Monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) induces apoptosis in primary renal cell carcinoma cells in vitro and inhibits tumor growth in vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. We investigate the susceptibility of human renal cell carcinoma (RCC) cells to TRM-1 and HGS-ETR2, 2 human monoclonal agonistic antibodies specific for TRAIL-R1 and TRAIL-R2, respectively. HGS-ETR2 effectively induced apoptotic cell death in 10 of 11 cell cultures, including 2 human RCC cell lines and 9 human primary RCC cell cultures, with a more pronounced effect after preincubation with anti-human IgG Fc. In contrast, TRM-1 was effective in only 1 primary RCC cell culture. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell-surface expression of the 2 TRAIL receptors, namely that TRAIL-R2 but not TRAIL-R1 was frequently expressed in most of the RCC cells tested. The activities of caspase-9, -8, -6, and -3 were increased with HGS-ETR2-induced apoptosis, and cell death could be blocked by specific caspase inhibitors for caspase-9, -8, and -3, and the general caspase inhibitor. In vivo administration of HGS-ETR2 with or without cross-linker significantly suppressed tumor growth of subcutaneously inoculated human RCC xenografts in immunodeficient mice. These results suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent in RCC.

Rational design, discovery, and synthesis of a novel series of potent growth hormone secretagogues.

In the joint experimental and computational efforts reported here to obtain novel chemical entities as growth hormone secretagogues (GHSs), a small database of peptides and non-peptides known to have GHS activity was used to generate and assess a 3D pharmacophore for this activity. This pharmacophore was obtained using a systematic and efficient procedure, "DistComp", developed in our laboratory. The 3D pharmacophore identified was then used to search 3D databases to explore chemical structures that could be novel GHSs. A number of these were chosen for synthesis and assessment of their ability to release growth hormone (GH) from rat pituitary cells. Among the compounds tested, those with a benzothiazepin scaffold were discovered with micromolar activity. To facilitate lead optimization, a second program, a site-dependent fragment QSAR procedure was developed. This program calculates a library of chemical and physical properties of "fragments" or chemical components in a known pharmacophore and determines which, if any, of these properties are important for the observed activity. The combined use of the 3D pharmacophore and the results of the site-dependent fragment QSAR analysis led to the discovery and synthesis of a novel series of potent GHSs, a number of which had nanomolar in vitro activity.

Rearrangements of actin cytoskeleton during infection with Escherichia coli O157 in macrophages.

The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as a fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as a fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.

Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157.

This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.

Detection of deoxyribonuclease I in a hormone-secretory pathway of pituitary cells in humans and rats.

High levels of deoxyribonuclease I (DNase I) were detected in the cytoplasm of hormone-secreting cells in anterior and intermediate lobes of human and rat pituitary glands. Tissue homogenate fractions of anterior and intermediate pituitary lobes showed strong DNase I-specific enzymatic activity. Immunofluorescence, with several specific antibodies against human and rat DNase I, indicated strong immunoreactivity in the cytoplasm of hormone-secreting cells within the gland. In situ hybridization demonstrated high levels of DNase I mRNA in the cells. By immunoelectron microscopy, immunogold particles were found in the rough endoplasmic reticulum, Golgi apparatus and secretory granules. Exocytotic secretory granules were also immunopositive. These results indicate that endocrine cells in the pituitary gland synthesize and secrete DNase I together with their respective pituitary hormones.

Detection of deoxyribonuclease I along the secretory pathway in Paneth cells of human small intestine.

The expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen.

Awareness of and attitude of elderly subjects regarding health care and welfare in rapidly ageing population in Japan.

OBJECTIVES: We aimed to obtain information on the degree of knowledge and understanding about the current systems of health care and welfare held by the elderly, in order to achieve comprehensiveness in family practice. METHOD: We conducted a study on the awareness of healthy elderly persons by direct interview. The study was carried out in Kuni Village in a remote mountainous region in Japan, where the elderly population accounts for 24.8% of the total population. The subjects were self-dependent in their daily living activities and were aged 65 years and older. RESULTS: The subjects' knowledge of health care and welfare systems was generally good, and the degree of their utilization of these systems was also good. But 83.3% of those who did not want to utilize the welfare system indicated their preference to depend on their family for support. CONCLUSION: Family physicians must endeavour to offer comprehensive care to their patients by including these systems for rapidly ageing communities.

Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A.

When AR42J cells, an amylase-secreting pancreatic exocrine cell line, were treated with activin A, cells extended neuritelike processes, and, concomitantly, amylase-containing vesicles disappeared. Immunofluorescence and immunoelectron microscopy revealed that these processes had neurite-specific cytoskeletal architectures: neurofilaments and microtubule bundles with cross-bridges of microtubule-associated protein 2. In addition to such morphological changes, activin-treated cells exhibited a marked increase in cytoplasmic free calcium concentration in response to depolarizing concentration of potassium. Moreover, activin-treated AR42J cells expressed mRNA for alpha 1 subunit of the neuroendocrine/beta cell-type voltage-dependent calcium channel. In naive AR42J cells, a sulfonylurea compound, tolbutamide, did not affect free calcium concentration, while it induced a marked elevation of free calcium in activin-treated cells. Single channel recording of the membrane patch revealed the existence of ATP-sensitive potassium channel in activin-treated cells. These results indicate that activin A converts amylase-secreting AR42J cells to neuronlike cells. Given that pancreatic endocrine cells possess neuronlike properties and express ATP-sensitive potassium channel as well as neuroendocrine/beta cell-type voltage-dependent calcium channel, activin treatment of AR42J cells may provide an in vitro model system to study the conversion of pancreatic exocrine cells to endocrine cells in islets.

Effect of nizofenone on experimental head trauma in mice.

The therapeutic effect of nizofenone, a cerebroprotective drug, on experimental traumatic head injury was investigated in mice. The animals under halothane anaesthesia were subjected to a severe concussive head trauma and their neurological status was assessed by a string grip-test for 6 consecutive days. The neurological function of the control groups fell markedly 1 hr after trauma and slowly recovered day by day, but some animals failed to restore to the normal level, even on the 6th day. The post-treatment of the head-injured mice with nizofenone (3 mg/kg/day, i.p.) significantly improved the neurological function. The neurological improvement by nizofenone was dose-dependent and a significant amelioration was observed at 0.3 mg/kg/day. Methylprednisolone (30 mg/kg/day, i.v.) did not exert any significant neurological improvement in this therapeutic, experimental condition. In this study, nizofenone treatment was found to beneficially influence the recovery of the neurological function, maybe by reducing the progress of brain injury following head trauma.

[Morphological methods for analysing hormone secretory mechanism in the pituitary cells].

We introduce here two morphological methods of the rat anterior pituitary cells. In the first experiment, we showed immunofluorescence combined with a confocal laser scanning microscope. Distribution of actin was examined in growth-hormone (GH) secreting cells and folliculo-stellate cells and compared between in vivo and in vitro cells. In tissue, actin immunoreactivity was mainly limited near the plasma membrane. In cultured cells localization of actin clearly changed and the cells sometimes showed stress fiber-like structures in the cytoplasm. The cells also showed a small amount of alpha-actinin- and myosin-like immunoreactivities along stress fiber-like structures, which cannot be detected in the tissue cells. These data clearly showed differences in the localization of cytoskeletal proteins between in vivo and in vitro. In the second experiment, we showed electron microscopic analysis combined with monitoring hormone secretion in vivo. A catheter was inserted through the left external jugular vein into the right atrium of the heart. Through the catheter GH-releasing factor (GRF) and/or somatostatin was administered to freely-moving rats. Only about 30% of rats showed an increase in blood GH after GRF injection. Such responded rats were selected for our morphological analysis. From 5 min after GRF injection, the rats were infused with somatostatin or saline for 10 min. The treatment of somatostatin clearly diminished exocytotic figures from the plasma membrane.

[Quality assurance for histodiagnosis and cytodiagnosis with a model of proficiency testing].

