Investigation of follicular helper T cells, as a novel player, in preeclampsia.

Preeclampsia (PE) is characterized by hypertension and proteinuria. It occurs in an around 3% to 5% of all pregnancies worldwide. The fetus is kind of semiallograft to the maternal host; immune system components encounter fetal antigens and develop adverse immune responses. Recently, it has been observed that the immune system plays an important role in PE. In the current study, we have tried to investigate the role of follicular helper T (Tfh) cells in the pathogenesis of PE. Blood samples of 49 PE women and 50 healthy controls were collected. Peripheral blood mononuclear cells were isolated, cells were cultured, and then RNA was extracted. Autoantibody and secretory cytokine levels were analyzed by ELISA. Tfh frequency and transcription levels of the related molecules and cytokine were assessed by flow cytometry and real-time PCR, respectively. The frequency of circulating Tfh cell in PE women was significantly higher compared with the healthy pregnant woman (Tfh cells with CD4+ ICOS + , P = 0.0064 and Tfh cells with CD4 + CXCR5 + , P = 0.029). Moreover, mRNA expression levels of CXCR5, BCL6, IL-21, and IL-6 ( P = 0.0006, P = 0.008, P = 0.0063, and P = 0.027, respectively) were upregulated in PE patients. Furthermore, IL-6 ( P = 0.0014) and IL-21 ( P = 0.0059) levels in both group were assayed and the results showed increased in patient group. We also measured autoantibody levels including antiphospholipid antibodies ( P = 0.0001), anticardiolipin antibodies ( P = 0.0004), anti-TPO ( P = 0.0008), anti-TG ( P = 0.001) in circulation of PE group, which were higher than the control group. This study provided insights into the involvement of Tfh cells in etiology and pathogenesis of PE, probably by developing autoantibodies.

The investigation of hereditary and acquired thrombophilia risk factors in the development of complications in pregnancy in Croatian women.

OBJECTIVES: To investigate the genetic and acquired thrombophilic risk factors in pregnancy-associated complications and venous thromboembolism (VTE) and evaluate the association between particular thrombophilic risk factors and thromboembolic complications. METHODS: In this study, pregnant women with pregnancy complications and VTE (N = 101) were the study group, and the control group were women with normal pregnancy (N = 102). All women underwent testing for factor V Leiden mutation (FVL), mutation of the coagulation factors II (FII20210), methylenetetrahydrofolate reductase (MTHFR), plasminogen activator inhibitor-1, antithrombin III (ATIII), protein C (PC) and protein S, lupus anticoagulant (LAC) antibodies, anticardiolipin antibodies and anti-beta-2-glycoprotein-1. RESULTS: In this study group, mutations of the FVL was 15.8% (16/101), FII20210 5.9% (6/101) and the MTHFR at locus 677 was TT in 19.8% (20/101). Deficiency of ATIII and PC were rare: 3.0% and 1.0%, respectively. LAC were significantly higher in the study group than in the control group: 32.7% versus 3.9%; p < 0.0005. Pregnant women with VTE have been more frequent for FVL (41.7%; p < 0.005), PC deficiency (25.0%; p < 0.005) and LAC (33.3%; p < 0.005). Combination of FVL and MTHFR mutation was related to the risk of recurrent fetal death and habitual abortion. CONCLUSION: The inherited and the acquired thrombophilic risk factors were found to be up to 10 times more common in the study group than in the control group.

Mutação G20210A no gene da protrombina, fator V de Leiden e anticorpos anticardiolipina não influenciam a sobrevida do enxerto renal após o transplante / Prothrombin G20210A gene mutation, factor V Leiden and anticardiolipin antibodies do not influence renal graft survival after transplantation

Introdução: Complicações tromboembólicas são importantes fatores de risco para perda do enxerto e pior evolução após o transplante renal. pacientes com defeito trombofílico apresentam maior risco de complicações tromboembólicas. Foram analisados, entre receptores de transplante renal, a prevalência de defeito trombofílico e o risco atribuído a esta condição para a perda do enxerto e para o desenvolvimento de tromboses intravasculares. Métodos: estudo do tipo coorte incluindo 388 receptores adultos analisados quanto à presença de trombofilia de acordo com a pesquisa de anticorpos anticardiolipidina (aCL) por ELISA e das mutações G1691A no gene do fator V (FV) e G20210A no gene da protrombina (PT) por PCR multiplex. Resultados: Defeito trombofílico foi identificado em 25,8% dos pacientes. As taxas de sobrevida de 2 anos do enxerto foram semelhantes entre os pacientes com e sem defeito trombofílico (94%, p=0,53), bem como a sobrevida dos enxertos livres de tromboses intravasculares (97% versus 97%, p=0,83). pacientes com defeito trombofílico apresentaram prevalência de tromboses intravasculares semelhante à do grupo-controle (3% versus 3,5%, p=0,82). O transplante renal anterior foi associado a maior risco de perda de enxerto (OR 20,8, p<0,001) e de ocorrência de trombose intravasculares (OR 6,8, p=0,008). Conclusões: As prevalências das mutações FVG1691A e PTG20210A na população estudada foram semelhantes às da população geral não transplantada, e a prevalência de anticorpos aCL superou a observada entre os indivíduos sadios. Não houve associação entre os marcadores de trombofilia estudados e a sobrevida em médio prazo do transplante renal.

