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International audience

International audience

Fibroblast growth factor 2 (FGF-2, basic fibroblast growth factor) has been reported to be elevated in tissues from benign prostatic hyperplasia (BPH), the most frequent neoplastic disease in aging men. This suggests that FGF-2 may play a significant role in the development of BPH. In this study the cellular distribution pattern of FGF-2 in tissues from BPH has been investigated by immunohistochemical and molecular biological methods. Radioimmunoassay revealed high concentrations of FGF-2, ranging between 450 and 950 ng per g tissue. Immunoblots confirmed the presence of a 18 kDa FGF-2 in tissue extracts. By immunohistochemistry done with a polyclonal antibody to recombinant FGF-2 on paraffin sections, FGF-2 was localized in fibroblasts, endothelial cells and smooth muscle cells of tissue samples of BPH. Nuclei of these cells were labelled distinctly. Moreover the cytoplasm of smooth muscle cells was labelled moderately. No immunostaining was seen in prostatic epithelium. Nonradioactive in situ hybridization with digoxygeninlabelled oligonucleotides revealed the presence of mRNA for FGF-2 in smooth muscle cells of the prostatic stroma. These results provide evidence that FGF-2 may be produced locally in the human prostate as a stromaspecific mitogen and may play a causal role in the development of BPH.

The duplication of genes for recognition molecules and the ensuing diversification of the members of such families generate complex groups of homologous proteins. One example are galactosidespecific lectins whose sequences display constant features related to sugar binding, the galectins. Based on the inverse abundance of the chicken galectins CG-14 and CG-16 in adult intestine and liver, these two lectins represent a model to comparatively study expression of the related proteins and the galectin-reactive sites (glycoproteins and glycolipids) biochemically and histochemically. Functional overlap andtor acquisition of distinct functions would be reflected in qualitative andlor quantitative aspects of ligand display. Using five different stages of embryogenesis, differential regulation of the two galectins was detected in liver and intestine. The clear preference for one galectin (CC-14) was observed in intestine already at rather early stages, whereas equivalence for both proteins was noted in liver from day 12 to day 18 prior to hatching, as seen by ELISA assays and Western blot analysis. Presentation of galectin-reactive glycoproteins showed a tendency for gradual increase in both organs. Galectin-blotting analysis revealed primarily very similar patterns of positive bands at the different stages of development and only few quantitative and qualitative changes. The reactivity of glycolipids in a solid-phase assay was more variable, even surpassing the response of extracts of the adult organ at several embryonic stages. While the localization patterns of the galectins and galectinreactive sites were nearly indistinguishable in the liver, intestinal tissue differed with respect to the placement and accessibility of binding sites. Thus, the results suggest a differential regulation of galectin activities in the two organs. As a sum they resemble the course of development of availability of glycoprotein ligands in vitro. These findings support the notion for a partial functional redundancy in this family. The described approach to employ galectin-specific antibodies and the labeled galectins as tools to assess presentation of ligands is suggested to be of general relevance to address the question of distinct vs. overlapping functions of related recognition molecules.

The display of cellular oligosaccharide chains is known to undergo marked developmental changes, as monitored histochemically with plant lectins. In conjunction with endogenous lectins respective ligand structures may have a functional role during fetal development. The assumption of a recognitive, functionally productive interplay prompts the study of the expression of a tissue lectin and of lectin-reactive glycoconjugates concomitantiy. Focusing on comrnon Bgalactosides as constituents of oligosaccharide chains and the predominant member of the farnily of galectins in marnrnals, namely galectin-1, the question therefore is addressed as to whether expression of lectin and lectinreactive glycoconjugates exhibits alterations, assessed in three morphologically defined fetal stages and in adult bovine organs. Using a sandwich ELISA, the leve1 of the rather ubiquitous galectin-1 is mostly increased in adult organs relative to respective fetal stages, except for the case of kidney. This developmental course is seen rather seldom, when the amounts of lectin-reactive glycoproteins or glycolipids are quantitated in solid-phase assays after tissue homogenization. Western blotting, combined with probing by labeled galectin-1, discloses primarily quantitative changes in the reactivity of individual glycoproteins. Perforrning the same assays on extract aliquots with a plant agglutinin, namely the galactoside-binding mistletoe lectin, whose fine specificity is different @m galectin-1, its reduced extent of binding in solid-phase assays and the disparate profile of lectin-reactive glycoproteins reveal a non-uniform developmental alteration within the group of stmctural variants of B-galactosides. Although sample preparation can affect ligand preservation andlor presentation and thus restricts the comparability of biochemical and histochemical results, especially for soluble reactants, the histochemical studies on frozen and paraffinembedded sections of bovine heart, kidney and liver demonstrate that the localization of the galectin and of lectin-reactive epitopes can show a sirnilar distribution, as seen in liver and heart, with organ-typical quantitative changes of a rather similar staining profile (heart, kidney) or notable changes in the spatial distribution (liver) in the course of development. This report emphasizes the potential value of combined monitoring of the lectin and its potential in vivo ligands to contribute to eventually unravel organ-related function(s) of a tissue lectin.

