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.

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.

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.

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.

In vivo expression of recombinant pregnancy-specific glycoprotein 1a inhibits the symptoms of collagen-induced arthritis.

PROBLEM: The contribution of Pregnancy-specific glycoproteins (PSG), the major variant of PSG released into the circulation during pregnancy, to the pregnancy-dependent improvement of rheumatoid arthritis (RA) has still not been elucidated. METHOD OF STUDY: Collagen-induced arthritis (CIA) was used to test the hypothesis that PSG1a when released into circulation has a modulatory role on the Th1-pathogenic response, thus improving the CIA symptoms. In vivo expression of PSG1a was induced by injection of the vaccinia (Vac)-based expression vector harboring the complete open-reading frame of PSG1a cDNA. RESULTS: In vivo PSG1a expression during the induction of CIA ameliorated the clinical symptoms, thereby reducing the arthritis score and incidence. Significantly lower levels of IL-17, IL-6, and IFN-γ, but higher levels of TGF-ß and IL-10 were secreted by collagen type II-stimulated spleen mononuclear cells from Vac-PSG1a-treated mice compared with control mice. Moreover, Vac-PSG1a treatment promoted the increase in splenic CD4+CD25+Foxp3+ Treg cells. CONCLUSION: Pre-clinical Vac-PSG1a treatment suppressed the Th1- and Th17-type-specific responses, leading to an increase in splenic Treg cells as well as IL-10- and TGF-ß-secreting cells, with the CIA symptoms being ameliorated.

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.

Anti-beta 2 glycoprotein 1 and anti-annexin V antibodies in women with recurrent miscarriage.

While it has been established that anti-phospholipid antibodies (aPL) are associated with recurrent miscarriage (RM), the importance of anti-beta2 glycoprotein 1 (GP1) IgG and anti-annexin V IgG antibodies as risk factors for RM is undefined. We have investigated the prevalence of anti-beta2 GP1 IgG and anti-annexin V IgG antibodies in 54 aPL-positive and 48 aPL-negative women with RM. The prevalence of IgG anti-beta2 GP1 antibodies was not significantly different in persistently aPL-positive women with RM (7%), aPL-negative women with RM (6%) and the normal parous control group (3%). Anti-annexin V IgG antibody prevalence was significantly increased in aPL-positive women with RM compared with aPL-negative women with RM (P = 0.01). The elevations were found in 35%, 19% and 16% of aPL-positive women with RM, aPL-negative women with RM and the control group respectively. No women showed positivity for both anti-beta2 GP1 IgG and anti-annexin V antibodies. Anti-beta2 GP1 IgG antibodies do not appear to be contributory to the investigation of women with RM. Anti-annexin V antibody positivity, although associated with aPL positivity in women with RM, is not an independent risk marker.

Purification and characterization of a bovine pregnancy-associated glycoprotein.

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).

Immunoturbidimetric determination of pregnancy-specific beta 1-glycoprotein (SP-1).

We describe a simple and rapid immunoturbidimetric end-point assay for measuring pregnancy-specific beta 1-glycoprotein (SP-1) in human serum with the Kone Progress clinical chemistry analyser, using a commercially available antiserum. The absorbance was measured at 340 nm 5 min after addition of antiserum. The assay range was 3-250 mg/l. Over this range the CV for between-day precision was below 6%. Bilirubin up to 160 mumol/l, haemoglobin up to 2 g/l and lipaemia up to 320 mg/l did not interfere. The reference interval in maternal serum at the 15th week of gestation, was 13 to 46 mg/l with a median of 25 mg/l (n = 78) and that of the 16th week was 6 to 60 mg/l with a median of 28 mg/l (n = 78). Comparison of results obtained by two commercially available radio-immunoassays and by the present method shows acceptable correlation. Rapid and reproducible, the immunoturbidimetric method is suitable for use in routine determination of SP-1.

[Evaluation of fetal antigenic stimulation in physiological and complicated pregnancy]. / Otsenka fetal'noi antigennoi stimuliatsii pri fiziologicheski protekaiushchei i oslozhnennoi beremennosti.

Fetal hemoglobin and trophoblastic beta 1-globulin levels have been determined in women with normal and complicated pregnancy in order to evaluate the rates of fetal antigenic stimulation. It was demonstrated that trophoblastic beta 1-globulin may not exactly represent fetal antigenic structures, but it is a marker of functional feto-placental activity. An elevated percentage of HbF in blood in threatened+ abortion and early toxemia of pregnancy confirmed feasibility of using it as an indirect marker of fetal antigenic stimulation and permeability of the fetoplacental barrier.

[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.

[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.

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.

The role of isotype and epitope specificity of monoclonal antibody mixtures in immunodiffusion reactions.

