, macrophages, T and to a lesser extent B cells in atherosclerotic plaques, together with the proinflammatory cytokine burden suggest mobilization of both innate and adaptive immune pathways in atherosclerosis pathobiology. The development of antibodies to oxidized low-density lipoprotein (ox-LDL), the experimental induction of atherosclerosis either via the transfer of T cells or immunization with autoantigens such as ß2 glycoprotein Ι (ß2-GPI) and heat shock proteins (HSP) further support the autoimmune nature of atherosclerosis. However, classical immunosuppressive and immune-modulatory drugs, successfully used in the therapy of autoimmune rheumatic diseases have shown limited benefits so far in the treatment of atherosclerosis.

An in vitro model to mimic the thrombotic occlusion of small vessels in catastrophic antiphospholipid syndrome (CAPS).

Platelet activation and decrease in platelet count characterize the development of the most feared form of antiphospholipid syndrome (APS), i.e. catastrophic APS (CAPS). We aimed to assess if immuno-affinity purified anti-ß2-glycoprotein I (aß2GPI) antibodies enhance platelet activation inducing a significant flow obstruction in a platelet function analyzer (PFA). Affinity purified aß2GPI antibodies were obtained from 13 triple positive patients with a strong lupus anticoagulant (LA) and high titers of IgG anticardiolipin antibodies (aCL) and IgG aß2GPI. Platelet activation stimulated by adenosine diphosphate (ADP) in the presence or absence of aß2GPI was measured by the expression of P-selectin on platelet surface using flow cytometry. P-selectin expression remained close to baseline when normal whole blood was incubated with aß2GPI alone. When stimulated using aß2GPI combined with ADP, P-selectin expression (28.42 ± 5.15% vs. 20.98 ± 3.94%, p = 0.0076) was significantly higher than ADP alone. Closure time of normal whole blood passed through the PFA was significantly shorter using affinity purified aß2GPI than control IgG both in Col/ADP (160.1 ± 62.1 s vs. 218.6 ± 43.8 s; p = 0.021) and Col/EPI cartridges (149.5 ± 26.7 s vs. 186.9 ± 45.5 s; p = 0.030). Thus, platelet activation is enhanced by aß2GPI antibodies with a consequent premature closure in a PFA, possibly resembling that in microcirculation in patients with CAPS.

Epidemiological, clinical and immune factors that influence the persistence of antiphospholipid antibodies in leprosy.

INTRODUCTION: Antiphospholipid antibodies (aPL) are described in individuals with leprosy without the clinical features of antiphospholipid antibody syndrome (APS), a condition involving thromboembolic phenomena. We have described the persistence of these antibodies for over 5 years in patients with leprosy after specific treatment. OBJECTIVES: To determine whether epidemiological, clinical and immunological factors played a role in the long-term persistence of aPL antibodies in leprosy patients after multidrug therapy (MDT) had finished. METHODS: The study sample consisted of 38 patients with a diagnosis of leprosy being followed up at the Dermatology and Venereology Outpatient Department at the Alfredo da Matta Foundation (FUAM) in Manaus, AM. ELISA was used to detect anticardiolipin (aCL) and anti-ß2 glycoprotein I (anti-ß2GPI) antibodies. Patients were reassessed on average of 5 years after specific treatment for the disease (MDT) had been completed. RESULTS: Persistence of aPL antibodies among the 38 leprosy patients was 84% (32/38), and all had the IgM isotype. Mean age was 48.1 ± 15.9 years, and 23 (72.0%) were male. The lepromatous form (LL) of leprosy was the most common (n = 16, 50%). Reactional episodes were observed in three patients (9.4%). Eighteen (47.37%) were still taking medication (prednisone and/or thalidomide). Mean IgM levels were 64 U/mL for aCL and 62 U/mL for anti-ß2GPI. In the multivariate binary logistic regression the following variables showed a significant association: age (p = 0.045, OR = 0.91 and CI 95% 0.82-0.98), LL clinical presention (p = 0.034; OR = 0.02 and CI 95% = 0.0-0.76) and bacterial index (p = 0.044; OR = 2.74 and CI 95% = 1.03-7.33). We did not find association between prednisone or thalidomide doses and positivity for aPL (p = 0.504 and p = 0.670, respectively). No differences in the variables vascular thrombosis, pregnancy morbidity, diabetes, smoking and alcoholism were found between aPL-positive and aPL-negative patients. CONCLUSION: Persistence of positivity for aPL antibodies was influenced by age, clinical presentation and bacterial index. However, further studies are needed to elucidate the reason for this persistence, the role played by aPL antibodies in the disease and the B cell lineages responsible for generation of these antibodies.

