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.     

Isotype distribution and clinical relevance of anti-beta2-glycoprotein I (beta2-GPI) antibodies: importance of IgA isotype.

The aim of this study was to evaluate the prevalence of IgG, IgA and IgM anti-beta2-GPI antibodies in anti-phospholipid syndrome (APS), and to establish the clinical significance of IgA type antibodies compared with the other isotypes. Anti-beta2-GPI antibodies were measured in the sera of 70 patients by solid-phase enzyme immunoassay in gamma-irradiated polystyrene plates coated with human purified beta2-GPI. Thirty-three out of the 70 patients were classified as having APS: three of them had primary, and 30 had secondary APS related to systemic lupus erythematosus (SLE). The remaining 37 patients had SLE without APS. Anti-beta2-GPI antibodies of IgG, IgA and IgM isotypes were present in 84.8%, 59.3% and 51.5% of patients with APS. Both the frequency and the level of each isotype were significantly higher in patients with APS. This association was very strong for IgA (P = 0.0004 for the antibody frequency and P < 0.0001 for the antibody level), as well as for IgG type antibodies (P < 0.0001 and P < 0.0001), whereas it was weaker for IgM (P = 0.01 and P = 0.04). A strong relationship was demonstrated between increased IgA anti-beta2-GPI antibody levels and a history of venous thrombosis, thrombocytopenia, heart valve disease, livedo reticularis and epilepsy. IgG anti-beta2-GPI antibodies were associated with the presence of lupus anticoagulant (LA) in addition to the main features of APS. However, antibodies of IgM isotype were related only to thrombocytopenia and heart valve disease. We recommend the evaluation of anti-beta2-GPI antibodies of IgA isotype in addition to IgG in patients with clinical suspicion of APS.     

Binding properties of antibodies to prothrombin and beta2-glycoprotein I (beta2-GPI) assayed by ELISA and dot blot

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards beta2-GPI (anti-beta2-GPI) or prothrombin (anti-II). They are mainly screened by ELISA using polyoxygenated plates. However, some authors have claimed that immunoblotting can also be used. Exposure of cryptic epitopes or increase of antigen density on its binding to either phospholipids or suitable plastic surfaces are the two hypotheses proposed for the interaction of beta2-GPI or prothrombin with their antibodies. Forty-five patients with aPL were studied: 25 with lupus anti-coagulant (LA) and anti-cardiolipin antibodies (aCL), 10 with LA alone and 10 with aCL but negative LA. All patients with LA and aCL were positive for anti-beta2-GPI by ELISA and dot blot, while 15/25 had anti-IIELISA and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti-beta2-GPI by ELISA or dot blot, whereas 6/10 had anti-IIELISA (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti-beta2-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to beta2-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with beta2-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 microg/ml) of beta2-GPI or prothrombin demonstrated that antibody binding reduction was more evident on dot blot than on ELISA. It was almost completely abolished on dot blot, while on ELISA a moderate inhibition was achieved even at the highest protein concentration. However, antibody binding on ELISA was virtually abolished when diluted sera were incubated with high protein concentrations applied to nitrocellulose membranes. We could infer that ELISA and dot blot detect antibodies with some differences in avidity but directed against native epitopes on beta2-GPI and prothrombin.     

High prevalence of anti-beta2 glycoprotein I antibodies in patients with ischemic heart disease.

Ischemic cardiac manifestations have been reported in a various percentage of patients with anti-phospholipid antibodies. As concerns the relationship between anti-beta2 glycoprotein I antibodies (anti-beta2-GPI) and ischemic heart disease (IHD), it was investigated in only one coronary primary prevention study. We investigated the prevalence of anti-beta2-GPI in a well characterized group of patients with different clinical manifestation of IHD. Sera from 37 patients (mean age 62.7 +/- 9.9) with IHD (20 with unstable angina-UA and 17 with effort angina-EA) and from 40 healthy subjects, matched for age and sex, were tested for the presence of IgG and IgM anti-beta2-GPI using an ELISA technique. Eleven/37 patients (29.7%) resulted positive for anti-beta2-GPI. A positivity for IgG anti-beta2-GPI was found in 10 patients, 1 patient was positive for IgM and 1 for both isotypes. The prevalence of anti-beta2-GPI in the control group resulted significantly lower (2.5%; p < 0.005) than in patients with IHD. Positivity for anti-beta2-GPI was found in 9/20 (45%) patients with UA and only in 2/17 patients (11.8%) with EA (p = 0.0365). IgG anti-beta2-GPI levels (median 7.7U/ml, range 2.6-24.1) were significantly higher in patients with UA compared to patients with EA (median 4.6 U/ml, range 2.3-11.5; p = 0.02) and controls (median 3.15 U/ml, range 2.3-9.0; p < 0.0001); also IgM levels resulted higher in patients with unstable angina. A positivity for anti-beta2-GPI was observed in 4/13 patients (30.8%) with a previous myocardial infarction (MI) and in 7/24 (29.2%) patients without a previous MI. Our findings suggest that anti-beta2-GPI could represent an expression of the T-cell activation detectable in patients with unstable angina. The lack of a significant difference in the prevalence of these antibodies in patients with or without a previous MI suggests that anti-beta2-GPI are not induced by tissue necrosis.     

