Gastric Pits Research Articles

Localization of TFF3 peptide in human esophageal submucosal glands and gastric cardia: differentiation of two types of gastric pit cells along the rostro-caudal axis

TFF3 (trefoil factor family 3), which is a major secretory product of the gastric antrum and the intestine, but which is nearly absent in the gastric corpus, plays a key role in the maintenance of mucosal integrity. Here, we have systematically investigated TFF3 expression in the esophagus and gastric cardia by the use of reverse transcription/polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Synthesis of TFF3, but not TFF1 or TFF2, is detectable in esophageal submucosal glands. The stratified squamous epithelium is devoid of TFF synthesis. Prominent TFF3 expression starts at the Z-line with a sharply decreasing gradient toward the cardia. Immunohistochemistry has localized TFF3 to surface mucous cells of the proximal cardia. TFF3 distribution differs characteristically from that of TFF1 (secreted primarily by superficial surface mucous cells), whereas TFF3, together with the mucin MUC5AC, is also found in deeper lying cells toward the isthmus. This is the first report of TFF3 as a typical secretory peptide of esophageal submucosal glands and gastric cardia. The different expression patterns of TFF3 and TFF1 in the cardia suggest a stepwise maturation of surface mucous cells from TFF3/MUC5AC-positive cells close to the isthmus to TFF1/TFF3/MUC5AC-positive cells at the pit. The gradient of TFF3 expression along the gastric rostro-caudal axis defines two types of gastric pit cells: those secreting TFF3 in the cardia and the antrum and those nearly devoid of TFF3 synthesis in the corpus. This indicates the special requirement, particularly of the esophagogastric junction, for TFF3-triggered protection and repair.

Expression of mutant type-p53 products inH pylori-associated chronic gastritis

To investigate the mutation of p53 immunohistochemically in non-tumorous gastric mucosa with H pylori infection before and after H pylori eradication therapy. 53 subjects (36 male, 17 female, mean age +/- SEM, 57.1 +/- 12.1) undergoing endoscopic examination were included in this study. 42 of 53 patients were H pylori-positive, and 11 were H pylori-negative. All H pylori-positive patients had successful eradication therapy. Biopsy specimens were taken from five points of the stomach, as recommended by the updated Sydney system. Immunohistochemical studies were performed by using primary antibodies against p53 (DO-7 and PAb240). p53 (DO-7 and PAb240) immunoreactivity was shown in the neck region of the gastric pits, however, quite a few cells were found to be immunopositive for p53 (PAb240) in the H pylori-infected gastric mucosa. The proportion of patients immunopositive for p53 (PAb240) was significantly reduced 6 mo after eradication [28/42 (66.7%) to 6/42 (14.3%)] (P < 0.05), while the biopsies taken from H pylori-negative patients showed no immunoreactivity for p53 (PAb240). p53 (PAb240)-positive patients were divided into two groups by the number of positive cells detected: one with more than six positive cells per 10 gastric pits (group A, n = 12), and the other with less than five positive cells per 10 gastric pits (group B, n = 30). Atrophy scores in group A were significant higher than those in group B at the greater curvature of the antrum (group A: 2.00 +/- 0.14 vs group B: 1.40 +/- 0.15, P = 0.012), the lesser curvature of the corpus (group A: 2.00 +/- 0.21 vs group B: 1.07 +/- 0.23, P = 0.017), and the greater curvature of the corpus (group A: 1.20 +/- 0.30 vs group B: 0.47 +/- 0.21, P = 0.031). Group A showed significant higher intestinal metaplasia scores than group B only at the lesser curvature of the antrum (group A: 2.10 +/- 0.41 vs group B: 1.12 +/- 0.29, P = 0.035). H pylori-associated chronic gastritis expressed the mutant-type p53, which was significantly associated with more severe atrophic and metaplastic changes. H pylori eradication led to a significant reduction in the expression of the mutant-type p53. It is considered that H pylori-infected chronic gastritis is associated with a genetic instability that leads to gastric carcinogenesis, and H pylori eradication may prevent gastric cancer.

