Induction of antiphospholipid antibodies and antiphospholipid syndrome-like autoimmunity in naive mice with antibody against human parvovirus B19 VP1 unique region protein

BACKGROUND: Previous studies have postulated a connection between human parvovirus B19 (B19) infection and anti-phospholipid antibodies (APhL). B19 infection and anti-phospholipid syndrome (APS) exhibit congruent symptoms. Recently, phospholipase A2 (PLA2)-like activity has been linked to the VP1 unique region (VP1u) of B19. However, the precise role of B19-VP1u in pathogenesis of autoimmunity is still obscure. METHODS: To elucidate the roles of VP1u in B19 infection and autoimmunity, the reactivity of B19-VP1u proteins with various autoantibodies were evaluated by ELISA and immunoblotting. Rabbits were immunized with purified recombinant B19-VP1u protein to generate anti-sera. Absorption experiments were conducted to determine the binding specificity of rabbit anti-sera against B19-VP1u, cardiolipin (CL) and beta-2-glycoprotein I (beta2GPI). Moreover, the effects of passive transfer of polyclonal rabbit anti-B19-VP1u IgG antibodies on platelets, activated partial thromboplastin time (aPTT), and autoantibodies were assessed. RESULTS: Autoantibodies against CL, beta2GPI, and phospholipid (PhL) in sera from patients with B19 infection, were cross-reactive with B19-VP1u. Consistently, sera from rabbits immunized with recombinant B19-VP1u protein displayed raised detectable immunoglobulins against B19-VP1u, CL, beta2GPI and PhL. Additionally, the mice immunized with anti-B19-VP1u IgG developed thrombocytopenia, prolongation of aPTT, and autoantibody against beta2GPI and PhL. CONCLUSIONS: These experimental results suggested the association between B19-VP1u and production of anti-beta2GPI antibodies, APhL, and APS-like autoimmunity. Altogether, it may provide a clue in understanding the role of B19-VP1u in inducing autoantibodies and B19-associated APS manifestations.     

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.     

The association of anti-parvovirus B19-VP1 unique region antibodies with antiphospholipid antibodies in patients with antiphospholipid syndrome

BackgroundHuman parvovirus B19 (B19) infection has been identified as a trigger of antiphospholipid syndrome (APS). However, the precise role of B19-VP1 unique region (VP1u) in patients with antiphospholipid syndrome remains unclear.MethodsIgM and IgG against B19-VP, and serum levels of antibodies directed against cardiolipin (CL), beta2-glycoprotein-I (β2GPI) and phospholipid (PhL) were determined using ELISA in 45 APS patients. Humoral responses of anti-B19-VP1u were assessed by Western blot and B19 DNA was detected by nested PCR. Absorption experiments were performed using B19-VP1u protein to determine the binding specificity of antiphospholipid antibodies (aPL).ResultsOne and 18 of 45 APS patients had detectable levels of anti-B19-VP IgM and anti-B19-VP IgG, indicating recent and past infection respectively. All serum samples from APS patients with diagnostic pattern DNA^?/IgM^?/IgG⁺ had anti-B19-VP1u activity. APS patients with anti-B19-VP1u antibody had a 4-fold increased risk for recurrent vascular thrombosis compared with those without anti-B19-VP1u antibody. The binding inhibition of CL, β2GPI, and PhL by absorption with B19-VP1u ranged from 31.4% to 91.1%, 0.8% to 59.8% and 20.2% to 72.1% respectively. Significantly higher inhibition to β2GPI by B19-VP1u absorption was observed in APS patients with anti-B19-VP1u antibody than in those without anti-B19-VP1u antibody.ConclusionsWe show a close association of B19 infection with aPL production and suggest B19-VP1u may be of pathogenetic importance in some patients with APS.     

