Human Gastric Biopsy Specimens Research Articles

Investigating the Interaction of Helicobacter pylori with the Gastric Mucosa.

Helicobacter pylori chronically infects the gastric mucosa of humans and diseases associated with infection include gastritis, peptic ulceration, and development of gastric cancer. The organism displays a distinct tropism for the gastric mucosa of humans and for the gastric mucin MUC5AC. While the majority of organisms are found in the mucus layer overlying the epithelial cells in the stomach, adherence of the organism to the gastric epithelium is necessary for the development of disease. The interaction of H. pylori with epithelial cells results in subversion of host cell signaling and induction of an inflammatory response. Factors that influence the outcome of infection include host genetics, environmental factors, and the phenotype of the infecting strain. In this chapter, we describe cell culture assays to assess the interaction of H. pylori with epithelial cells, immunofluorescent staining to detect H. pylori in infected human gastric biopsy specimens and the use of flow cytometry to detect mucin binding to H. pylori.

Loss of RNF43 Function Contributes to Gastric Carcinogenesis by Impairing DNA Damage Response.

Background & AimsRING finger protein 43 (RNF43) is a tumor suppressor that frequently is mutated in gastric tumors. The link between RNF43 and modulation of Wingless-related integration site (WNT) signaling has not been shown clearly in the stomach. Because mutations in RNF43 are highly enriched in microsatellite-unstable gastric tumors, which show defects in DNA damage response (DDR), we investigated whether RNF43 is involved in DDR in the stomach.MethodsDDR activation and cell viability upon γ-radiation was analyzed in gastric cells where expression of RNF43 was depleted. Response to chemotherapeutic agents 5-fluorouracil and cisplatin was analyzed in gastric cancer cell lines and xenograft tumors. In addition, involvement of RNF43 in DDR activation was analyzed upon Helicobacter pylori infection in wild-type and Rnf43ΔEx8 mice. Furthermore, a cohort of human gastric biopsy specimens was analyzed for RNF43 expression and mutation status as well as for activation of DDR.ResultsRNF43 depletion conferred resistance to γ-radiation and chemotherapy by dampening the activation of DDR, thereby preventing apoptosis in gastric cells. Upon Helicobacter pylori infection, RNF43 loss of function reduced activation of DDR and apoptosis. Furthermore, RNF43 expression correlated with DDR activation in human gastric biopsy specimens, and RNF43 mutations found in gastric tumors conferred resistance to DNA damage. When exploring the molecular mechanisms behind these findings, a direct interaction between RNF43 and phosphorylated H2A histone family member X (γH2AX) was observed.ConclusionsWe identified a novel function for RNF43 in the stomach as a regulator of DDR. Loss of RNF43 function in gastric cells confers resistance to DNA damage-inducing radiotherapy and chemotherapy, suggesting RNF43 as a possible biomarker for therapy selection.

Helicobacter pylori Activates Matrix Metalloproteinase 10 in Gastric Epithelial Cells via EGFR and ERK-mediated Pathways.

Helicobacter pylori colonizes the human stomach and increases the risk for peptic ulcer disease and gastric carcinoma. H. pylori upregulates the expression and activity of several matrix metalloproteinases (MMPs) in cell lines and in the gastric mucosa. The aim of this study was to explore the mechanisms leading to upregulation of MMP10 in gastric epithelial cells induced by H. pylori Infection of gastric cells with H. pylori led to an increase in levels of MMP-10 messenger RNA, protein secretion, and activity. cagA knockout mutants or CagA phosphorylation-defective mutants failed to increase MMP10 expression. These results were confirmed in infection experiments with clinical isolates with known cagA status and in human gastric biopsy specimens. Treatment of cells with chemical inhibitors of the receptor tyrosine kinase EGFR and the kinase Src abrogated H. pylori-induced MMP10 expression. Inhibitors of ERK1/2 and JNK kinases abolished and significantly decreased H. pylori-induced MMP10 expression, respectively, whereas inhibition of the kinase p38 had no effect. Finally, inhibition of MMP10 expression by small interfering RNA led to a decrease in the gastric cell-invasive phenotype mediated by the infection. In conclusion, CagA-positive H. pylori strains stimulate MMP10 expression. MMP-10 modulation occurs via EGFR activation in a process that involves Src, ERK, and JNK pathways. MMP-10 may be implicated in H. pylori-mediated extracellular matrix remodeling.

