Establishment and characterization of two cell lines (HEC-155, HEC-180) derived from uterine papillary serous adenocarcinoma.

Uterine papillary serous adenocarcinoma (UPSC) is an uncommon histologic subtype of endometrial cancer that characteristically behaves aggressively with a poor prognosis. We established two novel cell lines derived from UPSC designated HEC-155 and HEC-180. Both cell lines have been growing steadily in monolayer cultures for over ten years. Overexpression of p53, Ki67 and p27 was detected in both cell lines by immunohistochemistry. Using a DNA sequencing technique, a point mutation of p53 was detected in exon 8, codon 286 in HEC-155 and in exon 6, codon 195 in HEC-180. These newly established cell lines should be useful for investigating the characteristics of UPSC.

Disappearance of clonally expanded T cells after allogeneic leukocyte immunotherapy in peripheral blood of patients with habitual abortion.

Allogeneic leukocyte immunotherapy is often used to improve fertility of patients with habitual abortion (HA), which probably acts through immune-mediated mechanisms. However, the involvement of T cells is not clear. This study examined the effect of allogeneic immunotherapy on T cells of patients with HA. Peripheral blood mononuclear cells were obtained from 5 healthy women and 14 women with HA. RNA was isolated from mononuclear blood cells. Reverse transcription-polymerase chain reaction (RT-PCR), followed by single-strand conformational polymorphism (SSCP) were used to analyze the gene segments of T-cell receptor beta chain (TCRbetaV) variable regions. Oligoclonal accumulation of T cells was identified in peripheral blood of nonpregnant patients with a history of HA. It was also revealed, however, that immunostimulation reduced the number of accumulating T-cell clones (p = 0.0004). The results, together with the clinical effectiveness of immunotherapy, suggest that accumulation of T-cell clonotypes, which probably resulted from antigenic stimulation, is involved in the pathogenesis of HA.

Clonal expansion of T cells that are specific for autologous ovarian tumor among tumor-infiltrating T cells in humans.

OBJECTIVE: The purpose of this study was to determine whether oligoclonally expanding tumor-infiltrating lymphocytes (TIL) were tumor-specific. STUDY DESIGN: Peripheral blood lymphocytes (PBL) from an ovarian tumor-bearing patient were stimulated in vitro with an autologous cancer cell line (SMOV-2). Then genes coding for the third complementarity-determining region of the T cell receptor (TCR) beta chain were amplified by reverse transcription-polymerase chain reaction and separated by single-strand conformation polymorphism. Accumulated TCR clonotypes in vitro and in vivo in TIL were compared. RESULTS: Clonal expansion of T cells was generated from PBL by stimulation with SMOV-2. A portion of the proliferated clonotypes was found to be identical to those accumulated in TIL in vivo. CONCLUSION: This is the first demonstration that accumulating T cell clones in TIL recognize antigen(s) on an autologous tumor. Further characterization of such T cell clonotypes may lead to tumor antigen-specific immunotherapy.

Establishment and characterization of human ovarian clear cell adenocarcinoma cell line (SMOV-2), and its cytotoxity by anticancer agents.

A novel cell line derived from a surgically resected ovarian clear cell adenocarcinoma of 46 year-old Japanese woman was established and designated SMOV-2. Cells of this lineage were continuously propagated in vitro over 44 months and were grown in a mono-layered sheet with a doubling time of 48.2 hours. The histopathology of the transplanted tumor in nude mice showed two distinctive cell types, hobnail cells and clear cells, which demonstrated recognizable characteristics of clear cell adenocarcinoma, as compared to resected original tumors. At the molecular level, SMOV-2 cells had the wild type p53 genes that were free from missence mutations. Anticancer agents (cisplatin and paclitaxel) were examined for cytotoxity against these SMOV-2 cells in vitro. These examinations revealed that the chemotherapy-treated cells had decreased proliferation, cell cycle arrests, and induction of apoptosis by the anticancer agents. As can be gleaned from this research, SMOV-2 is a valuable model to study the mechanism of apoptotic responses of solid tumors to future anticancer agents.

Disturbed circadian core body temperature rhythm and sleep disturbance in school refusal children and adolescents.

We examined the circadian rhythm of core body temperature (CBT) in 22 school refusal patients, ages between 12 and 18 years, who did not have any physical or psychiatric disorders, but had indefinite complaints, and were suspected to have a circadian rhythm disturbance. To obtain normal data for analysis, CBT in 9 healthy age-matched school attendants who did not have any sleep, psychiatric, or medical disturbance were monitored. Circadian variation of CBT in school refusal patients did not present a clear rhythm, and appearance time of their lowest CBT was markedly delayed compared to healthy subjects. Amplitude of circadian CBT changes, fitted to a cosinor curve by the least square method, was significantly smaller in school refusals than in healthy subjects. These findings suggest that in school refusal patients who do not have physical and psychiatric disorders, clinical psychosomatic symptoms (e.g., fatigue and memory disturbance) and school refusal could be closely related to the desynchronization of their biorhythms, particularly the circadian rhythm of body temperature and sleep-wake rhythm.

[Establishment of a new human ovarian serous cystadenocarcinoma cell line (IM), and influence on cell proliferations by cisplatin with or without hyperthermia].

A new cell line, designated IM has been established from operation material derived from a woman with ovarian serous cystadenocarcinoma. The population doubling time of the 35th passage IM cell was 28.8 hours. And it was successively subcultured 165 times in over 7 years, moreover still kept CA125 production. The nuclear DNA and cell surface CA125 antigen were double stained by propidium iodide and anti CA125 monoclonal antibody-FITC. Then the two color cytogram obtained by flow cytometry was drawn up. For the most part of CA125 positive cell retained G0 + G1 of cell cycle, the lesser part was in G2 + M phase. The S phase rate of IM cell incubated with cisplatin at 37 degrees C or 41 degrees C for 1 hour that estimated by BrdU-propidium iodide double stain method of flow cytometry, it was suggested that the inhibition of DNA synthesis by cisplatin was increased with 41 degrees C low hyperthermia.

Time course analysis of alpha+ beta+ T cell clones during normal pregnancy.

During normal pregnancy, the fetus continues to mature inside the uterus without rejection. Inherited paternal antigens could be targeted by the maternal immune system. These reactions are believed to play a role in a number of habitual abortions. However, the precise maternal mechanisms preventing fetal tissue rejection are not well understood. Maternal T cells should recognize fetal antigens, so it is conceivable that antigen-specific T cell response to fetal antigens would occur by proliferation and accumulation of certain T cell clones in the pregnant mother. To elucidate the maternal immune response to the fetus we investigated the clonality of expanded T cells in peripheral blood lymphocytes in ten normal pregnant women. We employed reverse transcriptase-polymerase chain reaction for T cell receptor beta chain gene and subsequently analyzed the PCR product by single-strand conformation polymorphism analysis. A large number of distinctly expanded T cell clones were detected during pregnancy. These accumulations were observed as early as the ninth to tenth week post-conception and reached a maximum during the second trimester, suggesting the existence of dynamic antigen-specific T cell responses in the pregnant mother. However, after the 30th week of gestation, nearly all expanded T cell clones disappeared before parturition and the degree of clonality reached almost normal levels. Our results clearly indicate the existence of dynamic maternal T cell responses during pregnancy.