Galactosyltransferase (GalTase) has been discovered on a variety of cells where it is believed to be involved in cell-cell adhesion and cell-substratum adhesion as well as in metastasis of carcinoma cells. This immunohistochemical study was undertaken to identify the topography and the cellular distribution of GalTase in normal prostatic tissue, benign prostatic hyperplasia (11 cases), and prostatic carcinoma (26 cases). Immunoreactive GalTase was found to be exclusively associated with carcinoma cells and with premalignant epithelial cells in prostatic hyperplasia. In highly differentiated carcinomas, most of the carcinoma cells are positive for GalTase, whereas in poorly differentiated tumors, GalTase immunoreactivity was restricted to a subset of carcinoma cells with obviously invasive behavior. At the cellular level, GalTase was localized in the cytoplasm and at the cell membrane. In sections of normal prostatic tissue, as well as in unaltered acini of prostatic hyperplasic tissue, GalTase was not expressed. Fibroblasts, smooth muscle cells, and endothelial cells of the prostatic stroma were also consistently negative. With the use of immunoblots, we could confirm the presence of GalTase with a molecular mass of 45 kDa in the extracts of benign prostatic hyperplasic tissue and in prostatic carcinoma tissue but not in normal prostatic tissue. The results of our immunohistochemical study suggest that GalTase is a valuable marker to diagnose neoplastic transformation in prostatic tissue.

Plant and invertebrate lectins are valuable cyto- and histological tools for the localization of defined carbohydrate determinants. The well-documented ubiquitous occurrence of sugar receptors encourages functional considerations. Undoubtedly, analysis of the presence of vertebrate lectins in tissues and cells is required to answer the pertinent and tempting question on the physiological relevance of protein (lectin)-carbohydrate recognition in situ. Carrier-immobilized glycoligands, derived from custom-made chemical synthesis, enable the visualization of respective binding sites. Histochemically inert proteins or synthetic polymers with appropriate functional groups are suitable carrier molecules for essential incorporation of ligand and label. The resulting neoglycoconjugates can track down tissue receptors that are neither impaired by fixation procedures nor blocked by endogenous high-affinity ligands. Lectins, especially the receptors of the tissue under investigation (endogenous lectins), and appropriately tailored immobilized glycoligands or lectin-specific antibodies (when available) are complementary tools to test the attractive hypothesis that diverse, functionally relevant glycobiological processes within or between cells are operative. Concomitant evaluation of both sides of lectin histochemistry, namely lectins as tools and lectins as functionally important molecules in situ, will indubitably render desired progress amenable in our often still fragmentary understanding of the importance of tissue lectin and glycoconjugate expression and its regulation.