Diagnosis of Helicobacter spp. infection in canine stomach

A total of 75 biopsied samples of cardia, fundus, body, and pyloric antrum from necropsied dogs that were submitted to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University from April 2003 to June 2004 were investigated. The objectives of this study were to determine the prevalence of Helicobacter spp. in canine stomach by polymerase chain reaction (PCR) in comparison to histochemistry versus immunohistochemistry (IHC), and to correlate these diagnostic methods with the clinical significance in infected dogs. Histopathological results revealed 60.0% (45/75) of samples to be positive, and consisted of mild gastritis in 64.44% (29/45), moderate gastritis in 11.11% (5/45), and severe gastritis in 24.44% (11/45). The proportion showing no histopathological lesions was 40.0% (30/75). Helicobacter spp. were localized to the luminal crypt in 18.67% (14/75), gastric pit in 22.67% (17/75), gastric gland in 21.33% (16/75), and gastric epithelium in 8% (6/75). The percentages of positive samples of Helicobacter spp. diagnosed by hematoxylin and eosin stain (H&E), Warthin Starry stain (WSS), IHC with rabbit polyclonal anti-H. pylori antibody, and PCR were 17.3% (13/75), 46.7% (35/75), 30.7% (23/75), and 10.7% (8/75), respectively. No significant differences weree observed in histopathological changes in portions of the stomach (p>0.05). The diagnosis of Helicobacter spp. by PCR in comparison to that by WSS and IHC was not significantly different (p>0.05). There were no relationships between pathological studies using H&E, WSS, and IHC, and especially between PCR and clinical signs of Helicobacter spp. infections in canine stomachs (p>0.05). The present study revealed significantly different levels of correlation for Helicobacter spp. detection between H&E and WSS (p<0.001). Results indicate that the method of choice for diagnosis of Helicobacter spp. infection in canine stomach is dependent on the purpose of study and appropriate specimen collection.

Fluorescence diagnostics of Helicobacter pylori-infected human gastric mucosa: Establishing technique and validity

Objective. Bacterial factors, including strain type, anatomic distribution and density, and host responses are important determinants in the pathogenesis of erosive and neoplastic changes linked to gastric Helicobacter pylori (H. pylori) infection. The purpose of this study was to investigate the potential use of photodiagnostics in mapping H. pylori infection. The relationship between fluorescence in individual gastric pits of H. pylori(+) and H. pylori(−) subjects versus that in larger field views of the gastric mucosa and the use of fluorescence to determine H. pylori status in different gastric areas were studied. Material and methods. Antrum, corpus and fundus biopsies from 8 H. pylori(+) and 4 H. pylori(−) subjects taken during two gastroscopies were used for autofluorescence (535 nm excitation) and aminolevulinic acid (ALA)-induced protoporphyrin IX fluorescence (405 nm excitation) determinations. Results. In the antrum, corpus and fundus a close correlation between individual pit and full-field image (FFI) fluorescence was demonstrated for H. pylori status (R>0.85; R>0.75; R>0.80) and both excitation wavelengths (R>0.89; R>0.85; R>0.95), respectively. In the antrum, FFI in H. pylori(+) subjects exceeded that in H. pylori(−) subjects using 405 nm but not 535 nm excitation regardless of ALA treatment (p≤0.026). In the corpus and fundus, fluorescence using 405 nm excitation was greater in H. pylori(+) than in H. pylori(−) subjects only after ALA treatment (p<0.00005, p=0.03). The ratio of 535 nm:405 nm-excited fluorescence decreased from the fundus>corpus>antrum for both H. pylori(+) and H. pylori(−) subjects regardless of ALA treatment (p=0.03). Conclusions. Fluorescence-based identification of areas of H. pylori-infected gastric mucosa using 405 nm excitation combined with ALA treatment is feasible and, using a ratio of 535 nm:405 nm-excited fluorescence, it is possible to distinguish H. pylori status and the different areas of the stomach even without ALA.

