Molecular definition of human beta 2-glycoprotein I (beta 2-GPI) by cDNA cloning and inter-species differences of beta 2-GPI in alternation of anticardiolipin binding.

Human beta 2-glycoprotein I (beta 2-GPI) is involved in cardiolipin (CL) binding of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE). We examined the inter-species differences of beta 2-GPI in alternation of CL binding of aCL. beta 2-GPI preparations were obtained from human, bovine, and rat sera by sequential CL--polyacrylamide affinity, DEAE--cellulose, and anti-human IgG-conjugated Sepharose CL-4B column chromatography, and they had apparent molecular weights of 50, 53, and 55 kDa respectively. Human beta 2-GPI not only enhanced CL binding by aCL in SLE but also depressed it by those in syphilis. Either bovine and rat beta 2-GPI exerted no or quite small inhibition of the binding of syphilitic aCL compared with human beta 2-GPI whereas all three species of beta 2-GPI generated binding of aCL in SLE to a similar degree. Further, a complete cDNA clone, p beta 2-GPI, was isolated from a human hepatoma cell line, HepG2, and its nucleotide sequence was analyzed. The sequences of bovine and rat counterpart molecules (beta 2-GPI) are highly homologous to that of the deduced sequence, and their corresponding regions are 84.0 and 82.5% identical to the complete domain and to the amino acid sequence 53-326 of human beta 2-GPI respectively. One of major differences appears at position 154 in human beta 2-GPI, and might be associated with the inhibitory effect on the binding of syphilitic aCL. The sequencing analysis of these beta 2-GPI proteins might provide leads to functional sites of domains which would be associated with such serological phenomena.     

Anti-beta2-glycoprotein I (GPI) autoantibodies, annexin V binding and the anti-phospholipid syndrome

We examined the role of autoantibodies to beta2-GPI and prothrombin (PT) in the inhibition of annexin V binding to cardiolipin (CL) and the association with clinical manifestations of the anti-phospholipid syndrome (APS). Plasma samples from 59 patients with anti-phospholipid (aPL) antibodies were studied. Affinity purification of total IgG and IgG anti-ss2-GPI antibodies was performed using staphylococcal protein A and phospholipid liposomes. Annexin V binding to CL was significantly inhibited by 31/59 (53%) aPL+ plasma samples. There was a significant association between annexin V inhibition and elevated levels of IgG anti-cardiolipin (aCL) (r = -0.62; P < 0.001), IgG anti-ss2-GPI (r = -0.67; P < 0. 001) and a weaker association with lupus anti-coagulant (r = -0.27; P = 0.05). There was no association with other isotypes of aCL and anti-ss2-GPI or with anti-PT of any isotype. In patients with clinical manifestations of the APS there were higher levels of IgG aCL (median (range) Z score): 10.0 (0-17.6) versus 5.0 (0-16.1); P = 0.03), IgG anti-ss2-GPI (4.5 (0-11.3) versus 0.9 (0-9.7); P = 0.02) and greater inhibition of annexin V binding to CL (-3.4 (-11.4-0.6) versus -1.1 (-10.8-1.2); P = 0.22). Odds ratios for the laboratory assays and the presence of clinical manifestations of the APS varied between 0.38 and 4.16, with the highest values for IgG aCL (4.16), IgG anti-ss2-GPI (3.28) and annexin V inhibition (2.85). Additional experiments with affinity-purified IgG antibodies indicated that inhibition of annexin V binding was dependent upon the concentration of ss2-GPI and anti-ss2-GPI antibodies. These results indicate that inhibition of annexin V binding to procoagulant phospholipid surfaces is dependent upon anti-ss2-GPI antibodies and suggest a role for annexin V in the pathogenesis of the APS.     