Biosensor analysis of beta2-glycoprotein I-reactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies

BACKGROUND: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-beta2-glycoprotein I (beta2GPI) autoantibodies (anti-beta2GPI) and pathogen-specific beta2GPI cross-reactive antibodies that occur transiently during infections. METHODS: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-beta2GPI in serum samples, affinity-purified human beta2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to beta2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with beta2GPI-specific ELISA. RESULTS: Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50-500, resonance units (RU) for anti-beta2GPI ELISA-positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-beta2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized beta2GPI. In contrast to APS patient samples, no significant anti-beta2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection. CONCLUSIONS: The SPR biosensor system enables specific detection of APS-associated beta2GPI-reactive APL and differentiation from beta2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-beta2GPI responses in APS patient sera gives additional information regarding the influence of anti-beta2GPI IgG and IgM isotype distribution.     

The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study

Summary.  Background :   Diagnosis of the antiphospholipid syndrome (APS) is difficult as a result of limited specificity of existing assays for detecting clinically relevant antiphospholipid antibodies. Anti-beta2-glycoprotein I (beta2GPI) antibodies play a central role in the disease process of APS. Objectives :   We have investigated the relation between antiphospholipid antibodies with specificity for domain I of beta2GPI and thrombosis/pregnancy morbidity in an international multicenter study. Patients/methods :   Four hundred and seventy-seven patients derived from nine different centres met the inclusion criterion of having anti-beta2GPI antibodies in their plasma/serum. Clinical data and results of tests for lupus anticoagulant, anti-cardiolipin antibodies and anti-beta2GPI antibodies were established at the different centres of inclusion. After being re-tested for the presence of IgG and/or IgM anti-beta2GPI antibodies, the samples were tested for the presence of IgG-directed against domain I of beta2GPI and results were correlated with the thrombotic and obstetric history. Results :   Re-testing for the presence of anti-beta2GPI antibodies resulted in inclusion of 442/477 patients. IgG class anti-domain I antibodies were present in plasma of 243/442 patients (55%). 201/243 (83%) had a history of thrombosis. This resulted in an odds ratio of 3.5 (2.3–5.4, 95% confidence interval) for thrombosis. Anti-domain I IgG antibodies were also significantly correlated with obstetric complications [odds ratio: 2.4 (1.4–4.3, 95% confidence interval)]. Conclusion :   In this multicenter study, the detection of IgG antibodies that are directed against domain I of beta2GPI proved to be more strongly associated with thrombosis and obstetric complications than those detected using the standard anti-beta2GPI antibody assay.     

Laboratory diagnosis of parvovirus B19 infection.

The sensitivity and application of the polymerase chain reaction (PCR) for the diagnosis of parvovirus B19 (B19) infection was investigated by simultaneously assaying a collection of 279 consecutively received samples for presence of anti-B19 IgM and IgG antibodies by Western blot and for B19 DNA by PCR and dot-blot hybridization (dot-blot); samples were sera from patients with suspected B19 infection. PCR and dot-blot detected B19 DNA in 9% (16/179) and 1% (2/179), respectively of Ab-positive samples (IgM+/IgG-, IgM+IgG+, IgM-IgG+), and in 28% (15/54) and 2% (1/54), respectively, of IgM+ samples. PCR also detected B19 DNA in 2% (2/100) of IgM-/IgG- samples, both of which had normal total IgG and IgM levels. PCR is of unique value because it permits diagnosis of B19 infection even in the absence of specific acute phase (IgM) and in the presence or absence of convalescent-phase (IgG) Ab.     

广州地区献血人群人类细小病毒B19感染情况及病毒载量分析

目的了解广州地区献血者HPVB19感染状况。方法采用EIA对献血者血进行HPVB19 IgG、IgM筛查,并用PCR法对HPV B19抗体阳性血样进行HPV B19 DNA检测。结果HPV B19 IgG阳性率为38.6%(679/1760),HPVB19 IgM阳性率为1.9%(33/1760),两者有显著差异(P<0.001)。33例HPVB19 IgM阳性样本,HPVB19 DNA阳性检出率为63.6%(21/33);56例HPVB19 IgG阳性样本,HPVB19 DNA阳性检出率为1.8%(1/56),两者有显著差异(P<0.001)。21例HPV B19 DNA阳性样本中,病毒载量≥1×104Copies/ml占51.9%(13/21)。结论广州地区献血者HPV B19病毒既往感染率较高,但HPV B19病毒急慢性感染率较低。     

