In vitro cell cultures of Brunner's glands from male mouse to study GLP-1 receptor function.

Exocrine glands in the submucosa of the proximal duodenum secrete alkaline fluid containing mucus to protect the intestinal mucosa from acidic stomach contents. These glands, known as Brunner's glands, express high glucagon-like peptide 1 receptor (GLP-1R) levels. Previous studies have suggested that activation of the GLP-1R induces expression of barrier protective genes in Brunner's glands. Still, the lack of a viable in vitro culture of Brunner's glands has hampered additional studies of the functional consequences of GLP-1R activation. In this study, we established a procedure to isolate and culture cells derived from murine Brunner's glands. The isolated glandular cells retained functional GLP-1R expression in culture, making this in vitro system suitable for the study of GLP-1R activation. We found that cells derived from the Brunner's glands of mice pretreated with semaglutide contained significantly more mucus compared with Brunner's glands from vehicle-treated mice. Our data suggest a protective intestinal response upon semaglutide treatment, but further studies are required to leverage the full potential of cultured Brunner's gland cells.

Probiotic supplementation affects the glycan composition of mucins secreted by Brunner's glands of the pig duodenum.

The effect of a dietary probiotic blend on the carbohydrate composition of mucins secreted by the Brunner's glands in the duodenum of growing-finishing pigs was investigated by means of conventional (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) and lectin (15 lectins) histochemistry. Pigs were assigned to two dietary treatments: a control basal diet without the probiotic blend (No-Pro) and a test diet that included the probiotic blend (Pro). Duodenal tissue fragments were fixed in 4% phosphate-buffered-saline-buffered paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin wax. The secretory cells of the Brunner's glands from No-Pro pigs primarily produced neutral glycoproteins and a small amount of acidic non-sulphated mucins. This glycan pattern was opposite that of the Brunner's glands from Pro animals. A comparison of lectin-binding profiles of the secretory cells of Brunner's glands in these two groups showed that in Pro pigs, there was (i) a decrease in N-linked glycans containing α1,2-linked fucose (Con A, UEA I); (ii) a loss of complex types of N-glycans (PHA-L, PHA-E) terminating with lactosamine (RCA120), α1,6- and α1,3-linked fucose (LTA), and α-galactose (GSA I-B4), as well as of O-glycans with terminal Galß1,3GalNAc (PNA); and (iii) an increase in O-glycans containing GalNAc HPA. No-Pro and Pro samples showed no change in the expression of α2,6 sialoglycans and terminal GlcNAc residues and no affinity for MAL II, DBA, and SBA. These results indicate that probiotic supplementation affects the glycan composition of mucins produced in the Brunner's glands of growing-finishing pigs. These changes could effectively act on the gastrointestinal function and health status of these animals because the probiotic blend induced higher growth performance and meat quality in the test probiotic group than it did in the control basal diet group (Tufarelli et al., 2017).

Duodenal mucosal damage is associated with proliferative activity of Brunner's gland hamartoma: a case report.

BACKGROUND: Brunner's gland hamartoma is a rare tumor, predominantly found in the fifth to sixth decades of life. Generally, it is a single pedunculated polyp, rarely larger than 5 cm. Asymptomatic cases are found incidentally, but cases with a large polyp tend to have gastrointestinal bleeding and/or obstructive symptoms. Polyp size increases in a time-dependent manner, however, the growth mechanism is unknown. We report a Japanese male case in his mid-twenties with an over 6 cm sized polyp. CASE PRESENTATION: A 26-year-old man presented black stools and anemia. Endoscopic examination revealed a large pedunculated polyp at gastroduodenal junction. The polyp, subsequently resected by distal gastrectomy, was lobulated with random surface erosions and sized 6.4 × 3 cm. Histological examination revealed that the polyp arose from duodenal mucosa and was composed of hyperplastic Brunner's glands in lobules separated by fibromuscular septa, associated with lymphocytic infiltrate and lymphoid follicles. No evidence of malignancy was found. Thus, the lesion was diagnosed as Brunner's gland hamartoma. Further immunohistochemical studies indicated that gastric foveolar metaplasia is associated with surface epithelium covering upper two thirds of the polyp, showing immunohistochemical positivity for mucin 5 AC (MUC5AC). Below the metaplastic surface epithelium, Brunner's glands had high proliferative activity (MIB-1 labeling index: 7.9%). The similar staining pattern was observed at surface erosive sites (MIB-1 labeling index in Brunner's glands: 9%). On the other hand, surface epithelium in the lower side of the polyp still preserved intestinal nature, containing CDX2-positive nuclei and MUC2-positive goblet cells. Brunner's glands below the surface epithelium with intestinal characteristics showed low proliferative activity (MIB-1 labeling index: 0.77%). CONCLUSION: Proliferative activity of Brunner's glands was high at the sites with surface erosion and also below the epithelium showing gastric foveolar metaplasia. As gastric foveolar metaplasia occurs along with a mucosal repair process in the duodenum, mucosal damages underlay the hamartomatous proliferation of Brunner's glands and eventually resulted in a formation of large polypoid mass in this case.

