Lipid presentation by the protein C receptor links coagulation with autoimmunity.

Antiphospholipid antibodies (aPLs) cause severe autoimmune disease characterized by vascular pathologies and pregnancy complications. Here, we identify endosomal lysobisphosphatidic acid (LBPA) presented by the CD1d-like endothelial protein C receptor (EPCR) as a pathogenic cell surface antigen recognized by aPLs for induction of thrombosis and endosomal inflammatory signaling. The engagement of aPLs with EPCR-LBPA expressed on innate immune cells sustains interferon- and toll-like receptor 7-dependent B1a cell expansion and autoantibody production. Specific pharmacological interruption of EPCR-LBPA signaling attenuates major aPL-elicited pathologies and the development of autoimmunity in a mouse model of systemic lupus erythematosus. Thus, aPLs recognize a single cell surface lipid-protein receptor complex to perpetuate a self-amplifying autoimmune signaling loop dependent on the cooperation with the innate immune complement and coagulation pathways.

Antineutrophil properties of natural gingerols in models of lupus.

Ginger is known to have antiinflammatory and antioxidative effects and has traditionally been used as an herbal supplement in the treatment of various chronic diseases. Here, we report antineutrophil properties of 6-gingerol, the most abundant bioactive compound of ginger root, in models of lupus and antiphospholipid syndrome (APS). Specifically, we demonstrate that 6-gingerol attenuates neutrophil extracellular trap (NET) release in response to lupus- and APS-relevant stimuli through a mechanism that is at least partially dependent on inhibition of phosphodiesterases. At the same time, administration of 6-gingerol to mice reduces NET release in various models of lupus and APS, while also improving other disease-relevant endpoints, such as autoantibody formation and large-vein thrombosis. In summary, this study is the first to our knowledge to demonstrate a protective role for ginger-derived compounds in the context of lupus. Importantly, it provides a potential mechanism for these effects via phosphodiesterase inhibition and attenuation of neutrophil hyperactivity.

Detrimental roles of TNF-alpha in the antiphospholipid syndrome and de novo synthesis of antiphospholipid antibodies induced by biopharmaceuticals against TNF-alpha.

Antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by arterial and/or venous thrombosis and/or recurrent pregnancy losses. Obstetric APS (OAPS) is considered as a distinct entity from vascular APS (VAPS). In the absence of any additional disease, APS is designated as primary (PAPS), while the term secondary APS (SAPS) is used when other diseases are associated. Catastrophic APS (CAPS) is characterized by the rapid development of multiple thrombosis in various vital organs. The presence of antiphospholipid antibodies (aPL Abs) is considered as a laboratory criterion for APS diagnosis. aPL Abs cause an increase in systemic and decidual TNF-alpha levels in experimental model of APS (eAPS), while paradoxically, administration of TNF-alpha blockers has been associated with de novo synthesis of aPL Abs in patients with various autoimmune diseases. While eAPS provides evidence for the fact that application of TNF-alpha blockers has beneficial effects, lack of randomized prospective studies is the main obstacle for consideration of TNF-alpha blockers administration as a therapeutic option not for all, but at least for selected cases of APS patients despite compelling evidence for detrimental roles of TNF-alpha for both VASP and OAPS. This article represents a review of previously published reports on detrimental roles of TNF-alpha in APS, reports on the application of anti-TNF-alpha agents in eAPS and articles that reported de novo synthesis of aPL Abs induced by biopharmaceuticals against TNF-alpha.

The Role of TLR4 on B Cell Activation and Anti-ß2GPI Antibody Production in the Antiphospholipid Syndrome.

High titer of anti-ß2-glycoprotein I antibodies (anti-ß2GPI Ab) plays a pathogenic role in antiphospholipid syndrome (APS). Numerous studies have focused on the pathological mechanism in APS; however, little attention is paid to the immune mechanism of production of anti-ß2GPI antibodies in APS. Our previous study demonstrated that Toll-like receptor 4 (TLR4) plays a vital role in the maturation of bone marrow-derived dendritic cells (BMDCs) from the mice immunized with human ß2-glycoprotein I (ß2GPI). TLR4 is required for the activation of B cells and the production of autoantibody in mice treated with ß2GPI. However, TLR4 provides a third signal for B cell activation and then promotes B cells better receiving signals from both B cell antigen receptor (BCR) and CD40, thus promoting B cell activation, surface molecules expression, anti-ß2GPI Ab production, and cytokines secretion and making B cell functioning like an antigen presenting cell (APC). At the same time, TLR4 also promotes B cells producing antibodies by upregulating the expression of B-cell activating factor (BAFF). In this paper, we aim to review the functions of TLR4 in B cell immune response and antibody production in autoimmune disease APS and try to find a new way for the prevention and treatment of APS.

