Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.

Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors.

Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and mid- to late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 microm to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. (J Histochem Cytochem 46:911-934, 1998)

Immunohistochemical study of lymphoid tissue in human fetal salivary gland.

Immunoreactivity of lysozyme (LY), lactoferrin (LF), alpha 1-antichymotrypsin (alpha 1-ACT), alpha 1-antitrypsin (alpha 1-AT), keratin proteins KL1, PKK1, K8.12, S-100 protein, MAM-3, MAM-6, and epithelial membrane antigen (EMA) were evaluated in lymphoid and glandular tissues of developing salivary gland of human fetus (gestational age ranging from 17 to 40 wk to investigate the role of lymphoid tissue in developing salivary glands. In a total of 79 cases, lymphoid cell aggregations were noted in parotid (57 cases), submandibular (21 cases) and sublingual (5 cases) glands. Mononuclear cells showing intense activity of LY, alpha 1-ACT and alpha 1-AT were present in the lymphoid aggregation. The glandular ducts embedded in lymphoid tissue were negative to MAM-3, MAM-6, EMA and S-100 protein, but showed positive PKK1 and KL1 reaction during early stages of development, and showed degeneration and effacement upon increase in number and LY activity of the mononuclear cells. The lymphoid aggregations progressively emerged as lymph nodes.

Vimentin expression in benign and malignant lesions in the human mammary gland.

The distribution pattern of vimentin in mammary gland lesions, including benign lesions (36 cases) and adenocarcinomas (40 cases), was studied with the use of a monoclonal antibody. In myoepithelial cells of mastopathy, the positive vimentin staining was seen in 6 cases (33.3%). Gynecomastia showed strong vimentin expression in myoepithelial cells. Fibroadenomas (14 cases) expressed positive vimentin staining in 8 cases (57.1%) with various staining intensities in myoepithelial cells. Lactating mammary gland tissue was positive for vimentin staining in narrow myoepithelium. In adenocarcinoma, positive reactions in tumor cells were found in 17 cases (41.2%) of solid tubular, 12 cases (33.3%) of papillo-tubular, and 11 cases (54.5%) of scirrhous carcinomas. The staining intensity for vimentin was weak in modified myoepithelial cells and neoplastic cells. Malignant cells in mammary carcinomas with vimentin reactivity usually expressed keratin staining; thus a co-expression of vimentin and keratin was found in mammary carcinomas.

Neuron-specific enolase reduction in irradiated salivary glands of the rat--an immunohistochemical study.

Neuron-specific enolase (NSE) in the salivary glands of normal and irradiated rats was studied by immunohistochemical methods. The normal salivary gland of Sprague-Dawley rats (465 g) showed positive staining for NSE in striated ducts and granular convoluted tubule (GCT) cells. A single radiation, TDF (time, dose, fractionation factor) 100 and 200 (18.82 Gy and 27.97 Gy, respectively) was done, and 4 groups (1, 2, 3 and 4 weeks after radiation) of 5 rats each were used. Irradiated salivary glands indicated a remarkable reduction of NSE staining in GCT cells and a reduction but to a lesser degree in the striated duct of the submandibular gland. Immunohistochemical deposition of NSE was not changed in the sublingual glands of irradiated rats. The reduction of NSE immunodeposition was irradiation dose dependent.

Immunoreactivity of calmodulin, S-100 protein alpha and beta subunits in rat submandibular glands.

The calcium-binding proteins calmodulin and S-100 protein alpha and beta subunits were studied in Rat submandibular salivary gland using monoclonal antibodies and immunoperoxidase staining. Histologically, Rat submandibular gland consists of acinar and ductal components. The latter include intercalated, granular convoluted tubule, striated and excretory ducts. Ultrastructural studies of adult Rat granular convoluted tubule show granular cells, pillar cells and transition cells. Immunostaining for S-100 alpha and calmodulin was observed in granular convoluted tubule pillar cells and transition cells, whereas cells of striated ducts and intercalated ducts stained positive for calmodulin. S-100 beta staining in acinar cells was visible as a fine granular pattern. Although immunohistochemical localization in rat submandibular gland differed for S-100 alpha and beta subunits, expression was similar for S-100 alpha and calmodulin in the granular convoluted tubule segment. No immunodeposition of calmodulin, S-100 alpha or S-100 beta was found in granular cells, which contain growth factors such as EGF and NGF.

Vimentin expression in sweat gland tumours.

