Intrauterine fetal death in mice caused by cytomegalovirus-derived peptide induced aPL antibodies.

Immunization of mice with beta2 glycoprotein I (beta2GPI) and also with GDKV, a synthetic peptide representing the phospholipid (PL)-binding site of beta2GPI, induced pathogenic aPL antibodies that bind and activate endothelial cells, enhanced thrombus formation and caused fetal death in pregnant mice. TIFI is a PL-binding peptide spanning the Thr101-Thr120 of ulb0-hcmva from human cytomegalovirus (CMV), which shares structural similarity with the PL-binding site of beta2GPI. Immunization with this peptide induced pathogenic aPL and anti-beta2GPI antibodies in mice. These antibodies activated endothelial cells and enhanced thrombus formation in vivo, but whether these antibodies cause fetal death in mice is not known. The objective of this study was to examine the effects of these antibodies on pregnancy outcome in mice. Two groups of pregnant BALB/c mice were injected with either hybridoma supernatant containing D3/AC10, a CMV-peptide-induced monoclonal aPL, at days four, eight and 12 of the pregnancy, 100 microg per mouse (study group) or with culture media alone (control group). The litter size was significantly smaller in the study group (4.80 +/- 1.15 versus 7.28 +/- 0.18, t = - 2.526, P < 0.03). In conclusion, aPL induced by CMV peptides may have pathogenic properties similar to human autoimmune aPL. These findings further support the hypothesis that at least in some patients with APS, pathogenic aPL antibodies may be generated by immunization with CMV products during incidental exposure to the virus via a molecular mimicry mechanism.

The molecular basis of antiphospholipid syndrome.

We herein review evidence that the phospholipid-binding protein beta2 glycoprotein-1 (beta2GPI) is a causative autoantigen in APS. Recent work suggests that the molecular regions in beta2GPI that facilitate autoimmunization are those that promote binding to negatively charged phospholipids by means of strong positive (anionic) charge and hydrophobicity. Although many common infections can cause antiphospholipid antibodies to be produced in humans, such postinfectious aPL are rarely associated with thromboses or pregnancy morbidity, the central features of antiphospholipid syndrome (APS). We propose that the causes of APS include those infectious agents that mimic the above molecular domains in beta2GPI. In people who are susceptible to APS, tolerance to self-beta2GPI and phospholipids is likely to be broken by foreign bacterial or viral proteins that contain such beta2GPI-like epitopes.

Origin of antiphospholipid antibodies.

Our observations and those from others give further support to our hypothesis that "autoimmune aPL" may be generated by immunization with products from bacteria or viruses after incidental exposure or infection. We also were able to generate an APS-like syndrome in a strain of mice susceptible to autoimmunity, indicating that other factors such as genetic factors are likely to be involved in development of APS. Furthermore, not all aPL generated by immunization with bacterial or viral products were pathogenic. Based on the clinical experience and on the numerous reports indicating the presence of aPL in large number of infectious diseases, it may be expected that not all aPL produced during infection are pathogenic. We hypothesize that a limited number aPL induced by certain viral or bacterial products would be pathogenic in certain groups of predisposed individuals. Identification of those bacterial or viral agents may help to find strategies for the prevention of production of "pathogenic" aPL. Alternatively, free peptides may be used to induce tolerance against aPL production.

International classification criteria for antiphospholipid syndrome: synopsis of a post-conference workshop held at the Ninth International (Tours) aPL Symposium.

An international workshop on classification criteria for antiphospholipid syndrome (APS) was held on 16 September 2000 in Tours, France, following the Ninth International APS Symposium. The workshop addressed issues that were not resolved by the previous (1998) international workshop on the classification criteria. Participants at the workshop agreed that no changes should be made to the international (Sapporo) criteria at the present time. However, to improve the criteria, future efforts should be focused on the following: (1) further evaluation of the international (Sapporo) criteria for definite APS; (2) definition of other categories of APS such as 'probable' and 'possible' APS; (3) guidelines for the clinical diagnosis as distinct from classification of APS; (4) strategies to improve the compliance of laboratories worldwide, with recommended procedures for Lupus anticoagulant (LA) and anticardiolipin (aCL) assays; (5) development of monoclonal antibody standard reagents for aCL and LA assays; and (6) refinement and subsequent evaluation of antibeta2GPI assays for use in idenfitication of APS.

Mechanisms of pregnancy loss in antiphospholipid syndrome.

Antiphospholipid antibodies are associated with intrauterine fetal growth retardation and fetal distress leading to premature birth or fetal death. These complications are caused by uteroplacental insufficiency that is the result of multiple placental thromboses, infarcts, and spiral artery vasculopathy, which are almost certainly provoked by the hypercoagulable state induced by aPL antibodies. Available data indicate that the thrombogenic function of aPL antibodies involves their general effect on platelets, endothelial cells, anticoagulant mechanisms, and fibrinolytic pathways, as well as their local effect on trophoblasts and villi cells, leading to reduction of annexin V (placental anticoagulant protein-I) production and inhibition of its anticoagulant function.

Testing for antiphospholipid antibodies: problems and solutions.

