Effect of pregnancy-specific ß1-glycoprotein on indoleamine-2,3-dioxygenase activity in human monocytes.

The role of heterogenic human pregnancy-specific glycoprotein (PSG), obtained by the authors' technology, in the regulation of the indoleamine-2,3-dioxygenase (IDO) activity in female blood monocytes has been studied in vitro. PSG stimulated IDO activity under the conditions of induction of the monocytes by interferon-γ. Upon the induction of cell proliferation by lipopolysaccharides, the stimulating effect was obtained only with 10 µg/mL of PSG. Enhanced IDO activity is probably a factor of peripheral immunological tolerance and antimicrobial protection against intracellular infections in the gestation period.

Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

[The study of human pregnancy specific ß1-glycoprotein immunomodulating effects].

Effect of human Pregnancy specific ß1-glycoprotein (PSG) in physiological concentrations was analyzed against the expression of natural killer (NK)-, T-cells with natural killer functions (N KT-) and T-regulatory lymphocyte (Treg) markers, as well as on the activity of monocyte indolamine-2,3-dioxygenase (IDO) and lymphocyte apoptosis in vitro. It was revealed that PSG in high concentration (100 µg/mL) suppressed the CD16/56 expression by NK-cells, while inhibiting the cytolytic activity of these cells. Meanwhile, PSG in low concentrations (1 and 10 µg/mL) enhanced the CD16/56 expression by NKT-cells that was related to cytokine-producing activity. It was found that PSG increased the number of adaptive Tregs in culture (CD4+FOXP3+ and CD4+CD25(bright)FOXP3+). In addition, PSG promoted the IDO activity in peripheral monocytes, while further potentiating the Treg generation. In general, PSG rendered anti-apoptotic action on lymphocytes. Therefore, indicated effects can determine the PSG contribution to the development of immune tolerance in pregnancy.

Antiphospholipid syndrome and pre-eclampsia.

Antiphospholipid syndrome (APS) is defined as an autoimmune disorder characterized by recurrent thrombosis or obstetrical morbidity. These features are linked to the presence in blood of autoantibodies against negatively charged phospholipids or phospholipid-binding proteins. Obstetric morbidity includes recurrent abortion (early and late) and severe pre-eclampsia (P-EC)/hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, and/or severe placental insufficiency. Criteria that define the major clinical and laboratory events were published in revised forms in the Sydney recommendations in 2006. We analyzed the blood of patients with severe P-EC according to the subgroups based on the 2006 revised criteria definition and compared these results with women after uncomplicated pregnancy and delivery. We found 20% elevated antiphospholipid antibodies (APAs) in women with severe P-EC (group I, 7.5%; group IIa, 5.0%; group IIb, 5.0%; group IIc, 2.5%). The increased APAs were observed only in women with severe P-EC (odds ratio: 2.45; 95% confidence interval, 1.01 to 4.3) and not in patients with severe P-EC at >34 weeks of gestation. According to our retrospective observation, we recommend the determination of anticardiolipin antibodies, lupus anticoagulant, and ß-2 glycoprotein-1 antibodies in patients with severe P-EC at <34 weeks of gestation.

Antiphospholipid syndrome characteristics and adverse pregnancy outcomes after 20 weeks of pregnancy.

OBJECTIVE: To assess outcomes after 20 weeks of pregnancy according to autoantibody profile and clinical presentation of maternal antiphospholipid syndrome (APS). METHODS: The present retrospective cohort analysis included women diagnosed with APS at a tertiary medical center in Israel between January 1, 2012, and December 31, 2016. Anticardiolipin antibodies, anti-ß2-glycoprotein antibodies, and lupus anticoagulant were assessed. Participants were stratified by type of APS (obstetric vs thrombotic), antibody profile, and antibody titer (low vs high). Primary composite outcomes were rated as severe (stillbirth, fetal growth restriction at <34 weeks, severe pre-eclampsia, or delivery at <32 weeks) and mild (stillbirth, any fetal growth restriction, any pre-eclampsia, or delivery at <34 weeks). RESULTS: A total of 99 women were included in the analysis. The primary composite outcomes were similar regardless of stratification. Lupus anticoagulant positivity was associated with delivery before 37 weeks. When compared with low antibody titer, high antibody titer was associated delivery at or before 32 weeks (P=0.045) and 34 weeks (P=0.029). CONCLUSION: High antibody titer might be associated with an increased risk of severe prematurity among pregnant women with APS.