Regulated traffic of anion transporters in mammalian Brunner's glands: a role for water and fluid transport.

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.

Trefoil factor family (TFF) peptides of normal human Vater's ampulla.

Vater's ampulla is of great clinical relevance with regard to the influx of chyme, ascending inflammation, intubation during diagnostic and therapeutic endoscopic maneuvers, therapeutic papillotomy and, especially, the formation of malignancies. Little is known about the distribution of trefoil factor family (TFF) peptides in the ampulla. We have therefore examined TFF peptide distribution in the normal ampulla of Vater and compared it with that in duodenal mucosa and Brunner's glands. Expression and synthesis of TFF peptides in Vater's ampulla and duodenum was investigated by reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. The samples studied originated from 30 autopsy cases with short postmortem intervals. TFF3 was expressed in the ampulla of Vater. mRNA expression of TFF1 was detected in only approximately 25% of the investigated samples. Western blot revealed the production of TFF3 and immunohistochemistry showed that TFF3 was the product of goblet cells. TFF peptide composition of Vater's ampulla varied in comparison with that in the duodenum regarding TFF2 expression. The ampulla of Vater thus has a unique profile of TFF peptide production, supporting the hypothesis that the ampulla is an autonomous organ. The observed differences in the TFF peptide distribution between the duodenum and Vater's ampulla favour the investigation of TFF peptides as prognostic markers in the classification of ampullary carcinomas.

Metaplasia of the duodenum shows a Helicobacter pylori-correlated differentiation into gastric-type protein expression.

The origin of gastric metaplasia of the duodenum (GMD) remains enigmatic. We studied expression of mucins and trefoil peptides in GMD to gain insight into its phenotype and origin. We examined duodenal tissue of 95 patients (0 to 83 years old, 26 with gastric Helicobacter pylori infection) for the presence of GMD. Expression was examined immunohistochemically of secretory mucins (MUC2, MUC5AC, MUC5B, and MUC6), trefoil peptides (TFF1, TFF2, and TFF3), and sucrase-isomaltase (SI). GMD, found in 37 patients, correlated positively to gastric H. pylori infection, age, and villus atrophy. MUC2 and TFF3, expressed in normal goblet cells, were absent from 100% and 87% of GMD, respectively. GMD ubiquitously expressed MUC5AC, whereas MUC5AC expression in adjacent goblet cells was closely correlated with the extent of GMD. TFF1, TFF2, and MUC6 were found in 84%, 92%, and 65% of GMD, respectively. MUC5B was absent from epithelium and GMD. SI, expressed by villus enterocytes, was absent from GMD. Brunner's glands ubiquitously expressed MUC5B, MUC6, and TFF2. GMD was characterized by the expression of gastric-type proteins MUC5AC, MUC6, TFF1, and TFF2 and the absence of intestinal markers MUC2, TFF3, and SI. In terms of the location of metaplastic cells, our results suggest that epithelial cells migrating toward villus tips switch to gastric-type secretory cells. Positive correlation with infection suggests an inductive role H. pylori in the development of GMD.

Dynamic regulation of mucus gel thickness in rat duodenum.