人细小病毒B19IgM抗体间接ELISA检测方法的初步建立

目的：利用原核表达并纯化的人细小病毒B19（HPV B19）结构蛋白VP1初步建立HPV B19 IgM酶联免疫吸附试验（ELISA）的检测方法。方法以纯化的重组蛋白包被酶标板,建立IgM 间接ELISA检测方法,确定Cut-off值,并进行初步的应用。结果建立间接 ELISA方法Cut-off值为0．25,灵敏度为84．60％,特异性为99．70％,与对照试剂盒检测结果符合率为99．47％;用自产试剂盒检测115份孕妇和1700份健康献血志愿者血清中B19 IgM抗体,阳性率分别为12．17％和1．59％。结论通过包被 HPV B19-VP1蛋白建立的间接ELISA检测方法可用于 HPV B19早期感染或急性感染的辅助诊断。     

Increased seroprevalence of parvovirus B 19 IgG in complex regional pain syndrome is not associated with antiendothelial autoimmunity.

The etiology of complex regional pain syndrome (CRPS) is unclear yet. Recently autoantibodies and antecedent viral infections have been discussed to be involved in the pathogenesis of CRPS. We investigated sera from 39 CRPS patients and healthy controls for parvovirus B19 IgG and the occurrence of antiendothelial autoantibodies (AECA). CRPS patients showed a higher seroprevalence of parvovirus B19 IgG than controls (p < 0.01). All CRPS 2 patients were positive. 10.2% of the CRPS patients and 10.0% of the controls had AECA (n.s.) and AECA were not associated with parvovirus B19 seropositivity. Our findings suggest the involvement of parvovirus B19, but not autoantibody-mediated endothelial cell damage, in the pathogenesis of CRPS.     

The presence of IgG antibodies against β2-glycoprotein I predicts the risk of thrombosis in patients with the lupus anticoagulant

BACKGROUND: Lupus anticoagulant (LA) is a strong risk factor of thrombosis. However, a subgroup of patients positive for LA is unaffected by thrombosis and currently no predictive markers are available to identify patients positive for LA at increased risk for thrombosis. OBJECTIVE: The aim of the study was to investigate whether anti-beta-2-glycoprotein I (anti-beta2GPI) or anticardiolipin antibodies (ACA) are associated with an increased risk of thrombosis in patients persistently positive for LA. PATIENTS AND METHODS: A cohort of 87 consecutive patients persistently positive for LA was investigated, 55 with and 32 without a history of thrombosis. Immunoglobulin G (IgG) and M (IgM) antibodies against beta2GPI and cardiolipin were determined by enzyme-linked immunoassay. RESULTS: Patients positive for LA with thrombosis had significantly higher levels of anti-beta2GPI IgG (median 16.7 standard units, interquartile range 3.0-75.2, P = 0.002) and of ACA IgG (41.1 IgG phospholipid units per mL, 8.9-109.0, P = 0.002) than those without thrombosis (2.6, 1.4-7.9 and 9.7, 4.6-22.1, respectively). Levels of anti-beta2GPI IgM and ACA IgM did not differ significantly between LA patients with and without thrombosis (P = 0.25 and 0.12, respectively). Elevated anti-beta2GPI IgG was associated with an increased risk for thrombosis (OR = 4.0, 95% CI 1.2-13.1), especially for venous thromboembolism (OR = 5.2, 95% CI 1.5-18.0). CONCLUSIONS: Increased levels of anti-beta2GPI IgG were associated with thrombosis. We conclude that anti-beta2GPI IgG levels above normal predict an increased risk of thrombosis in patients persistently positive for LA.     

