We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

Docking results have enabled us to propose how resveratrol could act as a selective PGHS-1 peroxidase site inhibitor. The docking model has predicted a slightly less favorable ΔGbind (−17.9 kcal/mol) of the resveratrol to the PGHS-2 peroxidase site in comparison with its corresponding binding to the PGHS-1 (−20.4 kcal/mol). The formation of hydrogen bonds among the hydroxyl groups of the resveratrol phenyl rings, the backbone of Fe-heme and the carbonyl group of Leu294 inside the PGHS-1 peroxidase site, associated with the absence of His214 in the backbone of PGHS-1, are essential features that are required to maintain the aromatic rings of the natural product parallel to the Fe-heme group and transverse to the peroxidase access channel promoting a large steric hindrance at this site and its consequent selective inhibition.

Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAPK activation in PSTT, we established the first PSTT cell culture, IST-2, from a surgically resected PSTT. IST-2 cells expressed HLA-G and Mel-CAM but not E-cadherin, an immunophenotype characteristic of PSTT. IST-2 cells were highly motile and invasive in culture as compared to choriocarcinoma JEG-3 cells and normal extravillous trophoblastic cells. Based on wound assay, time-lapse videomicroscopy for cell tracking, and invasion chamber assays, we found that the motility and invasion of IST-2 cells were significantly reduced (P < 0.01) after treatment with the MEK inhibitors CI-1040 and PD59089, which prevent activation of MAPK. In contrast, neither compound had any effect on normal extravillous trophoblastic cells or JEG-3 cells. In conclusion, our findings demonstrate a functional role of MAPK activation in the motility and invasion of PSTT.

During the first trimester of pregnancy, the human placenta is an actively dividing and highly invasive tumour-like tissue, while near term, it represents a fully developed, non-invasive unit. In order to understand the molecular basis of this marked difference in the placental phenotypes, an approach based on a differential display–reverse transcription–polymerase chain reaction (DD–RT–PCR) was adopted to analyse changes in gene expression, using total RNA isolated from first-trimester and term placental villi. Using this approach, T-plastin was initially identified as being differentially expressed in the human first-trimester placenta. T-plastin is an actin-bundling protein and is known to be highly expressed in actively dividing cells and up-regulated in several carcinomas. Using a homogenous population of cytotrophoblasts and syncytiotrophoblasts isolated from human placentae, the present authors demonstrate the differential expression of T-plastin in cytotrophoblasts compared with the terminally differentiated syncytiotrophoblasts. The down-regulation of T-plastin expression is further demonstrated in human trophoblastic BeWo cells induced to differentiate using transforming growth factor (TGF)β1, a growth factor known for its anti-proliferative and anti-invasive response in placental cells. These studies suggest that expression of T-plastin in the placental context may indeed be associated with the enhanced replicative potential of placental trophoblasts.

In human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation.

Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, "i". Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen "i" was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with "i" in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

In human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation.