A quality assurance (QA) program for histopathology and cytology has not yet been completed although an inhouse quality control for technical standardization is being tested. The fact that a strict Performance Improvement Program of CAP (College of American Pathologists) for cytology is required separately from other laboratory tests emphasizes the importance of cytology, since erroneous cytology reports would directly cause incorrect clinical diagnosis. The results of questionnaires gathered by the QA Committee of the Japan Association of Registered Clinical Laboratories on preparation techniques, workload limits of pathologists and technologists, double diagnostic systems, error detection programs, and specialization of personnel are presented. In addition to pathology and cytology proficiency testing provided by CAP survey, a model of blind QC (quality control) in our laboratory is discussed; for diagnostic standardization of histopathology, reexamination of randomly selected cases by another pathologist and reexamination of previously diagnosed cases by the same doctor are periodically performed. For error detection of cytoscreening, cytologists are obligated to reexamine random samples of 1 to 10% of negative gynecological slides. Evaluation of sufficiency of slides as required by the Bethesda System is referred to diagnostic interpretation.

Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells.

Incubation of Caco-2 cells, a human intestinal cell line, with 25-hydroxycholesterol (25-HOC) marked enhanced cellular cholesteryl ester formation determined by incorporation of [14C]oleic acid into intracellular cholesteryl [14C]oleate. The stimulation by 25-HOC of cholesteryl ester formation was suppressed by staurosporine, a kinase inhibitor, but not by cycloheximide or actinomycin D. The specific activity of microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT) increased two-fold in cells treated with 10 microM 25-HOC for 5 h. ACAT activity decreased when microsomes were incubated without sodium fluoride, a phosphatase inhibitor, but the decrease in ACAT activity in cells stimulated with 25-HOC was more pronounced. The results suggest that protein phosphorylation may be involved in the stimulation of cholesteryl ester formation by 25-HOC in Caco-2 cells.

Morphological effects of somatostatin on rat somatotrophs previously activated by growth hormone-releasing factor.

Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.

A clinicopathologic study of tumors and tumor-like lesions of the penis.

A total of 147 specimens from 93 patients with penile lesions were examined at Nagasaki University Hospital during a 27-year period from 1961 to 1987. The most frequent malignant tumor was squamous cell carcinoma (SCC, 33 cases, 35.5%), followed by extramammary Paget's disease, transitional cell carcinoma, and Bowen's disease. The benign tumors and tumor-like lesions included condyloma acuminatum, cyst of the genitoperineal raphe, and lymphangioma. SCC of the penis accounted for less than 0.1% of all specimens and less than 0.62% of malignant tumors in men filed at our hospital. True phimosis accompanied 81.5% of the SCC cases. The 5- and 10-year survival rates for SCC were 77.2% and 71.3%, respectively. Two patients died of penile SCC. It was considered that an absence of both keratohyaline granules in the granular layer and melanin pigment in the basal layer can serve as a useful histologic indicator for diagnosis of well differentiated SCCs that are otherwise difficult to identify.

Thymolipoma with striated myoid cells. Histological, immunohistochemical, and ultrastructural study.

A large asymptomatic anterior mediastinal thymolipoma, discovered by chest radiograph during a regular check-up for company employees, was excised from a 33-year-old Japanese man. On immunohistochemical and electron-microscopic examination, clusters of myoglobin-positive cells with cytoplasmic Z band structures were found scattered in the medulla. Myoid cells have been previously seen in the normal thymus as well as in thymic hyperplasia, thymoma, and in the thymus of patients with myasthenia gravis. To our knowledge, this is the first reported instance of myoid cells in a thymolipoma.

[Histogenesis of the clear cytoplasm in the tumor cells of smooth muscle origin].

