Distribution of peptide-containing neurons and endocrine cells in the rabbit gastrointestinal tract,with particular reference to the mucosa

Summary The distribution patterns of peptide-containing neurons and endocrine cells were mapped in sections of oesophagus, stomach, small intestine and large intestine of the rabbit, by use of standard immunohistochemical techniques. Whole mounts of separated layers of ileum were similarly examined. Antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), enkephalins (ENK) and gastrin-releasing peptide (GRP) were used, and for each of these antisera distinct populations of immunoreactive (IR) nerve fibres were observed. Endocrine cells were labelled by the SP, SOM or NPY antisera in some regions.VIP-IR nerve fibres were common in each layer throughout the gastrointestinal tract. With the exception of the oesophagus, GRP-IR nerve fibres also occurred in each layer of the gastrointestinal tract; they formed a particularly rich network in the mucosa of the stomach and small intestine. Fewer nerve fibres containing NPY-IR or SOM-IR were seen in all areas. SOM-IR nerve fibres were very scarce in the circular and longitudinal muscle layers of each area and were absent from the gastric mucosa. The SP-IR innervation of the external musculature and ganglionated plexuses in most regions was rather extensive, whereas the mucosa was only very sparsely innervated. ENK-IR nerve fibres were extremely rare or absent from the mucosa of all areas, although immunoreactive nerve fibres were found in other layers.These studies illustrate the differences in distribution patterns of peptide-containing nerve fibres and endocrine cells along the gastrointestinal tract of the rabbit and also show that there are some marked differences in these patterns, in comparison with other mammalian species.     

The localization of macrophage subsets and dendritic cells in the gastrointestinal tract of the mouse with special reference to the presence of high endothelial venules

Summary This study concerns the distribution of macrophages and dendritic cells (DC) in the gastrointestinal tract of the mouse. Heterogeneity of macrophage population was found by using the MOMA-1, MOMA-2, ERTR-9, Mac-1 and F4/80 monoclonal antibodies. MOMA-1, ERTR-9, Mac-1 and F4/80+ cells were detected mostly at the villous cores in the lamina propria of the villi, whereas MOMA-2+ cells were primarily found around the crypts at the base of the villi. These MOMA-2+ cells revealed a granular appearance throughout the cytoplasm and displayed a strong acid phosphatase (AcPh) activity. Few MOMA-2+ cells were seen at the top of the villi in the epithelium. Although MOMA-1 and ERTR-9+ cells have similar morphology and the same distribution patterns in the lamina propria, they are likely different populations, because in Peyer's patches (PP), MOMA-1+ cells were present, whereas ERTR-9+ cells could not be detected. Both populations displayed AcPh activity. Strongly stained Mac-1+ cells were abundantly seen in the lamina propria of the small intestine. F4/80+ cells were rare. NLDC-145+ cells with AcPh activity and weak Ia staining were also found. In the PP-associated villi and in the T-dependent area of PP, dendritic NLDC-145+ cells, which were strongly Ia positive, were detected. MIDC-8+ cells were found only in the T-dependent area. Few NLDC-145+ cells (dendritic cells) were found in the upper part of the oesophagus. These cells were also stained with the MIDC-8 antibody. The MECA-325 monoclonal antibody recognized high endothelial venules (HEV) in PP and blood vessels at the base of the villi of the jejunumileum and caecum. Unlike in PP, the endothelium of the venules in the villi was flat.     

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.     

Cytologic observations on the pancreatic islets with reference to some endocrine-like cells of the gastrointestinal mucosa

Summary Some findings obtained from cytological studies on the pancreatic islet of man and different animal species by applying several appropriate methods are reported. Three islet cell types were found to be simultaneously demonstrated by toluidine blue after methylation and saponification of tissue sections; metachromasia appeared to be in some way a constant finding of argyrophil D-cells which, at least in duck and man, displayed also a weak PAS-positivity. No one of the three insular cell types was found to show all the morphologic patterns of enterochromaffine cells, nor enterochromaffine cells showed all the patterns of insular cells. The existence of possible morphologic relationships between insular A-cells and some endocrine-like cells in fundic mucosa and, on the other hand, between argyrophil D-cells and some cells in antro-pyloric mucosa of the stomach is emphasized.This work was supported by the Grant N. 04/57/4/7137 of the Consiglio Nazionale delle Ricerche, Rome.     