Amniotic fluid antiphospholipid antibodies: potential role in antiphospholipid syndrome-independent aberrant implantation process.

BACKGROUND: The direct role of antiphospholipid antibodies (aPL) at maternal-fetal interface has not been fully investigated, especially whether they are involved in physiological and pathological implantation conditions, in an antiphospholipid syndrome (APS)-independent manner. In fact, trophoblast cells and placental endothelial cells at the implantation site express potential aPL targeted-phospholipid antigens (PL Ags); thus, the local production and presence of their specific antibodies, not related to APS (characterized by aPL presence in the peripheral blood), could be a potential marker of aberrant invasion, implantation and fetal-maternal immune tolerance processes. METHODS: Anti-Beta2glycoprotein I (anti-ß2GPI) and anticardiolipin (aCL Ab) antibodies (the most clinically relevant aPL) were detected by immunoenzymatic assay (ELISA), in the amniotic fluid (AF) of 167 women with physiological and complicated common pregnancy conditions, sharing an aberrant implantation process, such as recurrent pregnancy loss (RPL), autoimmune hypothyroidism (ahT) and smoking. All women included in the study were negative to peripheral blood aPL. RESULTS: aCL and anti-ß2GPI antibodies were detectable in all the AF samples. RPL, ahT and smoking patients had higher level of anti-ß2GPI Abs (IgM) compared to women with physiological pregnancies (p < 0.0001). Since IgM cannot cross the placenta, their local production in response to maternal-fetal interface stimuli, could be hypothesized. CONCLUSIONS: The presence of aPL in the AF (not related to APS) could reveal a potential clinical significance at maternal-fetal interface in selected pregnancy complications, in which an aberrant implantation process, and in turn an impaired fetal-maternal immune tolerance cross-talk, could occur.

Effects of ß2/aß2 on oxLDL-induced CD36 activation in THP-1 macrophages.

AIMS: ß2-glycoprotein I/anti-ß2-glycoprotein I antibody complex (ß2/aß2) could promote oxLDL-induced endothelial inflammation through Toll-like receptor 4 (TLR4), therefore accelerates atherosclerosis in patients with anti-phospholipid syndrome (APS). However, effects of ß2/aß2 and TLR4 on oxLDL-induced CD36 activation in macrophages remain to be elucidated and are currently under investigation. MATERIALS AND METHODS: THP-1 macrophages with or without the pre-treatment of TAK-242, a TLR4 inhibitor, were treated with RPMI 1640, oxLDL, oxLDL+ß2/aß2 or oxLDL + LPS.CD36 expression and subsequent intracellular lipid accumulation, cholesterol-transportation-related proteins (ACAT1, ABCG1 and ABCA1) expression, inflammatory cytokines (IL-1ß, TNF-α and IL-6) secretion, focal adhesion kinases (FAK) activation and matrix metalloproteinases (MMP-2 and MMP-9) expression by these THP-1 macrophages were evaluated. Moreover, effects of TLR4 on oxLDL+ß2/aß2-induced peroxisome proliferators-activated receptor-γ (PPAR-γ) expression and CD36 translocation have also been observed. KEY FINDINGS: Compared with oxLDL-treated ones, CD36 expression, intracellular lipid accumulation and FAK activation were inhibited, whereas the levels of inflammatory cytokines and MMPs were upregulated in THP-1 macrophages treated with oxLDL+ß2/aß2 (p < 0.05). Moreover, observed differences between oxLDL-treated and oxLDL+ß2/aß2-treated THP-1 macrophages could be reversed by TAK-242 pre-treatment (p < 0.05). Furthermore, oxLDL+ß2/aß2 promoted PPAR-γ expression and CD36 cytoplasmic translocation in THP-1 macrophages, these effects could also be attenuated by TAK-242 (p < 0.05). SIGNIFICANCE: Through a TLR4 dependent manner, ß2/aß2 inhibited oxLDL-induced CD36 expression, lipid accumulation and FAK activation, while promoted inflammatory cytokines and MMPs expression in THP-1 macrophages, indicating the novel dual roles played by ß2/aß2 in APS-related atherosclerosis.

