The present study deals with the immunohistochemical evaluation of glial fibrillary acidic protein (GFAP) in salivary gland tumors and the results are compared with vimentin, cytokeratin (K8.12) and S100 protein. In normal tissue from tumors of the salivary gland, the myoepithelium did not express GFAP. In glandular tissues adjacent to tumor or invaded by carcinoma, GFAP was detected in flattened and spindle shaped cells that surrounded intercalated ducts and atrophic acinar structures. In pleomorphic adenoma, the nonluminal cells in tubulo-ductal structures and modified or neoplastic myoepithelial cells which formed solid, myxoid and chondroid structures showed intense immunoreactivity for GFAP. GFAP was also detected in basally located tumor cells in adenoid cystic carcinoma and papillary-cystadenocarcinomas. Those cells corresponding to the GFAP positive cells were also positive for vimentin, cytokeratin (K8.12) and S100 protein. The results of the present study allow us to suggest that the salivary gland tumors may originate from basal cells of intercalated ducts and the basal cells may be closely related with neural ectoderm.
Thrombomodulin (TM) is a kind of thrombin receptor which was identified originally on the endothelium and acts as a natural anticoagulant. However, we reported previously that TM was also expressed in the squamous epithelium mainly at the intercellular bridges (Am. J. Clin. Pathol. 88: 405-411, 1987). Our recent study revealed that TM was expressed in esophageal squamous cell carcinoma (SCC), and an inverse correlation of the TM expression with the lymph node metastasis was found: Tumor cells positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (Cancer Res. 55: 4196-4200, 1995). To investigate the detailed mechanism of TM expression in SCC cells and its relationship with metastasis, experimental studies using esophageal SCC cell lines are needed. The present preliminary study showed that esophageal SCC cell lines produced the same TM molecule as human endothelial cell line. Furthermore TM activity assay of esophageal SCC cell lines in this study demonstrated that the cancer cells positive for TM have anticoagulant activity. These findings may be useful for further experimental studies using esophageal SCC cell lines under various culture conditions or in animal models which may disclose a mechanism of the decreased TM expression in lymph node metastatic lesions.
Immunostaining of proliferating cell nuclear antigen (PCNA), which can be applied to routinely processed tissue sections, has extended the applications of cell kinetic studies. PCNA immunostaining intensity of cells varies among individual nuclei in tissue sections. To determine the changes in PCNA expression level during carcinogenesis, we employed an image analyzer to evaluate PCNA staining intensity in diisopropanolnitrosamine (DIPN) -induced neoplastic lesions of rat thyroid glands. In the normal thyroid gland, only small numbers of cells showed weak PCNA expression. The percentage of PCNA-positive cells increased during carcinogenesis, i. e. hyperplasia, adenoma, and then carcinoma. Analysis of PCNA staining intensity histograms revealed that the population of cells with stronger intensity increased during carcinogenesis, i. e. from adenoma to carcinoma. However, the percentage of cells with weak PCNA staining increased in hyperplasia, and did not change in intensity during further carcinogenic sequelae. These differences in PCNA expression level recognized by immunostaining intensity may reflect different cell kinetics between the process of hyperplasia and that of differentiation from adenoma to carcinoma.
Twenty seven cases of giant cell tumor of bone were evaluated for their cell kinetics by using proliferating cell nuclear antigen (PCNA) immunoreactivity and antiproteolytic and lysozymal activities in multinucleated giant cells and mononuclear cells in order to evaluate their cellular characteristics and possible histogenesis. The multinucleated giant cell nuclei were unlabeled by PCNA and the major proliferating cells in the tumor were identified as mononuclear cells. No significant correlation existed between the PCNA labeling index and histopathological gradings of the giant cell tumors. Mononuclear cells were either fibroblast-like cells or histiocyte-like cells. The fibroblast-like cells showed an intense immunoreactivity for vimentin and PCNA whereas the histiocyte-like cells were intensely reactive for lysozyme, alpha-1 antitrypsin, alpha-1 antichymotrypsin. The multinucleated giant cells, on the other hand, more frequently showed immunoreactivity of lysozyme, alpha-1 antitrypsin, alpha-1 antichymotrypsin mimicing the profile of histiocyte-like cells. The results of the present study allowed us to reasonably conclude that the fibroblast-like cells were the major proliferating cells in the giant cell tumors which may originate from the mesenchymal fibroblasts; the multinucleated giant cells do not form a proliferating population of cells but may be a fusion product of histiocyte-like cells possibly derived from the mononuclear phagocyte system. The conventional histopathological grading of the giant cell tumors of bone do not correspond with the cell proliferation potential in the tumor as assessed by PCNA immunohistochemistry.