Anti-Beta 2-glycoprotein I antibody isotype and IgG subclass in antiphospholipid syndrome patients

Beta 2-glycoprotein I (beta2-GPI) is an antigenic target recognised by antiphospholipid antibodies found in association with the antiphospholipid syndrome (APS). In this study, the prevalence of Immunoglobulin M (IgM) and IgA anti-beta2-GPI antibodies was examined in APS patients and compared with IgG antibodies. In addition the value of measuring antibody isotypes and IgG subclass was investigated in the laboratory diagnosis of APS. A solid phase enzyme linked immunosorbent assay was established to measure IgG, IgM and IgA and IgG subclass antibodies to beta2-GPI in patients with APS and a variety of other thrombotic and non-thrombotic disorders. Raised levels of IgM anti-beta2-GPI antibodies were observed in 65% of patients with APS, 21% with systemic lupus erythematosus (SLE), 23% with rheumatoid factor, 4% with stroke, 5% carotid artery stenosis (CAS), 17% with a biological false positive serology for syphilis, 43% with infectious mononucleosis (IM) and 27% with human immunodeficiency virus (HIV). The median value for IgM antibodies to beta2-GPI for all these groups ranged from 2 to 7 arbitrary units (AU). Elevated levels of IgA antibodies to beta2-GPI were found in patients with APS (47%), SLE (13%), rheumatoid factor (26%), CAS (48%), stroke (25%), VDRL false positive serology for syphilis (33%), IM (47%) and HIV (7%). The median value of IgA antibodies to beta2-GPI in all of these groups ranged from 2 to 4 AU. Conversely the median value for IgG anti-beta2-GPI in APS patients was 112 AU compared to 1-4 AU in the other conditions examined. The presence of IgM and IgA antibodies to beta2-GPI was much less specific and sensitive for APS than IgG, with raised levels of these isotypes seen in a variety of thrombotic and non-thrombotic disorders. Elevated levels of IgG1, IgG2, IgG3 and IgG4 antibodies to beta2-GPI were detected in APS patients. While all four IgG anti-beta2-GPI antibody subclasses were represented in APS patients there appeared to be a significant overall skewing towards to the IgG2 subclass.     

Anti-mitochondrial type M5 and anti-cardiolipin antibodies in autoimmune disorders: studies on their association and cross-reactivity.

In a series of 42 positive sera, anti-mitochondrial type M5 antibodies (AMA-M5) were found most frequently in patients with SLE (24) and SLE-like syndromes. Patients with AMA-M5 displayed a higher prevalence of thrombocytopenia, thrombosis, biological false positive seroreactions for syphilis, lupus-like anticoagulant activity and anti-cardiolipin antibodies in comparison with a group of 43 SLE AMA-M5 negative patients. The strong association between anti-phospholipid and AMA-M5 antibodies cannot be explained entirely by cross-reactivity between these two groups of antibodies, as indicated by absorption experiments and studies using affinity purified antibody preparations. However, cardiolipin liposomes were able to reduce partially the titres of AMA-M5 sera, suggesting that a small population of AMA-M5 antibodies exists that cross-reacts with cardiolipin. The existence of this population was further substantiated by our demonstration that an IgM monoclonal antibody, from a patient with Waldenström''s macroglobulinaemia, displayed both anti-cardiolipin and AMA-M5 activity, and AMA-M5 activity was completely inhibited by cardiolipin.     

Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus

Lyso(bis)phosphatidic acid (LBPA) is a novel antigenic target in anti-phospholipid syndrome (APS) and antibodies directed against LBPA (aLBPA) have been detected in sera from APS patients. In this study we first evaluated aLBPA in comparison with the most widely used methods (i.e. anticardiolipin [(aCL)-enzyme-linked immunosorbent assay (ELISA)] and antibeta-2-glycoprotein-I antibodies (abeta(2)-GPI-ELISA) utilized to detect antiphospholipid antibodies in patients with primary or secondary APS, systemic lupus erythematosus, chronic HCV infection and healthy subjects. We then assessed the relationship between aLBPA, lupus anticoagulant (LAC) and the main clinical manifestations of APS. Finally, we evaluated the presence of 'pure' (i.e. beta(2)-GPI-independent) aLBPA in patients with APS and controls. The results indicate that aLBPA as well as abeta(2)-GPI display higher specificity but lower sensitivity for APS compared to aCL. Moreover, serum aLBPA correlate closely with aCL and abeta(2)-GPI in APS patients and are strictly associated with LAC positivity. We demonstrate that beta(2)-GPI binds to LBPA with affinity similar to CL, and antibodies able to react with phosholipid-protein complex exist; however, 'pure' aLBPA can also be detected in sera of APS patients. Altogether these data confirm that LBPA may be an antigenic target in APS and that aLBPA are serological markers of APS with similar sensitivity and specificity compared to abeta(2)-GPI. However, the clinical utility of aLBPA detection alone or in combination with aCL and/or abeta(2)-GPI remains to be elucidated in larger and longitudinal studies.     

Use of monoclonal antibodies to identify shared idiotypes on anticardiolipin and anti-DNA antibodies in human sera.

Using hybridoma technology we produced monoclonal antibodies (MoAb) to idiotypic determinants on human anti-cardiolipin antibodies purified from a patient with SLE. Hybridomas were screened by inhibition of cardiolipin binding activity of sera from patients with SLE. Seven hybridomas were selected, two of which were studied extensively. Sera from a number of patients with SLE were found to share idiotypic determinants. This cross-reacting idiotype was not detectable on anti-cardiolipin antibodies in syphilis sera. The cross-reacting idiotype was present in sera with anti-ssDNA antibodies even though some of these sera had no anti-cardiolipin antibodies. We propose that these MoAb may recognize a regulatory idiotype.     

Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis.

The binding specificities were investigated of anti-phospholipid antibodies derived from sera from 55 patients with SLE and related diseases, and from 33 patients with syphilis. Antibodies from both these groups of patients bind strongly to cardiolipin in solid-phase immunoassays, but only antiphospholipid antibodies from patients with autoimmune diseases are associated with thrombotic complications and recurrent spontaneous abortions. IgG anti-phospholipid antibodies from both groups of patients cross-reacted with a range of negatively charged phospholipids, but binding to neutral phospholipids was largely restricted to sera from patients with syphilis. A monoclonal IgM lambda anti-cardiolipin antibody, derived from a patient with autoimmunity, was used to inhibit binding of anti-phospholipid antibodies to cardiolipin and to phosphatidic acid. This antibody inhibited the binding of autoimmune sera to cardiolipin more strongly than sera from syphilis patients, but the converse pattern of inhibition of binding to phosphatidic acid was observed. The VDRL titre correlated with anti-phospholipid antibody activity in sera from syphilis patients, but not from those with autoimmunity. Lupus anti-coagulant activity correlated weakly with IgG antibody levels to each of the negatively charged phospholipids among the patients with autoimmunity. Lupus anticoagulant activity did not correlate uniquely with any anti-phospholipid antibody specificity. These results provide further documentation of the great heterogeneity of anti-phospholipid antibodies associated with autoimmune disease and syphilis.     

Epitope studies with anti-beta 2-glycoprotein I antibodies from autoantibody and immunized sources

This paper examines the methodology of anti-beta(2)-glycoprotein I (beta(2)-GPI) epitope determination and provides further epitope studies using human sera containing anti-beta(2)-GPI autoantibodies. Studies in this field may be misleading as the antigen coating density using mutant forms of beta(2)-GPI may be below the threshold required for monogamous divalent binding by low affinity anti-beta(2)-GPI autoantibodies, while being easily detected by high affinity anti-beta(2)-GPI from immunized animals. The antigen density threshold effect is found in anti-beta(2)-GPI autoantibodies from humans and from monoclonal anti-beta(2)-GPI derived from mice with models of autoimmune disease. Anti-beta(2)-GPI from an autoimmune mouse and from 18/21 human sera did not bind above background levels to a domain-I-deleted mutant. In addition, single point mutations in domain I result in dramatic changes in the binding of many human sera containing anti-beta(2)-GPI. These findings support a conclusion that domain I of beta(2)-GPI contains significant epitopes for the anti-beta(2)-GPI antibodies found in the antiphospholipid syndrome.     

