Differences in immunoglobulin subclasses between dye exclusion and 51Cr release complement-dependent lymphocytotoxicity assays.

Paradoxical differences previously noted between lymphocytotoxicity detected by dye exclusion at room temperature (CDCE) or by 51Cr release (CDC51Cr) at 37 degrees C in maternal antipaternal complement-dependent lymphocytotoxicity have suggested that CDCE and CDC51Cr at 37 degrees C, but not at 20 degrees C, may detect different immunological antibody-antigen interactions. Reactions in the two test systems against the same target cells were compared in sera from known immune dialysis patients, secondary aborting women, and refractory platelet recipients before and after heat treatment of sera, absorption with solid-phase heparin, anti-light-chain augmentation, and the addition of murine monoclonal anti-IgG subclass antibodies. The results demonstrate significant differences between the two tests using the same target and sera. Further, the results imply the presence of an inhibitor and an inhibitor of inhibitor in sera. The involvement of different immunoglobulin subclasses was shown in the two tests. These data demonstrate the necessity for further study of the nature of the differences in the mechanisms of these clinically important antibody-detecting systems.

Vascular endothelial growth factor expression in transplanted human hearts.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen thought to play an important role in coronary collateral vessel formation. We used immunocytochemistry to determine VEGF expression in biopsies (n = 283) of transplanted human hearts (n = 109) with and without microvascular fibrin. Measures of vascular fibrin, alpha 2 plasmin-inhibitor (a2Pl), macrophages, neutrophils, and serum cardiac troponin T titers were used to evaluate myocardial damage. Antibody to T lymphocytes was used to evaluate cellular rejection, and HLA-DR, ICAM-1, and PAL-E antibodies were used to assess endothelial cell activation and phenotypic changes in the microcirculation. No VEGF immunoreactivity was detected in control donor hearts without fibrin, but the proportion of biopsies demonstrating VEGF immunoreactivity increased significantly in allografts with increasing fibrin and a2PI reactivity (P = 0.0001). VEGF immunoreactivity was confined to areas of fibrin deposition and was associated with infiltrates of macrophages and neutrophils (P < 0.0001), but not with T cells (P = 0.10). Biopsies with fibrin/VEGF reactivity were associated with increased capillary endothelial cell HLA-DR, ICAM-1, and PAL-E reactivity. In a subset of patients, serum cardiac troponin-T values were greater in patients with VEGF-positive (n = 21) than VEGF-negative (n = 19) biopsies (P = 0.05). Nested RT-PCR demonstrated that biopsies with and without fibrin/VEGF immunoreactivities expressed VEGF121, VEGF165, and VEGF189 variants, with VEGF165 being the dominate variant. These results indicate that endogenous VEGF is expressed locally following vascular thrombosis and myocardial cell damage, and that VEGF expression may be related to endothelial cell activation and phenotypic changes found in the microcirculation of cardiac allografts.

Deferential regulation of placenta growth factor (PlGF)-mediated signal transduction in human primary term trophoblast and endothelial cells.

Increasing evidence supports that many common obstetrical complications may involve the disruption of normal placental and/or uterine vascular function. Placenta growth factor (PlGF) is an angiogenic factor that is abundantly expressed in the placenta, with primary site of synthesis being trophoblast. Receptors for PlGF include products of the fms-like tyrosine kinase (flt-1) gene which is expressed in several cell types including endothelial cells and trophoblast. PlGF activation of flt-1 in trophoblast induces the stress activated protein kinase (SAPK) signal transduction pathways, JNK (c-Jun-N-Terminal Kinase) and p38, with little induction of the extracellular signal-regulated protein kinase (ERK)-1/2 pathways. In contrast, PlGF induces strong ERK-1/2 activation, but little JNK or p38 responses in human umbilical vein endothelial cells (HUVEC). To better understand the biochemical functions of PlGF in trophoblast, we studied upstream signal regulatory molecules to determine those that are responsible for directing the divergent PlGF signal transduction responses in these cell types. PlGF induced similar activation of Nck and PLC-gamma in trophoblast and HUVEC. In marked contrast, SHP-2 and Gab2 were strongly activated by PlGF in endothelial cells but not trophoblast. These results suggest a general role for Nck and PLC-gamma in mediating PlGF signal transduction responses independent of the different downstream MAPK pathways activated. However, SHP-2 and Gab2 are regulatory molecules involved in the PlGF induction of different terminal pathways in HUVEC and trophoblast.

