Immunohistochemical localization of kallikrein in human pancreas and salivary glands

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.     

Immunohistochemical studies of S-100 protein alpha and beta subunits in adenoid cystic carcinoma of salivary glands

Immunohistochemical studies were performed to explore the distribution of S-100 protein and its alpha, beta subunits in 76 adenoid cystic carcinomas (ACC) of the salivary glands. Histopathologically. ACC was divided into cribriform, tubular, basaloid and trabecular types which could be mixed in the same tumor. S-100 protein was usually positive in tumor cells forming cribriform structures; foci of strongly positive tumor cells were also distributed in the luminal layer of tubular structures, and in areas transitional between cribriform and tubular patterns. S-100 alpha staining was confined to some tumor cells in cribriform areas, to luminal tumor cells in tubular structures and to few tumor cells in basaloid structures. S-100 beta reaction was usually localized to luminal surfaces in a fine granular pattern in tubular and microtubular structures in a distribution somewhat similar to that in the normal salivary gland. Great heterogeneity in the immunohistochemical distribution of S-100, S-100 alpha and S-100 beta proteins was found in the various histologic types of ACC and the pattern was different from that seen in pleomorphic adenomas. It is possible that the ACC tumor cells positive for S-100 protein may be closely related to true or modified myoepithelial cells.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Tonin has been localized in salivary glands and kidney by the indirect immunofluorescence technique of Coons and by the unlabeled antibody technique of Sternberger. Both techniques gave identical results. Immunoreactive tonin was localized in the cytoplasm of granular convoluted tubular cells and on the apical surface of striated duct cells and collecting duct cells of the submandibular gland. In the parotid and sublingual glands, which lack granular cells, tonin was only found on the apical surface of striated duct and collecting duct cells. In the kidney, immunoreactive tonin was found only associated with cells of the distal convoluted tubules. After fixation with Bouin fluid or with ethanol, tonin was found not only on the apical surface of the cells but also in the apical and perinuclear cytoplasm. This cytoplasmic staining has been attributed to artefactual diffusion since, after fixation with formol-picric acid, the enzyme could only be localized on the apical surface of the tubular cells.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Summary As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H₂SO₄ to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H₂SO₄ resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c2c, 8c4c, and 8c2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 224 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.     

Immunohistochemical localization of activated EGF receptor in human eccrine and apocrine sweat glands.

Epidermal growth factor (EGF) is secreted into sweat from secretory cells of human sweat glands. The function of EGF in sweat is poorly understood. The biological function of EGF is exerted by the binding of EGF to the receptor (EGFR) and its activation. Therefore, we immunohistochemically localized the activated form of EGFR in human eccrine and apocrine sweat glands to assess the functional importance of the EGF-EGFR system in human sweat glands. Frozen sections of human skin were stained with a monoclonal antibody (MAb) specific for tyrosine-phosphorylated (activated) EGFR and with an MAb that stains both activated and non-activated EGFR. In the secretory portion of eccrine sweat glands, nuclei of the secretory cells were stained with the anti-activated EGFR MAb. In coiled and straight portions of eccrine sweat ducts, nuclei of luminal and peripheral cells were stained with the antibody specific for activated EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells possessed non-activated EGFR. In the secretory portion of apocrine sweat glands, activated EGFRs were present in cytoplasm and nuclei of secretory cells. These data suggest that EGF, already known to be present in the cytoplasm of secretory cells in eccrine and apocrine sweat glands, activates EGFR in the nuclei of secretory cells themselves in an intracrine manner. Because ductal cells do not express EGF, EGF in the sweat secreted from the secretory cells should activate EGFR in the ductal cells in a paracrine manner. (J Histochem Cytochem 49:597-601, 2001)     

Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.     

Immunohistochemical assessment of fractalkine, inflammatory cells, and human herpesvirus 7 in human salivary glands.

Human fractalkine (CX3CL1), a delta-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry.     

Comparative cytofluorometric DNA analysis of pleomorphic adenoma and adenoid cystic carcinoma of the salivary glands.

The nuclear DNA content in the tumor cells of 17 pleomorphic adenomas (PA) and 26 adenoid cystic carcinomas (ACC) was assayed by cytofluorometry to clarify the etiology of the differences in their biological nature. Aneuploidy was found in only two solid-pattern ACC samples; all the other samples had diploid stemlines. With respect to the polyploid cell rate and the S + G2M fraction, the differences between the four histological patterns of PA were not significant. In the ACC, the S + G2M fraction was significantly higher in solid pattern than in cribriform or trabecular pattern tumors, which may indicate that proliferative activity is higher in the solid pattern than in the other two. The polyploid cell rate in ACC was significantly higher in the solid pattern than in the cribriform pattern carcinomas. There was no significant difference in the mean polyploid cell rate between PAs and ACCs. The mean S + G2M fraction was significantly higher in all histological patterns of ACCs than in PAs. These results suggest that proliferative activity is greater in any one of the histological patterns of ACC than in PA. These findings may largely explain the biological differences between the two tumors.     

Immunohistochemical localization of the metalloproteinase meprin in salivary glands of male and female mice.

Meprin is a membrane-bound metalloproteinase which is expressed at high levels in the brush border membrane of proximal tubules of kidneys of some mouse strains (referred to as high meprin-activity mice). The mature active proteinase is not present in kidneys of many inbred strains of mice; however, these low meprin-activity mice possess a kidney protein that crossreacts with polyclonal antibodies prepared against meprin. In the present studies, immunohistochemical methods were used to determine the presence of meprin in liver, pancreas, spleen, testis, thymus, kidney, salivary glands, stomach, duodenum, and skin. Meprin crossreactivity was observed only in kidney and salivary glands. In salivary glands, the enzyme was found on the luminal surface of intercalated and striated ducts of submandibular and parotid glands and on interlobular ducts of the latter. In both kidney and salivary glands, the intensity of immunochemical staining was greater in males compared with females. For both sexes, immunoreactivity was markedly greater in the high meprin-activity mice compared to the low meprin-activity mice. These studies indicate that meprin has a limited tissue distribution, and that genetic and hormonal factors that regulate the proteinase are similar in kidney and salivary glands. The localization of the proteinase implies that the enzyme functions in modifying proteins and peptides that are secreted or re-absorbed in the ducts of these tissues.     

