Gastropathies in the Lundehund. II. A study of mucin profiles

The mucin profiles of the gastric mucosa in Lundehunds suffering from intestinal lymphangiectasia were examined and compared to the mucin profiles in control dogs from other breeds. A previous study performed on this material had shown that all examined Lundehunds had gastritis and about 30% had gastric carcinoma. Neutral and acid mucins were identified using the periodic acid-Schiff (PAS) and Alcian blue (pH 2.5) periodic acid-Schiff (AB-PAS) methods. The acid mucins were divided into sialomucins and sulfomucins based on their reaction with high-iron diamine Alcian blue, pH 2.5 (HID-AB). In Lundehunds with chronic atrophic gastritis in the fundic and body regions the surface and foveolar epithelium showed a predominantly normal mucin profile although some Lundehunds had a reduced mucin content. The mucous neck cells extended from below the gastric foveolae towards the muscularis mucosae. Morphometric examination showed that the abnormal presence of mucous neck cells occupied 41% of the height of the gastric mucosa in Lundehunds compared to only 19% in the control dogs (p < 0.05). Of the four Lundehunds with gastric carcinoma, two possessed neoplastic cells that contained minimal or no mucins. The amount and type of mucin in the neoplastic cells of the remaining two Lundehunds varied both between individuals and within a neoplasm. This study shows that the abnormal presence of mucous neck cells and the associated pseudopyloric metaplasia comprised the predominant changes in the gastric mucin profiles of Lundehunds.     

Cytology and microenvironment of somatostatin (D) cells in the gastric fundic mucosa of rodents

Somatostatin from gastric D-cells exerts an inhibitory effect upon the release of gastric acid, enzymes and gastrin. From previous investigations, a paracrine mode of action of somatostatin has been postulated. However, the exact route of the delivery of gastric somatostatin remains still uncertain and controversial. To obtain a closer view of fundic D-cells, the complete shapes and their microanatomical relationships to neighboring tissue elements were examined in immunostained serial semithin (0.5 mu m) sections of the fundic mucosa of rats, mice and golden hamsters, exemplarily by the combined utilization of computer-assisted 3D reconstructions. All the D cells examined in the present study were found to belong to the 'closed-type' of entero-endocrine cells lacking contiguity to the luminal surface. In their shape, the D cells in this region displayed an expressed 'pleomorphism'. A subpopulation of D cells, ovoid in shape, were thoroughly enclosed by single parietal cells. Most of the D cells appeared to be intimately juxtaposed to parietal cells and/or chief cells with their cell bodies or cytoplasmic processes, but simultaneously blood capillaries were regularly located in close vicinity to such D cells. Thus, somatostatin from the fundic D cells may act upon parietal cells and chief cells via both paracrine (direct cell to cell or diffusion) and endocrine (local circulatory system). The morphological heterogeneity of gastric fundic D cells may reflect certain functional states.     

The histologic spectrum of Barrett's esophagus

To define the histology of the columnarlined esophagus, we obtained esophageal biopsies from various levels with manometric control from 11 patients. There were three types of columnar epithelia above the lower esophageal sphincter: atrophic gastric-fundic-type epithelium with parietal and chief cells; junctional-type epithelium with cardiac mucous glands; and distinctive specialized columnar epithelium with a villiform surface, mucous glands and intestinal-type goblet cells. When present, specialized columnar epithelium was always the most proximal, and gastric fundic epithelium the most distal epithelium. Junctional epithelium was interposed between gastric fundic and specialized columnar or squamous epithelium. Four patients had unequivocal esophagitis in squamous epithelium, but its presence and severity did not correlate with inflammation in or length or type of distal columnar epithelium. Histoligic study of the columnar-lined esophagus demonstrated a spectrum of epithelial patterns. This heterogeneity helps to explain prior discrepant reports.     

