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The immunoreactivity pattern for different monoclonal antibodies to cytokeratins and to vimentin in epimyoepithelial islands typical for glands of patients with Sjögren's syndrome has been compared with that of normal parotid gland tissue. Two types of epithelial island cells were observed: one had an intermediate filament protein pattern similar to that of ordinary duct epithelial cells of normal parotid gland. The other had an intermediate filament protein pattern typical of myoepithelial and/or basal duct cells in normal glands. Thus, we conclude that the islands are composed of a mixed population of gland cells on the basis of their content of cytokeratins or of cytokeratins and vimentin. These cells might originate from pluripotential reserve cells or from ordinary duct, myoepithelial andlor basal duct cells which may have undergone metaplasia. [ABSTRACT FROM AUTHOR] more...

Objective: Bombesin (BN) and the mammalian homologue gastrin-releasing peptide (GRP) are known trophic factors, neurotransmitters and paracrine hormones. BN/GRP has not previously been demonstrated in synovial fluid. In this study, the amounts of BN/GRP and substance P (SP) present in synovial fluid from the knee joints of patients with rheumatoid arthritis (RA) and of healthy controls were measured., Methods: Synovial fluid from the knee joint was collected from patients with either longstanding RA (n = 32) or early arthritis (symptoms for < 12 months; n = 9) and from control subjects, i.e., individuals without known joint disease (n = 10). These samples were analyzed using radioimmunoassays., Results: Levels of BN/GRP-like peptide were below the assay detection limits in synovial fluid from controls. Detectable levels of immunoreactive BN/GRP were present in the majority of patients with either longstanding RA or early arthritis. The levels were significantly higher in the synovial fluid from patients classified as having early arthritis compared with those with longstanding RA (p < 0.05). There was a strong correlation between BN/GRP levels and the number of leukocytes in the synovial fluid in the patients with early arthritis. The levels of SP-like peptide in the patients, whether with early arthritis or longstanding RA, were significantly elevated compared with controls. However, there was no difference in the levels between these two patient groups., Conclusions: These observations show that BN/GRP-like peptide is present in the synovial fluid of joints affected by arthritis and that the pattern of BN/GRP increase differs from that of SP. It appears as if the presence of BN/GRP is particularly related to the early processes of joint involvement. These observations are of interest because BN/GRP has well-known trophic and paracrine effects and chondrocytes have recently been shown to produce neuropeptides such as BN/GRP. more...

We examined the effects of irradiation over the thorax of the rat on the mast cells, the neuropeptide-containing nerve fibers, and the expression of atrial natriuretic peptide in the heart and lung. The total doses were 20 to 36 Gy delivered as single doses or fractionated irradiation. Immunohistochemical and radioimmunoassay methods were used. The number of mast cells was much reduced in both the lung and heart in response to irradiation. A trend for lowering the atrial natriuretic peptide levels in plasma was noted both 1 day and 9 days after irradiation. In contrast to the situation in other organs (salivary and laryngeal glands, the intestine), no changes occurred in the immunohistochemical expression of neuropeptides. With these observations and those made in previous studies about the effects of radiotherapy on other organs, the functional significance and basis for further research in the fields are discussed. more...

Irradiation was administered to the upper abdomen of rats, whereupon the duodenum was examined. Numerous vasoactive intestinal peptide (VIP)- and substance P (SP)-like immunoreactive nerve fibers were seen in the damaged mucosa, often in close association to each other. The intensity of the SP- and VIP-like immunoreaction was increased in several of the tissue compartments and, as measured with radioimmunoassay, the contents of SP- and VIP-like materials were increased after 30 Gray. The results show that SP and VIP levels increase after irradiation and suggest that SP and VIP are involved in interactive reactions in the reorganization and inflammatory processes in the gut after abdominal irradiation. more...

The potential influence of corticosteroids on the bombesin (BN)-like peptide family is unknown. Therefore, the effects of adrenalectomy (ADX) on the nervous system of Sprague-Dawley rats, some of them being treated with high doses of the synthetic glucocorticoid dexamethasone (DEX), were investigated. After 8-10 days of treatment, the rats were sacrificed and tissues were prepared for radioimmunoassay (RIA) and immunohistochemical examination. We found an increase in BN-like immunoreactivity in the superficial layers of the dorsal horn of the lumbar spinal cord in the ADX + DEX animals. This increase was confirmed by RIA (P < 0.05). The observations show that the expression of BN-like peptides is influenced by glucocorticoids. The altered levels of BN-like peptides may be related to the trophic and antinociceptive effects previously reported for these peptides. more...

