A re-appraisal of the normal cut-off assignment for anticardiolipin IgM tests.

BACKGROUND: Recent reports show an apparent large number of individuals with low to moderate titers of anticardiolipin antibodies (ACA), particularly of the IgM isotype with no clinical signs of antiphospholipid syndrome (APS). The significance of these results is unknown. This study examined the prevalence of low positive titers of IgM ACA antibodies in a large number (n = 982) of normal blood donors (Group 1) and in a group of 159 individuals > 60 years of age (Group 2). The effect of re-defining the currently used cut-off values for the IgM ACA tests was also examined. METHODS: IgM ACA antibodies were tested in three ELISA assays: the Bindazyme Anti-IgM Cardiolipin EIA kit (assay A), an 'in-house' ACA test (assay B), and the APhL ELISA kit (assay C). RESULTS: THE normal range cut-offs were re-calculated using the 95th percentile of the data for Group 1 (12.4 MPL U mL(-1) for assay A, 5.4 MPL U mL(-1) for assay B and 9.5 MPL U mL(-1) for assay C) and Group 2 (9.9 MPL U mL(-1) for assay A, 5.5 MPL U mL(-1) for assay B and 13.2 MPL U mL(-1) for assay C). These values were not significantly different from the current cut-off values for each assay. The prevalence of low positive results in Group 1 relative to the re-defined cut-off for that group were: 1.0%, 1.1% and 0.9% in assay A, B and C; and in Group 2: 0.6%, 0.6% and 0.6%, respectively. An indeterminate zone (between the 95th and 99th percentile) was then established for the two groups. The prevalence in Group 1 was 3.8%, 3.9% and 3.9% for assays A, B and C, respectively, and for Group 2: 4.4% in all three assays. CONCLUSIONS: The data confirm that the current cut-off point for each of the three assays is correct. We suggest based on this study that the low positive range is re-assigned 'indeterminate' and recommend that samples falling in this category should be retested to confirm positivity at a later date.

Mitochondrial Dysfunction in the Liver and Antiphospholipid Antibody Production Precede Disease Onset and Respond to Rapamycin in Lupus-Prone Mice.

OBJECTIVE: Antiphospholipid antibodies (aPL) constitute a diagnostic criterion of systemic lupus erythematosus (SLE), and aPL have been functionally linked to liver disease in patients with SLE. Since the mechanistic target of rapamycin (mTOR) is a regulator of oxidative stress, a pathophysiologic process that contributes to the development of aPL, this study was undertaken in a mouse model of SLE to examine the involvement of liver mitochondria in lupus pathogenesis. METHODS: Mitochondria were isolated from lupus-prone MRL/lpr, C57BL/6.lpr, and MRL mice, age-matched autoimmunity-resistant C57BL/6 mice as negative controls, and transaldolase-deficient mice, a strain that exhibits oxidative stress in the liver. Electron transport chain (ETC) activity was assessed using measurements of oxygen consumption. ETC proteins, which are regulators of mitochondrial homeostasis, and the mTOR complexes mTORC1 and mTORC2 were examined by Western blotting. Anticardiolipin (aCL) and anti-ß2 -glycoprotein I (anti-ß2 GPI) autoantibodies were measured by enzyme-linked immunosorbent assay in mice treated with rapamycin or mice treated with a solvent control. RESULTS: Mitochondrial oxygen consumption was increased in the livers of 4-week-old, disease-free MRL/lpr mice relative to age-matched controls. Levels of the mitophagy initiator dynamin-related protein 1 (Drp1) were depleted while the activity of mTORC1 was increased in MRL/lpr mice. In turn, mTORC2 activity was decreased in MRL and MRL/lpr mice. In addition, levels of aCL and anti-ß2 GPI were elevated preceding the development of nephritis in 4-week-old MRL, C57BL/6.lpr, and MRL/lpr mice. Transaldolase-deficient mice showed increased oxygen consumption, depletion of Drp1, activation of mTORC1, and elevated expression of NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), a pro-oxidant subunit of ETC complex I, as well as increased production of aCL and anti-ß2 GPI autoantibodies. Treatment with rapamycin selectively blocked mTORC1 activation, NDUFS3 expression, and aPL production both in transaldolase-deficient mice and in lupus-prone mice. CONCLUSION: In lupus-prone mice, mTORC1-dependent mitochondrial dysfunction contributes to the generation of aPL, suggesting that such mechanisms may represent a treatment target in patients with SLE.

