Comparison of different enzyme-linked immunosorbent assay methods for avidity determination of antiphospholipid antibodies.

BACKGROUND: Avidity of antiphospholipid antibodies may be clinically useful as a valuable additional characteristic. The aim of this study was to compare several ELISA modifications with different chaotropic agents and calculation of avidity indices for the determination of anticardiolipin antibody (aCL) avidity. METHODS: We examined 28 serum samples with positive IgG aCL by adapted ELISA using various concentrations of urea and sodium chloride as chaotropic agents and different dilution of sera. We tested these conditions of ELISA-a single diluted serum sample with fixed concentration of a chaotrope and a serially diluted serum in the constant concentration of a chaotropic agent. RESULTS: We demonstrated that ELISA method for avidity determination based on a single dilution of serum in the presence of fixed concentration of chaotrope is convenient for determination of IgG aCL antibody avidity. Concentrations 6 and 8 mol/L of urea or 1 and 2 mol/L of NaCl were suitable for sufficient dissociation of immune complexes during ELISA procedure. CONCLUSION: This way was in good agreement with more demanding procedures. Both urea and sodium chloride may be used as chaotropic agents. Reference values of avidity indices essential for interpretation of patients' results must be established individually for distinct assay conditions.

Double positivity of the IgG isotype of both anticardiolipin and anti-ß2gpI antibodies is associated with the highest number of vascular impairment parameters in patients with primary antiphospholipid syndrome: preliminary data.

Although numerous studies investigated the association between homocysteine (Hcy), tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) and apolipoproteins (apos) with thrombosis and/or recurrent pregnancy losses, studies that analyzed the abovementioned parameters and multiple positivity of antiphospholipid antibodies (aPL Abs) in patients with primary antiphospholipid syndrome (PAPS) are lacking. Therefore, the aim of this study was to analyze the presence of various combinations of the abovementioned parameters and their associations with clinical and/or serological features of PAPS. High-pressure liquid chromatography (HPLC) was used for determination of Hcy, while apoAI, apoB, and lipoprotein (Lp) (a) concentrations were estimated by immunonephelometry. High-sensitivity C-reactive protein (hsCRP) was measured by immunoturbidimetry. Apo (a), TNF-α, and aPL Abs were measured by ELISA. Various combinations of analyzed parameters (Hcy/CRP/TNF/apoAI/apoB/apo (a)/Lp (a)) were not associated with a single presence of either aPL Abs. Double positivity for both isotypes of anticardiolipin (aCL) Abs (IgG + IgM) was associated with the increased apoB levels. The presence of the IgG isotype of both aCL + anti-beta2 glycoprotein I (aß2gpI) Abs was associated with the highest number of analyzed parameters (i.e., increased levels of hsCRP, Lp (a), and apo (a)). The presence of the IgG isotype of both aCL + aß2gpI Abs was associated with the highest number of vascular impairment parameters in patients with PAPS, and this combination confers the highest risk for the recurrence of thrombotic episodes. This is the first report that analyzed the association between various combinations of vascular impairment parameters with multiple aPL Ab positivity. Our results provide a rationale for further investigations of therapeutic approaches for PAPS patients.

Reversible cerebral vasoconstriction syndrome (RCVS) in antiphospholipid antibody syndrome (APLA): the role of centrally acting vasodilators. Case series and review of literature.

