Timothy W. Lyden

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.

Expression of inflammatory cytokines (interleukin-1β 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.

Modulation of phosphatidylserine epitope expression by BeWo cells during forskolin treatment

The adenylcyclase activator forskolin induces the human choriocarcinoma line, BeWo, to undergo differentiation and fusion within 48 to 72 h. Using three monoclonal antibodies that differentiate between the anionic phospholipids cardiolipin (CL) and phosphatidylserine (PS) and immunoperoxidase techniques we investigated the expression of PS by BeWo during 48 h of forskolin treatment. We observed that BeWo cells not exposed to forskolin express an epitope of pS that reacts strongly with monoclonal antibody BA3B5C4 (CL+/PS+), whereas following treatment with forskolin there is a decrease in reactivity with BA3B5C4 and a concurrent increased activity with a second PS-reactive monoclonal antibody, 3SB9b (CL-/PS+). A third monoclonal antibody, D11A4 (CL+/PS-), that reacted with all anionic phospholipids except PS did not bind to BeWo cells, whether forskolin treated or not. These observations support previous interpretations using human placenta that during cytotrophoblast differentiation two antigenic forms of PS are expressed. Based on the described relationship of PS with cellular fusion events in other systems and the association of naturally occurring antibodies against PS with pregnancy loss and intrauterine growth retardation in humans, we propose that altered expression of PS during normal placental development and in BeWo after exposure to forskolin may be critical in the cytotrophoblast differentiation process.

Antiphospholipid Antibodies—Lobsters or Red Herrings?

The antiphospholipid antibody (aPL) syndrome is characterized by the production of autoantibodies against negatively charged phospholipids and clinically associated with thrombocytopenia, thrombosis, pregnancy loss, or a combination ofthese events. 1---<; Patients with aPLs have an 80% to 90% pregnancy loss rate, two-thirds of which are lost in the first trimester. Even the few cases of successful pregnancy are at risk for intrauterine growth retardation (IUGR), placental abruption, prematurity, and early and severe pregnancy-induced hypertension (PIH).5,6The patients are also at risk for recurrent cerebrovascular disease (migraines, strokes, transient ischemic attacks), peripheral thrombosis, and cardiac disease,

I. Organ Culture of Amniochorionic Membrane In Vitro

PROBLEM: The purpose of the study was to develop a novel method of amniochorionic membrane culture aimed at maintaining tissue integrity.

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.

Antiphospholipid Antibodies and Placental Development

The antiphospholipid antibody syndrome (aPL syndrome) is characterized by elevated levels of autoantibodies against negatively charged phospholipids (aPLs) associated with several severe obstetrical complications, including recurrent pregnancy loss, pregnancy-induced hypertension and preeclampsia before 34 weeks’gestation, and intrauterine growth retardation (reviewed in 1, 2). The syndrome appears to be a prothrombotic state in which the patients are at risk for thrombosis at virtually any site. The most thoroughly studied aPLs are the lupus anticoagulant and antibodies against the phospholipid cardiolipin (aCLs). It is commonly proposed that aCLs, which are cross-reactive with other phospholipids, are primarily responsible for the thrombosis. The obstetric complications have been attributed to aCL-induced placental damage resulting from placental or decidual thrombosis.

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.

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.

A murine monoclonal antibody (RV3-27) raised against isolated human placental endogenous retroviral particles and reactive with syncytiotrophoblast

Particles with the characteristic shape of enveloped retroviral particles and maximal specific reverse transcriptase (RTase) activity at buoyant density of 1.15-1.17 g/ml have been isolated from human first-trimester chorionic villous tissue. Murine monoclonal antibodies (mAbs) to these isolated particles were generated. One IgM mAb (RV3-27) showed granular staining of cytoplasmic structures within syncytiotrophoblast by immunohistochemistry. Immunoelectron microscopic studies have demonstrated focal localisation to small submembranous regions of syncytiotrophoblast, as well as reaction with detergent-disrupted isolated placental retroviral-like particles. The RV3-27 mAb did not stain other human tissues in this focal manner, although increased generalised cytoplasmic staining was not uncommon; also, this mAb did not react strongly with the surface or cytoplasm of a variety of human cell lines (including choriocarcinoma cells). Immunoblotting and HPLC analyses have indicated the reactive placental antigen to be a 17-25 kDa protein. It is suggested that the RV3-27 mAb may be reactive with a syncytiotrophoblast antigen encoded by an endogenous retroviral sequence.