Complex cellular responses of Helicobacter pylori-colonized gastric adenocarcinoma cells

Helicobacter pylori is an important class I carcinogen that persistently infects the human gastric mucosa to induce gastritis, gastric ulceration, and gastric cancer. H. pylori pathogenesis strongly depends on pathogenic factors, such as VacA (vacuolating cytotoxin A) or a specialized type IV secretion system (T4SS), which injects the oncoprotein CagA (cytotoxin-associated gene A product) into the host cell. Since access to primary gastric epithelial cells is limited, many studies on the complex cellular and molecular mechanisms of H. pylori were performed in immortalized epithelial cells originating from individual human adenocarcinomas. The aim of our study was a comparative analysis of 14 different human gastric epithelial cell lines after colonization with H. pylori. We found remarkable differences in host cell morphology, extent of CagA tyrosine phosphorylation, adhesion to host cells, vacuolization, and interleukin-8 (IL-8) secretion. These data might help in the selection of suitable cell lines to study host cell responses to H. pylori in vitro, and they imply that different host cell factors are involved in the determination of H. pylori pathogenesis. A better understanding of H. pylori-directed cellular responses can provide novel and more balanced insights into the molecular mechanisms of H. pylori-dependent pathogenesis in vivo and may lead to new therapeutic approaches.     

Helicobacter (Campylobacter) pylori in gastric brushing cytology.

Helicobacter (formerly Campylobacter) pylori is frequently associated with chronic gastritis and peptic ulcer and has been implicated as an etiologic agent. Identification of H. pylori is important for specific treatment with antibiotics and bismuth compounds. We studied 27 patients who presented with symptoms of gastritis or peptic ulcer on whom paired gastric biopsies and gastric brushings for cytology had been performed. Biopsies were stained with H & E and Warthin-Starry or Giemsa for H. pylori. Previously, Papanicolaou-stained brushings were restained with Giemsa and reviewed blindly by two cytologists. Cytologic examination revealed the characteristic 1-3 mu curved or spiral gram-negative bacilli embedded in mucus in 12 of 27 (44%) of cases. Biopsies showed H. pylori in 13 of 27 (48%) of cases. Cytology and histology were concordant in 22 of 27 (81%) of cases. Three cases were positive on biopsy, negative on cytology; two of these were unsatisfactory cytology specimens. Two cases were positive on cytology, negative on biopsy, apparently sampling artifacts. Papanicolaou-stained slides were scored for several morphologic parameters; numbers of acute and chronic inflammatory cells and degree of cytologic atypia. None of these were predictive of the presence of H. pylori. We conclude that Giemsa-stained gastric brushings are a useful complement to gastric biopsies in establishing the diagnosis of H. pylori.     

Electron microscopic study of association between Helicobacter pylori and gastric and duodenal mucosa.

AIM--To study the ultrastructural appearances of Helicobacter pylori in antral and duodenal biopsy specimens and its relation with the epithelial cells. METHODS--Endoscopically obtained antral and duodenal biopsy specimens were examined using transmission electron microscopy and freeze fracture analysis. RESULTS--Most bacteria looked curved, but in the duodenal bulb coccoid bacteria were relatively common. Bacteria were often found around intercellular junctions. freeze fracture examination indicated abnormalities of the tight junction complexes in patients with H pylori infection. In many biopsy specimens bacteria were seen closely attached to the epithelial cell membrane by different forms of adhesion. In addition to what looked like intracytoplasmic penetration by bacteria, several examples of genuine penetration were observed. CONCLUSION--H pylori is commonly found adhering to epithelial cells. Occasionally, H pylori may also penetrate cells. These features may contribute to the pathogenic action of the organism.     

A simple method to demonstrate duodenal gastric metaplasia

AIMS: The diagnosis of duodenal gastric metaplasia (DGM) is based on the demonstration of periodic acid Schiff (PAS) positive mucin in duodenal columnar cells. Recently, groups of duodenal columnar cells were seen to be autofluorescent in haematoxylin and eosin (H&E) stained sections from a patient with DGM. MATERIALS AND METHODS: Consecutive archival gastric and duodenal H&E sections from 30 patients with chronic gastritis and DGM (CG+DGM), from 30 with chronic gastritis without DGM (control group I), from 30 with normal gastric and duodenal mucosa (control group II), and from five patients with coeliac disease (control group III) were reviewed on a fluorescent microscope. RESULTS: The surface epithelium of the gastric mucosa in all 95 cases had autofluorescent material. In 31 cases with DGM (including one unreported case in control group I), groups of columnar duodenal cells also had autofluorescent material. In the remaining 64 cases, duodenal columnar cells were not autofluorescent. Results were confirmed with the PAS stain. CONCLUSIONS: The method described detected apical mucin secretion in columnar cells, both in the stomach and in the duodenum from patients with DGM. The autofluorescence was induced by eosin (which binds to neutral mucin). Observing H&E stained duodenal biopsies under a fluorescence microscope may be sufficient to confirm DGM or to detect incipient DGM. Despite long observation periods and a long exposure time while photographing, the autofluorescence did not fade away. Because re-cuttings for special staining (PAS) are no longer required when this method is used, both final diagnosing time and laboratory costs can be reduced.     