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast.

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O2) yet PIGF expression decreased 73 +/- 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of function flt-1 on normal trophoblast suggests that VEGF/PIGF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.

Characterization of immunoglobulin class and subclass responses in secondary aborter sera.

Sera from secondary (2 degrees) aborters exhibit persistent, high-titred cytotoxicity against paternal as well as HLA dissimilar non-paternal lymphocytes. The majority of antipaternal complement-dependent cytotoxicity (CDC) and complement-independent antibody dependent cellular cytotoxicity (ADCC) was recovered in IgG enriched fractions following ion-exchange chromatography of 2 degrees aborter sera. The IgG subclasses mediating antipaternal reactivity were determined using murine anti-human IgG subclass specific monoclonal antibodies and Protein A affinity chromatography (SPA). Inhibition of paternal CDC by the anti-subclass reagents showed 75-90% of the reactivity mediated by maternal IgG1 antibodies. Anti-IgG3 inhibited 15-30% whereas anti-IgG2 produced little inhibition. SPA chromatography of 2 degrees aborter IgG supported the monoclonal antibody results in that greater than 80% of the CDC activity was recovered in the IgG1, 2, and 4 containing eluate and 20% was found in the IgG3 enriched effluent. Although the anti-IgG subclass specific monoclonals did not inhibit antipaternal ADCC, IgG3 did not appear to mediate this cytotoxicity as the ADCC was recovered in the eluate and not the effluent following SPA chromatography of 2 degrees aborter IgG enriched serum fractions. These data indicate that the humoral antipaternal and polyspecific CDC immune reactivities of 2 degrees aborters are due to the production of IgG1 and IgG3 antibodies.

Vascular endothelial growth factor expression in cycling human endometrium.

OBJECTIVE: To determine the spatial distribution of vascular endothelial growth factor protein in human endometrium and to assess temporal fluctuations in vascular endothelial growth factor gene expression and variant isoform production by stromal and epithelial cells during the menstrual cycle. DESIGN: Prospective study design. PATIENTS: Early proliferative endometrial biopsies were obtained from women undergoing gynecologic surgery for benign conditions; secretory stage biopsies were obtained from patients undergoing routine infertility investigations without evidence of luteal insufficiency. MAIN OUTCOME MEASURE: Immunohistochemical detection of vascular endothelial growth factor protein in endometrial biopsies, analyses of vascular endothelial growth factor RNA expression, and isoform production in intact endometrium and isolated endometrial stromal and epithelial cells. RESULTS: Strong vascular endothelial growth factor immunoreactivity was detected in the glandular epithelial cells of the secretory endometrium with no discernible immunoreactivity in stroma cells. The proliferative endometrium demonstrated prominent glandular immunoreactivity and faint, inconsistent stromal cell immunoreactivity. Preincubation of the antibody with excess cognate peptide abolished all immunoreactivity. A threefold to sixfold increase in vascular endothelial growth factor messenger RNA expression occurs in secretory versus proliferative endometrial samples. Endometrial stromal and epithelial cell isolates from both phases of the menstrual cycle express VEGF121, VEGF165, and VEGF189 isoforms, however, vascular endothelial growth factor variant 206 was not detected. CONCLUSIONS: Expression of vascular endothelial growth factor in the endometrium throughout the menstrual cycle suggests that vascular endothelial growth factor may promote the vascular growth, maintenance, and hyperpermeability required for adequate receptivity in the cycling human endometrium.

Trophoblast antigens in human seminal plasma.