Autoantibodies' titre modulation by anti-BlyS treatment in systemic lupus erythematosus.

OBJECTIVE: The objective of this study was to analyse autoantibodies' titres modulation during belimumab treatment in 50 patients with systemic lupus erythematosus (SLE). METHODS: Sera were collected at belimumab start (T0) and every six months until the 24th month. Disease activity index (SLEDAI-2K) was analysed at every timepoint. High avidity anti-dsDNA was detected by radioimmunological method, anti-ENA, anti-cardiolipin antibodies (aCL), anti-ß2 glycoprotein I (anti-ß2GPI) were analysed by ELISA. RESULTS: Fifty patients with SLE (mean SLEDAI-2K: 7.18 ± :3), mean age of 39 ± 11 years and mean follow-up of 13 ± 7.8 years were enrolled. A significant decrease of anti-dsDNA and anti-ß2GPI IgM titres was observed at all timepoints. IgG aCL titre showed significant decrease only at T18. Anti-dsDNA negativization was detected in 21%, anti-ß2GPI IgG in 33% and aCL IgG in 30% of sera, mostly at T6. Anti-ribosomal showed a significant titre decrease at T6 and T12, with negative seroconversion at T18. Anti-Sm titre significantly dropped down at T6, then remained stable during the time. Significant correlations were found between anti-dsDNA and anti-ribosomal titre and between SLEDAI ratio (SLEDAI value/SLEDAI T0) and anti-ribosomal titre ratio (value/value T0). CONCLUSIONS: Belimumab treatment induced a significant reduction of SLE-specific autoantibodies titre and IgM anti-ß2GPI. Anti-ribosomal titre decrease correlates with anti-dsDNA titre and disease activity improvement.

Role of TLR­4 in anti­ß2­glycoprotein I­induced activation of peritoneal macrophages and vascular endothelial cells in mice.

Anti­phospholipid syndrome (APS) is a systematic autoimmune disease that is associated with presence of antiphospholipid antibodies (aPL), recurrent thrombosis, and fetal morbidity in pregnancy. Toll­like receptor­4 (TLR­4), a member of TLR family, is known to have a fundamental role in pathogen recognition and activation of innate immunity. The ß2­glycoprotein I (ß2GPI), a protein circulating in the blood at a high concentration, is able of scavenging lipopolysaccharide (LPS) and clear unwanted anionic cellular remnants, such as microparticles, from the circulation. Our previous study demonstrated that TLR­4 and its signaling pathways contribute to the upregulation of pro­coagulant factors and pro­inflammatory cytokines in monocytes induced by anti­ß2GPI in vitro. The present study aimed to define the roles of TLR­4 in vivo. C3H/HeN mice (TLR­4 intact) and C3H/HeJ mice (TLR­4 defective) were stimulated with an intraperitoneal injection with anti­ß2GPI­immunoglobulin G(IgG), then peritoneal macrophages and vascular endothelial cells (VECs) were extracted from treated mice, and analyses were conducted on the expression profiles of pro­inflammatory cytokines and adhesion molecules. The results demonstrated that the expression of pro­inflammatory cytokines, including tumor necrosis factor­α (TNF­α), interleukin (IL)­1ß and IL­6, in the peritoneal macrophages, and adhesion molecules, including intercellular cell adhesion molecule­1 (ICAM­1), vascular cell adhesion molecule­1 (VCAM­1) and E­selectin, in VECs of C3H/HeN mice (TLR­4 intact) were significantly higher than those of C3H/HeJ mice (TLR­4 defective). The phosphorylation levels of p38 mitogen­activated protein kinase (MAPK) and nuclear factor­κB (NF­κB) p65 in peritoneal macrophages and VECs from C3H/HeN mice stimulated with anti­ß2GPI­IgG were significantly increased compared with those from C3H/HeJ mice (TLR­4 defective). The isotype control antibody (NR­IgG) had no such effects on peritoneal macrophages and VECs. Furthermore, the inhibitors of TLR­4, p38 MAPK and NF­κB may significantly reduce the anti­ß2GPI­IgG­induced TNF­α, IL­1ß and IL­6 mRNAs expression in the peritoneal macrophages from TLR­4 intact mice. The results indicated that a TLR­4 signal transduction pathway is involved in anti­ß2GPI­IgG­induced activation of peritoneal macrophages and VECs. This study has provided a basis for subsequent investigations to elucidate the pathological mechanisms underlying anti­phospholipid syndrome.