Plant and invertebrate lectins are valuable cyto- and histological tools for the localization of defined carbohydrate determinants. The welldocumented ubiquitous occurrence of sugar receptors encourages functional considerations. Undoubtedly, analysis of the presence of vertebrate lectins in tissues and cells is required to answer the pertinent and tempting question on the physiological relevance of protein (1ectin)-carbohydrate recognition in situ. Carrierimmobilized glycoligands, derived from custom-made chemical synthesis, enable the visualization of respective binding sites. Histochemically inert proteins or synthetic polymers with appropriate functional groups are suitable carrier molecules for essential incorporation of ligand and label. The resulting neoglycoconjugates can track down tissue receptors that are neither impaired by fixation procedures nor blocked by endogenous highaffinity ligands. Lectins, especially the receptors of the tissue under investigation (endogenous lectins), and appropriately tailored immobilized glycoligands or lectin-specific antibodies (when available) are complementary tools to test the attractive hypothesis that diverse, functionally relevant glycobiological processes within or between cells are operative. Concomitant evaluation of both sides of lectin histochemistry, namelylectins as tools and lectins as functionally important molecules in situ, will indubitably render desired progress amenable in our often still fragmentary understanding of the importance of tissue lectin and glycoconjugate expression and its regulation.

Compared with normal prostatic tissue, the level of basic fibroblast growth factor (bFGF) is elevated in prostatic tumors. This suggests that bFGF may play a role in the development of prostatic neoplasms. The current study was undertaken to identify the cellular distribution of bFGF in benign prostatic hyperplasia (BPH) and prostatic carcinoma using a polyclonal antiserum against recombinant bFGF. In paraffin sections of prostatic tumors immunoreactive bFGF was found in fibroblasts, smooth muscle cells, and endothelial cells. Distinct staining was seen in most nuclei of these cells and a less intense immunoreaction occurred in the cytoplasm of smooth muscle cells. No immunostaining was seen in prostatic epithelial cells of prostatic tumors whether benign or malignant. With digoxigenin-labeled oligonucleotides in nonradioactive in situ hybridization, the presence of mRNA for bFGF was shown in smooth muscle cells of the stroma, suggesting that these cells are the main source of bFGF in BPH. Because no immunostaining for bFGF was obtained in the carcinoma cells, a specific role for bFGF cannot be seen for the development of malignant prostatic tumors.

We used several biotinylated neoglycoproteins as tumor markers to detect and localize endogenous carbohydrate-binding proteins in cultured hepatoblastoma, melanoma, and bladder carcinoma tumor cells. The neoglycoproteins used consisted of cellobiose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine, lactose, maltose, mannose, melibiose, and xylose. In addition, naturally occurring asialofetuin that was chemically disialylated was also used. Binding to the cultured tumor cells was made visible with the avidin-peroxidase technique. Depending on the type of neoglycoprotein used, markedly different expression of cytoplasmic and nuclear receptors for sugars (endogenous lectins) was obtained from rat hepatoblastoma, human melanoma, and bladder carcinoma tumor cells. The most pronounced staining differences were documented for asialofetuin and the neoglycoproteins containing fucose, N-acetyl-galactosamine, and lactose.

Following the detection of individual members of the family of galectins it is an obvious challenge to define the extent of functional overlap/divergence among these proteins. As a step to address this issue a comparative profiling has been started in the mouse as a model organism, combining sequence analysis, expression patterns and structural features in the cases of the homodimeric galectins-1, -2 and -7. Close relationship was apparent at the level of global gene organization. Scrutiny of the proximal promoter regions for putative transcription-factor-binding sites by two search algorithms uncovered qualitative and quantitative differences with potential to influence the combinatorial functionality of regulatory sequences. RT-PCR mapping with samples from an array of 17 organs revealed significant differences, separating rather ubiquitous gene expression of galectin-1 from the more restricted individual patterns of galectins-2 and -7. Using specific antisera obtained by affinity depletion including stringent controls to ascertain lack of cross-reactivity these results were corroborated at the level of galectin localization in fixed tissue sections. Nuclear presence was seen in the case of galectin-1. In addition to nonidentical expression profiles the mapping of the carbohydrate recognition domains of galectins-1 and -7 by homology modelling and docking of naturally occurring complex tetra- and pentasaccharides disclosed a series of sequence deviations which may underlie disparate affinities for cell surface glycans/glycomimetic peptides. In view of applicability the presented data can serve as useful reference to delineate changes with respect to disease and in genetically engineered models. To enable more general conclusions on the galectin network it is warranted to further pursue this combined approach within this lectin family.

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell’s glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 µ g/ml. Under these conditions, we defined cell-type- and differentiationdependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAcdependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phytohaemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein’s glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.

