Effect of Pregnancy Specific ß1-Glycoprotein on the Replicative Potential of Naïve T Cells and Immune Memory T Cells.

We studied the effects of pregnancy-specific ß1-glycoprotein (PSG) on the replicative potential of naïve T cells (CD45RA+) and immune memory T cells (CD45R0+) in vitro by evaluating the expression of the hTERT gene in combination with the proliferative activity of cells. Human PSG was obtained by the author's patented method of immunopurification using a biospecific sorbent with subsequent removal of immunoglobulin contamination on a HiTrap Protein G HP column. We used monocultures of CD45RA+ and CD45R0+ lymphocytes isolated from peripheral blood mononuclear cells of reproductive-age women. It was found that PSG in physiological concentrations inhibited the expression of the hTERT gene mRNA in naïve T cells and immune memory T cells and simultaneously reduced the number of proliferating T cells estimated by the differential gating method. At the same time, PSG reduced CD71 expression only on naïve T cells without affecting this molecule on immune memory T cells. Thus, PSG decreased the replication potential and suppressed the proliferation of T cells and immune memory T cells, which in the context of pregnancy can contribute to the formation of immune tolerance to the semi-allogeneic embryo.

Epigenetic upregulation of large-conductance Ca2+-activated K+ channel expression in uterine vascular adaptation to pregnancy.

Our previous study demonstrated that pregnancy increased large-conductance Ca(2+)-activated potassium channel ß1 subunit (BKß1) expression and large-conductance Ca(2+)-activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BKß1 expression patterns in uterine arteries. Ovine BKß1 promoter of 2315 bp spanning from -2211 to +104 of the transcription start site was cloned, and an Sp1-380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BKß1 promoter activity. Estrogen receptor-α bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BKß1. The Sp1 binding site at BKß1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BKß1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BKß1 promoter and BKß1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BKß1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca(2+)-activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

Pregnancy-specific {beta}-1-glycoprotein 1 and human leukocyte antigen-E mRNA in human sperm: differential expression in fertile and infertile men and evidence of a possible functional role during early development.

BACKGROUND: Mature spermatozoa contain thousands of mRNA transcripts. It has been recently shown that human sperm can deliver RNA into the oocyte, suggesting that mRNAs might have a role before or after fertilization. Human embryos express PSG1 (pregnancy-specific beta-1-glycoprotein 1) and HLA-E (human leukocyte antigen-E), molecules playing a role in implantation and early development. We compared PSG1 and HLA-E sperm mRNA levels in fertile and infertile men and we tested the hypothesis that these transcripts are selectively retained in the newly formed zygote. METHODS: Real-time RT-PCR was used to analyze sperm mRNA levels (n = 11 fertile, n = 31 infertile patients) of PSG1, HLA-E and PRM2 (protamine 2). The presence of PSG1 and HLA-E proteins was evaluated by western blot in sperm protein extracts (n = 3). Using ICSI of human sperm into hamster oocytes we evaluated the permanence of these mRNAs at different time points (n = 10 for each time) after fertilization. RESULTS: PSG1, HLA-E and PRM2 transcripts were demonstrated in ejaculated sperm. The fertile group showed significantly higher levels of PSG1 and HLA-E mRNA (both P < 0.05) than the infertile group, whereas PRM2 levels were not significantly different. However, PSG1 and HLA-E proteins were not found in ejaculated sperm. Following ICSI, PRM2 was undetectable after fertilization; conversely, PSG1 and HLA-E transcripts remained detectable for at least 24 h of zygotic development. CONCLUSIONS: We provide new evidence that indicates that human sperm deliver transcripts that may have a role in early embryo development and decreased levels of these transcripts may be associated with infertility.

[The correlation between pregnancy specific beta-1 glycoprotein and cytokines in post-dated pregnancy].

We have simultaneously identified levels of pregnancy specific beta-1 glycoprotein (PSG) and cytokines, as well as determined correlation between them in women with post-dated pregnancy. We studied 165 women with post-dated pregnancy, aged form 20 to 37, mean age 29.0+/-7.4. According to the gestational age they have been divided into 3 groups: I group--40 (24.2%)--birth at term 41-42 weeks of pregnancy, II group--95 (57.6%)--birth at 43-44 weeks of pregnancy, III group--30 (18.2%)--birth at 43-44 weeks of pregnancy. The control group consisted of 30 patients with term pregnancy. The comparative study of characteristics of change of PSG revealed low levels of above mentioned protein in women with post-dated pregnancy. This could possibly influenced on activation of immune system of pregnant women and defended placenta from mothers' immune system Cytokines types Th-2, IL-4 and also anti-inflammatory cytokine IL-1beta were insignificantly reduced in post-dated pregnancy cases and the true value increased in concentration of IL-6. We have valued the correlation between cytokines in plasma serum and beta-1 trophoblastic glycoprotein, and revealed mean level of correlation between the PSGG, IL-1 and IL-6 values.

A placenta-specific enhancer of the human syncytin gene.