Bombesin (BN) and its mammalian counterpart gastrin-releasing peptide (GRP) act as neuroregulatory hormones and peripheral and central satiety-inducing agents. Previously, we demonstrated that irradiation induces an increase in the expression of BN/GRP in the innervation of the salivary glands in rats. We therefore carried out a study using radioimmunoassay (RIA) analysis and immunohistochemistry to examine whether saliva contains BN and whether irradiation affects the BN release to saliva in rats. Immunoreactivity for BN was detected not only in the innervation of the parenchyma but also in the duct cells and in the lumina of the ducts, suggesting entrance of BN into saliva. The RIA analysis confirmed that rat saliva contains a BN-like peptide. The observation shows that saliva contains this peptide but that there is no significant increase following the radiation schedule used. Nevertheless, the occurrence of an enhanced expression of BN in different peripheral tissues such as the salivary and laryngeal glands should be taken into consideration when discussing the clinically important problem of reduced food intake and anorexia in cancer patients. more...

The present investigation was designed to elucidate which role the sympathetic nerves play in the immunoexpression of atrial natriuretic peptide in the cardiac conduction system of the rat. In order to destroy the cardiac sympathetic nerve terminals, both surgical and chemical sympathectomy were performed. By use of immunohistochemical and radioimmunoassay techniques, the immunoreactivity and level of atrial natriuretic peptide in the conduction system and in the cardiac myocardium were determined. In contrast to the low degree of immunoreaction for atrial natriuretic peptide seen in control rats, the sympathectomized rats exhibited pronounced immunoreactivity for atrial natriuretic peptide in the atrioventricular bundle and bundle branches, which normally have high numbers of sympathetic nerve fibres. On the other hand, in the peripheral parts of the conduction system, where there are ordinarily few sympathetic nerve fibres, the degree of immunoreaction was unchanged. The quantitative measurements also showed that the entire ventricles, including the conduction system, contained increased levels of atrial natriuretic peptide in the treated hearts. The present study shows that destruction of the sympathetic nervous system leads to an increased level of atrial natriuretic peptide in the Purkinje fibres of bundle branches, which thus seem to have a dormant capacity for synthesis of this peptide. The results provide new evidence about the change in atrial natriuretic peptide levels that occurs when sympathetic innervation is altered., (Copyright 1998 Academic Press) more...

Previously, we have observed that the expression of the neuropeptides bombesin (BN-), the mammalian counterpart being gastrin-releasing peptide (GRP), and substance P (SP) in intact normal tissues, such as salivary and laryngeal glands, increases in response to irradiation. In the present study, the aim was to evaluate whether irradiation can have effects on individual cells that normally synthesize neuropeptides. In addition, since these neuropeptides are potentially mitogenic, we studied tumor cells. Therefore, the estrogen receptor-negative human breast cancer cell line MDA-MB-231 and its subline, with acquired doxorubicin resistance, MDA-MB-231 Dox were examined before irradiation and 4, 10, and 15 days after irradiation with 4 Gy (195 kV, 2 Gy fractions with 4 hours interval). Potential dose related changes were studied by delivering single doses of 2 or 9 Gy with the same technique. Immunohistochemical and radioimmunoassay (RIA) methods were used for detection of the SP and BN/GRP. Before, and at all time points following irradiation, a subpopulation in both cell lines displayed an intense immunostaining of SP and BN/GRP. A partial reorganization of the immunoreactive material was observed 10 days after irradiation. The RIA-analyses displayed signs of a dose-related increase, and a time-dependent transient and significant increase in the content of both peptides. The pattern of changes differed between the two peptides, and was especially pronounced in the doxorubicin resistant cells with regard to SP. Another neuropeptide, calcitonin gene related peptide (CGRP), was not detected in the cells used. The results suggest that irradiation has effects on a population of cultured neuropeptide-synthesizing cells. The occurrence and the specific changes obtained in the levels of neuropeptides, in response to irradiation, might imply an importance in the growth of breast cancer cells and in explaining repair processes following irradiation. more...

