Severe Asherman's syndrome complicated with placenta increta conceived by intracytoplasmic sperm injection following hysteroscopic surgery.

Although severe Asherman's syndrome is a disease that may cause infertility, pregnancy and childbirth are possible by performing hysteroscopic surgery. However, the obstetrical outcome is not always satisfactory. We report a case where severe Asherman's syndrome occurred following a cesarean section. Hysteroscopic surgery was performed due to secondary infertility, and pregnancy was achieved through a subsequent intracytoplasmic sperm injection. At 23 weeks of gestation, the patient was hospitalized due to the threat of premature labor, and a cesarean section was performed at 29 weeks of gestation after pregnancy-induced hypertension occurred. It was determined to be abnormal adherent placentation such as placenta increta through intraoperative findings, and a cesarean hysterectomy was performed. The pathological diagnosis of the uterus was placenta increta. Due to the risk of complications from placenta increta in pregnancies following hysteroscopic surgery in patients with severe Asherman's syndrome, it is important to realize the high risk involved in such cases during the pregnancy course, and careful perinatal management should be required.

Trends in the incidence of uterine cancer in Niigata, Japan: a population-based study from 1982 to 2007.

This study investigated trends in the incidence of uterine cancer in Japan. Data from the Gynecological Cancer Registry of Niigata comprising all new cases of uterine cancer registered for the entire female population aged 15 years and over a 25-year period were examined. The age-standardized ratio of carcinoma in situ has substantially increased among females < 40 years of age (from 3.8 (in the period of 1982-1989) to 40.9 (2000-2007). There was a significant trend in increasing incidence of invasive cervical cancer for those < 40 years of age (from 4.7 to 13.1), whereas a significant trend of decreasing incidence for the 50+ year age group. The ratios of corpus cancer were increased approximately two-folds both among the population aged < 50 years and those aged 50+ years and thus becoming equivalent to invasive cervical cancer. This prefecture-wide population-based study shows the practical trend in uterine cancer in Japanese females. The current health service must emphasize education among young adults concerning cervical cancer prevention while concentrating on screening. Avoiding risk factors, such as obesity, and increasing protective factors may lower risk for corpus cancer both in younger and older females.

Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo.

The role of T helper type 1 (Th1) and Th2 cells in tumor immunity was investigated using Th cells induced from ovalbumin (OVA)-specific T cell receptor transgenic mice. Although Th1 cells exhibited stronger cytotoxicity than Th2 cells, both cell types completely eradicated tumors when transferred into mice bearing A20 tumor cells transfected with the OVA gene (A20-OVA). Th1 cells eradicated the tumor mass by inducing cellular immunity, whereas Th2 cells destroyed the tumor by inducing tumor necrosis. Both Th1 and Th2 cells required CD8(+) T cells to eliminate tumors, and neither of these cells were able to completely eliminate A20-OVA tumors from T and B cell-deficient RAG2(-/-) mice. Mice cured from tumors by Th1 and Th2 cell therapy rejected A20-OVA upon rechallenge, but CD8(+) cytotoxic T lymphocytes were induced only from spleen cells prepared from cured mice by Th1 cell therapy. Moreover, we demonstrated that Th1 and Th2 cells used distinct adhesion mechanisms during tumor eradication: the leukocyte function-associated antigen (LFA)-1-dependent cell-cell adhesion step was essential for Th1 cell therapy, but not for Th2 cell therapy. These findings demonstrated for the first time the distinct role of antigen-specific Th1 and Th2 cells during eradication of established tumors in vivo.

The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells.

The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.

Humanizing bone marrow in immune-deficient mice.

Humanized mice are useful for studying human hematopoietic stem cells (HSCs) and their niche. In particular, clonal study of human HSC enables precise comparison of in vivo behavior between murine and human HSCs. A single HSC is able to reconstitute hematopoiesis even after serial transplantations in mice. While the life span of somatic cells is over that of individual in mice, this is not the case in humans. Clonal studies of human HSCs clearly demonstrated their aging in hosts. Since murine studies have demonstrated that HSCs are protected from aging by their niche in bone marrow, the humanizing niche model will reveal the precise mechanism by which human HSCs are protected from exhaustion in vivo. Direct transplantation of human mesenchymal stem cells into mouse bone marrow results in reconstitution of the functional human hematopoietic microenvironment comprised of pericytes, myofibroblasts, reticular cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells. These humanized mouse models are essential for testing whether the insights on hematopoiesis from mouse studies are applicable to humans before clinical application.