Bioweapons synthesis and storage: The venom gland of front-fanged snakes

A paradoxical task of the venom gland of snakes is the synthesis and storage of an instantly available suite of toxins to immobilize prey and the protection of the snake against its own venom components. Furthermore, autolysis of the venom constituents due to the action of venom metalloproteases is an additional problem, particularly among viperid venoms, which are typically rich in lytic enzymatic proteins. To address questions concerning these problems, the structure of the venom gland was investigated using light microscopy, SEM and TEM. The composition of the venom originating from the intact venom apparatus or from the main venom gland alone was analyzed by electrophoresis, and the pH of freshly expressed venom as well as pH optima of several representative enzymes was evaluated. Results from several species of rattlesnakes demonstrated that the venom gland is structurally complex, particularly in its small rostral portion called the accessory gland, which may be a site of activation of venom components. Secreted venom is stable in extremes of temperature and dilution, and several proximate mechanisms, including pH and endogenous inhibitors, exist which inhibit enzymatic activity of the venom during storage within the venom gland but allow for spontaneous activation upon injection into prey. Whereas acid secretion by the parietal cells activates digestive enzymes in the stomach, within the venom gland acidification inhibits venom enzymes. We propose that the mitochondria-rich cells of the main venom gland, which are morphologically and histochemically very similar to the parietal cells of the mammalian gastric pit, play a central role in the stabilization of the venom by secreting acidic compounds into the venom and maintaining the stored venom at pH 5.4. Hence, our results indicate yet another trophic link between the processes of venom production and of digestion, and demonstrate that the venom glands of snakes may represent an excellent model for the study of protein stability and maintenance of toxic proteins.

NADPH Oxidases in the Gastrointestinal Tract: A Potential Role of Nox1 in Innate Immune Response and Carcinogenesis

The gastrointestinal epithelium functions as physical and innate immune barriers against commensal or pathogenic microbes. NADPH oxidase 1 (Nox1) and dual oxidase 2 (Duox2), highly expressed in the colon, are suggested to play a potential role in host defense. Guinea-pig gastric pit cells and human colonic epithelial cells (T84 cells) express Nox1. With regard to activation of Nox1, the gastric epithelial cells are primed with Helicobacter pylori lipopolysaccharide, whereas T84 cells preferentially use the Toll-like receptor (TLR) 5, rather than TLR4, against Salmonella enteritidis infection. Thus, gastric and colonic epithelial cells may use different TLR members to discern pathogenicities among bacteria, depending on their environments and to activate Nox1 appropriately for host defense. Nox1-derived reactive oxygen species (ROS) have been implicated in the pathogenesis of inflammation-associated tumor development. The human stomach does not express Nox1. Helicobacter pylori infection alone does not induce it, whereas Nox1 is specifically expressed in gastric adenocarcinomas. In the human colon, Nox1 is differentiation-dependently expressed, and its expression is upregulated in adenomas and well-differentiated adenocarcinomas. Although Nox1 expression may not be directly linked to mitogenic activity, Nox1-derived ROS may exert a cancer-promoting effect by increasing resistance to programmed cell death of tumor cells.

Hedgehog Signal Activation in Gastric Pit Cell and in Diffuse-Type Gastric Cancer