Immune Complexes of Beta-2-Glycoprotein I and IgA Antiphospholipid Antibodies Identify Patients With Elevated Risk of Thrombosis and Early Mortality After Heart Transplantation.

Background: The presence of anti-Beta 2 glycoprotein antibodies (aB2GP1) of IgA isotype is common in patients with functional impairment of the organs in which B2GP1 is elaborated. Pretransplant IgA aB2GP1 has been associated with increased risk of thrombosis in kidney and heart transplanted patients and has also been related with early mortality after heart transplantation. Circulating immune complexes between IgA and B2GP1 (B2A-CIC) have been described in the blood of patients positive for IgA aB2GP1 with thrombotic clinical symptoms. In kidney transplanted patients, B2A-CIC is a biomarker that predicts which patients IgA aB2GP1 positive are at risk of thrombosis events following kidney transplantation and may lead to early prophylactic treatment. The prevalence of B2A-CIC and its relation with outcomes after heart transplantation is not known. Methods: Follow-up study based on 151 consecutive patients who received a heart transplant. Autoantibodies and B2A-CIC were quantified in pre-transplant serum samples. Three groups of patients were followed-up for 2 years: Group-1, positive for IgA aB2GP1 and B2A-CIC (N = 19). Group-2, only positive for IgA aB2GP1 (N = 28). Group-0 (control group): IgA aB2GP1 negative (N = 104). Results: Kaplan-Meir survival analysis showed that mortality in B2A-CIC positive was higher than group-0 at 3 months (HR:5.08; 95%CI: 1.36-19.01) and at 2 years (HR:3.82; 95%CI: 1.54-12.66). No significant differences were observed between group-2 and group-0. Multivariate analysis identified B2A-CIC as the most important independent risk factor for early mortality (OR = 6.12; 95% CI: 1.93-19.4). Post-transplant incidence of thrombosis was significantly higher in B2A-CIC positive patients than in the control group (OR: 6.42; 95%CI: 2.1-19.63). Multivariate analysis identified the presence of B2A-CIC (OR: 6.13; 95%CI: 2.1-19.63) and the pre-transplant habit of smoking actively (OR: 4.18; 95%CI: 1.35-12.94) as independent risk factor for thrombosis. The proportion of patients who had thrombotic events or died in the first trimester was significantly higher in group-1 (73.7%) than in group-0 (16.3%; p < 0.001) and in group-2 (39.3%; p = 0.02). Multivariate analysis identified B2A-CIC as the main independent risk factor for early outcomes (mortality or thrombosis) in the first 3 months after heart transplant (OR = 11.42, 95% CI: 1.69-9.68). Conclusion: B2A-CIC are a predictor of early mortality and thrombosis after heart transplant.

[oxLDL/ß2GPI/anti-ß2GPI antibody complex inhibits autophagy in RAW264.7 cells through the activation of AKT pathway].

Objective To investigate the effect of oxidative low-density lipoprotein/ß2 glycoprotein I/anti-ß2 glycoprotein I antibody (oxLDL/ß2GPI/anti-ß2GPI antibody) complex on autophagy in mouse macrophages (RAW264.7 cells) and its mechanism. Methods RAW264.7 cells were stimulated with medium, oxLDL, oxLDL/ß2GPI complex, oxLDL/ß2GPI/anti-ß2GPI antibody complex, oxLDL/anti-ß2GPI antibody complex and ß2GPI/anti-ß2GPI antibody complex separately. The protein levels of autophagy-associated proteins including LC3 and P62 were detected by Western blot analysis. The tandem mRFP-GFP-LC3 adernovirus was used to detect the patency of autophagosomes and autophagy flow. The AKT pathway inhibitor LY294002 (50 µmol/L) was used for investigating the role of AKT pathway in the autophagy in RAW264.7 cells treated by oxLDL/ß2GPI/anti-ß2GPI antibody complex. Results oxLDL/ß2GPI/anti-ß2GPI antibody complex reduced the level of LC3-II protein and the number of autophagosomes, increased the level of P62 protein and blocked the autophagy flow. In addition, the autophagy in RAW264.7 cells was partially restored by the pre-treatment of LY294002. Conclusion The oxLDL/ß2GPI/anti-ß2GPI antibody complex can inhibit the autophagy in RAW264.7 cells through activating the AKT pathway.