Incorporated bromodeoxyuridine (BrdU) into DNA was detected in glutaraldehyde and osmium-fixed regenerating rat liver at the electron microscopic level. Without HCl hydrolysis, BrdU was localized in the nuclei of regenerating hepatocytes by post-embedding immunogold technique using the monoclonal anti-BrdU antibody on ultrathin sections after etching of the epoxy resin with 1% sodium ethoxide or 10% H2O2 followed by trypsin digestion. This procedure localized BrdU in routinely prepared specimens for electron microscopy simultaneously at a much finer structure than ordinary immunoelectron microscopy.
This report concerns whether the otoconia of the utricle and saccule removed from the inner ear of amphibia, especially of the frog Rana nigromaculata, are composed of calcite or aragonite. These compounds were identified by selective staining with alizarine red S, Harris' hematoxylin and Feigl's solution. The result showed that both the saccular and the utricular otoconia were stained red and purple with alizarine red S and Harris' hematoxylin, respectively, while only the saccular otoconia were stained by black precipitates with Feigl's solution which stains aragonite. Furthermore, mineral aragonite, which is an inorganic substance, was stained with Feigl's solution for the purpose of comparison with the saccular otoconia, which contain organic substances. Thus, the saccular otoconia and the mineral aragonite had the same staining appearance. These observations provide evidence that the saccular otoconia consist of aragonite, the utricular of calcite. The present, selective staining methods may be useful for studies, such as those focused on phylogenetic development, of calcitic and aragonitic otoconia in otolithic organs.
The pattern of availability of free DNA phosphates, and the kind of DNA-protein complex arrangement, both induced by nuclear basic proteins, and the richness in arginine residues in these proteins were investigated cytochemically and cytophysically in spermatozoa of the South-American Hylidae species, Hyla fuscovaria and Hyla biobeba. The aim was to demonstrate differences at the level of sperm histones in two species of Hyla until recently considered to be congeneric. The results indicated differences in the spermatozoal nuclear basic proteins and DNA-protein complexes when the two species were compared. The spermatozoa of Hyla biobeba were assumed to be likely to contain a Bloch's “type 3” protein type (intermediate sperm basic protein), similarly to Hyla species of North and Central America. On the other hand, the data obtained for the spermatozoa of Hyla fuscovaria indicated that they contain a protamine or protamine-like protein, differing from Hyla biobeba and Hyla species of North and Central America. It is suggested that the differences reported here may be genusspecific, since Hyla fuscovaria has recently been reclassified as Scinax fuscovaria based on parameters other than sperm histone types. These findings are in agreement with the general view of a wide variability in sperm nuclear proteins in the Anura group.
In receptor autoradiography, the tissue distribution of receptors is visualized in the form of ligand bindings. However, this visualization is indirect because we can only observe and measure nonspecific bindings and total bindings. The distribution of receptors is obtained by subtracting the nonspecific bindings from the total bindings. In receptor autoradiography, it is important to compare the distribution of receptors between two groups of samples obtained from two different regions of tissues and/or organs. However, no appropriate method of statistical analysis has yet been used to compare the distribution of receptors of two different groups. In this paper, we present a statistical method for this purpose and apply this method to an example.
Growth cartilage (GC) cells comprise distinct cellular zones of proliferating, hypertrophic and calcifying stages. To obtain representative cell clones of each GC stage, the author immortalized primary cultured rat GC cells by retro virus carrying neo gene, cytomegalo virus promoter and c-myc oncogene. The GC cells were freed from rat costal GC by enzymatic digestion. Retro virus carrying neo gene, cytomegalo virus promoter and c-myc oncogene was infected onto the cultured GC cells. After screening and cloning, 22 clones with different characteristics were obtained, and examined for their morphology, nodule formation, proteoglycan formation, alkaline phosphatase activity, fiber formation, reactivity to late GC specific antibody and response to 1-34 PTH. Depending on these examined characteristics, they were assessed as fibroblasts (2 clones), resting cartilage cells (3 clones), or as GC cells (5 clones as proliferating GC cells, 5 clones as early hypertrophic GC cells, 6 clones as late hypertrophic GC cells, and 1 clone thought to be either late hypertrophic or calcifying GC cell).
