Neal S. Rote

Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation☆☆☆★★★

OBJECTIVE Trophoblast differentiation is associated with externalization of phosphatidylserine from the inner to the outer surface of the plasma membrane. In this study we tested the hypothesis that concurrent externalization and binding of annexin-V blocks the phosphatidylserine-rich surface from acting as a site for activation of coagulation and that antiphospholipid antibodies lead to a procoagulant state by preventing annexin-V binding. STUDY DESIGN A choriocarcinoma model of trophoblast differentiation, forskolin-activated BeWo cells and immunoperoxidase techniques were used to determine surface and cytoplasmic localization of annexin-V related to differentiation. Monoclonal immunoglobulin M antibodies against phosphatidylserine- and cardiolipin-dependent antigens were used to determine the effects of antiphospholipid antibodies on annexin-V localization and on the binding of prothrombin to the BeWo surface. RESULTS During differentiation BeWo cells externalized phosphatidylserine and increased the expression of surface annexin-V. Monoclonal antibody against phosphatidylserine removed annexin-V from the BeWo surface and increased binding of prothrombin. CONCLUSION Antiphosphatidylserine antibody induces sites for prothrombin binding on the surface of a BeWo model of trophoblast, most likely by removing annexin-V. This mechanism could explain the frequent observation of increased thrombosis at the maternal-fetal interface in miscarriages associated with antiphospholipid antibodies.

Association of lupus anticoagulant with antibody against phosphatidylserine

Lupus anticoagulant (LAC) is an antiphospholipid autoantibody identified by prolongation of in vitro phospholipid-dependent coagulation tests. Its presence is associated with thromboembolic disease and recurrent pregnancy loss in patients with or without clinical autoimmune disease. The purpose of this study was to identify the specific phospholipid(s) against which LAC is directed. The sera of 15 patients with LAC and of 41 LAC-negative controls were evaluated. Specific phospholipids were used to inhibit IgG binding in a partial thromboplastin ELISA, and serologic reactivity was measured with ELISAs which used specific phospholipids in the solid phase. Both phosphatidylserine and cardiolipin significantly inhibited IgG binding in the partial thromboplastin ELISA (76 and 75%, respectively); however, the serum of one patient who was strongly positive for LAC in coagulation assays was not inhibited by cardiolipin. In the specific phospholipid ELISAs, LAC-positive sera contained IgG (15 of 15 sera) and frequently IgM (9 of 15 sera; 60%) to phosphatidylserine. Most LAC-positive sera also contained IgG antibodies against other phospholipids: cardiolipin (10 of 11 sera; 91%), phosphatidylcholine (1 of 15 sera; 7%), phosphatidylethanolamine (12 of 15 sera; 80%), phosphatidylglycerol (12 of 15 sera; 80%), and phosphatidylinositol (10 of 15 sera; 67%). None of the LAC-negative controls had measurable IgG against any of these phospholipids. We conclude that LAC-positive sera contain antibody specificities against multiple phospholipids; however, anticoagulant activity is always associated with the presence of antibodies against phosphatidylserine.

Histocompatibility in couples with recurrent spontaneous abortion and normal fertility

Histocompatibility between husband and wife at the HLA locus has been suggested as a determinant of recurrent spontaneous abortion. We measured the incidence of HLA antigen sharing within 12 couples with histories of unexplained recurrent abortion and in a fertile control population of 77 couples. In the recurrent abortion group, 6 of 12 (50%) of the couples shared no HLA antigens, whereas only 3 of 12 (25%) shared one antigen, 1 of 12 (8.3%) shared two antigens, and 2 of 12 (17%) shared three antigens. In the fertile group, 27 of 77 (35.1%) shared no antigens, 33 of 77 (42.8%) shared one antigen, 14 of 77 (18.2%) shared two antigens, 2 of 77 (2.6%) shared three antigens, and 1 of 77 (1.3%) shared four antigens. In 50 of these control couples who were available for complete reproductive histories, there were no significant correlations between the incidence of antigen sharing and the numbers of offspring, the incidence of spontaneous abortion, or infertility problems. Six of the women in the recurrent abortion group became pregnant during the study. Three of these (50%) delivered live infants independent of the degree of antigen sharing and without the benefit of immunologic treatment. Therefore, the degree of HLA antigen sharing did not define a population with increased pregnancy wastage or predict subsequent pregnancy outcome.