Immunohistochemical localization of vimentin was studied in 93 cases of sweat gland tumours using a monoclonal anti-vimentin antibody. A strong immunoreactivity of vimentin was observed in modified myoepithelial or neoplastic myoepithelial cells of mixed tumour of the skin, syringoma, and sweat gland adenoma. Tumour cells in outer layers of tubular, ductal, and duct-like structures usually showed positive staining for vimentin, which coincided with modified myoepithelial cells. All tumour cells of clear cell hydroadenoma showed positive vimentin staining. Tumour cells of the luminal border of tubulo-ductal structures of mixed tumours were rarely immunoreactive for vimentin. Positive vimentin staining of tumour cells in the outer zone of tubulo-ductal structures in sweat gland tumours may be related to reactive proliferation of modified myoepithelial cells and simultaneous growth of luminal tumour cells.

Immunohistochemical study of neuron specific enolase expression in salivary-gland tumors.

The immunoreactivity of monoclonal anti-neuron-specific enolase (MoAb NSE) on 10% formalin-fixed sections of normal human salivary glands and tumors were examined by the avidin-biotin-peroxidase complex (ABC) method. MoAb NSE staining of ductal and acinar cells of the normal salivary glands were, negative, and peripheral nerve fiber in the gland tissue showed strongly positive staining. In salivary gland tumors, pleomorphic adenoma (34 in total 68), myoepithelioma (15 in 35), monomorphic adenoma (7 in 10), adenolymphoma (15 in 23), mucoepidermoid carcinoma (3 in 16), acinic cell carcinoma (1 in 7), adenoid cystic carcinoma (12 in 20) and sialocarcinoma (4 in 15), stained focally for MoAb NSE staining. Frequency of occurrence for positive NSE activity varies among benign and malignant salivary gland tumors. In addition to MoAb NSE, S-100 protein and GFAP also demonstrated positive reactions in pleomorphic adenoma (Simpson et al Cancer 54: 1364-1369, 1984). The outer layer cells and/or peripheral cells of tubulo-ductal structure as well as modified myoepithelial cells and the cells of solid structure coexpressed with NSE, S-100 protein and GFAP. It is postulated that the salivary gland tumors particularly pleomorphic adenoma may be neuroectodermal in origin, arising from stem cells in intercalated duct segments.

Immunohistochemical evaluation of mam-3 and mam-6 antigens in squamous-cell carcinoma of the oral cavity.

MAM-3 and MAM-6 antigens of human milk fat globule membrane designated as human polymorphic epithelial mucin (PEM) were detected immunohistochemically in a total of 86 specimens from 39 cases of oral squamous cell carcinoma (SCC) and 6 cases of normal oral mucosa. The two antigens were identified by MoAbs 67D11 and 115G3 (MAM-3 antigen) and by MoAbs 115D8 and 115F5 (MAM-6 antigen) in paraffin sections. MoAb 115G3 staining was found in well keratinized foci; whereas, MoAb 115D8 and 115F5 staining was very low in keratinized tumor cells. The cytochemical distribution of the antigens was classified into 3 types; (i) cell membrane positive type, (ii) whole cell positive type and (iii) antigen negative type. Reduction and disappearance of the two antigens were correlated to the mode of tumor invasion and suggested that glycocalyx complex in cell membrane properties was changed for malignant transformation.

Heterogeneity of keratin expression and actin distribution in benign and malignant mammary diseases.

Immunoreactivity of monoclonal anti-cytokeratin KL1, PKK1, K8.12 and anti-actin antibodies in 101 cases of diseased human breast lesions showed irregular keratin distribution in luminal cells of terminal ductal-lobular unit and basal layer cells of the interlobular and main duct. Actin staining was confined to myoepithelial cells. Benign lesions showed great heterogeneity in luminal cells of the terminal ductal-lobular units. Breast carcinoma showed a reduced staining for keratins, heterogeneity of keratin expression was found in solid tubular carcinoma, and actin was usually absent: however, papillo-ductal or comedo type had actin positive myoepithelial cells around carcinoma foci.

Immunohistochemical localization of MAM-3 and MAM-6 antigens in adenoid cystic carcinoma.