Mitochondrial Dysfunction in the Liver and Antiphospholipid Antibody Production Precede Disease Onset and Respond to Rapamycin in Lupus-Prone Mice.

OBJECTIVE: Antiphospholipid antibodies (aPL) constitute a diagnostic criterion of systemic lupus erythematosus (SLE), and aPL have been functionally linked to liver disease in patients with SLE. Since the mechanistic target of rapamycin (mTOR) is a regulator of oxidative stress, a pathophysiologic process that contributes to the development of aPL, this study was undertaken in a mouse model of SLE to examine the involvement of liver mitochondria in lupus pathogenesis. METHODS: Mitochondria were isolated from lupus-prone MRL/lpr, C57BL/6.lpr, and MRL mice, age-matched autoimmunity-resistant C57BL/6 mice as negative controls, and transaldolase-deficient mice, a strain that exhibits oxidative stress in the liver. Electron transport chain (ETC) activity was assessed using measurements of oxygen consumption. ETC proteins, which are regulators of mitochondrial homeostasis, and the mTOR complexes mTORC1 and mTORC2 were examined by Western blotting. Anticardiolipin (aCL) and anti-ß2 -glycoprotein I (anti-ß2 GPI) autoantibodies were measured by enzyme-linked immunosorbent assay in mice treated with rapamycin or mice treated with a solvent control. RESULTS: Mitochondrial oxygen consumption was increased in the livers of 4-week-old, disease-free MRL/lpr mice relative to age-matched controls. Levels of the mitophagy initiator dynamin-related protein 1 (Drp1) were depleted while the activity of mTORC1 was increased in MRL/lpr mice. In turn, mTORC2 activity was decreased in MRL and MRL/lpr mice. In addition, levels of aCL and anti-ß2 GPI were elevated preceding the development of nephritis in 4-week-old MRL, C57BL/6.lpr, and MRL/lpr mice. Transaldolase-deficient mice showed increased oxygen consumption, depletion of Drp1, activation of mTORC1, and elevated expression of NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), a pro-oxidant subunit of ETC complex I, as well as increased production of aCL and anti-ß2 GPI autoantibodies. Treatment with rapamycin selectively blocked mTORC1 activation, NDUFS3 expression, and aPL production both in transaldolase-deficient mice and in lupus-prone mice. CONCLUSION: In lupus-prone mice, mTORC1-dependent mitochondrial dysfunction contributes to the generation of aPL, suggesting that such mechanisms may represent a treatment target in patients with SLE.

Diagnostic criteria for systemic lupus erythematosus: a critical review.

Systemic lupus erythematosus is a multi-organ system autoimmune disease with clinical and serological heterogeneity. The formulation of initial criteria for SLE was first proposed by the American College of Rheumatology and appeared in 1971. Although the original purpose of the criteria was to classify the disease, it became widely used as a diagnostic criteria in clinical situations. Since then the ACR criteria have undergone at least two changes (in 1982 and 1997). Clinical manifestations that can differentiate SLE patients from healthy people such as skin lesions, arthritis, renal disorder, neurologic disorder, hematologic changes and others are included in these criteria. Serum anti-nuclear antibody, anti-ds-DNA antibody and anti-Sm antibody are important biomarkers of SLE patients. In 2012, the Systemic Lupus Collaborating Clinics proposed the SLICC criteria for SLE in view of new knowledge of autoantibodies and the importance of low complement. Future biomarkers may be useful in distinguishing SLE from other diseases and in monitoring of disease activity.

Antiphospholipid antibodies mediate autoimmunity against dying cells.

