Human Gastric Biopsies Research Articles

Biological variability of human intraepithelial lymphocytes throughout the human gastrointestinal tract in health and coeliac disease.

Intraepithelial lymphocytes are the first line of defence of the human intestinal immune system. Besides, their composition is altered on patients with coeliac disease (CD), so they are considered as biomarkers with utility on their diagnose and/or monitoring. Our aim is to address their variability through the human gastrointestinal tract in health and characterized them in further depth in the coeliac duodenum. Intraepithelial lymphocytes were isolated from human gastric, duodenal, ileal and colonic biopsies, then stained with specific antibodies and acquired by flow cytometry. Our results confirmed that the profile of Intraepithelial lymphocytes change through the length of the human gastrointestinal tract. Besides and given the central role that Interleukin-15 (IL-15) elicits on CD pathogenesis; we also assessed the expression of its receptor revealing that there was virtually no functional IL-15 receptor on duodenal Intraepithelial lymphocytes. Nevertheless and contrary to our expectations, the active IL-15 receptor was not increased either on Intraepithelial lymphocytes from CD patients. IL-15 might require additional stimulus to activate intraepithelial lymphocytes. These findings may provide novel tools to aid on a CD diagnosis and/or monitoring, at the time that provide the bases to perform functional studies in order of getting a deeper insight in the specific function that Intraepithelial lymphocytes elicit on CD pathogenesis.

Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus: A pilot bioinformatics study.

Helicobacter pylori (H. pylori) infection is related to various extragastric diseases including type 2 diabetes mellitus (T2DM). However, the possible mechanisms connecting H. pylori infection and T2DM remain unknown. To explore potential molecular connections between H. pylori infection and T2DM. We extracted gene expression arrays from three online datasets (GSE60427, GSE27411 and GSE115601). Differentially expressed genes (DEGs) commonly present in patients with H. pylori infection and T2DM were identified. Hub genes were validated using human gastric biopsy samples. Correlations between hub genes and immune cell infiltration, miRNAs, and transcription factors (TFs) were further analyzed. A total of 67 DEGs were commonly presented in patients with H. pylori infection and T2DM. Five significantly upregulated hub genes, including TLR4, ITGAM, C5AR1, FCER1G, and FCGR2A, were finally identified, all of which are closely related to immune cell infiltration. The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links. TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs, the largest number of TFs among the 5 hub genes. We identified five hub genes that may have molecular connections between H. pylori infection and T2DM. This study provides new insights into the pathogenesis of H. pylori-induced onset of T2DM.

Comprehensive microbial network analysis of gastric microbiome reveal key species affecting gastric carcinogenesis

Gastric cancer continues to be one of the most significant cancers globally. Changes in microbiome composition and diversity have been implicated in gastric carcinogenesis; however, little is known about microbial community interactions and keystone species that drive these changes toward carcinogenesis. The aim of this work was to elucidate essential microbial interactions and community drivers that affect gastric carcinogenesis. We mined and utilized publicly available gastric microbiome data of adult human gastric biopsies with specific gastric diseases (gastric cancer, gastritis (chronic, superficial, atrophic), intestinal metaplasia) and healthy controls. We analysed the combined dataset using standard microbiome data analytics. Network topological properties of the various networks for gastric conditions revealed that the clustering coefficient decreased with increasing carcinogenesis. Higher modularity and lower averaged path were observed in the healthy control group as compared to the pre-cancerous and cancer groups. Sucinnate-producing and utilizing species, Bacteroides coprocola, Blautia wexlerae, and Phascolarctobacterium succinatutens, were also identified as important hubs in the precancerous conditions (gastritis and intestinal metaplasia) while nitrate-reducing bacteria Rothia mucilaginosa, Haemophilus haemolyticus, Haemophilus parainfluenzae, and Veillonella dispar were revealed as hubs in the gastric cancer group. Haemophilus parainfluenzae, which is an opportunistic oral pathogen, was identified as a potential network connector in three gastric conditions (healthy, intestinal metaplasia, and gastric cancer). Sphingobium fontiphilum and Rothia mucilaginosa were also revealed as keystone species for the control and cancer groups, respectively. The results from the clustering revealed distinct clusters in different gastric networks, indicating that microbial communities formed different functional groups during carcinogenesis. The random forest models also revealed pathogenic species Fusobacterium nucleatum be highly discriminative between healthy controls and gastric cancer groups.

The cytosolic DNA sensor AIM2 promotes Helicobacter-induced gastric pathology via the inflammasome.

