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Antiphospholipid syndrome (APS) is characterized by thrombus formation, poor pregnancy outcomes, and a proinflammatory response. H3K4me3-related monocytes activation are key regulators of APS pathogenesis. Therefore, H3K4me3 CUT&Tag and ATAC-seq are performed to examine the epigenetic profiles. The results indicate that the H3K4me3 signal and chromatin accessibility at the FOXJ2 promoter are enhanced in an in vitro monocyte model by stimulation with β2GPI/anti-β2GPI, which mimics APS, and decreases after OICR-9429 administration. Furthermore, FOXJ2 is highly expressed in patients with primary APS (PAPS) and is the highest in patients with triple-positive antiphospholipid antibodies (aPLs). Mechanistically, FOXJ2 directly binds to the SLAMF8 promoter and activates SLAMF8 transcription. SLAMF8 further interacts with TREM1 to stimulate TLR4/NF-κB signaling and prohibit autophagy. Knockdown of FOXJ2, SLAMF8, or TREM1 blocks TLR4/NF-κB and provokes autophagy, subsequently inhibiting the release of inflammatory and thrombotic indicators. A mouse model of vascular APS is established via β2GPI intraperitoneal injection, and the results suggest that OICR-9429 administration attenuates the inflammatory response and thrombus formation by inactivating FOXJ2/SLAMF8/TREM1 signaling. These findings highlight the overexpression of H3K4me3-mediated FOXJ2 in APS, which consequently accelerates APS pathogenesis by triggering inflammation and thrombosis via boosting the SLAMF8/TREM1 axis. Therefore, OICR-9429 is a promising candidate drug for APS therapy., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Apolipoprotein H (APOH) downregulation can cause hepatic steatosis and gut microbiota dysbiosis. However, the mechanism by which APOH-regulated lipid metabolism contributes to metabolic dysfunction-associated steatotic liver disease (MASLD) remains undetermined. Herein, we aim to explore the regulatory effect of APOH, mediated through various pathways, on metabolic homeostasis and MASLD pathogenesis. We analyzed serum marker levels, liver histopathology, and cholesterol metabolism-related gene expression in global ApoH -/-  C57BL/6 male mice. We used RNA sequencing and metabolomic techniques to investigate the association between liver metabolism and bacterial composition. Fifty-two differentially expressed genes were identified between ApoH -/-  and WT mice. The mRNA levels of de novo lipogenesis genes were highly upregulated in ApoH -/-  mice than in WT mice. Fatty acid, glycerophospholipid, sterol lipid, and triglyceride levels were elevated, while hyodeoxycholic acid levels were significantly reduced in the liver tissues of ApoH -/-  mice than in those of WT mice. Microbial beta diversity was lower in ApoH -/-  mice than in WT mice, and gut microbiota metabolic functions were activated in ApoH -/-  mice. Moreover, ApoH transcripts were downregulated in patients with MASLD, and APOH-related differential genes were enriched in lipid metabolism. Open-source transcript-level data from human metabolic dysfunction-associated steatohepatitis livers reinforced a significant association between metabolic dysfunction-associated steatohepatitis and APOH downregulation. In conclusion, our studies demonstrated that APOH downregulation aggravates fatty liver and induces gut microbiota dysbiosis by dysregulating bile acids. Our findings offer a novel perspective on APOH-mediated lipid metabolic dysbiosis and provide a valuable framework for deciphering the role of APOH in fatty liver disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The expression of TTR and apolipoprotein H (APOH) genes and their relationship with prognosis in patients with colorectal cancer (CRC) metastasis by using bioinformatics analysis techniques are explored. The expression profiles of related genes in patients with CRC metastasis are retrieved from the Gene Expression Omnibus (GEO) database. The core genes transthyretin (TTR) and APOH are screened by constructing protein-protein interaction (PPI) network, and the corresponding patient data of 327 patients are extracted and included in the metastasis group. The TTR and APOH genes of 300 patients without CRC metastasis are screened and included in the control group. The relationship between the expression levels of TTR and APOH and the clinicopathological parameters of patients with CRC metastasis is analyzed. Kaplan-Meier survival curve is drawn to observe the influence of overexpression and low expression of TTR and APOH on the prognosis and survival of patients in the metastatic group. Receiver operating characteristic (ROC) curve is drawn to observe the prognostic efficacy of combined TTR and APOH detection in patients with CRC metastasis. The experimental results show that bioassay can confirm the close relationship between TTR, APOH, and patients with CRC metastasis. Regular detection of serum TTR and APOH expression can effectively assess the patient's condition and take measures to improve the prognosis of the patients., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Ye Lu et al.)

Objectives: Tissue factor (TF) and Human apolipoprotein H (APOH) seem to be significantly associated with a clinical manifestation in systemic lupus erythematosus (SLE) patients with or without APS, mostly because of thrombotic events and coagulation processes. Additionally, according to recent studies, these two factors appear to be an important part of immune response and inflammation., Methods: The objective of this study was to investigate three SNPs of APOH (rs4581, rs8178835 and rs818819) and three of TF (rs958587, rs3917615, rs1361600) in SLE patients and healthy subjects using TaqMan genotyping assay and their association with inflammatory cytokines level in serum and selected clinical parameters., Results: Present study revealed that TF rs3917615 and rs958587 and APOH rs4581 possibly predispose to joint involvement in SLE., Conclusions: Analysed genetic variants of TF and APOH may have an impact on inflammatory processes and clinical relevance in SLE patients in the Caucasian population.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Lp(a) (lipoprotein[a]) is an independent risk factor for cardiovascular diseases and plasma levels are primarily determined by variation at the LPA locus. We performed a genome-wide association study in the UK Biobank to determine whether additional loci influence Lp(a) levels. Approach and Results: We included 293 274 White British individuals in the discovery analysis. Approximately 93 095 623 variants were tested for association with natural log-transformed Lp(a) levels using linear regression models adjusted for age, sex, genotype batch, and 20 principal components of genetic ancestry. After quality control, 131 independent variants were associated at genome-wide significance (P ≤5×10 -8 ). In addition to validating previous associations at LPA , APOE , and CETP , we identified a novel variant at the APOH locus, encoding β2GPI (beta2-glycoprotein I). The APOH variant rs8178824 was associated with increased Lp(a) levels (β [95% CI] [ln nmol/L], 0.064 [0.047-0.081]; P =2.8×10 -13 ) and demonstrated a stronger effect after adjustment for variation at the LPA locus (β [95% CI] [ln nmol/L], 0.089 [0.076-0.10]; P =3.8×10 -42 ). This association was replicated in a meta-analysis of 5465 European-ancestry individuals from the Framingham Offspring Study and Multi-Ethnic Study of Atherosclerosis (β [95% CI] [ln mg/dL], 0.16 [0.044-0.28]; P =0.0071)., Conclusions: In a large-scale genome-wide association study of Lp(a) levels, we identified APOH as a novel locus for Lp(a) in individuals of European ancestry. Additional studies are needed to determine the precise role of β2GPI in influencing Lp(a) levels as well as its potential as a therapeutic target.

Aims: β2-glycoprotein I/anti-β2-glycoprotein I antibody complex (β2/aβ2) 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 β2/aβ2 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+β2/aβ2 or oxLDL + LPS.CD36 expression and subsequent intracellular lipid accumulation, cholesterol-transportation-related proteins (ACAT1, ABCG1 and ABCA1) expression, inflammatory cytokines (IL-1β, 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+β2/aβ2-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+β2/aβ2 (p < 0.05). Moreover, observed differences between oxLDL-treated and oxLDL+β2/aβ2-treated THP-1 macrophages could be reversed by TAK-242 pre-treatment (p < 0.05). Furthermore, oxLDL+β2/aβ2 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, β2/aβ2 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 β2/aβ2 in APS-related atherosclerosis., (Copyright © 2019. Published by Elsevier Inc.)