Reversible cerebral vasoconstriction syndrome (RCVS) is Raynaud's phenomenon of the brain. Changes in neurological function are dependent upon which areas of the brain are deprived of normal blood flow. Antiphospholipid antibody syndrome (APLA) is a common cause of Raynaud's phenomenon that can occur anywhere in the body, including the brain. Management of CNS vasospasm generally involves the use of centrally acting calcium channel blockers, which have been shown to relieve the associated headaches and transient neurological symptoms associated with it. Three patients with APLA and RCVS from our clinics are illustrated. It is demonstrated that the use of centrally acting calcium channel-blocking drugs, such as nimodipine, which prevent and reverse CNS vasospasm, led to clinical improvement in our patients over the course of 5-9 years. All of them had MRIs done at the initiation of therapy and 5-9 years after being on therapy. MRI measures of T2 lesion volumes (LVs) and number were obtained. All three patients had a good response in controlling clinical symptoms related to CNS vasospasm, Raynaud's phenomenon, visual disturbances, confusion, headaches, and hearing loss. There was also a resolution in the MRI findings of these patients. This case series of three patients shows a clinical improvement and decrease in T2 LV and number in patients with APLA and Raynaud's syndrome on centrally acting calcium channel blockers. RCVS is much more common than that currently appreciated. APLA is the common cause of RCVS. Further studies are needed to determine the optimal methods to diagnose RCVS and optimal therapies to treat it.

Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum.

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of ß2-glycoprotein I (ß2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of ß2-GPI to the cardiolipin surface. Using the switchSENSE sensor, ß2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between ß2-GPI-dependent autoimmune- and ß2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

Clinical analysis of primary Sjögren's syndrome complicating anemia.

This is a cross-sectional study to assess the prevalence and causes of anemia in the primary Sjögren's syndrome (pSS). One hundred and thirty-two consecutive patients with pSS were enrolled into the study. Standard hematological and immunological tests and examination of bone marrow were performed. Anemia occurred in 45 (34.1%) patients. The causes of anemia included anemia of chronic disease (69%), autoimmune hemolytic anemia (AIHA, 18%), iron deficiency anemia (9%) and other causes (4%), of which AIHA caused the most severe anemia. The prevalence of ANA, anti-Ro/SSA, and anti-La/SSB was much higher in patients with anemia than those without anemia. Anticardiolipin antibodies were most commonly detected in AIHA; the prevalence of IgG and hypocomplementemia in AIHA was much higher in patients without anemia. Abnormal bone marrow changes were observed in two cases with anemia, one with morphological changes in the myeloid, megakaryocytic, and erythroid lineages and one with hypocellularity in the erythroid lineage. Therefore, pSS patients with anemia may be associated with destruction of peripheral mature blood cells, impaired red cells production, and hematopoietic abnormalities due to an immune mechanism, although the concrete pathogenesis is still unclear.

Specificity and sensitivity of anti-beta2-glycoprotein I as compared with anticardiolipin antibody and lupus anticoagulant in Thai systemic lupus erythematosus patients with clinical features of antiphospholipid syndrome.

Antibodies to beta(2)-glycoprotein I (anti-beta(2)-GPI) have been reported to have stronger association with clinical antiphospholipid syndrome (APS) than anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC). We investigated the sensitivity and specificity of ELISA for anti-beta(2)-GPI in Thai systemic lupus erythematosus (SLE) patients with clinical features of APS and compared the results with IgG/IgM aCL and LAC to find the test with the best association. The hospital records of 151 Thai SLE patients whose sera had been sent for either IgG/IgM anticardiolipin antibodies or lupus anticoagulant testing were reviewed. Sera of patients either without complete clinical records or those with APS-related manifestations other than vascular thrombosis and pregnancy morbidity (according to the international consensus statement on preliminary classification criteria for definite APS) were excluded. For the remaining subjects (112 patients), their sera were tested for anti-beta(2)-GPI antibody, IgG and IgM anticardiolipin, and lupus anticoagulant. The sensitivity and specificity of each method were compared by using the chi-square test. Among the 112 (74.2%) SLE patients in the study, 35 (31.3%) presented with preliminary clinical criteria for APS (i.e., vascular thrombosis and pregnancy morbidity) whereas 77 (68.7%) did not. The sensitivity and specificity of anti-beta(2)-GPI determination were 57.1 and 79.2%, respectively, whereas those of IgG aCL were 25.7 and 94.8%, of IgM aCL were 5.7 and 98.7%, and of LAC were 44.8 and 77.3%, respectively. The accuracy of the four tests showed similar association with clinical APS (accuracy of test = 72.3, 73.2, 69.6, and 68.3%, respectively). Concerning the sensitivity, specificity, and difficulty of the methods, the combination of anti-beta(2)-GPI and IgG aCL tests was the best for the diagnosis of APS in Thai SLE patients.