Monoclonal antibodies in analysis of oncoplacental protein SP1 in vivo and in vitro.

Three monoclonal antibodies were developed to the placenta-specific glycoprotein SP1. The antibodies were used in the characterization of SP1 in placental tissue, pregnancy serum and urine, cancer serum, and cell culture. The three antibodies reacted similarly with purified SP1 of placental origin in radio- and enzyme immunoassay, and all three decorated the syncytiotrophoblast layer of placental villi in immunoperoxidase staining applied to fixed placental tissue. However, the three antibodies were found to have different and unique specificities and affinities. One antibody of relatively low affinity was used to isolate SP1 from placenta and fibroblast culture medium. The other two antibodies were used to develop a new and simple immunoassay for SP1. A panel of samples including cancer sera, pregnancy sera and urine, as well as cell culture fluids gave similar results in the monoclonal assay as in a conventional radioimmunoassay. Our work with monoclonal antibodies to SP1 provides further evidence for the production of authentic SP1 by fibroblastic cells in vitro and offers improved reagents for the isolation, characterization, and quantitation of an important marker of cancer and fetal development.

Pregnancy-specific glycoprotein expression in normal gastrointestinal tract and in tumors detected with novel monoclonal antibodies.

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members related to the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family and are encoded by 10 genes in the human. They are secreted at high levels by placental syncytiotrophoblast into maternal blood during pregnancy, and are implicated in immunoregulation, thromboregulation, and angiogenesis. To determine whether PSGs are expressed in tumors, we characterized 16 novel monoclonal antibodies to human PSG1 and used 2 that do not cross-react with CEACAMs to study PSG expression in tumors and in the gastrointestinal (GI) tract using tissue arrays and immunohistochemistry. Staining was frequently observed in primary squamous cell carcinomas and colonic adenocarcinomas and was correlated with the degree of tumor differentiation, being largely absent from metastatic samples. Staining was also observed in normal oesophageal and colonic epithelium. PSG expression in the human and mouse GI tract was confirmed using quantitative RT-PCR. However, mRNA expression was several orders of magnitude lower in the GI tract compared to placenta. Our results identify a non-placental site of PSG expression in the gut and associated tumors, with implications for determining whether PSGs have a role in tumor progression, and utility as tumor biomarkers.

Purification of pregnancy-specific beta 1-glycoprotein with a monoclonal antibody immunoadsorbent.

Hybrid cell lines secreting monoclonal antibodies against pregnancy-specific beta 1-glycoprotein (SP1) were produced by fusion of a mouse myeloma cell line with spleen cells from BALB/c mice immunized with purified placental SP1. The clones were further grown intraperitoneally in mice, and the ascites fluids contained anti-SP1 antibodies in high titers. One of the monoclonal antibodies was coupled to cyanogen-bromide-activated Sepharose and used for affinity chromatography of SP1 using late pregnancy serum as starting material. The purification of SP1 by means of immunoadsorption utilizing monoclonal antibodies offers remarkable advantages compared with the purification procedures used so far.

The effect of excess antigen on the enzyme-linked immunosorbent assay of onco-placental proteins.

The time course of the antigen-antibody reaction between human chorionic gonadotrophin, pregnancy specific beta 1 glycoprotein and pregnancy associated plasma protein A and their respective immobilised antibodies has been investigated. All 3 antigens showed enhanced binding with increasing length of incubation up to a maximum value, followed by a decrease. It is suggested that the diminution in binding at high antigen concentration is caused by the solubilisation of the immobilised antigen-antibody complex and that this is promoted by the high concentration of antigen.

Pregnancy-associated plasma protein A: a clinically significant predictor of early recurrence in stage II breast carcinoma.

The primary tumors and metastases from 30 patients with stage II breast carcinoma treated with low- or standard-dose combination chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil) were studied by the immunoperoxidase technique for pregnancy-associated plasma protein A (PAPP-A), pregnancy-specific beta-1-glycoprotein (SP-1), and placental protein five (PP-5). In addition to immunostaining, 25 traditional clinicopathologic features were assessed with respect to early (at less than two years) recurrence. Of the 11 patients with early recurrences, nine (82 per cent) were PAPP-A-positive, while 16 of the 19 patients without early recurrences (84 per cent) were PAPP-A-negative (P less than 0.0005). None of the other clinicopathologic features correlated with early recurrence. Immunostaining for PAPP-A is thus a clinically significant predictor of early recurrence in patients with stage II breast carcinoma.