The highly glycosylated ß-2-glycoprotein-1 (B2GP1), also called apolipoprotein H, is a 50 kDa human plasma protein with four or five N-glycosylation sites. Glycosylation of B2GP1 can impact auto antibody recognition leading to the development of antiphospholipid syndrome (APS), which can result in miscarriages or thrombosis. Next to its glycosylation different genetic variants are known to increase the risk of suffering from APS. Here we show that ESI-q/TOF-MS of intact B2GP1 can be used to analyze genetic variants and glycosylation simultaneously. After enrichment of B2GP1 from 16 different plasma samples and subsequent ESI-MS measurement of the intact protein, we detected five different SNPs in our samples either homozygous or heterozygous. The dominant glycan composition shows four biantennary, fully sialylated glycan structures, with a relative proportion of about 30%. We also detected compositions with one or two triantennary glycan structures in lower amounts and fucosylated species with one or two fucosyl residues. Two of our samples showed an unreported partially occupied fifth glycosylation site presumably arising from the presence of SNP variant S88N. Our method allows a fast determination of genetic variants and glycan compositions of human B2GP1 to be potentially used as diagnostic marker., (Copyright © 2019. Published by Elsevier B.V.)

We previously found that circulating β 2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of β 2 -glycoprotein I using structure-function analysis and mapped the critical region within the β 2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different β 2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified β 2 -glycoprotein I, as well as recombinant β 2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with β 2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for β 2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of β 2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of β 2 -glycoprotein I. This is the first study to show the therapeutic potential of β 2 -glycoprotein I D1 in the treatment of melanoma progression., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The study aimed to evaluate the clinical significance of laboratory findings in patients with catastrophic antiphospholipid syndrome (CAPS) and to report the effects of a well-defined treatment protocol in 14 consecutive cases. Thirteen patients (12 presenting one and one presenting two episodes of CAPS) were consecutively treated and monitored between 1986 and 2017. Antiphospholipid antibody (aPL) characteristics of the patients were compared with those of 64 matched controls (45 antiphospholipid syndrome patients and 19 aPL carriers) who did not develop CAPS during the same mean follow-up period (12 years ± 9.9 SD). Triple aPL positivity (IgG/IgM anticardiolipin + IgG/IgM anti-β2Glycoprotein I + lupus anticoagulants) significantly prevailed in the CAPS patients with respect to the controls (p = 0.003). IgG anticardiolipin and IgG anti-β2Glycoprotein I mean antibody titers of the CAPS patients were significantly higher than those of the controls (p = 0.0018 and p = 0.003, respectively). Triple therapy (anticoagulation + plasma exchange + steroids) was administered to all the CAPS cases except for one. Beginning in 2009, intravenous immunoglobulin infusion has also been included in the triple therapy protocol (six patients). All the patients recovered from CAPS; five showed renal failure and one a I-II class New York Heart Association (NYHA) dilated cardiomyopathy. Long-term outcomes of CAPS included a gradual worsening of renal failure in one patient who required hemodialysis 30 years after the acute episode. Renal function improved in the other four patients. The patient affected with dilated cardiomyopathy worsened to a II class NYHA over a five year period. Currently all the patients are alive. A specific antiphospholipid antibody profile could be considered a risk factor associated to CAPS. Early use of a defined treatment protocol based on triple therapy either or not associated with IVIG was associated with recovery in all CAPS patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

DNA vaccines have recently emerged as a therapeutic agent for treating autoimmune diseases, such as multiple sclerosis. Antiphospholipid antibody syndrome (APS) is an autoimmune disease characterized by β2-glycoprotein I (β2-GPI)-targeting antiphospholipid antibodies (APAs) and vascular thrombosis or obstetrical complications. To examine the therapeutic potential of a β2-GPI DNA vaccine, we administered a vaccine mixed with FK506 as an adjuvant to a mouse model of obstetric APS. First, the pCMV3-β2-GPI DNA vaccine, which encodes the full-length human β2-GPI gene, was constructed. Then, we administered the β2-GPI DNA vaccine in 0.1 ml of saline, mixed with or without 100 μg of FK506, intramuscularly to the mice on days 28, 35 and 42. Blood titers of the anti-β2-GPI antibody, platelet counts, activated partial thromboplastin times (aPTTs), and the percentage of fetal loss were measured. We also stimulated murine splenic T cells ex vivo with β2-GPI and determined the T helper cell proportion and cytokine secretion. The administration of the β2-GPI DNA vaccine mixed with FK506 reduced the blood IgG anti-β2-GPI antibody titers and suppressed APS manifestations in mice. The combination also suppressed interferon-γ and interleukin (IL)-17A secretion but increased the Treg cell proportion and IL-10 secretion in murine splenic T cells following ex vivo stimulation with β2-GPI. Our results demonstrated the therapeutic efficacy of a β2-GPI DNA vaccine and FK506 as an adjuvant in a murine model of obstetric APS. Possible mechanisms include the inhibition of Th1 and Th17 responses and the up-regulation of Treg cells., Competing Interests: The authors have declared that no competing interests exist.

Antiphospholipid antibody (aPL)‑mediated antiphospholipid syndrome (APS) is an autoimmune disease. Upon binding to aPL, the primary antigen of aPL, β2‑glycoprotein I (β2‑GP I), induces abnormal immune function, which further activates downstream signaling pathways in the cell and eventually leads to APS. The present study aimed to determine whether β2‑GP I antigen and anti‑β2‑glycoprotein I antibody (aβ2‑GP I), which belong to the aPL class of antibodies, may affect human chorionic epithelium cell (JEG‑3) proliferation, migration and invasion. Recombinant human (rh)β2‑GP I protein was expressed using a prokaryotic expression system and aβ2‑GP I antibody was purified from the blood serum of 10 patients with recurrent pregnancy loss. JEG‑3 cells were stimulated with rhβ2‑GP I and aβ2‑GP I separately or simultaneously, and serum immunoglobulin G of normal pregnant women was used as negative control. Using cell counting kit‑8, cell cycle and transwell assays in addition to EdU staining, it was determined that aβ2‑GP I/rhβ2‑GP I complex markedly increased JEG‑3 cell proliferation, migration and invasion. The results revealed that mRNA levels of inhibitor of nuclear factor (NF)‑κB kinase subunit (IKKβ), myeloid differentiation primary response protein MyD88 (MyD88), NF‑κB and NF‑κB inhibitor α (IκBα), as well as the protein levels of MyD88, IκBα and phospho(p)‑IκBα in JEG‑3 cells increased following incubation with the aβ2‑GP I/rhβ2‑GP I complex. The observed upregulation of p‑IκBα protein suggested that IκBα‑mediated inhibition of NF‑κB was weakened. Furthermore, JEG‑3 cells were transfected with PGMLV‑NF‑κB‑Lu vector. Luciferase activity in JEG‑3‑NFκB‑Luc1 and JEG‑3‑NFκB‑Luc2 cells was enhanced following treatment with aβ2‑GP I/rhβ2‑GP I complex. The present study demonstrated that aβ2‑GP I/rhβ2‑GP I complex activates NF‑κB through MyD88 signal transduction pathway, which further enhances JEG‑3 cell proliferation, migration and invasion.

This study aimed to explore the mechanism of a novel mutation (p.Lys38Glu) in apolipoprotein H (APOH) gene causing hereditary beta2-glycoprotein I (β2GPI) deficiency and thrombosis in a proband with thrombophilia. The plasma level of β2GPI was measured by ELISA and Western blotting, and anti-β2GPI antibody by ELISA. Lupus anticoagulant (LA) was assayed using the dilute Russell viper venom time. Deficiency of the major natural anticoagulants including protein C (PC), protein S (PS), antithrombin (AT) and thrombomodulin (TM) was excluded from the proband. A mutation analysis was performed by amplification and sequencing of the APOH gene. Wild type and mutant (c.112A>G) APOH expression plasmids were constructed and transfected into HEK293T cells. The results showed that the thrombin generation capacity of the proband was higher than that of the other family members. Missense mutation p.Lys38Glu in APOH gene and LA coexisted in the proband. The mutation led to β2GPI deficiency and thrombosis by impairing the protein production and inhibiting the platelet aggregation. It was concluded that the recurrent thrombosis of the proband is associated with the coexistence of p.Lys38Glu mutation in APOH gene and LA in plasma.

