Hirohisa Kurachi

Binding Sites for Interleukin-6 in the Anterior Pituitary Gland

Interleukin-6 (IL-6) binding site in the rat anterior pituitary gland was characterized using radioiodinated human recombinant (hr) IL-6. Results showed that the anterior pituitary gland contained 170 binding sites per cell of a single class with a dissociation constant of 2.7 x 10(-9) M. The binding of 125I-hrIL-6 to the rat anterior pituitary gland was competitively inhibited by unlabeled hrIL-6, but not by hrIL-1 alpha, hrIL-1 beta, hrIL-2 or hr-interferon-gamma, indicating these binding sites are specific for IL-6. We also demonstrated mouse IL-6 receptor gene expression in the rat anterior pituitary gland by Northern blot analysis. Furthermore, the IL-6 receptor was detected on human gonadotrophs by the double immunofluorescence method. Our findings demonstrate the presence and expression of IL-6 binding site in the rat anterior pituitary gland and the presence of IL-6 binding site on human gonadotrophs, suggesting the important role of IL-6 binding site in pituitary hormone release in both species.

Uterine Cervical Cancer Displaying Tumor-Related Leukocytosis : A Distinct Clinical Entity With Radioresistant Feature

BACKGROUNDnTumor-related leukocytosis (TRL) is occasionally found in patients with nonhematopoietic malignancies. We investigated the clinical implication of TRL and individualized treatment for TRL-positive cervical cancer, as well as the underlying biological mechanism.nnnMETHODSnClinical data from 258 cervical cancer patients treated with definitive radiotherapy were analyzed to investigate the association between TRL and treatment outcome. Clinical samples, cervical cancer cell lines, and a mouse model of cervical cancer were used to examine the mechanisms responsible for TRL in cervical cancer, focusing on the role of tumor-derived granulocyte colony-stimulating factor (G-CSF) and myeloid-derived suppressor cells (MDSCs). All statistical tests were two-sided.nnnRESULTSnTRL was statistically significantly associated with younger age (Wilcoxon rank sum test, P = .03), larger tumor size (Wilcoxon rank sum test, P = .006), advanced clinical stage (χ(2) test, P = .01), and shorter overall survival (Cox proportional hazard modeling and Wald tests, P < .001). Among cervical cancer patients, TRL was associated with upregulated tumor G-CSF expression (χ(2) test, P < .001), elevated serum G-CSF levels (Student t test, P = .03), larger spleens (Student t test, P = .045), and increased MDSC frequencies in the blood (Student t test, P < .001) compared with the TRL-negative patients. In vitro and in vivo experiments revealed that tumor-derived G-CSF was involved in the underlying causative mechanism of TRL and MDSCs induced by tumor-derived G-CSF are responsible for the rapidly progressive and radioresistant nature of TRL-positive cervical cancer. The administration of anti-Gr-1 neutralizing antibody or the depletion of MDSCs by splenectomy (n = 6 per group) inhibited tumor growth and enhanced radiosensitivity in TRL-positive cervical cancer xenografts (Wilcoxon rank sum test, P = .008 and P = .02, respectively).nnnCONCLUSIONSnTRL is associated with resistance to radiotherapy among cervical cancer patients, and MDSC-targeting treatments may have therapeutic potential in these patients.

Changes of bioactive luteinizing hormone after laparoscopic ovarian cautery in patients with polycystic ovarian syndrome

The serum levels of bioactive luteinizing hormone (LH), immunoreactive LH, follicle-stimulating hormone, androstenedione (A), and testosterone (T) were determined in nine anovulatory women with polycystic ovarian syndrome (PCOS) before and after laparoscopic ovarian cautery. Eight ovulated spontaneously and three conceived after treatment. Before treatment, the mean (+/- SEM) levels of bioactive LH, immunoreactive LH, A, and T were 51.4 +/- 8.6 mIU/mL, 36.0 +/- 4.5 mIU/mL, 1.98 +/- 0.35 ng/mL, and 1.18 +/- 0.13 ng/mL, respectively, which were significantly higher than those of five control women (19.2 +/- 1.6 mIU/mL, 21.4 +/- 1.2 mIU/mL, 0.54 +/- 0.03 ng/mL, 0.28 +/- 0.03 ng/mL). After treatment, the mean levels of these hormones had significantly decreased. Decreases in the levels of these hormones by laparoscopic ovarian cautery in women with PCOS may result in both restoration of the ovulatory cycle and achievement of pregnancy.