Characterization of progressive metaplasia in the gastric corpus mucosa of Mongolian gerbils infected with Helicobacter pylori

Spasmolytic polypeptide‐expressing metaplasia (SPEM) and intestinal metaplasia are considered neoplastic precursors of gastric adenocarcinoma in humans. Loss of parietal cells causes the development of SPEM in the gastric corpus and then chronic inflammation drives SPEM toward a more proliferative lineage. Mongolian gerbils infected with Helicobacter pylori develop chronic gastritis and metaplasia, mimicking aspects of human gastritis with H. pylori infection. We therefore examined metaplastic lineages in the gastric corpus mucosa of gerbils infected by H. pylori strain 7.13, which produces rapid onset of severe inflammation. Six weeks following H. pylori infection, Griffonia simplicifolia lectin II (GSII)‐positive SPEM developed in the base of oxyntic glands in association with parietal cell loss and inflammation. In association with severe inflammation, SPEM glands evolved into aberrant phenotypes, including branched lesions, dilated lesions, and penetrating invasive glands. Mucin 4 (MUC4) was up‐regulated in SPEM and progressive SPEM. Clusterin was expressed in the tips of branched and dilated lesions and throughout regions of invasive glands. Intriguingly, clusterin‐positive regions in these lesions expressed Ki67 and matrix metalloproteinase 7 (MMP‐7). These same regions were also positive for expression of phospho‐IkBα, suggestive of activated NFkB signalling. These findings suggest that clusterin‐positive regions in progressive phenotypes of SPEM have invasive characteristics. Thus, H. pylori infection in gerbils induces SPEM, which then can progress to further aberrant and invasive metaplastic phenotypes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Adherence of Helicobacter pylori to areas of type II intestinal metaplasia in Korean gastric mucosa.

The aim of this study was to examine whether Helicobacter pylori (H. pylori) attaches to areas of intestinal metaplasia in Korean patients. Gastric biopsy specimens with intestinal metaplasia from 8 gastric cancers, 24 gastric ulcers, 11 duodenal ulcers, and 57 chronic gastritis were examined. The specimens were stained with periodic acid-Schiff/alcian blue pH 2.5 and high-iron diamine/alcian blue pH 2.5 to identify the subtype of intestinal metaplasia, and then immunohistochemical stain was done with rabbit anti-H. pylori polyclonal antibody. In 17 patients, H. pylori attached to areas of type II intestinal metaplasia. All areas of intestinal metaplasia showing adherence contained sialomucin, and H. pylori was not detected in the areas of intestinal absorptive cells and sulfomucin-containing metaplastic cells.     

Bile reflux and intestinal metaplasia in gastric mucosa.

AIM: To determine associations between enterogastric bile reflux and gastric mucosal pathology. METHOD: A retrospective study using fasting gastric juice bile acid measurements and antral or prestomal biopsy specimens from 350 patients, 66 of whom had previously undergone surgery that either bypassed or disrupted the pyloric sphincter. RESULTS: Bile reflux was positively associated with reactive gastritis and negatively with Helicobacter pylori density. After stratification for previous surgery, age, and H pylori status, the histological feature most strongly associated with bile reflux was intestinal metaplasia, including all its subtypes. The prevalence of intestinal metaplasia was greatest in patients with both H pylori infection and high bile acid concentrations. Bile reflux was also positively associated with the severity of glandular atrophy, chronic inflammation, lamina propria oedema and foveolar hyperplasia. CONCLUSIONS: Bile reflux is a cause of reactive gastritis. It modifies the features of H pylori associated chronic gastritis. The changes are not confined to patients who have had surgery to their stomachs. The positive associations with atrophy and intestinal metaplasia have implications for models of gastric carcinogenesis.     

幽门螺杆菌与十二指肠疾病及胃上皮化生的关系

研究幽门螺杆菌与十二指肠疾病及其胃上皮化生的关系。方法：应用特殊染色方法对１６６例胃窦及十二指肠活检标本进行了形态学观察及ＨＰ检测。     

Loss of alkaline phosphatase activity in duodenal mucosa: a marker for precursors of gastric metaplasia?

