Phospholipid specificity and requirement of beta 2-glycoprotein-I for reactivity of antibodies from patients with primary antiphospholipid syndrome.

Some disease manifestations are associated with serum antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE) in what has been termed antiphospholipid syndrome (aPLS). There are patients with aPLS who do not have SLE or any other illness who have been grouped under the term primary antiphospholipid syndrome (PAPS). However, patients with diverse infections, notably syphilis, may have aPL but do not develop the associated clinical manifestations. This has been attributed, at least in part, to the immunochemical features of their aPL, including the requirement for beta 2-glycoprotein-I (beta 2GP-I) for binding of aPL to phospholipids, but these have not been studied in sera from patients with PAPS. By ELISA we studied 95 sera from 17 patients with PAPS and 100 sera from clinically normal individuals for IgG and IgM antibodies to the main anionic and zwitterionic phospholipids and their related compounds, phosphatidic acid (PA) and synthetic phosphorylcholine (PRC). beta 2GP-I was present, either in newborn calf serum (NBCS) or purified, to block wells and to dilute samples, or was substituted by 0.3% gelatin. Inhibition studies with phospholipid micelles were used to confirm reactivities with the corresponding phospholipids. All 17 patients had IgG and 11 had IgM antibodies to cardiolipin. Antibodies to anionic phospholipids were primarily IgG whereas those to zwitterionic phospholipids were mainly, and often exclusively, IgM. We found a statistically significant difference in the mean levels of antibodies to all anionic phospholipids except aPTS, and to the haptene PA (P < 0.001) between patients and controls. The difference between levels of IgM antibodies to zwitterionic phospholipids was statistically significant with sphingomyelin (P < 0.001) and the haptene (P < 0.001). Levels of most IgG and most IgM aPL correlated significantly among them. The pattern and titers of reactivity are variable between patients, but stable within each patient. Requirement of beta 2GP-I for this reactivity was not an all-or-nothing phenomenon in individual sera. In general, as in lupus sera, antibodies to anionic phospholipids require that this cofactor be present coating the ELISA plates, whereas those to zwitterionic phospholipids do not. It would appear that patients with PAPS have polyclonal mixtures of antibodies that react with various phospholipids and have different requirements for beta 2GP-I for such reactivity.     

Effect of Antiphospholipid Antibodies on β2Glycoprotein I-Phospholipid Interaction

PROBLEM: β₂ glycoprotein I (β₂GPI) physiologically binds to negatively charged phospholipids (PLs) and is a natural regulator of the coagulation cascade. Thrombotic clinical complications and recurrent fetal loss associated with autoimmune antiphospholipid (aPL) antibodies are thought to be related to their binding to β₂GPI-PL complex and interference with the physiological function of β₂GPI. METHOD OF STUDY: To investigate the effect of aPL on β₂GPI-PL interaction, we studied the binding of biotinylated β₂GPI to cardiolipin (CL) by enzyme-linked immunosorbent assay (ELISA) in the presence and absence of purified aPL immunoglobulin G (IgG) antibodies. RESULTS: Adding five different aPL IgG antibodies with different levels of aPL activity isolated from the sera of five patients with aPL-associated recurrent fetal death greatly increased the binding of biotinylated β₂GPI to CL-coated plates. The optical densities (ODs) were 0.635, 0.890, and 1.265 in the presence of three aPL IgG antibodies, compared to 0.425 in the absence of aPL IgG. In contrast, normal human IgG had no enhancing effect. The OD was 0.480 and 0.425, respectively. The enhancement of β₂GPI binding to CL by aPL IgG correlated with the titers of aPL antibodies. The use of phosphate-buffered saline with increasing salt concentrations as a washing buffer for the ELISA resulted in more stable binding of β₂GPI to PL in the presence of aPL IgG. CONCLUSIONS: These findings suggest that the binding of autoimmune aPL antibodies to β₂GPI-PL complex results in abnormally tighter interaction between β₂GPI and PLs, which may lead to physiological dysfunction of β₂GPI as a regulator of coagulation.     