The placenta provides nutrients and removes wastes from the fetus through various mechanisms of transfer, diffusion, carrier-mediated transport and active transport, and vesicular transfer.14 In humans the placental barrier is hemochorial (i.e., the placental trophoblast surface is in direct contact with the maternal blood, forming the maternal–fetal interface).The fertilized ovum implants directly into the maternal endometrium within a week of fertilization. Anchoring villi form at the end of the fourth postmenstrual week. Specialized invasive trophoblasts, at the tips of the cytotrophoblast columns of the anchoring villi, invade maternal spiral arterioles of the endometrium and the inner third of the myometrium. This vascular adaptation is noted as early as 16 days after ovulation.21 Communication between the developing intervillous space surrounding the villi and maternal blood supply is established by day 11 or 12, after ovulation. However, large amounts of maternal blood do not enter into the intervillous space until 8 weeks later.32 The intervillous space, at term, holds approximately 400 to 500 mL of blood.14, 192The trophoblast is an ectodermal covering of each villus. The villous barrier varies in thickness throughout gestation, initially being composed of syncytiotrophoblast, cytotrophoblast, and connective tissue stroma. Discontinuities begin to form in the syncytiotrophoblast layer at 9 to 12 weeks after menstruation.145 Cytotrophoblast covers 50% only of the villous circumference in the second trimester and diminishes to 20% at term.14 The formation of syncytial knots and vasculosyncytial membranes (VSM) beginning at 18 to 20 weeks gestation brings the fetal capillary in very close approximation to the maternal blood. The percentage of villous surface occupied by VSM varies from 0% at 12 weeks to nearly 30% at term.71 The VSM constitutes the effective metabolic barrier between the fetal and maternal circulations and is composed of syncytiotrophoblast cytoplasm, trophoblast basement membrane, stromal connective tissue, vascular basement membrane, and capillary endothelium (Fig. 1). The VSM becomes progressively thinner, measuring 1 to 3 μm at term, finally resulting in fusion of the capillary and trophoblast basement membrane.14The placental membranes are multilayered, composed of tissues from the inner cell mass (amnion), the trophoblast shell (chorionic mesoderm), with intervening connective tissue from the extraembryonic mesoderm. The chorion of the free membranes (chorion laeve) is intimately attached to the maternal decidua (Fig. 2). Decidua, the endometrial stromal fibroblast, under progestational influences becomes apparent by the 28th day after menstruation and completely surrounds the implanted blastocyst. With growth of the gestational sac, there is obliteration of the uterine cavity as a feto-maternal tissue junction is established around the whole conceptus at 17 weeks after menstruation.The decidua at the base of the placenta comes into direct contact with the invasive fetal trophoblast of the anchoring villi (Fig. 3). At this interface there is extensive mingling of fetal and maternal cells. The fetal trophoblast and maternal decidua are partially separated by a layer of fibrinoid. Increased fibrosis and acute or chronic inflammation may be seen with the implantation site particularly within basal villi.71Hofbauer cells are phagocytic cells of mesenchymal origin found in the stroma of villi, chorion, and amnion. They are present in the villi from the fourth week after ovulation, prior to the development of a bone marrow. They maintain their ability to undergo mitosis and self-replicate, in contrast to bone marrow-derived macrophages. These cells are relatively constant throughout gestation, comprise about 40% of the stromal cells,76 and express the leukocyte antigens, CD4, CD45, CD68, and CD1a.14, 143

Placental protein 19 (PP19) is a new placental tissue protein identified in extracts from the human term placenta by Bohn and Winkler (1985). We measured PP19 concentrations in body fluids and placental tissue culture media by radioimmunoassay. The minimum detectable dose of standard PP19 was 15 ng/ml. HCG and PP19 concentrations in media from placental tissue culture rose markedly at 144 hours, but were not increased by adding 0.1 mM cycloheximide. The circulating serum PP19 concentration was 4.5±1.1 ng/ml (mean±standard deviation) in the proliferative phase (n=8) and 5.1±1.6 ng/ml in the secretory phase (n=7) for nonpregnant women and 4.6±2.2 ng/ml from mean (n=12). Seminal plasma (n=8) contained 212.3±99.7 ng/ml. The maternal serum PP19 concentration in 291 normal pregnancies increased from 6.2 ng/ml (median) at 6–7 weeks of gestation to 34.1 ng/ml at 38–39 weeks. The mean PP19 concentration was higher in amniotic fluid and retroplacental blood but lower in umbilical cord blood than in circulating maternal serum. In hydatidiform mole, vesicular fluid contained high PP19 concentrations (1154.6±659.5 ng/ml), although these were not statistically higher than the normal range. The immunological identity of standard PP19 and PP19-like immunoreactive substances in the maternal serum, retroplacental blood, vesicular fluid from hydatidiform mole, and seminal plasma was indicated by parallelism between their dose-response curves in serial dilutions. An elevated (>10 ng/ml) serum PP19 concentration was observed in endometriosis (2 of 2 samples), squamous cell carcinoma of uterine cervix (2 of 11), ovarian adenocarcinoma (11 of 19), choriocarcinoma (1 of 5), invasive mole (3 of 11) and gastric adenocarcinoma (3 of 4). The level was low in myoma uteri (6 of 6), benign ovarian cyst (3 of 3), and endometrial carcinoma stage I (7 of 7). Ascites (2 of 5) and ovarian tumor fluid content (6 of 8) showed high PP19 concentrations. A metastatic gestational choriocarcinoma case showed elevated serum PP19 level in accordance with occurrence of DIC. In blood cell fractions separated by the Ficoll-Paque/Macrodex method, PMN fraction contained the highest PP19 concentration.