Introduction: Thromboembolic complications are important risk factors for graft loss and poor outcome after renal transplantation. patients with thrombophilic defects are at increased risk of thromboembolic complications. Were analyzed, among kidney transplant recipients, the prevalence of thrombophilic defects and the risk attributed to this condition for graft loss and the development of intravascular thrombosis. Methods: A cohort study including 388 adult recipients analyzed for the presence of thrombophilia according to anticardiolipidina antibodies (aCL) by ELISA and gene mutations G1691A in factor V (FV) and prothrombin gene G20210A (PT) by multiplex PCR. Results: thrombophilic defect was identified in 25.8% of patients. The survival rates of two years of the graft were similar between patients with and without thrombophilic defect (94%, p = 0.53), and the survival of free grafts of intravascular thrombosis (97% versus 97%, p = 0 , 83). patients with an increased prevalence of thrombophilic defect intravascular thrombosis similar to the control group (3% versus 3.5%, p = 0.82). Previous renal transplantation was associated with increased risk of graft loss (OR 20.8, p <0.001) and intravascular thrombosis (OR 6.8, p = 0.008). Conclusions: The prevalence of mutations and FVG1691A PTG20210A in this study were similar to those of the general population not transplanted, and the prevalence of aCL antibodies exceeded that observed among healthy individuals. There was no association between markers of thrombophilia studied and medium-term survival in renal transplantation.

Beneficial effects of treatment with transglutaminase inhibitor cystamine on macrophage response in NZB/W F1 mice.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder of unknown etiology. However, the definitive mechanisms remain obscure. Recently, transglutaminase 2 (TG2) was implicated in the pathogenesis of SLE. Cystamine, which inactivates TG2 activity by forming a mixed disulfide, may interfere with and inhibit other thiol-dependent enzymes such as caspases. To investigate the effects of cystamine in SLE pathogenesis, this in vivo study assessed the serum and macrophage response after administration of cystamine to NZB/W F(1) mice. The experimental results demonstrated for the first time a significant reduction in TG2 and matrix metalloproteinase (MMP)-9 activity; tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, TG2, tumor necrosis factor alpha, and tumor growth factor beta mRNA expression; and anticardiolipin autoantibodies (aCL) in NZB/W F(1) mice following cystamine administration. It strongly suggests the therapeutic potential of cystamine in SLE.

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.

Development of consensus guidelines for anticardiolipin and lupus anticoagulant testing.

Interlaboratory and intermethod variation in commercial and in-house tests used for the measurement of anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) limit the diagnostic value of the results from these tests. This short review summarizes published and unpublished guidelines (some developed using consensus procedures) on aCL and LA testing that are aimed at decreasing assay variation.

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin.

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.

Single nucleotide polymorphisms in the coding region of the apolipoprotein H (beta2-glycoprotein I) gene and their correlation with the protein polymorphism, anti-beta2glycoprotein I antibodies and cardiolipin binding: description of novel haplotypes and their evolution.

Apolipoprotein H (APOH), also known as beta2-glycoprotein I, is a major autoantigen for the production of antiphospholipid antibodies (APA) in autoimmune diseases. APA is also recognized by a cryptic epitope generated following the interaction of APOH with anionic phospholipids (PL). The prevalence of APA in the general U.S. white population is about 10%, but it ranges from 30-70% in patients with lupus and antiphospholipid syndrome. Since the structural characterization of APOH from different mammalian species is important to identify the evolutionary conserved regions that may be critical for its function, we have previously determined the chimpanzee APOH gene structure and the prevalence of APA. There are only two amino acid differences between the chimpanzee and human wild type APOH proteins. Chimpanzees have an unusually high prevalence (64%) of APA. There is a common protein polymorphism in the human APOH gene, with the occurrence of four alleles APOH*1, APOH*2, APOH*3 and APOH*4, the latter being present only in blacks. Based on its differential reactivity with an APOH monoclonal antibody, the APOH*3 allele is further divided into APOH*3(W) (present only in whites) and APOH*3(B) (present only in blacks). In this study we have screened a large African population (n = 755) to determine the prevalence of APA and the molecular basis of the protein polymorphism. Almost 50% of the Africans were found to be positive for APA. The APOH*3(B) allele was found to be identical to the chimpanzee's wild type APOH. Novel two-site or three-site haplotypes, encoded in the third domain of APOH, explained the molecular basis of the APOH*3(B), APOH*3(W) and APOH*4 alleles. Based on the comparison of the human and chimpanzee APOH DNA sequences, we suggest that the APOH*3(W) and APOH*4 alleles arose on the ancestral APOH*3(B) haplotype after the split of human races. We also found that these haplotypes are associated with the occurrence of APA. Recombinant APOH haplotypes, expressed in COS-1 cells, showed that these mutations also affect the binding of APOH to anionic PL.

