Hideharu Kanzaki

Suppression by human placental protein 14 of natural killer cell activity

ABSTRACT: Human decidua of early pregnancy contains considerable numbers of CD3− CD56+ natural killer (NK) cells. In this study, two major protein products of the decidua, placental protein 14 (PP14) and placental protein 12 (PP12), were tested for the ability to regulate human NK cell activity. In vitro overnight exposure to PP14 of blood lymphocytes or purified large granular lymphocytes (LGL) resulted in suppression of cytotoxicity against K562 target cells in a 4‐h 51Cr release assay. The NK inhibition was dependent on concentrations of PP14, being detectable at 5 μg/ml and reaching maximum at 50 μg/ml. Manifestation of PP14‐induced NK suppression required 18‐h contact with NK cells. The suppression of NK activity by PP14 was not abolished by indomethacin. In a target binding assay the number of PP14‐treated LGL binding to K562 was comparable to that of untreated ones. By contrast with PP14, PP12 produced no effects on NK cells. These results indicate that PP14 suppresses the function of NK cells, which might be involved in prevention of maternal immune rejection of fetus at the fetomaternal interface.

Monoclonal antiphosphatidylserine antibody reactivity against human first-trimester placental trophoblasts

OBJECTIVEnTo investigate the binding of antibodies against negatively charged phospholipids (antiphospholipid antibodies) to human placenta, we tested the reactivity of three mouse monoclonal antiphospholipid antibodies against first-trimester human placenta.nnnSTUDY DESIGNnFormalin-fixed and frozen sections of first-trimester placentas were stained by immunoperoxidase with three mouse monoclonal antibodies. Each monoclonal antibody reacted differently with cardiolipin and phosphatidylserine, 3SB9b reacted with phosphatidylserine, D11A4 reacted with cardiolipin, and BA3B5C4 reacted with both.nnnRESULTSn3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin-fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 reacted minimally or, more commonly, not at all.nnnCONCLUSIONnThe trophoblastic layer directly in contact with the maternal circulation is most reactive with antiphospholipid antibodies that react with phosphatidylserine rather than cardiolipin, suggesting that the trophoblasts may potentially be directly damaged by antiphospholipid antibodies through mechanisms unrelated to thrombosis. In addition, the differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving phosphatidylserine on the cytotrophoblast is altered concurrent with fusion into the syncytium.

Progesterone-induced smooth muscle—like cells in the subperitoneal nodules produced by estrogen: Experimental approach to leiomyomatosis peritonealis disseminata

In order to examine the pathogenesis and histogenesis of leiomyomatosis peritonealis disseminata (LPD), guinea pigs were treated with various doses of estradiol benzoate (E) (40, 80, 100, and 200 microgram/day, three times a week, by intramuscular injection). After treatment for 3 months, each dose of E produced lesions simulating the disseminated pattern of LPD. Ultrastructurally, the nodules were considered to be composed of cells resembling those of fibroblast. In guinea pigs, pretreatment with estradiol benzoate (100 microgram/day, three times a week intramuscularly for 3 months followed by combination treatment with E and progesterone (P) (0.5 and 1.0 mg/day, once a week, intramuscularly) for 1 to 3 months produced nodules similar to those with estrogen alone. Ultrastructurally, these nodules were composed of cells resembling smooth muscle and decidual cells. According to these results, we conjectured that estrogen differentiates and proliferates cells with the features of fibroblast-like cells from the subcoelomic totipotential mesenchyme, and these cells are differentiated into smooth muscle-like cells and decidua-like cells under the influence of both estrogen and progesterone, resulting in the production of multiple subperitoneal nodules.

Detection of p53 Gene Mutations in Human Ovarian and Endometrial Cancers by Polymerase Chain Reaction‐Single Strand Conformation Polymorphism Analysis

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162,175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.

Suppression of lymphocyte reactivity in vitro by supernatants of explants of human endometrium

To clarify the possible immunologic functions of the endometrium before implantation, supernatants from explant cultures of human endometrial tissues were examined for effects on mixed lymphocyte reaction and phytohemagglutinin M-stimulated cultures. Supernatants obtained from both proliferative and secretory phase endometria showed significant dose-related suppressive effects; the suppressive activity of secretory phase endometria was higher than that of proliferative phase endometria. On the other hand, no inhibitory effect was identified in supernatants from similar cultures of peritoneum and omentum, while supernatants of fallopian tubes showed slight suppressive activity. These results suggest the existence of soluble nonspecific immunosuppressive factor(s) released from human endometrium (especially during the secretory phase), and imply that these factor(s) may exert an important role in protecting the fertilized ovum from maternal rejection.

Effect of ovarian steroids on the secretion of immunosuppressive factor(s) from human endometrium.