Placental protein 19 (PP19) is one of the new placental tissue proteins identified in extracts from human term placenta by Bohn and Winkler [1]. We measured the PP19 concentration in body fluids and placental tissue by radioimmunoassay; the minimum detectable dose of standard was 1.5 ng/ml. Although ethylene diamine tetraacetic acid (EDTA-2K) inhibited the immunoreaction between PP19 (225/242) and anti-PP19 antibody (632 ZA), the PP19 concentration did not differ between serum and heparin and sodium citrate plasmas. The serum PP19 concentration was increased by hemolysis. In blood cell fractions separated by the Ficoll-Paque/Macrodex method, polymorphonuclear leukocyte fraction contained the highest PP19 concentration. The circulating serum PP19 concentration was 4.5±1.1 ng/ml (mean ± standard deviation) in the proliferative phase (n=8) and 5.1±1.6 ng/ml in the secretory phase (n=7) for nonpregnant women, and 4.6±2.2 ng/ml from men (n=12). Seminal plasma (n=8) contained 212.2±99.7 ng/ml. The maternal serum PP19 concentration in 291 normal pregnancies increased from 6.2 ng/ml (median) at 6–7 weeks of gestation to 34.1 ng/ml at 38–39 weeks. The mean PP19 concentration was higher in amniotic fluid and retroplacental blood, but lower in umbilical cord blood than that in circulating maternal serum. In hydatidiform mole, vesicular fluid contained high PP19 concentration (1154.6±659.5 ng/ml), although these maternal serum concentration was not statistically higher than normal range. The chorionic villous trophoblast contained more PP19 than decidua, chorion, and amnion. These results suggest that PP19 has an extraplacental source, even though the chorionic villous trophoblast may be the main source throughout pregnancy.

われわれは, 最近5年間に10例のクローン病を経験し, その内2年以上経過した4例につき報告した.これらは現在まで保存的治療により緩解が得られ社会復帰中である.症例は26歳女性, 20歳男性, 25歳男性, 22歳女性で, それぞれ直腸狭窄, 肛門周囲膿瘍, 腹痛そして右下腹部痛を主訴として来院した.当初, 人工肛門造設あるいは切除術が考慮されたが, サラゾピリン経口投与および半消化態栄養剤の投与が採用された.その後, それぞれ6カ月, 9カ月, 7カ月そして9カ月後に症状の改善を見, 緩解が得られた.

PP19, a new placental tissue protein, has α1-β1 electrophoretic mobility, a molecular weight of 36 500 and 3.9% carbohydrate. To study immunocytochemical PP19 localization in extravillous trophoblast, we obtained formalin-fixed specimens from extravillous tubal pregnancy at gestational weeks (GW) 7–9 (12 blocks); four early intrauterine pregnancies at GW 7–13 (12 blocks); four late pregnancies at GW 28–38 complicated with intramural uterine myoma, placenta increta and abruptio placenta (8 blocks); four invasive complete moles (9 blocks); and seven primary and metastatic gestational choriocarcinomas (12 blocks). Immunohistochemical staining was done for PP19, pregnancy-specific β1-glycoprotein (SP1) and human chorionic gonadotrophin (hCG) using the indirect-labeled antibody method [purified PP19 (Lot no. 225/242) and antibody against PP19 (Lot no. 632ZA) prepared by H. Bohn, antibodies against hCG (Behringwerke, Marburg, FRG) and SP1 (Dakopatts, Copenhagen, Denmark)]. In both early and late intrauterine pregnancies, the extravillous syncytiotrophoblastic cell (XST) showed positive staining for hCG and SP1 in the cytoplasm, as well as for PP19, which stained more intensively in the nucleus than in the cytoplasm. The three proteins were not seen in the evtravillous cytotrophoblastic cell (XCT) in the trophoblastic cell column and shell. The interstitial cytotrophoblast-like cell (ICT), which infiltrated into the decidua and myometrium, and their blood vessels, was immunoreactively positive for PP19 but negative for hCG and SP1 with the exception of SP1-positive ICT in the myometrium in late pregnancy. XST and ICT in the endosalpinx of tubal pregnancy stained for all three proteins. In invasive complete mole, XST stained for the three proteins, but ICT infiltrating into the decidua and myometrium stained more intensively for PP19 than for either hCG or SP1. XCT did not stain for the three proteins. Staining for the three proteins in gestational choriocarcinoma resembled that in invasive mole. By PP19 staining, XST and ICT infiltrating into surrounding tissue were clearly distinguishable from other cells of similar shape. PP19 staining thus can be a useful histochemical marker in assessing the cell viability of the trophoblastic tumor after chemotherapy.