IgG2 subclass restriction of anti-β2 glycoprotein 1 antibodies in autoimmune patients

The IgG subclass and light chain distribution of antiphospholipid antibodies (aPL) occurring in autoimmune patients were determined by means of two radioimmunoassays using either cardiolipinor β₂ glycoprotein 1 (β₂GP1)-coated microtitre plates and mouse MoAbs. Of 50 sera selected for positivity of anticardiolipin antibodies (ACA) of the IgG isotype, 32 (64%) possessed anti-β₂GPl antibodies and their presence was closely associated with clinical features of the antiphospholipid syndrome. Good correlations were found between ACA and anti-β₂GP1 antibodies when considering antibody level and patterns of light chain and IgG subclass, suggesting that, overall, the same antibodies were being measured. Light chain analysis showed the polyclonal origin of these antibodies and, in most sera, a trend towards use of λ chain. Among sera positive for anti-β₂GP1 antibodies, IgG2 was the major subclass reactive with β₂GP1 and cardiolipin (87% and 74β₂ of the IgG antibody activity, respectively). In contrast, in the group of 18 sera lacking anti-β₂GP1 antibodies, ACA were largely restricted to lgG3, with a lesser contribution by IgGl. A few selected sera from the anti-β₂GP1-positive group were shown to contain mixtures of antibodies that required β₂GP1 (restricted to IgG2 present in large amounts) and did not require this cofactor (restricted to IgG3 and/or IgG1 present in low amounts) for their reactivity with cardiolipin. There was no contribution of glycosylation to the epitopes recognized by anti-β₂GP1 antibodies, even though human anti-carbohydrate antibodies are restricted to the IgG2 subclass. These findings further emphasize the intra- and interindividual heterogeneity of aPL, and should help to discriminate clinically relevant specificies.     

Human reproductive failure is not a clinical feature associated with beta(2) glycoprotein-I antibodies in anticardiolipin and lupus anticoagulant seronegative patients (the antiphospholipid/cofactor syndrome)

It has been suggested that patients with clinical features suggestive of antiphospholipid syndrome but being lupus anticoagulant (LA) and anticardiolipin (aCL) negative, should be tested for antibodies to beta(2) glycoprotein-I (abeta(2)GP-I), a protein involved in the binding of antiphospholipid antibodies (aPL) to phospholipid surfaces. This was investigated in the present study where a total of 385 women aged 相似文献   

APhL antibody ELISA as an alternative to anticardiolipin test for the diagnosis of antiphospholipid syndrome

Background

Persistent levels of antiphospholipid (aPL) antibodies [lupus anticoagulant (LA), anticardiolipin (aCL), anti-beta 2 glycoprotein I (aβ₂GPI) IgG and/or IgM] in association with clinical features of thrombosis and/or pregnancy associated morbidity are indicative of antiphospholipid syndrome (APS). Of the aPL antibodies, aCL is the most sensitive for APS, however, their lack of specificity constitute a laboratory and clinical challenge. IgG/IgM antibodies directed against APhL (a mixture of phospholipids) has been reported to predict APS more reliably than aCL tests. The main objective of this study was to evaluate the performance characteristics of the APhL IgG/IgM ELISA, relative to the aCL and aβ₂GPI tests.

Methods

Sixteen (16) clinically confirmed APS and 85 previously tested serum (PTS) samples for aCL and aβ₂GPI IgG/IgM antibodies were evaluated with the APhL IgG/IgM ELISA. Clinical specificity was determined in 100 serum samples (50 healthy and 50 infectious disease controls [parvo- and syphilis-IgG/IgM positive]).

Results

The IgG antibody prevalence for aCL and APhL in the APS and PST groups was comparable with marginal differences in clinical specificities. In contrast to the aCL IgM ELISA, the APhL test showed improved clinical specificities (72% aCL vs 94% APhL in the healthy controls; 38% aCL vs 78% APhL in the infectious disease controls) with implications for increased reliability in the diagnosis of APS. The overall agreement of the APhL with the aCL or aβ₂GPI for the IgG tests was 89% and 85% respectively, and that of the APhL IgM to the aCL or aβ₂GPI IgM tests was 72% and 86% respectively.