Histochemical differentiation of endocrine gastrointestinal cells in test animals, with special reference to chicken embryos.

Based upon the characteristics of argent-affinity and argyrophilia and the application of specific immune histochemical methods, endocrine cells can be differentiated in the most prominent species of test animals. These methods are demonstrated in chicken embryos, because embryological studies made in order to reveal the still unknown endodermal or neuroectodermal origin of these cells are highly important.     

Methodological aspects on immunohistochemistry in dermatology with special reference to neuronal markers

Summary In an attempt to optimize the immunohistochemical procedure for visualizing neuronal markers, such as neuropeptides, in the human skin, different alternatives in all steps of the process were compared. We have studied the influence of type of immunohistochemical method, the biopsy technique, including the size of the punch biopsy, anaesthesia, the choice of fixative and the time of fixation, the storage process, the sectioning parameters, incubation procedure, the type of fluorophore-conjugated antibody and its dilution, mounting and storage, and, finally, microscopical examination.The following procedure was found to give the best result: punch biopsies of 3 mm, taken under local anaesthesia using lidocaine injected into the dermis-subcutis at the place of biopsy; fixation by a buffered 10% formalin solution containing 14% of saturated picric acid for 2 h at 4°C; storage in 10% sucrose buffer for at least 24 h up to 1 month at 4°C or deep-frozen to -70°C for 2 months (with only a minor structural deterioration); cryostat sectioning of the biopsies with a section thickness of 14 m and with the cutting edge perpendicular to the skin surface; rhodamine (TRITC)-conjugated, instead of fluorescein-isothiocyanate (FITC)-conjugated, secondary antiserum, since it gives a lower background fluorescence; and for the incubation and mounting procedures, our standard laboratory routines were applied. The result is an optimal indirect immunofluorescence technique, to be applied in dermatology. We also found that biopsies taken under local anaesthesia with chlorethyl spray lost almost all immunofluorescence for several neuronal markers in the epidermis-upper dermis.     

The noninvasive biochemical diagnosis of gastrointestinal disease, with special reference to children

Invasive tests to diagnose patients with gastrointestinal disease are rapidly being replaced by procedures which enable organ function to be assessed by monitoring the product of a metabolic reaction in readily available materials such as breath, blood, and urine. Examples of these approaches that will be assessed in this review include the hydrogen breath test for lactase deficiency, radioactive carbon dioxide breath measurements to test for fat digestion and absorption, and tests of pancreatic function based upon synthetic substrates from which fluorescein or para-aminobenzoic acid can be liberated by pancreas-specific enzymes. Significant advances have been made in improving the organ sensitivity of enzyme determinations. The determination of amylase isoenzymes has been less useful than the measurement of immunoreactive trypsin; this latter enzyme is greatly elevated in the blood of neonates with cystic fibrosis, whereas serum levels are greatly depressed in cystic fibrosis patients with pancreatic insufficiency as well as in most patients with steatorrhea due to chronic pancreatitis. Many of these tests are now becoming standard procedures in the investigation of infants with gastrointestinal disease.     

