Brunner's gland lesions in rats induced by a vascular endothelial growth factor receptor inhibitor.

Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner's gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner's gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner's glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner's gland lesion, we identify degeneration and necrosis of the Brunner's glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.

Expression of duodenase-like protein in epitheliocytes of Brunner's glands in human duodenal mucosa.

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G - Paneth cells located at the bottom of Lieberkuhn crypts - was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner's glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner's glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its co-localization with cathepsin G in Brunner's glands.

Novel localization of Rab3D in rat intestinal goblet cells and Brunner's gland acinar cells suggests a role in early Golgi trafficking.

Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, beta-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.

Use of taxol and collagenase for better three-dimensional visualization of microtubules in the enterocyte and Brunner's gland cell, with special reference to their relation to the Golgi apparatus.

Cytoskeletal microtubules were visualized in the mouse duodenal mucosa by an improved immunofluorescence method using a microtubule-stabilizing reagent, Taxol, and collagenase as an enzymatic epitope retriever. The improvement in immunostaining was shown morphologically and statistically by comparing fluorescence intensities of specimens prepared with or without Taxol and collagenase treatment. In free cells in the epithelium and in the lamina propria, microtubules radiated from the gamma-tubulin-immunostained organizing center. Enterocytes and Brunner's gland cells double-stained with an anti-alpha-tubulin antibody and a lectin (Helix pomatia agglutinin, soybean agglutinin or Ulex europaeus agglutinin-I) showed that microtubules ran along the cell axis and were abundant between the Golgi apparatus and the apical surface. The microtubules appeared to provide a structural support to hold the Golgi apparatus in position and to act as railways for secretory granules, which are transported towards the apical surface. In addition, gamma-tubulin-like immunoreactivity was associated with the Golgi apparatus in the enterocytes. These results show that the method using Taxol and collagenase is effective for visualizing microtubules in epithelial cells, and that microtubules may play important roles in both positioning of the Golgi apparatus and transport of secretory granules. Our results also support the idea that the Golgi apparatus may act as an organizing center for microtubules.

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.

Estimation and comparison of the contents of blood group B antigens in selected human tissues by microphotometric quantification of Griffonia simplicifolia agglutinin I-B4 staining with or without prior alpha-galactosidase digestion.

Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) has broader specificity for B antigen variants and can recognize the antigens in a wide variety of human tissues. Thus, the concentration range of GSAI-B4 required for staining and the susceptibility of staining to alpha-galactosidase digestion is presumed to correlate well with the density of B antigens in tissue sections. By microphotometric quantification of staining intensity at different concentrations of GSAI-B4 with or without alpha-galactosidase digestion, concentration of B antigens in selected tissues was evaluated and compared. Based on the present results and the previous ones of direct measurement of galactose of B antigens in sublingual glands and red blood cells (Ito et al., 1993), the order of concentration of B antigens in tissues examined was estimated as follows; mucous cells of sublingual glands from German nonsecretors < red blood cells and vascular endothelial cells (= 2.7 x 10(-3) nmole/cm2), thyroid papillary carcinomas and Hassall's corpuscles from nonsecretors < mucous cells of sublingual gland from Japanese nonsecretors < pancreatic acinar cells from both secretor and nonsecretors, Hassall's corpuscles and kidney collecting tubules form secretors < mucous cells of sublingual gland from secretors (> 8.5-11.7 nmole/cm2) and mucous cells of Brunner's gland from nonsecretors < mucous cells of Brunner's gland from secretors. From the above estimation, it is apparent that the expression of B antigen in Brunner's gland is partly dependent on the secretor status of individuals and that Japanese nonsecretors secrete substantial amounts of B antigens from sublingual gland while German nonsecretors do not. The present results also revealed an unexpected staining behavior of GSAI-B4 in some tissues, i.e. in mucous cells of sublingual glands and collecting tubules of kidney from secretors, staining intensity was markedly depressed at higher concentration of the lectin and this depression was recovered by prior alpha-galactosidase digestion. In addition, the present method was successfully applied for the estimation of the content of B antigens neo-expressed in thyroid papillary carcinomas, showing that the content of B antigen had a similar level to that of red blood cells and vascular endothelial cells.

Molecular anatomy of an endodermal gland: investigations on mucus glycoproteins and cell turnover in Brunner's glands of Didelphis virginiana using lectins and PCNA immunoreactivity.