In mouse pancreatic islets, whether in situ or transplanted to kidney, nerve fibers and a few perikarya expressed NPY-like immunoreactivity (NPY-LI). In 4-5 day old grafts, NPY-LI coexisted with VIP-LI in randomly distributed nerve fibers. By 2-52 weeks, NPY mainly co-existed with tyrosine hydroxylase in fibers emanating from the kidney parenchyma. Radioimmunoassays indicated that the NPY levels increased with time, while those of VIP decreased. The study shows that NPY is primarily present in the intrinsic VIP-ergic innervation of islet grafts but later is mainly a constituent of the ingrowing sympathetic innervation. more...

In the present study, the immunohistochemical expression of vasoactive intestinal polypeptide (VIP) in the parotid gland of rats exposed to fractionated irradiation was examined. VIP concentration was analyzed by radioimmunoassay (RIA). Irradiation lead to a marked increase in the immunohistochemical expression of VIP in the innervation of the gland parenchyme. VIP-like immunoreactivity (LI) frequently coexisted with SP (substance P)-LI in these nerve fibers. The pattern of VIP-innervation in association with large ducts and blood vessel walls was unchanged. RIA analysis revealed a more than three-fold elevation in VIP content in the gland in response to irradiation. The increase in VIP immunoreaction and VIP content was seen at examination ten days after cessation of a five-day treatment with a total dose of 30-40 Gray. The upregulation of VIP may be related to changes in the production of neurotrophic factors and to an increased demand for a potentiation of secretagogue effects of SP. more...

Purpose: The expression of different neuropeptides in the innervation of submandibular and parotid glands of the rats was examined 2 and 5 days after initiation of radiation treatment as well as 10 and 180 days following the termination of irradiation., Methods and Materials: The irradiation was given on 2 or 5 consecutive days with daily doses of 4-8 Gy up to a total dose of 20-40 Gy. Immunohistochemical methods were used for the demonstration of substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and, the rate-limiting enzyme in the catecholamine synthetic pathway, tyrosine hydroxylase (TH). The content of SP was also analyzed by the use of radioimmunoassay (RIA)., Results: In the parenchyma of both the submandibular and the parotid glands of control animals as well as after 2 days of irradiation treatment, a few nerve fibers showing SP-like immunoreactivity (LI) were observed. A marked increase in the expression of SP in the innervation of the parenchyma in both glands was observed 10 days after cessation of radiation treatment. The number of stained nerve fibers and the intensity of fluorescence in the fibers seemed to be dose dependent because the group subjected to a total dose of 40 Gy displayed a more pronounced staining intensity than that treated with 30 Gy. These results were supported by the RIA analysis. One hundred eighty days after treatment no obvious differences in SP-expression were seen between control and irradiated animals. No acute and long-term alterations were seen with regard to the other peptides and TH., Conclusions: These results suggest that specific dose- and time-dependent changes in the expression of SP in the parenchyma of both submandibular and parotid glands occur in response to fractionated irradiation. The observations add further aspects to the tissue differences in physiological response and sensitivity to irradiation. more...

It is previously well known that bombesin has effects as growth factor and that changes in bombesin content in air-way structures occur in various patho-physiological conditions. In the present study, the effects of radiotherapy on bombesin expression in the rat larynx were studied. Irradiation was given for five days, 6 or 8 gray daily. Ten days after cessation of irradiation, the subglottic part of the larynx from irradiated and control animals was dissected out, and processed for immunohistochemistry or radioimmunoassay. The radioimmunoassay analysis showed that the content of bombesin-like material increased 2-fold after irradiation. The immunohistochemical analysis displayed an increased bombesin-like immunoreactivity in local ganglionic cells and in nerve fibers in the submucosal glands. These nerve fibers are likely to be derived from the local ganglionic cells. On the other hand, there was no change in the pattern of immuno-reactivity in the innervation of the epithelium and the lamina propria, including the blood vessels. The observations show that radiotherapy can be added to the list of factors that influence bombesin expression in airway structures. more...

