Immunohistochemical localization of the α and β subunits of S-100 protein in pleomorphic adenoma of the salivary glands

The immunohistochemical expression of the alpha and beta subunits of S-100 protein in reactive, modified and transformed of myoepithelial cells, salivary pleomorphic was investigated using monoclonal antibodies. With S-100 alpha, normal salivary glands showed strong staining in serous acinar cells and moderate to slight staining in ductal segments, and with S-100 beta staining was slight or negative in acinar cells, but strong in nerve fibres. In pleomorphic salivary adenomas, the immunohistochemical distribution of S-100 alpha and beta proteins indicated great variation in the tumour cells. Some neoplastic cells gave similar staining for both S-100 alpha and beta, others were strongly positive for S-100 alpha and stained only slightly for S-100 beta, or vice versa. Yet other cells were positive for S-100 alpha and negative for S-100 beta, or vice versa. Pleomorphic salivary adenomas were classified both by histopathological criteria and by their staining pattern for S-100 alpha and beta proteins. Great heterogeneity in S-100 alpha and beta protein expression was found in individual tumour cells of both ductal and myoepithelial origin, and no regular pattern was identified. The cellular origin of salivary pleomorphic adenomas is discussed in terms of S-100 alpha and beta protein immunohistochemistry. Pleomorphic adenoma cells may be transformed from reserve cells into tumour cells displaying biologic properties of myoepithelial cells, ductal cells, or a mixture of both.     

Immunohistochemical localization of transforming growth factor-α and epidermal growth factor-receptor in the mesonephros and metanephros of the chicken

Summary Transforming growth factor-alpha (TGF-) is a polypeptide related to epidermal growth factor (EGF). Both bind to EGF-receptor (EGF-R) to carry out their function in a variety of tissues and cell lines. Several studies have shown their presence in mammalian kidney, however, nothing has to date been stated concerning their existence in avian kidney. Expression of TGF- and EGF-R is reported here for the first time during the development of the chicken kidney. Using immunohistochemical techniques, we identified a TGF- (but not EGF) in mesonephric distal tubule cells from day 8 to day 20 of embryonic development and in metanephric distal tubule cells from day 14 of embryonic development to the adult. The histochemical characteristics of these cells and their histological localization suggest that they may be the principal cells of the distal tubules. Similarly, EGF-R was found in mesonephric proximal tubule cells from day 7 to day 18 of embryonic development and in metanephric proximal tubule cells from day 13 of embryonic development up to adult stages. The coexistence of both TGF- and EGF-R from the onset of development of mesonephros and metanephros supports their possible role in mechanisms of proliferation and differentiation of the cells of these organs.     

Immunohistochemical localization of transforming growth factor β1 and β2 in mouse testes during postnatal development

We examined age-related changes in the expression of transforming growth factor-β(1) (TGF-β(1)) and transforming growth factor-β(2) in mouse testes. The mice were assigned to three age groups: 35, 50, and 75 days old. Paraffin embedded testis sections were processed for the standard streptavidin biotin peroxidase complex immunohistochemistry method. TGF-β(1) expression increased in aging round spermatids over the time studied. There was no expression in 35-day-old Leydig cells, whereas strong expression of TGF-β(1) was observed in 50-day-old Leydig cells. Expression decreased in 75-day-old Leydig cells. TGF-β(2) expression was weak in 35- and 50-day-old mouse spermatids, but expression was greater in 75-day-old elongated spermatids. In Leydig cells, TGF-β(2) expression was strong in both 35- and 50-day-old mice, whereas the expression of TGF-β(2) was less in 75-day-old Leydig cells. Our results suggest that TGF-β(1) and TGF-β(2) may play significant roles in testicular functions and germ cell development in mice.     

