Changes in expression and subcellular localization of nuclear retinoic acid receptors in human endometrial epithelium during the menstrual cycle.

The endometrium is a uniquely dynamic tissue in that it undergoes monthly cycles of proliferation and secretory activity, and is regulated by ovarian steroid hormones. In this study, we focused on retinoic acid receptors (RAR and RXR) which are ligand-dependent transcription factors belonging to the large family of steroid hormones and are expected to affect to cell growth and differentiation in the endometrium. We analysed the expression and subcellular localization of the RA receptors in 57 samples of human endometrium by immunohistochemistry and Western blotting. In the nuclei of the endometrial epithelium, the RA receptors were expressed strongly in the proliferative phase. However, RAR were drastically reduced in the epithelial nuclei during the secretory phase in association with changes in serum oestradiol and in the expression of the oestrogen receptor. The expression of RXR was localized in the epithelial nuclei throughout the menstrual cycle. Confocal laser scanning microscopical observation clearly showed the difference in the localization between RAR and RXR in the secretory phase. Furthermore the findings of immuno-electron microscopy showed pooled RAR around the rough endoplasmic reticulum, suggesting that transport of these receptors to the nuclei is inhibited. These findings suggest that RAR and RXR work mainly in the proliferative phase and that in the endometrium RXR may play a different role to RAR during the secretory phase.

Over-expression of heat shock proteins in carcinogenic endometrium.

We have previously shown that the subcellular localization of beta-catenin changes according to the cell proliferation status of the human endometrium, suggesting a role of intercellular transduction in cell growth control in human endometrium not only in the physiological but also in the carcinogenic condition. To further study the possible role of heat shock proteins (HSPs) in growth control, we immunohistochemically analyzed 92 endometrial samples, 30 of normal endometrium, 20 of endometrial hyperplasia and 42 of endometrial cancer, for expression of HSP27, HSP70, HSP90, estrogen receptor (ER) and progesterone receptor. HSP27 and HSP90 were detected in endometrial epithelium strongly in the proliferative phase and weakly in the secretory phase during the menstrual cycle according to the serum estradiol level. However, they were over-expressed in endometrial hyperplasia, especially HSP27. In endometrial cancer, HSP27 expression was heterogenic among the glands and lower than that in the proliferative phase and endometrial hyperplasia. HSP27 over-expression was also observed in samples including endometrial cancer and associated hyperplasia. Results of Western blotting followed those of immunohistochemistry. HSP70 was not changed during the menstrual cycle, as HSP27 and HSP90 were, and was rather stably expressed in endometrial hyperplasia and cancer. Our results suggest that HSP27 and HSP90 contribute to cell proliferation in endometrial epithelium and that over-expression of HSP27 in endometrial hyperplasia occurs as a result of the activated condition of ER, though in cancer it decreases according to the loss of function of ER.

HMB-45 staining for cytology of primary melanoma of the vagina. A case report.

BACKGROUND: Malignant melanoma in the vagina is very rare, but its diagnosis is usually easy if a melanin pigment is present. With cytodiagnosis, however, it is difficult to differentiate amelanotic melanoma or scantily pigmented melanoma from other conditions. In the present case, monoclonal antibody HMB-45, the efficacy of which has been established in histologic studies, was used in the cytodiagnosis of amelanotic melanoma in the vagina. CASE: A woman, aged 78 years, presented with a brownish, nodular tumor, diameter 3 cm, in the vagina. Scraping smears with Papanicolaou staining showed nonepithelial malignant cells without granules suggesting melanin. Smears stained with HMB-45 showed positive immunoreactivity. The diagnosis underwent histologic confirmation of amelanotic melanoma on the initial biopsy. CONCLUSION: Cytodiagnosis was made with HMB-45, which proved very effective in the differential cytodiagnosis of amelanotic melanoma and scantily pigmented melanoma, particularly because it obviated the need for tissue invasion.

Gene delivery systems using the Sendai virus.

Fusogenic liposome (FL) is a delivery system that can transfer encapsulated materials into living cells directly through membrane fusion. FL is a promising approach for gene therapy because it can deliver various genetic materials much more efficiently than other non-viral vectors without damaging the cell. FL-mediated gene transfer consists of two independent membrane fusion phenomena; generation of a FL by fusing a Sendai virus (SV) particle with a simple liposome encapsulating DNA, and successive fusion of the FL with cell membrane. The former requires viral F protein but no other special molecule on the liposomal membrane, whereas the latter may require the receptor (sialic acid) and unidentified assistant molecule(s) on the cell membrane. Further analysis suggests that these assistant molecule(s), not the receptor, may control the fusion and govern the cell specificity of FL-mediated delivery. This review has described a detailed analysis of these fusion phenomena and discussed possible applications of FL-mediated gene delivery to human gene therapy.