Primary retroperitoneal mucinous cystadenocarcinoma associated with pregnancy.

Primary retroperitoneal mucinous cystadenocarcinoma (PRMC) is an extremely rare tumor. Only 30 cases have been reported previously in the English literature, and little information is available concerning its treatment and prognosis. The patient was a 28-year-old woman, presenting with a right mid-abdominal tumor at 26 weeks of gestation. At 31 weeks of gestation, she underwent an exploratory laparotomy and was diagnosed with a PRMC. No disseminated tumor was observed, and an excision of only the tumor was performed. She had an uneventful vaginal delivery at 38 weeks of gestation and remains free of disease at 13 months after the operation. This report describes a case of PRMC associated with pregnancy. The optimal management of these retroperitoneal masses during pregnancy is discussed. Based on limited experience and the current literature, a PRMC with an intact capsule and no dissemination appears to have a good prognosis and can be treated by tumor excision alone in patients who wish to preserve fertility.

Prediction of myometrial invasion in patients with endometrial carcinoma: comparison of magnetic resonance imaging, transvaginal ultrasonography, and gross visual inspection.

This study evaluated the accuracy of magnetic resonance imaging (MRI) and transvaginal ultrasonography (TVUS) in preoperative detection of myometrial invasion by endometrial cancer. We also evaluated the results of gross visual inspection (GVI) of surgical specimens compared with histopathological diagnosis. One hundred and seventy-seven women underwent preoperative pelvic MRI, TVUS, and intraoperative GVI. Myometrial tumor invasion was evaluated histologically and classified as absent (depth a), superficial (depth b: < or = 50% invasion), or deep (depth c: > 50% invasion). The accuracy of MRI, TVUS, and GVI were 64.0, 66.9, and 63.8%, respectively. The positive predictive values of of each modality for depth a were 52.6, 51.4, and 52.2%, respectively. The accuracy of each in detecting deep myometrial invasion (depth c) were 84.0, 86.9, 83.1%. Although evaluation of depth a was limited with all modalities, MRI and TVUS were shown to be reliable for preoperative evaluation of deep myometrial invasion. The high accuracy of these three methods suggests that they are useful either interchangeably or in combination.

Potentiation of antitumor effect of NKT cell ligand, alpha-galactosylceramide by combination with IL-12 on lung metastasis of malignant melanoma cells.

The combined therapeutic effect of natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of alpha-GalCer or IL-12, the combined treatment of tumor-bearing mice with alpha-GalCer plus IL-12 caused a super-induction of serum IFN-gamma levels, though alpha-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-gamma production, the natural killing activity against YAC-1 cells and syngeneic B16-BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by alpha-GalCer plus IL-12 were enriched in both NK1.1+ TCRalphabeta+ NKT cells and NK1.1+ TCRalphabeta- NK cells. The preventing effect of alpha-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of alpha-GalCer was totally abolished in NKT-deficient mice. However, IL-12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that alpha-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands alpha-GalCer and IL-12.

Effects of progestins on the metabolism of cancellous bone in aged oophorectomized rats.

