Antiphospholipid antibodies and recurrent pregnancy loss.
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Abortion, Habitual↗
Biomedical subjects
N S Rote
Publications and source records attributed to N S Rote.
Antiphospholipid antibody binding to bilayer-coated glass microspheres.
Thrombosis, recurrent fetal loss, and thrombocytopenia are clinical manifestations associated with circulating antibodies that recognize cardiolipin (CL)- or phosphatidylserine (PS)-dependent antigens. Enzyme-linked immunosorbent assays (ELISAs) are generally used to determine the presence and specificity of antiphospholipid antibodies (aPLs). However, the presentation of the phospholipid antigen in the ELISA assay is unknown. In this study, we determined the specificity of three mouse monoclonal aPLs for phospholipid bilayer membranes. These monoclonal aPLs had been characterized by ELISA to have different specificities for CL and PS and were designated BA3B5C4 (CL+/PS+), 3SB9b (CL-/PS+), and D11A4 (CL+/PS-). Bilayers composed of 0-100% PS or CL in phosphatidylcholine (PC) were formed on the surface of 1.6 microns diameter glass microspheres to permit analysis by flow cytometry. BA3B5C4 and 3SB9b bound specifically to both PS- and CL-containing bilayers, and binding increased with increasing percentage of anionic phospholipid. The threshold for PS-dependent binding was 20 mol% PS for both BA3B5C4 and 3SB9b. For CL-dependent binding, the threshold was below 25 mol% CL for both of these antibodies. Binding to PS-containing bilayers was tested as a function of ionic strength for BA3B5C4 and 3SB9b. The ionic strength dependence of the binding suggested that the intermolecular attractive forces between anti-PS antibodies and PS-containing bilayers are predominantly multiple weak electrostatic bonds. D11A4 bound only to bilayers composed of 100% PS and 100% PC, and this antibody did not bind to CL-containing bilayers. The binding specificities of these aPLs to bilayer membranes suggest that, in this system, the conformation of the epitope involving CL, and perhaps PS, is different from that expressed in the routine clinical ELISA. Two of the monoclonal antibodies reacted in this model system at the low levels of PS typically externalized in the plasma membranes of activated platelets, apoptopic lymphocytes, and senescent red blood cells: thus, these surfaces are plausible candidates for the site of pathologically relevant antibody interactions.
Cardiolipins↗
Monoclonal antiphosphatidylserine antibody reactivity against human first-trimester placental trophoblasts.
OBJECTIVE: To investigate the binding of antibodies against negatively charged phospholipids (antiphospholipid antibodies) to human placenta, we tested the reactivity of three mouse monoclonal antiphospholipid antibodies against first-trimester human placenta. STUDY DESIGN: Formalin-fixed and frozen sections of first-trimester placentas were stained by immunoperoxidase with three mouse monoclonal antibodies. Each monoclonal antibody reacted differently with cardiolipin and phosphatidylserine, 3SB9b reacted with phosphatidylserine, D11A4 reacted with cardiolipin, and BA3B5C4 reacted with both. RESULTS: 3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin-fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 reacted minimally or, more commonly, not at all. CONCLUSION: The trophoblastic layer directly in contact with the maternal circulation is most reactive with antiphospholipid antibodies that react with phosphatidylserine rather than cardiolipin, suggesting that the trophoblasts may potentially be directly damaged by antiphospholipid antibodies through mechanisms unrelated to thrombosis. In addition, the differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving phosphatidylserine on the cytotrophoblast is altered concurrent with fusion into the syncytium.
Antibodies, Anticardiolipin↗
Expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) in amniochorionic membranes.