Sonic hedgehog (SHH) and Indian hedgehog (IHH) have cell-specific actions in some organs. Only SHH has been shown to regulate parietal cell differentiation. This study examined whether SHH, 2 other ligands IHH and Desert hedgehog, and receptors or downstream targets are expressed in normal gastric epithelium or in intestinal and diffuse-type gastric cancers. The effects of a Hedgehog (Hh) inhibitor, cyclopamine, were assessed in primary gastric epithelium cultures and gastric cancer cell lines. Reverse-transcription polymerase chain reaction and immunostaining compared expression and localization of Hh signaling molecules and phenotypic markers of pit, neck, and gland cells in situ and in cultured cells treated with cyclopamine. Bromodeoxyuridine staining assessed the effects of cyclopamine on proliferation. Hh signaling molecules were expressed differentially in pit, neck, and gland cells. IHH co-expressed with most downstream targets in the pit. IHH, SHH, Patched (PTCH), Smoothened (SMO), and downstream targets were expressed more frequently and highly in the diffuse as compared with intestinal type cancers. In diffuse cancers, IHH was expressed in cells with an epithelial phenotype and SHH in cells with a mesenchymal phenotype. Cyclopamine reduced the number of cells with a pit phenotype but not a gland phenotype in primary cultures. Cyclopamine had particularly potent effects of inhibiting the growth of cell lines that expressed high levels of SMO. Expression of IHH and downstream targets correlates with pit cells. IHH and SMO may be useful biomarkers of diffuse cancers that may show growth inhibition with Hh antagonists such as cyclopamine.

Histology of the digestive tract of the freshwater stingray Himantura signifer Compagno and Roberts, 1982 (Elasmobranchii, Dasyatidae)

We investigated the histology and histochemistry (of carbohydrates and proteins) of the digestive tract of the freshwater stingray Himantura signifier. The alimentary tract consists of a mouth, pharynx, esophagus, stomach (with a descending cardiac and ascending pyloric part), anterior intestine (with an initial portion and a spiral intestine) and posterior intestine, ending in a cloaca. Histologically, three layers--mucosa, muscularis and adventitia/serosa--were defined from the mouth to esophagus and in the posterior intestine, whereas in the stomach and anterior intestine four layers were present, including a submucosa. The epithelial lining of mouth, pharynx and cloaca was of the stratified cuboidal type, whereas that of the esophagus and posterior intestine was stratified columnar. The stomach and anterior intestine were lined by a simple columnar epithelium with microvilli. Goblet cells were observed along the alimentary tract, except in the stomach. In the descending cardiac portion of this organ, gastric glands composed of oxyntic, oxyntic-peptic and peptic cells were observed. The anterior intestine presented a spiral valve with 11 folds, formed by mucosa and submucosa. The posterior intestine was particular in displaying a three-layered muscularis. Mucosubstances secreted along the alimentary tract contained both neutral and acid mucins, but in the stomach only neutral mucins were detected. The stomach presented intense protein content in the epithelial lining of the gastric pits. Enteroendocrine cells were identified in the stomach and intestine. Overall, our data offer a baseline for comparative purposes and future detailed ultrastructural and immunohistochemical studies.

Histology and carbohydrate histochemistry of the alimentary canal in the rainbow trout Oncorhynchus mykiss

A histological and histochemical analyses were carried out on the entire alimentary canal of the rainbow trout Oncorhynchus mykiss. In particular the oesophageal region showed presence of terminal β‐D‐galactose(1–3)‐N‐acetylgalactosamine and α‐N‐acetylgalactosamine. In the anterior and posterior regions of the stomach, lining epithelium and gastric pits exhibited the presence of β‐gal and α‐GalNAc. In addition sialoglycoconjugates having sialic acid–β–galactose(1–3)‐N‐acetylgalactosamine and sialic acid‐N‐acetylgalactosamine as terminal tri‐ and di‐saccharides, were demonstrated. In proximal and distal intestine goblet cells showed the presence of sialoglyconjugates, having sialic acid‐β‐gal(1–3)‐GalNAc and sialic acid‐GalNAc as terminal sequences, belonging to N‐linked chains. In the enterocytes of the entire intestine, terminal GlcNAc, α‐Gal, α‐fucose were found.