A gold chloride method for demonstration of retinyl esters (vitamin A) -storing stellate cells (Ito cells) in liver by Wake et al (Stain Technol. 61: 193, 1986) was modified. The procedure utilizes a microslicer instead of a freezing microtome. With the use of this procedure, the reaction product was fine and stable. Sections of the same tissue-block can be observed at the light, fluorescence and electron microscopic leveles simultaneously. The black reaction product produced between retinyl esters and gold chloride is localized in the vitamin A lipid droplets. Using a low concentration of gold chloride staining solution (0.001%-0.0001%), non-specific precipitate was significantly reduced at the electron microscopic level.
We evaluated the immunohistochemical localization of xanthine oxidase in various human tissues. Xanthine oxidase was purified from cadaver liver. Polyclonal antibody against xanthine oxidase was raised in a rabbit. Immunoblot analysis showed that the raised antibody reacted specifically with one band whose position corresponded with that of the purified enzyme. Immunostaining of paraffin-embedded tissue sections showed intense reactivity in the following tissues: surface epithelium of tongue, esophagus, and trachea, sweat glands, and mammary glands. Weak, but positive, reactivity was observed in other tissues, such as glandular cells of the small and large intestine and renal tubules, skeletal muscle, gastric epithelial cells, alveoli of the lung, spleen, and liver cytoplasm. Xanthine oxidase staining was observed in infiltrating lymphocytes (probably T-lymphocytes but not in B-lymphocytes) in inflammatory lesions of the small and large intestine. Its ubiquitous localization suggests that xanthine oxidase is involved in cell proliferation/differentiation, the defense mechanisms, and in the pathogenesis of reperfusion tissue injury.
Enhanced polymer one-step staining for proliferating cell nuclear antigen (EPOS-PCNA) consists of monoclonal mouse anti-PCNA and horseradish peroxidase coupled to an inert polymer backbone. This reagent was developed to shorten immunostaining time. In order to clarify the characteristics of this new immunohistochemical reagent, we applied this reagent to the formalin-fixed, paraffin-embedded NR-S1 tumors of mouse tongues, and the staining results were compared with those from conventional PCNA and BrdU immunostaining. Positive reaction for EPOS-PCNA was observed to be granular or diffuse in the tumor cell nuclei. These positive cells were scattered in the NR-S1 tumor. The distribution and staining patterns of EPOS-PCNA were similar to those of PCNA. Their labeling indices (Lls) were also similar, although the Lls in EPOS-PCNA were higher than those in BrdU. We conclude that EPOS-PCNA is a simple, timesaving, and reliable method for assessing cell kinetics of tumor tissues.
The present study concerns the first trial of cultivation of neonatal rat cerebral cortical neurons containing the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase [NADPH-d (+) neurons] in the organotypic static slice cultures. NADPH-d (+) neurons were scattered throughout the cortical layers and were highly concentrated in layers VI and II/III at the initial period, i. e. day 0, in culture of 7-day-old cerebral cortex. This distribution pattern was essentially the same as those seen after 28days in culture and in 35-day old rats. There were no apparent changes in the number of NADPHd (+) cells in the cortex at each time period examined (0, 10, 19 and 28days in culture). The size of cortical NADPH-d (+) neurons increased significantly, particularly during the first 10days, in culture. The numbers of primary neurites and their branching points, and the total neuritic length all increased significantly up to 19 days, but no further increase was observed at 28 days in culture. Also, the morphological features of NADPH-d (+) neurons after 19 and 28days in culture were almost the same as those in 35-day-old rats. Thus, cortical NADPH-d (+) neurons developed and reached the maturate state after 19 days in the cultivation system used here. These findings suggest that the organotypic static slice culture technique provides successful cultivation of cortical NADPH-d (+) neurons with normal-like development in situ, and should therefore be useful for studying the biology of these cells.
An immunohistochemical study was performed to detect the localization of neural-type nitric oxide synthase (nNOS), endothelialtype NOS (eNOS) and inducible-type NOS (iNOS) in the mouse and human nasal mucosa. nNOS-immunoreactive nerve fibers were observed in the subepithelial layer and around the seromucous glands of mice. In these fibers, immunodouble staining revealed colocalization of nNOS and superoxide dismutase. In the human nasal cavity, strong eNOS immunoreactivity and weak iNOS immunoreactivity were found in the columnar epithelium.
Complement system and granulocytes are both important effecters in host defence mechanism. Murine peritoneal neutrophils elicited with Streptococcus preparation (OK432) was found to synthesize and secrete the third component of complement (C3). In this report the localization of C3 in the neutrophils was investigated by immunoelectron microscopy. C3 was found predominantly in vesicles which might be the counterpart of the most easily mobilizable secretory vesicles of human neutrophils. Murine eosinophils from peritoneum elicited with OK432 were found for the first time to contain C3 and to be localized in their crystalline granules.