Antigenic structures recognized by anti-beta2-glycoprotein I auto-antibodies.

Beta2-glycoprotein I (beta2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of beta2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193-K246, D222-K317 and E228-K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-beta2-GPI auto-antibodies to solid-phase beta2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-beta2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure.

Ischemic stroke subtypes and thrombophilia in young and elderly Brazilian stroke patients admitted to a rehabilitation hospital.

BACKGROUND AND PURPOSE: We sought to examine ischemic stroke subtypes and prevalence of thrombophilia in Brazilian stroke patients. METHOD: A total of 130 consecutive young and 200 elderly stroke patients were studied. RESULTS: Prevalence of thrombophilia was, respectively: protein S deficiency (11.5% versus 5.5%), protein C deficiency (0.76% versus 1%), resistance to activated protein C (2.3% versus 3.5%), mutation in V Leiden factor (1.5% versus 2%), antithrombin III deficiency (0% versus 0%), lupus anticoagulant (0% versus 0.5%), anticardiolipin antibodies (3% versus 10%; P=0.01), hyperhomocysteinemia (31.5% versus 53.5%; P=0.0001), mutation of the MTHFR gene in homocigosis (10% versus 5%), and heterocigosis (27.6% versus 41.9%; P=0.01). CONCLUSIONS: Prothrombotic conditions were more frequent in stroke of undetermined cause.

[Characteristics of blood dynamic parameters in pregnant women with anticardiolipin antibodies and history of adversely pregnant complications].

OBJECTIVE: To study the characteristics of blood dynamic parameters in pregnant women with anticardiolipin antibody (aCL) and a history of adversely pregnant complications. METHODS: The study included a prospective cohort of pregnant women with (272 cases) and without (186 cases) aCL. Among the aCL positive pregnant women, 91 cases had the history of adversely pregnant complications including spontaneous abortion, still fetus. Serum antibodies to cardiolipin (CL) were measured by a specific enzyme linked immunosorbent assay. Blood dynamics parameters were measured by non-invasive cardiovascular parameters surveyor (TP-CBS). RESULTS: The women were divided into three groups: group A (91 cases)included women with both the history of adversely pregnant complications and positive aCL, group B (181 cases) with positive aCL alone and group C (186 cases) without either of them. In group A maximum blood viscosity (Vmax) was (4.7 +/- 0.5) mPa x s and in group B it was (4.6 +/- 0.7) mPa x s. Both was higher than normal. In group B the Vmax was higher (P < 0.05) than in group C [(4.4 +/- 0.4) mPa x s]. The wave form coefficient(K) was different in three groups: group A was 0.41 +/- 0.04, group B was 0.39 +/- 0.05 and group C was 0.39 +/- 0.04. In group A their wave form coefficient was higher than in group B (P < 0.05). Their total peripheral resistance (TPR) was higher (P < 0.05) in group A [(1.3 +/- 0.4) mm Hg x s x ml(-1)] than in group B [(1.2 +/- 0.4) mm Hg x s x ml(-1)]. The cardiac index (CI) was in group A [(43 +/- 15) ml/(s x m(2))], in group B [(48 +/- 16) ml/(s x m(2))] and in group C [(48 +/- 14) ml/(s x m(2))], lower in group A than in group B (P < 0.05). CONCLUSIONS: An important change of blood dynamic parameters of pregnant women with aCL is the increase of Vmax. If Vmax, K and TPR were increased and CI was decreased simultaneously in women with aCL, it would result in more chances of adversely pregnant complications. Measuring the blood dynamics parameters will help us discover the sub-clinical hypercoagulable state of patients with aCL and guide us to treat them promptly, so that to improve successful pregnancy rate.