Unique Endoscopic Futures of Corpus Gastritis with H. Pylori Infection in Children and Adolescents; Close Observation By Conventional Endoscopy and Magnified Endoscopy

Backgrounds: Antral nodularity is the only reliable endoscopic feature in H. pylori (Hp) infected children. We reported that the regular arrangement of collecting venules (RAC) visible as numerous minute points by close observation of the gastric corpus with conventional endoscopy was indicative of Hp-negative normal stomachs. Aims: To evaluate the endoscopic futures of corpus gastritis with Hp infection in children and adolescents. Methods: From January 2001 to March 2005, 87 consecutive children and adolescents underwent EGD for UGI symptoms or anemia (38 boys, median age 13 y, range 9∼15 years). Hp positive was defined as both positive of 13C-UBT and histology. EGD was performed using a standard small caliber endoscope (GIF-XP 240 or 260; Olympus) and biopsy specimens were taken from the antrum, lower corpus lesser curvature, and upper corpus greater curvature for histopathology. After the routine EGD, close observation was performed at the point of lower corpus lesser curvature and upper corpus greater curvature (near biopsy sites). Each case was scored as RAC positive if the minute points were irregularly present or if absent, as RAC negative. In 8 patients (4 Hp negative and 4 positive) EGD was performed using magnifying endoscopy (GIF-Q240Z; Olympus) and the magnified view was compared to RAC pattern. Results: Of the 87 patients, 25 (29%) were Hp positive. By routine endoscopy, 7 patients were diagnosed as DU with Hp infection; 21 had antral nodularity with Hp gastritis. The RAC-negative pattern was observed in 27 patients and 21 (78%) were Hp positive. In the upper corpus greater curvature RAC was negative in 30 patients and 25 (83%) were Hp positive. The sensitivity, specificity, and accuracy of RAC-negative were 100%, 87%, and 91% in the lower corpus lesser curvature and 100%, 91%, and 94% in the upper corpus lesser curvature. In Hp negative and RAC-positive patients, magnifying endoscopy showed true capillaries forming around collecting venules in the gastric corpus. In Hp positive and RAC-negative patients, collecting venules and true capillaries were not seen and gastric pits and sulci were observed. Conclusion: The RAC negative pattern as identified by close observation of the gastric corpus using conventional endoscopy uniquely reflects corpus gastritis with Hp infection in children and adolescents.

Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium.

BackgroundHelicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry.MethodsRUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies.ResultsMUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2.ConclusionIn conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals.

Morphological and Immunohistochemical Features of Cryptosporidium andersoni in Cattle

Light and electron microscopic features and immunohistochemical features of Cryptosporidium andersoni (C. andersoni) and host reaction in the mucosa were studied. Although the affected cattle demonstrated no apparent clinical signs, a severe infection of C. andersoni was observed in the abomasum. C. andersoni were round in shape, measured 6-8 microm in size and were mainly observed to be freely located in the gastric pits, being attached in occasional cases to the surface of the abomasum epithelium. Frequent inflammatory cells had infiltrated the lamina propria of the affected mucosa, and frequent mitotic figures were observed in epithelial cells at the dilated isthmus. To access the cell kinetics, the number of epithelial cells infected with C. andersoni were counted and compared with noninfected cattle. The number of gastric pit cells in infected cattle was significantly higher than that in the controls. The number of proliferative cells determined by the Ki-67 antigen in C. andersoni infected cattle was also significantly higher than that in the controls. Transmission electron microscopy and scanning electron microscopy revealed that the morphology of the C. andersoni organism was common to those of other Cryptosporidium spp. Immunohistochemically, several commercial antibodies against Cryptosporidium spp. showed positive reactions at the wall of these oocysts or parasitophorous vacuoles. This report is possibly the first to discuss the prominent hyperplasia of the abomasum mucosa, as well as morphologic features of C. andersoni in cattle.

Eosinophil interactions with Haemonchus contortus larvae in the ovine gastrointestinal tract