Secondary recurrent spontaneous (2 degrees) aborters manifest persistent IgG, which show differential cytotoxicity patterns with lymphocytes from many donors. These are non-HLA-directed antibodies, which react allotypically with both trophoblast and lymphocytes. The antigens they recognize are designated trophoblast-lymphocyte crossreactive (TLX) antigens. Xenogeneic anti-TLX sera were studied with the use of enzyme-linked-immunosorbent (ELISA) and immunochemical assays to determine the TLX status of seminal plasma. The results showed 1) allotypic TLX antigens are present in seminal plasma; 2) seminal plasma TLX antigens may be membrane associated; 3) by immunoblotting, the molecular weights of antigens reactive with TLX antisera are 15, 22, 28, 33 kD and a smear between 180 and 340 kd; 4) by isoelectric focusing, TLX antigens show pI 4.0, 5.35, 5.9, 6.5, 6.8, and 7.2. Allotypic seminal plasma TLX antigens may provide the antigenic stimuli for persistent maternal humoral immunity.

Inhibitors of complement-mediated cytotoxicity in normal and secondary aborter sera.

Sera from patients with secondary (2 degrees) spontaneous abortions contain complement-dependent cytotoxic (CDC) antibodies with specificity for paternal lymphocytes. These lymphocytotoxins are not anti-HLA (human lymphocyte antigen) as shown by their polyspecificity on HLA select cell panels and by their removal following absorption with HLA-negative trophoblast membranes. They are predominantly IgG and have been designated as trophoblast-lymphocyte cross-reactive (TLX) antibodies. Normal and homologous 2 degrees aborter sera contain a CDC inhibitor that does not bind to paternal cells and must be present when complement is added to antibody. The inhibitor does not manifest anticomplement effects and appears to be species specific. Inhibitory capacity is increased by heating (56 degrees C for 30 min) and by absorption with heparin. When chromatographed on G-200 Sephadex, inhibitor appears in the void volume, suggesting a molecular weight of more than 250,000. It can be isolated from diethylaminoethyl cellulose into an euglobulin fraction that does not contain IgG, but does contain IgM, though no studies indicate the inhibitor to be IgM. We suggest that the inhibitor is under the control of a regulator molecule, probably an inhibitor-of-inhibitor, and that in 2 degrees aborter sera the equilibrium is unbalanced between antibody, inhibitor, and regulator.

Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast.

OBJECTIVES: Preeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known. STUDY DESIGN: Human primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O(2) or 1%O(2) conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA. RESULTS: Hypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O(2) culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression. CONCLUSIONS: NO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.

Differential regulation of human PlGF gene expression in trophoblast and nontrophoblast cells by oxygen tension.

OBJECTIVE: To determine the mechanism for differential effects of low oxygen tension on human PlGF gene transcription in trophoblast and nontrophoblast cells. STUDY DESIGN: Human PlGF reporter clones and real-time RT-PCR were used to compare the effects of hypoxia on gene transcription in human trophoblast and nontrophoblast cell lines. Overexpression of HIF-1alpha, inhibition of HIF-1 function and biochemical assessments of HIF-1 co-factor interactions were used to characterize hypoxia response mechanisms regulating PlGF transcription. RESULTS: PlGF transcription is specifically inhibited by low oxygen tension in trophoblast but is induced in some nontrophoblast cells. Overexpression of HIF-1alpha in normoxic cells or inhibition of HIF-1 function in hypoxic cells did not significantly alter transcription patterns of the PlGF gene in either cell type. CONCLUSIONS: These results suggest that transcriptional repression of PlGF gene expression occurs in human trophoblast exposed to low oxygen tension but that PlGF transcription is stimulated in certain hypoxic nontrophoblast cells. However, regulation of PlGF transcription is not mediated by functional HIF-1 activity in either cell type.

Proto-oncogenes and germ-cell differentiation.

