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.

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.

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.

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.

Development and characterization of monoclonal antibodies to pregnancy-specific beta 1-glycoprotein.

Six monoclonal antibodies were developed to pregnancy-specific beta 1-glycoprotein (PS beta 1G). Studies of ascitic fluid antibodies by a double-antibody radioimmunoassay (RIA) included an evaluation of titers, dose-response parameters, and mass action properties. Four of the antibodies demonstrated moderate to high titers ranging from 1/40 000 to greater than 1/120 000, as determined by the specific binding of 125I-labeled PS beta 1G. In inhibition studies utilizing a standard containing known quantities of placental PS beta 1G, two of the antibodies (AR#11 and B#2) were highly sensitive and only slightly lower in this regard than a high affinity polyclonal antiserum. The binding affinities of AR#11 and B#2 monoclonals were greater than 10(9) mol-1 which underline their importance as potential clinical reagents for the RIA of PS beta 1G. The Scatchard plots, for several of the antibodies, were linear and in full agreement with a single order of binding sites predicted for specific monoclonal reagents. Immunodiffusion results provide preliminary evidence that at least three distinct determinants on PS beta 1G are recognized by a number of the monoclonal antibodies. Further studies on the fine specificities of the antibodies by solid-phase RIA, as well as a detailed evaluation of their clinical applications, are in progress.

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.

[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.

The effect on pregnancy of intrauterine administration of antibodies against two pregnancy-associated murine proteins: murine pregnancy-specific beta 1-glycoprotein and murine pregnancy-associated alpha 2-glycoprotein.

Intrauterine administration of 100 arbitrary units (AU) of purified monospecific rabbit anti murine pregnancy specific beta 1-glycoprotein antibodies resulted in the loss of the fetuses in pregnant mice (14 out of 14). By contrast, a similar treatment with 100 AU of rabbit anti murine pregnancy-associated alpha 2-glycoprotein had an effect similar to saline on the outcome of pregnancy. Intravenous administration of 1000 AU of rabbit anti murine pregnancy specific beta 1-glycoprotein during pregnancy in mice had no effect on the outcome of pregnancy.

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.

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.

Monoclonal antibodies against pregnancy-specific beta 1-glycoprotein (SP1) in immunohistochemistry and radioimmunoassay.

Monoclonal mouse antibodies against pregnancy-specific beta-1-glycoprotein (SP1) have been studied for their suitability in immunoperoxidase staining and radioimmunoassay methodologies. These antibodies were useful in staining normal placentas, hydatidiform moles, invasive moles and choriocarcinomas. They showed good specificity, with minimal background staining, and will thus be superior to conventional polyclonal antisera in immunohistochemistry. However, the presently tested monoclonal anti-SP1 antibodies were found not to be suitable for radioimmunoassay.

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-associated beta 1-macroglobulin (beta 1-PAM): a new serum protein associated with pregnancy serum-derived immune complexes and ovarian cancer.

A new pregnancy-associated serum protein, beta 1-PAM, was identified following low pH dissociation and separation, by gel chromatography, of pregnancy serum-derived immune complexes. A sandwich-type micro-enzyme immunoassay was developed and blood from various groups of subjects examined for the presence of beta 1-PAM. In addition to pregnant women, only subjects with ovarian cancer (70%) had consistently high levels of the antigen. beta 1-PAM isolated from both pregnancy and ovarian cancer serum was shown to be antigenically identical and to share the same isoelectric point (pH 5.25) and ammonium sulphate precipitation range (1.1--1.5 M). The serum protein had a molecular weight of 1.5 x 10(6), especially when obtained from ovarian cancer patients. However, it was apparent that other molecular weight forms were present, particularly in material isolated from pregnancy serum, which were probably related to the subunit nature of beta 1-PAM and its association with immune complexes.

Pregnancy specific beta-1 glycoprotein (SP1) in patients with trophoblastic disease: molecular heterogeneity and a "SP1 consuming factor".

The reactivity of sera from 19 patients with trophoblastic disease was analyzed in analytic immunoelectrophoresis using rabbit and human pregnancy specific beta-1 glycoprotein (SP1) antisera. Molecular heterogeneity of SP1 was demonstrated in crossed immunoelectrophoresis and a "SP1 consuming factors" was found in serum from a patient with choriocarcinoma. The "SP1 consuming factor" was precipitated by 1.65 M (NH4)2SO4. The results are discussed in relation to quantification of circulating SP1 patients with trophoblastic disease.