A randomized clinical trial of high-intensity warfarin vs. conventional antithrombotic therapy for the prevention of recurrent thrombosis in patients with the antiphospholipid syndrome (WAPS).

BACKGROUND: The optimal intensity of oral anticoagulation for the prevention of recurrent thrombosis in patients with antiphospholipid antibody syndrome is uncertain. Retrospective studies show that only high-intensity oral anticoagulation [target international normalized ratio (INR) >3.0] is effective but a recent randomized clinical trial comparing high (INR range 3.0-4.0) vs. moderate (INR 2.0-3.0) intensities of anticoagulation failed to confirm this assumption. METHODS: We conducted a randomized trial in which 109 patients with antiphospholipid syndrome (APS) and previous thrombosis were given either high-intensity warfarin (INR range 3.0-4.5, 54 patients) or standard antithrombotic therapy (warfarin, INR range 2.0-3.0 in 52 patients or aspirin alone, 100 mg day(-1) in three patients) to determine whether intensive anticoagulation is superior to standard treatment in preventing symptomatic thromboembolism without increasing the bleeding risk. RESULTS: The 109 patients enrolled in the trial were followed up for a median time of 3.6 years. Mean INR during follow-up was 3.2 (SD 0.6) in the high-intensity warfarin group and 2.5 (SD 0.3) (P < 0.0001) in the conventional treatment patients given warfarin. Recurrent thrombosis was observed in six of 54 patients (11.1%) assigned to receive high-intensity warfarin and in three of 55 patients (5.5%) assigned to receive conventional treatment [hazard ratio for the high intensity group, 1.97; 95% confidence interval (CI) 0.49-7.89]. Major and minor bleeding occurred in 15 patients (two major) (27.8%) assigned to receive high-intensity warfarin and eight (three major) (14.6%) assigned to receive conventional treatment (hazard ratio 2.18; 95% CI 0.92-5.15). CONCLUSIONS: High-intensity warfarin was not superior to standard treatment in preventing recurrent thrombosis in patients with APS and was associated with an increased rate of minor hemorrhagic complications.

Primary, Secondary, Catastrophic Antiphospholipid Syndrome: is there a difference?

Following broad recognition of the disorder called Antiphospholipid Syndrome (APS), it has come to be subcategorized into Primary (PAPS), Secondary (SAPS), and Catastrophic Antiphospholipid Syndrome (CAPS). Primary utilized when there is no associated disorder, secondary with an associated autoimmune disorder such as systemic lupus erythematosus (SLE), and "catastrophic" when thrombosis occurs at multiple sites in a short space of time. Are these entities different? Such differences should be demonstrated in terms of their clinical presentation, disease course, pathogenesis, or management. If no differences exist, is there a basis for continued use of these terms? This manuscript will attempt to explore distinctions between subgroups of the APS and reasons for or against perpetuation of these classifications in the literature.

An adapted ELISA method for differentiating pathogenic from nonpathogenic aPL by a beta 2 glycoprotein I dependency anticardiolipin assay.

With the currently available commercial kits, as well as homemade assays for detecting anticardiolipin antibodies (aCL), it is not possible to discriminate nonpathogenic, beta 2 glycoprotein (GPI)-independent, infection-related antibodies from those of patients with the true autoimmune thrombotic syndrome, known as antiphospholipid syndrome (APS). We devised an assay that is able to differentiate these two types of antibodies by determining the beta 2 GPI requirements to bind in a cardiolipin ELISA. Beta 2 GPI was purified by perchloric acid precipitation, and fixed amounts were used in the dilution solutions of the tested samples that were also tested with no source of beta 2 GPI. The ELISA plates were coated with cardiolipin, as usual, and blocked with a chicken ovalbumin solution. The serum samples had to be highly diluted in order not to have beta 2 GPI from the patient serum. The reaction was detected with alkaline phosphate tablets and developed with pNp in diethanolamine buffer. The adapted ELISA aCL assay described here was able to discriminate infectious [syphilis, hepatitis C virus (HCV), dengue fever, human immunodeficiency virus (HIV) and leprosy] and autoimmune [primary APS and systemic lupus erythematosus (SLE) related APS]. Further testing should be performed to demonstrate that this method consistently differentiates pathogenic antibodies that bind in an aCL ELISA only in the presence of beta 2 GPI.