The Antiphospholipid Syndrome (APS) is characterized by thrombosis and pregnancy loss, clinical events mediated by pathogenic anti-phospholipid autoantibodies (aPL). ß2-glycoprotein I (ß2GPI) is the major autoantigens recognized by aPL. ß2GPI is a cationic protein that binds to negatively charged surfaces such as those of apoptotic cells. This feature may lead to two major events: i) immunization with ß2GPI fosters the Fc-receptor-mediated uptake by antigen presenting cells of apoptotic material decorated with ß2GPI and the activation of ß2GPI-specific T cells which in turn provide help to ß2GPI-specific B cells for the production of anti-ß2GPI; ii) apoptotic bodies decorated with ß2GPI can be opsonized by anti-ß2GPI and shifted towards a pro-inflammatory clearance by macrophages; epitope spread can occur with the generation of autoimmunity against nuclear autoantigens. In the presence of a predisposing genetic background and of a particular cytokine environment (type I interferons), the sequential emergence of autoantibodies can evolve into overt clinical disease. The spectrum of clinical phenotypes of the patients can be modulated by several factors affecting the pathogenicity of anti-ß2GPI (e.g. domain specificity). We conclude that dying cells may play a dual role in APS: (I) as immunogen for the induction of aPL (etiology) and (II) as targets of aPL for the chronification of inflammation and the development of autoimmune diseases (pathology).

Neonatal stroke associated with de novo antiphospholipid antibody and homozygous 1298C/C methylenetetrahydrofolate reductase mutation.

Antiphospholipid antibodies are a recognised prothrombotic risk factor associated with acute ischaemic infarction. Most autoimmune diseases are rare in infants, and in the neonatal period, autoimmunity is related to transplacental passage of maternal immunoglobulin G autoantibodies. Distinguishing between de novo and acquired autoimmunity has important therapeutic implications and is crucial for determining the prognosis. We present a case of a neonatal thrombotic stroke associated with de novo synthesis of antiphospholipid antibodies, a homozygous 1298C/C methylene-tetrahydrofolate reductase mutation and a double-homozygous plasminogen activator inhibitor 1 polymorphism (PAI-1 844A/A and 675 4G/4G), which may have increased the final thrombotic risk. Her mother was not positive for antiphospholipid antibodies. The authors highlight an unequivocal evidence of a de novo case of paediatric antiphospholipid antibody syndrome and emphasise the need for a thorough investigation in cases of neonatal stroke including molecular thrombophilia study.

Long-term outcome of 32 patients with chorea and systemic lupus erythematosus or antiphospholipid antibodies.

OBJECTIVE: The aim of this work was to describe chorea during systemic lupus erythematosus or antiphospholipid antibodies and its long-term outcome. METHODS: We retrospectively analyzed clinical features, laboratory findings, imaging characteristics, and outcome in a series of 32 patients. RESULTS: Most patients were women (28 of 32), and mean age at onset of chorea was 20.6 (9-62) years. Chorea was inaugural for 28 patients. Improvement was observed with various treatments. During follow-up (12.2 ± 11.3 years), severe manifestations of systemic lupus erythematosus were rare. Antiphospholipid antibodies were repeatedly positive for 90% of the patients. Twelve patients developed arterial thrombosis. Prophylactic treatment with antithrombotic therapy might reduce the risk of further thrombosis (8% versus 57%; P = 0.01). Cardiac valvulopathy occurred in 22 patients during follow-up. Chorea relapsed in 8 cases. CONCLUSIONS: Chorea had a good outcome in itself. This long-term follow-up shows, for the first time, that these patients have substantial risk for further arterial thrombosis.

Antiphospholipid syndrome in dermatology: an update.

Antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies, recurrent thrombosis, and fetal loss. Antiphospholipid antibodies are a family of autoantibodies that recognize various combinations of phospholipids, phospholipid-binding proteins, or both. APS can occur in the absence of underlying or associated disease (primary APS) or in combination with other diseases (secondary APS). The exact pathogenic mechanism by which these antibodies cause thrombosis is not known; however, several hypotheses, such as activation of platelet and endothelial cells and interference with the coagulation system, have been proposed. Diagnosis is based on the presence of at least one clinical and laboratory criterion each, according to International Consensus Statement on preliminary classification criteria. However, APS can be diagnosed in individuals even in the absence of some of the classification criteria. Clinical manifestations involve different organs and systems such as the blood vessels, central nervous system, skin, kidneys, gastrointestinal tract, heart, and placenta. The unifying mechanism of all these manifestations is thrombosis, either arterial or venous. Skin manifestations are varied and although not included in the diagnostic criteria, may be the presenting feature of this syndrome. Therefore all dermatologists should investigate the possibility of APS when cutaneous findings are related to venous or arterial thrombosis. The risk of thrombosis cannot be predicted, and therefore treatment is not initiated until a thrombotic event occurs. Indefinite anticoagulation is prescribed once a thrombotic event occurs. Prognosis depends on the severity of the clinical manifestations and so, knowledge of the presentation of this disease is important for early detection and prompt treatment to prevent life-threatening consequences of this catastrophic disease process.

