Immunohistological characterization of myoepithelial cells using monoclonal antibodies

Aim: The aim of this study was to investigate the expression of monoclonal antibody (mAbs) in the myoepithelial cells of the rat submandibular salivary glands (SSG) during postnatal development. Material and method: SSG were obtained from 1, 30 and 60 days old, male, AO rats. Streptavidin-biotin immunoperoxidase method was used for immunohistochemistry. Cryostat sections were fixed in acetone and incubated with mAbs which specifically reacts with cytokeratins (CK) polypeptides ( CK7, CK8, CK18, CK kidney, K8.12, K8.13), rats epithelial (PT13D11), rats collagen (RMC-23), laminin (α6β1) and rats sialoadhesin (ED3). Results: Immunohistochemical analysis of the myoepithelial cells showed phenotypic heterogeneity during the postnatal development. Myoepithelial cells of acini were stained by K8.13, PT13D11 and ED3 mAbs, whereas these cells were not stained by mAbs CK7, CK8, CK18, CK-kidney, RMC-23 and α6β1. In adult rats, myoepithelial cells of excretory duct were stained by K8.12 mAb. Conclusion: The obtained results show phenotypic heterogeneity of myoepithelial cells and might also be useful to further understanding of myoepithelial cells development and functions

Imunohistološke karaktersitike kolagena, laminina i sijaloadhezina u podviličnoj pljuvačnoj žlezdi pacova tokom ontogeneze

The aim of this study was to investigate the expression of collagen, laminin and sialoadhesin in the rat submandibular salivary gland during postnatal development (1st, 30th,and 60th day) by using various monoclonal antibodies (mAbs) RMC-23 (specific for collagen),a6ß1 (specific for laminin) and ED3 (specific for sialoadhezin). These components of extracellular matrix were detected. RMC-23 mAb showed strong positivity to the basal membranes of the ductal system (intercalated, striated and excretory ducts) and of intersticium. Increased expression in the basal membrane of acini during development of glands was noted. Similar immunoreactivity was shown byßmAb but the intersticium showed a negative reaction to 1a6 this antibody. Positive reactivity of ß1a6 mAb of epithelial ductal cells, particularly of the neonatal animals, was found. In contrast to ß 1a6 and RMC23 mAbs, ED3 mAb was increasingly expressed in the myoepithelial cells during ontogenesis. Our findings regarding the immunoreactivity of collagens and laminins are in accordance with the findings of other autors. The very interesting finding of sialoadhesin in myoepithelial cells of the rat submandibular salivary gland, which is not described in literature and needs further investigation. Our results suggest that adhesion molecules and extracellular matrix proteins have an important biochemical role during postnatal development of the submandibular salivary gland.Cilj ovog rada je bio da se ispita ekspresija kolagena, laminina i sijaloadhezina u podviličnoj pljuvačnoj žlezdi (PPŽ) pacova tokom postnatalnog razvoja (1, 30, 60 dana). Imunohistohemijski streptavidin-biotin peroksidaznom bojenjem, upotrebom različitih monoklonskih antitela RMC-23 (specifično za kolagen), aß1(specifično za laminin) i ED3 (specifično za sijaloadhezin) detektovane su ove komponente ekstraćelijskog matriksa. RMC-23 mAt se snažno vezuje za bazalne membrane izvodnih kanala (umetnuti, prugasti i ekskretorni kanali) i intersticijum. Ekspresija u bazalnim membranama acinusa se povećava u toku razvoja žlezde. Sličnu imunoreaktivnost ima aß1 mAt, ali je intersticijum pokazivao negativnu reakciju. Epitelne ćelije izvodnih kanala posebno neonatalnih životinja bile suaß+. ED3 mAt je za razliku odaßi RMC-23 mAt detektovano i u mioepitelnim ćelijama čija se reaktivnost povećava tokom ontogeneze. Veoma je interesantan nalaz sijaloadhezina u mioepitelnim ćelijama PPŽ pacova koji nije opisan u literaturi, što zahteva dalja ispitivanja. Ovi rezultati pokazuju da adhezioni molekuli i proteini ekstraćelijskog matriksa imaju značajnu biohemijsku ulogu u toku postnatalnog razvoja podvilične pljuvačne žlezde

Mesenchymal Stem Cells from Periapical Lesions Upregulate the Production of Immunoregulatory Cytokines by Inflammatory Cells in Culture