Lack of volatilization and escape of orally administered trichloroethylene from the gastrointestinal tract of rats

The sugar residues in glycoconjugates present in the parotid and mandibular glands of the adult fallow-deer were detected and characterized by using a battery of eight different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. Parotid secretory cells produced glycoconjugates with N-acetylgalactosamine, N-acetylglucosamine and mannose residues. Mucous acinar cells were the most reactive sites of the mandibular gland and contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(beta 1-->3)N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Mandibular mucous cells also presented N-acetylglucosamine and sialylated components with the terminal dimer sialic acid-N-acetylgalactosamine. Demilunar cells contained glycoconjugates with fucose and mannose residues. The apical surface of duct cells was stained by all the lectins.     

Changes in parietal and mucous cell mass in the gastric mucosa of normal subjects with age: a morphometric study

Whether or not the gastric mucosa undergoes significant changes in normal aging subjects is still open to debate. In 51 subjects undergoing endoscopy and lacking any significant endoscopic or histologic modification we evaluated mucosal thickness, gland number, numbers of parietal, chief and mucous cells at the fundus and of mucopeptic cells at the antrum, with a morphometric method, subgrouping the patients according to their age class. Our findings demonstrate that the number of parietal cells tends to increase with age and, on the other hand, the number of mucous cells is reduced in elderly subjects (p < 0.05). When considering the parietal-to-mucous cell ratio, this is significantly increased (p = 0.0005) with age. Acid secretion being an offensive factor and mucus a fundamental component of the gastric mucosal barrier, these findings suggest an increased susceptibility of the gastric mucosa to damage in the elderly.     

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells.     

Gastric gland metaplasia in the small and large intestine

Fifty-six surgical specimens with various ulcerative intestinal disorders were microscopically investigated for evidence of gastric gland metaplasia. Thiry-one specimens (55-4%) showed pyloric gland metaplasia. Among the 31 patients with pyloric gland metaplasia, five showed true gastric metaplasia, consisting of parietal cells, chief cells, and mucous neck cells. The percentage of true gastric metaplasia among pyloric gland metaplasia was as high as 16%, an overall frequency of 9% among various ulcerative intestinal disorders. The mechanism of pyloric gland metaplasia and true gastric metaplasia is not understood, but may occur secondary to submucosal response to ulcer healing and subsequent alteration of the intraluminal condition in the intestine.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na+/K+ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions.     

Histological effect of (R)-alpha-methylhistamine on ethanol damage in rat gastric mucosa: influence on mucus production

(R)-alpha-Methylhistamine, a selective agonist of histamine H3 receptors, prevents macroscopically visible gastric lesions by absolute ethanol in the rat. A further insight into its activity was the aim of our study. Rats were given saline or (R)-alpha-methylhistamine (100 mg/kg) intragastrically. After 30 min, absolute ethanol was given and gastric mucosa was sampled 60 min later. Histologic damage and intracellular and adherent mucus were quantified. Luminal surface and mucous cells were examined by scanning and transmission electron microscopy. (R)-alpha-Methylhistamine reduced the extent of lesions by ethanol from 96 to 18%. Surface mucous cells and mucous neck cells were increased in volume and number, packaging of intracellular mucus was modified, and the secretory processes were promoted by (R)-alpha-methylhistamine itself, although these modifications were mostly evident in stomachs subsequently exposed to ethanol. Adherent mucus layer thickness was increased by (R)-alpha-methylhistamine only after ethanol exposure. It is concluded that (R)-alpha-methylhistamine predisposes mucous cells to react to ethanol.     