Brunner's glands are located in the submucosa of the proximal duodenum and are unique to mammalian species. The North American opossum (Didelphis virginiana) is generally regarded as a prototype marsupial that closely resembles fossil didelphids which can be placed at the beginning of mammalian evolution. The current investigation provided an opportunity for the analysis of secretory products from these glands in a species thought to be more closely related to earlier evolutionary forms. Extracts of Brunner's glands were subjected to SDS-PAGE and Western blotting. The results indicate the presence of two high molecular weight PAS-positive glycoprotein bands. In addition to these two PAS-positive bands, several other glycoprotein bands were detected in the high molecular weight range that bind several lectins which typically recognize O-linked carbohydrates indicative of mucus type glycoproteins. The same lectins bind to glandular structures in tissue sections. Comparison of lectin binding sites with the pyloric glands of the stomach and duodenal goblet cells indicates that Brunner's glands carbohydrate residues resemble those of the pyloric glands more closely than those of the duodenal goblet cells. The low cell turnover rate in Brunner's glands is in contrast to the rapid turnover rate of goblet cell precursors in the duodenal crypts. The mucus composition and the cell turnover rate correlate well with embryological data and suggest that Brunner's glands of Didelphis evolved from an epithelium more closely associated with the stomach than that of the duodenum as the topography of the gland would suggest.

Initiated stem cells in murine intestinal carcinogenesis: prolonged survival, control by NK cells, and progression.

Weekly injections of dimethylhydrazine (DMH) (25 mg/kg), or azoxymethane (AOM) (8 mg/kg) to young adult male CDI mice for 1-2 months produced generalized intestinal crypt hyperplasia, which we measured in duodenum in terms of number of interphase and mitotic cells present in crypts. As shown earlier, the crypts expanded because of the presence of a hyperproliferative "initiated" crypt subpopulation which was also sensitive to natural killer (NK) cells. Hyperplasia was thus present as long as NK activity was suppressed by the carcinogen treatment. After interruption of the treatment for periods of 1, 2, 3, 6 and 10 months in the various groups, hyperplasia soon regressed as a result of elimination of the subpopulation by the recovering NK cells. When NK activity was once again eliminated during the terminal days of these "interruption periods" (by injections of anti-asialo GM-I antibody, alpha AGM-I), the original hyperplasia was fully reconstituted, apparently from stem cells of the subpopulation which survived up to 10 months in their crypt base location. These "initiated stem cells" represented, then, the original carcinogenic insult during the pre-cancerous period. They also appeared to be the source of the eventual neoplasia, as treating the animals with mutagens during the interruption periods produced specific changes in crypt base histology: new "crypt base basophilic" (CBB) cells appeared which produced large accumulations as well as microscopic tumors when NK activity was suppressed (by alpha AGM-I). Some of the initiated stem cells were apparently transformed into neoplastic ones which remained under NK control, the NK cells preventing the establishment of their progeny. Further experiments indicated that, although the initiated stem cells are not eliminated by normal NK activity, activated NK cells can kill them, thereby eliminating the potential source of neoplasia.

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.

[Changes in the duodenal glands with a modification of the diet (a histochemical study using lectins)]. / Izmeneniia duodenal'nykh zhelez pri modifikatsii ratsiona (gistokhimicheskoe issledovanie s ispol'zovaniem lektinov).

A set of lectins with different carbohydrate specificities were used for a histochemical analysis of the duodenal glands of rats which had different food rations during 3 months. Lectin receptors in glandulocytes of the duodenum had different spectra under different food regimens: protein diets of the animals correlated with the accumulation of glycopolymers in the glandular epithelium with the terminal residues of N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-fucose, higher content of cellulose in food was followed by reduction of fucosoglycans in combination with accumulation of mannose-containing glycopolymers. The regular specific features found point to quantitative and qualitative changes to the formed secretion of the duodenal glands under the influence of changing conditions of nutrition.

Eosinophilic bodies in pyloric and Brunner's gland cells.

A previously unreported cell phenotype occurred in the pyloric and Brunner glands in two gastrectomy specimens. The cells were characterised by homogeneous, eosinophilic material in the cytoplasm. The eosinophilic material had an abnormally strong reactivity for Cystatin C, a protein found recently in the normal secretion of pyloric and Brunner's gland cells. The reason for the apparent cytoplasmic accumulation of cystatin C in the two patients described remains unclear.

Ultrastructure of Brunner's glands in the horse.

Investigation of the duodenal submucosal glands of Brunner in the horse for the first time at the ultrastructural level has clarified some of the unique features of these equine glands. The horse is one of the very few mammals in which Brunner's glands are comprised of both mucous and serous tubuloacinar glands. Although the ultrastructural differences between the serous and mucous cell types are marked, particularly with respect to secretory granules and rough endoplasmic reticula, these cell types closely correspond to serous and mucous cells in the upper digestive system of other mammals. A minor and distinct population of goblet cell-like mucous cells, and endocrine-like cells were also observed in equine Brunner's glands. Both the serous and mucous cells appear to empty into common ducts which enter the base of the duodenal crypts. These submicroscopic cytologic data taken together with other physiologic data would suggest that, in the horse, Brunner's glands function both to provide mucosal protection in the proximal duodenum as well as to release digestive enzymes, such as lipase or other unidentified materials.

An immunohistochemical study of endocrine cells in the proximal duodenum of eight marsupial species.