Cardiovascular and thrombophilic risk factors for idiopathic sudden sensorineural hearing loss.

BACKGROUND: In recent years there has been a significant increase in the diagnosis of sudden sensorineural hearing loss (SSHL) in western, countries with an incidence of 20 of 100,000 people affected every year. No clear causes for this disease have been found thus far, but cochlear ischemia has been hypothesized in patients in whom an infectious episode or acoustic neurinoma have been excluded. OBJECTIVES: The aim of this case-control study was to investigate a number of acquired and inherited thrombophilic risk factors [antithrombin, protein C and S; factor V (FV) Leiden, FII polymorphism; lupus anticoagulant (LA); anticardiolipin (aCL) antibodies; fasting homocysteine (Hcy); lipoprotein(a) (Lp(a)); plasminogen activator inhibitor-1 (PAI-1)] in addition to cardiovascular risk factors in patients with idiopathic SSHL (ISSHL). PATIENTS AND METHODS: We investigated 155 patients (67 male/88 female; age: 55 (range 19-79 years) with a diagnosis of ISSHL within 30 days from the onset of symptoms, and 155 controls (67 male/88 female; age 54 (range 19-78 years). Fasting Hcy levels were significantly higher in patients than in controls [11.6 (6.7-60) micromol/L vs. 8.7 (5.0-24) micromol/L] as well as PAI-1 levels [19 (2-95) mg/dL vs. 14.5 (4.0-87) mg/dL]. Lupus anticoagulant was present in 13 of 155 (8.4%) patients; 20 patients (12.9%) had positivity of aCL (four IgM and 16 IgG). In no patient was a deficiency of physiological clotting inhibitors antithrombin, protein C and protein S found. No significant differences between patients and controls were observed for Lp(a) plasma levels [111 (1-1146) mg/L vs. 103 (11-695) mg/L] and for the presence of FV Leiden (4.5% vs. 4.5%) and FII variant G20210A (3.8% vs. 3.2%). RESULTS AND CONCLUSIONS: Independent risk factors for ISSHL at the multivariate analysis (adjusted for age, sex and the traditional cardiovascular risk factors) were the positivity of aCL: OR 5.6 (95% CI 2.0-15.3); cholesterol levels within the second and third tertiles (with respect to the first tertile): T2 = OR 4.8 (95% CI 1.9-12.6)/T3 = OR 19 (95% CI 7-50.1); PAI-1 and Hcy levels within the third tertile (with respect to the first tertile): OR 20 (95% CI 7.8-78) and OR 4.0 (95% CI 2.0-8.1), respectively. These preliminary data suggest that hypercholesterolemia, hyperhomocysteinemia, elevated PAI-1 levels and anticardiolipin antibodies are associated with ISSHL, so indirectly supporting the hypothesis of a vascular occlusion in the pathogenesis of the disease.

Lipoprotein (a) and other prothrombotic risk factors in Caucasian women with unexplained recurrent miscarriage. Results of a multicentre case-control study.

From 1998 to 2003, 133 Caucasian women aged 17-40 years (median 29 years) suffering from unexplained recurrent miscarriage (uRM) were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions (smoking and obesity) were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p<0.0001). Body mass index (BMI; p=0.78) and smoking habits (p=0.44) did not differ significantly between the groups investigated. Upon univariate analysis the heterozygous FV mutation, Lp(a) > 30 mg/dL, increased APA/ACA and BMI > 25 kg/m(2) in combination with a prothrombotic risk factor were found to be significantly associated with uRM. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0-10.7), the FV mutation (OR:3.8/CI:1.4-10.7), and increased APA/ACA (OR: 4.5/CI: 1.1-17.7) had independent associations with uRM.

Fetal calf serum in growth medium obscures the detection of early anticardiolipin antibody secreting clones.