The immune responses of males and females to bacterial infections display differences. The mechanisms that underlie this sexual dimorphism are multifactorial. Lipopolysaccharide (LPS) contributes to the pathogenesis of endotoxaemia. We have previously demonstrated that the plasma protein beta-2 glycoprotein-1 (β2GPI) reduces LPS-induced inflammation in male mice. In the present study using a more robust infection model of septicaemia the role of β2GPI is examined in both male and female wild type (WT) and β2GPI deficient (β2GPI -/- ) mice challenged with Escherichia coli (E. coli) intravenously. β2GPI deficiency led to an increase of E. coli colony forming units (CFU) in the circulation of both male and female mice. In male β2GPI -/- mice this was associated with a worse clinical severity score. This difference was not observed between female β2GPI -/- and female WT mice. Male WT mice had decreased levels of total and increased levels of free thiol β2GPI following administration of LPS or E. coli. This pattern of sexual dimorphic response was also observed in our cohort of humans with sepsis. These findings support a role for β2GPI in modulating the sex-specific susceptibility to gram-negative septicaemia.

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.

Homozygote genotype V 247 of the β 2 -glycoprotein-I (β 2 GP-I) gene has been associated with anti-β 2 GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to β 2 GP-I 247 has been little studied., Objective: To evaluate the immune cellular proliferation in response to native and non-native β 2 GP-I 247 valine/leucine phenotype from Mexican patients with PAPS., Methods: We studied 10 patients with PAPS and 10 healthy control subjects (HC). The polymorphism at position 247 of the β 2 GP-I gene was determined by PCR-RFLP and the corresponding β 2 GP-I protein was subsequently purified from normal human plasma by affinity chromatography. PBMC purified from patients and controls were stimulated with β 2 GP-I under native and in non native (reduced) conditions. We also determined the anti-β 2 GP-I production in vitro by B cell clones (EBV) generated in cocultures experiments. Differential Scanning Calorimetry (DSC) was studied to determine the structural differences between the β 2 GP-I 247 valine/leucine isoforms. Cytokine profile (IL-2, IL-4, IL-6, TNFα, INFγ) was evaluated in culture supernatants., Results: PAPS and healthy control PBMCs had a higher proliferative response when stimulated with β 2 GP-I under reduced cultures conditions compared to non-denatured conditions. PBMCs response from PAPS patients was higher. We observed more cell proliferation in response to β 2 GP-I 247 valine/leucine or valine isoforms in non-native conditions. In contrast, this response was not significant against β 2 GP-I 247 leucine. These findings were T CD4 + -dependent. Similar results were obtained with B cell clones derived from PAPS patients, which showed more pronounced proliferation in non native conditions and higher against β 2 GP-I 247 valine. No differences were found in anti-β 2 GP-I production, but high levels of IL-6 in vitro were identified. The structural analysis of both β 2 GP-I 247 isoforms by DSC showed a major conformational change due to a single mutation in the β 2 GP-I variants., Conclusions: PAPS PBMCs had a higher cellular response against β 2 GP-I 247 in non-native culture conditions preferentially to the β 2 GP-I 247 valine phenotype. This effect is T CD4 + dependent and appears to be driven by tertiary structural changes adopted by β 2 GP-I 247 polymorphism., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (β 2 GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL-β 2 GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of β 2 GPI in the fluid phase, suggesting that the pathogenic anti-β 2 GPI antibodies in this in vitro model target this domain.

Background: Immunoglobulin (Ig)A anti-β 2 -glycoprotein I (aB2GP1) antibodies are associated with thrombotic events, cardiovascular morbidity, and death in dialysis patients. About 30% of patients with chronic renal disease are positive for IgA aB2GP1; however, the origin of these antibodies is unknown. It has been speculated that dialysis membranes, age, or etiology of renal base disease are possible precipitating factors, although these factors do not appear to be the source of antibodies. B2GP1 is a protein of 326 amino acids grouped into five domains. Eight polymorphisms have been described; the most important are Val/Leu 247 , which appears to predispose aB2GP1 antibody production in patients with anti-phospholipid syndrome, and Trp/Ser 316 , which appears to have protective antibody production of aB2GP1., Methods: DNA samples from 92 patients with renal failure on hemodialysis were randomly collected with a 1:1 ratio for the positivity for IgA aB2GP1. Forty-six samples were positive for IgA aB2GP1 (group 1) and 46 negative for IgA aB2GP1 (group 2). All samples were anonymized to study polymorphism Val/Leu 247 and polymorphism Trp/Ser 316 ., Results: No significant differences were observed between those who were positive or negative for IgA aB2GP1 in patients with renal failure treated with hemodialysis and the polymorphism located in codons 247 and 316., Conclusions: The two groups of patients have the same prevalence in polymorphisms 247 and 316, and therefore there appears not to be a genetic predisposition in our population. New trigger factors must be studied., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To investigate the role of Toll-like receptor 2 (TLR2) in β2-glycoprotein 1/anti-β2-glycoprotein 1 (β2GP1/anti-β2GP1)-mediated tumor necrosis factor α (TNF-α) expression in mouse peritoneal macrophages., Methods: The peritoneal macrophages from BALB/c mice were treated with β2GP1/anti-β2GP1 complex, agonist of TLR2 (Pam3CSK4) and agonist of TLR4 [lipopolysaccharide (LPS)], inhibitor of TLR2 [monoclonal IgG to mouse TLR2 (anti-mTLR2-IgG)] and inhibitor of TLR4 (TAK-242) in vitro. The mRNA level of TNF-α in the peritoneal macrophages was tested by real-time quantitative PCR, the protein expression of TNF-α was detected by Western blotting and immunofluorescence cytochemistry, and the expression of TLR2 on the surface of the peritoneal macrophages was assessed by flow cytometry., Results: The mRNA and protein expression of TNF-α was significantly enhanced in mouse peritoneal macrophages treated with β2GP1/anti-β2GP1 complex, Pam3CSK4 and LPS. Anti-mTLR2-IgG could inhibit the effects of the above stimuli on TNF-α expression, but its effects were weaker than those of TAK-242. Meanwhile, the combination of anti-mTLR2-IgG and TAK-242 did not show much stronger inhibitory effects. Flow cytometry analysis showed that expression of TLR2 was enhanced by β2GP1/anti-β2GP1 complex, Pam3CSK4 and LPS in mouse peritoneal macrophages. However, anti-mTLR2-IgG, or TAK-242, or combination of both could not inhibit TLR2 expression in macrophages., Conclusion: Both TLR4 and TLR2 could increase the stimulating effect of β2GP1/anti-β2GP1 complex on the expression of TNF-α in mouse peritoneal macrophages.

Apolipoprotein H (ApoH) is a multi-functional plasma glycoprotein that has been associated with negative health outcomes. ApoH levels have high heritability. We undertook a genome-wide association study of ApoH levels using the largest sample to date and replicated the results in an independent cohort (total N = 1,255). In the discovery phase, a meta-analysis of two cohorts, the Sydney Memory and Ageing Study (Sydney MAS) and the Older Australian Twins Study (OATS) (n = 942) revealed genome-wide significant results in or near the APOH gene on chromosome 17 (top SNP, rs7211380, p = 1 × 10(-11)). The results were replicated in an independent cohort, the Hunter Community Study (p < 0.002) (n = 313). Conditional and joint analysis (COJO) confirmed the association of the chromosomal 17 region with ApoH levels. The set of independent SNPs identified by COJO explained 23% of the variance. The relationships between the top SNPs and cardiovascular/lipid/cognition measures and diabetes were assessed in Sydney MAS, with suggestive results observed for diabetes and cognitive performance. However, replication of these results in the smaller OATS cohort was not found. This work provides impetus for future research to better understand the contribution of genetics to ApoH levels and its possible impacts on health.