Prevalence of Coinfection with Gastric Non-Helicobacter pylori Helicobacter (NHPH) Species in Helicobacter pylori-infected Patients Suffering from Gastric Disease in Beijing, China.

The Helicobacter heilmannii sensu lato (H. heilmannii s.l.) group consists of long, spiral-shaped bacteria naturally colonizing the stomach of animals. Moreover, bacteria belonging to this group have been observed in 0.2-6% of human gastric biopsy specimens, and associations have been made with the development of chronic gastritis, peptic ulceration, and gastric MALT lymphoma in humans. To gain insight into the prevalence of H. heilmannii s.l. infections in patients suffering from gastric disease in China, H. heilmannii s.l. species-specific PCRs were performed on DNA extracts from rapid urease test (RUT)-positive gastric biopsies from 1517 patients followed by nucleotide sequencing. At the same time, Helicobacter pylori cultivation and specific PCR was performed to assess H. pylori infection in these patients. In total, H. heilmannii s.l. infection was detected in 11.87% (178/1499) of H. pylori-positive patients. The prevalence of H. suis, H. felis, H. bizzozeronii, H. heilmannii sensu stricto (s.s.), and H. salomonis in the patients was 6.94%, 2.20%, 0.13%, 0.07%, and 2.54%, respectively. Results revealed that all patients with H. heilmannii s.l. infection were co-infected with H. pylori, and some patients were co-infected with more than two different Helicobacter species. Helicobacter heilmannii s.l. infections are fairly common in Chinese patients. This should be kept in mind when diagnosing the cause of gastric pathologies in patients. Helicobacter suis was shown to be by far the most prevalent H. heilmannii s.l.species.

Helicobacter pylori infection introduces DNA double-strand breaks in host cells.

Gastric cancer is an inflammation-related malignancy related to long-standing acute and chronic inflammation caused by infection with the human bacterial pathogen Helicobacter pylori. Inflammation can result in genomic instability. However, there are considerable data that H. pylori itself can also produce genomic instability both directly and through epigenetic pathways. Overall, the mechanisms of H. pylori-induced host genomic instabilities remain poorly understood. We used microarray screening of H. pylori-infected human gastric biopsy specimens to identify candidate genes involved in H. pylori-induced host genomic instabilities. We found upregulation of ATM expression in vivo in gastric mucosal cells infected with H. pylori. Using gastric cancer cell lines, we confirmed that the H. pylori-related activation of ATM was due to the accumulation of DNA double-strand breaks (DSBs). DSBs were observed following infection with both cag pathogenicity island (PAI)-positive and -negative strains, but the effect was more robust with cag PAI-positive strains. These results are consistent with the fact that infections with both cag PAI-positive and -negative strains are associated with gastric carcinogenesis, but the risk is higher in individuals infected with cag PAI-positive strains.

A Role for the Vacuolating Cytotoxin, VacA, in Colonization and Helicobacter pylori–Induced Metaplasia in the Stomach

Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA+ background.

Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.

Helicobacter pylori dupAIs Polymorphic, and Its Active Form Induces Proinflammatory Cytokine Secretion by Mononuclear Cells

