Malgun (clear) cell change in Helicobacter pylori gastritis reflects epithelial genomic damage and repair

Cancers may develop in the background of genomic instability with accumulated mutations. Helicobacter pylori gastritis is characterized by acute foveolitis of the proliferative zone, which is found in any stage of the gastritis as long as the infection persists. Because acute foveolitis targets specifically the proliferative zone of pits, the proliferating epithelial cells are under severe and persistent mutagenic pressure. In H. pylori gastritis, a characteristic morphological change of epithelial cells, the malgun (clear) cell change is frequently present in association with acute foveolitis. Malgun cells have enlarged euchromatic nuclei and abundant cytoplasm. The expression of proliferating cell nuclear antigen and cytokeratin 8 are typically up-regulated in them indicating that they are mitotically and metabolically active. Here, we report evidence for DNA damage and repair in malgun cells. Significant double-strand DNA breaks were shown by the consistent terminal dUTP nick-end labeling in the nuclei of malgun cells. Proteins related to DNA damage and repair, such as Ku, poly(ADP-ribosyl) polymerase, OGG1, and MSH2 were selectively up-regulated in malgun cells. Inducible nitric oxide synthase was also up-regulated. There were occasional bcl2- and p53-expressing cells suggesting that further steps of carcinogenesis took place at the single cell level. Our results suggest that the malgun cell change represents a characteristic morphological sign of cellular genomic damage and repair, and may be implicated in an early stage of carcinogenesis. It is suggested that acute foveolitis of the proliferative zone is a major pathogenetic step of gastric carcinogenesis in H. pylori gastritis.     

Acute inflammation of the proliferative zone of gastric mucosa in Helicobacter pylori gastritis.

The neutrophilic infiltration has been regarded to represent the activity of Helicobacter pylori gastritis. It may involve the epithelium and/or lamina propria. The incidence and degree of the two types of infiltration do not correlate with each other frequently. We correlated the two types of neutrophilic infiltration with H. pylori infection and other pathologic parameters respectively in 300 randomly selected gastric biopsies as well as serial biopsies from a separate group of 95 patients who were treated for H. pylori infection. The "random biopsies" had chronic gastritis of various degrees, and the organisms were identified in 239 cases (79.7%); in the "treated group," the organisms disappeared completely in 62 cases (65.3%). Characteristically, the intraepithelial neutrophilic infiltration was predominantly localized to the proliferative zone of the gastric mucosa (zone 2) where the density of H. pylori was considerably lower than the surface epithelium. In the "random biopsies," both acute epithelial and interstitial neutrophilic infiltration correlated significantly (p < 0.01) with the H. pylori infection. In the "treated group," however, only acute epithelial inflammation correlated significantly (p < 0.01) with the eradication of infection while acute interstitial inflammation did not. Acute epithelial inflammation was no less frequently present in advanced chronic gastritis than in early chronic gastritis. Acute epithelial inflammation of the proliferative zone is a characteristic pathologic finding of H. pylori gastritis, and appears to be directly associated with the pathogenesis of H. pylori gastritis and its progression.     

Epithelial damage by Helicobacter pylori in gastric ulcers

On review of 136 consecutive biopsies of benign gastric ulcer, Helicobacter pylori was detected in 78 cases (57.3%). The gastric epithelium colonized by Helicobacter pylori showed a characteristic constellation of changes, including loss of apical mucous portion of individual cells, drop-out of epithelial cells, epithelial pits, erosions and cellular tufts, indicative of cellular injury and regeneration. Among the 58 Helicobacter-negative cases, similar changes were not observed in the ulcer edges, except for two cases which exhibited some cellular tufts. Thus, the topographic association of Helicobacter pylori with epithelial damage in the gastric ulcer edges in more than half of the cases suggests that this organism probably plays an aetiological role in ulcerogenesis, at least in these cases. Furthermore, the epithelial changes are so distinctive that they can serve as a helpful histological indicator for the presence of Helicobacter pylori in gastric biopsies.     