We have examined the effect of fetal calf serum (FCS) on the detection of early anticardiolipin antibody (ACL) secreting clones, using two well-established human IgG clones, LJ1 and AH2. By plating cells at 50/well and growing both clones simultaneously in standard growth (SG) medium containing 10% FCS, and in serum-free (SF) medium, we were able to measure by ELISA the total IgG and ACL levels in the supernatants. The mean OD values (x 1000) against cardiolipin for both LJ1 and AH2 were significantly higher for clones grown in SF than in SG medium: 331 OD units vs. 172, and 275 OD units vs. 166 respectively (p < 0.001). Importantly, the number of wells in which the OD value was > 0.25 units above background was: for LJ1 in SG only 3/36 vs. 30/36 in SF; similarly, for AH2 1/36 in SG vs. 22/36 in SF. In comparison, the total IgG assay using an OD value > 0.7 units above background, detected immunoglobulin secretion in all but one of the wells. We conclude that in ELISA procedures FCS in SG medium competes with solid phase cardiolipin for antibody binding. We suggest that these antibodies are binding to phospholipid from microvesicles found in FCS. We recommend that minimally 'positive' clones on testing should be re-tested and, if necessary, switched to SF medium in order to prevent such clones from being discarded prematurely.

The immunogenetics of the antiphospholipid syndrome, anticardiolipin antibodies, and lupus anticoagulant.

Whether a genetic predisposition to develop the antiphospholipid syndrome (APS) and to produce anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC) exists has been addressed by family studies and by population studies on primary APS and on aCL in diseases other than primary APS. Various studies suggest a familial occurrence of aCL and LAC, with or without clinical evidence of APS. This familial tendency could be genetically determined, because APS, aCL, and LAC occur in families carrying haplotypes which contain HLA-DR4, -DR7, and -DRw53. Population studies on primary APS also indicate that HLA genes have a role in conferring susceptibility to develop primary APS. Again, DR4, DR7, and DRw53 are the relevant loci. Population studies on aCL in diseases other than primary APS indicate that aCL are associated with DR4, DR7, and DRw53, at least when they are found in patients with systemic lupus erythematosus. Because HLA-DR4, -DR7, and -DRw53 are in linkage disequilibrium, the genetic association of aCL could be with DRw53 and, depending on the regional frequency of DR4 or DR7, it could be linked with either DR4 or DR7. HLA-DR4 seems to be more important in Anglo-Saxons, whereas DR7 emerges in populations of Latin origin. In this report we review our studies and the pertinent literature in this field.

Establishment of two distinct anti-cardiolipin antibody-producing cell lines from the same individual by Epstein-Barr virus transformation.

Two anti-cardiolipin antibody (ACA)-producing cell lines were established, using Epstein-Barr virus transformation followed by a repeated cluster-picking from the same individual with antiphospholipid syndrome who had a history of 8 consecutive fetal losses and deep venous thrombosis. Characterization of the two ACAs derived from these cell lines revealed that one (Ab-019, subclass IgM, kappa) reacted exclusively with cardiolipin and showed strong lupus anticoagulant activity, while the other (Ab-226, subclass IgM, lambda) reacted with negatively charged phospholipids such as phosphatidylserine and phosphatidylglycerol, as well as cardiolipin. Furthermore, Ab-226 showed reactivity with human umbilical vein endothelial cells whereas Ab-019 did not. It is suggested that ACA is heterogeneous even in the same individual, and that reactivity against negatively charged phospholipids corresponds to reactivity against endothelial cell.

Production of anti-cardiolipin antibody in AKR/J mice with streptozocin-induced insulitis and diabetes.

We herein report that anti-cardiolipin antibodies (ACA) were detected in AKR/J mice treated with multiple low doses of streptozocin (STZ)-induced insulitis and diabetes. Daily intraperitoneal (i.p.) injections of 40 mg/kg body wt. of STZ for five consecutive days in the AKR/J mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). ACA appeared on day 14, when hyperglycemia began to occur, at a rate of 13.3% (4/30). The rate increased to 83.3% (25/30) on day 21, when diabetes developed, and then fell to 10% (3/30) on day 28. Neither the diabetic AKR/J mice treated with a single high dose of STZ (200 mg/kg body wt.) nor the non-diabetic insulitis free Balb/c mice and B10.S(9R) mice treated with multiple low doses of STZ (40 mg/kg body wt.) produced ACA. The IgG subclass of the ACA belonged mainly to IgG2a. These findings suggest that ACA are produced in association with the development of insulitis, but not induced by either hyperglycemia or STZ.