Infection with Helicobacter pylori possessing a newly described virulence factor--duodenal ulcer-promoting gene A (dupA)--has been associated with duodenal ulceration and increased gastric inflammation. The dupA locus of 34 strains was sequenced. A panel of dupA mutants was generated and cocultured with human gastric epithelial cells and peripheral blood mononuclear cells; proinflammatory cytokine release was measured. IL8 expression was measured in human gastric biopsy specimens and related to the dupA and cagA status of infecting strains. Most H. pylori strains had a dupA allele that was longer (1884 bp; dupA1) than previously described dupA alleles, although some had truncated versions (dupA2). Unlike the best-characterized H. pylori virulence determinant, the cag pathogenicity island (cag PaI), neither dupA type induced release of interleukin (IL)-8 from gastric epithelial cells. However, infections due to dupA-positive strains were associated with higher-level mucosal IL-8 messenger RNA expression in the human stomach than were infections due to dupA-negative strains. To explain this paradox, we found that dupA1 (but not dupA2 or the cag PaI) substantially increased H. pylori-induced IL-12p40 and IL-12p70 production from CD14(+) mononuclear cells. Other T helper 1-associated cytokines were also modestly induced. We suggest that virulent H. pylori strains cause inflammation by stimulating epithelial cells through cag-encoded proteins and mononuclear inflammatory cells through dupA1 products.

The origin of non- H. pylori-related positive Giemsa staining in human gastric biopsy specimens: A prospective study

Background Upper gastrointestinal endoscopically biopsied specimens are usually sent for the diagnosis of Helicobacter pylori infection. The study aimed to determine the relationship between the origin of positive Giemsa staining and the grade of gastritis based on the updated Sydney system. Methods Gastric biopsy specimens taken at the lesser curvature and greater curvature sides of the corpus and greater curvature side of the antrum were stained with H&E, Giemsa, anti-East Asian-specific antibody and anti- H. pylori antibody stains. Pyrosequencing analysis was performed in cases that showed discrepancy between the Giemsa and anti- H. pylori antibody staining. Results Seventy-two out of 150 cases (48%) stained positive for anti- H. pylori antibody, of which 68 (94.4%) stained positive for anti-East Asian-specific antibody stain. Twelve of the 20 cases with discrepant results for Giemsa and anti- H. pylori antibody stains exhibited Campylobacter hyointestinalis infection. The grades of neutrophil activity ( p < 0.001) and chronic inflammation ( p < 0.001) were lower for Campylobacter infection than for East Asian CagA H. pylori-related infection. Conclusion C. hyointestinalis is the most common cause of non- H. pylori-related Giemsa positive infection, and is associated with lower grades of neutrophil activity and chronic inflammation than East Asian CagA H. pylori-related infection.

M1693 The Origin of Non- H. pylori-Related Positive Giemsa Staining in Human Gastric Biopsy Specimens: A Prospective Study in Comparison With East Asian cagA H. pylori Infection Based on the Updated Sydney System

M1693 The Origin of Non- H. pylori-Related Positive Giemsa Staining in Human Gastric Biopsy Specimens: A Prospective Study in Comparison With East Asian cagA H. pylori Infection Based on the Updated Sydney System

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA. Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.

Ultrastructure of Helicobacter pylori in human gastric mucosa and H. pylori-infected human gastric mucosa using transmission electron microscopy and the high-pressure freezing–freeze substitution technique

Conventional ultrastructural analyses of Helicobacter pylori and H. pylori-infected gastric mucosa by transmission electron microscopy (TEM) have limitations because of structural artifacts introduced during fixation. We used high-pressure freezing (HPF) followed by freeze substitution for TEM to investigate the ultrastructure of H. pylori and H. pylori-infected gastric mucosa. For HPF-freeze substitution, human gastric biopsy specimens were placed in the HPF instrument at a pressure of approximately 2,000 atm at the temperature of liquid nitrogen. Specimens were then transferred to an instrument for freeze substitution. Specimens were first placed in acetone containing 2% OsO4 at -85 degrees C. The temperature was increased to -3 degrees C, followed by embedding in Quetol 812. Ultrathin sections were double-stained by uranium acetate/lead nitrate. HPF and conventionally prepared samples were examined by TEM. The H. pylori envelope was clearly seen to consist of an outer membrane, periplasmic space, and plasma membrane. The periplasmic space was filled with electron-dense materials. A peptidoglycan layer was only occasionally visible. A thick, very fine filamentous or reticular fringe corresponding to the bacterial glycocalyx was seen surrounding the H. pylori cells. At the adhesion loci of H. pylori to gastric epithelial cells, H. pylori was connected to the epithelial cells by very fine, thickly arranged filaments or more closely, with a contact zone. The epithelial cells showed indentations or pedestals. The well-developed, thick bacterial glycocalyx of H. pylori appears to strongly interact with external cellular components and may play an important role in the adhesion of H. pylori to epithelial cells.