We examined the dynamic regulation of mucus gel thickness (MGT) in vivo in rat duodenum in response to luminal acid, cyclooxygenase (COX) inhibition, and exogenous PGE(2). An in vivo microscopic technique was used to measure MGT with fluorescent microspheres in urethan-anesthetized rats. Duodenal mucosa was topically superfused with pH 7.0 or pH 2.2 solutions with or without PGE(2) and indomethacin treatments. Glycoprotein concentration of duodenal loop perfusates was measured with periodic acid/Schiff (PAS) or Alcian blue (AB) staining. MGT and perfusate glycoprotein concentration were stable during a 35-min perfusion with pH 7.0 solution. Acid exposure increased MGT and PAS- and AB-positive perfusate glycoprotein concentrations. Indomethacin pretreatment increased both PAS- and AB-positive perfusate glycoprotein at baseline; subsequent acid superfusion decreased perfusate glycoproteins and gel thickness. PGE(2) (1 mg/kg iv) simultaneously increased MGT and PAS-positive perfusate glycoprotein concentrations followed by a transient increase in AB-positive glycoprotein concentration, suggesting contributions from goblet cells and Brunner's glands. Parallel changes in MGT and perfusate glycoprotein concentration in response to luminal acid and PGE(2) suggest that rapid MGT variations reflect alterations in the balance between mucus secretion and exudation, which in turn are regulated by a COX-related pathway. Luminal acid and PGE(2) augment mucus secretion from goblet cells and Brunner's glands.

Presence of sorbin in human digestive tract and endocrine digestive tumours.

BACKGROUND: Sorbin, a 153 amino acid peptide isolated from porcine intestine, was localised by immunohistochemistry in endocrine cells of the intestinal mucosa and pancreas and in the enteric nervous system in the pig. AIMS: To identify sorbin cells in normal human digestive tissues and to explore the expression of sorbin in 37 digestive endocrine tumours: 14 intestinal carcinoid tumours and 23 endocrine pancreatic tumours including six insulinomas. METHODS: Two polyclonal antibodies against the C-terminal and the N-terminal sequences of porcine sorbin raised in rabbit were used to evaluate sorbin expression by immunohistochemistry. RESULTS: In the human digestive tract, sorbin, characterised by both C-terminal and N-terminal immunoreactivity, was found in enterochromaffin cells of the gastric and intestinal epithelium from the pyloric junction to the descending colon. C-Terminal sorbin immunoreactivity alone was found in plexii from the enteric nervous system and in some insulin-containing cells of normal pancreas. C-Terminal and N-terminal antibodies disclosed sorbin in five of 14 intestinal carcinoid tumours; C-terminal antibody alone disclosed a C-terminal sorbin peptide in two of six insulinomas and three of 17 endocrine pancreatic tumours. The presence of sorbin was not associated with a specific clinical syndrome. CONCLUSIONS: Sorbin is present in the digestive tract in several forms. It is expressed in some intestinal and pancreatic endocrine tumours.

Brunner's glands: a structural, histochemical and pathological profile.

Brunner's glands are unique to mammalian species and in eutherians are confined primarily to the submucosa of the proximal duodenum. In the majority of species examined, they begin at the gastrointestinal junction and extend for variable distances distally in the wall of the proximal small intestine. Ducts of individual glands empty either directly into the intestinal lumen or unite with overlying intestinal glands (crypts of Lieberkühn) dependent on the species. Secretory units of Brunner's glands consist of epithelial tubules that show frequent distal branchings. The secretory units, with the exception of those found in rabbits and horses, consist primarily of a mucin producing cell type. However, other cell types normally associated with the overlying intestinal epithelium may be encountered scattered within the secretory units reflecting the developmental origin of these glands. Secretion from Brunner's glands contributes to a layer of mucus that forms a slippery, viscoelastic gel that lubricates the mucosal lining of the proximal intestinal tract. The unique capacity of this mucus layer to protect delicate underlying epithelial surfaces is due primarily to the gel-forming properties of its glycoprotein molecules. Mucin glycoproteins produced by Brunner's glands consist primarily but not exclusively of O-linked oligosaccharides attached to the central protein core of the glycoprotein molecule. Human Brunner's glands produce class III mucin glycoproteins and are thought to be the product of mucin gene MUC6 which is assigned to chromosome 11 (11p15-11p15.5 chromosome region). In addition to mucin glycoproteins and a limited amount of bicarbonate, numerous additional factors (epidermal growth factor, trefoil peptides, bactericidal factors, proteinase inhibitors, and surface-active lipids) have been identified within the secretory product of Brunner's glands. These factors, incorporated into the mucus layer, guard against the degradation of this protective barrier and underlying mucosa by gastric acid, pancreatic enzymes, and other surface active agents associated with this region. Yet other factors produced by Brunner's glands function to provide active and passive immunological defense mechanisms, promote cellular proliferation and differentiation, as well as contribute factors that elevate the pH of luminal contents of this region by promoting secretion of the intestinal mucosa, pancreatic secretion and gall bladder contraction. Additional insights concerning the role of Brunner's glands in the mammalian gastrointestinal tract as well as their possible evolution in this class of vertebrates have been gained from a basic understanding of their pathobiology.