Oncogenes are identified functionally by their ability to induce neoplastic transformation of susceptible cells. The first oncogenes to be characterized were isolated from acutely transforming retroviruses. Subsequently, it was determined that the retroviral oncogenes were formed from normal, progenitor genes. These cellular homologs of the viral oncogenes are termed proto-oncogenes. The derivation of oncogenes from proto-oncogenes is the consequence of mutations that remove regulatory constraints from the proto-oncogene. The ability of oncogenes to induce transformation implies that proto-oncogenes may function in growth and differentiation pathways in normal cells. Although many proto-oncogenes have been defined, the normal physiological function of most is not known. Studies of proto-oncogene expression during normal gametogenesis have determined that some genes are expressed in a stage-specific manner. The use of germ cells to provide homogeneous and defined normal cell populations facilitates identifying the roles proto-oncogenes have in regulating cell growth and differentiation.

Angiogenesis and the expression of vascular endothelial growth factor in endometrium and placenta.

PROBLEM: The demand for increased angiogenesis and microvascular permeability during cyclical changes in the endometrium and during placentation raises the possibility that aberrations in these events could lead to suboptimal reproductive performance. However, relatively little is presently known regarding the regulation of vascular growth and permeability in these tissues. METHOD OF STUDY: This review of current literature focuses on the expression, regulation, and potential physiological effects of vascular endothelial growth factor (VEGF) within endometrial and placental tissue. RESULTS: Spatial and temporal expression of VEGF as well as its restricted specificity, essential role in vasculogenesis/angiogenesis, and ability to induce vascular permeability makes VEGF an attractive regulator of vascular growth and permeability in the endometrium and placenta. CONCLUSION: A better understanding of the production, regulation, and physiological responses of the vasculature to angiogenic growth factors may lead to new therapeutic strategies for reproductive disturbances secondary to vascular insufficiencies within the female reproductive tract.

Proto-oncogenes in development and cancer.

Although analogies are often made comparing development to cancer, there is of course a major difference. Normal development requires complex patterns of rigidly controlled cell proliferation and differentiation. In contrast, cancer represents the pathological condition that results when normal cell growth patterns are uncoupled from their regulatory influences. Genetic studies of RNA tumor viruses have provided insights into the relationships and differences of the genes responsible for normal development and cancer. The presence of discrete genes (oncogenes) within the genome of oncogenic retroviruses is responsible for their tumorigenic potential. Molecular genetic studies have found that normal eukaryotic cells possess genes that are quite homologous to the retroviral oncogenes. These normal cellular genes (proto-oncogenes) are involved in the regulation of proliferation and differentiation. However, if mutated, proto-oncogenes have the potential for inducing neoplastic transformation. The conversion of a proto-oncogene to an oncogene is called activation. Proto-oncogenes can become activated by a variety of genetic mechanisms including transduction, insertional mutagenesis, amplification, point mutations, and chromosomal translocations. In each instance the genetic aberration results in a proto-oncogene that is now free of its normal regulatory constraints. Such deregulation of function imparts a distinct growth advantage to the cell.

Regulation of immunity to extraembryonic antigens in human pregnancy.

Pregnancy results in the immunologic challenge of the female to a wide variety of allogeneic antigens. Particular attention has been given to antibodies directed to allotypic trophoblast antigens (TLX), for trophoblast form the true allograft interface between mother and fetus. Studies found that antibodies to paternal TLX allotypes are produced in women suffering from secondary recurrent abortions. These TLX antibodies are not directed to classical HLA private epitopes. In this report, treatment of lymphocytes with papain to remove HLA Class I did not decrease TLX antigen densities. These results suggest TLX antibodies are not directed to Class I epitopes, public or private. The allotypic nature of TLX antigens requires that a pregnant female must be able to regulate TLX immune responses to avoid rejection of the conceptus. One mechanism to specifically and systemically regulate TLX immunity is the idiotype anti-idiotype network. We provide preliminary evidence in this report for the presence of TLX idiotype network in a normal primigravida. Initially, no antipaternal TLX antibodies were detected in the serum of the primigravida, suggesting no TLX immunization had occurred. However, separation of Ab1 from Ab2 by absorption of primigravida serum with 2 degrees aborter Ab1 resulted in seroconversion. The primigravida's Ab1 was cytotoxic for paternal and 3rd-party lymphocytes in a non-HLA-restricted pattern. Primigravida's Ab2 was recovered from the Ab1 matrix by competitive elution by using platelets as source of TLX antigen. The Ab2 was found to inhibit cytotoxicity by 2 degrees aborter Ab1 as well as primigravida Ab1. This is evidence that the Ab2 recognizes a cross-reactive idiotype (CRI) on TLX antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of serum versus plasma on agglutination of antibody-coated indicator cells by human rheumatoid factors.