The pathophysiology of periapical lesions (PLs) is under control of pro-inflammatory and anti-inflammatory (mainly immunoregulatory) cytokines. We have recently established mesenchymal stem cells (MSCs) from PLs and showed their suppressive effects on the production of proinflammatory cytokines from PLs inflammatory cells (ICs). In this work we studied the production of interleukin (IL)-10, IL-27 and transforming growth factor (TGF)-β, by PL-ICs in direct or indirect contacts with PL-MSCs. PL-ICs, which were isolated from four different asymptomatic PLs, predominantly composed of lymphocytes, followed by neutrophil granulocytes, macrophages and plasma cells. PLMSCs, expressing typical MSC markers, were co-cultivated with PL-ICs at 1:10 ratio, either in direct contact or in a transwell-system, for 24 hours. The levels of cytokines in cell-culture supernatants were tested by ELISA. The results showed that PL-MSCs up-regulated the production of all three immunoregulatory cytokines by PL-ICs. PL-MSCs stimulated the production of IL-10 and IL-27 via soluble factors, whereas the up-regulation of TGF-β required direct cell-to-cell contacts. In conclusion, our results showed for the first time the involvement of PL-MSCs in restriction of inflammation in PLs by up-regulation of immunoregulatory cytokines

Different Roles of a Rat Cortical Thymic Epithelial Cell Line In Vitro on Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis, Induction of Apoptosis, Nursing and Growth Promoting Activities

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells

Imunohistohemijska identifikacija citokeratina u submandibularnoj pljuvačnoj žlezdi pacova u toku ontogeneze

Cytokeratins (CK) are a group of proteins comprised of at least 20 different skeletal polypeptides specific for simple and complex epithelia of almost all tissues. Until recently only a few antibodies for cytoskeletal proteins were available and thus it was very difficult to visualize all the components of postnatal cytodifferentiation of salivary glands. Therefore, morphologic and immunophenotypic features of the CK polypeptides, have been analized within the rat submandibular salivary glands (SSG) during postnatal development. SSG were obtained from 1, 30 and 60 days old, male, AO rats. Streptavidin-biotin immunoperoxidase method was used for immunohistochemistry. Cryostat sections were fixed in acetone and incubated with monoclonal antibodies (mAbs) which specifically react with CK polypeptides (CK7, CK8, CK18, CK kidney, K8.12, K8.13) and rat epithelium (PT13D11). Immunohistochemical analysis of the rat SSG showed phenotypic heterogeneity of particular components of this gland during postnatal development. Regarding CK profiles it was shown that epithelial cells of the acini expressed CK18, whereas epithelial cells of the duct expressed K8.12 and K8.13 mAbs. Epithelial cells of the acini were not stained by K8.12 and PT13D11 mAbs. Intercalated ducts were strongly CK kidney+ and PT13D11+. Myoepithelial cells of the acini were stained by K8.13 mAbs, whereas these cells were not stained by mAbs specific for simple epithelia. This immunohistological study showed that the heterogeneity expression of CK polypeptides might also be useful to further understand the origin of epithelial SSG cells, as well as their development and function.Citokeratini (CK) su grupa složenih proteina koji se sastoje od najmanje 20 različitih polipeptida citoskeleta, specifični za običan i složeni epitel skoro svih tkiva. Do skoro je bilo dostupno samo nekoliko antitela specifičnih za proteine citoskeleta i zbog toga je bila otežana vizualizacija komponenti postnatalne citodiferencijacije pljuvačnih žlezda. U ovoj studiji su, pomoću panela monoklonskih antitela (mAt), analizirane morfološke i imunofenotipske osobine CK polipeptida, u submandibularnoj pljuvač noj žlezdi (SPŽ) pacova tokom ontogeneze. Submandiblarne pljuvačne žlezde uzimane su od soja AO pacova, muškog pola, starosti 1, 30 i 60 dana. U ovom radu korišćena je imunohistohemijska metoda streptavidin-biotin peroksidaznog bojenja. Tkivni preseci fiksirani su u acetonu i inkubirani sa mAt specifičnim za CK polipeptide (CK7, CK 8, CK 18, CK bubrega, K8.12, K8.13) i epitel pacova (PT13D11). U ovoj imunohistohemijskoj analizi, ekspresija anti -CK antitela na SPŽ pacova, pokazuje fenotipsku heterogenost određenih komponenti ove žlezde tokom postnatalnog perioda. Epitelne ćelije acinusa su CK18+, dok epitelne ćelije kanalića imaju izražene CK definisane sa K8.12 i K8.13 mAt. Epitelne ćelije acinusa nisu bile obojene sa K8.12 i PT13D11 mAt. Umetnuti kanali su bili izrazito CK bubrega+ i PT13D11+. Mioepitelne ćelije acinusa su bile obojene sa K8.13 i PT13D11 mAt, dok su ove ćelije imale negativnu reakciju sa mAt specifičnim za obični epitel. Ova imunohistohemijska studija pokazuje heterogenost u ekspresiji CK polipeptida i može poslužiti za razumevanje porekla, razvoja i funkcije epitelnih ćelija SPŽ pacova