Biopsy specimens from duodenal mucosa in 34 patients with upper gastrointestinal symptoms and endoscopically abnormal mucosa (including duodenitis, active duodenal ulcer, and healed duodenal ulcer) and in 9 patients with histologically normal mucosa were examined histologically for gastric metaplasia and endogenous alkaline phosphatase (AP) activity. Using haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS), we found gastric metaplasia in 91.2 per cent (31 out of 34) of patients with altered duodenal mucosa and in 33.3 per cent (3 out of 9) of patients with histologically normal, non-inflamed duodenal mucosa (P less than 0.001). To characterize gastric metaplasia further, histochemical methods for AP activity were applied to duodenal mucosa specimens. No AP activity was detected in complete metaplastic cells, but focal or diffuse loss of AP activity was frequently shown in otherwise normal appearing enterocytes next to metaplastic cell groups. Focal loss of AP activity was also detected in seven out of nine healthy controls (= 77.8 per cent) which appeared normal when stained with H&E. Our results suggest that the loss of AP activity in enterocytes may be an early marker of developing gastric metaplasia or at least a morphological manifestation of cell damage.     

Helicobacter-associated duodenitis and gastric metaplasia in duodenal ulcer patients.

Biopsy specimens were taken from the duodenal bulb and the distal duodenum in 45 duodenal ulcer patients before and after treatment with histamine-2 antagonists, prostaglandin analogues or antacids. After four weeks of treatment, the ulcer had healed in 31 patients. The treatment did not lead to a reduced frequency of helicobacter-associated duodenitis or gastric metaplasia of the duodenal epithelium. We found gastric metaplasia in 52.3% of all biopsy specimens from the duodenal bulb, chronic active duodenitis in 71.9% and helicobacter-like structures in 15.9%. The helicobacter organisms were found only in areas of gastric metaplasia, and an accompanying chronic active duodenitis was found in 94.1%. In the distal duodenum, we observed chronic active duodenitis in 15.0% of the specimens. Here the inflammation was not associated with gastric metaplasia or helicobacter-like structures. These observations support the hypothesis that Helicobacter pylori colonizes the duodenal mucosa only in areas of gastric metaplasia, and that such colonization may lead to an active duodenitis.     

Bacteriophages in Helicobacter (Campylobacter) pylori

Bacteriophages in different stages of maturation were found in thin sections of a clinical isolate of Helicobacter (Campylobacter) pylori. Mature phage heads measured 70 x 60 nm and the tail at least 120 nm. Lysogeny was maintained during subculture on blood agar for more than 3 months after isolation from a gastric biopsy.     

Intestinal metaplasia in endoscopic biopsy specimens of gastric mucosa

A total of 1412 consecutive cases of endoscopic gastric biopsy, carried out over a four year period, were reviewed and specimens were examined histochemically to determine the prevalence of intestinal metaplasia and its variants. Three types were characterised: complete intestinal metaplasia and two classes of incomplete intestinal metaplasia (type IIa and type IIb) depending on the absence or presence, respectively, of sulphomucins within mucin secreting columnar cells. Type IIb intestinal metaplasia was significantly more common in patients with gastric carcinoma (p less than 0.001) and in those with dysplasia (p less than 0.001) than in patients with benign gastric pathology. No such association was found with either type I or type IIa intestinal metaplasia. In addition to those present in the columnar cells of type IIb intestinal metaplasia, sulphomucins were also commonly found in goblet cells of all three types of metaplasia. The presence of sulphomucins in goblet cells, however, was not significantly associated with gastric carcinoma or dysplasia. The significance of the different types of intestinal metaplasia in relation to the pathological findings is discussed.     

