High prevalence of dna from non-H. pylori helicobacters in the gastric mucosa of venezuelan pet dogs and its histological alterations

Non-H. pylori helicobacters (NHPH) have been demonstrated as gastric spiral-shaped bacteria in specimens obtained from dogs; however, their roles in the pathogenesis of upper gastrointestinal disease have not yet been clearly established. The purpose of this study was to evaluate the prevalence of NHPH DNA in the gastric mucosa of dogs and its association with histopathology. Helicobacter was detected through histopathological techniques, PCR, and FISH analysis from fundic biopsies of twenty dogs with or without signs of gastrointestinal disease. PCR and FISH were based on partial 16S rRNA gene sequences. Nineteen dogs showed mild to marked gastritis in the fundus, and only one dog had a healthy gastric mucosa. NHPH DNA was detected in 18 dogs with gastritis and one with normal gastric mucosa. However, there was no significant correlation between the presence of NHPH DNA and the degree of gastritis. These results show a high prevalence of NHPH DNA in the gastric mucosa of dogs from Venezuela. Further studies are necessary to determine a possible association between a specific NHPH species and the degree of gastritis.

Use of polymerase chain reaction and enzymatic cleavage in the identification of Helicobacter spp. in gastric mucosa of human beings from North Paraná, Brazil

Helicobacter pylori is the most common gastric bacteria of human beings. Animal-borne helicobacter have been associated with gastritis, ulceration, and gastric mucosa-associated lymphoid-tissue lymphoma in people. We attempted to identify the species of Helicobacter spp. that infect human beings in north Paraná, Brazil. Samples of gastric mucosa from 38 dyspeptic patients were analyzed by optic microscopy on silver stained slides, polimerase chain reaction (PCR), and enzymatic cleavage. Genus and species-specific primers to H. pylori, H. heilmannii, H. felis, and consensual primers to H. bizzozeronii or H. salomonis were used. The PCR products were submitted to enzymatic cleavage by VspI (Helicobacter spp. product) and HinfI (species products) enzymes. Thirty-two out of 38 patients evaluated had 3.2 to 5 µm long bacteria that resembled H. pylori in Warthin-Starry stained slides and were positive to the genus Helicobacter by PCR. In 30 of these patients the bacteria were identified as H. pylori. Two samples positive by silver stain were negative to all species tested by PCR. None of the 38 samples was positive to animal-origin helicobacter species. These results show that PCR and enzymatic restriction are practical methods to identify the species of helicobacters present in gastric mucosa of human beings. People in north Paraná appear to be infected mostly with H. pylori.

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.

Immunoexpression of CD95 in chronic gastritis and gastric mucosa-associated lymphomas

CD95 (Fas/APO-1)-mediated apoptosis plays an important role in immunological regulation and is related to the pathogenesis of autoimmune diseases. Immunoexpression of CD95 has been reported to frequently occur in low grade non-Hodgkin lymphomas, especially of post-germinal center histogenesis, among which those originating in mucosa-associated lymphoid tissue (MALT lymphomas). However, there is no report comparing in situ immunoexpression of this marker in lymphomas and the hyperplastic lymphoid reaction (chronic gastritis) related to Helicobacter pylori infection. The purpose of the present research was to compare the intensity of lymphoid CD95 immunoexpression in 15 cases of H. pylori-related chronic gastritis and 15 gastric MALT lymphomas. CD95 (anti-CD95) was detected by an immunoperoxidase technique in paraffin sections using the catalyzed amplification system. Graduation of reaction intensity (percentage of CD95-positive cells) was semiquantitative, from 1+ to 4+. Nine cases of chronic gastritis were 4+, five 2+ and one 1+. Three lymphomas were 4+, three 3+, four 2+, four 1+, and one was negative. Although 14 of 15 lymphomas were positive for CD95, the intensity of the reaction was significantly weaker compared to that obtained with gastric tissue for patients with gastritis (P = 0.03). The difference in CD95 immunoexpression does not seem to be useful as an isolated criterion in the differential diagnosis between chronic gastritis and MALT lymphomas since there was overlapping of immunostaining patterns. However, it suggests the possibility of a pathogenetic role of this apoptosis-regulating protein in MALT lymphomas.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma-/- C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4-/- than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4-/- than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 ± 3.07 pg/mL, mean ± SD) and -negative (11.07 ± 3.5 pg/mL) WT BALB/c mice, in the group of IL-4-/- animals, the serum concentration of IFN-g was significantly higher in the infected ones (38.16 ± 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

Over-expression of cyclooxygenase-2 in endoscopic biopsies of ectopic gastric mucosa