Immunohistochemical localization of β1-adrenergic receptors in the liver of male and female F344 rat

The distribution of beta(1)-adrenergic receptors in the liver of Fischer 344 (F344) rat has been examined by an immunohistochemical method. The study was carried out on formalin-fixed and paraffin-embedded livers from young adult, middle-aged, and old female and male F344 rats. An antibody specific for the beta(1)-adrenoreceptor subtype was used. A positive reaction was found in the liver parenchyma of female and male rats from all age groups. Within the liver lobule, a clear zonation is observed, with the beta(1)-adrenoreceptor positivity most evident in pericentral zone hepatocytes and a gradual fading of the immunostaining from pericentral to periportal zone hepatocytes, which may be completely negative. Immunoreactivity is localized on the cell membrane and on the membrane of peripheral cytoplasmic vesicles, and is mostly confined to the cell side facing vascular space. The intensity of immunostaining seems to be slightly higher in the 6- and 10-month-old female rats as compared to the matched male rats and to the senescent female rats. No age-related changes in the intensity of immunostaining are appreciable in male rats. However, no definite conclusion could be drawn about the existence of gender-related differences or age-related changes in the density of beta(1)-adrenoreceptors. A low density of beta1-adrenoreceptor was observed in the spontaneous preneoplastic lesions of the livers from senescent rats.     

Immunohistochemical studies of S-100 protein α and β subunits in adenoid cystic carcinoma of salivary glands

Immunohistochemical studies were performed to explore the distribution of S-100 protein and its α,β 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 α 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-100β 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 a and S-100β 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.     

Immunocytochemical localization and concentrations of the α and γ subunits of 7S-nerve growth factor in the submandibular gland of the mouse

Summary For unexplained reasons, nerve growth factor (NGF) exists in very high concentrations in the submandibular gland of the mouse. The NGF in the gland, called 7S-NGF, is a non-covalent complex of three protein subunits, named -, - and -NGF. All the known biological activity resides in the -NGF subunit, and previous studies have shown that -NGF is present in much greater concentrations in the male submandibular gland than in the female gland. The higher concentration in the male is due to the fact that -NGF is synthesized in the granular tubule cells of the submandibular gland. These cells are much more numerous in the male gland.In contrast to -NGF, neither the concentrations of and subunits nor their cellular localization in the mouse submandibular gland have been established. In this study, radioimmunoassays specific for . and subunits determined that both are present in much higher concentrations in the male gland. Immunocytochemical work localized both subunits in the granular tubule cell in the male and female submandibular gland. This indicates that all the components of 7S-NGF exist in a single cell type in the gland and suggests that 7S-NGF can be formed within this cell and secreted as a complex into the saliva.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

Quantitative mapping of cocaine-induced ΔFosB expression in the striatum of male and female rats

ΔFosB plays a critical role in drug-induced long-term changes in the brain. In the current study, we evaluated locomotor activity in male and female rats treated with saline or cocaine for 2 weeks and quantitatively mapped ΔFosB expression in the dorsal striatum and nucleus accumbens of each animal by using an anti-FosB antibody that recognizes ΔFosB isoforms preferentially. Behavioral analysis showed that while there was little difference between males and females that sensitized to cocaine, nonsensitizing rats showed a large sex difference. Nonsensitizing males showed low behavioral activation in response to cocaine on the first day of treatment, and their activity remained low. In contrast, nonsensitizing females showed high activation on the first day of treatment and their activity remained high. Western blot and immunohistochemical analyses indicated that basal levels of ΔFosB were higher in the nucleus accumbens than the dorsal striatum, but that the effect of cocaine on ΔFosB was greater in the dorsal striatum. Immunostaining showed that the effect of cocaine in both the dorsal striatum and nucleus accumbens was primarily to increase the intensity of ΔFosB immunoreactivity in individual neurons, rather than to increase the number of cells that express ΔFosB. Detailed mapping of ΔFosB-labeled nuclei showed that basal ΔFosB levels were highest in the medial portion of the dorsal striatum and dorsomedial accumbens, particularly adjacent to the lateral ventricle, whereas the cocaine-induced increase in ΔFosB was most pronounced in the lateral dorsal striatum, where basal ΔFosB expression was lowest. Sex differences in ΔFosB expression were small and independent of cocaine treatment. We discuss implications of the sex difference in locomotor activation and regionally-specific ΔFosB induction by cocaine.     