Antibody reactivity against trophoblast and trophoblast products.

Sensitive immunoassays have been applied to WHO reference bank sera from fertile and infertile women in order to assess any naturally occurring antibody reactive with isolated human placental trophoblast membranes or two separate trophoblast protein products (hCG and SP1). A very low incidence of antibody reactive with solubilised trophoblast membrane was detected, and no significant antibody to either hCG or SP1 could be detected. Infertile states represented within this serum bank appear unlikely to involve adverse immune reactions to trophoblast.

Interference of an alpha 2 component in immunological determinations of pregnancy-specific beta 1 glycoprotein in serum.

Pregnancy-specific beta 1 glycoprotein (SP1-beta) was purified from human retroplacental blood by sequential anion exchange chromatography, gel chromatography and affinity chromatography. The final preparation appeared to be electrophoretically and immunochemically pure and was in particular free from any component with alpha mobility. The preparation was used as immunogen in rabbits as well as tracer and standard for radioimmunoassay and for cross- and rocket-immunoelectrophoresis. It was shown that this radioimmunoassay procedure, allowed quantitative determination of SP1-beta glycoprotein without interference by the alpha component.

Immunochemical and biological studies on two molecular variants of pregnancy specific beta 1 glycoprotein (SP1) SP1 alpha and SP1 beta.

Analytical crossed immunoelectrophoresis was performed to examine the relationship of a high molecular mass alpha 2 mobile glycoprotein (SP1 alpha) which is immunochemically identifiable with pregnancy specific beta 1 glycoprotein (SP1 beta). There was no evidence for any reaction of SP1 alpha with antisera to normal human serum protein, nor for complex formation following incubation with normal serum proteins. The half-life of SP1 alpha was apparently shorter than that of SP1 beta after delivery of the placenta.

The variable influence of an alpha component in pregnancy plasma on four different assay systems for the measurement of pregnancy-specific beta1 glycoprotein.

Four methods (radioimmunoassay, electroimmunoassay, laser nephelometry and kinetic immunoturbidimetry) have been evaluated for the measurement of proteins with pregnancy-specific beta1 glycoprotein immunoreactivity. The influence of two maternal plasma proteins with different electrophoretic mobilities (alpha2 and beta1) but with similar antigenic determinants has been assessed in each assay system. The radioimmunoassay method, which utilises a homogeneous preparation of pregnancy-specific beta1 glycoprotein for the preparation of iodinated tracer, and the kinetic immunoturbidimetric assay which measures antigen-antibody complex formation over the first minute of the reaction, were both considerably more specific for the beta1 component of pregnancy specific beta1 glycoprotein immunoreactivity than the laser nephelometric or electroimmunoassay methods.

[Localization of antigenic determinants on a molecule of trophoblast-specific beta 1-glycoprotein]. / Lokalizatsiia antigennykh determinant na molekule trofoblastspetsificheskogo beta 1-glikoproteina.

Spatial localization of antigenic determinants of trophoblast-specific beta I-glycoprotein (TSG) has been elucidated using chemical modifications of the sugar and protein moieties of the molecule. Various deglycosylation procedures of TSG afforded fragments slightly soluble even in the presence of powerful detergents. Treatment of TSG with boric acid and its salts, accompanied with a conformational change of the sugar moiety, failed to alter conformation of the protein portion as evidenced by CD spectral data. This modification was found to increase the antigenic activity of TSG only scarcely. Modification of tryptophane or tyrosine residues of TSG changed spatial structure of the protein portion to can be a considerable loss of the TSG antigenic activity. The data obtained led to the conclusion that antigenic determinants of TSG are localized at the protein portion of the molecule and are topographic. A tryptophane residue is an indispensable constituent of the antigenic determinants.

[Relation between the spatial structure and antigenic activity of trophoblast-specific beta1-glycoprotein]. / Issledovanie vzaimosviazi prostranstvennoi struktury i antigennoi aktivnosti trofoblastspetsificheskogo beta1-glikoproteina.

