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.

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.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

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.

Expression of TERT in precancerous gastric lesions compared to gastric cancer

The objective of this study was to determine the levels of TERT mRNA and TERT protein expression in stomach precancerous lesions such as intestinal metaplasia (IM) and gastric ulcer (GU) and compare them to gastric cancer (GC). Real-time PCR was performed to detect TERT mRNA expression levels in 35 biopsies of IM, 30 of GU, and 22 of GC and their respective normal mucosas. TERT protein was detected by immunohistochemistry in 68 samples, 34 of IM, 23 of GU, and 11 of GC. Increased TERT mRNA expression levels were observed in a significant number of cases, i.e., 46% of IM, 50% of GU, and 79% of GC. The relative mean level of TERT mRNA after normalization with the β-actin reference gene and comparison with the respective adjacent normal mucosa was slightly increased in the IM and GU groups, 2.008 ± 2.605 and 2.730 ± 4.120, respectively, but high TERT mRNA expression was observed in the GC group (17.271 ± 33.852). However, there were no statistically significant differences between the three groups. TERT protein-positive immunostaining was observed in 38% of IM, 39% of GU, and 55% of GC. No association of TERT mRNA and protein expression with Helicobacter pylori infection or other clinicopathological variables was demonstrable, except for the incomplete type vs the complete type of IM. This study confirms previous data of the high expression of both TERT mRNA and protein in gastric cancer and also demonstrates this type of changed expression in IM and GU, thus suggesting that TERT expression may be deregulated in precursor lesions that participate in the early stages of gastric carcinogenesis.

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.

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.

Frequência e distribuição de Helicobacter spp. na mucosa gástrica de cães

A associação das helicobacterias com doença gástrica em humanos e em alguns animais domésticos e selvagens sugere a participação dessas na patogênese da gastrite em cães. Neste artigo procurou-se verificar a presença de Helicobacter spp. na mucosa gástrica de cães e avaliar sua associação com os achados macro e microscópicos, considerando a idade. Coletaram-se amostras das regiões cárdica, fúndica, do corpo e pilórica dos estômagos de 60 cães para a realização de exame histopatológico, utilizando-se as colorações pela hematoxilina-eosina (HE) e carbolfucsina (CF). Tais exames revelaram Helicobacter spp. em 96,7% dos animais, sendo observados infiltrados inflamatórios, predominantemente mononucleares (100%), hiperplasia de nódulos linfoides (98,3%), erosões/ulcerações (6,7%), hemorragia (5%) e hiperemia (95%) em amostras coradas por HE. Não houve correlação da infecção por helicobacter spp. com a idade do animal e da idade com alterações inflamatórias na presença da bactéria. As amostras das regiões do corpo e pilórica apresentaram maior presença de bactérias a histopatologia (CF) (ambos 95%), em seguida vindo as regiões fúndica (91,7%) e cárdica (56,7%). Houve correlação do número de Helicobacter spp. com o de células inflamatórias e nódulos linfoides nessas regiões, sugerindo que as bactérias encontradas na mucosa gástrica dos cães poderiam ser responsáveis pelas alterações que caracterizam a gastrite.

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.

Hepatic changes in mice chronically infected with Helicobacter trogontum

We infected NIH germ-free female mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the lesions it induced in the liver of this host. Fifteen mice were challenged with a single dose of H. trogontum (test group) and killed 6, 12 and 18 months after inoculation (5 animals/group). Nine animals were challenged with 0.85% saline alone (control group) and killed at the same times. Fragments from the liver, cecum and colon were obtained for microbiologic and histologic examination. Stool samples were also collected. H. trogontum was detected in the cecum, colon and/or stool samples of all test mice. As expected, the bacterium was not isolated from any specimen obtained from the control animals. On the other hand, although we could not cultivate the bacterium from the liver, 13 test animals (86.7%) presented histological changes in this organ. The 6-month group presented infiltration of mononuclear and polymorphonuclear cells in the hepatic parenchyma and the two other groups presented foci of mononuclear cells. The results suggest that H. trogontum can elicit a hepatic inflammatory response in mice since the only difference between control and test animals was the presence of H. trogontum in the latter. This result, together with the growing number of related reports in the literature, reinforces the possible role of Helicobacter infection in the pathogenesis of hepatobiliary diseases.

Infectious agents in coronary atheromas: a possible role in the pathogenesis of plaque rupture and acute myocardial infarction

In this review we report our recent findings of histopathological features of plaque instability and the association with Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP) infection, studying thrombosed coronary artery segments (CAS) of patients who died due to acute myocardial infarction. Vulnerable plaques are known to be associated with fat atheromas and inflammation of the plaque. Here we demonstrated that vulnerability is also related with focal positive vessel remodeling that maintains relatively well preserved lumen even in the presence of large atheromatous plaques. This phenomena may explain why the cinecoronariography may not detect large and dangerous vulnerable plaques. Greater amount of these bacteria in vulnerable plaques is associated with adventitial inflammation and positive vessel remodeling: the mean numbers of lymphocytes were significantly higher in adventitia than in the plaque, good direct correlation was obtained between numbers of CD20 B cells and numbers of CP infected cells in adventitia, and between % area of MP-DNA in the plaque and cross sectional area of the vessel, suggesting a cause-effect relationship. Mycoplasma is a bacterium that needs cholesterol for proliferation and may increase virulence of other infectious agents. In conclusion, co-infection by Mycoplasma pneumoniae and Chlamydia pneumoniae may represent an important co-factor for plaque instability, leading to coronary plaque thrombosis and acute myocardial infarction, since larger amount of these bacteria strongly correlated with histological signs of more vulnerability of the plaque. The search of CMV and Helicobacter pilori in these tissues resulted negative.

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

Application of isotope-selective non-dispersive infrared spectrometry for the evaluation of the 13C-urea breath test: comparison with three concordant methods

The aim of this work was to compare the performance of isotope-selective non-dispersive infrared spectrometry (IRIS) for the 13C-urea breath test with the combination of the 14C-urea breath test (14C-UBT), urease test and histologic examination for the diagnosis of H. pylori (HP) infection. Fifty-three duodenal ulcer patients were studied. All patients were submitted to gastroscopy to detect HP by the urease test, histologic examination and 14C-UBT. To be included in the study the results of the 3 tests had to be concordant. Within one month after admission to the study the patients were submitted to IRIS with breath samples collected before and 30 min after the ingestion of 75 mg 13C-urea dissolved in 200 ml of orange juice. The samples were mailed and analyzed 11.5 (4-21) days after collection. Data were analyzed statistically by the chi-square and Mann-Whitney test and by the Spearman correlation coefficient. Twenty-six patients were HP positive and 27 negative. There was 100% agreement between the IRIS results and the HP status determined by the other three methods. Using a cutoff value of delta-over-baseline (DOB) above 4.0 the IRIS showed a mean value of 19.38 (minimum = 4.2, maximum = 41.3, SD = 10.9) for HP-positive patients and a mean value of 0.88 (minimum = 0.10, maximum = 2.5, SD = 0.71) for negative patients. Using a cutoff value corresponding to 0.800% CO2/weight (kg), the 14C-UBT showed a mean value of 2.78 (minimum = 0.89, maximum = 5.22, SD = 1.18) in HP-positive patients. HP-negative patients showed a mean value of 0.37 (minimum = 0.13, maximum = 0.77, SD = 0.17). IRIS is a low-cost, easy to manage, highly sensitive and specific test for H. pylori detection. Storing and mailing the samples did not interfere with the performance of the test.