Vitamin B12 R-binder (R-binder), a binding protein for cobalamin, is distributed preferentially in the normal endocervical epithelium. Endometrial epithelium is virtually negative for R-binder. The Rbinder expression in the uterine cervical and endometrial adenocarcinomas has not been reported. We investigated the immunohistochemical localization of R-binder in surgically resected cervical adenocarcinomas (CAs) and endometrial adenocarcinomas (EAs). Immunoreactivity was graded semiquantitatively. Seventeen of 20 (85%) CAs and 22 of 31 (71%) EAs showed R-binder immunoreactivity. Eight of 11 (73%) well-differentiated CAs and 8 of 23 (35%) well-differentiated EAs were strongly positive for R-binder (P<0.05). Immunoreactivity was seen in the membrane, cytoplasm and secretions of the tumor cells. Secretions in the well-differentiated CAs (9/10, 90%) were stained more frequently than those in well-differentiated EAs (8/17, 47%) (P<0.05). R-binder positivity may be more useful in the diagnosis of well-differentiated CAs than of well-differentiated EAs.
A murine monoclonal antibody, AD117m, was produced by fusion of murine myeloma cells with spleen cells obtained from mice immunized with intact OMC-4 (a human adenocarcinoma cell line derived from uterine cervix). The hybridoma that produced AD117m was selected from five colonies showing strong reactivity with OMC-4 but no reactivity with OMC-1 (a human squamous cell carcinoma cell line from uterine cervix). Since antigenic activity was reduced by treatment of the cells with periodate and enhanced by treatment of them with sialidase, the epitope of the antigen seemed to be expressed as a carbohydrate moiety. The AD117m-defined antigen was found not only in the glycoproteins and glycolipids of OMC-4 cells but also in a human meconium neutral glycolipid fraction. We purified the antigen from glycolipids of human meconium, and its structure was characterized by methylation analysis, mass-spectrometry, and sequential glycosidase treatment. The antigen structure was concluded as Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (lactotetraosylceramide). Immunohistochemical study using formalin-fixed and paraffin-embedded tissue sections showed that in the uterine cervix, all cases of adenocarcinomawere stained with AD117m (20/20), but AD117m reacted in only two out of ten cases of squamous cell carcinoma. In an effusion cytological study, AD117m was found to distinguish metastatic carcinoma cells from reactive mesothelial cells, and showed increased specificity and sensitivity when compared with antiCEA antibody. These observations indicate that AD117m is able to improve the differential diagnosis between reactive mesothelial cells and metastatic carcinoma cells in effusion cytology.
In the present study, we examined SBA lectin binding sites and patterns in hyperplastic dental epithelium overlying the crowns of pathologically unerupted molar teeth from congenital osteopetrotic (OP/OP) mice, and compared our findings with the pattern seen in the normal littermates. The hyperplastic dental epithelium of OP/OP mice disclosed preferential and intense staining of SBA. The lectin binding sites were demonstrated on membrane of the epithelial cells that interconnected with superficial gingival epithelium. At ultrastructural level, SBA labeling was detected over the desmosomal complexes of the epithelial cells. In normal mice, the reducing dental epithelium, as well as junctional epithelial cells, likewise demonstrated the lectin staining, but to a significantly lesser extent. The results of this study, taken together with suggested property of SBA binding, indicate that the hyperplastic dental epithelium in OP/OP mice might overexpress sugar sequences with N-acetyl-D-galactosamine on their cell surface, and this may be attributed to a functional alteration or degradation of such cell surface, due secondary to the pathological aberrations residing in OP/OP mice.
Rat prostate was stained immunohistochemically using polyclonal androgen receptor antibody. A rabbit polyclonal antibody referred as NH27 was raised against human androgen receptor. Male SpragueDawley rats were divided into four experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrified two days after castration. In group 3, rats were administered subcutaneously 1mg/ animal of testosterone daily for three days after two days of castration. In group 4, rats were given 10mg/ kg of TZP-4238, an anti-androgen agent, orally by gavage once a day for 1 week. Frozen sections of the prostates were fixed in Zamboni's fixative. The sections were incubated overnight at 4°C with NH27. In the control rat prostate, positive staining for the antibody appeared in nuclei of the glandular epithelium. In addition, immunodetectable androgen receptor remarkably decreased 2 days after castration and returned to intact levels after testosterone-administration in the castrated rats. These findings are in agreement with results of previous work on the prostate. Furthermore, nuclear immunostaining of the androgen receptor remarkably decreased after the treatment with TZP-4238. The precise mechanism which produces atrophy of the prostatic glandular epithelium is not yet clear from the present study. We further speculated that TZP-4238 was considered to be a steroidal androgen receptor antagonist. It is concluded that immunohistochemical analysis using NH27 may be a very useful method for predication of the effects of antiandrogen treatment for rats.