Libman-Sacks endocarditis associated with antiphospholipid syndrome and infection.

INTRODUCTION: Antiphospholipid syndrome (APS) is a systemic autoimmune disease, associated not only with a hypercoagulable state and recurrent fetal loss but with many diverse clinical manifestations including heart involvement, neurological manifestations, as well as skin, kidney and hematologic abnormalities. Cardiac manifestations include coronary by-pass graft and angioplasty occlusions, cardiomyopathy, cyanotic congenital heart disease, intracardiac thrombus and complications of cardiovascular surgery. The valvular heart disease was defined as Libman-Sacks nonbacterial endocarditis. Previously, we have shown a linear subendothelial deposition of anti-cardiolipin/beta2 glycoprotein I (beta2GPI) antibodies in the valve specimens derived from APS patients. The involvement of complement C3c in the pathogenesis was documented. We assessed the beta2GPI-related target epitope recognized by the anti-beta2GPI Abs on the valves. MATERIALS AND METHODS: In order to find the beta2GPI-related target epitopes recognized by the anti-beta2GPI antibodies on the valves, we used beta2GPI-related synthetic peptides. The presence of anti-beta2GPI Abs on the studied valves was detected by anti-idiotypic antibody, followed by immunoperoxidase analysis. Biotin attached to the N-terminal of beta2GPI-related synthetic peptides and control peptide were used to identify the epitope addressed by the anti-beta2GPI Abs deposited on the patient's valve. The binding was probed by streptavidin-peroxidase and appropriate substrate. The specificity was confirmed by competition assays with control peptide and anti-idiotypic antibody. RESULTS: Among the beta2GPI-related synthetic peptides, two peptides were found in previous studies to mimic common pathogens either bacteriae or viruses, which raised a possible infectious origin for APS. One of these peptides, TLRVYK, is a specific target for anti-beta2GPI Abs deposited on the APS valves. This synthetic peptide was able to displace the anti-anti-beta2GPI anti-idiotypic Abs for binding the anti-beta2GPI Abs on the valve by a competition assay. CONCLUSION: We point to the possibility that Libman-Sacks nonbacterial endocarditis may have an infectious origin.

Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

The antiphospholipid antibodies.

Antiphospholipid antibodies (APLAs) are a group of autoantibodies directed against certain phospholipids, or their protein cofactors. Assay of APLAs is important because their interaction with anionic phospholipid-protein cofactors can generate a syndrome of hypercoagulability associated with a wide variety of thromboembolic events. This article presents the characteristics of some APLAs [anticardiolipin antibodies (aCLAs), lupus anticoagulant (LA) and anti-beta2-glycoprotein I antibodies (anti-beta2-GPIAs)], their action, and their interaction with blood and endothelial cells. The presence of APLAs has been reported in many diseases (autoimmune diseases, atherosclerosis, infections, malignancies), being related to pathogenic mechanisms and/or to a more severe evolution of the disease.

Structural characterization and optimization of antibody-selected phage library mimotopes of an antigen associated with autoimmune recurrent thrombosis.