Sheep were immunized by weekly oral infections with Haemonchus contortus for 9 weeks followed by anthelmintic treatment. They were challenged either 9 or 22 weeks later with PBS (sham controls) or one million exsheathed L3 surgically injected in the abomasum, and killed 24 h or 48 h later. Sheep challenged 9 weeks after immunization displayed varying degrees of tissue eosinophilia that showed a significant inverse relationship with the number of intra-epithelial mast cells (globule leucocytes). Close association of eosinophils with tissue larvae was observed mainly in the gastric pits (24 h) or on the mucosal surface (48 h). All L3-challenged sheep in this group had detectable globule leucocytes and tissue IL-4 mRNA, as measured by Southern blot RT-PCR. In contrast, sheep challenged 22 weeks after immunization had no detectable globule leucocytes or IL-4 mRNA and although they exhibited consistent tissue eosinophilia, eosinophils were not closely associated with tissue larvae. Scanning and transmission electron microscopy of sheep sensitized and rested for 9 weeks before challenge showed that L3 surrounded by eosinophils were at varying stages of damage and structural collapse. These studies strongly indicate that eosinophils can damage and probably kill gastrointestinal nematode larvae in vivo. In addition, they also suggest that effective killing by tissue eosinophils may depend on other microenvironmental factors such as intra-epithelial mast cells and IL-4.

Increased expression of sonic hedgehog and altered methylation of its promoter region in gastric cancer and its related lesions

The hedgehog (Hh) signaling pathway plays an important role in foregut development, and its activity is increased in various tumors, including those of the digestive tract. Our objective in the present study was to determine the pattern and extent of Sonic hedgehog (Shh) expression in gastric cancer and related lesions as well as the methylation status of its promoter region, in an attempt to clarify the regulatory mechanism of Shh expression. A total of 237 gastric cancers and related lesions (89 carcinomas, 22 high-grade dysplasia, 21 low-grade dysplasia, 47 intestinal metaplasia, 38 chronic gastritis, and 20 normal epithelia) were subjected to immunohistochemical analysis with the Shh monoclonal antibody. The methylation status of Shh was determined by methylation-specific PCR (MS PCR), involving bisulfate treatment of DNA from 150 tissues followed by amplification using specific primer pairs designed by our group. Shh was completely absent in the upper part of normal gastric epithelia (gastric pit cells), and no significant differences were observed among the lower parts of normal epithelia, chronic gastritis, and intestinal metaplasia. However, Shh expression was significantly elevated in neoplastic lesions, such as carcinoma and high low-grade dysplasia, compared to non-neoplastic lesions. In carcinomas, Shh expression was associated with clinical stage, direct tumor invasion, and differentiation of tumor cells. Methylation of the Shh promoter region was frequent in normal gastric pit cells (11/18, 61.1%), but very rare in gastritis (0/18), intestinal metaplasia (0/19), dysplasia (0/10), and carcinoma (1/63, 1.6%), and correlated significantly with expression (P<0.001). Our results suggested that the increased and constitutive Shh expression is implicated in gastric carcinogenesis, and that promoter methylation may be an important regulatory mechanism of Shh expression.

Role of surfactant protein D (SP-D) in innate immunity in the gastric mucosa: evidence of interaction with Helicobacter pylori lipopolysaccharide

Surfactant protein D (SP-D) is a collagenous glycoprotein, a collectin, which functions as a pathogen-associated molecular pattern (PAMP) recognition receptor in the innate immune response. Although originally identified in the lung as a component of surfactant, SP-D also occurs in the gastric mucosa at the luminal surface and within gastric pits of mucus-secreting cells. Infection with the gastroduodenal pathogen Helicobacter pylori up-regulates expression of SP-D in human patients with gastritis, and its influence on colonization has been demonstrated in a Helicobacter SP-D-deficient (SP-D—/ —) mouse model. SP-D binds and agglutinates H. pylori cells in a lectin-specific manner, and has been shown to bind H. pylori lipopolysaccharide. Furthermore, evidence indicates that H. pylori varies LPS O-chain structure to evade SP-D binding which is speculated aids persistence of this chronic infection.