The cis- and trans-acting factors that are critical for placenta-specific expression of the human syncytin gene are unknown. We identified a 146-base pair (bp) region of the 5'-flanking region of the human syncytin gene from nt-294 to -148 that is essential for basal gene expression in human BeWo and JEG3 choriocarcinoma cell lines but not in hepatoblastoma and kidney cell lines. Ligation of the 146-bp fragment to a SV40 promoter or a human beta-globin minimal promoter markedly enhanced promoter activity in the placenta cells but not in the liver and kidney cells. DNase I footprint assays indicated that nuclear extracts from BeWo cells but not HepG2 cells protected four regions (FP1-FP4) of the 146-bp fragment. Site-directed mutagenesis of an SP1-binding site in FP3 and a GATA-binding site in FP4 significantly repressed promoter activity in the placenta cells. Overexpression of SP1 (Sp1 transcription factor) and GATA2 (GATA binding protein 2) and GATA3 induced syncytin promoter activity but had little or no effect on the activities of syncytin promoter fragments containing mutations in the SP1- and GATA-binding sites. GATA2 and -3 mRNA levels increased markedly during spontaneous in vitro differentiation of human cytotrophoblast cells when the cytotrophoblast cells fused to form a syncytium. These findings strongly suggest that the 146-bp region of the 5'-flanking region (nt-294/-148) of the human syncytin gene acts as a placenta-specific enhancer. Binding of SP1 and GATA family members to this enhancer is critical for cell-specific expression of the syncytin gene.

The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid.

BACKGROUND: Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB) specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells. RESULTS: Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug. CONCLUSIONS: These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation.

It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.

Pregnancy-specific glycoprotein gene expression in recurrent aborters: a potential correlation to interleukin-10 expression.

PROBLEM: The expression of the pregnancy-specific glycoprotein (PSG) genes in the uterine endometrium of women experiencing recurrent first-trimester abortions, and potential correlations to cytokine expression were examined. METHOD OF STUDY: Endometrial RNA, isolated from women with a history of either repetitive first-trimester pregnancy losses or uncomplicated pregnancies, was isolated and analyzed for PSG transcripts by the reverse transcriptase-polymerase chain reaction method. PSG genes showing different patterns of expression were expressed in baculovirus, and the purified proteins examined for their effects on cytokine expression. RESULTS: The expression of PSG11 in the endometria of recurrent aborters was significantly lower than in that of controls (P < 0.01). When tested on monocytes, PSG11 stimulated secretion of interleukin (IL)-10. CONCLUSIONS: The level of expression of the PSG11 gene in the uterine endometrium, during the peri-implantation period, correlates with the risk of pregnancy loss in some women experiencing recurrent spontaneous abortions. The ability of PSG11 to influence the secretion of IL-10 suggests that PSG11 may contribute to the local modulation of the inflammatory T helper-1 response seen in the endometrium of these women.

Effect of pregnancy-specific beta 1-glycoprotein on the development of preimplantation embryo.

The objective of this study is to test the hypothesis that members of the pregnancy-specific beta 1-glycoprotein (PSG) family enhance the growth and maturation of embryos. cDNA encoding two members of the PSG family, namely PSG1 and PSG3, were expressed in Chinese Hamster Ovary (CHO) cells with the expression vector pH beta APr-1-neo. Two-cell stage mouse embryos were co-cultured in a two-chamber system with CHO cells expressing either recombinant PSG1 (rPSG1) or PSG3 (rPSG3) in the presence and absence of neutralizing PSG antibodies. The cleavage and maturation stage of the embryos was assessed at 12-hr intervals. Mouse embryos co-cultured with transfectants expressing rPSG1 showed a significant enhancement of cleavage and maturation rate compared to controls with P < 0.005-0.004. In co-cultures with CHO cells expressing rPSG3, no significant difference from the controls was observed in the early stage of development until late blastocyst formation. At that stage, there was a statistically significant enhancement of development by rPSG3 when compared to controls with P < 0.001. These results suggest that PSG1 and PSG3 exhibit embryotropic activity at different stages of development in the mouse model.

[Effects of trophoblastic beta 1-glycoprotein (TBG) on the functional activity of different cell lines]. / Vliianie trofoblasticheskogo beta 1-glikoproteina (TBG) na funktsional'nuiu aktivnost' razlichnykh kletochnykh linii.

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.

The hormonal basis of ectopic pregnancy.

The endocrine origin of EP revolves around certain endocrine events that modify reproductive tract function. The reproductive tract and the embryo are highly dependent on the proper function. Changes in this environment from either external or internal origin can lead to abnormalities in implantation including EP. Based on the examples presented above, it seems that both a high estrogenic or a high progestogenic environment can contribute to an increased risk of EP. This observation stresses the importance of the proper hormonal balance rather than an absolute value of a single hormone in the normal implantation process.

A murine model for the assessment of placental and fetal development in teratogenicity studies.

During normal pregnancy in the mouse, maternal serum levels of the analogues to human schwangerschaftsprotein-1 and alpha-fetoprotein correlate significantly with the growth of the placenta and fetus respectively. This relationship has been utilized in the analysis of the effect of sodium selenite on placental and fetal growth in mice. Moderate doses of sodium selenite did not affect the growth of the placenta and fetus significantly, whereas high doses of selenite resulted in a large percentage of abortions. The protein markers were found to be useful in the prediction of placental and fetal growth, and they are suggested to be of general use in the study of the impact of teratogenic substances, since they reflect the status of the fetoplacental mass during gestation.

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.

An animal model for the study of the function of pregnancy-specific beta 1-glycoprotein and pregnancy zone protein: a review.

Two pregnancy-associated murine proteins, PAMP-1 and PAMP-2, have been found to be immunologically cross-reacting analogues to the human proteins pregnancy zone protein (PZP) and pregnancy-specific beta 1-glycoprotein (SP-1), respectively. In this study, the physiochemical and biological properties of the proteins are compared and the effect on pregnancy of administration of monospecific antibodies against the proteins is reported.