The embryonic development of the intestinal mucosa with special reference to its epithelium

Electron microscopic examinations were made of different parts of the bovine intestine (n = 13) up to the 10th week of embryonic development. During the 'phase of undifferentiated epithelium' the embryonic intestinal epithelium can be classified as stratified and is perhaps a pool of cells. Microvilli of the apical plasmalemma appear at first in neighboring and opposing cells in the centre of the epithelium. They already show microfilaments as well as a glycocalix. The supranuclear cytoplasm shows many granules, vesicles and arciform structures which may be used in the process of microvilli formation. The importance of infranuclear basal granules in the peripheral epithelial cells is still unknown; perhaps they are merely phylogenetic remnants of a principle of development common to all vertebrate intestines. Single cilia which are formed in the periluminal cytoplasm presumably suppress mitotic activities of the epithelial cells and induce their ensuing differentiation. Epithelial proliferation is the initial event of villigenesis, giving rise to epithelial primary villi. Immediately following is the formation of secondary villi during proliferation of the mesenchyme.     

Studies on mycobacteria isolated from animals, with special reference to the agglutination test

Ninety-three strains of slowly-growing mycobacteria were studied biochemically. Ninety of these were isolated from animals (pigs, cattle, dog and poultry) and three from dust and sawdust-bedding in a pighouse. One strain from a lymph node of a pig was identified as M. gordonae. Ninety-two strains fitted into the M. aviam-intracellulare complex. Of the 92 biochemically confirmed M. avium-intracellulare strains, 78 were tested serologically ad modum Schaefer. Of 73 strains from pigs, one was serotype 1, fifty serotype 2 and eight serotype 8, while two could not be type and twelve were autoagglutinable. Three strains from pighouse environment were serotype 8 and two from cattle and a dog were both serotype 2. A slight modification of Schaefer's agglutination method, using smaller amounts of antigen and antiserum, was developed.     

Membrane-bound glycoconjugates of fetal mouse erythropoietic cells with special reference to phagocytosis by hepatic macrophages

Using lectin and colloidal iron (CI) stainings in combination with neuraminidase digestion, glycoconjugates on the surface of erythropoietic cells of the yolk sac and liver in fetal mice were examined. Fetal hepatic macrophages were capable of distinguishing between phagocytozed and non-phagocytozed erythroid elements as described in our previous study. Marked differences between these two elements could be ultrahistochemically detected on their cell surface. The phagocytozed elements, such as nuclei expelled from erythroblasts and degenerating primitive erythroblasts, faintly bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a weak peanut agglutinin (PNA) binding. In contrast, erythroblasts at various maturation stages, erythrocytes and normal primitive erythroblasts heavily bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a moderate PNA binding. No differences in binding of either concanavalin agglutinin,Ricinus communis agglutinin-I or PNA were noted between phagocytozed and non-phagocytozed erythroid elements. Desialylation appears to be one of the most important signs for the recognition mechanism of fetal macrophage phagocytosis. During maturation of hepatic erythroblasts, sialic acid changes its affinity forLimax flavus agglutinin from strong to weak, and soybean agglutinin binding sites disappear at the basophilic erythroblast stage. Glycoconjugates on polychromatophilic erythroblasts acquire similar compositions to those of erythrocytes.     

Localization of chromogranin A-immunoreactivity in bovine gastrointestinal endocrine cells with special reference to Grimelius silver stain.

Immunohistochemical studies of the gastrointestinal tract were carried out to characterize the cells exhibiting immunoreactivity for chromogranin A (CGA), a glycosylated protein primarily found in secretory granules of the adrenal medulla. Double immunostaining for gastrointestinal hormones and CGA revealed that in the bovine gastrointestinal tract CGA immunoreactivity occurs in mucosal epithelial cells containing gastrin, glucagon, substance P or motilin, but not in those containing somatostatin. Combined staining with anti-CGA serum and Grimelius' silver demonstrated frequent association of the two stains in a variety of endocrine cells. However, intracellular distribution of the two stains was different: CGA-immunoreactivity was detected in both supra- and infranuclear cytoplasm, whereas Grimelius' silver was mostly localized in the infranuclear region. These results suggest that CGA is the target of Grimelius' silver, as postulated recently (Rindi et al., 1986), but that some subcellular structure-related modification of molecules such as sialation is necessary for the positive Grimelius reaction.