Role of surfactant protein D (SP-D) in innate immunity in the gastric mucosa: evidence of interaction with &lt;I&gt;Helicobacter pylori&lt;/I&gt; lipopolysaccharide

Surfactant protein D (SP-D) is a collagenous glycoprotein, a collectin, which functions as a pathogen-associated molecular pattern (PAMP) recognition receptor in the innate immune response. Although originally identified in the lung as a component of surfactant, SP-D also occurs in the gastric mucosa at the luminal surface and within gastric pits of mucus-secreting cells. Infection with the gastroduodenal pathogen Helicobacter pylori up-regulates expression of SP-D in human patients with gastritis, and its influence on colonization has been demonstrated in a Helicobacter SP-D-deficient (SP-D(-/-)) mouse model. SP-D binds and agglutinates H. pylori cells in a lectin-specific manner, and has been shown to bind H. pylori lipopolysaccharide. Furthermore, evidence indicates that H. pylori varies LPS O-chain structure to evade SP-D binding which is speculated aids persistence of this chronic infection.

A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract

This report describes the development and the clinical evaluation of a novel confocal endomicroscope for obtaining fluorescence images of cellular morphology of the mucosae of the upper- and the lower-GI tract in vivo. The work assessed the feasibility of performing in vivo microscopy at endoscopic examination and evaluated fluorescence imaging protocols. Images were collected in real time by using two prototype endoscope configurations, featuring slightly different miniaturized fiber-optic confocal microscopes, fitted integrally into the tips of conventional endoscopes. Confocal scanning was performed at 488 nm illumination for excitation of exogenously applied fluorophores (topical acriflavine and intravenous fluorescein). The images were compared with conventional histology of biopsy specimens and the findings of white-light endoscopy. Confocal endomicroscopy enabled imaging of cellular and subcellular structures (i.e., nuclei) of the GI tract. The crypts of the colonic mucosa, the villi of the terminal ileum and duodenum, the gastric pits of the stomach, and the squamous epithelium of the distal esophagus could be clearly visualized. Acriflavine strongly contrasted the cell nuclei of the surface epithelium, including the absorptive epithelial cells and the mucous secreting goblet cells. Fluorescein stained the extracellular matrix of the surface epithelium and also the subepithelial layers of the lamina propria. Images at increasing depth beneath the epithelium showed the mucosal capillary network. The findings correlated with the histology of biopsy specimens. The development of a fluorescence confocal endomicroscope makes it practical to examine the upper- and the lower-GI mucosa in cellular detail during otherwise routine endoscopic examination. The results represent a major technical advance in the development of this new optical imaging modality for the in vivo examination of GI tissue.

Gastric Pit Cell Hyperplasia and Glandular Atrophy in Carbonic Anhydrase IX Knockout Mice: Studies on Two Strains C57/BL6 and BALB/C

Carbonic anhydrase (CA) isoenzyme IX is a hypoxia-inducible enzyme, which is expressed in the human and rodent gastrointestinal tract and overexpressed in several different tumors. Functionally, it has probably an effect on proliferation and differentiation of gastrointestinal epithelial cells. It may also participate in gastric morphogenesis, since a recent study has shown gastric pit cell hyperplasia and glandular atrophy in CA IX-knockout mice. However, it is not known whether CA IX produces morphological changes in the gastric mucosa, which can turn into a dysplasia or malignancy in the presence of some carcinogenic factors. High-salt diet is considered such a factor which has been shown to modulate Helicobacter pylori-associated carcinogenesis. We produced two strains of CA IX-knockout mice, C57/BL6 and BALB/c, and the mice ate either standard or high-salt feed for 20 weeks. Stomach samples were collected from 40 Car 9(-/-) knockout mice and 37 wildtype littermates, and the tissue sections were examined for histology. CA IX-deficiency caused gastric pit cell hyperplasia and glandular atrophy in both BALB/c and C57/BL6 strains. Excess dietary salt had no significant effect on the severity of pit cell hyperplasia. No dysplasia was found in any of the groups. In C57/BL6 mice, CA IX-deficiency was associated with gastric submucosal inflammation. The results indicate that CA IX-deficiency provides a useful model to study the mechanisms of gastric morphogenesis and epithelial integrity. Further studies are needed to see whether CA IX has a role in the regulation of immune response. The findings suggest that although CA IX-deficiency is not a tumor-promoting factor per se, it induces glandular atrophy in the body mucosa, a lesion which is considered to be a preneoplastic alteration in the stomach.