A model for the antiphospholipid antibody syndrome: Monoclonal antiphosphatidylserine antibody induces intrauterine growth restriction in mice

OBJECTIVE Antiphospholipid antibodies are associated with clinical intrauterine growth restriction. In this study we investigated whether immunoglobulin M monoclonal antibodies against phosphatidylserine or cardiolipin or cross-reactive with both phospholipids would induce intrauterine growth restriction in an experimental model of the antiphospholipid antibody syndrome. STUDY DESIGN Balb/c or CD-1 mice were injected intraperitoneally on day 8 of pregnancy with three immunoglobulin M monoclonal antibodies that differentiated between cardiolipin- and phosphatidylserine-dependent antigens or with control immunoglobulin M monoclonal antibodies against irrelevant antigens. The animals were killed on day 15 of pregnancy and placental and fetal weights were measured. RESULTS Monoclonal antibody 3SB9b, which reacted in enzyme-linked immunosorbent assays with phosphatidylserine but not cardiolipin, induced a significant reduction in both fetal and placental weights. Monoclonal antibodies BA3B5C4, which was cross-reactive with cardiolipin and phosphatidylserine, and D11A4, which reacted with cardiolipin, did not alter fetoplacental weights. CONCLUSION An antiphospholipid antibody that reacts with phosphatidylserine induces significant fetal and placental intrauterine growth restriction in a mouse model for the antiphospholipid antibody syndrome, but those that react with cardiolipin do not.

Protective efficacy of a modified immune serum globulin in experimental group B streptococcal infection

In spite of aggressive antimicrobial therapy and extensive support measures, the mortality rate in early-onset group B streptococcal infection continues to be exceedingly high. In previous studies, we have demonstrated that passive immunotherapy with fresh whole blood containing opsonic antibody-improved survival in human neonates with group B disease. Transfusion of whole blood, plasma, or other blood products has a number of drawbacks, however. In the present study, we have evaluated immune serum globulin and a preparation of ISG modified for intravenous use for levels of type-specific antibody, opsonic activity, and protective efficacy against type Ia, II, and III group B streptococci. Type-specific antibody was detected in most of the preparations tested. In general, the level in MISG was less than that in the comparison ISG lot. Opsonic activity was also detected in these preparations against the more antibody-sensitive group B strains but was not present for opsonin resistant strains of type Ia, II, and III. Both ISG and MISG provided protection in neonatal rats infected with group B streptococci; in most cases MISG was more efficacious than the ISG from which it was made. These studies suggest that passive immunotherapy with MISG may be a valuable adjunct to current regimens used in the management of early-onset group B disease. This would be especially so if donors could be selected whose serum or plasma contained high levels of opsonic and protective activity against both antibody-sensitive and antibody-resistant group B strains.

The demonstration of lupus anticoagulant by an enzyme-linked immunoadsorbent assay

Lupus anticoagulant, an autoantibody associated with thromboembolic disease and pregnancy loss, is currently identified by its capability to prolong phospholipid-dependent coagulation tests, such as the activated partial thromboplastin time (APTT). The use of a coagulation assay for the detection of an antibody has several inherent disadvantages: fresh plasma is required for accurate testing, prolongation of coagulation test(s) is not specific for lupus anticoagulant, and coagulation assays are not easily manipulated for the characterization of antigen-antibody interactions. Using partial thromboplastin an the antigen, we have developed an enzyme-linked immunoadsorbent assay (ELISA) for the detection of lupus anticoagulant. Fifteen women with lupus anticoagulant (being evaluated for recurrent pregnancy loss or autoimmune disease) and 40 lupus anticoagulant-negative controls (including 20 with recurrent pregnancy loss, 12 parous women, and 8 with known autoimmune disease) were evaluated using the APTT and ELISA assays for lupus anticoagulant. All patients with lupus anticoagulant, as defined by the APTT, had significantly elevated IgG (sensitivity and specificity, 100%) or IgG + IgM (sensitivity 93%, specificity 95%) levels compared to controls.