A prospective study of antibodies to β2-glycoprotein I and prothrombin, and risk of thrombosis

Antiphospholipid syndrome (APS) is a clinical autoimmune disorder characterized by thrombosis/pregnancy morbidity associated with the persistence of lupus anticoagulant (LA) and/or anticardiolipin (aCL) antibodies. We assessed the contribution of antibodies to beta2-glycoprotein I (anti-beta2GPI) and prothrombin (anti-PT) to the thrombotic risk in a cohort of 194 consecutive patients with persistent LA and/or aCL. Median follow-up was 45 months. A total of 39 patients (20.1%) had one documented episode of thrombosis during follow-up. Eleven of these patients had no previous thrombosis before enrollment in the study and 28 had recurrences of thrombosis. There were 21 venous and 18 arterial thrombotic events and the overall incidence of thrombosis was 5.6% per patient-year. After multivariate analysis, the male sex (P = 0.025), a previous thrombosis (P < 0.01), the presence of anti-beta2GPI (P = 0.001), and the presence of anti-PT (P = 0.03) remained as independent risk factors for recurrent thrombosis. Only IgG anti-beta2GPI and anti-PT were associated with an increased risk of thrombosis (P < 0.01 and P = 0.005). Patients testing positive for anti-beta2GPI had a higher rate of thrombosis than did antiphospholipid patients without anti-beta2GPI (8.0% vs. 3.1% per patient-year). Similarly, a higher rate of thrombosis was found in patients with positive anti-PT compared with patients without anti-PT (8.6% vs. 3.5% per patient-year). Considering only the group of 142 LA positive patients, the highest incidence of thrombosis was found in LA patients positive for both anti-beta2GPI and anti-PT (8.4% per patient-year). In conclusion, the presence of IgG anti-beta2GPI and anti-PT in patients with LA and/or aCL and mainly in those with LA predicts a higher risk of thromboembolic events.     

佛山地区无偿献血人群人细小病毒B19感染情况分析

目的了解佛山地区无偿献血人群人细小病毒(HPV)B19感染现状。方法采用ELISA检测血液中的HPV B19IgG和IgM抗体,并用PCR检测抗体阳性标本HPV B19DNA。结果 368例无偿献血者标本中检出HPV B19IgG阳性92例,阳性率为25.00%;检出HPV B19IgM阳性2例,阳性率为0.54%,两者比较差异有统计学意义(P0.01)。94例抗体阳性标本中,检测HPV B19DNA阳性4例,阳性率为4.26%。结论佛山地区无偿献血人群存在较高的HPV B19既往感染率,急、慢性感染率较低,慢性感染者HPV B19病毒载量较低。     

Immune reconstitution to parvovirus B19 and resolution of anemia in a patient treated with highly active antiretroviral therapy

Immune reconstitution inflammatory syndrome (IRIS) is an unsolved problem in the treatment of human immunodeficiency virus (HIV)-1 infection. Despite the high seroprevalence of parvovirus B19 (PVB19) among HIV-1-positive patients, reports on PVB19-induced anemia, especially that associated with PVB19-related IRIS, in these patients are limited. We present the case of a man with acquired immunodeficiency syndrome who developed severe transfusion-dependent anemia and was seropositive and borderline positive for immunoglobulin-M and IgG antibodies against PVB19, respectively. PVB19-DNA was also detected in his serum. The patient was diagnosed with pure red cell anemia (PRCA) caused by a primary PVB19 infection and was treated with periodical blood transfusions. However, he subsequently tested negative for IgG antibodies and developed chronic severe anemia with high levels of PVB19 viremia. This indicated a transition from primary to persistent infection. After initiation of highly active antiretroviral therapy, the patient showed an inflammatory reaction with rapid deterioration of anemia and seroconversion of the IgG antibody to PVB19. Subsequently, PRCA was completely resolved, but the patient’s serum still contained low levels of PVB19-DNA. Thus, this was a case of IRIS associated with PVB19 infection. Our report highlights the significance of seroconversion to PVB19 in the diagnosis of IRIS and re-emphasizes the finding that persistently high levels of PVB19 viremia after primary infection are probably because of the lack of protective antibodies.     

Seroprevalence of human parvovirus B19 amongst North Indian blood donors - do current donor testing guidelines need a relook?

Objectives

The present study was aimed to find out the prevalence of parvovirus B19? amongst healthy blood donors and blood transfusion recipients so as to determine the feasibility of providing seronegative blood components to vulnerable recipients.

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.     