The proximal duodenum of eight marsupial species, (koala, common brushtail possum, ring-tailed possum, common wombat, great grey kangaroo, parma wallaby, short-nosed bandicoot and tiger cat) were investigated immunohistochemically using 12 specific antisera for gut hormones. Several types of immunoreactive cells were seen on the intestinal villi and in crypts of these species: 9 types in the koala; 8 types in the common brushtail possum; 7 types in the common wombat; 6 types in the short-nosed bandicoot and 5 types in the ringtailed possum, great grey kangaroo, parma wallaby and tiger cat. Gastrin-, somatostatin-, motilin- and serotonin-immunoreactive cells were seen in all species examined. A few BPP-, enteroglucagon-, CCK-, secretin-, GIP- and neurotensin-immunoreactive cells were seen but only in few species. A few substance P-immunoreactive cells were detected only in the koala. Immunoreactive cells were also seen in Brunner's glands: 5 types in the parma wallaby; 3 types in the great grey kangaroo and tiger cat; 2 types in the koala and common wombat; 1 type in the short-nosed bandicoot. No immunoreactive cells were found in Brunner's glands of the common brushtail possum.

[Quantitative characteristics of the endocrine cells of the duodenal glands of Carnivora]. / Kolichestvennaia kharakteristika éndokrinnykh kletok duodena'l- nykh zhelez mlekopitaiushchikh otriada khishchnykh (Carnivora).

In the duodenal glands of the Carnivora investigated endocrine elements have been revealed, a part of them is presented as serotonin-producing EC-cells. Endocrine cells are situated in terminal parts and in glandular ducts, among them elements of open and close types are distinguished. Distribution of these cells in the glandular lobules is subjected to the distal gradient regularity, specific for the gastrointestinal tract mucosal membrane. Amount of endocrinocytes in the glands is much less than in the gut crypts. There is no correlation between distribution of the endocrine cells in the glands and in the crypts. The results of unifactor analysis of variance demonstrate a slight effect of the taxonomic position of the species on the number of endocrine cells in the duodenal glands. The proper endocrine apparatus of the duodenal glands is supposed to produce a local regulatory influence on the secretory activity of exogenic glandulocytes, as well as ensure humoral connections of the duodenal glands with other parts of the gastrointestinal tract.

Morphometry of normal duodenal mucosa.

The morphometric variability of normal duodenal mucosa was assessed in bioptic specimens obtained from bulb, second and third duodenum. Biopsies were obtained from 82 cases ranging in age from 18 to 67 years. The results demonstrate a great variability in villous shape and height without mean height differences in relation with age of subjects or site of biopsies. Subvillous thickness of lamina propria, intraepithelial lymphocyte count, inflammatory cell count in lamina propria and goblet cell number did not show significant differences. Superficial gastric metaplasia was seen in 8.5% of bulbar biopsies, 3.6% of biopsies taken in the second duodenum but it was constantly absent in the third duodenum. Brunner's glands above the muscularis mucosa were seen in 45% of bulbar biopsies, 23% of biopsies of second duodenum and in 4.6% of biopsies of third duodenum.

[Duodenal glands in normal and vagotomized rats]. / Duodenal'nye zhelezy krys v norme i pri vagotomii.

In Wistar rats by means of the stereo- and morphometry methods, using semithin sections, histotopography of the duodenal glands (DG) and effect of vagotomy (VT) on them have been studied. The zone of the DG arrangement in the submucosal tela of the duodenum is about 5,000 mcm long. In the proximo-distal direction of the zone mentioned three parts can be distinguished--oral, middle and caudal. In the DG light-optically four varieties of mucocytes are identified. VT results in decreasing thickness of the zone of the DG arrangement, in outgrowth of the connective tissue and in a lowered amount of secrete in mucocytes.

Carbohydrate histochemistry of human Brunner's glands.

Brunner's glands are known to produce neutral mucins. In order to achieve a better knowledge of their carbohydrate profile, we used five peroxidase-labeled lectins on surgical specimens of human duodenum. This method allowed us to identify at least two different types of neutral mucins in Brunner's glands secretion, thus demonstrating a heterogeneous mucin production. The structure of terminal oligosaccharidic chains in these glycoproteins has also been hypothesized.

[Effect of a diet with excess cellulose on the morphofunctional status of the duodenal glands in the rat]. / Vliianie ratsiona s izbytkom kletchatki na morfofunktsional'noe sostoianie duodenal'nykh zhelez krys.

The developmental degree of the duodenal glands and their functional activity have been studied in rats given food with an increased contents of cellulose. In 3 months in the duodenum of the test animals extent of the glandular field increases significantly in comparison to the intact animals. This increase is connected with new formation of the gland terminal parts from epithelium of the intestinal crypts in the more caudal parts of the gut. Simultaneously, functional activity of the glandular cells increases; this is evident from elevated concentration of RNA in nucleoli, share of the nucleoli with predominant contents of euchromatin, and also from decreased concentration of PAS-positive secretion in the terminal parts. These differences can be connected with changes in pH of the stomach contents towards acidic value, when excess of cellulose is taken.