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.

Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals.

Antiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL.

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.

[Cerebral venous thrombosis and familial prothrombin gene G20210a mutation]. / Trombosis venosa cerebral y mutación G20210A del gen de la protrombina familiar.

INTRODUCTION: The prothrombin gene variant G20210A was first described as a risk factor for deep vein thrombosis, and recently for cerebral venous thrombosis, although reported cases had other concomitant risk factors. CLINICAL CASES: A 33 years old woman, with no previous vascular nor thrombotic risk factors, was admitted with thrombosis of superior longitudinal, lateral and sigmoid right sinus. The father had deep venous thrombosis 3 years before. One year later, the 29 year old sister of the proband, developed massive deep venous thrombosis, when she was 8 months pregnant. Laboratory investigations showed elevated anticardiolipin antibodies titer in the proband. Prothrombin activity was in the normal range in the 3 patients. Prothrombin gene mutation G 20210A was detected in the 3 patients. CONCLUSION: As the presence of more than one thrombophilic factor (in the reported case, prothrombin G20210A mutation and anticardiolipin antibodies) increases the likehood of a thrombotic event, it is useful to screen for thrombotic genetic conditions, even when other vascular risks are present, and vice versa.

Chimpanzee apolipoprotein H (beta2-glycoprotein I): report on the gene structure, a common polymorphism, and a high prevalence of antiphospholipid antibodies.

Apolipoprotein H (apoH, protein; APOH, gene) is a 50-kDa glycoprotein that binds to negatively charged substrates, including phospholipids. ApoH is a main target antigen for the binding of antiphospholipid antibodies that are associated with thrombotic events. We have previously characterized the structural organization of the human APOH gene. Because of the significant structural homology between the human and chimpanzee genomes, we have employed oligonucleotides from the human APOH gene sequence to amplify chimpanzee DNA covering the entire transcribed region together with flanking sequence in the 5' region. As in humans, the chimpanzee APOH gene consists of eight exons and seven introns and encodes for a 326-amino-acid protein. The deduced amino acid and nucleotide sequence show 99.4% and 99.6% similarity between human and chimpanzee APOH, respectively. Using isoelectric focusing (IEF) and immunoblotting, we screened 155 chimpanzees (128 unrelated captured parents and 27 captive-born offspring) for the apoH protein polymorphism. The most common IEF pattern in chimpanzees was identical to a previously described APOH*3 allele in humans. In addition, an anodally shifted pattern was observed in chimpanzees with an allele frequency of 0.168, and the corresponding allele was designated as APOH*4. DNA sequencing of APOH*4 carriers revealed a missense mutation in exon 6 (A-->G) at codon 210, which replaces the amino acid lysine by glutamic acid. This mutation does not affect the binding of apoH to cardiolipin as revealed by cardiolipin/enzyme-linked immunosorbent assay (ELISA). We also evaluated the prevalence of anti-apoH antibodies in chimpanzee plasma by using human-apoH-based ELISA and the association of the Lys210Glu mutation with the occurrence of anti-apoH antibodies. The prevalence of anti-apoH antibodies in chimpanzees (64%) was found to be unusually high compared with that found in humans. However, the Lys210Glu mutation showed no association with the occurrence of anti-apoH antibodies. The prevalence of anti-apoH antibodies in chimpanzees may serve as a useful animal model for the human antiphospholipid syndrome, where these antibodies are associated with clinical manifestations.

The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity.

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins. (Blood. 2001;97:3820-3828)

Overlap of the anti-cardiolipin and anti-nucleosome responses of the (NZW X BXSB)F1 mouse strain: a new pattern of cross-reactivity for lupus-related autoantibodies.

