Transduction of antisense cyclin D1 using two-step gene transfer inhibits the growth of rat hepatoma cells.

Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.

Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter.

We previously reported that the retroviral vector expressing the herpes simplex virus-thymidine kinase gene under the control of 0.3-kb human alpha-fetoprotein (AFP) gene promoter (AF0.3) provided the cytotoxicity to ganciclovir (GCV) in high-AFP-producing human hepatoma cells but not in low-AFP-producing cells. Therefore, specific enhancement of AFP promoter activity is likely to be required to induce enough cytotoxicity in low-AFP-producing hepatoma cells. In this study, we constructed a hybrid promoter, [HRE]AF, in which a 0.4-kb fragment of human vascular endothelial growth factor 5'-flanking sequences containing hypoxia-responsive element (HRE) was fused to AF0.3 promoter. By means of the reporter gene transfection assay, hypoxia-inducible transcriptions that were mediated by [HRE]AF promoter were detected in low- and non-AFP-producing human hepatoma cells, but not in nonhepatoma cells. When the herpes simplex virus-thymidine kinase gene controlled by [HRE]AF promoter was transduced into hepatoma and nonhepatoma cells by a retroviral vector, the exposure to 1% O2 induced GCV cytotoxicity specifically in the hepatoma cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors consisting of the virus-infected hepatoma cells gradually disappeared by GCV administration. These results indicate that the hypoxia-inducible enhancer of the human vascular endothelial growth factor gene, which is directly linked to human AFP promoter, involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.

Effect of a high cholesterol diet on lipid metabolizing enzymes in spontaneously hypertensive rats.

A high cholesterol diet induced a fatty liver and an increase in cholesterol oleate in spontaneously hypertensive rats. The activity of microsomal glycerophosphate acyltransferase in liver increased 2-3-fold to meet the increased supply of oleate, the synthesis of which was stimulated by a 10-fold increase in microsomal delta 9-desaturase activity. Hepatic fatty acid synthetase and diacylglycerol acyltransferase activities were decreased somewhat. These results, together with the fact that the large increases in hepatic cholesterol ester and triacylglycerol were not correspondingly reflected in plasma, indicated that the fatty liver resulted from decreased secretion of lipoprotein rather than increased lipogenesis. Endogenous cholesterol in liver microsomes increased 2-fold and hepatic acyl-CoA:cholesterol acyltransferase activity increased 3-fold, whereas plasma lecithin:cholesterol acyltransferase activity was unchanged. Thus, the increase in cholesterol oleate seen in spontaneously hypertensive rats fed a high cholesterol diet is due mainly to increases in acyl-CoA:cholesterol acyltransferase and delta 9-desaturase activities.

Biotin-containing intranuclear inclusions in endometrial glands during gestation and puerperium.

Intranuclear inclusions sometimes are observed in endometrial glands, especially during puerperium, and superficially they resemble those from herpesvirus-infected cells. To study the significance of the inclusions, endometrial and placental tissue from 215 subjects, including 69 pregnancy-associated samples, were examined. With screening by peroxidase-labeled avidin alone, followed by the peroxidase reaction, only pregnancy-related endometrium (in 32 cases) demonstrated reactivity in the intranuclear inclusions. These intranuclear inclusions demonstrated positive immunostaining for biotin using the peroxidase-antiperoxidase method in all but six cases, whereas there was no reaction for Herpes simplex in any of the cases. The endometrial tissues of the 32 cases were obtained from the 16th gestational week to the 37th postpartum day. Although the histogenesis of these intranuclear inclusions has not been clarified, they should not be mistaken for those in endometrial cells infected by herpesvirus because of intranuclear biotin and a false-positive histochemical reaction in the avidin-biotin-peroxidase complex method.

Effect of dietary alpha-linolenate/linoleate balance on mean survival time, incidence of stroke and blood pressure of spontaneously hypertensive rats.

Following the suckling period, stroke-prone spontaneously hypertensive rats (SHR-SP) were fed semi-purified diets supplemented either with safflower seed oil (rich in linoleic acid) or with perilla seed oil (rich in alpha-linolenic acid). The mean survival time of male SHR-SP fed the perilla diet was longer than that fed the safflower diet by 17% (p less than 0.001) while the difference was 15% in female SHR-SP (p less than 0.05). The mean survival times of female SHR-SP were more than 40% longer than those of male SHR-SP in both dietary groups. Post-mortem examinations of brains revealed apoplexy-related symptoms as the major cause of the death in both dietary groups. The systolic blood pressure was lower by ca. 10% (21 mmHg) in the perilla group than in both the safflower group and conventional diet group. The eicosapentaenoate (20:5 n-3)/arachidonate (20:4 n-6) ratio of platelet phospholipids in spontaneously hypertensive rat (SHR), a measure of platelet aggregability, was much higher in the perilla group than in the safflower group. Thus, increasing the dietary alpha-linolenate/linoleate ratio resulted in an increased mean survival time of SHR-SP rats, possibly by lowering blood pressure and platelet aggregability.

