Immunocytochemical identification and localization of peptide hormones in the gastro-entero-pancreatic (GEP) endocrine system of the mouse and a stomachless fish,Barbus conchonius

Summary A large number of antisera mainly raised against mammalian hormones are tested immunocytochemically on the GEP-endocrine system of mouse and fish (Barbus conchonius). The endocrine pancreas of mouse and fish appeared to contain the same four endocrine cell types; insulin-, glucagon-, PP- and somatostatin-immunoreactive cells.In mouse about 13 GEP endocrine cell types are distinguished 1. insulin-, 2. somatostatin-, 3. glucagon-, 4. PP-, 5. (entero)glucagon-/PP-like, 6. CCK-like, 7. substance P-, 8. neurotensin-, 9. VIP-, 10. gastrin-, 11. secretin-, 12. -endorphin-, 13. serotonin-immunoreactive cells.Based on this and a previous study at least 13 GEP endocrine cell types seems to be present in stomachless fish: 1–9 as described for mouse, 10. (entero)glucagon-like, 11. met-enkephalin, 12. VIP-like, 13. unspecific immunoreactive endocrine cells.Coexistence of glucagon and PP-like peptides is found in the gut and pancreas of mice and in the gut of B. conchonlus. In mouse pancreas and fish gut, endocrine cells showing only PP-or glucagon-like immunoreactivity are found too. In mouse stomach some endocrine cells, showing only PP-immunoreactivity are demonstrated. In the same region coexistence of C-1-gastrin-and FMRF-amide-immunoreactivity is found in endocrine cells. The importance of these phenomena are discussed.Enteric nerves immunoreactive with antisera raised against substance P and GRP are found in mouse, against somatostatin and met-enkephalin in both mouse and fish and against VIP in fish.In honour of Prof. P. van Duijn     

Immunohistochemical localization of (neuro)peptide hormones in endocrine cells and nerves of the gut of a stomachless teleost fish,Barbus conchonius (Cyprinidae)

Summary Enteroendocrine cells containing glucagon-, substance P-, neurotensin- and VIP-like substances have been demonstrated immunocytochemically in the gut of Barbus conchonius. Mainly based on the distribution of the immunoreactive endocrine cells in this and a previous^* study, at least eight different enteroendocrine cell types appear to be present in this stomachless fish: 1. C-terminal-gastrinimmunoreactive cells^*, predominantly present in the upper parts of the folds of the proximal part of the intestinal bulb. 2. Metenkephalin-immunoreactive cells^*, basally located in the folds of the first segment. 3. Pancreatic polypeptide (PP)-immunoreactive cells^*, mainly present in the first half of the first segment. 4. Glucagon-like-immunoreactive (GLI) cells that are basally located in the folds of the first segment and that contain a different polypeptide (possibly glicentin) than pancreatic glucagon cells. 5. Substance P-immunoreactive cells, present in the upper parts of the folds throughout the gut. 6. C-terminal-neurotensin-immunoreactive cells, basally located in the folds throughout the first segment. 7. Vasoactive intestinal polypeptide (VIP)-immunoreactive cells, present in small numbers in the proximal part of the intestinal bulb. 8. Nonspecifically-immunoreactive cells^*, found throughout the intestinal bulb. Many VIP-immunoreactive nerves have been demonstrated in the smooth muscle layer and myenteric plexus of the gut; furthermore some of them are peptide histidineisoleucine (PHI)-immunoreactive. Substance P-, somatostatin-, neurotensin- and met-enkephalin-immunoreactive nerves are also found. Thus, at least partial sequences of four different mammalian neuropeptide hormones (VIP, substance P, neurotensin, met-enkephalin) occur both in endocrine cells and enteric nerves of the gut of B. conchonius.     