Ectopic gastric mucosa (EGM) is considered to be a congenital condition. Rare cases of adenocarcinoma have been described. There are no data justifying regular biopsies or follow-up. Cyclooxygenase-2 (COX-2) is a protein involved in gastrointestinal tumor development by inhibiting apoptosis and regulating angiogenesis. The aim of this prospective study was to evaluate COX-2 expression in EGM and compare it with normal tissue and Barrett's esophagus. We evaluated 1327 patients. Biopsies were taken from the inlet patch for histological evaluation and from the gastric antrum to assess Helicobacter pylori infection. Biopsies taken from normal esophageal, gastric antrum and body mucosa and Barrett's esophagus were retrieved from a tissue bank. EGM biopsies were evaluated with respect to type of epithelium, presence of H. pylori, and inflammation. COX-2 was detected by immunohistochemistry using the avidin-biotin complex. EGM islets were found in 14 patients (1.1%). Histological examination revealed fundic type epithelium in 58.3% of cases, H. pylori was present in 50% and chronic inflammation in 66.7%. Expression of COX-2 was negative in normal distal esophagus, normal gastric antrum and normal gastric body specimens (10 each). In contrast, EGM presented over-expression of COX-2 in 41.7% of cases and Barrett's esophagus in 90% of cases (P = 0.04 and 0.03, respectively). COX-2 immunoexpression in EGM was not related to gender, age, epithelium type, presence of inflammation or intestinal metaplasia, H. pylori infection, or any endoscopic finding. Our results demonstrate up-regulation of COX-2 in EGM, suggesting a possible malignant potential of this so-called harmless mucosa.

P53 and Rb tumor suppressor gene alterations in gastric cancer

Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma. METHOD: Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using 35S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone. RESULTS AND DISCUSSION: No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P = .90). There was no correlation of gene alterations with the disease severity. CONCLUSIONS: The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

Gastric ulcers in pigs affected with postweaning multisystemic wasting syndrome

Samples of gastric lymph nodes and the stomachs from 24 pigs selected from herds affected by postweaning multisystemic wasting syndrome (PMWS) and sudden death associated with gastric ulcers were studied. Pigs were selected on the basis of unthriftiness, decreased feed intake, and wasting. The stomachs were opened, inverted, and classified into 0-3 score according the severity of the gross lesions present in pars oesophagica (non-glandulargastric mucosa). Selected samples were processed for paraffin embedding and stained with hematoxylin and eosin. Immunohistochemistry using anti-PCV2 (porcine circovírus type 2) antibody, anti-Helicobacter pylori antibody and a wide-spectrum anti-cytokeratin antibody was performed. Gross changes in pars oesophagea were classified according to the severity of lesions as score 3, 2, and 1 in 8, 6, 5 stomachs respectivelly. Microscopically, hyperplastic lymphoid follicles, lymphohistiocytic inflammatory infiltrates and focci of necrosis in the gastric mucosa were common findings. Large amounts of PCV2 antigen were observed in the cytoplasm and nuclei from intralesional cells and debris from the gastric glandular mucosal zone; however, in the fundus, anti-PCV2 immunostaining was restricted to the surface mucosal cells and foveolar compartment. All gastric lymph nodes were positive for PCV2 antigen. Anti-H. pylori immunostaining was seen in eleven cases, mainly in the antrum, on the mucosal surface and foveolar compartment. The association of the anti-PCV2 immunostaining with the glandular mucus-producing cells suggests a role for PCV2 as an additional factor for the swine ulcer development.

Distribution of collagen types I, III, and IV in gastric tissue of marmosets (Callithrix spp., Callitrichidae: Primates)

Extracellular matrix (ECM) components such as fibrillar collagens play a fundamental role in wound repair and have also been studied in association with the gastric ulcer healing process in gastroenterology. Nevertheless, there have been no studies in the literature to date regarding the description and characterization of ECM components, neither in normal nor in injured gastric tissue of primate species. The objective of this study was to investigate the expression of gastric collagen types I, III, and IV in marmosets (Callithrix sp.). Histological specimens from the stomach of 6 Callithrix jacchus, 12 C. kuhli, and 12 C. geoffroyi were evaluated. The specimens were immunostained with anti-types I and III collagen polyclonal antibodies and anti-type IV collagen monoclonal antibody. Collagen types I and III were detected in the submucosa and lamina propria between the mucosal glands while collagen type IV was detected in the muscularis mucosae, muscular layers, blood vessels, and gastric mucosa between the mucosal glands. It is hoped that these findings can contribute to future studies on the gastric extracellular matrix components in primates and to comparative studies in the area of gastroenterology.