Kinetic differentiation mode chromatography using 8-quinolinol and fluorimetric detection for sensitive determination of aluminum adhering to the gastric mucosa

A highly sensitive method of kinetic differentiation (KD) mode high-performance liquid chromatography (HPLC) with fluorimetric detection was established using 8-quinolinol to measure aluminum adhering to the gastric mucosa. After sucralfate was hydrolyzed by 1 mol/l hydrochloric acid, an 8-quinolinolate–aluminum complex was produced by reacting aluminum with an 8-quinolinol solution. Then contaminants in the gastric mucosa and sucralfate were removed by liquid–liquid extraction with chloroform. Next, the 8-quinolinolate–aluminum complex was separated on a reversed-phase column that was specifically designed to detect aluminum (50×4.6-mm I.D.). Separation was done at a flow-rate of 0.8 ml/min, using BES buffer containing sodium dodecyl sulfate (pH 7.0) as the mobile phase. Fluorescence was detected at 370 nm (excitation) and 504 nm (emission). The sensitivity of this method was more than 1000 times greater than that of absorptiometry using 8-quinolinol. The detection and quantitation limits were 1.68 and 5.11 ng/ml, respectively. When tested with aluminum solutions of 10, 30, and 90 ng/ml, the intra-assay and inter-assay coefficients of variation were below 7.1%, with an error of less than 8.3%. Aluminum adhering to the gastric mucosa was determined by HPLC and absorptiometry after administration of sucralfate to rats. The HPLC method showed that aluminum levels were higher at sites of ulceration than in the normal mucosa at all times after sucralfate administration. When the values above zero obtained for absorptiometry were assessed, there was a significant correlation ( r=0.993, P<0.0001) between the aluminum concentrations measured by the two methods. This new HPLC method could be applied to the determination of aluminum in small samples, such as human gastric mucosal biopsy specimens.

Helicobacter pylori: an invading microorganism? A review

In this review we evaluate the pros and cons of Helicobacter pylori invasion of epithelial cells as part of the natural history of H. pylori infection. H. pylori is generally considered an extracellular microorganism. However, a growing body of evidence supports the controversial hypothesis that at least a subset of H. pylori microorganisms has an intracellular (intraepithelial) location. Most significant is the fact that H. pylori invades cultured epithelial cells with invasion frequencies similar to Yersinia enterocolitica and better than Shigella flexneri; furthermore, studies of invasion mechanisms suggest that H. pylori invasion of and survival within epithelial cells is not merely a passive event, but requires active participation of the microorganism. Although many studies of human gastric biopsy specimens have failed to demonstrate any intracellular H. pylori, some studies have revealed a minor fraction of H. pylori inside gastric epithelial cells, with possible linkage to peptic ulceration and epithelial cell damage. In conclusion, these data encourage further research to establish whether intracellular H. pylori does play a role in H. pylori colonization of the human stomach and in peptic ulcer pathogenesis.

Correlation between Helicobacter pylori infection and IL-18 mRNA expression in human gastric biopsy specimens.

Our data indicate that H. pylori infection is associated with active interleukin-18 production in patients with chronic gastritis. Different cell types appear to be involved in this activity and may play a role in the development of immunopathologic damage.

Molecular Evidence of Helicobacter cinaedi Organisms in Human Gastric Biopsy Specimens