Avidity of anti-beta-2-glycoprotein I antibodies

The terms affinity and avidity are often used indiscriminately, despite clearly differing. Since affinity refers to monovalent binding of antibodies to a monovalent epitope, the majority of data on the binding of anti-beta2-glycoprotein I antibodies (anti-beta2-GPI) characterized their avidity rather than affinity. Anti-beta2-GPI were generally believed to be of low avidity, but heterogeneous avidity of patients' IgG anti-beta2-GPI has been demonstrated. High avidity anti-beta2-GPI monoclonals were reported to possess higher pathogenicity than low avidity anti-beta2-GPI. Polyclonal high avidity anti-beta2-GPI were found to be more common in patients with antiphospholipid syndrome (APS) and associated with thrombosis. Some conformational changes of beta2-GPI are required for the binding of polyclonal anti-beta2-GPI to the antigen: neither high density of the antigen nor high avidity of the anti-beta2-GPI alone is sufficient for the recognition. Avidity of anti-beta2-GPI should be considered in any attempt of inter-laboratory standardisation and/or evaluation of anti-beta2-GPI enzyme-linked immunosorbent assay (ELISA).     

IgG class anti-cardiolipin antibody as a possible marker for evaluating fetal risk in patients with systemic lupus erythematosus

To identify the correlation between incidence of anti-phospholipid antibodies and fetal prognosis in pregnant SLE patients, we measured the amount of anti-cardiolipin antibody in their sera, using solid-phase enzyme immunoassay (EIA) methods. Findings in the group having poor obstetric results (fetal loss group) and in those with a history of full-term births (live birth group) were compared with regard to other anti-phospholipid antibodies. The incidence of IgG class anti-cardiolipin antibody was 60% in the fetal loss group and 19% in the live birth group, (P less than 0.05). The incidence of the other anti-phospholipid antibodies, including lupus anticoagulant and biological false-positive serological test for syphilis (BFP-STS), did not differ significantly between the two groups. Therefore, the presence of IgG class anti-cardiolipin antibody may prove to be a useful marker for evaluating fetal risk in SLE patients.     

Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody

We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.     

Anti-mitochondrial type 5 antibodies and anti-cardiolipin antibodies in systemic lupus erythematosus and auto-immune diseases.

Twenty sera from patients with systemic lupus erythematosus (SLE) and high titre of IgG anti-cardiolipin antibodies (ACA) were studied in order to evaluate the prevalence of anti-mitochondrial type 5 antibodies (AMA 5). None of these sera were found to be AMA 5 positive but five of 18 were positive for VDRL. Twenty sera from patients with AMA 5 were studied in order to evaluate the prevalence of ACA: only six of 20 were positive for ACA. In contrast to this finding, 15 of the 20 sera positive for AMA 5 were also positive for VDRL (P less than 0.001). The six sera positive for ACA and AMA 5 were absorbed with cardiolipin micelles. This absorption eliminated the ACA activity but not the AMA 5 activity. Despite the clinical similarities between the two groups of patients with AMA 5 or ACA, these data suggest that patients with AMA 5 and patients with ACA belong to two different subsets of SLE or SLE-like syndromes and that AMA 5 antigen is different from cardiolipin.     

Thin-layer chromatography immunostaining in detecting anti-phospholipid antibodies in seronegative anti-phospholipid syndrome