Electron microscopic localization of 5′-nucleotidase in rat salivary glands

Summary The localization of 5-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5-nucleotidase activity and compare it with the traditional lead-ion method.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Effects of transforming growth factor β, tumor necrosis factor α and interferon γ on pancreatic islet β-cell responsiveness to transforming growth factor α

The insulin-producing pancreatic islet -cell, characterized by low proliferative potential, is normally not responsive to the polypeptide epidermal growth factor (EGF) or its homolog transforming growth factor (TGF-). Since EGF receptors in other tissues can be up-regulated by other growth factors and by cytokines, we have in this paper investigated whether such a -cell responsiveness to TGF-, or EGF, can be conferred by co-culture with interferon (IFN-), tumor necrosis factor (TNF-) or transforming growth factor (TGF-) in various combinations. To this end, fetal rat pancreatic islets enriched in -cells were isolated and cultured for 3 days with or without 200 pM or 20 nM TGF-. It was found that neither of these TGF- concentrations affected -cell mitogenesis, insulin content or insulin secretion. However, IFN- (1000 U/ml) evoked a modest stimulation of -cell replication, while suppressing insulin secretion and leaving the islet insulin content unaltered. TNF- (1000 U/ml), on the other hand, affected none of these parameters either alone or in any combination with TGF- or IFN-. However, when TNF- or IFN-, either alone or in combination, were combined with the cytokine interleukin-1, this resulted in islet disintegration, whereas the latter cytokine alone did not exert any gross necrotic changes evident by light microscopy. TGF- (500 pM) stimulated insulin secretion but did not influence islet insulin content or -cell mitogenesis either alone or in combination with TGF- (200 pM or 20 nM). In no instance could any mitogenic or secretory response to low or high concentrations of TGF- be conferred by IFN-, TNF- or TGF- whether used alone or in combinations. Hence, responsiveness to TGF- or EGF in the -cell obviously cannot be achieved by any of these peptides.Abbreviations EGF epidermal growth factor - IFN- interferon - TGF- transforming growth factor - TGF- transforming growth factor - TNF- tumor necrosis factor      

Expression of transforming growth factor-β isoforms in the rat male accessory sex organs and epididymis

We studied the expression and distribution of transforming growth factor-β (TGF-β) isoforms in the rat male accessory sex glands and the epididymis. Our data demonstrate the expression of both TGF-β1 and -β3 isoforms in ventral prostate (VP), seminal vesicle (SV), coagulating gland (CG), and epididymis (E) by Northern blot analysis. In addition, there was differential expression of TGF-β3 in the three regions of epididymis, the corpus region being the highest. Immunostaining data showed intense staining for latent TGF-β1 in all the male accessory glands. In contrast, no staining using antibodies specific for active TGF-β1 was observed. No expression of TGF-β2 was evident either by immunohistochemistry or Northern blot analysis. The presence of mature TGF-β3 protein was observed in the secretory epithelium of VP, CG, and corpus E. There was no detectable staining of TGF-β3 in the seminal vesicle and caput and cauda regions of epididymis. These data suggest possible differential regulation of TGF-β isoform expression in the male reproductive system and predict unique roles for individual TGF-β isoforms in sperm maturation and maintenance.     