The expression of pepsinogen c mRNA in normal gastroduodenal mucosa and the gastric ulcer margin of the rat.

During the healing of experimental gastric ulcers in the oxyntic mucosa, there is a dedifferentiation of the glands in the ulcer margin: previous studies have shown that the parietal cells lose their capacity to produce HCl, and mucous cells replace the zymogen cells. Primarily, we wished to investigate whether or not the glands of the ulcer margin transcribe mRNA for pepsinogen; secondly we also wanted to locate such transcription in other parts of the gastroduodenal epithelium. For this purpose, we first established the baseline for distribution of pepsinogen mRNA in normal rats. We then studied its location in the margin of ulcers in the corpus region after 1-15 days of healing. Formaldehyde-fixed paraffin sections were used for in situ hybridization of mRNA for pepsinogen C, utilizing radioactive riboprobes. The normal gastroduodenal mucosa showed widespread hybridization: the signal was particularly strong in the zymogen cells; weaker signals were obtained from the mucous neck cells, and the cells of the cardiac, antral, and Brunner glands. Specific hybridization was weak or absent in the ulcer margin during the entire period studied. It is concluded that the capacity to produce pepsinogen C is significantly reduced or absent in the gastric ulcer margin during the first 15 days of healing; this should reduce the risks of peptic attack on the delicate scar and margin tissues during ulcer healing.

Expression of trefoil peptides pS2 and human spasmolytic polypeptide in gastric metaplasia at the margin of duodenal ulcers.

Duodenal ulcers are associated with gastric metaplasia in the duodenum, both at the ulcer margin and at more distant sites in the duodenal bulb. pS2 and human spasmolytic polypeptide (hSP) are secretory peptides expressed in gastric epithelial cells and in gastric metaplasia. As these peptides may be important in ulcer healing, this study investigated the possibility that the expression of pS2 and hSP is increased in gastric metaplasia at the margin of duodenal ulcers. Duodenal bulb biopsy specimens from 12 duodenal ulcer patients were assessed. Sections were immunostained with monoclonal antibodies for pS2 and hSP. Cytoplasmic stain intensities were measured by an image analysis system and expressed as integrated optical density (IOD) units, In situ hybridisation for pS2 and hSP mRNA was carried out on parallel sections. Duodenal sections were also stained with diatase periodic acid Schiff/alcian blue to localise areas of gastric metaplasia. pS2 antigen staining in the duodenum was restricted to surface epithelial cells, and hSP to acinar and ductular components of Brunner's gland. mRNA localisation corresponded to immunostaining cells. In gastric metaplasia, pS2 expression was greater at the ulcer margin than away from the ulcer, as judged by the intensity of antibody staining (mean IOD units (SEM), 20.6 (3.3) v 9.5 (3.0); p < 0.001). There was a trend towards greater hSP staining at the ulcer margin but this did not achieve statistical significance. These findings support the putative role of pS2 and possible hSP in mucosal healing and providy further evidence for an autocrine 'ulcer-gastric metaplasia-repair' loop involving these trefoil peptides.

Immunohistochemical study of the distribution of endocrine cells in the gastrointestinal tract of the lesser mouse deer (Tragulus javanicus).

The occurrence and distribution of endocrine cells in the gastrointestinal tract of the lesser mouse deer, Tragulus javanicus, were studied immunohistochemically. Fourteen types of endocrine cells immunoreactive for serotonin, somatostatin, enteroglucagon, pancreatic glucagon, bovine pancreatic polypeptide (BPP), gastrin, substance P, motilin, gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), methionine-enkephalin-Arg6-Gly7-Leu8 (MENK-8), secretin, neurotensin, peptide tyrosine tyrosine (PYY) and chromogranin were revealed. Chromogranin-, serotonin-, somatostatin- and enteroglucagon-immunoreactive cells were detected in all regions examined, while pancreatic glucagon-immunoreactive cells, except in the proper gastric gland region, were not found in other regions of the gastrointestinal tract. Few BPP-immunoreactive cells in either the proper gastric gland or pyloric gland regions and abundant gastrin-immunoreactive cells in the pyloric gland region were observed. Restricted distributions of substance P-, GIP-, gastrin-, motilin-, CCK-, MENK-8-, secretin-, neurotensin- and BPP-immunoreactive cells in the small intestine, and BPP-, substance P-, PYY- and motilin-immunoreactive cells in the large intestine were noted. The important findings include the presence of BPP-immunoreactive cells in the abomasum, pancreatic glucagon-immunoreactive cells in the proper gastric gland region, and substance P- and motilin-immunoreactive cells in the large intestine. It is suggested that the distribution pattern of gut endocrine cells in the lesser mouse deer is more similar to that in the pig than in the domestic ruminants so far reported.