Altered expression of aquaporin 4 and H+/K+‐ATPase in the stomachs of peptide YY (PYY) transgenic mice

The hormone PYY (peptide YY), synthesized by endocrine cells in the pancreas, ileum, colon and stomach has widespread inhibitory effects on gastrointestinal and pancreatic fluid secretion. Transgenic mice expressing a viral oncoprotein under the control of the PYY gene 5'-flanking region develop well-differentiated colonic endocrine tumours producing mainly PYY and enteroglucagon. In the present study, we investigated the expression of AQP4 (aquaporin 4) water channel and H(+)/K(+)-ATPase in stomachs from both control and transgenic mice. Semi-quantitative RT (reverse transcriptase)-PCR showed an increase in the AQP4 transcript compared with control mice. Quantitative Western-blot analysis of stomachs from control and transgenic mice confirmed a significant increase in the 30 kDa AQP4 protein in transgenic mice. In control mice, AQP4 is specifically expressed in the basolateral membrane of gastric parietal cells, located in the basal region of the fundic glands. This particular location suggests that parietal cells in the base region of gastric pits might have a major role in water transport when compared with the more superficial parietal cells. Interestingly, immunofluorescence studies on transgenic mice revealed that the quantitative increase of AQP4 expression was actually due to an increase in the number of AQP4-expressing epithelial cells rather than to a higher expression of AQP4 in parietal cells. In fact, immunofluorescence experiments using the specific antibody raised against the AE2 isoform of Cl(-)/HCO3- exchanger specifically expressed in parietal cells confirmed that the number of parietal cells was comparable in both PYY and control stomachs. Moreover, in transgenic mice, a parallel significant decrease in the expression of H(+)/K(+)-ATPase was observed, as revealed by RT-PCR, quantitative immunoblotting and immunofluorescence. In the present study, we demonstrate that the sustained inhibition of gastric secretion due to tumours producing PYY/enteroglucagon in transgenic mice is associated with an increase in AQP4 expression and a down-regulation of H(+)/K(+)-ATPase in parietal cells that acquire the characteristics of basal parietal cells. The absence of H2 receptors-mediated signalling due to the inhibition of histamine release from ECL (enterochromaffin-like) cells by PYY may be in part responsible for the observed increase in the number of parietal cells expressing AQP4.

Gastric immunolocalization and plasma profiles of acyl-ghrelin in fasted and fasted-refed prepuberal gilts

Ghrelin is a peripheral circulating hormone, mainly released from the stomach, which can stimulate food intake. We studied fed, fasted and fasted-refed prepuberal gilts in order to outline possible changes in gastric mucosal ghrelin cells and in plasma ghrelin profiles in response to food deprivation. Acyl-ghrelin-immunoreactive cells were numerous in oxyntic glands, less abundant in cardiac glands and least frequent in pyloric glands, with the addition of a minor population of labelled cells in the gastric pit mucosa. When fed and fasted animals were compared (72-h fast versus fed; n = 4 each), no clear-cut differences were revealed in labelled cell numbers, nor in their staining intensity. An RIA for plasma porcine acyl-ghrelin (n-octanoylated at Ser-3), not recognizing des-acyl-ghrelin, was validated. Plasma acyl-ghrelin progressively increased upon fasting (over 6, 12, 24 and 48 h); ghrelin levels significantly (P<0.05) higher than those prefast were reached at 72 h. After refeeding, plasma ghrelin was rapidly restored to basal values by 6 h. In the same animals, plasma insulin was significantly reduced throughout the fasting period (6-72 h), while rapidly increasing after refeeding. Non-esterified fatty acid levels increased during fasting (12-72 h) and rapidly returned to low values after refeeding. In conclusion, the present study demonstrates that starvation and refeeding influence ghrelin plasma level in prepuberal gilts. The absence of detectable changes in ghrelin cells, as seen in immunohistochemistry, could be due to a large intracellular storage of potentially releasable acylghrelin.