Heat-labile serum inhibitor of anti-phospholipid antibody binding in ELISA

Normal human sera (NHS), heat-inactivated at 56 degrees C for 30 min, demonstrated positive ELISA reactions for anti-cardiolipin (aCL) antibodies. The heat-induced reactivity in ELISA was inhibitable by the cardiolipin antigen and was abolished by prior IgG depletion of the heated NHS with a protein A preparation. The heat-potentiated aCL also cross-reacted selectively with phosphatidic acid and phosphatidylserine, but not with phosphatidylcholine or phosphatidylethanolamine.     

Naturally occurring V region connected antibodies inhibit anti-dsDNA antibody reactivity with dsDNA

The production of autoantibodies against a vast array of self antigens, most notably double stranded (ds) DNA, characterized systemic lupus erythematosus (SLE). The purpose of this work is to study specific Ig fractions isolated from normal human serum (NHS) and their effect on the binding of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies (Abs) to dsDNA. A fraction named immunoglobulin G (IgG)-reactive IgG was purified from total NHS IgG by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules (IgG-Sepharose column). IgG-reactive IgG was co-incubated with systemic lupus erythematosus (SLE) patient's serum and binding of the anti-dsDNA Abs to dsDNA was measured by enzyme-linked immunosorbent assay (ELISA). Co-incubation of SLE patient's serum with IgG-reactive IgG resulted in a dose-dependent reduction in binding of anti-dsDNA Abs to dsDNA. A reduction greater than 70% was observed at a concentration of 300 μg of IgG-reactive IgG per mL of a 400-fold diluted SLE patient's serum whereas total NHS IgG, at the same concentration, resulted in a 10% reduction in binding. The purification process used to isolate IgG-reactive IgG was based on interactions between intact Ig rather than on interactions between F(ab’)₂ portions. IgG₂ is the predominant immunoglobulin (Ig) subclass in IgG-reactive IgG. Thus, IgG₂ might have an important role in the connectivity characteristics of NHS IgG. The capacity of IgG-reactive IgG to inhibit anti-DNA Ab binding to dsDNA may have potential application in the treatment of SLE. This targeted biological approach may provide an alternative strategy to immunosuppressants.     

Hidden anti-phospholipid antibodies in normal human sera circulate as immune complexes whose antigen can be removed by heat,acid, hypermolar buffers or phospholipase treatments

Heat treatment of normal human serum reveals otherwise masked anti-cardiolipin antibodies (aCL). We studied the mechanism of masking and the nature of the inhibitor of these aCL IgG. Other forms of treatment, besides heating for 30 min at 56 °C, can also unmask hidden aCL IgG. These include acid pH, hypermolar buffers and phospholipase digestion. When unmasked, these aCL recognize other anionic and zwitterionic phospholipids, but do not react with DNA, cell antigens or IgG. Using thin layer chromatography we demonstrate that the heat-labile inhibitor(s) of these aCL are phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. These antibodies are not β₂ -glycoprotein-I dependent and actually compete with this protein for phospholipid binding. The hidden antibodies are comprised of two populations of IgG autoantibodies: one reactive with cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine and sphingomyelin, and the other reactive almost exclusively with phosphatidylcholine and phosphorylcholine on enzyme-linked immunosorbent assay plates or when exposed by bromelain on the erythrocyte surface. Our data suggest that hidden aCL are natural oligoreactive IgG anti-phospholipid autoantibodies that circulate masked by their antigen.     

Inhibition of histone/anti-histone reactivity by histone-binding serum components; differential effect on anti-H1 versus anti-H2B antibodies.