Reperfusion after a period of ischemia results in reperfusion injury (IRI) which involves activation of the inflammatory cascade. In cardiac IRI, IgM natural antibodies (NAb) play a prominent role through binding to altered neoepitopes expressed on damaged cells. Beta 2 Glycoprotein I (β2GPI) is a plasma protein that binds to neoepitopes on damaged cells including anionic phospholipids through its highly conserved Domain V. Domain I of β2GPI binds circulating IgM NAbs and may provide a link between the innate immune system, IgM NAb binding and cardiac IRI. This study was undertaken to investigate the role of Β2GPI and its Domain V in cardiac IRI using wild-type (WT), Rag-1 -/- and β2GPI deficient mice. Compared with control, treatment with Domain V prior to cardiac IRI prevented binding of endogenous β2GPI to post-ischemic myocardium and resulted in smaller myocardial infarction size in both WT and β2GPI deficient mice. Domain V treatment in WT mice also resulted in less neutrophil infiltration, less apoptosis and improved ejection fraction at 24 h. Rag-1 -/- antibody deficient mice reconstituted with IgM NAbs confirmed that Domain V prevented IgM NAb induced cardiac IRI. Domain V remained equally effective when delivered at the time of reperfusion which has therapeutic clinical relevance.Based upon this study Domain V may function as a universal inhibitor of IgM NAb binding in the setting of cardiac IRI, which offers promise as a new therapeutic strategy in the treatment of cardiac IRI.

Background: Antiphospholipid antibodies (aPL) have been advocated as potential mediators of unexplained female infertility, but no evidence has yet been raised to support such an association., Objectives: To test the hypothesis that aPL might interfere with uterine decidualization, a gene expression study was performed on decidual stromal cells treated with different aPL preparations., Methods: Decidual stromal cells were isolated from first-trimester deciduas obtained from two women undergoing elective abortion, and treated with: (i) a β2GPI-dependent aPL monoclonal antibody (IS3); (ii) IS3 plus TIFI, a synthetic peptide mimicking PL-binding region of β2GPI; and (iii) IgG from healthy subjects (NHS). Gene expression data were acquired using human HT-12 v3 beadchip arrays (Illumina). Differential expression analysis was performed by fitting a gene-wise linear model using the treatment group and decidual source as covariates., Results: In the comparison of IS3 versus IgG NHS-treated decidual cells, gene ontology (GO) enrichment was expressed in terms relating to well-characterized aPL-mediated cellular effects: "inflammatory response," "immune response," "response to stress," "oxydoreductase activity," "metalloendopeptidase activity," and "cytokine/chemokine activity." As expected, almost all genes were up-regulated by IS3 treatment. The same GO categories appeared to be differentially expressed when IS3 treatment was compared to IS3 + TIFI, but with most genes being down-regulated., Conclusions: Given the inflammatory response evinced on gene expression analysis of decidual stromal cells treated with a β2GPI -dependent aPL monoclonal antibody, it is feasible that aPL might interfere with uterine decidualization, affecting the early stages of implantation and ultimately resulting in female infertility.

The human brain and human cognitive abilities are strikingly different from those of other great apes despite relatively modest genome sequence divergence. However, little is presently known about the interspecies divergence in gene structure and transcription that might contribute to these phenotypic differences. To date, most comparative studies of gene structure in the brain have examined humans, chimpanzees, and macaque monkeys. To add to this body of knowledge, we analyze here the brain transcriptome of the western lowland gorilla (Gorilla gorilla gorilla), an African great ape species that is phylogenetically closely related to humans, but with a brain that is approximately one-third the size. Manual transcriptome curation from a sample of the planum temporale region of the neocortex revealed 12 protein-coding genes and one noncoding-RNA gene with exons in the gorilla unmatched by public transcriptome data from the orthologous human loci. These interspecies gene structure differences accounted for a total of 134 amino acids in proteins found in the gorilla that were absent from protein products of the orthologous human genes. Proteins varying in structure between human and gorilla were involved in immunity and energy metabolism, suggesting their relevance to phenotypic differences. This gorilla neocortical transcriptome comprises an empirical, not homology- or prediction-driven, resource for orthologous gene comparisons between human and gorilla. These findings provide a unique repository of the sequences and structures of thousands of genes transcribed in the gorilla brain, pointing to candidate genes that may contribute to the traits distinguishing humans from other closely related great apes., (© 2015 Wiley Periodicals, Inc.)

We identified eight candidate thinness predisposition variants from the Illumina HumanExome chip genotyped on members of pedigrees selected for either healthy thinness or severe obesity. For validation, we tested the candidates for association with healthy thinness in additional pedigree members while accounting for effects of obesity-associated genes: NPFFR2, NPY2R, FTO, and MC4R. Significance was obtained for the interaction of FTO rs9939609 with APOH missense variant rs52797880 (minor allele frequency 0.054). The thinness odds ratio was estimated as 2.15 (p < 0.05) for the combination of APOH heterozygote with the homozygote for the non-obesity FTO allele. Significance was not obtained for any other combination of a candidate variant with an obesity gene or for any of the eight candidates tested independently.

Introduction: The identification of the genetic risk factors that could discriminate non- thrombotic from thrombotic antiphospholipid antibodies (aPLA) carriers will improve prognosis of these patients. Several human studies have shown the presence of aPLAs associated with atherosclerotic plaque, which is a known risk factor for thrombosis. Hence, in order to determine the implication of atherosclerosis in the risk of developing thrombosis in aPLA positive patients, we performed a genetic association study with 3 candidate genes, APOH, LDLR and PCSK9., Material & Methods: For genetic association study we analyzed 190 aPLA carriers -100 with non-thrombotic events and 90 with thrombotic events- and 557 healthy controls. Analyses were performed by χ2 test and were corrected by false discovery rate. To evaluate the functional implication of the newly established susceptibility loci, we performed expression analyses in 86 aPLA carrier individuals (43 with thrombotic manifestations and 43 without it) and in 45 healthy controls., Results: Our results revealed significant associations after correction in SNPs located in LDLR gene with aPLA carriers and thrombotic aPLA carriers, when compared with healthy controls. The most significant association in LDLR gene was found between SNP rs129083082 and aPLA carriers in recessive model (adjusted P-value = 2.55 x 10-3; OR = 2.18; 95%CI = 1.49-3.21). Furthermore, our work detected significant allelic association after correction between thrombotic aPLA carriers and healthy controls in SNP rs562556 located in PCSK9 gene (adjusted P-value = 1.03 x 10-2; OR = 1.60; 95%CI = 1.24-2.06). Expression level study showed significantly decreased expression level of LDLR gene in aPLA carriers (P-value <0.0001; 95%CI 0.16-2.10; SE 0.38-1.27) in comparison to the control group., Discussion: Our work has identified LDLR gene as a new susceptibility gene associated with the development of thrombosis in aPLA carriers, describing for the first time the deregulation of LDLR expression in individuals with aPLAs. Besides, thrombotic aPLA carriers also showed significant association with PCSK9 gene, a regulator of LDLR plasma levels. These results highlight the importance of atherosclerotic processes in the development of thrombosis in patients with aPLA.