Conclusion

Routine use of the APhL IgG/IgM ELISA may substantially reduce the high number of false positives associated with the aCL test without loss in sensitivity for APS.     

Affinity-purified cardiolipin-binding antibodies show heterogeneity in their binding to oxidized low-density lipoprotein

Antiphospholipid antibodies in autoimmune sera have been shown to react with a complex of phospholipids (cardiolipin) and a plasma phospholipid-binding protein, β₂-glycoprotein I (apolipoprotein H). The binding of these antibodies was inhibited by oxidized low-density lipoprotein (LDL) in sera from patients with systemic lupus erythematosus (SLE), suggesting cross-reactivity between antiphospholipid antibodies and antibodies binding to oxidized LDL. We purified antiphospholipid antibodies by cardiolipin-polyacrylamide column from seven SLE sera and studied the reactivity of eluted fractions with cardiolipin–β₂-glycoprotein I complex and oxidized LDL (malondialdehyde-conjugated LDL) in solid-phase enzyme immunoassay. In four sera the binding of IgG antibodies to cardiolipin–β₂-glycoprotein I complex and to oxidized LDL appeared in the same fractions, whereas in three sera reactivities against cardiolipin and oxidized LDL were observed, at least in part, in separate fractions. The binding to solid-phase cardiolipin was dependent on the presence of exogenous β₂-glycoprotein I in all fractions. Our findings show that antiphospholipid antibodies are heterogeneous in their binding to oxidized LDL, indicating that these two antibodies may have different subspecificities. Some eluted fractions reacted only with oxidized LDL, and did not show binding to cardiolipin–β₂-glycoprotein I complex, suggesting that the lipid part in the antigenic complex might be responsible for the cross-reactivity of these antibodies. Accordingly, the biological functions of antibodies against phospholipid–β₂-glycoprotein I complex and antibodies against oxidized LDL may also be different.     

The effect of hydroxychloroquine on thrombosis prevention and antiphospholipid antibody levels in primary antiphospholipid syndrome: A pilot open label randomized prospective study

BackgroundSporadic studies suggest hydroxychloroquine (HCQ) may be effective for thrombosis prevention in patients with primary antiphospholipid syndrome (PAPS) and may lead to antiphospholipid antibody (aPL) titer reduction but data from randomized studies are lacking.MethodsWe conducted a pilot open-label randomized prospective study aiming to evaluate the safety and efficacy of HCQ for thrombosis prevention in 50 patients with PAPS allocated 1:1 to HCQ plus standard care (systemic anticoagulation and/or antiplatelet therapy) vs. standard care alone, as well as the effect of HCQ on aPL titers of 50 PAPS patients and 15 asymptomatic aPL carriers.ResultsHCQ use plus standard care was associated with lower incidence rate of thrombosis than standard care alone (0.001 vs. 0.007, log-rank p =0.048) over an average 2.6-year follow-up, and a multivariate hazard ratio of 0.09 (95% CI = 0.01–1.26, p = 0.074) after adjusting for the effect of age, sex, traditional cardiovascular risk factors, triple aPL positivity, history of recurrent thrombotic events at baseline, and poor anticoagulation quality (INR levels within therapeutic range for ≤80% of follow-up). No significant difference in safety outcomes was observed between the two groups. Long-term HCQ use was associated with a decrease in aPL titers except for IgM anticardiolipin antibodies, which tended to decrease overtime regardless of treatment allocation.ConclusionsHCQ may represent an effective adjuvant treatment for thrombosis prevention in patients with PAPS, which may be mediated via a reduction in aPL titers. Larger randomized trials are needed in order to corroborate this finding and investigate the thromboprotective role of HCQ in asymptomatic aPL carriers.     

Detection of antiphospholipid antibodies by automated chemiluminescence assay

The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2-glycoprotein I IgG or IgM antibodies (anti-β2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI by ELISA, were also positive for IgG aCl by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (p<0.0001). Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-β2-GPI IgG (ELISA). Thus, the new technology of automated chemiluminescence assay for measuring aPL may represent an useful tool to identify "true" APS patients.     