Temperature- and pH-dependence of spatial structure of a native trophoblast-specific beta-glycoprotein (TSG), its desialated and deglycosylated derivatives, as well as of a fragment obtained by partial acid hydrolysis on the temperature and pH variations has been studied using CD and UV spectroscopy. Within the range 45-50 degrees C a conformational transition of the protein moiety of TSG, leading to partially reversible alterations in tertiary and secondary structures of this glycoprotein after cooling the solution to 20 degrees C has been found out. The results of spectral studies of the spatial structure of the TSG protein component have been compared with the data on antigen activity of native, temperature- and pH-denaturated, desialated, and deglycosilated TSG. It has been concluded that the protein moiety of TSG consists mainly of beta-structures; the greater part of antigen determinants of TSG is topographic and belongs to the protein component of TSG, and only 15% of antigen determinants are not connected with TSG's spatial structure.

[Immunochemical and physico-chemical characteristics of glycoprotein from retroplacental blood having several common antigenic determinants with trophoblastic beta 1-glycoprotein]. / Immunokhimicheskaia i fiziko-khimicheskaia kharakteristika glikoproteina iz retroplatsentarnoi krovi, imeiushchego riad obshchikh antigennykh determinant s trofoblasticheskim beta 1-glikoproteinom.

A glycoprotein (TSG-fs) isolated from retroplacental blood is shown to have common antigenic determinants with trophoblast-specific beta 1-glycoprotein (TSG, SP-1). Antigenic activity of TSG-fs constitute 3.4% of TSG's activity. Like TSG, glycoprotein TSG-fs contains four intramolecular disulfide bonds and belongs to proteins with predominantly beta-structure. CD-spectroscopy data give evidence of high stability of the secondary structure of TSG-fs. Immunochemical identity of TSG-fs with the reduced and carboxymethylated derivative of TSG has been demonstrated. The above data allow to suggest that TSG-fs is a fragment of the TSG molecule.

[Non-specific immunological depression in pregnancy]. / La dépression immunitaire non spécifique de la grossesse.

The inhibitory effect of maternal plasma on in vitro lymphocyte responses shows that non specific inhibitory factors appear in early pregnancy. During the last decade the surge of interest in pregnancy proteins accompagnied by the advancement in immunological methodology has led to the isolation and characterization of these inhibitory factors. These pregnancy associated proteins play an important role in survival of the foetal allograft. It is significant that low plasma levels of inhibitory factors in early pregnancy are detected in women in whom spontaneous abortion occur.

[Placental proteins as regulators of immunological reactions in pregnancy]. / Plastsentarnye belki kak reguliatory immunologicheskikh reaktsii pri beremennosti.

The effect of two specific placental proteins, trophoblastic beta 1-glycoprotein (TBG) and chorionic alpha 1-microglobulin (CAG), on the immunological reactions was studied in vitro. TBG in physiological doses suppressed the proliferation of lymphocytes induced by plant mitogens and allogenic cells in the unidirectional mixed cultures, strengthened the effect of concanavalin A upon the induction of cells-suppressors in the culture and, in low concentrations, decreased the percentage of E- and EAC-rosette-forming cells. In none of the tests used CAG was effective. But when studying the effect of TBG and CAG mixture on PHA-induced proliferation of lymphocytes the inhibiting effect of TBG was weakened and, in some cases, completely relieved.

[Comparison of the action of the sera of pregnant women and of trophoblastic beta 1-glycoprotein in a number of immunological tests]. / Sopostavlenie deistviia syvorotki beremennykh zhenshchin i trofoblasticheskogo beta 1-glikoproteida v riade immunologicheskikh testov.

The effects of blood sera of pregnant women in the I, II and III trimesters (SPWI, SPWII, SPWIII) were compared with those of purified preparations of trophoblastic beta 1-glycoproteid (TBG) on phytohemagglutinin (PHA) induced proliferative response of lymphocytes of the intact donors and on the production of a factor inhibiting the migration of leucocytes (FIM-L). SPWI and purified TBG preparations in doses corresponding to its level in SPWI were shown to produce similar effects. With the increase of TBG concentration, however, the inhibiting effect of its preparations on the proliferation of lymphocytes increased much more intensively than that of SPWII and SPWIII containing the same dose of this protein. The stimulating effect of TBG on the production of FIM-L increased with the dose as well while SPWII and SPWIII did not exert any effect on the migration of leucocytes. A suggestion is put forward that SPWII and SPWIII contain factors modifying the effect of TBG.