Analytical and clinical performance of a new,automated assay panel for the diagnosis of antiphospholipid syndrome

Summary. Background: Anticardiolipin (aCL) and anti‐β₂glycoprotein I (aβ₂GPI) antibodies are part of the criteria for antiphospholipid syndrome (APS). Therefore they are widely measured and about 30 commercial kits are available. Objectives: To investigate the analytical and clinical performances of four fully automated, chemiluminescent assays: HemosIL AcuStar aCL IgG, HemosIL AcuStar aCL IgM, HemosIL AcuStar aβ₂GPI IgG, and HemosIL AcuStar aβ₂GPI IgM. Methods: Cut‐off values were assessed by testing 250 blood donors. Total coefficients of variation (CV) were determined with six plasma pools and two controls ranging from 4.3 to 2694 U mL^?1 depending on the assay. Samples from 218 well‐characterized patients and 103 controls were measured in three laboratories to determine inter‐laboratory variation. The results of the 321 samples were compared with three commercial assays (REAADS, INOVA and VARELISA). Results: Cut‐off values were assigned to 20 arbitrary units for all the tests. Total CV ranged from 4.3 to 11.2%. No interference of hemoglobin, bilirubin, triglycerides, heparins and rheumatoid factor was observed. Inter‐laboratory variability was low and no sample changed status. Overall status agreement between HemosIL assays and the comparator kits ranged from 82 to 96%. Sensitivity, specificity, agreement when predicting APS and the odds ratios when predicting a thrombotic or obstetric event gave comparable results between HemosIL AcuStar and the three other assays. Conclusions: Our study demonstrates that the fully automated HemosIL AcuStar aPL assay panel showed similar performances to the three commercial ELISAs commonly used by various laboratories to detect antiphospholipid antibodies.     

Anti-beta 2 glycoprotein I antibodies and the risk of myocardial infarction in young premenopausal women

BACKGROUND: Contrasting data have been reported on the association between the presence of anti-phospholipid antibodies (aPL) and arterial thrombotic events, particularly those in coronary arteries. This discrepancy is perhaps related to the confounding effect of traditional risk factors. Among them, coronary atherosclerosis appears to be the most important in studies conducted in middle-aged and elderly patients. OBJECTIVE: To minimize such confounding effects, a multicenter case-control study on the association between aPL and myocardial infarction (MI) was carried out in a rare cohort of young premenopausal women. METHODS: We evaluated 172 cases hospitalized for a first MI before the age of 45 years and 172 controls individually matched with cases for age, sex and geographical origin. Clinical and laboratory data were collected and levels of anti-cardiolipin (aCL), anti-beta2 glycoprotein I (anti-beta2GPI) and anti-nuclear antibodies (ANA) were measured. RESULTS: A significant association between MI and IgG/IgM anti-beta2GPI antibodies was observed; the results were confirmed after adjusting for smoking and hypertension (anti-beta2GPI IgG OR = 2.47, 95% CI 1.81-3.38; anti-beta2GPI IgM 4th quartile OR 3.68, 95% CI 1.69-8.02). The association between anti-beta2GPI antibodies and MI was detected in both subgroups with and without coronary artery stenosis. Whereas the association of aCL IgG with MI was modest, ANA showed no significant association with MI. No aPL were found in unselected patients (mainly males) who recently developed acute MI. CONCLUSIONS: Anti-beta2GPI antibodies are a significant risk factor for MI in young premenopausal women independently of other risk factors, including the degree of coronary artery stenosis.     

In vivo inhibition of antiphospholipid antibody‐induced pathogenicity utilizing the antigenic target peptide domain I of β2‐glycoprotein I: proof of concept

Summary. Objectives: In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N‐terminal domain I (DI) of β₂‐glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL‐induced pathogenicity in vivo. Methods: C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG‐APS, 500 μg) or IgG from normal healthy serum (IgG‐NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10–40 μg). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule‐1 (VCAM‐1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. Results: IgG‐APS significantly increased thrombus size as compared with IgG‐NHS. The IgG‐APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P ≤ 0.0001) and DI(D8S/D9G) (P ≤ 0.0001), but not in those treated with DI(R39S) or control peptide. This inhibitory effect by DI was dose‐dependent, and at lower doses DI(D8S/D9G) was a more potent inhibitor of thrombosis than wild‐type DI (P ≤ 0.01). DI also inhibited IgG‐APS induction of VCAM‐1 on the aortic endothelial surface and TF production by murine macrophages. Conclusion: Our findings in this proof‐of‐concept study support the development of recombinant DI or the novel variant DI(D8S/D9G) as a potential future therapeutic agent for APS.     