Background: Human genetic factors influence the outcome of pegylated interferon and ribavirin hepatitis C therapy. We explored the role of IL28B, APOH and ITPA SNPs on the outcomes of triple therapy including telaprevir or boceprevir in patients with compensated cirrhosis chronically infected with HCV-1., Patients and Methods: A total of 256 HCV-1 Caucasian treatment-experienced patients with compensated cirrhosis from the ANRS CO20-CUPIC cohort were genotyped for a total of 10 candidate SNPs in IL28B (rs12979860 and rs368234815), APOH (rs8178822, rs12944940, rs10048158, rs52797880, rs1801689 and rs1801690) and ITPA (rs1127354 and rs7270101). We tested the association of IL28B and APOH SNPs with sustained virological response and of ITPA SNPs with anemia related phenotypes by means of logistic regression assuming an additive genetic model., Results: None of the six APOH SNPs were associated with sustained virological response. The favorable alleles of the IL28B SNPs rs12979860 and rs368234815 were associated with sustained virological response (rs12979860: OR = 2.35[1.50-3.70], P = 2x10(-4)). Refined analysis showed that the effect of IL28B SNPs on sustained virological response was restricted to prior PegIFN/RBV relapse (OR = 3.80[1.82-8.92], P = 8x10(-4)). We also confirmed the association between ITPA low activity alleles and protection against early hemoglobin decline in triple therapy (P = 2x10(-5))., Conclusion: Our results suggest that the screening of rs12979860 may remain interesting for decision making in prior relapse HCV-1 Caucasian patients with compensated cirrhosis eligible for a telaprevir- or boceprevir-based therapy.

Background: In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (β2GPI). β2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS). Patients develop autoantibodies targeting an epitope located on the N-terminal Domain I of β2GPI rendering this domain of interest as a possible therapeutic., Results: This new method of production of Domain I of β2GPI has increased the production yield by ~20 fold compared to previous methods in E.coli. This largely scalable, partially automated method produces 50-75 mg of pure, folded, active Domain I of β2GPI per litre of expression media., Conclusion: The application of this method may enable production of Domain I on sufficient scale to allow its use as a therapeutic.

Multiple pathologic conditions, including hemorrhage, tumor angiogenesis, and ischemia-reperfusion events, will result in hypoxia and subsequent reperfusion. Previous studies have analyzed the lipid changes within whole tissues and indicated that ischemia-reperfusion altered tissue and cellular phospholipids. Using an in vitro cell culture model of hypoxia and reoxygenation, we examined the endothelial lipid changes. We hypothesized that phospholipid scramblase 1, a protein that regulates bilayer asymmetry, is involved in altering the phospholipids of endothelial cells during hypoxia, a component of ischemia, leading to β2-glycoprotein I and IgM binding and subsequent lipid-mediated, inflammatory responses. We have completed the first comprehensive study of steady-state phospholipid scramblase 1 mRNA levels, protein expression, and activity under conditions of hypoxia and reoxygenation. Phospholipid scramblase 1 regulates phosphatidylserine exposure in response to oxygen stress, leading to β2-glycoprotein I and IgM binding and lipid-mediated, inflammatory responses., (© Society for Leukocyte Biology.)

The objective of the present study was to detect the association of the Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene and serum lipid levels in the Mulao and Han populations. A total of 879 subjects of Mulao and 844 subjects of Han Chinese were included. The levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and ApoA1 in Mulao, and triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), ApoA1 and the ratio of ApoA1/ApoB in Han were different among the three genotypes of the rs1801690 SNP (P < 0.05-0.01). Subgroup analyses showed that the levels of TC, TG, LDL-C, and ApoA1 in Mulao males; ApoA1 in Mulao females; TC, TG, HDL-C and ApoB and the ApoA1/ApoB ratio in Han males; and HDL-C, ApoA1 and the ApoA1/ApoB ratio in Han females were associated with the genotypes of rs1801690 (P < 0.05-0.001). Serum lipid parameters were also associated with several environmental factors (P < 0.05-0.001). The Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene was associated with some serum lipid parameters in the two ethnic groups, but the trends of association suggest that the Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene might have racial/ethnic-and/or gender-specificity.

Objective: To prepare a polyclonal antibody against human and murine β2 glycoprotein 1 (β2GP1) antigen with chemically synthesized β2GP1 peptides, and identify its specificity and pathogenicity., Methods: The peptides from the NH₂-terminal 35th-51th amino acids of β₂GP1 were synthesized by standard Fmoc assay, and then used to immunize New Zealand white rabbits after coupling with keyhole limpet hemocyanin (KLH). The polyclonal antibody in the rabbit sera was purified by protein G column. The titer and specificity of the polyclonal antibody were determined by ELISA and Western blot analysis. The total RNA was extracted and the protein lysates were collected from C3H/HeN mouse peritoneal macrophages treated with the above anti-β₂GP1 peptides antibody/β₂GP1 complexes in vitro. And the tissue factor (TF) mRNA and protein expression in the peritoneal macrophages were detected by real-time quantitative PCR and Western blotting. The activation of p38 and NF-κB p65 induced by anti-β₂GP1 peptides antibody/β₂GP1 complexes was determined by Western blotting using phosphor-specific antibodies. Experimental antiphospholipid antibody syndrome (EAPS) mouse model was established in C3H/HeN mice by intraperitoneal injection of anti-β₂GP1 peptides antibody in vivo. The titers of anti-β₂GP1 antibodies in the mouse peripheral blood and the activated partial thromboplastin time (APTT) were detected., Results: The purity of chemically synthesized β₂GP1 peptides was 94%, which met the immunogen standard. The titer of antiserum of the rabbit immunized with β₂GP1 peptide coupling with KLH was over 1:32 000. Western blotting showed that the anti-β₂GP1 peptides antibody could specifically recognize both human and mouse β₂GP1. Furthermore, ELISA showed that the antibody could specifically bind to β₂GP1 cryptic epitope. In vitro experiments demonstrated that the anti-β₂GP1 peptides antibody/β₂GP1 co mplexes could enhance p38 and NF-κB p65 phosphorylation in mouse peritoneal macrophages and induce TF mRNA and protein expression. Moreover, EAPS mouse model induced by anti-β₂GP1 peptide antibody was successfully established, in which the titers of anti-β₂GP1 antibody in mouse peripheral blood were greater than 1:3200 and APTT was significantly shorter that that of control group., Conclusion: The anti-β₂GP1 peptide antibody we prepared could specifically recognize both human and mouse β₂GP1 and specifically bind to β₂GP1 cryptic epitope. It was also proved to have the pathogenic effect.

Introduction: To determine the polymorphism at position 247 of the β2-glycoprotein I (β2GPI) gene in patients with systemic lupus erythematosus (SLE) and healthy controls in the Chinese Han Population and elucidate the relationship between β2GPI polymorphisms and anti-β2GPI levels, and furthermore investigate the correlation between β2GPI polymorphisms and thrombosis in patients with SLE., Methods: The β2GPI polymorphisms of 300 patients with SLE and 300 healthy controls were examined by single-specific-primer polymerase chain reaction (SSP-PCR), the efficacy of which was confirmed by sequencing technology. Anti-β2GPI antibodies were tested by enzyme-linked immunosorbent assay. β2GPI polymorphisms associated with thrombosis and the presence of anti-β2GPI antibodies were also statistically evaluated with SPSS software., Results: The genotype expressions and the allele frequencies were obtained in both patients with SLE and healthy controls. The SLE patients with thrombosis had significantly higher frequencies of the VV genotype and V allele than those without thrombosis and the controls, and there were no differences in VV genotype and V allele expression between the SLE patients without thrombosis and the controls. In contrast, the presence of anti-β2GPI antibody was related to the VV genotype of β2GPI, and the anti-β2GPI-positive patients had obviously higher frequencies of the VV genotype than the negative ones and the controls., Conclusion: The study results suggested that the V/V genotype and the V-encoding allele at position 247 of the β2GPI gene had strong correlation with the occurrence of thrombosis and the production of the anti-β2GPI antibodies, showing that the Val(247) β2GPI allele may be one of the genetic risk factors for the development of thrombosis in patients with SLE., (© 2014 John Wiley & Sons Ltd.)