Increased apoptosis in gastric mucosa adjacent to intestinal metaplasia

BACKGROUND: The biological processes involved in the development of gastric mucosal atrophy and intestinal metaplasia are still incompletely understood. Reports testing the hypothesis that apoptosis leads to atrophy have yielded conflicting results. The availability of new antibodies for the detection of apoptotic cells in tissue sections has facilitated the analysis of the role of apoptosis in the gastritis-atrophy-intestinal metaplasia sequence. METHODS: Archival material from 40 gastric resection specimens with normal mucosa (n = 5), chronic active gastritis (n = 17), or intestinal metaplasia (n = 18) was studied. Immunohistochemistry was performed using antibodies directed against cleaved cytokeratin 18 and active caspase 3. Slides were scored on a 0-3 scale for the presence of apoptotic cells. RESULTS: Normal gastric mucosa contained low numbers of apoptotic cells at the surface epithelium (mean score, 0.20). This number was significantly increased in cases with chronic gastritis (mean score, 1.06) and in those with intestinal metaplasia (mean score, 2.56). Within the intestinal metaplasia cases, 44 different foci of intestinal metaplasia were identified. In 39 of these 44 areas, concentrations of apoptotic cells were seen immediately adjacent to the foci of intestinal metaplasia, but not in the metaplastic epithelium itself. CONCLUSIONS: Apoptosis is uncommon in normal gastric mucosa. Chronic inflammation and intestinal metaplasia are associated with increased apoptosis, but occur mainly at the mucosal surface and not in the deeper layers. These findings do not support the concept that apoptosis underlies the loss of gastric glands and leads to atrophy, but the observed concentration of apoptotic epithelial cells adjacent to foci of intestinal metaplasia could be related to heterogeneity of epithelial damage, causing apoptosis, to which intestinal metaplasia is a response.     

Hydrophobic characterisation of Helicobacter (Campylobacter) pylori

Cell-surface hydrophobicity of Helicobacter (formerly Campylobacter) pylori was tested by aqueous two-phase partitioning and hydrophobic interaction chromatography. The hydrophobicity of H. pylori greatly exceeded that of Campylobacter fetus subsp. fetus, C. jejuni and Bacillus subtilis. A partition coefficient (PC) of hydrophobicity in the two-phase system was determined for H. pylori. PC was dependent on pH and the PC value was increased by greater than 20-fold at pH 2.5. Lithium cations increased PC, indicating a net negative surface charge. The presence of urea prevented the relative loss of hydrophobicity at raised pH. Exposure of H. pylori to proteolytic enzymes reduced the ability of the bacteria to adhere to human polymorphonuclear neutrophils (PMN). These findings suggest that H. pylori possesses protein-associated hydrophobic factors that are responsible for the non-opsonic adherence to PMN cell membranes.     

Urease production by Helicobacter (Campylobacter) pylori

Urease activity of 50 Helicobacter pylori (H. pylori) strains was assessed employing a photometric assay. Urea hydrolysis reached a maximum in the late log-phase and during the plateau phase of bacterial growth. The reaction time of H. pylori urease was significantly shorter than that of other urease producing bacteria (P. mirabilis, P. vulgaris, K. pneumoniae, K. oxytoca). Increasing the reaction temperature hardly led to an acceleration of the quick urea hydrolysis of H. pylori, in contrast to the situation with P. mirabilis. Acetohydroxamic acid showed a dose-dependent non-competitive suppression of urease production, whereas 9 antibiotics in subinhibitory concentrations did not influence urease production of H. pylori.     

Gastrointestinal endocrine cells in metaplasia of the gastric mucosa and duodenum

Grimelius reaction and immunohistochemical PAP method were used to study endocrine cells producing gastrin (G-cells), somatostatin (D-cells) and gamma-endorphin (GER-cells) in gastric and duodenal mucosa of 95 males with atrophic gastritis with intestinal and pyloric metaplasia. The number of cells was counted per 1 mm2 of the mucosa. In the cases of marked intestinal metaplasia the number of G-, GER- and especially D-cells in the pyloric region non-metaplastic epithelium decreases and is approaching to its number in the duodenum of the control group. In the foci of marked pyloric metaplasia of gastric corpus the number of G- and GER-cells is almost the same as in the zones of gastric metaplasia of duodenum, and is approximating their number in the pyloric region of controls, thus allowing the designation of pyloric metaplasia as a complete one.     

小儿胃黏膜肠上皮化生病理组织学特点

一、材料和方法1.材料:对1990—2004年我科及浙江大学附属儿童医院病理科共12746例胃镜活检胃黏膜组织进行回顾性研究,其中诊断胃炎伴胃黏膜肠上皮化生55例,男性23例,女性32例,年龄最小者3岁,最大16岁,平均年龄(9.1±2.8)岁。随机抽取200例经病理诊断胃炎不伴肠化患儿作为对照组。2.标本处理:分别在胃窦近幽门小弯侧3～5cm处钳取黏膜标本2块及十二指肠球部钳取黏膜标本1块。1块胃黏膜标本行快速尿素酶试验。其余经4%中性甲醛固定,常规石蜡垂直包埋3μm连续切片3张,分别用于HE染色、PAS染色和胃幽门螺杆菌(Hp)亚甲蓝法染色。取胃黏膜肠上…