Laboratory evaluation of anti-phospholipid syndrome: a preliminary prospective study of phosphatidylserine/prothrombin antibodies in an at-risk patient cohort

Immunoglobulin (Ig)G/IgM autoantibodies to phosphatidylserine/prothrombin (aPS/PT) were evaluated individually and in combination with criteria anti-phospholipid (aPL) tests in a prospectively ascertained cohort of patients at risk for anti-phospholipid syndrome (APS). One hundred and sixty (160) consecutive requests for lupus anti-coagulant (LAC) from the University of Utah Health Sciences Center were identified during 8 weeks. Of these, 104 unique patients had additional requests for cardiolipin (aCL) and/or beta2 glycoprotein I (aβ₂GPI) IgG and/or IgM; samples were retained and analysed for aPS/PT, aCL and/or aβ₂GPI IgG and IgM antibodies. Following testing, a comprehensive chart review was performed and patients categorized according to their clinical diagnosis. Individual and combined sensitivities, specificities, odd ratios (OR), diagnostic accuracy for specific tests or combinations by receiver operating characteristic (ROC), area under the curve (AUC) analyses and correlations between test results were determined. The sensitivities of aPS/PT IgG/IgM (54·6/45·5%) were lower than LAC (81·8%) but higher relative to aCL IgG/IgM (27·3/0%) or aβ₂GPI IgG/IgM (27·3/0%). The best correlation between LAC and any aPL test was observed with aPS/PT (P = 0·002). There was no significant difference in the diagnostic accuracies for any panel with LAC: LAC/aβ₂GPI IgG/aCL IgG [AUC 0·979, OR 475·4, 95% confidence interval (CI) 23·1–9056·5, P = 0·0001 and LAC/aβ₂GPI IgG/aPS/PT IgG or LAC/aPS/PT IgG/aCL IgG (AUC 0·962, OR 265·3, 14·2–4958·2, P = 0·0001). The high correlation between LAC and aPS/PT IgG/IgM in this preliminary study suggest that this marker may be useful in the evaluation of APS. More studies to determine the optimal aPL antibody tests combination are needed.     

Antibodies to β2-glycoprotein I—a specific marker for the antiphospholipid syndrome

The antiphospholipid syndrome is a disorder characterized by recurrent thrombosis and the presence of antibodies specific to phospholipids. However, the diagnosis of this syndrome is hampered by the lack of a specific laboratory test. In this study an ELISA for the measurement of antibodies to solid-phase β₂-glycoprotein I (β₂-GPI) was established and compared with anticardiolipin antibodies for diagnosis of antiphospholipid syndrome. Significantly elevated levels of antibodies to β₂-GPI were found in all patients with definite antiphospholipid syndrome (median = 91 AU). Marginally elevated levels of antibodies to β₂-GPI were observed in 5% of patients with systemic lupus erythematosus (SLE; median = 4 AU), 1% with stroke (median = 3 AU), 13% with infectious mononucleosis (median = 3 AU), 10% with HIV infection (median = 3 AU) and 8% with VDRL false-positive serology for syphilis (median = 4 AU), but not in patients with rheumatoid factor, syphilis or carotid artery stenosis. In contrast, significantly raised levels of anticardiolipin antibodies were observed in 100% of patients with definite antiphospholipid syndrome, 30% with SLE, 88% with HIV infection, 94% with syphilis, 62% with infectious mononucleosis, 9% with rheumatoid factor-positive sera, 74% VDRL false-positive serology for syphilis, 47% with stroke and 0% with carotid artery stenosis. This solid-phase assay for antibodies to β₂-GPI is highly specific for the antiphospholipid syndrome and represents an advance in the laboratory diagnosis of this disorder.     