Superantigens and experimental SLE induced by idiotypic dysregulation.

OBJECTIVE: The effects of the superantigens (SAgs) Staphylococcal Enterotoxin B (SEB), Toxic Shock Syndrome Toxin-1 (TSST-1) and Mycoplasma Arthritidis Mitogen (MAM) were examined on the induction and on the course of experimental SLE-like disease. METHODS: Immunization of BALB/c mice with human anti-DNA mAb (MIV-7) carrying the pathogenic idiotype 16/6 emulsified in complete Freund's adjuvant (CFA), followed by a boost of MIV-7/PBS 3 weeks later, generated an experimental SLE via an idiotypic dysregulation. RESULTS: After immunization with MIV-7/SAg, replacing the MIV-7 boost by SAg, and then injecting SAg 7 weeks after the regular induction of the SLE-like disease, the mice failed to produce anti-hIgM and dsDNA Ab up to 6 months after the induction. The mice immunized with MIV-7/CFA and boosted with the SAg had high titers of anti-hIgM but no detectable anti-dsDNA Ab. In both experimental groups low titers of anti-CL Abs developed in 25/40 (62%) and 30/38 (79%) of the mice respectively, including the control mice immunized with non-pathogenic human IgM/SAg or PBS/SAg. The mice immunized according to the "classical" protocol showed increased titers of anti-dsDNA Ab (22%) and anti-CL Ab (28%) during 10 weeks of observation. In contrast SEB, TSST-1 and MAM induced a 29%, 1% and 17% reduction in the anti-DNA titers and a 32%, 15% and 12% reduction in the anti-CL titers, respectively. CONCLUSIONS: These data suggest that the SAg tested here cannot replace the effect of CFA in the induction of the primary humoral response. The SAgs TSST-1, SEB and MAM did not induce the SLE-like disease following idiotypic modulation. Moreover, they may have had a suppressive effect on the idiotypic network in our model. The appearance of anti-CL Abs in almost all the experimental groups including the naive mice supports the possibility that microbial SAgs can induce the production of autoantibodies by different mechanisms. The SAgs TSST-1, SEB and MAM reduced autoantibody production in the serologically established idiotypic-induced experimental SLE-like murine model. This beneficial effect may indicate new directions for research on the management of SLE.

HIV infection and antiphospholipid antibody: literature review and link to the antiphospholipid syndrome.

There is a high incidence of antiphospholipid antibodies, detected by assays for anticardiolipin or lupus-like anticoagulant, in HIV disease. However, a link to the antiphospholipid syndrome, with clinical thrombosis, is tenuous. We report a case of a 25-year-old man with undetermined risk factors for HIV presenting with possible antiphospholipid syndrome manifesting as necrotic skin lesions as the initial clinical presentation for HIV. We also review the literature exploring the association between HIV and antiphospholipid syndrome.

Elevated serum interleukin-33 levels in patients with Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is the most common systemic vasculitis and is known as an immunoglobulin (Ig) A related immune complex-mediated disease. However, the molecular mechanisms in the development of HSP are not yet fully understood. Herein, we investigated the serum levels of Interleukin (IL)-33 and soluble ST2 (sST2) in HSP patients and their association with disease severity and IgA autoantibodies production. The serum levels of IL-33 and sST2 were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) in the serum of 33 patients with HSP and 22 controls. Serum levels of IgA anti-endothelial cell antibodies (AECA) and IgA anticardiolipin antibodies (ACA) in HSP patients were detected by double antigen sandwich ELISA. Our results indicated that serum levels of IL-33 but not sST2 were significantly elevated in patients with HSP in acute stage and restored to normal levels in convalescent stage. Moreover, serum IL-33 levels were correlated with the severity of HSP and serum concentrations of AECA-IgA and ACA-IgA. Taken together, we show firstly that serum IL-33 is abnormally elevated in HSP patients. IL-33 might be associated with the IgA autoantibodies production in the pathogenesis of HSP.

Anti-phospholipid antibodies following vaccination with recombinant hepatitis B vaccine.