In clinical practice it is possible to find patients with clinical signs suggestive of anti-phospholipid syndrome (APS) who are persistently negative for the routinely used anti-phospholipid antibodies (aPL). Therefore, the term proposed for these cases was seronegative APS (SN-APS). We investigated the clinical usefulness of thin-layer chromatography (TLC) immunostaining in detecting serum aPL in patients presenting clinical features of SN-APS. Sera from 36 patients with SN-APS, 19 patients with APS, 18 patients with systemic lupus erythematosus (SLE), 20 anti-hepatitis C virus (HCV)-positive subjects and 32 healthy controls were examined for aPL using TLC immunostaining. Anti-β(2) -glycoprotein-I, anti-annexin II, anti-annexin V and anti-prothrombin antibodies were tested by enzyme-linked immunosorbent assays (ELISA). Eahy926, a human-derived endothelial cell line, was incubated with immunoglobulin (Ig)G fraction from SN-APS patients and analysis of phospho-interleukin (IL)-1 receptor-associated kinase (IRAK) and phospho-nuclear factor (NF)-κB was performed by Western blot, vascular cell adhesion molecule 1 (VCAM-1) expression by cytofluorimetric analysis and supernatants tissue factor (TF) levels by ELISA. TLC immunostaining showed aPL in 58·3% of SN-APS patients: anti-cardiolipin in 47·2%, anti-lyso(bis)phosphatidic acid in 41·7% and anti-phosphatidylethanolamine in 30·5%. Six of 36 patients showed anti-annexin II. Incubation of Eahy926 cells with IgG from SN-APS induced IRAK phosphorylation, NF-κB activation, VCAM-1 surface expression and TF cell release. TLC immunostaining could identify the presence of aPL in patients with SN-APS. Moreover, the results suggest the proinflammatory and procoagulant effects in vitro of these antibodies.     

Anti-cardiolipin antibodies in infectious mononucleosis react with the membrane of activated lymphocytes

To elucidate the mechanisms of autoantibody induction in infectious mononucleosis (IM), we have studied sera from 35 patients with IM with enzyme-linked immunosorbent assays using purified antigens. In the IM group 37% had IgM antibodies to cardiolipin above the normal range (mean plus 2 standard deviations of control sera). Significantly elevated frequencies of antibodies to actin (26%) and cytoskeletal antigens (97% versus 29% in normal sera) were also found, but levels of IgM rheumatoid factors, IgM antibodies to single-stranded DNA and antibodies to ribonucleoproteins (nRNP/Sm, Sm and La) were normal. Affinity purified anti-cardiolipin antibodies reacted with the cell membrane of transformed lymphocytes but not with resting cells, suggesting that cell activation was required for the expression of antigenic epitopes. Our data suggest that the autoantibody response in IM is restricted to two classes of autoantigens: cytoskeletal and cell membrane antigens. The appearance of antigenic epitopes on EBV-transformed lymphocytes could be a mechanism for the generation of anti-cardiolipin antibodies in infectious mononucleosis. Similar mechanisms could operate in autoimmune rheumatic disease.     

Anti-argonaute2 (Ago2/Su) and -Ro antibodies identified by immunoprecipitation in primary anti-phospholipid syndrome (PAPS)

Objectives. Primary anti-phospholipid syndrome (PAPS) is an autoimmune condition defined by anti-phospholipid antibodies (aPL) and thrombotic or obstetric events. Some PAPS can evolve into systemic lupus erythematosus (SLE) during follow-up. Few studies systematically examined lupus autoantibodies and their clinical significance in PAPS. The aim of our study is to analyze the clinical and laboratory correlations with lupus-related autoantibodies, detected by immunoprecipitation (IP), a technique not yet systematically applied to investigate autoantibodies in this condition.

Methods. Sera from 52 PAPS patients were screened by indirect immunofluorescence (IIF) antinuclear antibodies (ANA), IP of ³⁵S-labeled K562 cell extract, and ELISA [anti-Argonaute2 (Ago2, Su), 60kRo, 52kRo, La, dsDNA)]. Anti-Ago2/Su positive sera were also tested for anti-GW bodies (GWBs) by IIF double staining, using rabbit anti-Rck/p54 serum.

Results. First, 56% of PAPS patients (29/52) were ANA positive, mainly with speckled pattern. Anti-Ago2/Su antibodies were found in 13% (7/52), anti-Ro/SSA in 10% (5/52), anti-La in one case. The clinical profile of patients did not seem to be related to the presence of these antibody specificities. However, levels of IgG anti-β2 glycoprotein I antibodies were lower in anti-Ago2/Su positive patients (p = 0.02). None of anti-Ago2/Su or —Ro patients developed SLE during a 2-year follow-up. Ago2 is a key component of GWBs, however, only 1/7 anti-Ago2/Su serum showed a typical cytoplasmic GWBs staining.

Conclusions. Anti-Ago2/Su and -Ro antibodies are the two autoantibodies detected by IP in our PAPS cohort. Clarifying why Ago2/Su and Ro are specific targets of autoimmunity may help to understand the mechanisms of autoantibody production.     