Clonal analysis of high-grade squamous intra-epithelial lesions of the uterine cervix

We previously reported that invasive squamous cell carcinomas of the uterine cervix are of monoclonal composition. In the current study, we extended our previous work to determine the clonal composition of cases of high‐grade squamous intra‐epithelial lesion (HSIL). Clonal analysis targeting the HUMARA locus was performed on cervical tissue from 9 cases, 8 showing heterozygosity at the HUMARA locus and being, therefore, informative for clonality analysis. Uterine cervices were cut into 12 blocks, fixed with formalin and embedded in paraffin, and DNA was extracted from targeted lesions of each block. A total of 30 samples of cervical intra‐epithelial neoplasia 3 (CIN3) (14 samples of carcinoma in situ and 16 samples of severe dysplasia) and 1 sample of CIN2 (moderate dysplasia) were analyzed. Monoclonal composition of the lesions was demonstrated in 30/30 cases of CIN3. Polyclonal composition was seen in the single case of CIN2. In 6 uterine cervices, in which dysplastic lesions were present in more than 3 blocks, the pattern of X‐chromosome inactivation was the same in all lesions, suggesting that these individual lesions were derived from a single cell, with intra‐epithelial extension within the cervical mucosa. By contrast, one uterus contained 2 discontinuous dysplastic foci with different patterns of X‐chromosome inactivation, indicating that the 2 lesions developed independently from each other. Our results demonstrate that (i) lesions of CIN3 (severe dysplasia and carcinoma in situ) are composed of a clonal neoplastic population of cells and (ii) most cases of HSIL are unifocal in origin. Int. J. Cancer 73:339–344, 1997.

MR imaging of cervical carcinoma: comparison among T2-weighted, dynamic, and postcontrast T1-weighted images with histopathological correlation

Abstract.Background: To identify the reasons for misdiagnosis of the degree of stromal invasion by uterine cervical cancer with various magnetic resonance sequences.nnMethods: T2-weighted, dynamic, and postcontrast T1-weighted images were obtained in the sagittal plane in 20 patients with uterine cervical cancer. After evaluating these sequences for the degree of stromal invasion, histologic specimens were directly correlated with these images.nnResults: The degree of stromal invasion was correctly diagnosed in 15 of the 20 cases on T2-weighted images, in 12 on dynamic images, and in eight on postcontrast T1-weighted images. All misdiagnoses were due to overestimation. Histologically, peritumoral stroma showed inflammation or edema in two patients, whereas no histological abnormality was found in the other patients. A hyperintense rim, i.e., a peritumoral enhanced ring-shaped structure, was observed on the enhanced images of five patients. The hyperintense rim corresponded to the periphery of the tumor in three patients and to the cervical stroma in two patients.nnConclusion: T2-weighted images permitted the most accurate evaluation of stromal invasion by uterine tumors. Overdiagnosis may be due to an abnormal intensity of the cervical stroma, which was observed more frequently on dynamic and postcontrast T1-weighted images than on T2-weighted images.nn

Cervical Invasion of Endometrial Carcinoma — Evaluation by Parasagittal MR Imaging:

Twenty-seven consecutive patients were examined by T2-(1 800/70 ms) and postcontrast T1-weighted (600/15) spin echo (SE) or dynamic (200/15) SE MR imaging to determine the usefulness of parasagittal MR imaging in assessing cervical invasion of endometrial carcinoma. The images were obtained in a direction parallel to the longitudinal axis of the uterus (parasagittal). The cervical epithelium, being hyperintense on the late phase dynamic and postcontrast T1-weighted SE images, had disappeared partially or totally in all 4 patients with cervical invasion. The enhanced cervical epithelium was completely seen in one patient with the tumor protruding into the cervical canal in a polyp-like form without cervical epithelial invasion. The same was also seen in the 22 patients with the tumor remaining in the corpus cavity. The enhanced parasagittal MR images facilitated the evaluation of the extent of the endometrial carcinoma.

Treatment of endometriosis with a decreasing dosage of a gonadotropin-releasing hormone agonist (nafarelin): a pilot study with low-dose agonist therapy (“draw-back” therapy)