Plasma and serum contain an inhibitor (I) of the agglutination of rabbit IgG-coated sheep erythrocytes by human rheumatoid factor (RF). Plasma but not serum contains an inhibitor of the inhibitor (I/I) which allows RF to interact with its target. In normal blood, there is more I than I/I, and I can be removed by solid-phase chromatography through concanavalin A (Con A). Plasma I/I is heat labile being eliminated by heating at 56 degrees C for 30 min. Addition of exogenous calcium clots EDTA plasma, causing an irreversible loss of I/I, and suggesting its involvement in the clotting cascade. The absence of I/I from Factor V-deficient plasma and destruction of I/I by Russell's viper venom indicate I/I either is associated with or is a part of Factor V. These findings suggest a balanced interplay between I and I/I, and indicate results of immunological tests done in vitro may not accurately reflect immune function in vivo. This seems to represent an unexplored link between hemostasis and immunity.

Uteroplacental vascular involvement in recurrent spontaneous abortion.

Spontaneous abortion is common in human pregnancy. Recent advances in pregnancy immunology and vascular biology are reviewed with emphasis upon the events associated with recurrent fetal losses. Certain treatment options used to alleviate or prevent some miscarriages are presented and discussed.

Trophoblast immunity in human pregnancy defined by antiidiotype.

Successful reproduction in mammals requires the mother to immunologically accept genetically disparate tissues. Allotypic trophoblast antigens (TLX) are thought to be responsible for influencing maternal acceptance of the feto-placental graft, and faulty regulation of immunity to TLX antigens has been associated with recurrent pregnancy losses. In this report, rabbit antiidiotype (RAb2) was produced to a human TLX antibody (Ab1). This RAb2 detected TLX cross-reactive idiotypes (CRI) on antitrophoblast IgG from women with normal and abnormal pregnancies. These findings support an hypothesis that women respond immunologically to allotypic trophoblast antigens, and that idiotype-antiidiotype regulation of this response is characteristic of normal pregnancy.

Placenta growth factor: potential role in pregnancy.

PROBLEM: In spite of the known requirement for adequate vascularity during placentation, little is known regarding the regulation of angiogenic growth factor production by trophoblast. Placenta growth factor (PIGF) is a recently discovered angiogenic growth factor whose expression is relatively limited to trophoblast. METHOD OF STUDY: Current literature of PIGF was reviewed, with emphasis on its expression, regulation, role in angiogenesis, and potential function(s) at the maternal-fetal interface. RESULTS: PIGF is abundantly expressed by trophoblast, which implies that it could act in a paracrine manner to modulate vascular development, stability, and/or function within the decidua and placental villi. In addition, expression of the PIGF receptor, fms-like tyrosine kinase (flt-1) receptor, on trophoblast raises the potential for an autocrine role of PIGF in regulating trophoblast growth and/or function. CONCLUSIONS: The potential for PIGF to influence both vascular endothelial cells and trophoblast suggests that aberrant trophoblast production of PIGF could compromise cellular function during gestation and contribute to the vascular and placental pathologies noted in many obstetric complications.

Signal transduction and biological function of placenta growth factor in primary human trophoblast.

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon gamma and tumor necrosis factor alpha. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.