Inhibitor(s) of natural anti-cardiolipin autoantibodies.

IgG fractions were purified on Sepharose anti-human IgG column from eight sera of healthy donors, having no anti-cardiolipin (aCL) activity as measured by anti-cardiolipin ELISA assay (aCL-ELISA). All the IgG fractions, after elution with 4.9 M MgCl2, reacted with CL. The antigen-binding characteristics of the IgG fractions purified from normal human serum (NHS) were similar to those of IgG fractions purified from sera of four patients with the anti-phospholipid syndrome (APLS). Competition assay confirmed the specificity of the binding of the purified IgG fractions to CL. The same results have been achieved with IgG fractions purified on Sepharose Protein-A column. The binding to CL was completely inhibited by either whole NHS and sera from various animal species, or by beta 2-glycoprotein I (beta 2-GPI). Our results support the notion of the existence of both natural anti-CL antibodies and serum inhibitor(s) in sera of healthy individuals. It is conceivable that in part the pathogenesis of APLS entails defects in the natural inhibitors of aCL antibodies.     

Anti-beta(2)-glycoprotein I autoantibody expression as a potential biomarker for strokes in patients with anti-phospholipid syndrome

Anti-phospholipid syndrome (APS) is an autoimmune disease. Cerebral ischemia associated with APS occurs at a younger age than typical atherothrombotic cerebrovascular disease, is often recurrent, and is associated with high positive IgG anti-phospholipid (GPL) unit levels. This study sought to determine the frequency rates of anti-cardiolipin (aCL) dependent on the presence of beta(2)-GPI, anti-beta(2)-glycoprotein I (abeta(2)-GPI), and anti-phosphatidyl serine (aPS) IgG autoantibodies among stroke patients, and thus demonstrate the importance of testing for abeta(2)-GPI autoantibodies. For these study, stroke patients and control subjects recruited from Mosul, Erbil, and Dohuk provinces in Northeren Iraq between March 2004 and March 2005 were evaluated. All cases were under 50 years-of-age and had no recognizable risk factors. Using ELISA to evaluate the presence of IgG isotype of aCL, abeta(2)-GPI, and aPS autoantibodies in their blood, the results indicated that the frequency of abeta(2)-GPI was 14/50 (28%), aCL was 11/50 (22%), and aPS was 9/50 (18%) among stroke patients. In contrast, aCL was detected in 2/30 (6.7%) of control subjects; each of the other anti-phospholipid antibodies (APLA) was never observed. Of all the abeta(2)-GPI(+) cases, the incidence of stroke patients having the combined profile of abeta(2)-GPI + aCL was 11/14 (78.6%) and of abeta(2)-GPI + aPS was 9/14 (64.3%). Only 2/14 (14.3%) of these abeta(2)-GPI(+) patients also expressed aCL in the absence of aPS. The frequency of patients expressing all three markers was only 9/14 (64.3 %). In none of the APS/stroke patients were aCL or aPS expressed in the absence of the abeta(2)-GPI. Conversely, abeta(2)-GPI as a sole marker was seen in 3/14 (21.4%) of these patients (i.e., in absence of either other marker). It can be concluded from these studies that the among the three major forms of APLA examined, the presence of abeta(2)-GPI IgG autoantibodies appeared to correlate best with stroke in patients who were concurrently suffering APS.     

Relationship between anti-cardiolipin and anti-endothelial cell antibodies in systemic lupus erythematosus

Anti-endothelial cell antibodies (AECA) have been detected in 51 lupus sera by cell surface radioimmunoassay with a prevalence of 39.2% for IgG and 45.1% for IgM-AECA. No correlations were found between AECA and different clinical or laboratory parameters, including the presence of anti-cardiolipin antibodies and signs associated with the presence of anti-phospholipid antibodies. However, absorption with cardiolipin liposomes partially inhibited endothelial cell binding, and affinity purified anti-cardiolipin antibodies were able to react with intact human endothelial cells. The binding did not occur via Fc receptors since blocking of Fc receptors with rabbit IgG did not affect the endothelial cell reactivity. Taken together our findings support the hypothesis that antibodies directed against negatively charged phospholipids can be part of the anti-endothelial cell antibodies in certain lupus sera. The possible role of these cross-reacting antibodies in the pathogenesis of vascular thrombosis in lupus patients is discussed.