Adult and developing salivary glands were investigated using five monoclonal antibodies against cytokeratins (CKs) and vimentin. Acinar cells displayed mainly CK 18 whereas CKs 7, 17 and 19 were only detected in duct and myoepithelial cells. All epithelial and myoepithelial cells were unreactive for one vimentin antibody (Vim 9) whereas with the other (Vim 24), myoepithelial cells and basal cells of excretory ducts were stained. Fetal cells showed the CK pattern of duct cells. At gestational week 18, a reaction for both vimentin antibodies could be found in basal cells of terminal tubules. Although vim 9 reactivity has been shown for a number of salivary neoplasms, it has not been detected in any adult epithelial salivary tissue. The finding of this reactivity in the fetal gland indicates that the expression of this intermediate filament protein in certain salivary neoplasms may be a sign of dedifferentiation resulting in the expression of a filament pattern found in an earlier stage of gland development. more...

In order to study the importance of myoepithelial cells in the formation of parotid epi-myoepithelial islands of the autoimmune sialoadenitis in Sjögren's syndrome, the distribution of cytoskeletal elements has been investigated by electron and immunofluorescence microscopy. Antibodies to intermediate filament proteins--prekeratins, desmin and vimentin--were used as well as a 'DNase I/anti-DNase I' immunohistochemical procedure to detect actin microfilaments for characterization of myoepithelial cells compared to the other types of parotid epithelial cells. Two types of prekeratin antibodies were raised. One of these reacted preferentially with myoepithelial cells. The same type of antiserum also showed a bright fluorescence in cells located peripherally in the epi-myoepithelial islands. These island cells, however, exhibited only a very low actin microfilament content and were thus not ordinary myoepithelial cells. The other type of prekeratin antibodies reacted with all types of parotid epithelia, except acinar secretory epithelium. They also stained all island epithelial cells, but the reactivity was less in cell regions with pronounced lymphocyte infiltration. The heterogeneity between the island epithelial cells, immunohistochemically as well as ultrastructurally, indicates different stages of island formation. Interepithelial lymphocytes showed less vimentin and actin filament content in comparison with lymphoid cells outside the islands. Conclusively, typical epi-myoepithelial islands are composed of metaplastic epithelial cells of intercalated duct origin and invaded lymphoid cells, and ordinary myoepithelial cells are lacking. more...

Oncocytomas and Warthin's tumors were studied ultrastructurally and with immunofluorescence microscopic techniques against cytokeratins, desmin, vimentin, and actin, the latter characterizing myoepithelial cells. The cell types were found within oncocytomas, one type packed with mitochondria and virtually without filaments, the other contained fewer mitochondria but large amounts of cytokeratins. In Warthin's tumors, all cells contained cytokeratins, the basal cell to a much higher degree. No tumor cells contained vimentin or desmin. Actin was found only close to the apical cell surface in the cylindrical cells in the Warthin's tumors and around the few acinar lumina found in the oncocytomas; thus, a myoepithelial origin of oncocytic tumors of salivary glands seems less plausible. The similarity in staining pattern, namely the pronounced dimorphism of both tumor types, indicates a common progenitor of oncocytomas and the epithelial component in adenolymphomas. more...

Four cases of malignant mixed tumor (carcinoma in pleomorphic adenomas) were studied for ultrastructural appearance and for the presence of cytokeratins and vimentin. Ultrastructurally, both squamous and glandular epithelial differentiation were found not only in the same tumor but also within the same cell. One tumor showed mainly mesenchymal differentiation with fibroblast-like cells. The intermediate filament expression of benign mixed tumors (i.e., both cytokeratin and vimentin content) were found in two of the three malignant tumors investigated. In the third tumor, only cytokeratins were found. Thus, the filament content of mixed tumors may change when the tumor becomes malignant. This change does not always parallel a change in morphology. Although one tumor was clearly epithelial ultrastructurally and another mostly mesenchymal, both did contain both cytokeratins and vimentin. more...

The differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart has been investigated. Several aspects of some of the structural proteins, that means the intermediate filament subunit skeletin, myosin, and the myofibrillar M-line proteins MM-creatine kinase and myomesin have been studied by using gel electrophoresis as well as enzyme and immunohistochemical techniques in combination with electron microscopy of both plastic and cryosectional material. We show that marked changes take place in the organization of the intermediate filament system and in the contractile apparatus, both in atria and in ventricles of the rat heart during postnatal development. In the newborn rats no dense myofibrillar M-bands were present in the M-region and the sarcomeric bands were irregular while in the four-week-old rats dense M-bands composed of a set of five crossbridges interconnecting the thick filaments were present. The sarcomeric bands were now regular. These observations are related to the presence of different isomyosins in the atria and in the ventricles of the newborn and the four-week-old rats, to the observation that MM-creatine kinase was only present in the M-region in the four-week old rats and to the physiological maturation of the heart. more...