The presence of high titers of anti-cardiolipin antibodies (ACA's) of autoimmune origin, which are known to bind to plasma beta2-glycoprotein I (aka apolipoprotein H), correlates clinically with autoimmune recurrent thrombosis. Soluble beta2-glycoprotein I binds to solid-phase ACA (immobilized on a surface plasmon resonance chip) with a Kd of 1.4 microM, but if the reactants are reversed and beta2-glycoprotein I is on the solid-phase support, then the Kd is 52 nM. This 27-fold difference in affinity reflects the avidity/entropic advantage obtained for an antibody binding to an antigen that is made multivalent because it is attached to a solid phase. A mimotope of this antigen, selected from a phage display peptide library screen with an ACA, has been shown to bind to solid-phase ACA as a phage, using surface plasmon resonance. This peptide is representative of the motif from 37 peptides obtained in a previously reported phage library screen with this ACA (1). A synthetic version of this peptide, referred to as P4, has the sequence: A1G2P3C4I5L6L7A8R9D10R11C12P13G14, and binds to its selecting antibody with a Kd of 42 nM. NMR data indicate that proline-13 is present in both cis and trans configurations, and that these two geometries dramatically affect the overall tertiary structure of the molecule. The peptide lacking this proline binds severalfold better to the ACA, consistent with at least one of these structures having low affinity for binding ACA. Replacement of the arginine-9 position with a proline decreases binding affinity to ACA 10-fold. Another phage library-selected peptide has a proline in position 9, but also has a leucine in position 5, instead of isoleucine. Since its affinity for ACA is nearly as good as that for peptide P4, the phage library screening must have selected for a non-beta-branched amino acid in this position to compensate for the adverse effects of the arginine-9 to proline-9 substitution. The solution structure of a modified version of the antibody-selected phage peptide P4 with the central proline was determined. This peptide has one turn comprised of Ala-Pro-Asp-Arg, with the proline peptide bond in the cis configuration, and another turn that contains the disulfide and adjacent residues. If the disulfide is replaced by a thioether, and the central proline by an alpha-methyl proline, in an attempt to make the peptide more biologically stable, there is little adverse effect on affinity for ACA. The thioether bond/turn is fairly well defined with a Calpha to Calpha separation of 4.9 +/- 0.8 A. The alpha-methyl proline adopts the trans configuration, and this central Ala-(alpha-methyl-Pro)-Asp-Arg turn adopts a distorted type I turn conformation with a probable i to i+3 hydrogen bond. Modeling studies suggest that the proline peptide bond configuration switched from cis to trans in the presence of the alpha-methyl group on proline because of steric hindrance with the beta-carbon of the preceding residue. Overall, this peptidomimetic molecule is structurally very similar to the peptide with natural amino acids, with an rmsd difference of only 1.37 A, when comparing backbone atoms.

[Synthesis and biological properties of artificial antigens based on 280-289 fragment of beta2-gpI]. / Sintez i biologicheskie svoistva iskusstvennykh antigenov na osnove peptida, sootvetstvuiushchego posledovatel'nosti 280-289 belka beta2-gpI.

A series of artificial antigens were synthesized on the basis of the FC(Acm)KNKEKKC(Acm)S peptide from the beta 2-glycoprotein I sequence: lipophilic analogues, the peptide-BSA conjugate, and multiple antigen peptide (MAP) containing eight copies of the peptide on an oligolysyl core. The solid phase method for acylation of the peptide with fatty acids and the HPLC analysis of the acylpeptides were described. Antigenic properties of the resulting compounds were evaluated by CL-ELISA.

Oxidized low-density lipoprotein (Ox-LDL) but not LDL aggravates the manifestations of experimental antiphospholipid syndrome (APS).