Tumor necrosis factor-alfa and interleukin-4 in cerbrospinal fluid and plasma in different clinical forms of multiple sclerosis

Background/Aim. Multiple sclerosis (MS) is an immunemediated central nervous system disease characterized by inflammation, demyelination and axonal degeneration. Cytokines are proven mediators of immunological process in MS. The aim of this study was to investigate whether there is a difference in the production of the tumor necrosis factor alpha (TNF-alpha) and interleukin-4 (IL-4) in cerebrospinal fluid (CSF) and plasma in the MS patients and the controls (other neurological non-inflammatory diseases) and to determine a possible difference in these cytokines in plasma and CSF in different clinical forms of MS. Methods. This study involved 60 consecutive MS patients - 48 patients with relapsing-remitting MS (RRMS) and 12 patients with secondary progressive MS (SPMS). The control group consisted of 20, age and sex matched, nonimmunological, neurological patients. According to the clinical presentation of MS at the time of this investigation, 34 (56.7%) patients had relapse (RRMS), 14 (23.3%) were in remission (RRMS), while the rest of the patients, 12 (20.0%), were SPMS. TNF-alpha and IL-4 concentrations were measured in the same time in CSF and plasma in the MS patients and the controls. Extended disability status score (EDSS), albumin ratio and IgG index were determined in all MS patients. Results. The MS patients had significantly higher CSF and plasma levels of TNF-alpha than the controls (p < 0.001 for both samples). IL-4 CSF levels were significantly lower in the MS patients than in the controls (p < 0.001), however plasma levels were similar. The patients in relapse (RRMS) and with progressive disease (SPMS) had higher concentrations of CSF TNF-alpha levels than the patients in remission (p < 0.001). IL-4 CSF levels in relapse (RRMS) and SPMS groups were lower than in the patients in remission. The patients in remission had an unmeasurable plasma TNF-alpha level and the patients with SPMS had significantly lower IL-4 levels in plasma than the patients in relapse and remission (p < 0.001). The only significant correlation between cytokine level with either EDSS, or albumin ratio, or IgG index, was found between CSF TNF-alpha levels and albumin ratio in the patients with relapse (R square = 0.431, p < 0.001). Conclusion. According to the obtained data MS relapse was characterized by high concentrations of TNF-alpha in CSF and plasma and low concentrations of IL-4 in CSF. Remission was characterized by high concentrations of IL-4 and low concentrations of TNF-alpha both in CSF and plasma. SPMS was characterized with lower concentrations of TNF-alpha and IL-4 compared to relapse, both in CSF and plasma

Gold in the past, today and future

This paper deals with gold, which is described as a chemical element. Special attention is paid to its physical-chemical properties and, furthermore, where or in what form it can be found in nature. We discuss the role it has played through history and we inform how gold has been developed to the level it has reached today's value. Still more, when gold is broken into nanoparticles, this form could be highly useful for a wide range of processes, including general nanotechnology, electronics manufacturing and the synthesizing of different functional materials. It is important that we know that gold is also used in industry in many engineering applications (contacts in micro-electronics) and medicine (dental alloys, implants)

Zajedničke imunofenotipske odlike podvilične pljuvačne žlezde i grudne žlezde pacova