Studies on "end-stage" kidneys. II. Embryonal hyperplasia of Bowman''s capsular epithelium

Clusters of deeply staining "embryonal-type" epithelial cells surround hyaline glomeruli in some patients with end-stage renal disease treated with dialysis. The kidneys of 40 patients, dialyzed for 2 months to 2 years, were studied with a variety of histologic and histochemical staining techniques. In 4 of these cases such "embryonal-type" cell clusters, arising from the parietal epithelium of Bowman's capsule and here termed "embryonal hyperplasia of Bowman's capsule epithelium" (EHBCE), are identified. In 6 additional cases there are similar cells in a tubular pattern, resembling the adenomatoid or pseudotubule structures of chronic, cresentic glomerulonephritis. The largest lesions are often found in groups within localized areas of cortex, giving their distribution a "field" effect. In such cases they have the appearance of new growths, suggesting the induction of a less differentiated cell type in these diseased adult tissues.     

Light microscopic and ultrastructural evidence of in vivo phagocytosis of Helicobacter pylori by neutrophils

Helicobacter pylori is believed to cause chronic active gastritis. Infection/colonization of the gastric mucosal surface induces a mucosal inflammatory reaction in the form of lymphocytic aggregates, plasma cells and, particularly, neutrophils, which may, in turn, damage the mucosal epithelium. In vitro studies demonstrate that, in culture, the bacilli are readily phagocytosed by neutrophils, this evoking a neutrophilic oxidative burst. However, it has been claimed that neutrophils do not phagocytose H. pylori in vivo. In this study of 19 endoscopic biopsies of gastric mucosa with H. pylori-associated gastritis, Cresyl violet staining for light microscopy and electron microscopy are used to demonstrate that, in vivo, neutrophils actively phagocytose and destroy the bacilli in the epithelial intercellular space and in the mucin on the surface of the mucosa. Direct contact of neutrophils with H. pylori was observed in 17 of 17 cases by light microscopy and in 4 of 15 cases by electron microscopy. Phagocytosis by neutrophils was seen in 14 of 17 cases by light microscopy and in 3 of 1 5 cases by electron microscopy. It was most evident in the surface mucus coat where "wolf packs" of neutrophils were seen attacking the microbes. Ultrastructurally, neutrophil phagolysosomes contained both intact and partially digested bacteria, convincing evidence that the primary function of neutrophils in chronic active gastritis is to destroy H. pylori organisms. This study leaves open the question of whether, or how, neutrophils damage the gastric mucosa.     

Histopathological changes in gastric mucosa colonized by H. pylori

Helicobacter pylori (H. pylori), infection has been linked to acute and chronic gastritis, non-ulcer-dyspepsia, peptic ulcer, gastric adenocarcinoma and gastric non-Hodgkin's lymphoma of mucosa-associated lymphoid tissue (MALT). The epithelial changes in H. pylori colonized gastric mucosa are easy to recognize in routine Haematoxylin & Eosin stained sections and are so distinctive that they can serve as a helpful histological indicator for the presence of H. pylori in gastric biopsies. The histopathology of seventy-five gastric biopsies showing colonization by H. pylori was studied. Histologically, the H. pylori colonized gastric epithelium showed characteristic changes that were topographically related to the bacteria. These changes included irregular surface, epithelial pits, individual cell dropout and microerosion, which were specific for H. pylori colonization. These were absent in areas not colonized by H. pylori and in 20 consecutive H. pylori negative gastric biopsies seen during the same study period. As specific treatment for H. pylori infection is available, identification of H. pylori colonization in gastric biopsies should be attempted in all cases of gastritis, peptic ulcers and non-ulcer-dyspepsia.     