OBJECTIVE: This study was designed to investigate the expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) by fetal membranes in response to infection in vivo and to endotoxin in organ culture. STUDY DESIGN: Amniochorionic membranes were collected from infected and uninfected women and analyzed for cytokine messenger ribonucleic acid and protein. Normal membranes were cultured and exposed to endotoxin. Messenger ribonucleic acid expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular localization of messenger ribonucleic acid and protein was determined by in situ hybridization and immunocytochemical evaluation, respectively. RESULTS: Messenger ribonucleic acid for interleukin-1 beta appeared to be increased in infected or endotoxin-stimulated amniochorionic membranes, whereas interleukin-6 messenger ribonucleic acid was only observed in infected membranes or after endotoxin stimulation. Interleukin-1 beta messenger ribonucleic acid was localized exclusively to chorionic cells, whereas protein was observed in both chorion and amnion. Interleukin-6 messenger ribonucleic acid and protein were produced in both amniotic and chorionic cells. CONCLUSION: Amniochorionic membranes are a site of inflammatory cytokine production. These findings may have significance in preterm labor or premature rupture of membranes.
Amnion↗
Expression of endogenous HIV-1 crossreactive antigens within normal human extravillous trophoblast cells.
Expression of intact endogenous retroviruses by normal placental villous trophoblast and immuno-crossreactivity of villous trophoblast with anti-retroviral antisera have been documented. The nature and/or potential function of these particles/proteins has not yet been fully defined. We previously reported that monoclonal antibodies directed against HIV-1 envelope and gag proteins react with normal human villous trophoblast. In this study, we report that extravillous trophoblast (EVT) from second- and third-trimester tissue are also cross-reactive with anti-HIV-1 gp120/160 and p17/18 antibodies. We document a differential expression of such cross-reactive epitopes between mononuclear EVT and placental bed giant cells. Mononuclear EVT principally displayed reactivity throughout the cytoplasm with little or no difference between cells, whereas placental bed giant cells displayed distinct localization of labeling to limited areas of cytoplasm. This pattern of reactivity apparently correlates with trophoblast morphological differentiation and with our earlier observations concerning villous trophoblast. These data illustrate that retrovirus-associated epitopes are expressed by trophoblast throughout the normal human placenta and that this distribution is related to morphologic differentiation of these cells.
Chorionic Villi↗
Monoclonal antiphosphatidylserine antibody inhibits intercellular fusion of the choriocarcinoma line, JAR.
Naturally occurring antiphospholipid antibodies are strongly associated with placental dysfunction and severe obstetrical complications. We have produced three monoclonal antiphospholipid antibodies that differentiate between phosphatidylserine (PS)- and cardiolipin (CL)-dependent antigens, 3SB9b (CL-/PS+), BA3B5C4 (CL+/PS+), and D11A4 (CL+/PS-). We tested these monoclonal antiphospholipid antibodies in an assay for intertrophoblastic fusion. A JAR choriocarcinoma cell line was induced to undergo intercellular fusion by forskolin in the presence or absence of monoclonal antiphospholipid antibodies. The amount of syncytium formation was quantified by using fluorescein isothiocyanate (FITC)-conjugated anti-desmosome antibody to visualize intercellular membranes and propidium iodide to stain nuclei and by counting those cells with multiple nuclei. Without the presence of antiphospholipid antibodies, and in cultures containing BA3B5C4 (CL+/PS+) or D11A4 (CL+/PS-), approximately 70% of JAR formed syncytial cells after 24 h of forskolin treatment. Less than 13% of the cells formed synctia in 2-day cultures that were not exposed to forskolin or that contained forskolin in the presence of 3SB9b (CL-/PS+). These data suggest that phosphatidylserine is externalized during intertrophoblastic fusion and that antiphospholipid antibody with reactivity against PS, but not CL, can affect placental development by interfering with the normal formation of syncytiotrophoblast.
Antibodies, Anticardiolipin↗
Monoclonal IgM antiphosphatidylserine antibody reacts against cytoskeleton-like structures in cultured human umbilical cord endothelial cells.