The effects of progestins on bone loss in female oophorectomized (ovx) rats were evaluated. One-year-old Sprague-Dawley rats were divided into eight groups: (1) beginning controls (control); (2) sham-operated controls (sham); (3) ovx; (4) ovx treated with estrogen (ovx + E); (5) ovx treated with progesterone (ovx + P); (6) ovx treated with estrogen and progesterone (ovx + E + P); (7) sham group treated with estrogen (sham + E); and (8) sham group treated with progesterone (sham + P). Immediately after surgery, the rats in the hormone injected groups were subcutaneously (s.c.) injected daily for 15 weeks with estrogen (17-beta-estradiol, 0.01 mg/kg in ethanol), or progesterone (4-pregnene-3,20-dione, 0.1 mg/kg in ethanol), or both. At the end of 15 weeks, the bone mineral density (BMD) and bone histomorphometry of the rats' lumbar vertebrae and serological parameters were measured. In the sham, ovx, and ovx + P groups, treatment with progesterone alone did not maintain the BMD in the lumbar vertebrae, but in the ovx + E and ovx + E + P, sham + E, and sham + P groups, progesterone did not inhibit the action of estrogen in the aged ovx rat model. BMD in the sham + P group was significantly higher than in the sham group (270.8+/-10.8 mg/cm2 versus 253.6+/-10.2 mg/cm2; p < 0.01). Bone histomorphometry revealed that bone volume (BV/TV) increased more in the ovx + E + P group than in the ovx + E group and more in the sham + P group than in the sham group, but not significantly. The ovx + E, ovx + E + P, sham + E, and sham + P groups showed no significant differences in the bone formation and resorption parameters, but the bone formation variables tended to increase in the ovx + E + P and sham + P groups. We concluded that progesterone alone cannot prevent bone loss or the increase in turnover after ovx and that estrogen, not progesterone, accounted for all of the bone activity in this study. It seems doubtful that progesterone inhibits the action of estrogen, and in fact may have a beneficial effect on bone metabolism.

Manipulation of Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells.

We have investigated the possibility that the Th1/Th2 balance in vivo may be modulated by adoptive transfer of Th1 or Th2 cells induced in vitro. Thl cells were induced from I-Ad-binding OVA323-339-specific T-cell receptor-transgenic (TCR-Tg) mouse spleen cells by culturing with OVA323-339 peptide and antigen presenting cells (APC) in the presence of IL-2, IL-12 and anti-IL-4 mAb. Th2 cells were induced from TCR-Tg mouse spleen cells by culturing with IL-2, IL-4 and anti-IL-12 mAb in addition to OVA323-339 plus APC. Immunomodulating activities of both Th1 and Th2 cells were determined by their effect on delayed type hypersensitivity (DTH) responses or cytokine production. No significant DTH responses (footpad swelling) were observed in untreated BALB/c mice following a single injection of OVA323-339-pulsed syngeneic spleen cells. However, adoptive transfer of Th1 cells into BALB/c mice induced strong dose dependent DTH responses in response to I-Ad-bound OVA323-339 but not unrelated peptide. In contrast, only slight DTH responses were detected in BALB/c mice transferred with Th2 cells. In parallel with the DTH responses, increased levels of serum IFN-gamma were demonstrated in mice adoptively transferred with Th1, while no significant increase was observed in Th2-transferred mice. In vitro analysis also demonstrated that both spleen cells and popliteal lymph node cells prepared from Th1-transferred mice showed Th1-type cytokine production, while cells obtained from Th2-transferred mice revealed Th2-dominant cytokine production. Such immune deviation induced by antigen-specific Th1 cells was demonstrated up to three months after cell transfer. Therefore, it may be possible to manipulate the Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells.

Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells.

Naive Th cells obtained from OVA(323-339)-specific DO11.10 TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells.

Technetium-99m-labeled monoclonal antibody with preserved immunoreactivity and high in vivo stability.

Recent availability of monoclonal antibodies (MoAb) and their radiolabeling through the use of the bifunctional chelating agents (BCA) have become an alternative procedure for in vivo radioimmunodetection. Using a newly synthesized BCA, a p-carboxyethylphenylglyoxal-di(N-methylthiosemicarbazone) (CE-DTS), the coupling and technetium-99m (99mTc) labeling of monoclonal IgG against hCG were carried out. In the system presented, factors affecting stability and immunoreactivity were examined. Immunoreactivity of the original IgG (56C) was preserved by conjugating one CE-DTS molecule per molecule of IgG (56C) using the phosphorylazide method, however, 99mTc labeling pH affected the immunoreactivity and limited the 99mTc labeling reaction between pH 4.5 and 6.2. A screening of labeling conditions, such as pH, reaction time, and reducing agent system were then carried out. Technetium-99m-labeled IgG (56C), [99mTc]CE-DTS-IgG (56C), showed good stability upon incubation with mice sera and comparable mice biodistribution to that of indium-111 (111In) DTPA-IgG (56C). Thus, these results indicate the excellent potential of CE-DTS as a BCA for labeling MoAb with 99mTc.