The association of anti-nuclear antigen (ANA) and anti-cardiolipin (CL) antibodies is often observed during systemic lupus erythematosus (SLE) or the primary anti-phospholipid syndrome, thereby raising the possibility of a relationship between these two autoantibody populations. To determine whether ANA and anti-CL antibodies can overlap, we derived, from a male (NZW x BXSB)F1 mouse, 14 hybridomas selected based on their capacities to react with CL and to label HEp-2 cell nuclei. Four of these anti-CL were IgG and bound to CL and phosphatidylserine in a cofactor-dependent manner and reacted strongly with nucleosomes. Variable region sequence analysis indicated that these four monoclonal antibodies (mAb) were derived from three independent B cell clones that used recurrent heavy and/or light chain immunoglobulin rearrangements, as assessed by comparison with each other and prototypic anti-CL mAb previously derived from different lupus mouse strains. These results indicate that anti-CL mAb can have overlapping cross-reactivities with nucleosomes, thereby defining a new category of SLE-related autoantibodies characterized by their capacities to recognize distinct supramolecular complexes, formed by the association of an anionic structure and a protein, that exert a strong selective pressure on autoreactive B cell clones.

Evidence that immunoglobulin specificities of AIDS-related lymphoma are not directed to HIV-related antigens.

Chronic B-cell stimulation may be a predisposing event in the early pathogenesis of the acquired immunodeficiency syndrome (AIDS)-related lymphoma (ARL). ARL-derived immunoglobulin (Ig) genes are significantly diversified from germline, suggesting that antigenic stimulation via Ig receptors may occur prior to malignant transformation. We have evaluated 6 ARL-derived antibodies for binding to human immunodeficiency virus (HIV) and cell surface epitopes. Five cases expressed IgM, and 1 case expressed IgG. Expressed V genes were significantly diversified (3%-15%) from known germline V genes. A non-Ig producing mouse myeloma cell line was transfected with expression vectors containing the lymphoma-derived V genes. By enzyme-linked immunosorbent assay and Western blot assay, the lymphoma-derived Ig's showed no reactivity against HIV recombinant proteins. Also, no specific HIV reactivity was observed by flow cytometry with lymphoma-derived Ig's against the T-cell line infected with T-tropic HIV-1 or peripheral blood mononuclear cells infected with M-tropic HIV strains, indicating lack of binding to native HIV epitopes. However, 2 of the lymphoma-derived Ig's (ARL-7 and ARL-14) bound strongly to non-HIV-infected cells of various tissue origins. Thus, these findings suggest that the transformed B cells of AIDS-associated lymphomas may not arise from the pool of anti-HIV specific B cells but, rather, may develop from B cells responding to other antigens, including self-antigens. (Blood. 2000;95:1393-1399)

Anticardiolipin antibodies in first-degree relatives of type 1, insulin-dependent, diabetic patients.

First-degree relatives (FDRs) of diabetic patients are at risk of IDDM, and frequently present several autoantibodies. We detected anticardiolipin antibodies (aCL) in 42 FDRs, aged 12.4 +/- 4.2 years and in 52 controls. aCL (IgG and IgM) were measured by ELISA and their results expressed in arbitrary units. All FDRs underwent islet cell antibodies (ICA) measurement, intravenous glucose tolerance test and HbA1c levels. HLA typing and HLA-DQ molecular analysis were performed in all FDRs. Positive levels of aCL-IgG were observed in 8/42 FDRs and no control subject (p = 0.04); aCL-IgM values were similar in FDRs and controls. No correlation was found between aCL levels and chronologic age or HbA1c levels. No association was observed between aCL frequency and immunologic (ICA), metabolic or genetic (HLA) parameters. No FDR showed any feature of antiphospholipid syndrome. aCL-IgG presence in FDRs is suggestive of a need to carry out a follow-up study to establish the significance of these antibodies.

Heterogeneity of anti-phospholipid and anti-endothelial cell antibodies.

The role of the anti-phospholipid antibodies (APLA) and anti-endothelial cell antibodies (AECA) in the pathogenesis of anti-phospholipid syndrome (APS) is unclear. Differences in the reported involvement of APLA may be due, in part, to the polyclonal nature of these antibodies and the use of serum and serum fractions for analysis. To circumvent this issue, we generated monoclonal antibodies (MAB) from three patients with APS and two healthy controls. We then compared the antigen binding patterns and the heavy chain variable region (VH) DNA sequences of the MAB derived from patients with APS to those from healthy controls. The results of this study indicate that APLA and AECA comprise a highly heterogeneous population of antibodies with respect to the antigens they recognize, as well as VH gene usage. MAB derived from patients with APS do not differ from those derived from normal individuals based on either antigen recognition or VH gene usage. These results suggest the importance of additional predisposing factors in the pathogenesis of APS.