[OxLDL/ß2GPI/anti-ß2GPI complex promotes calcification and inflammatory cytokine expression in A7r5 cells].

Objective To evaluate the effect of oxidized low-density lipoprotein/ß2-glycoprotein I/anti-ß2 glycoprotein I antibody (oxLDL/ß2GPI/anti-ß2GPI) complex on the calcification and inflammatory cytokine expression in A7r5 rat vascular smooth muscle cells, and to find out the role of Toll-like receptor 4 (TLR4) signal in this process. Methods The A7r5 cells were intervened with oxLDL, oxLDL/ß2GPI complex, oxLDL/anti-ß2GPI complex, ß2GPI/anti-ß2GPI complex, and oxLDL/ß2GPI/anti-ß2GPI complex for different time, with or without TLR4 inhibitor (TAK-242). Alizarin red staining was used to observe the calcification. Real-time quantitative PCR was applied to detect the total mRNA levels of actin-associated protein SM22α (trangelin) and Runt-related transcription factor 2 (RUNX2). The protein levels of SM22α and RUNX2 were measured by Western blotting. Tumor necrosis factor α (TNF-α) was detected by ELISA. Different concentrations of TNF-α were adopted to stimulate A7r5 cells, and the above methods were operated to examine the changes of SM22 and RUNX2. Results When A7r5 cells were incubated by oxLDL/ß2GPI/anti-ß2GPI complex, more calcified nodules were observed, the expression of SM22α was reduced and RUNX2 expression was enhanced at both mRNA and protein levels. And the complex increased the expression of inflammation cytokine TNF-α. TLR4 inhibitor TAK-242 reversed the phenomenon. Different concentrations of TNF-α could reduce mRNA and protein expression of SM22α while raise RUNX2 expression. Conclusion oxLDL/ß2GPI/anti-ß2GPI complex can increase the expression of cytokine TNF-α, and thus quicken calcification procedure in A7r5 cells, along with the change of the traditional contraction phenotype into the osteogenic phenotype. TLR4 receptor participates in this process.

Brief Report: How Do Patients With Newly Diagnosed Systemic Lupus Erythematosus Present? A Multicenter Cohort of Early Systemic Lupus Erythematosus to Inform the Development of New Classification Criteria.

OBJECTIVE: Systemic lupus erythematosus (SLE) presents with nonspecific signs and symptoms that are also found in other conditions. This study aimed to evaluate manifestations at disease onset and to compare early SLE manifestations to those of diseases mimicking SLE. METHODS: Academic lupus centers in Asia, Europe, North America, and South America collected baseline data on patients who were referred to them during the previous 3 years for possible SLE and who had a symptom duration of <1 year. Clinical and serologic manifestations were compared between patients diagnosed as having SLE and those diagnosed as having SLE-mimicking conditions. Diagnostic performance of the 1997 American College of Rheumatology (ACR) SLE classification criteria and the 2012 Systemic Lupus International Collaborating Clinics (SLICC) SLE classification criteria was tested. RESULTS: Data were collected on 389 patients with early SLE and 227 patients with SLE-mimicking conditions. Unexplained fever was more common in early SLE than in SLE-mimicking conditions (34.5% versus 13.7%, respectively; P < 0.001). Features less common in early SLE included Raynaud's phenomenon (22.1% versus 48.5%; P < 0.001), sicca symptoms (4.4% versus 34.4%; P < 0.001), dysphagia (0.3% versus 6.2%; P < 0.001), and fatigue (28.3% versus 37.0%; P = 0.024). Anti-double-stranded DNA, anti-ß2 -glycoprotein I antibodies, positive Coombs' test results, autoimmune hemolytic anemia, hypocomplementemia, and leukopenia were more common in early SLE than in SLE-mimicking conditions. Symptoms detailed in the ACR and SLICC classification criteria were significantly more frequent among those with early SLE. Fewer patients with early SLE were not identified as having early SLE with use of the SLICC criteria compared to the ACR criteria (16.5% versus 33.9%), but the ACR criteria demonstrated higher specificity than the SLICC criteria (91.6% versus 82.4%). CONCLUSION: In this multicenter cohort, clinical manifestations that could help to distinguish early SLE from SLE-mimicking conditions were identified. These findings may aid in earlier SLE diagnosis and provide information for ongoing initiatives to revise SLE classification criteria.