The first aCL test was developed in 1983 and subsequently standardized. Although new and more specific tests have become available, the aCL ELISA and the LA tests are still the first choice to be used in diagnosis of APS. Newer tests such as the anti beta 2 GP1 ELISA and the APhL ELISA Kit (Louisville APL Diagnostics) use somewhat different antigens and likely provide a more specific (and possibly more reliable) diagnosis of APS while retaining good-to-excellent sensitivity. Other tests, such as ELISA for prothrombin antibodies and annexin V antibodies, are still undergoing development and will require standardization and extensive evaluation. We thank Dr Isabel Abreu and Dr Mittermeyer B. Santiago for performing some of the studies reported in this review.

New developments in viral peptides and APL induction.

The associations of antiphospholipid antibodies (aPL) with thrombosis and fetal death are well recognized, but the mechanism(s) that induce their production are not. We demonstrated induction of pathogenic aPL antibodies by immunization with foreign beta(2)-GPI, or synthetic peptides representing the PL-binding site of the beta(2)-GPI. These antibodies caused intrauterine fetal death and transverse myelopathy due to spinal cord infarction in mice, and activated endothelial cells in vitro. We also introduced aPL in mice by immunization with PL-binding viral peptides and observed their pathogenic effects. This study demonstrated that pathogenic effects of aPL antibodies induced by immunization with a human CMV-derived PL-binding synthetic peptide. We hypothesize that in APS patients aPL is induced by beta(2)-GPL-like PL-binding products of human common bacteria or viruses.

Hughes syndrome associated with cytomegalovirus infection.

We report the case of a patient with an acute cytomegalovirus (CMV) infection who developed Hughes syndrome, manifested by a common iliac vein thrombosis. IgM anticardiolipin antibodies (aCL) appeared with the onset of the infection, followed later by IgG aCL. Five months later, both IgM and IgG aCL levels disappeared from the serum. This is the second case of Hughes syndrome associated with CMV infection to be reported in the literature.

Induction of antiphospholipid antibodies by immunization with synthetic viral and bacterial peptides.

We previously induced pathogenic antibodies against anionic phospholipids (PL) in experimental animals by immunization with lipid-free purified human beta2glycoprotein I (beta2GPI). We hypothesized that antiphospholipid antibodies (aPL) are induced by in vivo binding of foreign beta2GPI to self-PL, thus forming an immunogenic complex against which aPL antibodies are produced. If this hypothesis is true, other PL-binding proteins that are products of ubiquitous viral/bacterial agents may also induce aPL. To test this hypothesis, groups of NIH/Swiss mice were immunized with synthetic peptides of viral and bacterial origin that share structural similarity with the putative PL-binding region of beta2GPI. Compared with the control groups, animals immunized with the peptides produced significantly higher levels of aPL and anti-beta2GPI antibodies. These findings demonstrate that some PL-binding viral and bacterial proteins function like beta2GPI in inducing aPL and anti-beta2GPI production, and are consistent with a role for such viral and bacterial proteins in inducing aPL antibody production in humans.

GDKV-induced antiphospholipid antibodies enhance thrombosis and activate endothelial cells in vivo and in vitro.

Antiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). beta2-Glycoprotein I (beta2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with beta2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of beta2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274-Cys288 in the fifth domain of human beta2GPI, which represents the phospholipid-binding site of beta2GPI. The Abs generated had aPL and anti-beta2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of beta2GPI are thrombogenic and activate endothelial cells.

A role for the polymorphism at position 247 of the beta2-glycoprotein I gene in the generation of anti-beta2-glycoprotein I antibodies in the antiphospholipid syndrome.

OBJECTIVE: To determine the frequencies at which either a valine or leucine occurs at position 247 in the beta2-glycoprotein I (beta2GPI) gene of normal individuals of the Caucasian, African American, and Asian ethnic groups and to compare these data with those in patients with the antiphospholipid syndrome (APS), with and without anti-beta2GPI antibodies. METHODS: The DNA segment containing the position-247 polymorphism was amplified by seminested polymerase chain reaction, and the polymorphism was detected by restriction endonuclease digestion. DNA samples from 370 healthy controls of different racial backgrounds were analyzed, and the results were compared with those from 149 APS patients (66 primary; 83 secondary). Allele and genotype frequencies were compared using Fisher's exact test. When significant differences were detected, pairwise comparisons were made using Fisher's exact test with a Bonferroni adjustment. RESULTS: Allele and genotype expression was significantly different (P < 0.0001 for both) among the 3 races, with the V allele and the VV genotype occurring most often among Caucasians, less among African Americans, and least among Asians. Conversely, the V allele and the VV genotype were found more frequently among Asian APS patients than among controls (P = 0.0028 and P = 0.0023, respectively). No significant differences in allele or genotype frequencies were seen in comparisons of the Caucasian or the African American patients with appropriate controls. The differences in allele and genotype frequencies seen in the Asian APS patients were restricted to the anti-beta2GPI-positive patients (P = 0.0018 and P = 0.0005, respectively). CONCLUSION: In Asian patients with APS, expression of a V at position 247, especially in the homozygous state, is significantly associated with the presence of anti-beta2GPI antibodies and, therefore, can be viewed as a major risk factor in this ethnic group (odds ratio 9.19 and 16.33, respectively).