Adult T cell leukemia clinically manifested with ascites and pleural effusion.

A 62-year-old man with adult T cell leukemia (ATL) presented with complaints of anorexia and abdominal fullness. Evaluation revealed ascites and pleural effusion, but no lymph node swelling, hepatosplenomegaly, or skin involvement. The diagnosis of ATL was made by the detection of specific surface markers for T lymphocytes in pleural effusion and ascitic fluid, and by determination of human T cell leukemia virus type I (HTLV-I) proviral DNA integration in mononuclear cells of pleural effusion. This case was considered a rare type of ATL with infiltration of the pleura and peritoneum.

[A case of tracho-bronchial aspergillosis complicated with acute myeloblastic leukemia].

A 59-year-old male was admitted to our hospital in Jan. 1991 with complaints of general malaise and palpitation. Laboratory findings on admission showed anemia, thrombocytopenia and leukopenia consisted of 2.0% myeloblasts with Auerbodies. The bone marrow study showed granuloid hyperplasia with 45.5% myeloblasts. The diagnosis of acute myeloblastic leukemia (M1) was made. After BHAC-AMP therapy, he obtained complete remission. However, he complained of fever and cough, and his chest X ray film showed a focal infiltrative shadow in the right upper lung field. Antibiotics for bacteria and fungus were administered and the abnormal shadow improved in a week. However, as he had hemosputum, the bronchoscopic examination was performed, and multiple ulcers covered by yellow-white tissue were revealed on the wall of the trachea and bilateral main bronchi. Biopsy specimens obtained by transbronchial biopsy showed bronchial aspergillosis. Though intravenous infusion and inhalation of amphotericin B were effective for aspergillosis, he had a relapse of the leukemia and died in autumn, 1991.

[A case of malignant mesothelioma of the tunica vaginalis testis].

A case of malignant mesothelioma of tunica vaginalis testis experienced in an 83-year-old male who presented with a complaint of left intrascrotal pain is reported. The tentative diagnosis was acute epididymitis. Left epididymectomy was performed following the unsuccessful treatment with antibiotics. The histology of the surgical specimen revealed malignant mesothelioma. Radical excision of the left spermatic cord and scrotal content indicated locally advanced disease with positive surgical margin. Metastatic workup, including abdominal and chest computerized tomography scan, magnetic resonance imaging and bone scan demonstrated presence of metastasis to the paraaortic lymph nodes and the lumber spine. The patient is being followed conservatively in consideration of his age and advanced stage of the disease.

[A case of gastric cancer with recurrent bone metastases successfully treated with induced hypertension chemotherapy using cisplatin].

A sixty-eight-year-old female with bone metastases from gastric cancer successfully treated with induced hypertension chemotherapy using cisplatin is reported. She had undergone R2 curative subtotal gastrectomy in June 1985, and had orally taken tegafur 600 mg/day and then changed to doxifluridine 800 mg/day as postoperative adjuvant chemotherapy. Five months after the operation she had back pains and both 99mTc-MDP and 67Ga-citrate scintigram showed L1 vertebra and rib bone metastasis. Induced hypertension chemotherapy using cisplatin was then intermittently performed from January 1986 to September 1990, a single course of which was 25 mg/body div x 2/week for serial 4 weeks; a total of seven courses were carried out and consequently the total volume of the administered cisplatin reached 1,100 mg. Neither medullar nor renal toxicities were observed, but mild gastrointestinal symptoms were noted. The patient no longer has back pains, and no signs of bone metastases were seen on both scintigrams for two years and eight months from December 1988 to August 1991. This case is very rare because her bone metastases were successfully treated with induced hypertension chemotherapy using cisplatin. However, metastatic bone tumors from gastric cancer usually resist any treatments. It is expected that the successfully treated patients even with bone metastasis will be increasingly reported from now as various new approaches including induced hypertension chemotherapy are introduced.

[Pancoast syndrome in a patient with malignant lymphoma arising from the pleura].