The distribution of neurons containing the calcium-binding protein parvalbumin was mapped in the subcortical auditory system of the mongolian gerbil. All nuclei of the auditory pathway expressed numerous neurons, which are imunoreactive with parvalbumin antiserum, with the exception of the meidal geniculate body, which is characterized by parvalbumin-positive neuropil. Soma area measurements of parvalbumin-positive neurons, their distribution patterns within a given auditory nucleus and in some cases also the configuration of their basal dendrites, indicate that parvalbumin-immunoreactivity is not restricted to one morphologically defined neuron population, but rather labels subpopulations of morphologically and/or physiologically defined neuron types. We speculate that parvalbumin-immunoreactive neurons may represent a network of highly active units with fast, highly synchronized firing patterns, which are adapted to rapid and precise processing and analysis of acoustic stimuli.
Immunohistochemical study of expression of calmodulin, S-100 α, β protein and cytokeratin K8.12 was carried out on normal and duct-ligated submandibular (SMG) and sublingual glands (SLG) of rats. The normal rat SMG showed calmodulin and S-100 α immunoreactivity in the pillar and transition cells of the granular convoluted tubule (GCT) and striated duct cells. S-100 β staining was positive in acinar cells of SMG as fine granular deposition. A trace immunoreaction product of keratin K8.12 was observed in GCT segments and striated ducts. Three days following duct ligation, immunostaining for calmodulin and S-100 β disappeared in both SMG and SLG, however, S-100 α staining persisted in pillar, transition and striated duct cells. The altered GCT cells of SMG and duct-like structures of SLG in the duct-ligated glands also showed S-100 α staining. In addition, K8.12 immunoreactivity was noted in the duct-like structures, particularly in those cells located luminally. The day 5 duct-ligated specimen of SMG retained positive S-100 α protein in the newly formed duct-like structures and the day 7, 10 and 14 specimens showed squamous metaplasia with positive K8.12 staining at the luminal side of the ducts. Myoepithelial cells in duct-ligated SLG stained positively for K8.12. It is concluded that loss of calmodulin from the ductal cells of SMG and SLG, and S-100 β from acini of SMG marks the earliest and predominant pathological alteration followed by increased proliferation of duct-like structures with cells containing cytokeratin K8.12 as pathological alteration of duct ligation of the salivary glands.
Metallothionein (MT) induction after cadmium administration in the seminal vesicle, coagulating gland, ampullary gland and Cowper's gland of the rat was immunohistochemically investigated. 1 week after castration, ten-week-old male Wistar rats were injected with testosterone propionate (1 mg/rat) once a day until the end of the experiment. After 3 weeks, rats were injected daily with a physiological saline, 0.3 mg/kg of Cd, and 0.9 mg/kg of Cd. There was no significant difference between each of the four tissue weights in the three groups. MT localization was observed mainly in the basal cells of the seminal vesicle, ampullary gland and Cowper's gland, and in the epithelial cells of the coagulating gland. MT was induced by cadmium administration mainly in the basal cells of the seminal vesicle, ampullary gland and Cowper's gland, and in the epithelial cells of the coagulating gland. Immunohistochemically, differences were observed in MT induction in the cells between the coagulating gland and other three glands, and MT was thought to prevent cellular damage from toxic metals.
In order to expand the possibility combining enzyme cytochemistry and immunocytochemistry, the post-embedding immunogold technique was applied to tissue sections stained by either the cerium-based or lead-based method for phosphatases. After enzyme cytochemical staining for alkaline phosphatase (ALPase) and acid phosphatase (ACPase), rat liver sections were dehydrated and embedded in the LR White without osmication. The ultrathin sections were labeled by post-embedding immunogold technique for anti-cathepsin D. Both the lead-based and cerium-based methods were sufficient to localize enzyme activities in the tissue before the immunoreaction. However, lead phosphate deposits, which were reaction products of phosphatases detected by the lead-based method, largely disappeared from the tissue embedded in LR White during the post-embedding immunoreaction. In contrast, cerium phosphate deposits, reaction products by the cerium-based method, were retained in the tissue even after the immunoreaction. Moreover, from the clear-cut difference in particle sizes, the cerium phosphate precipitates were readily distinguished from the immunogold particles within the same organelle. The preceding enzyme cytochemical procedures by the cerium-based method did not hinder the sensitivity and specificity of post-embedding immunogold labeling. These results indicate that the cerium-based method has the advantage of combining pre-embedding enzyme cytochemistry with post-embedding immunocytochemistry.
In the present work we have localized at the electron microscope level acetylcholine (ACh) in frog neuromuscular junctions, simultaneously in storage (synaptic vesicles) and releasing space (synaptic cleft) by a cytochemical method, previously described (19), which involved the precipitation of ACh-like cations with silicotungstic acid. The fixation took place in different physiological states (resting, during electrical stimulation and recovery). At rest, ACh was localized in the synaptic vesicles as point-like precipitates and in the synaptic cleft beneath some but not all the “active zones” as laminar precipitates. We think that the precipitates seen in the synaptic vesicles correspond to stored ACh, while those in the synaptic cleft to ACh released through the “active zones”. After a prolonged tetanic electrical stimulation, laminar precipitates covered the whole axoplasm of the nerve terminals, and it appeared that they extend beyond the nerve terminal membrane in contact with Schwann cells. The laminar precipitates found in the nerve terminals might correspond to a newly synthetized axoplasmic ACh (or leaked into the axoplasm from the synaptic vesicles) while the precipitates which filled whole synaptic cleft are presumed to be ACh released by electrical stimulation of the motor nerve. During the recovery period, the distributions of both types of precipitates became similar to those observed at rest. This cytochemical fixation method of ACh-like cations appears to be a useful “tool” for the quick capture of the neurotransmitter ACh in the synaptic vesicles, axoplasm and synaptic cleft, and therefore helpful for identification of cholinergic nerve terminals.