Growth and differentiation of the mammary gland during development and lactation are controlled by complex hormonal mechanisms. Additionally growth factors are supposed to act as local mediators of the hormonally controlled developmental processes. Mammary tissue for this study was obtained from non pregnant control heifers, primigravid heifers (second part of pregnancy), around parturition, during lactation (early and late) and from dry cows. Using RT-PCR and ribonuclease protections assay (RPA) the expression of the following growth factors was studied in the different phases bovine mammary gland development: Insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), fibroblast growth factor 1 (FGF-I), fibroblast growth factor 2 (FGF-2), transforming growth factor alpha (TGF-alpha). Additionally the expression of fibroblast growth factor receptor (FGFR) and growth hormone receptor (GHR) was investigated. The cellular distribution pattern of several of these growth factors and GHR was obtained using Immunocytochemical techniques. The detailed expression and localization pattern of these growth factors are presented and their role in the local regulation of the bovine mammary gland is briefly discussed.

Monocytes/macrophages play a critical role in the pathogenesis of HIV infection, both as targets for virus replication and as sources of production of multifunctional cytokines. Endothelins, peptides with potent vasoconstricting activities originally isolated from endothelial cells, are also produced and secreted by macrophages in a manner similar to that of other cytokines. In an attempt to explore the potential role of endothelins in HIV-infection, we investigated the effect of the HIV-1 envelope glycoprotein, glycoprotein 120, on monocytic endothelin-1 production. This glycoprotein has been identified as a potent stimulator of monokines such as TNF-alpha and IL-6, which have been implicated as potential mediators of HIV-encephalopathy. We found that glycoprotein 120, similar to LPS, stimulates the secretion of endothelin-1, as well as TNF-alpha, from macrophages in a concentration-dependent manner. Using reverse transcriptase polymerase chain reaction, we found that circulating monocytes in HIV-infected individuals show a distinct expression of the endothelin-1 gene that is not detectable in healthy controls, indicating chronic activation of this gene in HIV-infection. In addition, cerebral macrophages in patients with HIV-encephalopathy were strongly positive for endothelin. Thus, monocytic endothelins appear to be stimulated during HIV infection. Their potent vasoactive properties render them potential candidates for mediating alterations in the cerebral perfusion pattern associated with the AIDS dementia complex.

alpha-Galactosyl residues in the carbohydrate part of cellular glycoconjugates can serve as cell type-associated markers and are implicated in intercellular adhesion and biosignaling. This biological significance explains the interest to characterize probes with respective specificity as the Griffonia simplicifolia I-isolectin B4. Due to the documented occurrence of an alpha-galactoside-binding immunoglobulin G fraction in human serum we compared the extent of binding and its pattern for the lectin and the antibody using surface-immobilized extract proteins and fixed sections of bovine testicular tissue with known lectin reactivity. The antibody fractions were obtained either solely from affinity chromatography isolation on immobilized melibiose or after an additional step to deplete this fraction of galactoside-binding activities without pronounced specificity to the alpha-anomeric linkage. They yielded a rather indistinguishable reactivity in comparison to that of the lectin, when an indirect approach was used. Labeling of the antibodies with a hydrazide derivative of biotin did not affect the pattern of binding. However, significant differences were noted, when conjugation of label was targeted to amino groups via N-hydroxy-succinimide esters of biotin and digoxigenin despite performance of the modification under activity-preserving conditions. Notably, the apparent strong staining of Leydig cells and nuclei of primary spermatocytes, respectively, was not inhibitable by sugar. These differences were corroborated by a nonidentical response of the various probes in solid-phase assays with extract proteins. Thus, care should be exercised in the interpretation of histochemical data, obtained with this type of modified antibody. When these precautions are fulfilled, this immunoglobulin fraction from human serum has the potential as an alpha-galactosyl-specific histochemical tool.

Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors, which trigger a phosphorylation cascade resulting in the modulation of numerous signalling pathways, and dictate gene expression. Immunohistochemical techniques were used to demonstrate the presence of growth hormone receptors in 126 formalin-fixed, paraffin-embedded melanocytic tumours comprising melanocytic naevi, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma and metastatic melanomas. The relative proportion of positive cells and intensity of staining was higher in neoplastic cells, compared to normal cutaneous cells. Of the 76 cases of common melanocytic naevi (CMN) studies, 46 were weakly reactive with MAb 263. Heterogeneity of immunoreactivity was found in primary melanoma lesions with a variable range of positive cells. Of 37 cases studied, 34 were moderately to strongly positive. Immunoreactivity showed subcellular localization of the GH-receptor in cell membranes, was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. The nuclear localization of immunoreactivity is the result of nuclear GH-receptor/binding protein, identically to the cytosolic and plasma growth hormone binding protein. Intense immuno-reactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH-receptor synthesis. In the primary lesions, dermal tumour cells tended to be more immunoreactive relative to those seen in the dermal region. Metastatic lesions in various organs also expressed growth hormone receptors in secondary tumour cells and all of the metastatic cases were positive. The expression of GH-receptors in human melanoma cells means that these cells are directly responsive to GH action and that GH may stimulate local production of IGF-I, which then acts in an autocrine mechanism.

Introduction: Anatomical courses on the limb osteology provide basic facts and anatomical terms necessary for the veterinarian profession. Within the course most students encounter an extensive anatomical terminology for the first time that has to be addressed during a period limited by the course duration. Additional self-learning hours can be difficult if the access to bones is restricted and term explanations are limited to textbooks and figures. Materials and Methods: 3D models of thoracic and pelvic limb bones of the horse were created in 3DSOM pro ver3, 2011 Creative Dimension Software Ltd. using on average 30 to 45 shots from different angles of every limb bone. 3D models were improved in Meshmixer, 2014 Autodesk Inc. and Blender 2.7, Blender Foundation. Latin terms from Nomina Anatomica Veterinaria 2012 were used to label the models with the help of FinalMesh 1.0, Pelikan Software Kft. Results: 3D models of thoracic and pelvic limb bones of the horse were developed and labelled with 354 anatomical terms in total. Each bone can be rotated and inspected from different angles. The corresponding area appears labelled on the bone model after a klick on an anatomical term from the list. The bone models were exported into WebGL format that enables them to be processed in most commonly used internet browsers (Mozzila, Firefox, Google Chrome etc.) with no additional programs. 3D models are intended for the use on PCs, tablets, and smartphones and will be available at the web site of the Faculty of Veterinary Medicine University of Zagreb after termination of the standard publication procedure. Conclusion: 3D labelled bone models should prove valuable asset during self-study of the thoracic and pelvic limb osteology. Their small size enables easy and quick processing while the Latin labelling and free access ensure the worldwide usage.

Introduction: This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. In this study, we have determined and compared expression pattern of: 1) Thy1 - YFP positive cells during in vitro differentiation of neural stem cells (NSC), 2) during embryonic development of Thy1 – YFP mouse embryo and 3) after transplantation of Thy1 – YFP cells into the stroke-affected mouse brain. Materials and Methods: Neural stem cells were isolated from the forebrain of 14.5 old mouse embryos and cultured as neurospheres. Immunocytochemistry and RT-PCR were performed after cells differentiation. Embryonic slices were prepared for immunohistochemistry. In third part of this study, PKH26 labeled NSC were transplanted into the stroke-affected mouse brain obtained by MCAO method. Results: THY1 - YFP expression was described during differentiation of NSC and observed in neural progenitors as well as in mature neurons. Neural progenitors were nestin positive while mature cell were not. THY1 – YFP positive cells were positive for mature neuronal markers (MAP2, β3-tubulin and NeuN) during the whole differentiation period, while astrocytes which were GFAP positive were not expressing YFP. During embryonic development THY1 – YFP positive cells were observed from E12.5 and the expression steadily increased to the end of pregnancy. Positive cells were present in both, central and peripheral nervous system. After transplantation, cells survived and incorporated into the stroke-affected brain. Conclusion: Our results showed that mature neurons as well as neuronal progenitor cells do express THY1 – YFP construct in in vitro conditions, during embryonic development and after transplantation into the stroke-affected mouse brain. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.