Determination of diagnostic threshold in harmonization and comparison of clinical utility for five major antiphospholipid antibody assays used in Japan

BackgroundAnticardiolipin antibodies (aCL) and anti‐β₂‐glycoprotein I antibodies (aβ₂GPI) are essential in diagnosing antiphospholipid syndrome (APS) according to the international APS guideline. Five commercial assays for aCL and aβ₂GPI are available in Japan, but their test results are quite discordant. For harmonization of diagnosing APS, upper reference limit (URL) and diagnostic accuracy of each assay were evaluated and compared by testing common sets of specimens across all assays.MethodsWe evaluated two manual and three automated assays for aCL and aβ₂GPI of IgG‐ and IgM classes. 99%URL (the upper limit of reference interval: as per guideline) together with 97.5%URL were determined by testing sera from 198 to 400 well‐defined healthy subjects. Both URLs were compared with the cutoff values, which were determined based on ROC analysis by testing 50 each of plasma specimens from patients with/without APS. Diagnostic accuracy was evaluated as area under curve (AUC) of the ROC curve.ResultsA variable degree of discrepancy between URLs and the cutoff values was observed, which was partly attributable to between‐year assay variability. 97.5%URLs were set lower and closer to the cutoff values than 99%URLs. For all assays, diagnostic accuracies of both aβ₂GPI‐IgG and aCL‐IgG were generally high (AUC: 0.84−0.93); whereas those for IgM‐class assays were low (AUC: 0.57−0.67), implicating its utility is limited to rare IgG negative APS cases.ConclusionTo ensure harmonized APS diagnosis, the diagnostic thresholds of the five assays were evaluated by common procedures. Contrary to the guideline, 97.5%URL is rather recommended for diagnosing APS, which showed a closer match to the cutoff value.     

Combined search for anti-beta2-glycoprotein I and anticardiolipin antibodies in antiphospholipid syndrome: contribution to diagnosis

In this study we sought to assess (1) the diagnostic value of a combined search for anti-beta(2)-glycoprotein (abeta(2)-GPIs) and anticardiolipin antibodies (aCLs) in primary (APS I) and secondary (APS II) antiphospholipid syndrome and (2) the influence of the beta(2)-GPI preparation in the ELISA's results. abeta(2)-GPI and aCL concentrations were assessed in 70 patients with APS and compared with those in 65 patients with systemic lupus erythematosus (SLE) without clinical features of APS. In APS patients (38 with APS I, 32 with APS II), the diagnosis had to have been made at least 3 years earlier; in subjects with SLE, the diagnosis had to have been made at least 5 years earlier. All serum samples were tested for abeta(2) -GPI with the use of an in-house ELISA with an abeta(2) -GPI preparation from human plasma. Samples negative for abeta(2) -GPI were controlled with 2 additional beta(2)-GPI preparations, 1 from human serum and 1 from bovine serum. In APS, abeta(2)-GPIs were more frequent than in SLE (76% and 15%, respectively; P <.0001), mainly with IgG isotype and with significantly higher levels than those found in SLE. The specificity for APS was 92% for IgG abeta(2)-GPIs and 68% for IgG aCLs. The highest association with APS was found for the combination of the 2 markers (odds ratio 29; 95% confidence interval 10-76; P <.0001). Among the APS patients, 6 were positive for aCL only and remained negative regardless of which beta 2 -GPI preparation was used; 1 patient was aCL-negative and only positive with human beta 2 -GPI. These data emphasize the heterogeneity of the APS immunologic profile and the diagnostic possibilities of both antibodies.