Anti-RNP/Sm antibodies in patients with systemic lupus erythematosus and its role in thrombosis: a case-control study.

OBJECTIVE: To validate the association of thrombotic events with positive lupus anticoagulant (LA) and co-presence of anti-RNP/Sm, as well as the diagnostic accuracy of this combination of antibodies for thrombosis. METHODS: Case-control study of patients with systemic lupus erythematosus (SLE) who presented thrombosis after SLE diagnosis and controls with SLE without thrombosis. Comorbidities, traditional risk factors, clinical variables, and treatment were evaluated. Antiphospholipid (aPL) and anti-RNP/Sm antibodies were determined. RESULTS: Sixty-three cases and 63 controls were studied, 88% women, median age of 40 years, and disease duration of 135 months at study inclusion. No differences were found between groups regarding age, comorbidities, or clinical characteristics at SLE diagnosis. Patients with thrombosis were more frequently positive for anti-RNP/Sm (p = 0.001), IgG aCL (p = 0.02), IgG anti-B2GPI (p = 0.02), IgM anti-B2GPI (p = 0.02), LA (p < 0.001), the combination of anti-RNP/Sm + LA (p < 0.001), and aPL triple marker (p = 0.002), compared to controls. The combination of anti-RNP/Sm + LA, SLEDAI-2 K, and prednisone dose was associated with thrombosis (p < 0.05). The combination of anti-RNP/Sm + LA showed 56% sensitivity, 79% specificity, 73% positive predictive value, 64% negative predictive value, positive likelihood ratio (LR) 2.69, and negative LR 0.56 for predicting thrombosis. No difference was found in the comparison of area under the curve between LA alone and the combination of anti-RNP/Sm + LA (p = 0.73). CONCLUSION: Thrombosis was associated with disease activity, dose of prednisone, and the combination of anti-RNP/Sm antibodies and LA. This combination of antibodies could be useful in the identification of SLE patients at risk of thrombosis.

Anti-ß2 -glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression.

Autoantibodies characteristic for anti-phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are anti-ß2 -glycoprotein I (ß2 GPI) antibodies and anti-DNA antibodies, respectively, and almost half of APS cases occur in SLE. Anti-ß2 GPI antibodies are recognized to play a pivotal role in inducing a prothrombotic state, but the precise mechanism has not been fully elucidated. In a widely accepted view, binding of anti-ß2 GPI antibodies to cell surface ß2 GPI in monocytes and endothelial cells triggers the Toll-like receptor 4-myeloid differentiation primary response 88 (TLR)-4-MyD88) signaling pathway which leads to activation of p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinases (MEK-1/ERK) and/or nuclear factor kappa B (NF-κB) and expression of tissue factor (TF). However, resting cells do not express substantial amounts of TLR-4. Previously, we generated a mouse monoclonal anti-ß2 GPI antibody WB-6 and showed that it induced a prothrombotic state - including TF expression on circulating monocytes - in normal mice. In the current study, we aimed to clarify the mechanism of interaction between WB-6 and resting monocytes, and found that WB-6 exhibits binding activity to DNA and enters living monocytes or a monocytic cell line and, to a lesser extent, vascular endothelial cells. Treatment of the cells with DNase I reduced the internalization, suggesting the involvement of cell surface DNA in this phenomenon. Monocytes harboring internalized WB-6 expressed TF and tumor necrosis factor (TNF)-α which, in turn, stimulated endothelial cells to express intercellular adhesion molecule 1 (ICAM-I) and vascular cell adhesion molecule 1 (VCAM-I). These results suggest the possibility that a subset of anti-ß2 GPI antibodies with dual reactivity to DNA possesses ability to stimulate DNA sensors in the cytoplasm, in addition to the cell surface receptor-mediated pathways, leading to produce proinflammatory and prothrombotic states.