Proliferation and migration kinetics of stem cells in the rat fundic gland

The proliferation and migration of stem cells in the developing and adult rat fundic gland have been studied using BrdU immunohistochemistry and BrdU-GSA II (Griffonia-simplicifolia agglutinin-II) double staining. In the developing rat fundic gland, stem cells were first scattered throughout all levels of the epithelia and then concentrated in the depth of the pits. With the elongation and maturation of the fundic glands, stem cells left the gland base and moved upward. By 4 weeks after birth, the development of the fundic gland was completed and stem cells were confined to a narrow proliferative zone in the isthmus, reaching the adult distribution pattern. In the adult rat fundic gland, stem cells in the isthmus differentiated and migrated upward and downward, replacing the surface mucous cells and glandular cells respectively. For upward migration, it took about one week for stem cells to migrate from the isthmus to the surface. For downward migration, it took about two weeks for stem cells to migrate from the isthmus to the neck, and it took 30-36 weeks to reach the gland unit's blind end. Finally stem cells were lost at the deepest level of the glands. The results obtained by simple topographical distribution in the present experiment agreed well with those obtained by quantitative analysis, suggesting the usefulness of BrdU immunohistochemistry for cell kinetic studies.     

An IgG monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates in human tissues

Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice.     

Chronic atrophic fundic gastritis diagnosed by a modified Congo red test

BACKGROUND AND STUDY AIMS: Chronic atrophic fundic gastritis (CAFG) is associated with several diseases, such as gastric cancer, gastric ulcer, pernicious anemia, and bacterial overgrowth. In spite of recent technical improvements, the gastroscopic diagnosis of CAFG remains uncertain. Congo red chromogastroscopy is capable of visualizing acid-producing normal fundic mucosa, but has hitherto not been suitable for routine use. The aim of our study was to establish a reliable endoscopic technique with which to diagnose CAFG. PATIENTS AND METHODS: This prospective study comprises 124 consecutive patients (71 women, 53 min) with a mean age of 65 years (range 36-92). Macroscopic evaluation of the gastric fundic mucosa in routine endoscopy using video techniques was compared with evaluation by means of a modified endoscopic Congo red test (MCRT). In routine gastroscopy, CAFG was recognized by the thin, friable mucosa, with a marked visible vascular pattern and fold atrophy. With MCRT, the diagnosis of CAFG was made within five minutes' observation when no red-to-blue color shift in the fundic mucosa could be induced by 0.2 mu g/kg intravenous pentagastrin. The results were then compared with the histological examination of biopsies from the fundic mucosa. RESULTS: CAFG was confirmed by histology in 40 of 124 cases. The diagnostic sensitivity of MCRT was 1.0 (40/40), with a positive predictive value of 0.90, whereas the values for macroscopic gastroscopic evaluation were 0.25 (10/40) and 0.50, respectively. CONCLUSIONS: We conclude that MCRT is a sensitive, fast, and cost-effective method of identifying patients with CAFG, and well suited for use in routine gastroscopy.     

Transplantation. Abstract from 1970

We previously suggested that hydroxyl free radical (-OH) production may play a role in carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG-induced gastric cancer in rats and human gastric carcinoma occur most often in the antral mucosa and rarely in the normal fundic mucosa. We hypothesized that regional differences in anti-oxidant activity may be responsible. In the present study, we examined anti-oxidant activity by comparing the relative rates of reduction of a nitroxide free radical, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol), in the antral and fundic mucosa of male Wistar rats using ESR. The relative rate of Tempol reduction was significantly slower in the antral portion of the wall than in the fundic portion when Tempol [4 x 10(-6) mole/mg wet weight of gastric wall] in HEPES buffer (pH 7.4) was spread over the mucosal surface of a section of the gastric wall. Addition of a sulfhydryl group modulator, N-ethylmaleimide, to the mucosal surface before treatment with Tempol removed the significant difference observed in the rates of reduction in the antral and fundic portions of the gastric wall. No signals were detected in the muscle layer. Our results indicate that the relative rate of free radical reduction by sulfhydryl groups was significantly slower in the antral mucosa than in the fundic mucosa. We therefore conclude that a regional difference in the rates of reduction of free radicals by sulfhydryl groups may result in the site susceptible to development of MNNG-induced gastric cancer.     