A 79-year-old man was admitted to the hospital because of left shoulder pain. He presented with signs and symptoms typical of Pancoast syndrome. A chest roentgenogram and computed tomogram revealed a mass lesion that was connected to old pleural thicking due to tuberculosis in the left superior sulcus. A cervical lymph node biopsy was done and non-Hodgkin's lymphoma was diagnosed. Chemotherapy was effective but the patient died of severe pneumonia. Pancoast syndrome associated with malignant lymphoma is very rare. Care must be taken in the management of patients with shoulder pain, chest pain, and old pleural tuberculosis.

Malignant neurofibroma with glandular differentiation (glandular schwannoma).

A case of malignant shwannoma is reported with unusual elements in an 89-year-old female. A large mass was located in the subcutaneous tissue of the right lateral chest wall and measured 5 cm in the greatest diameter. Histologically the tumor was composed of neurofibroma and malignant schwannoma with glandular differentiation. Neurofibroma characterized by numerous hyaline neural nodules was located in the peripheral portion of the tumor, whereas malignant schwannoma occupied a large part of the central portion of the tumor. The glandular elements observed in some areas of malignant schwannoma consisted of cuboidal and columnar shaped cells and were arranged in tubular or tubulo-medullary fashion in which rosettes or pseudorosettes were found. Mucicarminophilic material was observed, both in the cytoplasm and in the lumen. Seven reported cases of peripheral nerve tumor with glandular differentiation are reviewed briefly.

Ultrastructural localization of endogenous peroxidase activity in benign thyroid diseases.

Ultrastructural localization of endogenous thyroid peroxidase under benign pathological conditions such as toxic diffuse goiter, non-toxic multinodular goiter, and adenoma, and in normal tissue was studied. Peroxidase activity was visualized by a cytochemical reaction for electron microscopy. In toxic diffuse goiters and most non-toxic multinodular goiters, reaction product for peroxidase was observed not only in the cytoplasm but also at the external surface of microvilli of follicular cells. In normal thyroid tissues and adenomas, peroxidase was visualized only in the cytoplasm. Peroxidase activity at the external surface of microvilli of the follicular cells was found in the tissues obtained from the goiters which showed "hot" radioiodine scintigram. These findings suggest that follicles with peroxidase activity at the external surface of microvilli in non-toxic multinodular goiter are "autonomous follicles" and that peroxidase at the external surface of microvilli plays some role in active iodine uptake.

Heterotopic brain of the tongue.

A 6-week-old male infant with lingual mass suffered from feeding difficulty and respiratory distress. Resected specimens revealed brain tissue composed of astroglia, oligodendroglia, ependyma, choroid plexus, neuronal cells, and primitive cells but no other elements suggestive of teratoma, therefore it was diagnosed as heterotopic brain of the tongue.

Effect of dietary alpha-linolenate/linoleate balance on brain lipid compositions and learning ability of rats.

Spontaneously hypertensive rats (SHR) and normotensive control, Wistar/Kyoto (WKY) rats through two generations were fed a semipurified diet supplemented either with safflower oil (rich in linoleate) or with perilla oil (rich in alpha-linolenate). The cerebral lipid contents and phospholipid compositions did not differ between the two dietary groups of SHR rats. There were also no differences in the unsaturated/saturated ratios of individual phospholipids or the proportions of plasma-logens. However, the proportions of (n-3) and (n-6) fatty acids were significantly different. Decreases in the proportions of docosahexaenoate [22:6 (n-3)] in phosphatidylethanolamine and phosphatidylserine in the safflower oil group were compensated for with increases in the proportions of docosatetraenoic [22:4 (n-6)] and docosapentaenoic [22:5 (n-6)] acids as compared with the perilla oil group. These differences in phospholipid acyl chains were much smaller than the difference in the proportions of linoleate and alpha-linolenate of the diets. In a brightness-discrimination learning test, the total number of responses to the positive and negative stimuli were less in the groups fed perilla oil. However, the alpha-linolenate-deficient group took longer to decrease the frequency of R- responses and therefore longer to learn the discrimination. Consequently, the correct response ratios were higher in the perilla oil groups than in the safflower oil groups. Thus, the dietary alpha-linolenate/linoleate balance influenced the (n-3)/(n-6) balance of polyenoic fatty acids differently among brain phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Localized primary amyloid tumor of the thyroid developing in the course of Hashimoto's thyroiditis.