Phenotypic and functional characteristics of in vivo-induced interleukin-12-activated killer cells.

A single i.p. administration of IL-12 (2000 U/mouse) into the mice caused the elevation of serum IFN-gamma activity and the generation of killer cells which can lyse various kinds of tumor cells including both NK-sensitive and -resistant tumor cells. Such in vivo induced killer cells were not detected in the mice treated with the same dose of IL-2. The generation of IL-12-activated killer cells (IL-12AK) peaked at day 1 and sustained their cytotoxicity until day 3 after IL-12 administration. The generation of IL-12AK was inhibited by in vivo administration of anti-asialo GM1 (ASGM1) Ab but not anti-CD4 or anti-CD8 mAbs, suggesting that the precursor cells for IL-12AK were ASGM1+CD4-CD8- NK cells. The phenotypic characterization of in vivo induced effector cells with IL-12AK activity was carried out by separating the cells with FACStar. The IL-12AK activity was highly enriched in ASGM1+CD4-8- or NK1.1+CD4-8- NK cells, but not in CD8+ T cells and CD4+ T cells. The IL-12AK cells were also generated in tumor-inoculated mice. In parallel with the in vivo generation of IL-12AK generation, the growth of i.p. inoculated MBL-2 lymphoma cells was markedly inhibited by the administration with IL-12. The in vivo antitumor activity of IL-12 was blocked by the administration of anti-ASGM1 but not anti-CD4 or anti-CD8 mAbs in concomitant with the decrease of IL-12AK generation. From these results, it was indicated that ASGM1+NK1.1+CD4-8- NK type IL-12AK cells might play an important role in IL-12-induced local therapy of tumor in vivo.

The restraint stress drives a shift in Th1/Th2 balance toward Th2-dominant immunity in mice.

When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-gamma production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders.

Functional characterization of NK1.1 + Ly-6C+ cells.

It was found that NK1.1+ cells were subdivided by their different expression pattern of Ly-6C antigen. To characterize their functional significance in immunoregulation, we separated NK1.1 + Ly6C+ cells and NK1.1 + Ly-6C- cells from C57BL/6 mouse nylon-passed spleen cells by FACStar. Both NK1.1 + Ly-6C+ and NK1.1 + Ly-6C- cells responded to the stimulation with IL-2 plus IL-12 and showed strong cytotoxicity against YAC-1 cells. However, these cells revealed different ability in terms of IFN-gamma production. Only NK1.1 + Ly-6C+ cells, but not NK1.1 + Ly-6C- cells, cultured with IL-12 alone or IL-2 plus IL-12, produced high levels of IFN-gamma. Flow cytometric analysis demonstrated that NK1.1 + Ly-6C+ cells consisted of NK1.1 + CD3-Ly-6C+ NK cells and NK1.1 + CD3 + Ly-6C+ NKT cells. Therefore, we further separated these two populations from NK1.1 + Ly-6C+ cells to define their functions. Although, both NK1.1 + CD3-Ly-6C+ NK cells and NK1.1 + CD3+ NKT cells showed the same level of cytotoxicity. It was clearly demonstrated that NK1.1 + CD3+Ly-6C+ NKT cells were major immunoregulatory cells to produce IFN-gamma in respond to IL-12 alone or IL-2 plus IL-12.

Reconstitution of immune systems in RAG2-/- mice by transfer with interleukin-12-induced splenic hematopoietic progenitor cells.

The administration of a high dose of IL-12 into the mice resulted in the induction of splenomegaly. From the flow cytometry analysis of cellularity in an enlarged spleen, it was demonstrated that Thyl.2-CD45RB-c-Kit + Sca-1 + Lin- hematopoietic progenitor cells markedly increased in IL-12-administered mouse spleen compared with untreated mouse spleen. The IL-12-induced hematopoietic progenitor cells showed a greatly enhanced colony-forming activity in CFU-granulocyte/macrophage (CFU-GM), blast-forming units-erythroid (BFU-E) and CFU-spleen (CFU-S) assay. Moreover, it was initially demonstrated that the transfer of IL-12-induced splenic hematopoietic progenitor cells into immunodeficient RAG2-/- mice caused a complete reconstitution of their immune functions including T- and B-cell-mediated immunity. Thus, the evidence that IL-12 has a capability of inducing hematopoietic progenitor cells possessing stem cell-like activity in vivo, indicated another important immunomodulating activity of IL-12 in immunotherapy.