To elucidate the effect of ovarian steroids on the secretion of immunosuppressive factor(s) from endometrium, supernatants from human endometrial cultures with or without the addition of progesterone or estrogen were compared for their effects on mixed lymphocyte reaction and phytohemagglutinin-induced lymphocyte reactivity. The suppressive activities of supernatants from the proliferative endometria were significantly increased by the addition of progesterone into their explant cultures. The addition of both progesterone and estrogen was also effective in increasing the inhibitory degree of proliferative endometria but that of estrogen alone showed no influence. Supernatants from the secretory endometria had higher suppressive activities than those from proliferative ones and showed no change in suppressive activity by the addition of ovarian steroids. The concentrations of progesterone in supernatants were too low to inhibit immune reactivity. These findings suggest that exogenous or endogenous progesterone may indirectly play an important role in the release of immunosuppressive factor(s) from human endometrium.

Developmental potential of frozen-thawed human blastocysts

PurposeTo examine the possibility of freezing human embryos at late cleaved stages (morula or blastocyst stage), we cryopreserved human embryos 5 days (day 5) or 6 days (day 6) after insemination and investigated their developmental potential after thawing.Materials and MethodsOne hundred nineteen morphologically good-quality human embryos from 43 women undergoing in vitro fertilization treatment between 1991 and 1992 were frozen using dimethylsulfoxide as a cryoprotectant. The embryos were cryopreserved for 5 to 30 months. After thawing they were then cultured in vitro for 24 hr to investigate their developmental potential. Survival rates and developmental rates were morphologically assessed after 24 hr of in vitro culture.ResultsDevelopmental rates were significantly (P <0.01 or P <0.05) lower than survival rates at every developmental stage. There was no difference in total survival rates between embryos frozen 5 days after insemination (78.2%; 54/69) and embryos frozen 6 days after insemination (70.0%; 35/54). However, the developmental rates after 24 hr of culture was significantly (P <0.05) lower for embryos frozen 6 days after insemination (6.0%; 3/50) than for embryos frozen 5 days after insemination (18.8%; 13/69). Only two embryos developed into fetuses after transfer into the uterus (1.7%; 2/119).ConclusionsFrom the results, the developmental potential of frozen-thawed human blastocysts was found to be significantly reduced, even though the blastocysts were of morphologically good quality. Longer in vitro exposure of embryos appears to reduce their developmental potential.

Studies on T-Lineage Cells in Human Decidua of First Trimester Pregnancies

ABSTRACT: T‐lineage cells in human decidua of early pregnancies were tested for surface markers, proliferative response, interleukin‐2 (IL‐2) production, and natural killer (NK) activity. T‐lineage (CD2+) cells that were obtained from decidua by the use of E‐rosette formation contained fewer CD3+ mature T cells and CD4+ cells than those from the peripheral blood of the same donors, while no differences were seen in the frequencies of CD8+ cells. P55 molecules of IL‐2 receptor (IL‐2R/p55, Tac antigen) were hardly detected on fresh decidual T‐lineage cells, though approximately 20% were positive for HLA‐DR. More than a half of decidual T‐lineage cells expressed CD56 molecules on their surface and killed K562 cells, the prototype target of NK cells, while most of them were negative for CD16 and CD57. Upon stimulation with IL‐2, decidual T‐lineage cells demonstrated dose‐dependent proliferative response. In addition, they were induced to produce high amounts of IL‐2 by stimulation with mitogens but not with alloantigens. These results suggest that human decidua contains high numbers of CD2+3‐CD16+56+ lymphocytes and that this population responds to IL‐2, produces IL‐2 and mediates NK activity.

Malignant fibrous histiocytoma of the uterus

A primary, malignant pleomorphic giant cell tumor of the uterus was studied by light and electron microscopy. The tumor was characterized by spindle cells, plump epithelioid cells, pleomorphic giant cells, osteoclast-like giant cells, and foamy xanthomatous cells. Histochemically, tumor cells did not show either myogenic or epithelial characteristics. Immunohistochemically, tumor cells were devoid of evidence of desmin, cytokeratin, myoglobin, and lysozyme (muramidase), but vimentin was weakly positive, and alpha 1-antichymotrypsin was weakly positive in the cytoplasm of pleomorphic giant cells. Ultrastructurally, tumor cells did not show either myogenic or epithelial features, but they resembled a variant of malignant fibrous histiocytoma. The present case was classified as a storiform-pleomorphic and giant cell type of malignant fibrous histiocytoma of the soft tissues. The uterus is considered to be an additional possible site of malignant fibrous histiocytoma.

Hyporeactivity to mitogens of retroplacental blood lymphocytes in human pregnancy

Retroplacental blood lymphocytes (RPL) obtained from women without complications at the term delivery were studied for proliferative response to mitogens and surface phenotype and were compared with autologous peripheral blood lymphocytes (PBL). Proliferation of RPL induced by phytohemagglutinin (PHA) was significantly lower than that of PBL in 11 of 13 cases. Also RPL proliferated poorly in response to concanavalin A (Con A) in 8 of 10 cases. On the other hand, flow cytometry analysis of T cell subsets revealed that RPL contain comparable or higher number of CD3+ cells and CD4+ cells and similar numbers of CD8+ cells as compared with PBL in most cases. These results indicate that T cell proliferative response in retroplacental space is impaired, which could be due to a functional defect of RPL.