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Venous thrombosis (VT) is a worldwide medical problem. In order to identify individuals at high risk early, it is necessary to find more genetic risk factors. Nowadays, the studies on genetic factors of thrombosis are mainly focused on coagulation and anticoagulation factors. The exploration of other proteins involved in thrombosis and hemostasis may lead to a breakthrough., Objectives: We used APOH as a candidate gene to investigate the existence of genetic variation that could increase the risk of thrombosis., Methods/results: In the current study, with a resequencing method followed by a case-control study, four polymorphisms (c.-32C>A, c.422T>C, c.461G>A, and c.1004G>C) in APOH (encoding β2 -glycoprotein I) were found to be in high linkage disequilibrium, which could result in three haplotypes. The H2 heterozygotes and H3 homozygotes had approximately 1.5-fold and seven-fold increased risks for VT, respectively. The minor allele frequency in the general population was ~ 10%. In addition, H3 individuals showed a significantly decreased level of β2 -glycoprotein I, but an increased level of thrombin generation. Functional tests indicated that the mutant β2 -glycoprotein I had a significantly lower capacity to extend thrombin clotting time and increase thrombin generation potential., Conclusions: This study revealed APOH as a new candidate gene associated with thrombosis, and further genetic research on this gene in patients in whom the cause of thrombophilia is unknown is therefore warranted., (© 2014 International Society on Thrombosis and Haemostasis.)

Background & Aims: HCV requires host lipid metabolism for replication, and apolipoproteins have been implicated in the response to treatment., Methods: We examined plasma apolipoprotein concentrations in three cohorts of patients: mono-infected patients with symptomatic acute hepatitis C (aHCV); those undergoing treatment for chronic hepatitis C (cHCV); and HIV/HCV co-infected patients being treated for their chronic hepatitis C. We also evaluated associations between apolipoproteins and IL28B polymorphisms, a defined genetic determinant of viral clearance., Results: Plasma apolipoprotein H (ApoH) levels were significantly higher in patients who achieved spontaneous clearance or responded to pegylated-interferon/ribavirin therapy. Strikingly, patients carrying the IL28B rs12979860 CC SNP correlated with the plasma concentration of ApoH in all three cohorts. Both ApoH and IL28B CC SNP were associated with HCV clearance in univariate analysis. Additional multivariate analysis revealed that the association between IL28B and HCV clearance was closely linked to that of Apo H and HCV clearance, suggesting that both belong to the same biological pathway to clearance. The association between IL28B CC SNP and ApoH was not observed in healthy individuals, suggesting that early post-infection events trigger differential ApoH expression in an IL28B allele dependent manner., Conclusions: This relationship identifies ApoH as the first induced protein quantitative trait associated with IL28B, and characterises a novel host factor implicated in HCV clearance., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

β2-glycoprotein I (β2-GPI) is a plasma protein that interacts with oxidized low-density lipoproteins (oxLDL) via β2-GPI domain V to form oxLDL/β2-GPI complexes, potential autoantigens promoting atherogenesis in patients with antiphospholipid syndrome (APS). Such a interaction would expose β2-GPI domain I or/and IV, structures recognized by anti-β2-GPI autoantibodies. IgG immune complexes with oxLDL/β2-GPI complexes can interact with macrophages via Fcγ receptor, causing oxLDL/β2-GPI endocytosis and foam cell formation, contributing to atherosclerosis. Here, we use recombinant domain V to study the interaction between oxLDL and β2-GPI and hypothesized that domain V would interfere with this interaction thereby reducing oxLDL macrophage uptake and foam cell formation. The β2-GPI domain V sequence was expressed by using the Pichia pastoris expression system to obtain recombinant domain V of β2-GPI (P.rβ2-GPI DV). ELISA tests demonstrated that P.rβ2-GPI DV interacted with oxLDL via 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), a negatively charged lipid moiety of oxLDL. The ω-carboxyl residue of oxLig-1 is required for the interaction. Serologic tests showed a significant increase in oxLDL and oxLDL/β2-GPI levels in patients with APS (p < 0.05 compared to controls). P.rβ2-GPI DV was able to bind oxLDL in high affinity and competitively inhibited native β2-GPI (nβ2-GPI) binding to free oxLDL as well as to oxLDL from the oxLDL/β2-GPI complexes. These observations suggest that P.rβ2-GPI DV may be used to inhibit the formation of the oxLDL/β2-GPI complexes, a potential approach for reducing foam cell development and mitigating atherogenesis in patients with APS. The present work provides a new effective strategy to prevent the progression of atherothrombotic vascular complications in APS patients.

Objective: The BXSB.Yaa mouse strain is a model of systemic lupus erythematosus that is dependent on duplication of the Toll-like receptor 7 gene. The objective of this study was to systematically describe the amplified autoimmune phenotype observed when the soluble plasma protein β2 -glycoprotein I (β2 GPI) gene was deleted in male BXSB.Yaa mice., Methods: We generated BXSB.Yaa and NZW mouse strains in which the β2 GPI gene had been knocked out by backcrossing the wild-type strains with C57BL/6 β2 GPI(-/-) mice for 10 generations. Sex- and age-matched mice of the various strains were housed under identical conditions and were killed at fixed time intervals. Serum and tissue specimens were collected at various time points. Lupus-associated autoantibodies, inflammatory cytokines, and the type I interferon (IFN) gene signature were measured. Flow cytometric analyses of lymphocyte populations were performed. The severity of glomerulonephritis was graded by 2 independent renal histopathologists., Results: Male BXSB.Yaa β2 GPI(-/-) mice developed significant lymphadenopathy and splenomegaly compared with age-matched controls. Male BXSB.Yaa β2 GPI(-/-) mice also had significantly higher levels of autoantibodies, increased levels of inflammatory cytokines including tumor necrosis factor α, interleukin-6, and BAFF, and more severe glomerulonephritis. The type I IFN gene signature in male BXSB.Yaa β2 GPI(-/-) mice was significantly higher than that in control mice. Male BXSB.Yaa β2 GPI(-/-) mice also had marked dysregulation of various B cell and T cell populations in the spleens and lymph nodes and a disturbance in apoptotic cell clearance., Conclusion: Deletion of β2 GPI accelerates and potentiates the autoimmune phenotype in male BXSB.Yaa mice., (Copyright © 2014 by the American College of Rheumatology.)

Objective: Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. β2 glycoprotein I (β2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, β2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to β2GPI and its relationship with atherosclerotic process., Approach and Results: Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte β2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to β2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte β2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte β2GPI and TF with IMT or FMD was detected. β2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to β2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm., Conclusions: A significant percentage of patients with SLE and PAPS show a β2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

Background: Pemphigus and bullous pemphigoid are two autoimmune diseases that have similar pathogenesis. Both have a genetic predisposition, which promotes the production of auto antibodies targeted against different components of the epidermal desmosome and hemidesmosome. Antiphospholipid antibodies (aPL) are heterogeneous group of antibodies found in patients with autoimmune diseases and inflammatory conditions and are associated with thrombotic events., Objective: We sought to determine the expression profile of eight non classical aPLs in ABD patients., Methods: A cohort of 266 serum samples of patients with pemphigus, bullous pemphigoid and controls was screened for the presence of eight aPL antibodies, using the Bio-Rad BioPlex™ 2200 system., Results: Phosphatidylserine-beta-2-glycoprotein-I (aPS-β2GPI) and anti prothrombin complex (aPT-PT) serum profiles were significantly more prevalent among ABD patients; 20.7% patients with ABD compared to 5.9% of control patients were positive for aPS-β2GPI IgM. In addition, aPT-PT IgM was more prevalent among ABD patients (31% vs. 14.8%)., Conclusion: aPL auto antibodies are more prevalent in ABD. Any clinical association should be further determined.