Histologic changes of the gastric mucosa associated with primary gastric lymphoma in endoscopic biopsy specimens

CONTEXT: Recently, we have observed intestinal metaplasia, atrophy, and dysplasia in the mucosa adjacent to primary gastric lymphoma (PGL) in gastrectomy specimens. OBJECTIVE: To determine the frequency and type of epithelial disorders at the histopathologic level in the mucosa adjacent to PGL in endoscopic specimens. DESIGN: We studied 54 endoscopic biopsies from patients harboring PGL. We searched for the following morphologic changes in the gastric mucosa: intestinal metaplasia; atrophy; dysplasia; epithelial erosion; and atypical regeneration of the glandular epithelium. Other nonepithelial findings such as lymphoid follicles, Helicobacter pylori, and lymphoma grade, were also recorded. For comparative purposes, 50 endoscopic biopsies with gastric adenocarcinoma and 50 biopsies with chronic gastritis associated with H pylori infection were also studied. RESULTS: The 54 biopsies included 28 (52%) low-grade and 26 (48%) high-grade PGLs. We found intestinal metaplasia in 32 biopsies (59%), atrophy in 20 biopsies (37%), dysplasia in 2 biopsies (4%), erosion of the epithelium in 33 biopsies (61%), and atypical regenerative changes of the glandular epithelium in 10 biopsies (19%). Lymphoid follicles were found in 21 biopsies (39%), and H pylori was demonstrated in 31 biopsies (57%). When groups were compared, the frequency of epithelial changes in biopsies from patients with PGL and adenocarcinoma was similar. Intestinal metaplasia or atrophy were present in only 10% of biopsies from patients with gastritis, and dysplastic glands were not identified. CONCLUSIONS: Biopsies from patients with PGL showed chronic damage of the gastric mucosa at diagnosis, including precancerous conditions. Intestinal metaplasia and atrophy were among the most frequent disorders, but dysplasia was also occasionally present. Endoscopists and pathologists must be acquainted with such changes and look for them in the initial biopsy, as well in subsequent samples. This practice is particularly important when reviewing biopsies from patients with low-grade mucosa-associated lymphoid tissue (MALT)-lymphomas who are eligible for eradication treatment for H pylori.     

"Thick" cell membranes revealed by immunocytochemical staining: a clue to the diagnosis of mesothelioma

The distinction of malignant mesothelioma from metastatic adenocarcinoma in pleural effusions and biopsies is frequently a diagnostic problem. Immunocytochemical staining of 13 malignant mesotheliomas, eight primary adenocarcinomas of the lung, five metastatic adenocarcinomas of the lung, and 20 primary adenocarcinomas in extrapulmonary sites with a monoclonal antibody to epithelial membrane antigen (EMA) revealed "thick" cell membranes in all cases of mesothelioma. This distinctive pattern of staining was seen in the periphery of cell clusters and circumferentially around individual cells in cytologic preparations, cell blocks, and tissue sections. Intracellular and intercellular acini were also outlined by anti-EMA, and long intraluminal microvillous projections were demonstrated. Weak cytoplasmic staining was only rarely seen in mesothelioma cells. This membranous staining pattern was not observed in adenocarcinomas, which displayed strong and diffuse cytoplasmic staining. The immunocytochemical demonstration of thick and spiky membranes circumferentially disposed around individual cells corresponds to aberrant microvilli, a diagnostic clue in the recognition of malignant mesothelioma.     

Solitary epithelial cells in B cell gastric MALT lymphoma

BACKGROUND: Gastric mucosa associated lymphoid tissue (MALT) lymphoma is a low grade B cell lymphoma histologically characterised by neoplastic B cells surrounding follicles in a marginal zone pattern and selectively infiltrating epithelium to form characteristic lymphoepithelial lesions. AIMS: To identify solitary epithelial cells in gastric MALT lymphoma and investigate their nature. METHODS: Anonymised endoscopic biopsies from eight B cell gastric MALT lymphomas and 10 control biopsies from chronic atrophic gastritis were selected. The numbers of solitary cytokeratin positive epithelial cells were assessed both semiquantitatively and quantitatively in immunostained sections. Chromogranin A expression was studied in sections consecutive to those stained for cytokeratin. RESULTS: Statistical analysis of the quantitative data confirmed that solitary epithelial cells were significantly more common in the lymphomas. The study of consecutive sections showed that the single cells express chromogranin A. CONCLUSIONS: The presence of solitary, cytokeratin positive epithelial cells within the neoplastic infiltrate is a characteristic feature of gastric B cell lymphoma. These solitary epithelial cells are of neuroendocrine origin.     

