Antonio R. Cabral

The antiphospholipid/cofactor syndromes: A primary variant with antibodies to β2-glycoprotein-I but no antibodies detectable in standard antiphospholipid assays*****

BACKGROUND Most systemic lupus erythematosus (SLE) patients with two or more clinical manifestations of the antiphospholipid syndrome (APS) and negative antiphospholipid antibodies (aPL) have antibodies to beta 2-glycoprotein-I (a beta 2 GP-I). Herein we describe a similar set of circumstances, but in patients without evidence of SLE. PATIENTS AND METHODS We studied 6 patients with recurrent venous and/or arterial thromboses without aPL as detected by routine assays nor clinical or serological evidence of other autoimmune disease. Immunoglobin (Ig) G and IgM antibodies to bovine and human phospholipid-free beta 2 GP-I were studied by Western blot test and by enzyme-linked immunosorbent assay (ELISA) utilizing radiated and nonirradiated plates. We also tested antibodies to cardiolipin, phosphatidylserine, and phosphatidylethanolamine by ELISA. As controls, 54 normal sera were studied. RESULTS All 6 patients had recurrent arterial and/or venous thromboses. Three also had thrombocytopenia, 1 had livedo reticularis, and 2 had valvular heart disease. None of the patients had aPL, but all had serum IgG reactivity against human and bovine beta 2 GP-I (P < 0.001 versus controls for both). Titers of anti-bovine beta 2 GP-I were higher when studied in irradiated plates but were also higher than normal in nonirradiated plates (P < 0.001). These antibodies did not recognize human or bovine beta 2 GP-I bound to cardiolipin in solid phase. We confirmed by Western blot that these autoantibodies recognize human beta 2 GP-I. We found no IgM a beta 2 GP-I. CONCLUSIONS We describe a primary condition akin to the antiphospholipid syndrome with negative aPL, but with serum IgG antibodies to human and bovine beta 2 GP-I. These antibodies recognize beta 2 GP-I epitopes that are not accessible when beta 2 GP-I is bound to cardiolipin.

Autoantibodies in systemic lupus erythematosus.

Lupus anti-DNA may have higher homology with germline than those from normal subjects. However, in NZB/NZW mice, bacterial DNA is more antigenic than mammal DNA, which could indicate an antigen-driven origin. High-affinity antibodies to double-stranded DNA cross-react with small nuclear ribonucleoprotein and ribosomal P proteins. These cross-reactive anti-DNA may penetrate live cells. Antibodies to ribosomal P proteins are associated with neuropsychiatric, renal, and hepatic lupus involvement. IgG antibodies to (H2A-H2B)-DNA complexes antedate procainamide-induced lupus. Autoantibodies to some La/Ro peptides in a mother indicates that her children may develop neonatal lupus and determine who will have congenital heart block. Perinuclear antineutrophil cytoplasmic antibodies are present in 25% of systemic lupus erythematosus patients without correlation with anti-DNA or disease activity. Different antiphospholipid antibodies require different protein cofactors for reactivity. Those to anionic phospholipids require beta 2-glycoprotein I, whereas anti-phosphatidylethanolamine antibodies require kininogen or its binding protein. Antibodies to phospholipid-free beta 2-glycoprotein I are associated more strongly with clinical antiphospholipid syndrome than are antiphospholipid antibodies.

Serum anti-β2-glycoprotein-I and anticardiolipin antibodies during thrombosis in systemic lupus erythematosus patients

Abstract PURPOSE: Antibodies to β2-glycoprotein-I are more strongly associated with clinical antiphospholipid syndrome than are anticardiolipin antibodies. We previously found a decrease in anticardiolipin antibodies at the time of thrombosis in 6 patients with systemic lupus erythematosus (SLE). We therefore sought to determine the prevalence and levels of antibodies to β2-glycoprotein-I and to cardiolipin before, during, and after thrombosis in patients with SLE, and to compare them with patients who did not have thrombosis. METHODS: We studied 24 patients with SLE who had at least one episode of thrombosis and 102 patients with SLE without thrombosis. Serum anticardiolipin antibodies were measured by conventional enzyme-linked immunosorbent assay (ELISA) using newborn calf serum as the blocking agent. Serum anti-β2-glycoprotein-I antibodies were measured by ELISA on nonirradiated plates, using purified human β2-glycoprotein-I without phospholipid. RESULTS: All patients with thrombosis had anti-β2-glycoprotein-I antibodies, compared with only 17% of controls (P CONCLUSION: Anti-β2-glycoprotein-I antibodies are strongly associated with thrombosis in patients with SLE. The decrease of anti-β2-glycoprotein-I levels at the time of thrombosis may indicate a pathogenic role. This antibody may also be a marker of predisposition for thrombosis in these patients.

Hemolytic anemia related to an IgM autoantibody to phosphatidylcholine that binds In vitro to stored and to bromelain-treated human erythrocytes

Both normal and autoimmune mice have IgM natural autoantibodies to bromelain-treated erythrocytes in which phosphatidylcholine (PTC) becomes exposed. At one stage this antibody may participate in the genesis of autoimmune hemolytic anemia in the NZB mouse. We have recently studied a patient with hemolytic anemia who had persistently high serum titers of IgM anticardiolipin antibodies (aCL) that were also demonstrated in a hemolysate of his erythrocytes obtained at the time of the anemia. We affinity-purified the antibody and sought its binding to normal human bromelain-treated erythrocytes (BrE) because of the IgM isotype of the antibody, since cardiolipin is not a constituent of the erythrocyte wall, and because the anionic phospholipids, with which aCL are known to cross-react, are not located at the outer leaflet of the erythrocyte membrane. We found binding of the antibody to HBrE in their hemolysates and by flow cytometry. We also demonstrated that the aCL cross-reacted extensively with PTC, as well as with other anionic or zwitterionic phospholipids. The purified IgM antibody lysed BrE in the presence of complement and also bound to in vitro-aged erythrocytes. Because this patient had no other evidence of systemic lupus erythematosus or any other autoimmune condition, his disease may represent a variant of the recently described primary antiphospholipid syndrome.