A case of localized primary amyloid tumor of the thyroid gland developing in the course of Hashimoto's thyroiditis was studied using histochemistry, immunohistochemistry and electron microscopy. The patient was diagnosed as having Hashimoto's thyroiditis by histological examination of the thyroid and by the presence of a high titer of serum thyroglobulin and thyroid microsomal antibodies. In addition, the thyroid gland exhibited multiple nodular deposits of amyloid which were resistant to prior incubation with potassium permanganate. The amyloid deposits were surrounded by numerous histiocytes and multinucleated giant cells which contained small amyloid droplets in their cytoplasm. However, no amyloid deposits were observed in the walls of blood vessels. Immunohistochemistry showed that the amyloid was strongly positive for amyloid P component, IgG and kappa light chains. Ultrastructurally, the amyloid was composed of straight fibrils with a diameter of 7 to 10 nm. Histiocytes extended slender cytoplasmic processes in a radial fashion into amyloid fibrils, which exhibited a highly organized star-like pattern. This was considered to be an extremely rare case of localized primary amyloidosis of the thyroid, in which IgG, especially kappa light chains (AL), was present as a precursor protein.

Development of an inveterate gastroduodenal ulcer caused by antral G-cell hyperplasia of the stomach (pseudo-Zollinger-Ellison Syndrome): report of a case.

We describe herein the case of a 54-year-old Japanese woman in whom an inveterate peptic ulcer developed in association with pseudo-Zollinger-Ellison Syndrome (pseudo-ZES). The patient presented with weight loss and abdominal distension caused by antral and duodenal stenosis due to an inveterate peptic ulcer. Her serum gastrin level was very high; however, no evidence of a gastrinoma or carcinoid tumor was detected by preoperative examinations or surgery. A total gastrectomy and double-tract reconstruction was performed, and pathological examination revealed a gastric ulcer (UL-IV) with no histopathological evidence of a neoplasm. Immunohistochemical staining showed an obvious increase in the number of endocrine cells that were positive for chromogranin A, and marked G-cell hyperplasia was observed in the antral mucosa. Furthermore, the number of enterochromaffin-like cells was remarkably high. From the results of the immunohistochemical examination, the patient was diagnosed as having hypergastrinemia due to antral G-cell hyperplasia. Postoperatively, the patient's serum gastrin level fell rapidly to within the normal range, her nutritional status improved, and her weight increased by about 10 kg within 1 year.

[A case of pulmonary lymphangiomyomatosis diagnosed by biopsy of retroperitoneal tumor and treated with hormonal therapy].

A 34-year-old woman was admitted to our hospital because of exertional dyspnea and ascites. Chest roentgenogram showed hyperinflation of the lung and diffuse reticular shadows. Abdominal CT showed retroperitoneal tumors. Ascites was considered to be chylous. These findings strongly suggested the diagnosis of pulmonary lymphangiomyomatosis. Under lumbar anesthesia, laparotomy was performed, and the histological finding of the retroperitoneal tumor revealed angiomyoma. Bilateral oophorectomy was performed. Medroxyprogesterone at 15 mg/day was administered postoperatively. In advanced cases of this disease, open lung biopsy or even transbronchial lung biopsy is often avoided because of severe cardiopulmonary dysfunction. However, such cases associated with retroperitoneal tumor, laparotomy is quite useful not only in making the diagnosis but also in the treatment of this disease.

Intraplacental choriocarcinoma with fetomaternal transfusion.

Intraplacental choriocarcinoma is very rare, and is usually found only after maternal and fetal metastatic disease is identified. The purpose of this case report is to review the incidence and findings of intraplacental choriocarcinoma. A term placenta was investigated because the newborn was born with severe anemia (Hb 3.0 g/dL). A 2 cm nodule was noted on the surface of the amniotic membrane and grossly resembled an infarction. The tumor was examined microscopically with immunohistochemical staining for the alpha- and beta-human chorionic gonadotropin (alpha-hCG, beta-hCG) subunits, human placental lactogen (hPL) and Ki-67. Microscopically, the tumor consisted of necrotic areas with proliferation of atypical trophoblastic cells and destruction of the villi and capillaries. The cells were positive for the alpha-hCG, beta-hCG subunits, hPL and Ki-67, consistent with intraplacental choriocarcinoma. The mother and newborn were investigated for the presence of metastatic disease. Computed tomography scans and magnetic resonance imaging of the mother and infant were negative for metastatic disease. Choriocarcinoma, limited only to the placenta with no evidence of metastatic disease is very rare. Primary intraplacental choriocarcinoma may frequently be overlooked or missed, and choriocarcinoma may possibly arise in the placenta more often than in retained or persistent trophoblast following pregnancy.