Unusual Helicobacter pylori in gastric resection specimens: an old friend with a new look

Immunohistochemical staining is useful in the diagnosis of Helicobacter pylori-induced gastritis. The authors encountered gastric resection specimens with an unusual pattern of reactivity on H pylori immunostains where the typical morphology of the organism was not recognizable, but the characteristic chronic gastritis associated with infection was present. The authors sought to explore this phenomenon by retrospectively reviewing and immunostaining 28 gastric resection specimens for H pylori. Six cases with large clumps of immunohistochemically positive but morphologically unrecognizable material were identified on light microscopy, corresponding on electron microscopy to clusters of predominantly coccoid H pylori, some located intracellularly. Such organisms were not identifiable without immunohistochemistry, and the phenomenon was not encountered in gastric biopsies. The authors conclude that this staining pattern reflects true H pylori infection that is not diagnosable without immunohistochemistry. Based on its occurrence only in resections, it may be the result of hypoxic or other stress induced when the mucosa is not promptly fixed.     

Helicobacter pylori infection produces expression of a secretory component in gastric mucous cells

Helicobacter pylori infection induces the expression of a secretory component (SC) in gastric epithelial cells. We investigated the cell lineage of the SC- and immunoglobulin (Ig) A-expressing epithelial cells in H. pylori-infected gastric mucosa. Materials were obtained by means of gastric biopsy from H. pylori-infected patients (24 cases) before and after the eradication of H. pylori, from five normal uninfected volunteers, and from three gastrectomy cases. Acetic acid-ethanol-fixed and paraffin-embedded specimens were examined using histochemical staining for gastric mucins (periodic acid oxidation-thionine Schiff reaction-concanavalin A-horse radish peroxidase staining) by means of immunostaining for gastric mucins (45M1 and HIK1083), intestinal cells (MUC2 and CD10), Ki67, H. pylori, SC, and IgA. The SC and IgA were not found in normal gastric mucosa. The expressions of the SC and IgA in gastric surface mucous cells and mucous neck cells in the generating zone of the gastric mucosa of H. pylori-infected patients were significantly higher before eradication of H. pylori than after the eradication. These mucous cells have the potential for SC-mediated translocation of IgA into the gastric lumen, and this may act as part of the antibacterial defense system against H. pylori infection in the gastric generating zone.     

Genotoxic and oxidative damage induced by Helicobacter pylori in Meriones unguiculatus.

Infection with Helicobacter pylori has been shown to be at the origin of various gastric pathologies. However, it has not yet been established whether the etiology of such diseases, particularly of gastric cancer, is related to the production of free radicals or to mutagenesis. The aim of this study was to determine whether a six-month infection with Helicobacter pylori increased the amount of lipid peroxidation, nitric oxide, and DNA damage in Mongolian gerbils (Meriones unguiculatus). H. pylori was characterized genotypically and administered orally to the animals. Four tests were applied to identify the presence of bacteria at one, two, four, and six months after the inoculation, namely, isolation and identification in culture, the urease test, the ELISA assay, and immunohistochemical staining of gastric biopsies. The infection was considered to be successful when three of the above-mentioned tests were positive. The infection occurred in 30% of the animals in the first month after the H. pylori inoculation and in 60-70% of the animals in the later stages. Levels of malondialdehyde, nitric oxide, and DNA damage (using the "comet" assay) were determined in the gastric tissue of the animals at one, two, four, and six months. We found statistically significant increases in malondialdehyde and nitric oxide levels from the second month on. The comet assay in animals infected with H. pylori showed a significant increase in the mean tail length throughout the observation period. We conclude that our results support the assumption that oxidative damage and DNA breakage produced by the infection with H. pylori are some of the initial alterations occurring in the development of gastric diseases.     