One hundred twenty-six urease-negative gastric biopsy specimens were evaluated for the presence of Helicobacter genus-specific 16S ribosomal DNA (rDNA) and H. pylori-specific glmM DNA sequences by PCR. The species specificity of the glmM PCR assay was demonstrated, as H. pylori was the only Helicobacter species that yielded the expected glmM amplicon. Most urease-negative specimens (118 of 126 specimens) lacked Helicobacter DNA. However, 8 of 126 urease-negative specimens contained Helicobacter 16S rDNA. In order to identify the Helicobacter species present in urease-negative gastric biopsy specimens, 16S rDNA amplicons were cloned and sequenced. Sequence comparisons were performed by analyses of the sequences in public sequence databases. Two samples contained 16S rDNA that was identified as H. cinaedi with 100% identity and that spanned approximately 400 bp (398 and 398 bp, respectively). In contrast, multiple differences (97% identity; 390 of 398 bp) were observed with H. pylori 16S rDNA in this region. This finding was verified by sequencing an overlapping 537-bp fragment within the 5' portion of 16S rDNA. Although the clinical findings were consistent with H. pylori infection (e.g., duodenal ulcer disease), rapid urease testing and DNA sequence analyses suggested the presence of H. cinaedi organisms and the absence of H. pylori in two human antral biopsy specimens. This study represents the first report of an enteric urease-negative helicobacter in the human stomach. Although these organisms were previously associated with extragastric infections, the roles of these organisms in the pathogenesis of chronic gastritis or peptic ulcer disease remain unclear.

Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.

Since Helicobacter pylori was first cultivated from human gastric biopsy specimens in 1982, it has become apparent that many related species can often be found colonizing the mucosal surfaces of humans and other animals. These other Helicobacter species can be broadly grouped according to whether they colonize the gastric or enterohepatic niche. Gastric Helicobacter species are widely distributed in mammalian hosts and are often nearly universally prevalent. In many cases they cause an inflammatory response resembling that seen with H. pylori in humans. Although usually not pathogenic in their natural host, these organisms serve as models of human disease. Enterohepatic Helicobacter species are an equally diverse group of organisms that have been identified in the intestinal tract and the liver of humans, other mammals, and birds. In many cases they have been linked with inflammation or malignant transformation in immunocompetent hosts and with more severe clinical disease in immunocompromised humans and animals. The purpose of this review is to describe these other Helicobacter species, characterize their role in the pathogenesis of gastrointestinal and enterohepatic disease, and discuss their implications for our understanding of H. pylori infection in humans.

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells.

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky's solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry.

A virulence factor of Helicobacter pylori: role of heat shock protein in mucosal inflammation after H. pylori infection.

Among the various virulence factors of Helicobacter pylori the role of its heat shock protein 60 (HSP60, HspB) in mucosal inflammation after H. pylori infection was examined. In flow cytometric analysis, the expression of HSP60 on the cell surface was different, depending on the H. pylori strain used. The HSP60 epitope was also detected on the surface of both human gastric cancer cells (MKN45, KATOIII, and MKN28) and human gastric biopsy specimens. The intensity of the expression of HSP60 on the cell surface correlated significantly with the adhesion of H. pylori to MKN45 cells, but not with urease activity and production of vacuolating cytotoxin. A monoclonal antibody to H. pylori HSP60 inhibited the adhesion of H. pylori to MKN45 cells. These results suggest that HSP60 of H. pylori might act as an important virulence factor after H. pylori infection.

Nonopsonic activation of neutrophils and cytotoxin production by Helicobacter pylori: ulcerogenic markers.

Fifty-four clinical isolates of Helicobacter pylori from human gastric biopsy specimens were tested for cytotoxin production, as determined by intracellular vacuolization of HeLa cells, and for the induction of oxidative burst in human polymorphonuclear leukocytes (PMNLs), as measured by luminol-enhanced chemiluminescence (CL). Nonopsonized, 20 of the H. pylori strains induced a rapid and strong CL response, in contrast to 30 other strains, which showed only weak and slow responses. Another four strains gave inconclusive results. Cytotoxin production was demonstrated in 10 of the 20 strains with rapid responses but only in 3 of the 30 strains with slow and low responses (p = 0.0027, Fisher's exact test, two-tailed). Eleven of the 15 cytotoxin-producing strains (p = 0.0135) and 13 of the 20 strains with strong CL responses (p = 0.0209) were from 22 patients with peptic ulcer disease (PUD). The ability of some nonopsonized H. pylori to activate PMNLs showed co-variation with their ability to produce cytotoxin, but these two properties seem to be independent markers of PUD.