Perinuclear clear cytoplasm observed in the light microscopic specimens of the tumor cells of smooth muscle origin is in general, understood as the artefact caused by the formalin fixation. However, the precise mechanisms of the histogenesis of clear cytoplasm are still not clear. We observed the clear cytoplasm directly by mean of the electron microscopy of materials detached from light microscopic specimen. Furthermore, we observed the light microscopic specimens made by varying types of methods, examining whether the clear cytoplasm was present or not. The electron microscopy of materials detached from light microscopic specimens revealed the band-like defects of cytoplasm along the long axes of tumor cells. These defects were thought to result from the falling off of cytoplasm. The 1 mu section of the epon embedded block derived from the paraffin embedded block for light microscopic specimen presented no clear cytoplasm, suggesting that the cytoplasm falls off at the procedure of deparaffinization and staining. Although the specimen of conventional frozen section showed no clear cytoplasm, the specimen made by the frozen sectioning after formalin fixation revealed clear cytoplasm. Consequently, it is thought that the fixation of the tissue before the sectioning makes the cytoplasm fragile, thereafter, the cytoplasm falls off at the procedure of deparaffinization and staining.

Morphological analysis of growth hormone release from rat somatotrophs into blood vessels by immunogold electron microscopy.

The ultrastructure of GH release from the somatotrophs of the rat anterior pituitary was examined in vivo by immunogold electron microscopy. Under pentobarbital anesthesia, injection of GH-releasing factor clearly induced an increase in both plasma GH content and the number of exocytotic GH-immunopositive granules in the cells. The exocytotic events occurred from a part of the plasma membrane facing endocrine cells, including other somatotrophs, and other portions facing folliculo-stellate cells or the walls of blood vessels. When released from the plasma membrane, the GH-immunopositive secretory granules sometimes appeared to aggregate with each other and showed an irregular shape surrounded by a single unit membrane. The exocytotic secretory granules were released into the extracellular space, and then flowed into the sinusoids as irregularly shaped GH-immunopositive electron-dense masses. After reaching the vascular space via the intercellular spaces between the endothelial cells, the contents of each mass became diffusely dispersed into the blood stream, with concomitant disappearance of immunopositivity. The present study thus revealed the morphological aspects of the process of GH secretion from somatotrophs into the blood vessels.

[A case of primary intracranial malignant melanoma with characteristic magnetic resonance imaging].

The authors reported a rare case of primary malignant melanoma in the central nervous system and discussed the findings of MRI. A 60-year-old male was admitted for examinations to discover the cause of his generalized tonic convulsions. On admission, he had neither neurological deficits, nor were there any abnormalities revealed during physical examination. Ct scan disclosed a slightly high-density mass with perifocal edema in the right parietal cortex which enhanced markedly after injection of contrast material. This lesion was hypointense on T1 weighted image of MRI (GE: 1.5 Tesla) and a hyperintense band was observed on the surface of the tumor. On T2 weighted image, the tumor showed iso-hypointensity surrounded by an increased signal area compatible with edema. On August 31, 1988, a gross total removal of the tumor was performed. Microscopically, it was identified as a malignant melanoma. No melanoma was found in other parts of the body during careful examinations, especially in dermatologic and ophthalmologic examinations. Characteristic findings of the hyperintense band on T1 weighted image coincided well with the pathological findings of excessive melanin deposition on the surface of the tumor, and which resulted from the paramagnetic free-radicals in the melanin. MR image may be useful for differentiation of intracranial malignant melanomas from other mass lesions.

Somatostatin-containing neurons in the mouse brain: an immunohistochemical study and comparison with the rat brain.

The distribution of somatostatin-containing neurons in mice of both sexes was immunohistochemically examined and compared with that in rats. In radioimmunoassay the relative somatostatin content in the mouse brain was 2-3 times higher than that in the rat. The overall immunohistochemical staining for somatostatin was much stronger and more prominent in the mouse than in the rat. Although the distribution pattern of somatostatin immunoreactivity was basically the same between the two animals, several regions, especially the nucleus anterior hypothalami and the nucleus interpeduncularis, were found to contain large aggregates of somatostatin-immunoreactive neurons in the mouse brain but not in the rat. The electrolytic lesions to the nucleus anterior hypothalami caused a marked decrease in somatostatin immunoreactivity of the outer layer of the median eminence in the mouse. This suggests that the nucleus anterior hypothalami is an additional source of somatostatin for the median eminence in the mouse. The differences recognized between the species are interesting from functional and evolutionary points of view.