Developmental expression of transforming growth factor-α and epidermal growth factor receptor proteins in the human pancreas and digestive tract

This study was designed to localize transforming growth factor alpha (TGF-) and epidermal growth factor receptor (EGFR) expression in the developing human gastrointestinal tract and pancreas. Immunohistochemical techniques using specific antibodies against human TGF- and EGFR were performed on digestive tissues of fetuses from 9 to 10 to 24 weeks of gestation, children and adults. In fetuses, TGF- and EGFR proteins were expressed in all epithelial tissues studied with a good correlation and from an age as early as 9 to 10 weeks of gestation, except for TGF- in the esophagus. The strongest TGF- immunostaining was noted in the stomach and the proximal colon. Unexpectedly, immunoreactive gut endocrine cells were observed with the two antibodies used. Relatively numerous in fetuses, they decreased in number with age and were rare in adults particularly along the colon. Enteroglucagon-secreting cells were shown to express TGF- while some gastrin, somatostatin and pancreatic glucagon cells were immunostained with EGFR antibodies. The presence of TGF- and of its recetor in digestive tract epithelium and pancreatic tissues early in fetal life suggests a functional role for TGF- during the developmental process of the digestive system. We demonstrate that TGF- is also produced by endocrine cells and might have an additional mode of action other than paracrine, at least during fetal life.     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.     

Apyrase and α-glucosidase in the salivary glands of Aedes albopictus

An apyrase and an α-glucosidase were detected in the salivary glands extracts of adult Aedes albopictus. The apyrase is a 61,000 Da secreted protein that hydrolyses ATP and ADP. This protein is synthesized in adults and is preferentially accumulated in the distal lateral lobes of the female salivary glands. The α-glucosidase is a secreted 67,000 Da protein. This enzyme is synthesized during adult life and accumulated in the proximal-lateral lobes of both males and females. The results are discussed and compared with data previously obtained with Aedes aegypti salivary glands.     

Expression of type I and II receptors for transforming growth factor β in the adult rat testis

 After having established the specificity of the antibodies for the rat testis by western blot analysis, the potential target cells for transforming growth factors (TGFβs) were identified by immunohistochemical detection of both type I (TβRI) and type II (TβRII) transducing receptors for TGFβs in the adult rat testis in situ. Leydig cells showed a strong TβRII immunoreactivity whereas the TβRI staining was weak. Only TβRII was detectable in Sertoli cells. In germ cells, staining for TβRI was stronger than for TβRII and the expression of both receptors depended on the seminiferous cycle stage. TβRI first appeared in pachytene spermatocytes and was absent in elongated spermatids from stage XIV onwards. Labelling for TβRII was observed as early as the spermatogonia stage; it increased in pachytene spermatocytes at the onset of TβRI and disappeared in elongating spermatids from stage XI onwards. These results show that TGFβs can affect somatic cells functions and suggest that these factors are involved in the control of meiosis and early spermiogenesis, exerting a direct effect on germ cells. Accepted: 18 June 1998     

Acid phosphatase and peroxidase in ‘resting’ acinar cells of the major salivary glands of cats and their possible movement into secretory granules

Synopsis After fixation by perarterial perfusion using an aldehyde mixture, salivary tissues were prepared for ultrastructural cytochemistry of acid phosphatase or peroxidase. Great variations in the distributions of the reaction products occurred, often within the same cell. Acid phosphatase staining occurred not only in lysosomes and sometimes in a GERL system, but a diffuse cytoplasmic component was also found in submandibular central acinar cells and to a lesser extent in parotid acini and variable staining occurred in the secretory granules of these cells. Peroxidase was variably associated with rough endoplasmic reticulum in submandibular demilunar cells, parotid acini, and more strongly in some sublingual cells. The secretory granules of the latter were darkly stained, but in parotid granules there was varibale staining and least staining occurred in the granules of submandibular demilunes.These results are thought to indicate that not all enzymes present in secretory granules have reached there by an elective secretory process. Sometimes they appear to have entered the granules haphazardly, possibly having been enzymes associated with intracellular cisternal channels for transport or metabolism of other secretory substances and ultimately to have passed into the cisternal channels by chance or as part of a natural removal of redundant material.