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

[Circulating immune complexes and anti-mite specific IgG antibody in status asthmatics--effects of methylprednisolone and analysis of an anti-complementary factor].

Serial changes of circulating immune complexes and anti-mite specific IgG antibody in the peripheral blood were measured eight times in six status asthmatics treated with high doses of methylprednisolone (MPS). Circulating immune complexes decreased to normal levels three hours after MPS administration. By contrast, anti-mite specific IgG antibody increased to beyond the normal range 14 days after MPS administration. Serum levels of IgE and allergen specific IgG4 antibody did not show any significant changes. In vitro experiments, circulating immune complexes in the sampled sera were decreased by an absorption test with anti-mite specific IgG antibody, and increased by reaction with an adequate volume of mite allergen. These facts indicate that circulating immune complexes are an anti-complementary factor working on the complement system, as previously reported, and that mite allergen and anti-mite IgG antibody relate to their conformation in status asthmatics. Clinically, MPS might possess an inhibitory effects on the formation of immune complexes and/or accelerate complement-dependent solubilization of antigen-antibody complexes, so that anti-mite specific IgG antibody increased markedly in the peripheral blood.

[Inhibition of Cls activity by methylprednisolone].

From a clinical study it was found that serum ClINH activity increased 6 hours after methylprednisolone (MP) pulse therapy in all the patients we studied with connective tissue diseases. Furthermore plasma ClINH-Cls complex decreased after pulse therapy. These phenomena lead us to investigate the effect of MP on Cls. 1) When a constant amount of Cls (8 micrograms/ml) was incubated with several concentrations of MP (2-50 mg/ml), the Cls activity of consuming C4 hemolysis was inhibited by MP in a dose-dependent manner. 2) MP inhibited consumption of C2 as well as C4 by Cls in a dose-dependent manner, even when MP had been removed by dialysis following incubation with Cls. These experimental data suggested that the trace amounts of Cls generated by immune complexes could be inhibited by long circulation of MP even at low concentrations in vivo, and resulted in a decrease of ClINH consumption and ClINH-Cls complex formation. These results indicated that the inhibition of Cls activity is one of the most important mechanisms in the process of the anti-inflammatory effect of MP in vivo.

[Successful bronchial arterial infusion (BAI) of ACNU in the treatment of pulmonary infiltration of acute non-lymphocytic leukemia (ANLL) cells].

A 35-year-old man was admitted to our hospital because of lumbago on March 25, 1988. On admission white blood count was 1,200/microliters with neutrophils of 9% and lymphocytes of 91%, hemoglobin level was 11.2g/dl and platelet count was 55 x 10(3)/microliters. Bone marrow smear showed 77% leukemic cell including non-specific or specific esterase-positive cells. Chest X-rays showed the presence of mediastinal tumor and diffuse reticular shadows. A diagnosis of ANLL was made and a hematological remission was obtained after one course of combination chemotherapy consisting of BH-AC, daunorubicin and prednisolone, but the enlarged mediastinal tumor and pulmonary infiltration worsened rapidly followed by marked dyspnea. This radiographic abnormal shadow was confirmed to be leukemic infiltration from the finding of transbronchial lung biopsy. We hesitated to give systemic chemotherapy because he also had had liver abscess. Accordingly we performed BAI of ACNU at a dosage of 150 mg which led to a dramatic improvement in dyspnea. 60Co therapy was performed on the mediastinal tumor. On May 30, when he had a relapse, he was unsuccessfully treated with systemic chemotherapy. The leukemic cells invaded most of the organs and the patient died on July 19, 1988. It is likely that BAI of ACNU for leukemic pulmonary infiltration was effective.

[Prognostic implications of the ploidy pattern of the nuclear DNA in hepatocellular carcinomas].

The nuclear DNA contents of paraffin-embedded specimens of 41 cases of a hepatocellular carcinoma have been measured by means of flow cytometry. Results have indicated that 25 cases (61%) were diploid and 16 cases (39%) were aneuploid. In the aneuploid cases, the serum AFP level was found to be higher and stage more advanced. We also found that patients with aneuploid tumors had a poorer prognosis than those with diploid tumors, this fact uncovered by means of a Cox's proportional hazard model. In conclusion, the ploidy pattern of the nuclear DNA may serve as a useful prognostic marker for a hepatocellular carcinoma.