Carbonic anhydrase in the monkey stomach and intestine

The distribution of carbonic anhydrase in the stomach and intestine of the cynomolgus monkey (Macaca fascicularis) was studied by the histochemical method of Hansson. In the gastric surface epithelium high enzyme activity was found in the cytoplasm and at the lateral cell borders. The parietal cells in the gastric glands also showed high enzyme activity, while the chief cells were less active. The mucous cells in the pyloric glands and in the Brunner's glands demonstrated a staining pattern similar to that of gastric surface cells. The mucosa of the duodenum and the jejunum was less intensely stained than the gastric mucosa. The enzyme activity was located at the lateral cell borders of the enterocytes, with weak or no cytoplasmic activity. Goblet cells and Paneth cells were unstained. In the ileum a small number of epithelial cells displayed high enzyme activity; their identity is not clear at present. In the cecum and colon large amounts of cytoplasmic carbonic anhydrase were found in the surface epithelium and in the upper part of the glands. Capillaries showing clear enzyme staining were found in the mucosa of all tissues; they were often located close to the surface epithelium and the glands. In the stomach, cecum and colon the distribution of the enzyme in the monkey appears very similar to that reported for other mammalian species, but in the small intestine clear differences exist. The functional role of carbonic anhydrase at the various sites in the gastrointestinal tract is only partly understood.     

Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells

Helicobacter pylori infection is associated with altered gastric epithelial cell turnover. To evaluate the role of oxidative stress in cell death, gastric epithelial cells were exposed to various strains of H. pylori, inflammatory cytokines, and hydrogen peroxide in the absence or presence of antioxidant agents. Increased intracellular reactive oxygen species (ROS) were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay, and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Infection with H. pylori or exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Basal levels of ROS were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals. H. pylori strains bearing the cag pathogenicity island (PAI) induced higher levels of intracellular oxygen metabolites than isogenic cag PAI-deficient mutants. H. pylori infection and hydrogen peroxide exposure resulted in similar patterns of caspase 3 and 8 activation. Antioxidants inhibited both ROS generation and DNA fragmentation by H. pylori. These results indicate that bacterial factors and the host inflammatory response confer oxidative stress to the gastric epithelium during H. pylori infection that may lead to apoptosis.     

Alterations in the proliferating compartment of gastric mucosa during Helicobacter pylori infection: the putative role of epithelial cells expressing p27(kip1).

The proliferating zone contains stem cells that give rise to all epithelial cells of the gastric mucosa. In the present study, we investigated the turnover of gastric epithelial cells in the proliferating zone of Helicobacter pylori-infected mucosa, with or without intestinal metaplasia, before and after eradication of the microorganism. In addition, we studied the topographical distribution of the cyclin dependent kinase inhibitor p27(Kip1), which plays a critical role in cell cycle progression and differentiation programs. Twenty-eight patients (22 male), aged 32-78 years and with dyspeptic symptoms, were endoscoped, and gastric biopsies were obtained from antrum and corpus for histopathological examination and the Campylobacter-like organisms test; eradication therapy was given to infected patients, and all patients were re-endoscoped after 105 +/- 33 days (mean +/- SD). The kinetics of gastric epithelial cells and p27(Kip1) status was assessed by means of immunohistochemistry and TUNEL (Tdt-mediated dUTP-biotin nick end labeling) assay. Twenty-one (21) of 28 patients were H. pylori positive, and 7 were found H. pylori negative and served as controls. In antrum, intestinal metaplasia was detected in 7/21 (33.3%). In H. pylori gastritis, Ki67 expression was found increased in the proliferating zone, compared with normal (P =.03); analogous results were obtained with the other proliferation markers, namely retinoblastoma protein and topoisomerase IIalpha. An inverse relationship between proliferation index and atrophy was disclosed (P =.02). A reduction in the proliferation index was observed after eradication, albeit not significant. Apoptotic epithelial cells were found significantly increased (P <.01) in H. pylori gastritis, and a significant reduction was observed after eradication (P <.01). In addition, apoptotic index was found to correlate with H. pylori density. The topographical study of p27(Kip1) revealed a p27(kip1)-positive epithelial cell population that resided deep in the proliferating zone; these cells were considered to be stem cells and were found significantly increased in areas with intestinal metaplasia (P <.05); in H. pylori gastritis, there was also an increase that did not reach statistical significance. H. pylori infection induces apoptosis and increases proliferation in the proliferating zone. The increased cellular turnover, together with the increased number of putative p27(Kip1)-positive stem cells in the context of intestinal metaplasia, provides further evidence for the role of H. pylori infection in gastric carcinogenesis.     