Release of neutrophil extracellular traps by neutrophils stimulated with antiphospholipid antibodies: a newly identified mechanism of thrombosis in the antiphospholipid syndrome.

Antiphospholipid antibodies (aPL), especially those targeting β2‐glycoprotein I (β2GPI), are well known to activate endothelial cells, monocytes, and platelets, with prothrombotic implications. In contrast, the interaction of aPL with neutrophils has not been extensively studied. Neutrophil extracellular traps (NETs) have recently been recognized as an important activator of the coagulation cascade, as well as an integral component of arterial and venous thrombi. This study was undertaken to determine whether aPL activate neutrophils to release NETs, thereby predisposing to the arterial and venous thrombosis inherent in the antiphospholipid syndrome (APS).

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.

Phospholipid specificity and requirement of β2-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.

Antiphospholipid Antibodies in Patients with Idiopathic Autoimmune Haemolytic Anemia

Isolated cases of anti-phospholipid antibody (aPL)-associated idiopathic autoimmune haemolytic anemia (IAHA) have been recently described. To assess the significances of this association, we studied by ELISA the presence of aPL in sera from 18 patients with IAHA and 14 patients with non-autoimmune haemolysis (NON-AH). Four IAHA cases and none of the NON-AH controls showed IgM anticardiolipin antibodies (aCL) that crossreacted extensively with zwitterionic as well as with other anionic phospholipids. IgG aCL were detected in 6 patients with IAHA and in 1 patient with NON-AH; there was little cross-reactivity with other phospholipids. Our results suggest that antiphospholipid antibodies are present in a substantial number of patients with IAHA. This humoral response does not seem to be secondary to the haemolysis proper. The potential pathogenic significance of this finding is discussed.

Decrease in serum antiphospholipid antibody levels upon development of nephrotic syndrome in patients with systemic lupus erythematosus: Relationship to urinary loss of IgG and other factors

PURPOSE Having observed a decrease in antiphospholipid antibodies (aPL) upon the development of nephrotic syndrome, as well as a negative association between nephrotic syndrome and secondary antiphospholipid syndrome, in patients with systemic lupus erythematosus (SLE), we sought to determine if this could be due to urinary loss of aPL and/or other factors. SUBJECTS AND METHODS IgG and IgM aPL as well as other autoantibodies were studied by enzyme-linked immunosorbent assay with cardiolipin as antigen in serum and urine from six patients with SLE who had elevated serum aPL levels and developed nephrotic syndrome (cases). For controls, we studied: (1) three SLE patients with nephrotic syndrome but low aPL levels; (2) three patients with non-SLE nephrotic syndrome; (3) three SLE patients with high-titer aPL but no proteinuria; and (4) 10 healthy volunteers. RESULTS We found urinary IgG, but no IgM, aPL in all cases and in one control from Group 2. Serum IgG aPL had gradually decreased after the development of nephrotic syndrome and had become normal. IgM aPL had also decreased in the four patients who had elevated levels, having reached normal levels at the time of the study in two. There was an apparent correlation between serum and urine IgG aPL levels but not between urinary IgG aPL and total proteinuria. By Farrs method, we found no urinary anti-DNA despite high serum titers in three cases. The two cases and one of the controls in Group 1 who had serum antibodies to extractable antigens also had these antibodies in the urine. CONCLUSION Urinary loss of IgG aPL during nephrotic syndrome does not completely explain the reduction in serum aPL, since IgM also decreases. There could also be decreased synthesis and/or increased catabolism of immunoglobulins.

Anti-Cardiolipin, Anti-Cardiolipin Plus Bovine, or Human β2Glycoprotein-I and Anti-Human β2Glycoprotein-I Antibodies in a Healthy Infant Population

Abstract Background Pathogenic antiphospholipid antibodies studies are usually conducted on populations of adults. Studies involving normal children are scant. Methods Antibody reactivity against CL alone (true aCL), CL-complexed to bovine β 2 GP-I (aCL-bovine β 2 GP-I), or human (aCL-human β 2 GP-I) β 2 GP-I, or to phospholipid-free human β 2 GP-I (anti-human β 2 GP-I) was determined by ELISA in serum samples from 360 Mexican children ranging from 1 month through 8 years of age. Results Statistical analysis of variance and rankings of Kruskal-Wallis demonstrated no significant difference in all tested antibody activities between ages and genders of the study population. Values are presented as a percentile distribution included between 5 and 99, corresponding to the percentages of the studied population. Normal arbitrary units (AU) for IgG, IgA, and IgM true aCL that correspond to the 95 and 99 percentiles are as follows: 2.15 and 3.5; 2.35 and 5.0, and 3.15 and 4.5, respectively. IgG, IgA, and IgM aCL-bovine β 2 GP-I activities are 2.6 and 5.0, 3.0 and 5.0, and 2.7 and 6.0 AU, respectively, while IgG activities of aCL-bovine and human β 2 GP-I are 1.45 and 1.80, respectively. Normal values for IgG anti-human β 2 GP-I are 1.85 AU. Conclusions While elevated serum levels of antibodies to CL and/or β 2 GP-I have been associated with thrombotic and hematologic manifestations, the majority of reports deal with adult populations. We report the cut-off values (in AU, international PL units, and international units for β 2 GP-I) of the specific serologic response of true aCL, aCL-bovine β 2 GP-I, aCL-human β 2 GP-I, and anti-human β 2 GP-I in healthy Mexican children.