Induction and intracellular localization of a 72-kDa heat shock protein in rat gastric mucosa after water-immersion stress

We investigated the expression and changes in the intracellular localization of a 72-kDa heat shock protein (HSP72) in rat gastric pyloric and fundic mucosa before and after water-immersion stress. Severe mucosal damage was found in the fundic mucosal area of the stomach after this stress. However, no mucosal lesion developed in the pyloric mucosal area. HSP72 in both the soluble and insoluble fractions of the pyloric and the fundic mucosal areas was significantly increased after water-immersion stress, peaking 6h after the initiation of the stress. The increase in HSP72 was more significant in the pyloric mucosal area than in the fundic mucosal area under both normal and stress conditions. The increase of HSP72 in the pyloric mucosal cells occurred prior to the formation of the mucosal lesions, whereas the increase of HSP72 in the fundic mucosal cells was observed after ulcer formation. An immunohistochemical study showed that HSP72 was constitutively expressed in the cytoplasm of the gastric mucosal cells, and that the intranuclear induction of HSP72 was remarkably intense in the pyloric mucosal cells, especially in the proliferative zone, compared with the fundic mucosal cells. Our results may suggest that HSP72 has an important cytoprotective function in gastric mucosal cells and that there is a "biophysical" difference between pyloric and the fundic mucosal cells.     

Uremia in the rat affects gastric cell growth and differentiation

The aim of this study was to determine if hypertrophy of different tissues seen in uremic rats included gastrointestinal hypertrophy and an increase in parietal cell mass that might explain the increased acid secretion we previously reported. Chronic renal failure was induced by subtotal nephrectomy. Despite a lower total body weight, uremic rats had a significantly greater stomach weight (33%), corpus area (13%), corpus mucosal height (19%), and parietal (32%) and enterochromaffin-like (ECL, 54%) cell density, but a 16% decrease in mucous neck cell region height. These findings suggest that uremia leads to gastric stem cell stimulation with differentiation favoring parietal and ECL cells over mucous cells. In addition, in uremic rats there was an increase in height of the duodenal mucosa, but not of the ileal or transverse colon mucosa. In conclusion, the present study shows that uremia in the rat promotes hypertrophy of the stomach with cell differentiation favoring parietal cells over mucus cells. The increase in parietal cell mass may explain the increased acid secretion in these rats.     

Proteolytic release and partial characterization of human sperm-surface glycopeptides

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.     

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross-reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand -Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.     

Profiling glycoprotein n-linked oligosaccharide by capillary electrophoresis

A method for analysis of N-linked oligosaccharides derived from glycoproteins including sialic acid-containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. Glycoproteins were heat-denatured in the presence of a reducing agent and the N-linked oligosaccharides were released by peptide N-glycosidase (PNGase F; EC3.5.1.52)-catalyzed hydrolysis. The released N-linked oligosaccharides were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N-linked oligosaccharide, desialylated, galactosylated biantennary, core-substituted with fucose (A2F) was tested for APTS-based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N-linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resolution of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.     

Effect of marzulene on the restitution of rat gastric mucosa after NaOH-induced injury

BACKGROUND/AIMS: The purpose of this study was to assess the effect of marzulene (L-glutamine plus azulene) on the repair of NaOH-induced gastric mucosal injury in rats. METHODOLOGY: Gastric mucosal injury was induced with intragastric instillation of 3.0 ml of 5% NaOH for 1 minute. From 2 days after the operation, the rats were orally given chow pellets containing 0%, 0.25%, or 0.5% of marzulene for 25 weeks. RESULTS: Oral administration of marzulene at both dosages significantly increased the mucosal heights of the fundic and antral mucosa at week 25. Marzulene also increased the labeling indices of the fundic and antral epithelial cells, but not the mucosal blood flow. CONCLUSIONS: These findings indicate that marzulene stimulates repair mechanisms of rat gastric mucosa after NaOH injury. This effect of marzulene may be associated with a stimulation of gastric epithelial cell proliferation.