PROBLEM: It has been proposed that antibodies against phospholipid-dependent antigens (aPLs), induce recurrent pregnancy loss and thrombosis through modulation of endothelial cell function, yet aPLs have not been conclusively shown to bind with endothelial cells. METHOD: Using indirect immunofluorescence we investigated the anti-endothelial cell reactivity of three monoclonal antibodies that differentiate between the phospholipids cardiolipin (CL) and phosphatidylserine (PS): BA3B5C4 (CL+/PS+); 3SB9b (CL-/PS+); and D11A4 (CL+/PS-). Cultured umbilical cord endothelial cells were prepared without fixation or with cold acetone fixation. RESULTS: None of the aPLs reacted with endothelial cells prepared without fixation. 3SB9B reacted strongly with cytoskeletal-like components in acetone-fixed cells, whereas BA3B5C4 and D11A4 were unreactive. The cytoskeletal-like binding of 3SB9b was completely blocked by a monoclonal antibody against vimentin, whereas antibodies against tubulin or actin were not inhibitory. Lipid extraction of the cells destroyed the 3SB9b reactive antigen without affecting the reactivity of anti-vimentin. CONCLUSION: These results suggest that phospholipid-dependent antigenic determinants are not expressed on the surface of resting endothelial cells but that a PS-dependent antigenic determinant is associated with endothelial cell intermediate filaments.
Animals↗
Expression of phosphatidylserine-dependent antigens on the surface of differentiating BeWo human choriocarcinoma cells.
PROBLEM: Antiphospholipid antibodies (aPLs) are associated with pregnancy loss, pregnancy-induced hypertension, and intrauterine growth retardation. We have previously reported that phosphatidylserine (PS)-dependent antigens are expressed in formalin-fixed cells concurrent with differentiation in a choriocarcinoma model (BeWo) of cytotrophoblast. That study, however, could not differentiate between cytoplasmic or surface antigen expression. METHOD: Three monoclonal aPLs that differentiate between PS- and cardiolipin (CL)-dependent antigens were reacted with BeWo, with or without forskolin activation, before fixation, and antibody binding was evaluated by immunoperoxidase techniques. RESULTS: Activation with forskolin induced a PS-dependent antigenic determinant on the surface on BeWo cells. CL-reactive monoclonal antibodies did not react with the cell surface, whether forskolin treated or not. CONCLUSION: These observations demonstrate that a PS-dependent antigen is expressed on the surface of a model of differentiating cytotrophoblastic cells and should be accessible in vivo to circulating aPLs.
Antibodies, Antiphospholipid↗
Ultrastructural characterization of endogenous retroviral particles isolated from normal human placentas.
Human placental endogenous retroviral (ERV) particle isolates were investigated by ultrastructural evaluation. Although retrovirus-like structures in normal human placental tissue sections have been described, the precise nature of these particles has not previously been defined. By direct electron microscopic (EM) observation of placental ERV isolates that display retroviral reverse transcriptase (RTase) activity and a buoyant density consistent with type-C retroviruses (1.17 g/ml on sucrose), we have shown these to contain particles with characteristic retroviral ultrastructural features. Samples were stained with 1.0% phosphotungstic acid (PTA), 0.5% uranyl acetate (UA), and low-angle-shadowed with platinum/palladium. Isolated placental ERV particles have an apparent diameter of approximately 120 nm, are membrane-bound with a short surface fringe, and contain capsid particles (about 90 nm in diameter) within an internal matrix structure. These observations support the view that human placental cells normally express ERV particles.
Centrifugation, Density Gradient↗
Immunologic detection of phosphatidylserine externalization during thrombin-induced platelet activation.
The antiphospholipid antibody syndrome is characterized by circulating antiphospholipid antibodies against cardiolipin (CL) and phosphatidylserine (PS) and clinically associated with a high risk of spontaneous thrombosis. Three monoclonal antibodies that differentiate between CL or PS were tested against resting and thrombin-activated platelets by flow cytometry. Each antibody reacted differently with CL and PS; 3SB9b reacted with PS, D11A4 reacted with CL, and BA3B5C4 reacted with both CL and PS. Activated platelets bound BA3B5C4 and 3SB9b, but not D11A4. The BA3B5C4-reactive epitope appeared earlier during activation than the epitope reactive with 3SB9b. These data suggest that antibodies against PS are reactive with activated platelets and that two immunoreactive forms of PS are sequentially expressed on platelets during activation.