IgG fractions were purified on a protein G-agarose column from sera of both systemic lupus erythematosus (SLE) patients and healthy donors. All IgG fractions, after elution with 0.5 M acetic acid, reacted with histones in an anti-histone ELISA assay, and IgG anti-histone activity was in all instances higher in the IgG fraction than in the corresponding whole serum. This was shown to be due to the presence in serum of histone-binding components that inhibited IgG binding to histones. Both normal human and SLE patients' sera had these histone-binding components, and disparity between serum-positive and -negative anti-histone antibody (AHA) tests was not dependent on differences in the blocking capacity but on IgG antibody levels and avidity. Interaction of normal serum IgG fraction with all five histones was of low avidity, whereas interaction of IgG from AHA-positive SLE sera with both H1 and H2B had high avidity. Low-affinity antibodies to every histone fraction, but also high-affinity anti-H1 antibodies, were preferentially inhibited. Our data indicate that several serum protein components are inhibiting histone/anti-histone interaction and may play a protective role against both high-affinity anti-H1 antibodies present in SLE patients, and natural, low-affinity, anti-histone antibodies. As some acute phase proteins, notably C-reactive protein, bind to histones, it is conceivable that they play such a role. High-affinity anti-H2B antibodies, present in some SLE patients, and not inhibited by these serum components, may, on the other hand, participate in the pathogenesis of the disease.     

Antiphospholipid antibodies bind ATP: a putative mechanism for the pathogenesis of neuronal dysfunction

Antiphospholipid antibodies (aPL) generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (> 50%) inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound beta2-glycoprotein-I (beta2-GPI, also known as apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of beta2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of beta2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through beta32-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters.     

Expression of anticardiolipin cofactor, human beta 2-glycoprotein I, by a recombinant baculovirus/insect cell system.

A full-length cDNA coding a human beta 2-glycoprotein I (beta 2-GPI) was introduced into the baculovirus genome to construct a recombinant baculovirus. Spodoptera frugiperda (Sf9) cells were infected with the recombinant baculovirus. A protein (mol. wt 43,000) reactive with anti-beta 2-GPI antisera was produced in the insect cells and secreted into the culture medium. The recombinant beta 2-GPI was purified from the culture supernatant by sequential cardiolipin (CL)-affinity column chromatography and gel filtration. The N-terminal amino acid sequence of the protein was identical to that of the native beta 2-GPI purified from human sera, and a putative signal peptide was cleaved from the secreted form of the recombinant protein. The purified recombinant protein had a cofactor activity which enhances CL binding of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE) patients, as well as the native beta 2-GPI. Thus, the beta 2-GPI expressed in insect cells is an immunologically active cofactor.     

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.     

Anticardiolipin Antibodies in Patients With Pregnancy Loss Induce Factor Xa Production in the Presence of (β2-Glycoprotein I

PROBLEM : Anticardiolipin antibodies (aCL) are commonly associated with recurrent pregnancy loss, though the mechanism is uncertain. Some investigators have indicated that aCL may be directed at a complex made up of cardiolipin and a blood anticoagulant, β2-glycoprotein I (β2GPI). We therefore investigated the effects of β2GPI-dependent aCL IgG enriched fractions, isolated from sera of patients with pregnancy losses, on blood coagulation. METHOD : β2GPI-dependent aCL were prepared from sera of three women with second trimester pregnancy losses, by cardiolipin affinity column chromography, following by anti-β2GPI affinity column chromatography. The effects of β2GPI and β2GPI-dependent aCL on the activation of factor X in vitro were examined. RESULTS : β2GPI inhibited the activation of factor X and β2GPI-dependent aCL blocked this inhibitory effect in a dose dependent manner. CONCLUSION : These results imply the possibility of β2GPI-dependent aCL induce hypercoagulation or thrombus by blocking the inhibitory effect of β2GPI on activation of factor X, which may result in pregnancy loss.     