Introduction. Submandibular salivary gland is a part of the neuro-immune-endocrine system. It contains biological factors which regulate a number of functions in the body including the modulation of thymus function. Objective. The aim of the study was to investigate immunophenotypic characteristics of submandibular salivary glands of rats during ontogenesis, using the panels of monoclonal antibodies and to compare with the phenotypic characteristics of epithelial components of the thymus. Methods. Submandibular salivary glands and thymus were obtained from 1, 30 and 60 days old male AO (Albino, Oxford) rats. Streptavidin-biotin peroxidase method was used for staining. Results. Immunohistochemical analysis of rat submandibular salivary glands showed phenotypic heterogeneity of particular components of this gland during the postnatal development. We demonstrated that rat submandibular salivary glands share common antigens with rat thymic epithelial cells, but the observed phenotypic similarity between the individual regions was considered much more significant. Our data showed that the phenotypic similarity between duct epithelial cells and subcapsular epithelial cells and most medullary cells, whereas cortical epithelial cells are phenotypically similar to acinar cells. Conclusion. This immunohistological study showed phenotypic complexity of the submandibular salivary gland and similarity to the thymus that opens new perspectives in studying phenotypic similarities between this gland and lymphatic organs.Uvod. Submandibularna pljuvačna žlezda je deo neuroimunoendokrinog sistema koja posredstvom bioloških faktora reguliše mnoge funkcije u organizmu, uključujući i modulaciju funkcije timusa. Cilj rada. Cilj rada bio je da se ispitaju imunofenotipske odlike submandibularne pljuvačne žlezde pacova tokom ontogeneze pomoću panela monoklonskih antitela i uporede s fenotipskim svojstvima epitelnih komponenti timusa. Metode rada. Istraživanja su rađena na pacovima soja AO (Albino Oxford), muškog pola, starosti od jednog, 30 i 60 dana. Za fenotipsku analizu korišćena je streptavidin-biotin- peroksidazna metoda bojenja. Rezultati. Ova imunohistološka studija ukazuje na heterogenost fenotipskih odlika pojedinih komponenti submandibularne pljuvačne žlezde tokom postnatalnog razvoja. Takođe je ustanovljeno da submandibularna pljuvačna žlezda i timus imaju zajedničke antigene, ali je mnogo značajnija fenotipska sličnost između pojedinih regiona u oba ispitivana organa. Epitelne ćelije izvodnih kanala submandibularne pljuvačne žlezde su fenotipski slične epitelnim ćelijama subkapsularne i većini ćelija u meduli timusa. Epitelne ćelije acinusa su fenotipski slične epitelnim ćelijama korteksa timusa. Zaključak. Fenotipska kompleksnost submandibularne pljuvačne žlezde i sličnost s timusom otvara nove mogućnosti za analizu fenotipske sličnosti između ove žlezde i limfatičnih organa

Role of β2 Integrins in the Binding of Thymocytes to Rat Thymic Macrophages

A role of β2 integrins and one of their ligands, ICAM-1, in thymic macrophage (TMF)/thymocyte interactions was studied. TMF were isolated as adherent cells from 4-day old culture of thymic-cell suspensions either from normal or hydrocortisone-treated rats. Adherent cells were 94-98% positive with ED1 (a pan-macrophage marker). The majority of them (75-95%) expressed the CD11b and CD18 molecules, and 60-70% expressed CD54 (ICAM-1). A low proportion of TMF (10-20%) expressed CDlla (LFA-1). The expression of all these antigens was upregulated by IFN-α and TNF-α. The effect of these mAbs on TMF/thymocyte binding was studied using a simple rosette assay by incubating unstimulated or IFN-γ or TNF-α stimulated TMF, grown on microscopic slides with resting or ConA +IL-2 activated thymocytes. It was found that LFA-1/CD18 and ICAM-1 play a significant role in the TMF/thymocyte adhesion. In addition, a LFA-l-dependent/ICAM- 1-independent adhesion pathway was observed, suggesting that LFA-1 might use another ligand. The inhibitory effect of anti-CD18 mAb (WT-3) was higher than the effect of anti-LFA-1 mAb (WT-1) and was a consequence of blocking the CD18 chain both on thymocytes and TMF. No significant difference in the expression and function of adhesion molecules was found between TMF obtained from normal or hydrocortisone-treated rats. The involvement of CD1 1b in these processes was of lesser importance than the role of the CD11a molecule. By using mAbs to different epitopes of the CD11b molecule, such as OX-42 (anti-CD11b/CD11c), ED7, and ED8 (anti-CD11b), it was found that they were either slightly or moderately inhibitory under certain experimental conditions or did not significantly modulate TMF/thymocyte binding. Oχ-42 was slightly stimulatory in some experiments. Cumulatively, these results show that 2 integrins play a significant role in TMF/thymocyte interactions and probably contribute to T-cell development in vivo