Animals↗
Monoclonal antiphospholipid antibody reactivity against human placental trophoblast.
Naturally occurring antibodies against the negatively charged phospholipids cardiolipin (CL) and phosphatidylserine (PS) have been associated with recurrent pregnancy loss. One prevalent hypothesis proposes that antiphospholipid antibody (aPL) mediated pathophysiology is through increased placental thrombosis. In this study we investigated the reactivity of three mouse monoclonal aPLs with term and 26 week human placental preparations. Each monoclonal antibody reacted differently with CL and PS; 3SB9b reacted with PS (CL-/PS+), D11A4 reacted with CL (CL+/PS-) and BA3B5C4 reacted with both CL and PS (CL+/PS+). 3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 had only weak reactivity in the subtrophoblastic stromal region of the placenta in frozen sections. aPL staining was also observed against extravillous cytotrophoblast. BA3B5C4 stained cytoplasmic structures, whereas 3SB9b stained the plasma membrane region with little cytoplasmic staining. These data suggest that the trophoblastic layer is reactive with aPLs and may potentially be directly damaged through mechanisms unrelated to thrombosis. In addition, the trophoblastic layer directly in contact with the maternal circulation is most reactive with aPLs that are PS+ rather than CL+. The differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving PS on the cytotrophoblast is altered concurrent with fusion into the syncytium.
Antibodies, Monoclonal↗
Antiphospholipid antibodies--lobsters or red herrings?
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Antibodies, Antiphospholipid↗
Lack of an association between late fetal death and antiphospholipid antibody measurements in the second trimester.
To examine the relationship between elevated antiphospholipid antibody levels in the maternal circulation and late fetal death, we carried out a case-control study in which levels of anticardiolipin and antiphosphatidylserine antibodies were measured in banked second-trimester sera from 309 pregnancies ending in fetal death and from 618 viable control pregnancies. The sera were obtained from a population base of approximately 22,000 pregnancies enrolled for maternal alpha-fetoprotein screening between 15 and 20 weeks' gestation. The anticardiolipin immunoglobulin G level was markedly elevated (15.6 SD) in one serum sample associated with a fetal death. Otherwise, the anticardiolipin and antiphosphatidylserine measurements were similar in the two populations. Several other factors known to be associated with fetal death were also examined, and these all demonstrated the expected relationship. Antiphospholipid antibody measurements obtained at 15 weeks' gestation or later in the general pregnancy population are not helpful in identifying pregnancies at risk for fetal death.
Adult↗
Antiphospholipid antibodies and recurrent pregnancy loss: correlation between the activated partial thromboplastin time and antibodies against phosphatidylserine and cardiolipin.
Concordance was determined among the presence of the lupus anticoagulant measured by prolongation of the activated partial thromboplastin time and IgG and IgM antibodies against phosphatidylserine and cardiolipin in 47 patients selected for study because of histories of recurrent spontaneous pregnancy loss and a positive test for at least one antiphospholipid antibody. Forty-five of 47 patients (96%) had a prolonged activated partial thromboplastin time, ranging from 46 to 150 seconds. Elevated levels of IgG antiphosphatidylserine antibodies and IgM antiphosphatidylserine antibodies were present in 41 (87%) and in 19 (40%) of samples, respectively. Antibodies against cardiolipin were less commonly observed; IgG anticardiolipin antibodies in only 32 (68%) of 47 samples and IgM anticardiolipin antibodies in 15 (36%) of 42 samples. Neither the level of IgG antiphosphatidylserine antibodies nor the level of IgG anticardiolipin antibodies correlated well with the degree of prolongation of coagulation in the activated partial thromboplastin time (R = 0.312, p = 0.032 for IgG antiphosphatidylserine antibodies versus activated partial thromboplastin time; R = 0.251, p = 0.088 for IgG anticardiolipin antibodies versus activated partial thromboplastin time). Concordance with the activated partial thromboplastin time, however, was observed in 41 (87%) samples for IgG antiphosphatidylserine antibodies and in only 32 (68%) samples for IgG anticardiolipin antibodies. Our conclusion is that the antiphosphatidylserine assay correlates best, although not totally, with the presence of lupus anticoagulant and that the antiphosphatidylserine assay is more sensitive than testing for anticardiolipin.