Antiphospholipid antibody profiles in lupus nephritis with glomerular microthrombosis: a prospective study of 124 cases.

INTRODUCTION: Glomerular microthrombosis (GMT) is a common vascular change in patients with lupus nephritis (LN). The mechanism underlying GMT is largely unknown. Although several studies have reported the association of antiphospholipid antibodies (aPL) with GMT, the relation between GMT and aPL remains controversial. Previous studies have demonstrated that some aPL could bind to several hemostatic and fibrinolytic proteases that share homologous enzymatic domains. Of the protease-reactive aPL, some can inhibit the anticoagulant activity of activated protein C and the fibrinolytic function of plasmin, and hinder the antithrombin inactivation of thrombin. The purpose of this study was to investigate the prevalence of GMT in LN patients and examine the relation between the aPL profiles (including some protease-reactive aPL) and GMT. METHODS: Renal biopsy specimens were examined for the presence of glomerular microthrombi. Plasma samples from 25 LN patients with GMT (LN-GMT group) and 99 LN patients without GMT (LN-non-GMT group) were tested for lupus anticoagulant and antibodies against cardiolipin, beta2 glycoprotein I, plasmin, thrombin, tissue plasminogen activator, and annexin II. RESULTS: The prevalence of GMT in LN patients was 20.2%. Compared with the LN-non-GMT group, the LN-GMT group had an elevated systemic lupus erythematosus disease activity index; elevated renal tissue injury activity and chronicity indices; elevated serum creatinine, blood urea nitrogen, and proteinuria levels; a lower serum C3 level and much intense glomerular C3, C1q staining; and a higher frequency of hypertension (P < 0.05 for all). Additionally, the detection rate of lupus anticoagulant, immunoglobulin G (IgG) anti-beta2 glycoprotein I and anti-thrombin antibodies were higher in the LN-GMT group than in the LN-non-GMT group (P < 0.05 for all). No statistical differences were found in the detection rates of IgG anti-cardiolipin, plasmin, tissue plasminogen activator, or annexin II antibodies (P > 0.05 for all). No detectable difference in IgM autoantibodies to the above antigens was observed between the two groups. CONCLUSIONS: GMT occurs in approximately 20.2% of LN patients. Patients with GMT have severer renal tissue injuries and poorer renal functions than patients without GMT. The lupus anticoagulant and antibodies against beta2 glycoprotein I and thrombin may play a role in GMT.

The high prevalence of antiphospholipid antibodies in refractory focal epilepsy is related to recurrent seizures.

BACKGROUND: Previous studies have shown the association between antiphospholipid antibodies with epilepsy but there are no studies addressing the effect of seizure frequency, duration of epilepsy, epilepsy type and aetiology on the prevalence of these antibodies in well-evaluated refractory epilepsy. METHODS: Anticardiolipin, anti-beta2-glycoprotein I and antinuclear antibody levels were measured in 105 well-evaluated patients with refractory focal epilepsy. Clinical determinants included the patient history, electroclinical classification and high resolution brain magnetic resonance imaging. RESULTS: Patients with seizures during the month prior to sampling (recent seizures) had increased prevalence of immunoglobulin (Ig) G class anticardiolipin antibodies (29%) compared with healthy controls [13%; age-adjusted odds ratio (OR): 3.09, 95% confidence interval (CI): 1.30-7.34] and patients with no recent seizures (11%; age-adjusted OR: 4.00, CI: 0.84-19.02). The patients with recent seizures had increased prevalence of moderate positive IgG class anticardiolipin antibodies (12%) compared with the controls (4%) and the patients with no recent seizures (0%; age-adjusted OR: 4.45, CI: 1.14-17.36). The prevalence of IgG class anticardiolipin antibodies was not associated with epilepsy type, duration or aetiology. CONCLUSION: The presence of antiphospholipid antibodies is associated with recurrent seizures in patients with refractory focal epilepsy. The measurement of these antibodies may be useful in evaluating the outcome of epilepsy.

Predictive, protective, orphan autoantibodies: the example of anti-phospholipid antibodies.