Aim: It has been reported that oxidized low-density lipoprotein (oxLDL) forms a stable and non-dissociable complex with β2-glycoprotein I (β2GPI) and that IgG anti-β2GPI autoantibodies are able to recognize this complex, thus facilitating macrophage-derived foam cell formation in patients with antiphospholipid syndrome (APS). However, the immunopathological mechanisms of oxLDL/β2GPI complexes in promoting foam cell formation are not fully understood. In this study, we examined the role of toll-like receptor 4 (TLR4) in the oxLDL/β2GPI/anti-β2GPI complex-induced transformation of mouse peritoneal macrophages to foam cells., Methods: Oil red O staining and optical density (OD) measurements of intracellular stained oil red O solution were used to monitor the transformation of peritoneal macrophages to foam cells in TLR4-competent C3H/HeN and TLR4-mutant C3H/HeJ mice. During foam cell formation induced by the oxLDL/β2GPI/anti-β2GPI complex, the expression of TLR4 and activation of nuclear factor kappa B (NF-κB) were confirmed by analyzing the protein and mRNA levels of these compounds. Furthermore, the related active molecule expression during foam cell formation induced by the oxLDL/β2GPI/anti-β2GPI complex was examined in the presence or absence of TLR4., Results: The data showed that treatment with the oxLDL/β2GPI/anti-β2GPI complex markedly increased foam cell formation, the TLR4 expression, NF-κB activation, the tissue factor (TF) expression and tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) secretion in the C3H/HeN mice. However, the transformation of macrophages to foam cells and the expression levels of phosphorylated NF-κB, TF, TNF-α and MCP-1 were significantly reduced in the C3H/HeJ mice treated with the oxLDL/β2GPI/anti-β2GPI complex. In addition, compared with that achieved by oxLDL alone, the oxLDL/β2GPI complex decreased foam cell formation and the related signaling molecule expression in the C3H/HeN mice., Conclusions: Our results indicate that TLR4 plays an important role in the process of oxLDL/β2GPI/anti-β2GPI complex-induced transformation of macrophages to foam cells, which may accelerate the development of atherosclerosis in the setting of APS. However, β2GPI alone functions as an antiatherogenic protein by preventing the foam cell formation induced by oxLDL.

Antiphospholipid syndrome (APS) is an autoimmune disease with clinical manifestations of thrombosis and pregnancy complications. Beta2-glycoprotein I (β2GPI) is the major antigen for the APS-related antibodies. Heparin, low-molecular weight heparin and the synthetic pentasaccharide fondaparinux are commonly used for prophylaxis and treatment of thrombosis in patients with antiphospholipid syndrome. These antithrombotic drugs bind and activate antithrombin III to inactivate blood clotting proteases. Heparin and heparin derivatives might have a direct beneficial effect in APS via binding to β2GPI and interfering with prothrombotic properties of β2GPI/antibody complexes. We compared fondaparinux to heparin regarding its ability to bind β2GPI and inhibit the binding of β2GPI/antibody complexes to negatively charged phospholipids and endothelial cells. Although heparin and fondaparinux bind β2GPI at therapeutically relevant doses, neither fondaparinux nor heparin was efficient in inhibition of the binding of β2GPI/antibody complexes to negatively charged phospholipids and endothelial cells. Our studies suggest that these drugs do not act on pathological properties of β2GPI/antibody complexes, emphasizing the need for a new treatment specific for β2GPI-related thrombosis in APS. We observed that the binding interface of fondaparinux on β2GPI does not include the lysine residues known to be critical for binding of heparin. The docking model of the β2GPI complex with fondaparinux is in agreement with multiple experimental observations., (© 2013.)

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta2-glycoprotein I (β2GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β2GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β2GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β2GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.

Background: β2 -Glycoprotein I (β2 -GPI), also designated apolipoprotein H, is a 50-kDa protein that circulates in blood at high concentrations, playing important roles in autoimmune diseases, hemostasis, atherogenesis, and angiogenesis, as well as in host defense against bacteria and in protein/cellular waste removal. Plasma β2 -GPI levels have a significant genetic component (heritability of ~ 80%)., Objectives: To present the results of a genome-wide association study for plasma β2 -GPI levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project., Patients/methods: A total of 306 individuals for whom β2 -GPI plasma measurements were available were typed for 307,984 single-nucleotide polymorphisms (SNPs) with the Infinium 317k Beadchip (Illumina). Association with the β2 -GPI phenotype was investigated through variance component analysis, and the most significant results were followed up for association with coronary artery disease (CAD) in an independent in silico analysis involving 5765 CAD cases from the PROCARDIS Project and 7264 controls from the PROCARDIS Project and the Wellcome Trust Case Control Consortium (WTCCC) collection., Results: After correction for multiple testing, three SNPs located in/around two genes (ELF5 and SCUBE2) reached genome-wide significance. Moreover, an SNP in the APOH gene showed suggestive association with the β2 -GPI phenotype. Some of the identified genes are plausible biological candidates, as they are actually or potentially involved in inflammatory processes., Conclusions: Our results represent a first step towards identifying common variants reflecting the genetic architecture influencing plasma β2 -GPI levels, and warrant further validation by functional experiments, as the functions of some of the discovered loci are still unknown., (© 2013 International Society on Thrombosis and Haemostasis.)

Introduction: Previous studies have suggested that the possession of the Val/Val genotype of the Val247Leu polymorphism of the β(2)-glycoproteinI (β(2)-GPI) gene may be associated with antiphospholipid syndrome (APS), and, among patients with APS, with the production of anti-β(2)-GPI antibodies or the development of thrombosis. Given the controversial results reported, the aim of this work is to combine previous findings by means of a systematic review and a meta-analysis., Methods: We retrieved studies analyzing the genotype of the above-mentioned polymorphism among patients with APS by means of electronic database search. A meta-analysis was conducted in a random effects model and calculations of odds ratio (OR) and confidence intervals (CI) were done. Sensitivity analysis and tests for heterogeneity of the results were performed., Results: Eight previous studies analyzed the association of APS, anti-β(2)-GPI antibodies and/or thrombosis with the Val247Leu polymorphism. After meta-analysis, patients with APS had a significantly higher prevalence of the Val/Val genotype of this genetic variant when compared with controls (OR=2.04; 95% CI: 1.12, 3.73; P=0.02). Among patients with APS, those with anti-β(2)-GPI antibodies had a higher prevalence of this genotype (OR=1.73; 95% CI: 1.04, 2.87; P=0.03). No significant results were found for the presence of arterial or venous thrombosis., Conclusions: Val/Val genotype of β(2)-GPI gene is associated with a significant excess risk to suffer from APS and, among patients with APS, to have anti-β(2)-GPI antibodies. No definite conclusions can be made regarding the association of this polymorphism with thrombosis among APS patients., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: The objective of this paper is to explore whether the valine/leucine(247) (Val/Leu(247)) polymorphism of β(2)-glycoprotein I (β(2)GPI) confers susceptibility to anti-phospholipid syndrome (APS) and thrombosis and predicts positivity for anti-β(2)GPI antibodies., Methods: A meta-analysis was conducted on the associations between the β(2)GPI Val/Leu(247) polymorphism and susceptibility to APS and thrombosis and positivity for anti-β(2)GPI., Results: A total of 1507 patients with APS and 1450 controls in 12 comparative studies were included in this meta-analysis. Meta-analysis of the β(2)GPI Val/Leu(247) polymorphism showed significant associations between the β(2)GPI Val allele and APS, thrombosis, and anti-β(2)GPI positivity (odds ratio (OR) 1.316, 95% confidence interval (CI) 1.068-1.621, p = 0.010; OR 1.908, 95% CI 1.195-3.046, p = 0.007; OR 1.630, 95% CI 1.018-2.609, p = 0.042, respectively). A direct comparison between anti-β(2)GPI-positive and -negative patients revealed that the frequency of the Val allele was significantly higher in anti-β(2)GPI-positive patents (OR 1.514, 95% CI 1.017-1.253, p = 0.041). Furthermore, a direct comparison between thrombosis-positive and -negative patients also indicated that the Val/Val + Val/Leu and the Val/Val vs. Leu/Leu genotypes of the β(2)GPI polymorphism were significantly elevated in patients with thrombosis (OR 2.817, 95% CI 1.200-6.610, p = 0.017; OR 3.312, 95% CI 1.338-8.200, p = 0.010, respectively)., Conclusion: This meta-analysis shows that the β(2)GPI Val/Leu(247) polymorphism is associated with susceptibility to APS and thrombosis and with anti-β(2)GPI positivity.