Accumulation of IL-12-activated antitumor effector cells into lymph nodes of tumor-bearing mice.

Simultaneous administration of high dose of IL-12 into tumor-inoculated mice resulted in a marked reduction of tumor growth in parallel with the augmented generation of cytotoxic T-cells, natural killer (NK) cells and IFN-gamma-producing Th cells. We found that these IL-12-activated antitumor effector cells preferentially accumulated in peripheral lymph nodes concomitantly with lymphadenopathy. However, IL-12 rather induced disappearance of antitumor effector cells including CD4+ T, CD8+ T and NK cells from spleen in spite of inducing splenomegaly. Lymph node cells obtained from IL-12-treated B16F0-bearing mice showed a marked IFN-gamma production in response to not only IL-2, IL-12, anti CD3 mAb but also B16F0 melanoma cells. Moreover, they could lyse B16F0 melanoma cells in a long-term cytotoxicity assay. It was also confirmed that IL-12-activated IFN-gamma producing Th1 cells were accumulated in tumor local site. Thus, IL-12 appeared to have a capability of stimulating selective migration of antitumor cells into lymph nodes and tumor local sites.

Decrease in the proportion of granulated CD56+ T-cells in patients with a history of recurrent abortion.

We investigated levels of CD56+ T-cells (CD3+ CD56+ cells) in peripheral circulation, which express one of the natural killer (NK) cell markers, by flow cytometry and with monoclonal antibodies in patients with a history of recurrent abortion. We compared these values with those obtained in normal women who were not pregnant as well as in normal pregnant subjects. The percentage of CD56+ T-cells in the peripheral blood of patients with a history of recurrent abortion was less than that in the non-pregnant or pregnant women. These results suggest that CD56+ T-cells with extrathymic properties may be associated with the maintenance of normal pregnancy in humans.

Application of interleukin 12 to antitumor cytokine and gene therapy.

In vivo administration of interleukin 12 (IL-12) at 2000 U/mouse induced IL-12-activated killer (IL-12AK) cells in parallel with an elevation in serum interferon-gamma (IFN-gamma) activity. Although NK1.1+CD3- natural killer cells are the major precursor of IL-12AK cells, asialoGM1+CD8+ T-cells were also demonstrated to be novel precursors. Such anomalous killer cells may play an important role in the early stages of the host defense mechanisms against tumors. It was also shown that IL-12 is effective in inducing tumor-specific cytotoxic T-lymphocytes. Consistent with these data, IL-12 had marked activity against various kinds of established tumors when given systemically. Mice cured of tumors by IL-12 treatment acquired tumor-specific T-cell immunity. Moreover, we initially demonstrated that IL-12 was effective in preventing and inhibiting the growth of primary tumors induced by the chemical carcinogen methylnitrosourea using c-Ha-ras transgeneic mice. Finally, we investigated the application of IL-12 to antitumor gene therapy. Transfer of the IL-12 gene into A20 B-lymphoma cells resulted in the continuous production of IL-12 and caused abrogation of in vivo tumorigenicity. Tumor cells transfected with the IL-12 gene are potentially a good tool as a tumor vaccine, as they effectively induced IL-12AK cells, IFN-gamma production, and tumor-specific protective immunity. Although B16-BL-6 melanoma cells, which are a highly metastatic subclone of B16 melanoma cells, showed resistance to IL-12 gene therapy, combination therapy with the B7-1 gene and systemic IL-12 administration almost completely inhibited tumor metastasis. Similar results were obtained using B16-BL-6 melanoma cells transfected with both B7-1 and IL-12 genes. These results suggest that IL-12 is a promising cytokine for antitumor cytokine and gene therapy.