Expression of the CD 15 antigen is a marker of cellular differentiation in cervical intra-epithelial neoplasia (CIN)

The CD 15 antigen (3-fucosyl N-acetyllactosamine), present on the outer cell membrane of cervical squamous epithelial cells, is recognized by the monoclonal antibody MC2, which is similar to several commercially available antibodies. Staining sections of cervical biopsies with MC2 clearly demonstrates the zone of supra-basal differentiated cells in the normal squamous epithelium. Staining with MC2 also demonstrates the diminished proportion of this zone occurring with grades of CIN, reflecting progressive de-differentiation of the epithelium. In immature squamous metaplastic epithelium, absence of cytoplasmic differentiation is reflected by lack of staining. As expression of the CD 15 antigen by cervical squamous cells mirrors cytoplasmic maturity and is a marker of cellular differentiation, staining colposcopic biopsies with MC2 may aid the routine histopathological grading of CIN. A comparison is made between the staining pattern observed using MC2 with that of two commercially available antibodies (Leu M1 and Dako M1), and a possible role for the CD 15 antigen in cellular adhesion in squamous mucosae is discussed.     

Adherence of Helicobacter pylori to cultured human gastric epithelial cells.

Experiments were performed to demonstrate that adherence of Helicobacter pylori to gastric epithelial cells causes alterations in the cell cytoskeleton. H. pylori intimately attached to cultured human gastric epithelial cells on small cellular projections, while there was no intimate association of H. pylori with cultured human esophageal epithelial cells. Fluorescein-conjugated phalloidin staining of gastric epithelial cells showed that H. pylori adherence stimulated actin polymerization; this stimulation was not observed with esophageal cells. Also, this organism's selectivity for gastric mucosa was supported by rare binding of bacteria to esophageal epithelial cells and gastric fibroblasts.     

A method for the isolation of human gastric mucous epithelial cells for primary cell culture: A comparison of biopsy vs surgical tissue

We have developed a method for the isolation and growth of normal human gastric mucous epithelial cells using biopsies or surgically resected tissues as the source of the cells. The attachment and growth of cells were dependent upon: (1) cell planting density, 50,000 cells/cm²; (2) extracellular matrix (fibronectin); and (3) and the use of a porous filter. In all experiments we found better cells attachment and growth of human gastric mucous cells isolated from surgical specimens compared with those gastric mucous cells isolated from gastric biopsies. The initial cell viability (as measured by Trypan-blue) was the same in both populations of gastric mucous epithelial cells isolated from either gastric biopsies or surgical specimens. After 4–5 days in culture one could detect various amounts of mucin in all the cells using either periodic acid Schiff (PAS) staining or a specific anti-mucin antibody. A similar pattern of much straining was also found in primary cultures of guinea pig gastric mucous epithelial cells. Immunohistochemical staining for chief cells (anti-pepsinogen) or parietal cells (anti-H⁺/K⁺ ATPasc) in the gastric mucous cuboidal-like epithelial cells with tight junctions, desmosomes,short microvilli, a filamentous terminal web, mucous granules, and basal lamina-like structure. We could not detect the presence of fibroblasts during the 7–9 days that the primary cells were in culture. This cell culture method will prove useful in the isolation of normal human gastric mucous epithelial cells for in vitro studies of gastric mucosal injury and repair.