MAM-3 and MAM-6 antigens were detected immunohistochemically in 34 cases of adenoid cystic carcinomas (ACC) of the salivary glands and these patterns were compared to these of epithelial membrane antigen (EMA) and laminin. ACC was histologically divided into three types; the cribriform pattern, the tubular and trabecular pattern, and the solid cluster pattern. Immunostaining of EMA and MAM-6 antigen had a similar distributions in the luminal borders of luminal tumor cells, whereas the MAM-3 antigen was slight or negative in luminal borders. Myoepithelial derived tumor cells of ACC accompanying hyaline stroma demonstrated positive staining for the MAM-6 antigen (whole cell positive type), and luminal tumor cells of microcysts showed strong staining for the MAM-3 antigen. Laminin staining was confined to the basement membrane and surface borders in pseudocyst cavities. In salivary gland ACC, laminin staining can be used as a marker of pseudocyst surfaces and immunostaining of EMA and the MAM-6 antigen as a marker of luminal borders of cyst. These two histochemical markers were useful for discriminating pseudocyst and cyst.

Immunohistochemical localization of lysozyme, lactoferrin, alpha 1-antichymotrypsin, and alpha 1-antitrypsin in salivary gland of human fetuses.

26 human fetuses were examined to elucidate the immunohistochemical distributions of lysozyme, lactoferrin, alpha 1-antichymotrypsin, and alpha 1-antitrypsin in prenatal salivary glands. Development of fetal salivary glands was divided into 4 stages: The early developmental stage (EDS), the early intermediate developmental stage (EIDS), the late intermediate developmental stage (LIDS), and the late developmental stage (LDS) and were used to compare antigen localization during salivary gland development. Lysozyme (LY) staining was prominent in serous or demilune cells of the mucous acinar compartment. Lactoferrin (LF) was rarely seen in the fetal glands; only trace amounts were seen in serous cells, alpha 1-antichymotrypsin (alpha 1-ACT) was diffusely positive particularly in glandular ducts, alpha 1-antitrypsin (alpha 1-AT) was also diffusely distributed in all salivary gland elements and was more abundant in ductal cells than acinar cells. During the EDS, immunohistochemical staining of LY, LF, alpha 1-ACT, and alpha 1-AT could be observed with glandular intensity increases corresponding to the advance of cytodifferentiation of granular epithelium occurring in the subsequent EIDS and LIDS. Staining intensities were continuous during the LDS even though the amount of those materials in the fetal salivary glands was not of the extent seen in the adult salivary gland. These results suggest that production of LY, LF, alpha 1-ACT, and alpha 1-AT was positive during prenatal development of human salivary glands. The present study discusses the protective roles and defense mechanisms of LY, LF, alpha 1-ACT, and alpha 1-AT in developing human salivary glands.

Prenatal development of human major salivary glands and immunohistochemical detection of keratins using monoclonal antibodies.

The major salivary glands were examined from 69 human fetuses ranging from 10 to 40 weeks of gestation. Prenatal growth curves of developing salivary glands could be established by histological scoring, and development was divided into the early developmental stage (EDS) from 10 to 18 weeks, early intermediate developmental stage (EIDS) from 19 to 24 weeks, late intermediate developmental stage (LIDS) from 15 to 32 weeks, late developmental stage (LDS) from 33 to 40 weeks. Characteristic morphogenesis and cytodifferentiation occurred in glandular duct cells during the period of EIDS and LIDS. In the LDS, acini and ducts of the salivary glands histologically developed into a mature state similar to adult glands. Immunohistochemical staining with monoclonal antibodies (MoAbs) PKK1, KL1, K8.12, K8.13, K4.62, RPN 1160, 1162, 1163, 1164, and 1165 was performed. During the fetal period, keratin expression as revealed by MoAbs PKK1, KL1, K8.12 was well established, and the staining pattern for each of these antibodies was comparable. Other antibodies showed rare or negative staining except K8.13 which had a diffuse, non-specific staining pattern. Accordingly, the proliferation and cytodifferentiation of fetal stage keratin staining in ductal cells as revealed by MoAbs PKK1, KL1, and K8.12 showed a heterogenic distribution in both luminal and basal cells. It is a characteristic finding that the cytodifferentiation of ductal luminal cells precedes ductal basal cells. Ductal basal cells stained with MoAb K8.12 and show heterogeneity of keratin distribution continuously until the full term of gestation. The keratin staining of oral epithelium was also examined to compare with distribution of salivary gland ductal cells and oral epithelial cells. In the present study, the developmental sequence of salivary gland cells and the immunohistochemical properties of keratin proteins in these cells were described in relation to the histogenesis of salivary gland tumours.