This study was undertaken to evaluate the possible role of hepatitis B recombinant vaccine inducing the synthesis of IgG and IgM anti-cardiolipin antibodies (aCL), antibodies against beta(2)GPI (anti-beta(2)GPI), lupus anti-coagulant (LA), anti-nuclear antibodies and antibodies against extractable nuclear antigens (anti-ENA). The study population consisted of 85 healthy students (63 female, 22 male; mean age 20.8 years), vaccinated with three doses of recombinant DNA hepatitis B vaccine. One month after vaccination with the first dose of hepatitis B vaccine a minority of vaccinated individuals showed changes in IgG or IgM aCL or anti-beta(2)GPI or LA activity (P < 0.001). Among subjects in whom changes of IgG anti-beta(2)GPI were observed, a significantly higher number of increased (8/85) than decreased (2/85) values were found (P < 0.01). Analyses of paired data showed that differences in aCL or anti-beta(2)GPI levels before vaccination or 1 month later did not reach statistical significance. In two people aCL transitorily reached medium positivity after the first dose of hepatitis B vaccine with a drop 5 months later. Similar evident anti-beta(2)GPI fluctuation was also observed in one person. Another participant was initially low positive for IgG anti-beta2GPI and the levels were increasing after vaccination. Two participants became positive for anti-nuclear antibodies during 6 months' follow-up. There were no sex-dependent differences in tested antibodies observed and no associations between levels of aPL and levels of anti-HBV antibodies. We conclude that HBV can induce aPL, although rarely. In genetically susceptible individuals or together with some other triggers such combination might confer the risk of developing a continuous autoimmune response in an individual.

Diagnostic and prognostic significance of anticardiolipin antibodies in patients with recurrent spontaneous abortions.

PROBLEM: The role of ACA in unexplained RSA is controversial. In the present study, diagnostic and prognostic aspects were investigated. METHOD: One hundred five nonpregnant patients with primary, 29 with secondary RSA, and 209 controls were investigated for IgG-ACA. Follow-up studies were done during pregnancy in 76 individuals. IgM-ACA were tested in a subset of patients. RESULTS: Elevated ACA levels were significantly more frequent in both patient groups (26 and 24%) than in controls (16%). However, there was no correlation of ACA with various parameters including pregnancy outcome. In ACA-positive patients with successful pregnancy a significant decrease of ACA values during pregnancy was observed, while ACA remained high in aborting patients. IgG- and IgM-ACA correlated well. CONCLUSIONS: Although the data from nonpregnant RSA patients does not allow diagnostic or prognostic conclusions to be drawn, sequential testing of ACA-positive individuals provides the possibility to foresee pregnancy outcome.

Induction of high levels of anticardiolipin antibodies in mice by immunization with beta 2-glycoprotein I does not cause fetal death.

OBJECTIVE: Our purpose was to determine whether anticardiolipin antibodies induced by immunization with beta 2-glycoprotein I cause fetal death in mice. STUDY DESIGN: Female BALB/c mice were immunized with beta 2-glycoprotein I in a carbohydrate adjuvant or with carbohydrate adjuvant alone. The mice were mated with BALB/c males and killed on day 11 to 13 of pregnancy, and the fetal status was determined. Posttreatment blood samples were obtained for measurement of anticardiolipin and anti-beta 2-glycoprotein I antibodies and platelet counts. RESULTS: Anticardiolipin and anti-beta 2-glycoprotein I antibodies developed in all mice immunized with beta 2-glycoprotein I. Fetal death occurred in 17 of 145 gestational sacs (12%) in 18 mice immunized with beta 2-glycoprotein I compared with 24 of 177 (14%) sacs in 21 control mice. There were no morphometric or histologic differences between gestational tissues, and platelet counts were similar for each group. CONCLUSIONS: The induction of high levels of anticardiolipin antibodies in BALB/c mice by beta 2-glycoprotein I immunizations did not result in fetal death or thrombocytopenia. These nonpathogenic beta 2-glycoprotein I-induced anticardiolipin antibodies should prove useful in the characterization of clinically relevant epitopes for antiphospholipid syndrome.

Antiphosphatidylserine antibodies are elevated in normal tension glaucoma.