IgA anticardiolipin and anti-beta2-glycoprotein I are the most prevalent isotypes in African American patients with systemic lupus erythematosus.

BACKGROUND: Ethnicity plays a role in the prevalence, isotype distribution, and clinical significance of anticardiolipin (aCL) and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies in patients with systemic lupus erythematosus (SLE). Few studies have been done in the African American population. METHODS: Serum samples from 100 African American patients with SLE were tested for IgG, IgM, and IgA aCL and anti-beta2-GPI antibodies by enzyme-linked immunosorbent assay (ELISA). Computerized clinical data on these patients were reviewed with a specific focus on clinical manifestations of antiphospholipid syndrome (APS). RESULTS: Positivity for at least one isotype of aCL antibodies was found in 33% of the patients, whereas 28% were positive for at least one isotype of anti-beta2-GPI antibodies. IgA was the most prevalent isotype for both antibodies; 24% of the patients in the aCL ELISA and 19% in the anti-beta2-GPI ELISA were positive for IgA. Positivity for both aCL and anti-beta2-GPI in the same patient was seen more frequently with the IgA isotype. Fewer than half of the patients positive for aCL antibodies had medium-to-high levels of antibodies. A few patients had presented thrombotic manifestations, and these patients were positive for aCL (P = 0.01) and anti-beta2-GPI antibodies (P = 0.02). No other manifestations of APS could be significantly correlated with the presence of these antibodies. CONCLUSIONS: Our results show that IgA is the most prevalent isotype among the African American patients with SLE studied. The predominance of the IgA isotype and the low prevalence of medium-to-high levels of aCL antibodies may account for the low frequency of clinical manifestations of APS in these patients.

Antiphospholipid (Hughes') syndrome in African-Americans: IgA aCL and abeta2 glycoprotein-I is the most frequent isotype.

Antiphospholipid (Hughes') syndrome (APS) has not been reported in African-Americans (A-A) as frequently as in other ethnic groups. We describe eight A-A female patients with APS, including two cases of primary APS (PAPS), four with APS secondary to systemic lupus erythematosus (SLE), one with Sjögren's syndrome, and one with overlap connective tissue disease (CTD). Their mean age was 34 y (range 24-47 y). Patients were followed for a mean of 6 y (range 0.3-11 y). During follow up, both anticardiolipin (aCL) and anti-beta2glycoprotein-I (abeta2GPI) antibodies were measured in stored sera by enzyme-linked immunosorbent assay (ELISA). IgA was the most frequent isotype of aCL and abeta2GPI, and co-occurred with the IgM isotype in three of four patients with neurologic manifestations.

Antibodies to cardiolipin and beta2-glycoprotein-1 in HTLV-1-associated myelopathy/tropical spastic paraparesis.

Anticardiolipin and anti-beta2GP1 antibodies were measured in 50 patients with HTLV-1-associated Myelopathy-Tropical Spastic Paraparesis (HAM-TSP) and the results were compared with those obtained for 34 HTLV-1-positive and 35 HTLV-1-negative controls, as well as 128 SLE patients. aCL but not anti-beta2GP1 was associated with HTLV-I infection. aCL was more prevalent than anti-beta2GP1 (32% vs. 8%) and was not associated with anti-beta2GP1 in HAM-TSP. IgA was the dominant isotype of aCL and anti-beta2GP1. The data suggest that tin HAM-TSP, IgA aCL are frequent and are associated with HTLV-1 infection.

Anticardiolipin and anti-beta2glycoprotein-I antibodies in patients with systemic lupus erythematosus: comparison between Colombians and Spaniards.

We studied the prevalence, isotype distribution, and clinical significance of anticardiolipin (aCL) and anti-beta2glycoprotein I (anti-beta2GPI) antibodies in two populations of patients with systemic lupus erythematosus (SLE), 160 Colombians and 160 Spaniards. All sera were tested in our laboratory by enzyme-linked immunosorbent assay (ELISA) for IgG, IgM, and IgA aCL, as well as IgG and IgM anti-beta2GPI. Positive results for at least 1 of the 3 aCL isotypes were found in 40 Colombians (25%) and 55 Spaniards (34%). IgG aCL was the predominant isotype in both populations. Positive results for at least 1 of the anti-beta2GPI isotypes were found in 34 Colombians (21%) and 29 Spaniards (18%). IgG anti-beta2GPI was the dominant isotype in Colombians, while IgM was predominant in Spaniards. Positivity for anti-beta2GPI in aCL-positive patients was present in 77% in the Colombian group and 50% in the Spaniard group. Among Colombians, IgG aCL and anti-beta2GPI correlated with thrombosis, fetal loss, and thrombocytopenia. Among Spaniards, IgG aCL and IgG anti-beta2GPI correlated with thrombosis, fetal loss, and livedo reticularis. For detecting thrombosis and fetal loss, aCL ELISA was more sensitive than anti-beta2GPI in Spaniards, and anti-beta2GPI ELISA was more specific than aCL in both populations.