Antibodies against beta2-glycoprotein I complexed with an oxidised lipoprotein relate to intima thickening of carotid arteries in primary antiphospholipid syndrome

To explore whether antibodies against beta2-glycoprotein I (beta2GPI) complexed to 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) and to oxidised low-density lipoproteins (oxLDL) relate to paraoxonase activity (PONa) and/or intima media thickness (IMT) of carotid arteries in primary antiphospholipid syndrome (PAPS). As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL) without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: beta2GPI-oxLDL complexes, IgG anti-beta2GPI-oxLig-1, IgG anti-beta2GPI-oxLDL antibodies (ELISA), PONa, (para-nitrophenol method), IMT of common carotid (CC) artery, carotid bifurcation (B), internal carotid (IC) by high resolution sonography. Beta2GPI-oxLDL complex was highest in the control group (p < 0.01), whereas, IgG anti-beta2GPI-oxLig1 and IgG anti-beta2GPI-oxLDL were highest in PAPS (p < 0.0001). In healthy controls, beta2GPI-oxLDL complexes positively correlated to IMT of the IC (p = 0.007) and negatively to PONa after correction for age (p < 0.03). PONa inversely correlated with age (p = 0.008). In PAPS, IgG anti-beta2GPI-oxLig-1 independently predicted PONa (p = 0.02) and IMT of B (p = 0.003), CC, (p = 0.03) and of IC (p = 0.04). In PAPS, PONa inversely correlated to the IMT of B, CC and IC (p = 0.01, 0.02 and 0.003, respectively). IgG anti-beta2GPI-oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity.     

Characterization of monoclonal anti-beta2-glycoprotein-I and anti-prothrombin antibody fragments generated by phage display from a patient with primary antiphospholipid syndrome

The molecular structure of antibodies associated with autoimmune thrombosis is beginning to be understood. We describe the binding specificities and sequence analysis of anti-beta2-glycoprotein-I (anti-beta2GP-I) or anti-prothrombin (anti-PT) antibody fragments generated by phage display from a patient with primary antiphospholipid syndrome (APS). We obtained 39 positive clones, two that had the correct size reacted with beta2GP-I (Beta 1 and Beta 2). Ten clones with the same restrictive pattern recognized PT (Prot 1) and cross-reacted with beta2GP-I. All three clones recognized anionic and zwitterionic phospholipids. The V(H) regions of both anti-beta2GP-I clones are members of the VH4 family. Prot 1 has a V(H) segment of the VH3 family. The Beta 1 J(H) segments are J(H)5b and J(H)4b for Beta 2 and Prot 1. V(L) genes are V(lambda)1, 3 and 1, respectively. No J(L) was identified for Beta 1, while Beta 2 and Prot 1 carry J(lambda)3b genes. Beta 1 and Beta 2 carry highly conserved germ-line V(H) and V(L) genes. Mutations of the Prot 1 gene appear to be antigen-dependent, most are hotspot mutations located in the CDR 1 and 2 regions. Our work suggests that some anti-beta2GP-I from patients with primary APS are natural autoantibodies. Our work may also help to explain the frequent coexistence of anti-beta2GP-I and anti-PT in the same patient.     

Persistence of antiphospholipid antibodies over time and its association with recurrence of clinical manifestations: A longitudinal study from a single centre

PurposeTo analyze the antiphospholipid antibody (aPL) persistence over time in patients with antiphospholipid syndrome (APS) and its association with clinical recurrence and to identify predictors of aPL persistence over time.Patients and methods200 patients with a diagnosis of APS and at least three follow-up aPL determinations were included. Persistent aPL profile was defined as the presence of lupus anticoagulant (LAC) and/or IgG/IgM anticardiolipin (aCL) and/or IgG/IgM anti-β2 glycoprotein-I (aβ2GPI) (> 99th percentile) antibodies in at least 66% of follow-up measurements. Multilevel mixed-effect generalized linear models with logit link were used.Results112 (56%) patients maintained persistent aPL profiles over time, while 88 (44%) were transient. Median follow-up time was 172.5 months. Follow-up time did not affect the odds of aPL persistence in multivariate analysis (p = 1.00). Baseline triple aPL positivity [OR 78 (95%CI 16.9–359.7, p < 0.001)] and double aPL positivity [OR = 7.6 (95%CI 3.7–15.7, p < 0.001)] correlated with persistent aPLs over time, while isolated LAC [OR = 0.26 (95% CI 0.08–0.49, p = 0.002)] or isolated IgG/IgM aCL [OR = 0.20 (95% CI 0.11–0.59, p = 0.004)] positivity, were predictors of transient aPL profile. Patients with persistent aPLs had higher rate of clinical recurrence in comparison to patients with transient aPLs [OR = 2.48 (95%CI 1.34–4.58, p = 0.003)].ConclusionsMore than half of patients with baseline medium-high titer aPL positivity had persistent positive aPLs over time. Patients with persistent aPLs were more prone to present recurrence of clinical manifestations. Multiple aPL positivity increased the odds of a persistent aPL profile over time, while isolated LAC and aCL positivity decreased it.     