Mouse inoculation for the detection of non-cultivable gastric tightly spiralled bacteria

In the present study we compared the inoculation of swine gastric mucus into the stomach of mice, the urease test and carbolfuchsin-stained smears for the diagnosis of the infection with "Gastrospirillum suis" ("Helicobacter heilmannii" type 1), an uncultivated tightly spiralled gastric bacterium. Fragments obtained from the antral and oxyntic mucosa of the stomach of 50 slaughtered pigs were used for urease test, for carbolfuchsin-stained smears and for obtaining scrapings of mucus for mouse inoculation. The mice were killed by spinal dislocation 10 days after inoculation and fragments of the antral and oxyntic mucosa were used for spiral bacterium identification (urease test and carbolfuchsin-stained smears). Among the methods employed for the diagnosis of "H. heilmannii" infection, the inoculation of gastric mucus into the stomach of mice was the most sensitive and demonstrated bacterial positivity in 31 (62.0%) swine. Direct examination showed tightly spiralled bacteria in the gastric mucosa of only 4 (8.0%) of the 50 pigs studied. Among them, 3 (6.0%) presented a positive preformed urease test. Spiral bacteria were not seen in the gastric mucosa of any control mice. These results show that the use of the mouse inoculation method improved the detection of "H. heilmannii" in swine

Methylation status of ANAPC1, CDKN2A and TP53 promoter genes in individuals with gastric cancer

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80°C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4% of gastric cancer samples, with 35% methylation in diffuse-type and 26.9% in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30% diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.

Helicobacter pylori adhesion to gastric epithelial cells is mediated by glycan receptors

Helicobacter pylori adhesion to gastric epithelial cells constitutes a key step in the establishment of a successful infection of the gastric mucosa. The high representation of outer membrane proteins in the bacterial genome suggests the relevance of those proteins in the establishment of profitable interactions with the host gastric cells. Gastric epithelial cells are protected by a mucous layer gel, mainly consisting of the MUC5AC and MUC6 mucins. In addition to this protective role, mucins harbor glycan-rich domains that constitute preferential binding sites of many pathogens. In this article we review the main players in the process of H. pylori adhesion to gastric epithelial cells, which contribute decisively to the high prevalence and chronicity of H. pylori infection. The BabA adhesin recognizes both H-type 1 and Lewis b blood-group antigens expressed on normal gastric mucosa of secretor individuals, contributing to the initial steps of infection. Upon colonization, persistent infection induces an inflammatory response with concomitant expression of sialylated antigens. The SabA adhesin mediates H. pylori binding to inflamed gastric mucosa by recognizing sialyl-Lewis a and sialyl-Lewis x antigens. The expression of the BabA and SabA adhesins is tightly regulated, permitting the bacteria to rapidly adapt to the changes of glycosylation of the host gastric mucosa that occur during infection, as well as to escape from the inflammatory response. The growing knowledge of the interactions between the bacterial adhesins and the host receptors will contribute to the design of alternative strategies for eradication of the infection.

Argyrophil cell density in the oxyntic mucosa is higher in female than in male morbidly obese patients

Obesity is a multifactorial disorder often associated with many important diseases such as diabetes, hypertension and other metabolic syndrome conditions. Argyrophil cells represent almost the total population of endocrine cells of the human gastric mucosa and some reports have described changes of specific types of these cells in patients with obesity and metabolic syndrome. The present study was designed to evaluate the global population of argyrophil cells of the gastric mucosa of morbidly obese and dyspeptic non-obese patients. Gastric biopsies of antropyloric and oxyntic mucosa were obtained from 50 morbidly obese patients (BMI >40) and 50 non-obese patients (17 dyspeptic overweight and 33 lean individuals) and processed for histology and Grimelius staining for argyrophil cell demonstration. Argyrophil cell density in the oxyntic mucosa of morbidly obese patients was higher in female (238.68 ± 83.71 cells/mm2) than in male patients (179.31 ± 85.96 cells/mm2) and also higher in female (214.20 ± 50.38 cells/mm2) than in male (141.90 ± 61.22 cells/mm2) morbidly obese patients with metabolic syndrome (P = 0.01 and P = 0.02, respectively). In antropyloric mucosa, the main difference in argyrophil cell density was observed between female morbidly obese patients with (167.00 ± 69.30 cells/mm2) and without (234.00 ± 69.54 cells/mm2) metabolic syndrome (P = 0.001). In conclusion, the present results show that the number of gastric argyrophil cells could be under gender influence in patients with morbid obesity. In addition, gastric argyrophil cells seem to behave differently among female morbidly obese patients with and without metabolic syndrome.

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg,ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.