Immunohistochemical evidence for the presence of progesterone receptor in rat submandibular glands.

Immunohistochemical analysis of progesterone receptor was carried out in rat submandibular glands. Immunoreactivity to progesterone receptors was found in cell nuclei of the intralobular duct system within male and female rat submandibular glands. The female glands contained more immunoreactive cells than the male glands. In ovariectomized rats progesterone receptor-containing cells decreased in number while testectomized glands revealed an increase. When estradiol was administered to gonadectomized rats of both sexes, the immunoreactivity in cells of the intralobular duct system markedly increased. These results suggest the possibility that progesterone may modulate various metabolic functions in the rat submandibular glands.     

Immunohistochemical localization of tonin and its relation to kallikrein in rat salivary glands

Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.     

Immunocytochemical localization of PTHrP in human and rat salivary glands

Parathyroid hormone-related protein (PTHrP) was isolated from tumours and is thought to represent the main factor responsible for humoral hypercalcaemia, which accompanies neoplastic diseases. At present, the protein is known to reside in multiple tissues and organs of both humans and animals. Our study was aimed at demonstrating the presence of PTHrP in normal salivary glands (parotid and submandibular) of rats and humans. Application of immunocytochemical techniques permitted to document the presence of PTHrP in the human and in the rat salivary glands. In all cases, an intense reaction was observed in intra- and interlobular ducts. In rat salivary glands, PTHrP was also present in cells of mucous acini. In our opinion, the presence of PTHrP in the ducts indicates participation of the protein in electrolyte transport across the epithelial cells. The positive reaction noted in mucous acini of rat salivary glands may indicate accessory role of PTHrP in the secretory processes in the glands.     

Histochemical studies on pseudocysts in adenoid cystic carcinoma of the human salivary gland

Summary Pseudocysts are unique structures found in adenoid cystic carcinomata of human salivary glands. They were studied in 13 such cases by histochemical and immunohistochemical means. The pseudocysts contained an abundance of mucoid materials which reacted strongly with both Alcian Blue and dialysed iron ferrocyanide. The mucoid material was digested with chondroitinase ABC and heparitinase, but was resistant toStreptomyces hyaluronidase. The inner surfaces of the pseudocysts were strongly reactive for laminin, whereas the interface between the tumour cell nests and the outer stromal area was intensely reactive for fibronectin. Numerous fibronectin-reactive fibrils and blood coagulation factor XIII (F-XIII)-positive cells were distributed extensively in the outer stromal area. The F-XIII-positive cells were also found within some pseudocysts. The results obtained in the present study have shown that the pseudocysts represent a peculiar structure consisting of basement membrane components; laminin, fibronectin, heparan sulphate and chondroitin sulphate.     

Identification of acid-sensing ion channels in adenoid cystic carcinomas

Tissue acidosis is an important feature of tumor. The response of adenoid cystic carcinoma (ACC) cells to acidic solution was studied using whole-cell patch-clamp recording in the current study. An inward, amiloride-sensitive Na(+) current was identified in cultured ACC-2 cells while not in normal human salivary gland epithelial cells. Electrophysiological and pharmacological properties of the currents suggest that heteromeric acid-sensing ion channels (ASICs) containing 2a and 3 may be responsible for the proton-induced currents in the majority of ACC-2 cells. Consistent with it, analyses of RT-PCR and Western blotting demonstrated the presences of ASIC2a and 3 in ACC-2 cells. Furthermore, we observed the enhanced expression of ASIC2a and 3 in the sample of ACC tissues. These results indicate that the functional expression of ASICs is characteristic feature of ACC cells.     

Subcellular localization of blood group substances ABH in human salivary glands

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.     

Effects of progesterone receptor blockers on human granulosa-luteal cell culture secretion of progesterone, estradiol, and relaxin

We have developed culture methods for human luteinizing granulosa cells (GLC) that support the timely and dynamic secretion of estrogen (estradiol-17beta; E(2)), progesterone (P(4)), and relaxin (Rlx) in patterns that mimic serum hormone concentrations during the luteal phase of the menstrual cycle. Additional hCG, to simulate rescue of the corpus luteum, prevented the normal decline in GLC hormone production. To test the importance of the P(4) receptor in P(4) production, GLC were treated in vitro with two P(4) receptor antagonists. Human GLC received one of two hCG support protocols: a Baseline group simulating the normal luteal phase or a Rescue group simulating early pregnancy. Baseline and Rescue groups were treated with either RU-486 or HRP2000 either early or late in the cell culture period. The effects of treatments or control on ovarian steroid and peptide hormone production were determined (significant difference was P < 0.05). In the Rescue group, late treatment resulted in an immediate and dramatic decline in E(2), P(4), and Rlx secretion to nearly nondetectable levels within 1 day after treatment, and hormones remained depressed for the remaining 10 days of culture. In contrast, early treatment resulted in a decline in steroid hormone secretion that returned to control levels within 5 days of cessation of treatment, and Rlx secretion was delayed for approximately 5 days more than in controls. The data support the hypothesis that P(4) may be a required autocrine factor, not only for its own production but also for the maintenance of full endocrine function of the corpus luteum.