Patients with atherosclerotic syndrome, negative in anti-cardiolipin assays, make IgA autoantibodies that preferentially target domain 4 of beta2-GPI

Autoantibodies targeting beta2-glycoprotein l (beta2-GPI), a component of the atherosclerotic plaque, are commonly found in patients with acute ischemic syndromes. Serum samples from APS (antiphospholipid syndrome) patients and from cardiovascular patients exhibiting acute atherosclerotic syndromes were analyzed for IgG and IgA antibodies in both anti-beta2-GPI and anticardiolipin (aCL) ELISA assays. All of the APS samples used here were positive in both assays. Serum samples from 382 atherosclerosis patients were also analyzed for IgG and IgA antibodies in the same assays. In sharp contrast to the APS samples, we found that only 1% of the samples from atherosclerosis patients were positive for IgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive for IgA anti-beta2-GPI and only 1.6% for IgG anti-beta2-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human beta2-GPI and full-length human beta2-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-beta2-GPI ELISA assays. Domain-deleted mutants D--345 and D--45 inhibited the binding in the IgA anti-beta2-GPI assay, suggesting that these autoantibodies recognize domain 4 of the beta2-GPI molecule. These results clearly show that IgA anti-beta2-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.     

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.     

Antibodies to β2-Glycoprotein-I: Urea Resistance, Binding Specificity, and Association with Thrombosis

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-2-glycoprotein I (anti-2GPI) antibodies using standard anticardiolipin (aCL) and anti-2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D _o) corresponding to the same optical density was defined as residual activity (RA = 100 D/D _o). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120–1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA 30%. Anti-2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA 30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-2GPI negative and three non-APS anti-2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when 2GPI was incubated with CL in the ELISA plates; thus some anti-2GPI negative sera from APS patients recognized the CL/2GPI complex, rather than CL or 2GPI alone. In conclusion, anti- 2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/2GPI complex and not CL or 2GPI. Antibodies to either 2GPI or the CL/2GPI complex derived from APS sera present a high resistance to urea. Anti-2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease.     

Standardization of Elisa for the Detection of Anti-Cardiolipin Antibodies - Effect of Non-Specific IgG Binding

Various frequencies of anti-cardiolipin antibodies reported in patients with systemic lupus erythematosus and other autoimmune and infectious diseases necessitates the need for standardization of the immunoassay. We report here the usefulness of “no-antigen” control for each serum in determining the actual value of the anti-phospholipid antibodies. This non-specific “no-antigen” binding was quite variable from serum to serum and in general was higher in the patient population than in healthy individuals which served as controls. This non-specific binding may be associated with the increased IgG content of the sera as shown by the linear association of the absorbance of the back-ground readings to increases in IgG concentration (correlation coefficient 0.98).     

Heat inactivation of serum potentiates anti-cardiolipin antibody binding in ELISA

Heat treatment of sera at 56 degrees C for 30 min results in positive ELISA reactions for anti-cardiolipin antibody (aCL) in sera that had undetectable or low levels of aCL before heat inactivation. The positive, potentiated reactivity of the heated sera in the aCL ELISA could be inhibited with the cardiolipin antigen and was abolished by prior IgG depletion using staphylococcal protein A. The heat-potentiating effect of aCL binding in ELISA was evident in both normal human sera and clinical sera including sera from patients with systemic lupus erythematosus and syphilis.     

Enrichment of antibodies against phospholipids in circulating immune complexes (CIC) in the anti-phospholipid syndrome (APLS)

The aim of this study was to analyse and characterize immunoglobulins in CIC and serum from patients with anti-cardiolipin antibodies. CIC from five patients were isolated by gradient centrifugation and gel filtration. The distribution between serum and CIC of immunoglobulin reactivity against different phospholipids was determined. Serum and CIC contained antibodies against cardiolipin and other negatively charged phospholipids. The relative concentration of these antibodies was higher in the immune complexes than in corresponding sera, and the avidity of antibodies in immune complex form was higher. The presence of high concentrations of antibodies to negatively charged phospholipids in CIC from patients with anti-cardiolipin antibodies could be of pathogenic significance in APLS by conferring characteristics to the complexes of importance for binding to membrane components. This could have special implications with regard to platelet and complement activation, thrombocytopenia and thrombophilia.     