β₂-GPI (β₂-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of the present study was to assess the contribution of β₂-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β₂-GPI inhibited endothelial cell migration, which was restored by its neutralizing antibody. NF-κB (nuclear factor κB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-κB significantly attenuated the inhibitory effect of β₂-GPI on cell migration. Moreover, β₂-GPI was found to induce IκBα (inhibitor of NF-κB) phosphorylation and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS [endothelial NO (nitric oxide) synthase] and NO production were all increased by β₂-GPI and these effects were remarkably inhibited by NF-κB inhibitors and siRNAs of p65 and p50. Furthermore, β₂-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. The findings of the present study provide novel insight into the ability of β₂-GPI to inhibit endothelial cell migration predominantly through the NF-κB/eNOS/NO signalling pathway, which indicates a potential direction for clinical therapy in vascular diseases.

The significance of beta2-glycoprotein I (β2GPI) polymorphisms in the production of anti-β2GPI and other antiphospholipid autoantibodies (aPL) and in the pathogenesis of primary antiphospholipid syndrome (PAPS) is not well understood. We performed a study comparing the distribution of polymorphisms at codons 247 (Val247Leu) and 316 (Trp316Ser) of the β2GPI gene in a Caucasian Spanish population of PAPS patients and healthy controls, and then making correlations with the development of anti-β2GPI antibodies and other aPL and associated clinical manifestations. A total of 57 PAPS patients and 100 control subjects were included. In the analysis of Val247Leu polymorphism, alleles (V and L) and genotypes (V/V, V/L, L/L) were similarly distributed in PAPS patients and controls (P = 0.66 and P = 0.22, respectively). Regarding Trp316Ser polymorphism, we found a higher percentage of patients with respect to controls expressing S allele (11.4 vs. 5%, P = 0.02) and T/S genotype (22.8 vs. 10%, P = 0.02). However, when we compared T/T and T/S genotypes in PAPS patients, we found no differences regarding generation of anti-β2GPI, other aPL and clinical manifestations favoring any genotype. Our findings suggest that among Spanish Caucasians, polymorphisms at codon 247 (Val247Leu) do not seem to influence PAPS pathogenesis. On the contrary, polymorphisms at codon 316 (Trp316Ser), by means of an increased S allele and T/S genotype presence in Spanish Caucasian patients, might play a role in the pathogenic development of PAPS, although mechanism would not involve an increased production of anti-β2GPI and other aPL.

β₂-Glycoprotein I (β₂GPI) is a highly abundant plasma protein and the major antigen for autoantibodies in the antiphospholipid syndrome. Recently, we have described a novel function of β₂GPI as scavenger of lipopolysaccharide (LPS). With this in mind we investigated the conservation of β₂GPI in vertebrates and set out to identify the binding site of LPS within β₂GPI. The genome sequences of 42 species were surveyed. Surface plasmon resonance (SPR) was performed with peptides to characterise the binding site of β₂GPI for LPS. β₂GPI could be identified in most tested vertebrates with a high overall amino acid homology of 80% or more in mammals. SPR revealed that a synthesised peptide (LAFWKTDA) from domain V of β₂GPI was able to compete for binding of β₂GPI to LPS. The AFWKTDA sequence was completely conserved in all mammals. The peptide containing the LPS binding site attenuated the inhibition by β₂GPI in a cellular model of LPS-induced tissue factor expression. Other important sites, such as the binding site for anionic phospholipids and the antiphospholipid antibody binding epitope, were also preserved. β₂GPI is highly conserved across the animal kingdom, which suggests that the function of β₂GPI may be more important than anticipated.

β(2) -Glycoprotein I (β(2) -GPI) is a protein that circulates in blood at high concentrations. The function of β(2) -GPI has long been an enigma. More than 20 years ago, it was discovered that β(2) -GPI is the major antigen for the circulating antibodies in the antiphospholipid syndrome. However, this knowledge has not advanced our understanding of the physiologic role of the protein. In recent years, new insights have suggested an important function of this protein in innate immunity. β(2) -GPI was found to scavenge lipopolysaccharide and was able to clear unwanted anionic cellular remnants such as microparticles from the circulation. The function of β(2) -GPI seems to depend on the structural conformation of the protein, and it has been established that β(2) -GPI can exist in at least two conformations. In this review, we will highlight and summarize the current knowledge on this protein., (© 2011 International Society on Thrombosis and Haemostasis.)

Beta 2 glycoprotein I (β2GPI) is the major auto antigen in the antiphospholipid syndrome but also interacts with fibrinolytic and angiogenic proteins. The aim of this study was to examine the angiogenic potential of β2GPI in vivo in β2GPI deficient mice utilizing angiogenic assays. β2GPI deficient mice show increased microvessel formation in comparison to β2GPI replete controls when injected with growth factor free-matrigel implants. However, microvessel formation in matrigel plugs of β2GPI deficient mice was less than in β2GPI replete mice when basic fibroblast growth factor (bFGF) was included in the matrigel. Hemoglobin content was higher in vascular endothelial growth factor (VEGF) containing-matrigel plugs in the β2GPI deficient mouse demonstrating that the lack of β2GPI led to increased extravasation by VEGF. Melanoma B16F10 tumour growth was enhanced in β2GPI deficient mice. Melanoma microvessel density was increased in β2GPI deficient mice but the proliferation rate of tumour cells (determined by Ki67 immunohistochemistry) was unaffected by the presence or absence of β2GPI. Subcutaneous delivery of native human β2GPI by the ALZET osmotic pump did not affect melanoma tumour growth in β2GPI deficient mice. We conclude that the in vivo unopposed action of β2GPI is anti-angiogenic however this function is modified in the presence of a strong angiogenic stimulus into stabilization of vessel formation. Although the presence of β2GPI attenuates vessel sprouting in certain tumours, no survival benefit is conferred to tumour bearing animals. This does not preclude the potential benefit of modified or fragments of β2GPI in anti-angiogenesis research., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: This study investigated the effect and clinical significance of beta2-GPI in hepatocellular carcinoma (HCC)., Methods: Double fluorescent immunostaining analysis was performed in paraffin wax-embedded histological sections of nine HCC parenchyma, seven adjacent non-cancerous tissues and seven control liver tissues from hepatitis B virus (HBV) infected patients using a beta2-GPI polyclonal antibody and a HBV surface antibody. NF-κB activation was assessed by a non-radioactive electrophoretic mobility shift assay (EMSA) and immunofluorescence assay in SMMC-7721 HCC cells exposed to various treatments. The cells were transiently transfected with vectors expressing beta2-GPI (group one), HBsAg (group two), both beta2-GPI and HBsAg (group three), or with a control vector (group four). Untransfected cells (group five) were also used as a control. Alpha fetoprotein (AFP) expressions were also detected by ELISA in all groups., Results: The highest degree of co-localization of beta2-GPI and HBsAg proteins was seen in the endochylema and occurred at the nuclear border in the cancer tissues. Weak beta2-GPI protein staining was present in the endochylema, with a strong signal for HBsAg protein in HBV control samples. Adjacent non-tumorous liver tissue samples also showed HBsAg staining but stronger beta2-GPI signals in the endochylema. In experiments with SMMC-7721 HCC cells, groups one and two had induced activation of NF-κB with the relative NF-κB DNA-binding activities of 55.84 and 51.12, respectively. However, the highest relative NF-κB DNA-binding activity was observed in group three (80.5). The percentages of cells with NF-κB translocated from the cytoplasm to nucleus in groups one, two, three, four and five compared with total cells were 13.5, 8.7, 24.9, 5.7 and 0.95%, respectively. The mean AFP levels were significantly higher in group three (0.0640 ± 0.0059) than in group five (P < 0.001). It appeared higher in group three than in group one (0.0562 ± 0.0060, P < 0.05) and group four (0.0585 ± 0.0040, P < 0.05), while no significant differences were seen between groups three and two, and between groups four and five., Conclusions: Beta2-GPI may play a role in the development of HBV-related HCC by activating NF-κB via interaction of beta2-GPI and HBsAg.