In order to cytochemically demonstrate the endogenous glandular source of hydrogen-peroxide (H2O2), which is a component of the salivary peroxidase antimicrobial system, we applied the cerium method to the Mongolian gerbil parotid gland. When NADH and NADPH were used as substrates of the H2O2 generating enzyme, reaction products were observed in association with the plasma membrane and pinocytotic vesicles of the myoepithelial cell. No reaction product was formed in the absence of these substrates. The reaction was inhibited by the addition to the complete medium of catalase (H2O2 scavenger) or of p-benzoquinone (O2-scavenger), or by heating prior to incubation. These results suggest that NADH- and NADPH-dependent H2O2 generating oxidase may be present in the myoepithelial cell. In addition, electron-dense precipitates, formed by the reaction of cerous ions, were also observed on the outer surface of the luminar plasma membrane of acinar and intercalated duct cells, and in the cristae and outer compartment of mitochondria mainly of duct cells. However, these reaction products were formed whether the substrates were contained in the cerium medium or not. Since the reaction was also weakened by catalase or p-benzoquinone, or by preheating, it is likely that some endogenous H2O2-generating factors, independent bf the exogenous substrates, may exist in these sites.
The localization of fucosylceramide recognized by a monoclonal antibody (MAb), PC47H, was examined immunohistochemically using normal and cancerous tissues. Immunoreactive fucosylceramide was observed in pancreas, parathyroid, submandibular gland, placenta, ovary, intestine, kidney, heart, plasma cells, adenocarcinomas of various organs and multiple myeloma, but not in lymphocytes and in squamous cell carcinoma. The immunoreactive fucosylceramide was localized mainly in the rough endoplasmic reticulum (ER) and plasma membranes of thes cells. Selective expression of fucosylceramide in particular organs and adenocarcinomas may reflect a role of fucosylceramide.
The localization of proteasomes (multicatalytic protease complexes) in human intestine was studied immunohistochemically. Positive staining for proteasomes was seen in absorptive epithelial cells, connective tissue cells in the tunica propria, smooth muscle cells in the lamina muscularis mucosae and the muscle layer, endothelial cells and nerve cells. A positive reaction was seen in the nuclei of the crypt cells, which were growing, and in the cytoplasm of the epithelial cells of the upper half of intestinal villi of the small intestine and the cells facing the intestinal cavity of the large intestine, which were not growing. These findings show that proteasomes are widely distributed in various types of cells suggesting that these protease complexes have essential roles for cell viability. Their intracellular localization suggests that they may be involved in cell growth and differentiation.
The localization of metallothionein (MT), a small molecular weight heavy metal-binding protein in the female human breast, was investigated using an immunohistochemical technique. Immunohistochemically, MT was found in myoepithelial cells of normal glands, ducts or cysts with fibrocystic disease, fibroadenoma and intraductal papilloma, and rarely in cancer cells. Positive staining for immunoreactive MT of myoepithelial cells was observed both in the cytoplasm and nucleus. Myoepithelial cells in the resting gland of a normal breast were strongly positive for immunoreactive MT, but their staining of the active glands was weaker than that of the resting glands. Myoepithelial cells in fibrocystic disease, fibroadenoma and intraductal papilloma of the breast showed strong positive staining for MT, were round or oval-shaped and arranged compactly in one layer and occasionally in two or more layers. Carcinoma was usually devoid of myoepithelial cells, which, however, were rarely found around the tumor. Cancer cells were slightly positive for immunoreactive MT and some tumor cells showed a strong positive staining.
Estrogen receptor (ER) -bearing parenchymal cells in the rat anterior pituitary gland were identified and classified immunocytochemically using antibodies against various anterior pituitary hormones as well as ER. The percentage of the respective pituitary hormone-positive cells among the total number of ER-immunoreactive cells (RH/ER) was higher in the order of somatotrophs, lactotrophs and gonadotrophs in both sexes. The proportion of ACTH- and TSHβ-positive cells among ER-immunoreactive cells was below 3%. The proportion of somatotrophs was significantly higher in the male. The RH/ER ratio of gonadotrophs and lactotrophs fluctuated during the sexual cycle of the female and both were highest at the proestrus stage. The present study provides immunocytochemical confirmation of many reports that estrogen regulates LH and PRL secretion via an estrogen receptor protein in all hormone-producing cells such as gonadotrophs and lactotrophs, and suggests that somatotrophs, which are more abundant in ER-immunoreactive paren-chymal cells, may also be under the control of estrogen.