Anti-phospholipid antibodies (aPL) are one of the most recent examples of autoantibodies that can appear even long time before any clinical manifestation can be associated with them. There is a general agreement that they may represent a strong risk factor for recurrent thrombosis and/or fetal losses. Anti-phospholipid antibodies represent a necessary but not sufficient factor (first hit) for thrombosis, and require additional triggering factors (second hit) to disclose the thrombogenic activity. Several factors may affect the predictive value of aPL, including titre, immunoglobulin isotype, fine antigenic specificity and affinity binding activity. Their careful evaluation is suggested in order to characterize the true predictive value of aPL.

Pediatric antiphospholipid antibodies and antiphospholipid syndrome.

Antiphospholipid syndrome (APS) can occur in children, like adults, with the same diverse spectrum of thrombotic sites but predominately with deep vein thrombosis and stroke. In contrast with adults, however, transient nonthrombogenic antiphospholipid (aPL) antibodies are seen more commonly, usually after childhood infections. In those with "true" aPL antibodies, recurrent thrombotic events seem less frequent than in adults, perhaps reflecting the less prothrombotic hemostatic state of childhood. Children with thrombotic events in APS present difficult management problems, as there is little evidence-based medicine. The duration and intensity of anticoagulation are unresolved management issues, but a target international normalized ratio of 2 to 3 is used by most. Multicenter randomized controlled trials would provide answers to some of these issues but are difficult to organize due to ethical issues and the rarity of the condition. A pediatric APS registry such as the Ped-APS Register is more easy to organize and can yield informative data.

Recurrent pregnancy loss and frequency of eight antiphospholipid antibodies and genetic thrombophilic factors in Czech women.

PROBLEM: The aim of this study was to investigate frequencies of eight antiphospholipid antibodies (aPLs) in serum, four genetic thrombophilic factors and their mutual relation in 206 patients with repeated pregnancy loss (RPL). METHOD OF STUDY: Enzyme-linked immunosorbent assay was used for detection of aPLs against ph-serine, ph-ethanolamine, ph-inositol, DL-glycerol, phosphatidic acid, anti-annexin V, cardiolipin, and beta2-GPI. FV 1691G>A (Leiden mutation), FII 20210G>A mutation, MTHFR 677C>T and MTHFR 1298A>C variant genotypes were determined using a melting curve analysis of the PCR amplification product detected by the fluorescence resonance energy transfer. Genotypic distribution and allelic frequencies were calculated. Correlation between aPLs and thrombophilic factors was tested by chi-square and Fisher exact test. RESULTS: Our results show significantly increased prevalence of aPLs against ph-inositol (17-19.6% dependent on number of spontaneous miscarriages) and against ph-serine (18-25%). aPLs in IgG prevail. In 96% of the studied group, at least one risk factor was found (either aPLs positivity or thrombophilic factor). Both aPLs and thrombophilic factors were present in 43%. In the group of women with three or more RPLs, strong positive correlation of aPLs positivity and thrombophilic risk factors was observed. CONCLUSION: Antiphospholipide antibodies and genetic thrombophilic factors are important risk factors in the pathogenesis of RPL. Both autoantibodies against various kinds of phospholipides and genetic thrombophilic factors must be studied together in diagnosis of RPL for appropriate treatment.

Standardization, regulation, quality assurance and emerging technologies in hemostasis: issues, controversies, benefits, and limitations.

There are many benefits to the overall process of standardization for tests of hemostasis and thrombosis. Nevertheless, it should also be recognized there are several specific problems and limitations to this process, as highlighted in this review. Some of the issues are formidable, but it is hoped that they are not insurmountable. Sometimes, clinical pressures drive diagnostic test processes before they are formally proven to be of clinical value. Such clinical pressures drive diagnostic changes in the hemostasis laboratory, including the incorporation of new and emerging technologies, which in turn drives the need for evolving and effective external quality assurance. Incorporated into the diagnostic or test-performance process are a large number of organizations involved in driving standardization, with the ultimate intention of improving diagnostics, but this process will also have unintended potential for adverse outcomes. Although this review notes many of the benefits to the process, it focuses primarily on those negative factors that are often less obvious but still require attention and process review.

Immunization of LDL receptor-deficient mice with beta2-glycoprotein 1 or human serum albumin induces a more inflammatory phenotype in atherosclerotic plaques.