Twenty monoclonal antibodies to human alpha-fetoprotein have been characterized in terms of IgG subclass, epitope specificity and immunodiffusion properties. The Ig molecules consisted of six G1, ten G2a and four G2b isotypes. The epitope analysis was conducted by a solid-phase RIA employing 125I-labelled AFP. The RIA analysis resulted in the identification of seven determinants, two of which were specific for one antibody each, while others were specific for two or more antibodies. In Ouchterlony tests with the antibodies, none formed bands of immunoprecipitate when diffused individually against the antigen. Tests with all possible mixtures of pairs of the antibodies (190) resulted in positive immunodiffusion responses with only two mixtures. These each contained antibodies of the 2b isotype and demonstrated distinct epitope specificities. The immunodiffusion of mixtures of three antibodies from a group of 12 that were selected to represent equal numbers of isotypes resulted in 84 positive responses and 136 negative responses, i.e. from a total of 220 mixtures. A striking correlation was noted between positive immunodiffusion tests and composition of antibody isotypes and of epitope specificities of the mixtures. Each mixture was found to contain antibodies of at least one 2b isotype and of different epitope specificities. None of the mixtures that lacked a 2b isotype (56) responded in immunodiffusion tests. Similarly, in instances (46) in which the epitope specificities of the antibodies in the mixture were the same, i.e. duplicated or triplicated, the results were again negative despite the presence of a 2b isotype. The comparison of these studies with similar studies of a smaller group of antibodies to pregnancy-specific beta 1-glycoprotein strongly suggests that the G3 antibody isotype may have an immune precipitation enhancing effect similar to the G2b isotype.

[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.

Immunohistochemistry in the diagnosis of malignant mesothelioma.

The histological diagnosis of malignant mesothelioma of the pleura, especially the distinction from peripheral adenocarcinoma of the lung, may be difficult. The immunohistochemical reports previously published on this subject show diverging results mainly because a variety of antibodies and staining techniques have been used by the different authors. To obtain comparable and reproducible results standard techniques and commercialized antibodies should be applied in routine pathology. In order to investigate the value of immunohistochemistry for the separation of the two entities formalin fixed and paraffin embedded blocks of 47 mesotheliomas and 22 adenocarcinomas were investigated with the PAP technique and commercially available antibodies to carcino-embryonic antigen (CEA), keratin, vimentin, epithelial membrane antigen (EMA), pregnancy specific antigen (SP1), S-100 protein and monoclonal antibody lu-5 (mAB lu-5). CEA positivity was found in all 22 adenocarcinomas examined, but only 2/47 (4%) of all mesotheliomas showed a positive result. SP1 was positive in 13/22 (59%) of the adenocarcinomas, whereas only 3/47 (6%) mesotheliomas were positive for this marker. No significant difference in the rate of positive cases in the adenocarcinoma and mesothelioma group could be found with the other above mentioned antigens. The results of our study indicate that especially CEA, but also SP1 are valuable markers in the diagnosis of malignant mesothelioma.

Immunosuppressive properties of pregnancy serum on the mixed lymphocyte reaction.

The human fetus may escape immunological attack because of serum factors which have immunomodulatory influence on maternal cellular effector responses. Paired peripheral and retroplacental sera were shown to inhibit the allogenic mixed lymphocyte reaction (MLR) used as an in-vitro model of cell-mediated immunity. There was no correlation between the suppressive effect of the peripheral and retroplacental sera and the serum concentrations of four pregnancy-related proteins (alpha-fetoprotein, pregnancy-associated alpha 2 glycoprotein, pregnancy-associated plasma protein A and Schwangerschaftsprotein 1) to which immunosuppressive properties have been ascribed, but there was a negative correlation between peripheral AFP and MLR inhibition (r = -0.62, P less than 0.001). Hence, the factor or factors responsible for suppressing the MLR are not those investigated in the present study.

An immunochemical study of trophoblast-specific beta 1-glycoprotein and its fragments.

Trophoblast-specific beta 1-glycoprotein (TSG, SP-1) has been isolated from retroplacental blood serum by the usual techniques. Together with the purified TSG, an antigen (TSGfs) possessing partial immunochemical identity with TSG, has been isolated. This antigen, with molecular mass 25 KD, contains virtually no fucose nor neuraminic acid and differs from TSG in a lower content of arginine, tyrosine and aspartic acid. In addition, a low-molecular weight fragment of the TSG molecule (TSGhs), partially identical immunochemically with TSG and fully identical immunochemically with TSGfs, has been obtained by partial acid hydrolysis of TSG. The partial acid hydrolysis of TSG yields TSGhs and high-molecular weight fragments with molecular masses greater than 160 KD which also exhibit a partial identity with TSG. The results provide evidence for the occurrence of a labile linkage bonding the two parts of the TSG molecule which carry different immunochemical determinants.