Phenotypic identity of gastric mucous neck cells and mucous cells of cardiac, pyloric, and Brunner's glands.

AIM: To investigate the tissue specificity of a novel monoclonal antibody raised to a tissue fraction of normal human liver and which identified certain cells of gastric and duodenal mucosa. METHODS: A total of 155 samples of various tissues obtained from 100 surgical specimens were fixed in cold ethanol-paraformaldehyde, embedded in paraffin wax, and 3 microns sections were studied by immunohistochemical and lectin staining procedures. RESULTS: Immunohistochemical staining showed a major tissue specific component which was strongly expressed by mucous neck cells of the body of the stomach, glands of the cardia and pyloric antrum, and by Brunner's glands. Staining for antigen in the periductal glands of normal major biliary and pancreatic ducts was variable and relatively weaker. It was not detected elsewhere in normal intestine or in the other normal tissues tested. Barrett's mucosa of gastric cardia type, and pyloric gland metaplasia in the gall bladder and small bowel affected with Crohn's disease stained for the antigen. The tissue distribution of the antigen was identical with that of a glycoprotein, demonstrated by an induced affinity for concanavalin A following treatment of tissue sections with periodic acid. The antigen was not sensitive to sialidase. CONCLUSIONS: The tissue component identified (designated here as antigen D10) seems to be characteristic of certain differentiated epithelial cells derived from that part of foregut giving rise to stomach, duodenum, and biliary and pancreatic ducts. The antibody will be of use in investigating pathological processes involving tissue differentiation at these sites, and in the oesophagus and intestines.

Determination of the histological distribution of insulin like growth factor 1 receptors in the rat gut.

The histological distribution of insulin like growth factor 1 (IGF 1) receptors in the rat gut was studied. Immunostaining of IGF 1 receptors identified localisation on the villus epithelium, in the crypts, and in Brunner's glands of the small intestine. These tissues represent areas of high cell growth/differentiation, division, and macromolecular synthesis respectively, which constitute biological activities long associated with IGF 1. Cellular localisation of IGF 1 receptors was seen in the lamina propria by IGF 1 receptor immunostaining and ligand binding of biotinylated IGF 1. IGF 1 receptor immunostaining in the spleen showed receptor localisation to the splenic pulp thus pointing to macrophages as the possible IGF 1 receptor positive cells in the lamina propria. The results further implicate IGF 1 as an important growth factor in gut maintenance.

Ulcer-associated cell lineage ('pyloric metaplasia') in Crohn's disease: a lectin histochemical study.

Chronic intestinal ulceration in Crohn's disease is associated with the development of an epidermal growth factor-secreting cell lineage, or 'ulcer-associated cell lineage' (UACL). Expression of oligosaccharides by UACL was studied using a panel of 25 biotinylated lectins with an avidin peroxidase revealing system and compared with that of adult and fetal Brunner's glands, gastric antral mucosa, and 'gastric metaplasia' within the duodenum, in order to clarify further the interrelationships of these lineages. UACL was obtained from ileal resections performed for Crohn's disease. Lectin binding of the glandular component of UACL closely resembled that of antral mucosal glands and also that of fetal and adult Brunner's glands. Lectin binding of the ductal component of immature UACL, in which a surface component had not developed, resembled that of the gland. The surface and ductal components of mature UACL showed a distinct lectin-binding profile, which was very different from that of the gland, but closely resembled that of antral foveolar epithelium and 'gastric metaplasia' within the duodenum. It is concluded that there is differentiation of UACL from the glandular to surface components and that oligosaccharide expression of the lineage reflects that of normal Brunner's gland and gastric antral mucosa.