Antiphospholipid antibodies are a risk factor for venous and arterial thrombosis and may contribute to the development of atherosclerosis.The aim of this study was to investigate whether antibodies to human beta2-glycoprotein 1 (beta2GP1), as a model of antiphospholipid antibodies, modify the phenotype of atherosclerotic lesions. LDL receptor-deficient mice were immunized with human beta2GP1, human serum albumin (HSA), or not immunized, and fed a high-cholesterol diet for 14 weeks. Some mice also received pravastatin. Immunization with human beta2GP1 or HSA resulted in formation of autoantibodies recognizing murine beta2GP1 or murine albumin, respectively. We quantified atherosclerotic lesion development and mRNA levels of inflammation associated proteins in the thoraco-abdominal aorta as well as lesion development,cellular composition and collagen content in the aortic roots. Immunization with beta2GP1 or HSA had no effect on lesion size,but modified the expression in plaque areas of several inflammation-associated proteins. Expression of matrix metalloproteinase-9, tissue factor, interferon-gamma and CD25 was highest in the thoraco-abdominal aorta of beta2GP1-immunized mice, lowest in non-immunized mice and intermediate in HSA-immunized animals. Immunization with beta2GP1, but not HSA, resulted in a lower smooth muscle cell and collagen content of lesions in aortic roots. Statin treatment partially reversed the effects of beta2GP1 immunization. We conclude that immunization with beta2GP1, and to a lesser extent with HSA, leads to modifications in the cellular and protein composition of atherosclerotic plaques, which are associated with a more inflammatory phenotype. Statin treatment partially prevents these changes.

Sphingosine 1-phosphate regulates peritoneal B-cell trafficking for subsequent intestinal IgA production.

Sphingosine 1-phosphate (S1P) is known to play a pivotal role in the regulation of lymphocyte emigration from organized lymphoid tissues such as the peripheral lymph nodes and thymus, but its immunologic role in unorganized and diffused tissues remains to be elucidated. Here we show that the trafficking of peritoneal B cells is principally regulated by S1P. All peritoneal B cells including B1a, B1b, and B2 B cells express comparable levels of the type 1 S1P receptor. Thus, treatment with FTY720, an S1P receptor modulator, caused the rapid disappearance of peritoneal B cells by inhibiting both their emigration from parathymic lymph nodes and their recirculation from the blood into the peritoneal cavity without affecting their progenitor populations. These changes did not affect natural plasma antibody production or phosphorylcholine (PC)-specific antibody production in serum after peritoneal immunization with heat-killed Streptococcal pneumoniae (R36A). However, FTY720 dramatically reduced peritoneal B cell-derived natural intestinal secretory IgA production without affecting the expression of J-chain and polyimmunoglobulin receptors. Additionally, FTY720 impaired the generation of PC-specific fecal IgA responses after oral immunization with R36A. These findings point to a pivotal role for S1P in connecting peritoneal B cells with intestinal B-cell immunity.

The association of VP1 unique region protein in acute parvovirus B19 infection and anti-phospholipid antibody production.

BACKGROUND: Previous studies have postulated a connection between human parvovirus B19 (B19) infection and anti-phospholipid antibodies (aPL). Recently, the phospholipase domain of B19 has been linked to B19-VP1 unique region (VP1u). To elucidate the roles of VP1u in B19 infection and aPL production, the major reactivity of anti-B19-VP1u, anti-cardiolipin antibody (aCL), and anti-beta2-glycoprotein I (beta2GPI) antibody was evaluated. METHODS: Sera from 102 clinically suspected cases of B19 infection were analyzed by nested PCR and ELISA. Humoral responses of anti-B19-VP1u and anti-B19-VP1uD175A IgM/IgG antibodies, aCL and the anti-beta2GPI antibody were assessed by Western blot and ELISA. Absorption experiments were also performed to determine the binding specificity of immunoglobulins to B19-VP1u, CL and beta2GPI. RESULTS: Sera from patients with the diagnostic pattern DNA+/IgM+/IgG+ had a high frequency (57%) for recognition of CL and beta2GPI. Furthermore, adsorption experiments were performed by adding purified B19-VP1u, which partially suppressed the reactivity of anti-B19VP1u to CL and beta2GPI. CONCLUSIONS: Serum from patients with acute B19 infection has a high frequency in recognition of CL and beta2GPI, and the phospholipase domain observed in the B19-VP1u may have contributed to the production of aPL. These findings may provide a clue for understanding the roles of B19-VP1u in B19 infection and aPL production.