The two main entities of open-angle glaucoma are primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG). Both diseases may be associated with autoimmune processes. Therefore, IgG and IgM antibodies to phospholipids (APL) and their subspecies cardiolipin (ACL), phosphatidylserine (APS) and beta2-glycoprotein (beta2GP) were determined in 43 NTG patients, 40 POAG patients and 40 healthy controls in a prospective study. The most prominent observation was the increase in APS concentrations in NTG patients (IgG 20.6 +/- 2.7 U/ml, IgM 24.4 +/- 3.4 U/ml) compared with POAG patients (IgG 8.8 +/- 1.2 U/ml, IgM 11.0 +/- 1.7), and controls (IgG 7.7 +/- 1.3 U/ml, IgM 12.8 +/- 1.5 U/ml). APS may be important due to their binding specificity to phosphatidylserine molecules which become accessible during apoptosis; this in turn may lead to local thrombosis.

Antiphospholipid antibodies in Chilean patients with systemic lupus erythematosus.

Antiphospholipid antibodies (aPLs) are a heterogeneous family of antibodies found in autoimmune disorders, infectious diseases, and other situations. The presence of different aPLs has been associated with various clinical manifestations of the antiphospholipid syndrome (APS). The objective of this study was to investigate the prevalence of aPLs in a group of 90 Chilean patients with systemic lupus erytematosus (SLE) and 90 healthy controls. We measured anticardiolipin antibodies (aCLs), antiphosphatidylserine antibodies (aPSs), anti-beta(2) glycoprotein I antibodies (anti-beta(2)GPIs), and antiprothrombin antibodies (aPTs) with an enzyme-linked immunosorbent technique using "in-house" assays. Fifty-four of 90 SLE patients (60.0%) had some type of aPL. Forty of 90 (44.4%) were positive for aCLs, 9 of 61 (14.8%) had aPSs, 21 of 90 (23.3%) had anti-beta(2)GPIs, and 18 of 90 (20.0%) had aPTs. In the control group, prevalences were as follows: aCLs, 3.3%; aPSs, 1.1%; anti-beta(2)GPIs, 1.1%; aPTs, 2.2%. In most cases, values were in the low-positive range. Of all aPL detected, 29.5% was of the IgG isotype, 37.5% IgM, and 33.0% IgA. We observed a correlation between aCLs and aPSs and of these antibodies with anti-beta(2)GPIs and aPTs but not between anti-beta(2)GPIs and aPTs. Our results show a high prevalence of aPLs in SLE patients. An association between different specificities and isotypes of aPLs was also observed.

Antibodies to cardiolipin in normal C57BL/6J mice: induction by estrogen but not dihydrotestosterone.

Autoantibodies against cardiolipin, a phospholipid, have been demonstrated in a variety of pathological states including several autoimmune conditions in humans and in certain lupus-prone mice. In this study we detected antibodies reactive to cardiolipin in normal C57BL/6J mice by ELISA. The autoantibodies are detected less frequently in the serum of male than in female mice, suggesting the influence of sex hormones. The relative refractoriness of normal male mice to the induction of anticardiolipin antibodies is not due to the suppressive effects of male hormones, since prepubertal orchiectomy has little influence on this autoantibody. Further, dihydrotestosterone treatment of orchiectomized mice has minimal effect on anticardiolipin antibodies. However, orchiectomized mice when given estrogen develop a marked increase in the incidence as well as the levels of these autoantibodies. Similarly, estrogen treatment of female mice further augments the incidence and the levels of these autoantibodies. Estrogen-treated mice also have antibodies reactive against other membrane phospholipids including phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The intensity of binding of autoantibodies to the above phospholipids varies among individual mice. To our knowledge, this is the first report on the demonstration of antiphospholipid autoantibodies in normal mice and induction of these antibodies by estrogen.

Induction of anti-phospholipid autoantibodies by beta2-glycoprotein I bound to apoptotic thymocytes.