Effect of Normal Human Serum on the Binding of Specific Antibodies and Platelet-unrelated Immune Complexes to Human Platelets

Radiolabelled staphylococcal protein A was used to quantitate the binding of IgG on stored human platelets from human sera containing specific antibodies reactive with platelets and rabbit serum containing immune complexes (IC). Normal human serum (NHS) inhibited the binding of IC onto platelets and to various extents also the binding of specific antibodies. The attachment of inhibitors to platelets seemed to be reversible. The considerable difference in the inhibitory capacities of IgG-deficient sera and monomeric IgG indicates that IgG is the major inhibitory component of NHS. The binding of IgG from NHS onto platelets evidently hampers the detection of weak platelet antibodies even with the most sensitive tests. Purified Clq, known to modify the reactions of IC with fresh platelets did not alter the binding of IC onto stored platelets. A monoclonal, antiglobulin-active rheumatoid factor of IgM class displayed only moderate inhibition. Therefore, the application of RF or Clq for the differentiation of the binding induced by IC or antibodies is not useful in this assay system. The heterogeneity of immunologic receptors of platelets provides an explanation of the inhibitory inefficiency of Clq.     

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.     

Anticardiolipin antibodies in patients with chronic hepatitis C virus infection: characterization in relation to antiphospholipid syndrome

The antiphospholipid syndrome (APS) is usually defined by the association of clinical manifestations that comprise venous and/or arterial thrombosis, recurrent fetal losses, and thrombocytopenia, along with the presence of anticardiolipin (aCL) antibodies and/or lupus anticoagulant. Various infectious diseases can induce aCL; however, these antibodies are not usually associated with thrombotic events, as happens with autoimmune diseases, in which these antibodies need the presence of beta(2)-glycoprotein I. Levels of immunoglobulin G (IgG) and IgM aCL antibodies were determined by enzyme-linked immunosorbent assay for 243 patients with chronic hepatitis C virus (HCV) infection and 100 healthy controls. Clinical events of APS, the level of beta(2)-glycoprotein dependence of aCL, the presence of cryoglobulins and other autoantibodies, and cross-reactivity between purified aCL and HCV were evaluated. Positive results for aCL antibodies were found more frequently (3. 3%) for the patients with HCV infection than for healthy controls (0%). All positive aCL antibodies were beta(2)-glycoprotein I independent. No significant association was found between aCL antibodies and clinical manifestations of APS, neither was one found between the presence of other autoantibodies or cryoglobulins and that of aCL. Finally, no cross-reactivity between aCL antibodies and HCV antigens was observed. As previously reported, aCL antibodies seem to be an epiphenomenon, and they do not have clinical or laboratory significance in HCV patients.     

Clinical relevance of multiple antibody specificity testing in anti-phospholipid syndrome and recurrent pregnancy loss

We wanted to evaluate whether testing for anti-phosholipid antibodies other than anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (abeta2GPI) immunoglobulin (Ig)G and IgM identifies patients with recurrent pregnancy loss (RPL) who may be positive for anti-phospholipid syndrome (APS). In a cross-sectional study comprising 62 patients with APS, 66 women with RPL, 50 healthy blood donors and 24 women with a history of successful pregnancies, we tested IgM and IgG antibodies to phosphatidic acid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol and phosphatidyl serine with and without beta-2 glycoprotein I (beta2GPI) from a single manufacturer as well as aCL and abeta2GPI antibodies. Diagnostic accuracies of individual and combined anti-phospholipid (aPL) assays were assessed by computing sensitivities, specificities, positive predictive values and negative predictive values together with their 95% confidence intervals. There was a general trend for increased sensitivities in the presence of beta2GPI co-factor with significant effect for certain specificities. The overall combined sensitivity of the non-recommended aPL assays was not significantly higher than that of the aCL and aB2GPI tests. Multiple aPL specificities in RPL group is not significantly different from controls and therefore of no clinical significance.