American Society for Reproductive Immunology Report of the Committee for Establishing Criteria for Diagnosis of Reproductive Autoimmune Syndrome

Coulum CB, Brunch O W , Clark DA, Gleicher N , Kuttek W, Lockslzin MD, Rote NS. Anierican Societj, , for Reproductire Imniinology Report of the Coninlittee f a r Establish ing Criteria for Diagnosis of Reproductice Autoimr?iune Slwilronie. AJRI 1999; 41:121132 0 M~inksgaurd, Copenhagen

Circulating immune complexes in pregnancy, preeclampsia, and autoimmune diseases: Evaluation of Raji cell enzyme-linked immunosorbent assay and polyethylene glycol precipitation methods

Sera from 86 individuals were tested for circulating immune complexes by the polyethylene glycol precipitation method and a Raji cell enzyme immunoassay (Raji-ELISA). These included normal nonpregnant control subjects, nonpregnant patients with autoimmune diseases, healthy women in the second and third trimesters of pregnancy, patients with preeclampsia, and women with pregnancies complicated by preexisting autoimmune diseases. Diseases such as systemic lupus erythematosus and rheumatoid arthritis were associated with increased levels of immune complexes in both pregnant and nonpregnant individuals. Circulating immune complexes were not observed in normal pregnancies or in preeclampsia. Although pregnancy itself is not an immune complex-associated state, the presence of immune complexes in autoimmune diseases may explain some of the complications observed during pregnancy in these patients.

Detection of circulating immune complexes with a Raji cell enzyme immunoassay

The Raji cell assay for circulating immune complexes (CIC) is frequently the method of choice when the detection of large, complement fixing complexes is desired. We have developed an enzyme-linked immunosorbent assay modification (Raji-ELISA) of the Raji cell technique which is easy to perform, uses commercially available reagents and is more convenient than the conventional Raji cell radioimmune assay (Raji-RIA). Fourteen samples were tested by both assays and a good correlation was observed (P = 0.05). Sera from patients with suspected immune complex associated diseases were tested in the Raji-ELISA. Unsensitized Raji cells gave a value of 1.25 micrograms of immune complex associated IgG/ml. Using 2 standard deviations as a cutoff to determine positivity, 2 of 32 healthy controls (6.2%) had elevated levels of circulating immune complexes. In our study population, 9 of 23 cancer patients (39.1%), 10 of 13 patients with autoimmune diseases (76.9%), 3 of 17 patients with positive rheumatoid factor titers (17.6%), 1 of 23 pregnant patients (4.3%), 1 of 5 preeclamptic patients (20%) and 9 of 30 other patients with suspected immune complex associated diseases (30%) had elevated levels of CIC.

Demonstration of Lupus Anticoagulant Antigens Using an Enzyme‐Linked Immunoadsorbent Assay (ELISA)

Lupus anticoagulant (LAC) is an autoantibody that is associated with thromboembolic disease and recurrent pregnancy loss.’ It is termed an “anticoagulant” because it prolongs phospholipid-dependent coagulation tests [such as the activated partial thromboplastin time (APTT)] by binding to the phospholipid portion of the prothrombin-prothrombinase complex.2 Carreras has demonstrated that LAC-containing plasma fractions inhibit the generation of prostacyclin by vascular tissues and has hypothesized that this is the mechanism of the associated thrombosis and pregnancy loss.’ It has been suggested that the antiphospholipid activity of LAC, which is demonstrable in coagulation assays, is also involved in the inhibition of prostacyclin generation. However, the specific phospholipid(s) against which LAC is directed is unknown. In order to evaluate LAC-antigen interactions, we have developed an ELISA for the detection of LAC4 and have used it to study the antigenic specificity of LAC by binding-inhibition assays.