Ox-LDL is thought to play a major role in atherogenesis. The mechanisms mediating the deleterious influences of Ox-LDL include foam cell formation and cell cytotoxicity. The production of anti-Ox-LDL antibodies results in the formation of immune complexes which are taken up at enhanced rate by macrophages, leading to foam cell formation. APS is characterized by repeated venous and arterial thromboembolic phenomena, recurrent fetal loss and thrombocytopenia, associated with the presence of antibodies to negatively charged phospholipids (aPL) (i.e. cardiolipin, phosphatidylserine). Phospholipids bear structural resemblance to LDL, and several studies have indeed proved that aPL display cross-reactivity with anti-Ox-LDL antibodies. In this study we assessed the capacity of oxidized and native forms of LDL to aggravate the clinical picture of experimentally induced APS in naive mice. Mice were actively immunized intradermally with anticardiolipin antibodies and developed a clinical picture resembling APS in humans. Subsequently, the mice were infused with either Ox-LDL, native LDL or PBS, and similar regimens were applied to controls. APS mice infused with Ox-LDL were found to exhibit a significantly more severe form of the disease in comparison with native LDL- and PBS-infused mice, expressed by lower platelet counts (261,000/mm3, 535,000/mm3 and 455,000/mm3, respectively), longer activated partial thromboplastin time (aPTT) (99 +/- 12 s, 63 +/- 8 s and 74 +/- 8 s, respectively) and higher fetal resorption rates (72.7%, 34.4% and 32.6%, respectively). The results of this study show that Ox-LDL, compared with native LDL, aggravates the clinical manifestations of experimental APS and suggest that cross-reactivity of Ox-LDL with phospholipids may provide a pathogenic explanation for this effect.

Characterization of estrogen-induced autoantibodies to cardiolipin in non-autoimmune mice.

Antibodies to cardiolipin, in humans, have been associated with a variety of autoimmune disorders including anti-phospholipid syndrome, systemic lupus erythematosus and Sjögren's syndrome. These antibodies have also been demonstrated in autoimmune-prone MRL-Mp-lpr/lpr (MRL/lpr), BXSB-Mp-(+yaa) (BXSB) and (NZW x BXSB)F1 mice. In previous work, we had shown that gonadectomized or intact male and female non-autoimmune C57BL/6 mice, upon treatment with estrogen, express autoantibodies to cardiolipin. In this study, we extend these findings and show that the expression of these antibodies persists for months even after the exposure to exogenous estrogen has been terminated. These antibodies are of IgM and IgG, but not IgA, isotypes, and the predominant IgG subisotype is IgG2b. Estrogen-induced antibodies to cardiolipin only minimally cross-reacted with DNA, actin or ovalbumin. The binding of antibodies to cardiolipin from autoimmune human patients in general has been shown to depend upon the presence of a cofactor, beta2-glycoprotein I. We found that in estrogen-treated C57BL/6 mice, as well as in SLE-prone MRL/lpr and BXSB mice, the binding of anti-cardiolipin antibodies to cardiolipin was not enhanced, but rather reduced, in the presence of human beta2-glycoprotein I. Further, addition of exogenous human beta2-glycoprotein I to purified immunoglobulin fractions containing anti-cardiolipin antibodies reduces, rather than enhances, the binding to cardiolipin. Together, these data show that persistent detectable levels of IgG and IgM autoantibodies specific for cardiolipin can be induced in normal mice by estrogen treatment alone (i.e. without administration of autoantigens). Further, we characterize these antibodies regarding their kinetics, cross-reactivity, isotype distribution and cofactor (beta2-glycoprotein I) requirements.

Anti-beta 2-glycoprotein I antibody: specificity and clinical significance.

Cardiolipin binding of IgG-class anticardiolipin antibody (aCL) depends on the existence of beta 2-glycoprotein I (beta 2-GPI). We developed an EIA system that enables detection of antibodies against beta 2-GPI, without the presence of cardiolipin. This system involves use of irradiated polystyrene plates, in which oxygen atoms are introduced onto the surfaces of the plates. beta 2-GPI bound to the surface of these plates is assumed to undergo a conformational change that exposes normally cryptic epitopes. Anti-beta 2-GPI antibody measured using this EIA system showed good correlation with aCL measured by conventional EIA methods and may prove useful in evaluating the risk of thrombosis and monitoring the clinical course in patients with SLE. Utilizing this EIA system and beta 2-GPI-deleted mutants, we found that the fourth domain of beta 2-GPI is involved in expression of one of the cryptic epitopes recognized by aCL. We also found that oxidized LDL are sequentially targeted by beta 2-GPI and aCL.