OBJECTIVEnTo evaluate the efficacy of half-dose GnRH agonist therapy for endometriosis.nnnDESIGNnProspective, longitudinal pilot study.nnnSETTINGnOsaka University Hospital.nnnPATIENT(S)nPatients with symptomatic endometriosis.nnnINTERVENTION(S)nFifteen patients were randomized to receive either full-dose nafarelin treatment (200 microgram b.i.d.) for 24 weeks (n = 7) or full-dose nafarelin treatment for 4 weeks followed by half-dose nafarelin treatment (200 microgram daily) for 20 weeks (n = 8).nnnMAIN OUTCOME MEASURE(S)nClinical symptoms and the results of physical examinations. Serum E(2) and carcinoma antigen 125 (CA125) levels, lipid profiles, and urinary levels of the N-telopeptide of type I collagen. Bone mineral density of the lumbar spine.nnnRESULT(S)nSubjective and objective manifestations of endometriosis were decreased to a similar extent in both study groups. Adverse effects were markedly reduced with half-dose administration. In the half-dose group, the mean serum E(2) level was significantly suppressed by 4 weeks of treatment with full-dose nafarelin and remained at approximately 30 pg/mL with half-dose nafarelin. Loss of bone mineral density was significantly less with half-dose treatment.nnnCONCLUSION(S)nHalf-dose administration of nafarelin after pituitary down-regulation with full-dose nafarelin (draw-back therapy) is a new protocol for the treatment of endometriosis that is effective and associated with fewer adverse effects.

fhit Alterations in endometrial carcinoma and hyperplasia.

The fhit (fragile histidine triad) gene on chromosome 3p14.2 is a candidate tumour‐suppressor gene; its abnormal transcripts are detected in several human cancers. To define the role of the fhit gene in the development of endometrial cancer, we examined 39 endometrial carcinomas for the presence of fhit gene alterations. fhit transcripts were analyzed by RT‐PCR and direct sequencing. Loss of fhit transcript was observed in 6/39 (15%) tumours. Aberrant fhit transcripts, with deletions and/or insertions, were observed in 7/39 (18%) tumours but not in any normal endometrium. Allelic losses at D3S1300 and D3S4103, both located within intron 5 of fhit, were detected in 6/25 (24%) and 5/22 (23%) informative cases respectively. Expression of fhit protein was detected by immunohistochemistry; fhit protein was strongly expressed in 8/8 proliferative phase and 5/5 secretory phase endometria, also in 5/5 atrophic endometria; and it was strongly expressed in 6/6 simple hyperplasias without atypia, 6/6 complex hyperplasias without atypia, and 11/11 complex hyperplasias with atypia. In contrast, loss or reduced expression of fhit protein was observed in 13/27 (48%) endometrial adenocarcinomas. The impaired expression of the fhit protein was significantly correlated with the histological grade of the tumours. The present data suggest that inactivation of the fhit gene is an important genetic event associated with the genesis of endometrial carcinoma, especially with tumours of higher histological grade, which are believed to emerge directly from an atrophic endometrium. Int. J. Cancer 85:306–312, 2000. ©2000 Wiley‐Liss, Inc.

Expression of Epidermal Growth Factor and Transforming Growth Factor-Alpha in Fallopian Tube Epithelium and Their Role in Embryogenesis

We studied the expression of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha in human fallopian tube epithelium at various menstrual stages. Immunohistochemical staining using anti-EGF and anti-TGF-alpha antibodies showed a specific staining in ampullary tube epithelium at late follicular and luteal stages but the staining was very weak at the early follicular stage. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) using beta-actin mRNA as an internal standard revealed the menstrual-stage-specific expression of EGF and TGF-alpha gene transcripts: amounts of EGF and TGF-alpha mRNA relative to those of beta-actin were significantly higher at late follicular and luteal stages than at the early follicular stage. To clarify the biological role of these growth factors, mouse 2-cell embryos were cocultured with human fallopian tube epithelial cells with or without blocking the action of these growth factors. Cocultures significantly promoted blastocyst formation, but this promotive effect of the tubal epithelial cells was completely abolished by the addition of anti-EGF and/or anti-TGF-alpha monoclonal neutralizing antibodies to the coculture system. These results demonstrated that EGF and TGF-alpha were synthesized and expressed in fallopian tube epithelium at specific menstrual stages, and may be involved in early embryonic development.

Ovariectomy increases the level of estrogen receptor mRNA and estrogen receptor binding sites in female rat adipose tissue

The roles of estrogen in the changes in estrogen receptor (ER) mRNA and ER binding sites in rat adipose tissue were studied in female rats. To elucidate the mechanism(s) behind ovariectomy (OVX)-induced obesity, the levels of ER mRNA and ER binding sites in adipose tissue were analyzed three weeks after OVX using Northern blot analysis of ER mRNA and the [3H]E2 binding assay, respectively. OVX induced an increase in body weight, and replacement of estradiol (E2) prevented that increase. Significant increases in the amounts of ER mRNA and in [3H]E2-specific binding were observed after OVX, and E2 replacement reduced both of those increases. These results suggest that E2 may regulate rat obesity directly through ER in adipose tissues.