Immunologic characterization and functional properties of murine antibodies raised against deleted mutants of human beta 2-glycoprotein I

beta 2-Glycoprotein I (beta 2GPI) is a 50 kDa molecule proposed as a principal target of 'autoimmune' antiphospholipid antibodies (aPL). We have used deleted mutants (DM) representing different domains of beta 2GPI (I-IV, IV-V and V) for immunization of naive mice and studied the characteristics of the respective murine IgG preparations in comparison with affinity-purified IgG from two patients with primary antiphospholipid syndrome. Immunization with beta 2GPI and with the DM produced anti-beta 2GPI antibodies, part of which reacted with negatively charged phospholipids (PL), whereas reactivity with cardiolipin was evident only in the IgG from mice immunized with beta 2GPI. These results are consistent with the presumption that aPL are induced following the in vivo association of beta 2GPI (used for immunization) with resident negatively charged PL. Accordingly, DM which either lack the PL binding site or aPL attachment locus did not elicit, upon immunization, antibodies reactive with PL. Further, murine anti-beta 2GPI IgG and human 'autoimmune' aPL were similar, albeit not identical, in terms of DM requirement for PL binding and charge dependency. Murine antibodies and human aPL, regardless of their binding characteristics, were found to bind significantly to platelets upon their activation with thrombin and to promote platelet activation. The results of the current study emphasize the dissimilarities between human 'autoimmune' aPL and murine anti-beta 2GPI. Thus, anti-beta 2GPI antibodies to different DM as well as human aPL are capable of binding and activating human platelets provided beta 2GPI is present.      

Bacterial immunity and immunogenesis of normal human salivary IgA and serum IgG2 antiphospholipid autoantibody: a link?

The anti-phospholipid antibody (aPL) in 26 heat-inactivated normal human sera (NHS) was tested for IgG subclass in ELISA. The specific antibody in NHS included all four IgG antibody subclasses, as well as IgA. The incidence of IgG subclasses ranged from 50% (13/26) for IgG1 to 92% (24/26) for IgG2. Specific IgA anti-phospholipid antibody (aPL) was detected by ELISA in 38% (28/73) of normal human saliva. The salivary IgA aPL bound preferentially to anionic phospholipids including cardiolipin, phosphatidylserine and phosphatidic acid but not to phosphatidylcholine or sphingomyelin. Unlike aPL in normal human sera, aPL in saliva was predominantly not associated with the previously described heat-labile inhibitor of aPL. This may indicate a role of salivary IgA aPL in local immunity by binding to cross-reactive bacterial cell surface components including phospholipids.     

The clinical relevance of IgA anticardiolipin and IgA anti-β2 glycoprotein I antiphospholipid antibodies: A systematic review

The antiphospholipid syndrome (APS) is diagnosed in patients with thromboembolic events and/or pregnancy loss in the presence of persistent laboratory evidence for antiphospholipid antibodies (aPL). Diagnostic tests for the detection of antiphospholipid antibodies include laboratory assays that detect anticardiolipin antibodies, lupus anticoagulants, and anti-β(2)-glycoprotein I antibodies. Most studies on aPL have mainly focused on the estimation of the IgG and IgM isotypes, with only a few studies reporting on the pathogenic significance of IgA aPL.In this review we aimed to summarize and analyze the evidence published in the literature on the prevalence and the clinical significance of IgA aPL.     