Proliferation and differentiation of intestinal epithelial cells during development of Barbus conchonius (Teleostei,Cyprinidae)

Summary The processes of proliferation, cell division and differentiation of intestinal epithelial cells have been studied during development of the fish, Barbus conchonius. On the 3rd day, nearly all cells of the presumptive gut proliferate. Once the intestinal epithelium begins to differentiate, a decreasing percentage of proliferative cells can be found. On the 7th day, when intestinal folds start to develop, the proliferative cells become restricted to the future basal parts of the folds.Ultrastructural examination of ³H-thymidine-labeled cells and mitotic cells of 6-day-old larvae shows that functional enterocytes are proliferative. The same feature is suggested for older fish. Proliferating undifferentiated dark cells, characterized by many free ribosomes and a few organelles, are also present in the intestinal epithelium of larval fish; they are considered to be stem cells, mainly for goblet cells. Proliferating goblet cells and enteroendocrine cells were not observed. The latter cell type is scarce and has a long turnover time.A common feature of all these dividing cells is the presence of isolated spherical to cylindrical lamellar structures which may have lost contact with the cell membrane during prophase; they probably regain this contact by fusion with the cell membrane at the end of mitosis.     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Summary Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP-or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas.Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide-and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide-or glucagon-immunoreactivity were also found. Glucagon-and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Arrayed adenoviral expression libraries for functional screening

With the publication of the sequence of the human genome, we are challenged to identify the functions of an estimated 70,000 human genes and the much larger number of proteins encoded by these genes. Of particular interest is the identification of gene products that play a role in human disease pathways, as these proteins include potential new targets that may lead to improved therapeutic strategies. This requires the direct measurement of gene function on a genomic scale in cell-based, functional assays. We have constructed and validated an individually arrayed, replication-defective adenoviral library harboring human cDNAs, termed PhenoSelect library. The adenoviral vector guarantees efficient transduction of diverse cell types, including primary cells. The arrayed format allows screening of this library in a variety of cellular assays in search for gene(s) that, by overexpression, induce a particular disease-related phenotype. The great majority of phenotypic assays, including morphological assays, can be screened with arrayed libraries. In contrast, pooled-library approaches often rely on phenotype-based isolation or selection of single cells by employing a flow cytometer or screening for cell survival. An arrayed placental PhenoSelect library was screened in cellular assays aimed at identifying regulators of osteogenesis, metastasis, and angiogenesis. This resulted in the identification of known regulators, as well as novel sequences that encode proteins hitherto not known to play a role in these pathways. These results establish the value of the PhenoSelect platform, in combination with cellular screens, for gene function discovery.     

Ultrastructural characterization of 6 immunoreactive enteroendocrine cells in Barbus conchonius (Teleostei, Cyprinidae)

Using the semi-thin/ultra-thin technique six different immunoreactive endocrine cell types are ultrastructurally identified in 0.5% glutaraldehyde fixed gut of B. conchonius. In addition two of them (gastrin- and PP-immunoreactive cells) are also characterized with the immunogold method, showing that the immunoreactivity is only restricted to the secretory granules. Size distribution histograms and the average diameters of 30% (d30) of the largest granules are given, showing a gradual increase in granule size from unspecific immunoreactive cells, (d30 = 110 nm) via gastrin- (119 nm), VIP-like- (127 nm), met-enkephalin- (143 nm) and PP- (174 nm) to glucagon-immunoreactive cells (178 nm). The presence of PP- and glucagon-immunoreactivity in the same cells and the consequence for their granule size is discussed. In the distal part of the gut endocrine cells are found showing no immunoreactivity with the antisera used; their granules (d30 = 144 nm) were, although not significantly, larger then those of VIP-like-immunoreactive cells, also found in that part of the gut. It is supposed that they represent substance P-immunoreactive cells. Unfortunately, secretory granules of several cell types showed about 20% more shrinkage in 0.5% glutaraldehyde fixed tissue, than in osmicated tissue.     

Immunological importance of the second gut segment of carp. I. Uptake and processing of antigens by epithelial cells and macrophages

Uptake and transport of soluble (ferritin) and particulate ( Vibrio anguillarum ) antigen from intestinal lumen to mucosal macrophages was studied using immunocytochemical and electron-microscopical techniques. Both antigens were taken up by epithelial cells of the second gut segment, reached the supranuclear vacuoles and were finally transported to large intraepithelial macrophages. In contrast with particulate antigen, antigenic determinants of ferritin were demonstrated in the basal part of the epithelium and appeared to be released at the mucosal site.
After anal intubation many small macrophages penetrated the intestinal epithelium, took up antigen and after 24 h disappeared again from the intestinal mucosa. This feature was antigen independent and also occurred after anal PBS-intubation. The larger, less mobile macrophages stayed in the intestinal epithelium and finally exposed antigenic determinants (of both antigens) at their outer surface, suggesting an antigen-presenting function. Whether these large macrophages can induce a local or mucosal immune response and whether the smaller mobile macrophages are involved in a systemic response is discussed.