The target of many anti-phospholipid autoantibodies (aPL) has been shown to be a complex between anionic phospholipid and the plasma protein beta2-glycoprotein I (beta2GPI) or the protein beta2GPI alone. As aPL binding studies have been performed almost exclusively in vitrothe identity of the natural target and/or immunogen for aPL in vivo remains undetermined. The anionic phospholipids of cell membranes represent an important potential target and immunogen for aPL. Although anionic phospholipids are normally absent from the extracellular surface of cell membranes, they redistribute from the inner to the outer leaflet during apoptosis. We have previously shown that beta2GPI binds selectively to the surface of apoptotic, but not viable, cells, and that binding of beta2GPI to the surface of apoptotic cells generates an epitope recognized by aPL from patients with primary aPL syndrome and systemic lupus erythematosus. We show here that immunization of non-autoimmune mice with beta2GPI combined with, or bound to, apoptotic cells induces aPL and lupus anticoagulant activity. Generation of aPL required heterologous beta2GPI, and occurred upon immunization with apoptotic cells and beta2GPI by three different routes of administration. Importantly, for intravenous immuniz-ations, generation of aPL occurred only when apoptotic cells and beta2GPI were injected together, but not when either was injected alone, suggesting that cell-bound beta2GPI is the true immunogen for production of aPL. Unlike other models of induced aPL, adjuvant was not an absolute requirement. Induced aPL reacted with murine, as well as bovine, beta2GPI, suggesting that heterologous beta2GPI bound to apoptotic cells can break tolerance and induce auto-antibodies reactive with autologous beta2GPI. Combined with our previous data, these results show that apoptotic cells can serve as both immunogens and natural targets for aPL.

Antiphospholipid antibodies and antiphospholipid syndrome in patients presenting with immune thrombocytopenic purpura: a prospective cohort study.

The pathogenetic role and the clinical importance of the presence of antiphospholipid antibodies (APAs) in patients with immune thrombocytopenic purpura (ITP) are not clear. In this study, the prevalence and clinical significance of APAs were investigated in patients with ITP. Eighty-two newly diagnosed ITP patients were prospectively studied. They were evaluated for the presence of lupus anticoagulant (LA) and immunoglobulin G/M anticardiolipin antibodies (ACAs). Thirty-one patients (37.8%) were APA positive at diagnosis. No statistically significant differences were found between the APA-positive and APA-negative groups regarding gender, initial platelet counts, or response to methylprednisolone therapy. After 5 years of follow-up, cumulative thrombosis-free survival of APA-positive (n = 31) and APA-negative (n = 51) ITP patients was 39% and 97.7%, respectively. A significant difference was found between these groups by log-rank test (P =.0004). In addition, LA was an important risk marker for the development of thrombosis in ITP patients. After a median follow-up of 38 months, 14 ITP patients (45%) who had APA positivity developed clinical features (thrombosis or fetal losses) of antiphospholipid syndrome (APS). There were no differences between the APA-positive patients with and without APS regarding the initial platelet counts, response to the therapy, or ACA positivity. The positivity rate for LA was significantly higher in those patients with ITP who developed APS (chi(2): P =.0036; relative risk 7.15; 95% confidence interval, 1.7-47). In conclusion, this study indicates that a significant proportion of patients initially presenting with ITP and APA positivity developed APS. In patients with ITP, the persistent presence of APAs is an important risk factor for the development of APS.

Characterization of a human monoclonal autoantibody directed to cardiolipin/beta 2 glycoprotein I produced by chronic lymphocytic leukaemia B cells.

We determined the specificity and sequence of immunoglobulin molecules synthesized by monoclonal B cells from a patient with chronic lymphocytic leukaemia (CLL) who presented with a number of clinical and biological autoimmune symptoms. Heterohybrids obtained by fusion of CLL cells with the mouse X63-Ag 8.653 myeloma produced IgM lambda MoAbs directed to the cardiolipin/beta 2 glycoprotein I (beta 2GPI) complex and ssDNA. They were devoid of polyreactivity. Nucleotide sequence analysis of the variable domain of the mu chain indicated the utilization of the VH4 71.2 gene or one allotypic variant, DXP4 and JH3 segments. The lambda light chain used the single gene from the V lambda 8 subfamily, J lambda 3 and C lambda 3 genes. The VH gene displayed 11 nucleotide changes in comparison with its putative germline counterpart. However, these nucleotide changes correspond to variations observed in other published VH4 sequences, suggesting gene polymorphism rather than somatic mutation. DXP4 and JH3 were also in germline configuration. The VL gene exhibited a single replacement mutation in CDR1. These data suggest that the monoclonal CLL B cells in this patient retained VH and VL genes in germline configuration although they secreted a pathogenic anti-cardiolipin antibody associated with clinical symptoms, vasculitis and thrombosis, which may be provoked by antibodies to the phospholipid/beta 2GPI complex.