Anti-phospholipid antibodies in HIV infection and SLE with or without anti-phospholipid syndrome: comparisons of phospholipid specificity, avidity and reactivity with beta2-GPI.

Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent assay (ELISA) methods. Sera from 30 patients with systemic lupus erythematosus (SLE), but without features of anti-phospholipid syndrome (APS) (SLE/non APS), six with SLE and secondary APS, (SLE/APS) and 11 with primary APS (PAPS) were also evaluated as controls. The resistance of the aPL antibody binding to dissociating agents was evaluated by treating the ELISA wells, after serum incubation with 2 M urea or 0.6 M NaCl for 10 min. An anti-beta2-glycoprotein-I (beta2-GPI) ELISA was used to assess serum reactivity against beta2-GPI, a plasma protein considered as the true antigen of aCL antibodies occurring in APS and SLE patients. The prevalence of aCL, aPS, aPI and aPC antibodies in HIV-1 infection was 36%, 56%, 34% and 43% respectively, which was comparable to that found in SLE/APS and PAPS patients and significantly higher than that observed in SLE/non-APS patients. Anti-beta2-GPI antibodies occurred in 5% of HIV-1 infected vs. 17% in SLE/non-APS (P=0.11), 50% in SLE/APS (P=0.009) and 70% in PAPS patients (P=0.0014). A significant decrease of aPL binding after urea and NaCl treatment was observed in the sera of HIV-1-infected, compared to that of APS patients, indicating that aPL antibodies from HIV-1 infected individuals have low resistance to dissociating agents. In conclusion, aPL antibodies (1) occur in HIV-1 infection; (2) tend to recognize various phospholipids but not beta2-GPI; and (3) are of low resistance to dissociating agents-a finding probably reflecting low antibody avidity. Finally, these, like the autoimmune-type aCL antibodies, tend to recognize the oxidized CL-a finding probably indicating autoantibody generation as a result of neoepitope formation by oxidized PLs.     

An analysis of gastric parietal cell antibodies and thyroid cell antibodies in patients with pernicious anaemia and thyroid disorders

An analysis of immunoglobulins responsible for gastric parietal cell antibodies and for antibodies to thyroid cell has been performed in twenty-four patients with pernicious anaemia and thirteen patients with thyroid disorders. Immunofluorescent technique with conjugated antisera specific to each of the three main immunoglobulins and to β_1c-globulin was used.

IgG-globulin was responsible for gastric parietal cell antibodies in all patients investigated; IgA was found in gastric parietal cell antibodies of four thyroid patients but in none of the pernicious anaemia patients; IgM antibodies were found in two pernicious anaemia patients and in six thyroid patients. With regard to thyroid cell antibodies, IgG and IgA were both found responsible for antibody activity in thirteen cases and IgM in ten cases. In six patients, antibodies to thyroid cell were not β_1c-fixing, whereas all sera with gastric parietal cell antibodies fixed β_1c-globulin.

An obvious relationship could not be found either between the titres of the different types of antibodies within the same category of antibody, or between the titres of gastric parietal cell antibodies and thyroid cell antibodies in the same individual. An occasional relationship was found between the titres of IgG-parietal cell or thyroid cell antibodies and the capacity to bind β_1c-globulin.

These results demonstrate that each of the three main immunoglobulins may contribute to gastric parietal cell antibodies and to antibodies to thyroid cell.

     

Classification of primary antiphospholipid syndrome as systemic lupus erythematosus: Analysis of a cohort of 214 patients

Objectives

To assess the limitations of the SLICC (Systemic Lupus International Collaborating Clinics) classification criteria for systemic lupus erythematosus (SLE), in patients with primary antiphospholipid syndrome (PAPS).

Detection of Human Toxoplasma-Specific Immunoglobulins A, M, and G with a Recombinant Toxoplasma gondii Rop2 Protein

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2_196–561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2_196–561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG⁺ IgA⁻ IgM⁻; n = 35), group B (IgG⁺ IgA⁺ IgM⁺; n = 21), group C (IgG⁺ IgA⁺ IgM⁻; n = 5), and group D (IgG⁺ IgA⁻ IgM⁺; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2_196–561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2_196–561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.