Abortion, Habitual↗
IgG1 and IgG2 are the predominant subclasses of antiphospholipid antibody in women with the lupus anticoagulant.
We studied the sera of 36 patients with lupus anticoagulant and IgG antibodies against both phosphatidylserine and cardiolipin. Most sera also had IgG antibodies against other phospholipids: 97% against phosphatidylinositol, 91% against phosphatidylglycerol, and 82% against phosphatidylethanolamine. IgG2 was the predominant subclass against cardiolipin and phosphatidylserine; 35 of 36 patients (98%) had IgG2 against both phospholipids. Most patients also had the IgG1 subclass; 32 of 36 (89%) against cardiolipin and 25 of 36 (69%) against phosphatidylserine. IgG3 and IgG4 subclasses were present at very low concentrations and in only a minority of the sera. The antibody response against phosphatidylserine was characterized by significantly less IgG1 than was the response against cardiolipin (P less than 0.01), although the IgG2 responses against each phospholipid were not different. IgG subclasses were unrelated to any other aspect of the patients' history, including a history of thrombocytopenia or thrombosis, a positive antinuclear antibody test, or a diagnosis of systemic lupus erythematosus.
Blood Coagulation Factors↗
Production of monoclonal antibodies specific for platelet activation antigens and their use in evaluating platelet function.
Monoclonal antibodies (MAb) were used to identify platelet membrane molecules that are expressed after platelet activation. Balb/C mice were immunized with fixed thrombin-activated human platelets and their spleen cells were fused with the murine myeloma cell line NS1-Ag4/1. The resulting hybridomas were screened for antibody production against fixed thrombin-activated platelets and fixed resting platelets by flow cytometry. Two MAbs 2C8 (an IgM) and 1E3 (an IgG2a) demonstrated significant binding to fixed thrombin-activated platelets while reacting minimally with fixed resting platelets. The reactivity of 2C8 and 1E3 were compared to MAb's S12 and AC1.2, both of which have known specificity for an alpha-granule membrane protein (GMP-140) expressed on the surface of activated platelets. In radioimmunoprecipitation studies, both 2C8 and 1E3 immunoprecipitated a protein of approximately 140 kDa similar to that precipitated by S12 and AC1.2. Immunodepletion studies, with S12, AC1.2, 1E3, and 2C8 confirm that they all react with the same antigen. 2C8 may recognize the same epitope as S12, whereas 1E3 appears to recognize a different epitope of the same molecule. The use of these MAbs to measure platelet activation in whole blood correlates well with the results of conventional platelet aggregometry.
Animals↗
Pregnancy-associated immunological disorders.
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Abortion, Habitual↗
The association of antiphospholipid antibodies with severe preeclampsia.
Over a 3-year period, we studied 43 women who presented with severe preeclampsia prior to 34 weeks' gestation. Seven (16%) had significant levels of antiphospholipid antibodies, whereas none of the normotensive controls of similar gestational age had antiphospholipid antibodies (P less than .001). Three of the seven women with antiphospholipid antibodies suffered the following complications during the peripartum period: 1) cerebral infarction and episodes of transient monocular blindness; 2) pulmonary embolism, deep venous thrombosis, and an autoimmune flare in the postpartum period; and 3) transient monocular blindness and amnesia after delivery. Our experience suggests that antiphospholipid antibodies are found in a substantial proportion of cases of early-onset severe preeclampsia and have important clinical implications. We suggest that patients with early-onset severe preeclampsia be screened for antiphospholipid antibodies; if antibodies are detected, these women should be considered for prophylactic anticoagulation therapy.
Autoantibodies↗