In order to examine the relationship between the intermediate filaments from Purkinje fibres of the cow heart conduction system and five proposed subclasses of mammalian intermediate filaments, the gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) has been used to examine the specificity and crossreactivity of our antibodies against the Purkinje fibre intermediate filament protein, skeletin. Bovine tissues known to contain intermediate filaments of the five main subclasses were examined with antiskeletin and with preimmune serum and the specific antiserum absorbed with pure skeletin as controls. The antibodies raised against Purkinje fibre skeletin reacted with all three polypeptides of the "neurofilament triplet", with glial fibrillary acidic protein (GFAP), with smooth muscle desmin and also slightly with some prekeratin subunits and with endothelial vimentin. From studies with monoclonal antibodies and amino acid sequencing, certain regions of all intermediate filaments are suggested to be structurally related. Here we show that Purkinje fibre skeletin seems to share antigenic determinants with the proposed five main classes of intermediate filaments. Our antibody is the first carefully controlled experimentally induced antibody having such properties. This might be due to the special attributes of the intermediate filament system in Purkinje fibres, which themselves have unique properties. more...

A three-dimensional cytoskeleton, morphologically composed of intermediate filaments, desmosomes, and Z-disks, remains in bovine heart Purkinje fibres after extraction with Triton X-100 and low and high ionic strength solutions. Biochemically this cytoskeleton mainly consists of skeletin (Mr approximately 55 000) and of alpha-actinin (Mr approximately 95 000). Minor high molecular weight components are also present. By gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) we show here that a 150 000-dalton component is a complex of alpha-actinin and skeletin. Furthermore, this 150 000-dalton component has now been purified and can be converted into 95 000- and 55 000-dalton subunits by reduction with a high concentration of beta-mercaptoethanol and heating. We also show that solubilized pure skeletin and pure alpha-ctinin from Purkinje fibres, after mixing, partly form a stable complex with a molecular weight of about 150 000 daltons. more...

The intermediate filament (IF) composition of muscle cells of various sources is still a controversial issue. In the present study, the IF composition of bovine Purkinje fibres (PFs), atrial and ventricular myocardium, and gastric smooth muscle (SM) has been compared using biochemical and immunocytochemical methods. The Mr of the major IF subunit protein in all four tissues was 55,000. In two-dimensional (2-D) electrophoresis gels of Triton-treated ordinary atrial and ventricular myocardium and the gastric muscular wall, two or three isoelectric isoforms were seen, whereas in PFs up to seven isoforms caused by phosphorylation were observed. In immunofluorescence studies antibodies against the Mr 55,000 subunit of PFs and gastric SM, respectively, both showed identical reactivity with PFs, atrial and ventricular myocytes, gastric SM cells and some SM cells in intramyocardial and gastric muscular wall blood vessels. A small amount of vimentin (Mr 57,000) was also detected in 2-D gel electrophoresis in all four tissues as well as in immunoblotting of PFs with antibodies to vimentin. Immunofluorescence studies using both polyclonal and monoclonal antibodies to vimentin showed that vimentin was present in the endothelium and SM cells of both intramyocardial and gastric muscular wall vessels, sometimes together with desmin in the vascular SM cells, but was never seen in PF, atrial, ventricular or gastric SM cells proper. As expected, vimentin was present in interstitial tissue, i.e., fibroblasts and capillaries. However, interestingly, the monoclonal antibodies, which recognized different antigenic determinants of vimentin, did not give identical staining patterns. Especially the staining of the vascular SM cells differed. Since this staining pattern did not change upon denaturation and unmasking experiments, it seems that the organization of vimentin in different mesenchymal cell types varies. Vimentin was also detected in isolated PFs but here it was located solely in the contaminating interstitial tissue. Thus, desmin is the sole IF protein expressed in PFs, in atrial and ventricular myocytes and in gastric SM cells proper; vimentin alone being present in the interstitial tissue cells, whilst in vascular SM cells desmin and vimentin are coexpressed in various proportions. The variation in number of isoforms of desmin and the heterogeneity in staining of mesenchymal tissues with monoclonal vimentin antibodies probably indicates that the IF cytoskeletons are differently organized in various cell types, even though they contain IFs of the same class. more...