Helicobacter pylori (H. pylori) infection can trigger chronic gastric inflammation perpetuated by overactivation of the innate immune system, leading to a cascade of precancerous lesions culminating in gastric cancer. However, key regulators of innate immunity that promote H. pylori-induced gastric pathology remain ill-defined. The innate immune cytosolic DNA sensor absent in melanoma 2 (AIM2) contributes to the pathogenesis of numerous autoimmune and chronic inflammatory diseases, as well as cancers including gastric cancer. We therefore investigated whether AIM2 contributed to the pathogenesis of Helicobacter-induced gastric disease. Here, we reveal that AIM2 messenger RNA and protein expression levels are elevated in H. pylori-positive versus H. pylori-negative human gastric biopsies. Similarly, chronic Helicobacter felis infection in wild-type mice augmented Aim2 gene expression levels compared with uninfected controls. Notably, gastric inflammation and hyperplasia were less severe in H. felis-infected Aim2-/- versus wild-type mice, evidenced by reductions in gastric immune cell infiltrates, mucosal thickness and proinflammatory cytokine and chemokine release. In addition, H. felis-driven proliferation and apoptosis in both gastric epithelial and immune cells were largely attenuated in Aim2-/- stomachs. These observations in Aim2-/- mouse stomachs correlated with decreased levels of inflammasome activity (caspase-1 cleavage) and the mature inflammasome effector cytokine, interleukin-1β. Taken together, this work uncovers a pathogenic role for the AIM2 inflammasome in Helicobacter-induced gastric disease, and furthers our understanding of the host immune response to a common pathogen and the complex and varying roles of AIM2 at different stages of cancerous and precancerous gastric disease.

Mass Spectrometry-Based Proteomics of Minor Species in the Bulk: Questions to Raise with Respect to the Untargeted Analysis of Viral Proteins in Human Tissue.

(1) Background: Untargeted mass spectrometry (MS)-based proteomic analysis is highly amenable to automation. Software algorithms translate raw spectral data into protein information obtained by a comparison to sequence databases. However, the technology has limitations, especially for analytes measured at the limit of detection. In a protein expression study of human gastric biopsies, the question arose whether or not it is possible, as well as sensible, to search for viral proteins in addition to those from the human host. (2) Methods: Experimental data-independent MS data were analyzed using protein sequences for oncoviruses, and BLAST analyses were performed to elucidate the level of sequence homology to host proteins. (3) Results: About one hundred viral proteins were assigned, but there was also up to 43% sequence homology to human proteins. (4) Conclusions: There are at least two reasons why the matches to viral proteins should be used with care. First, it is not plausible that large amounts of viral proteins should be present in human gastric biopsies, so the spectral quality of the peptides derived from viral proteins is likely low. As a consequence, the number of false assignments is high. Second, homologous peptides found both in human and virus proteomes contribute to matching errors. Thus, though shotgun proteomics raw data can technically be analyzed using any database, meaningful results cannot be always expected and a sanity check must be performed. Both instrumentation and bioinformatic processing in MS-based proteomics are continuously improving at lowering the limit of detection even further. Nevertheless, data output should always be controlled in order to avoid the over-interpretation of results.

Targeting Host Sulphonyl Urea Receptor 2 Can Reduce Severity of Helicobacter pylori Associated Gastritis.

While most Helicobacter pylori-infected individuals remain asymptomatic throughout their lifetime, in a significant proportion, the resulting severe chronic gastritis drives the development of gastric cancer. In this study, we examine a new therapeutic target, a host potassium channel regulatory subunit, SUR2 (encoded by ABCC9), with potential to protect against H pylori-associated diseases. SUR2 gene (ABCC9) expression in human gastric biopsies was analyzed by quantitative polymerase chain reactions. Helicobacter-infected mice were administered the SUR2-channel agonists, pinacidil and nicorandil, then gastric tissues analyzed by histology, immunohistochemistry and quantitative polymerase chain reaction, and splenic tissues by enzyme-linked immunosorbent assays. Invitro studies were performed on human and mouse macrophages, human gastric epithelial cells and mouse splenocytes. ABCC9 expression in human and mouse stomachs is downregulated with H pylori infection. Treatment of Helicobacter-infected mice with SUR2 channel modulators, pinacidil or nicorandil, significantly reduced gastritis severity. In gastric epithelial cells, nicorandil-induced opening of the SUR2 channel increased intracellular K+ and prevented H pylori-mediated Ca2+ influx and downstream pro-inflammatory signaling. SUR2 is a novel host factor that regulates Helicobacter pathogenesis. Pharmacological targeting of SUR2 provides a potential approach for reducing the severity of H pylori-associated gastritis, without eradicating infection.

A20 undermines alternative NF-\u03baB activity and expression of anti-apoptotic genes in Helicobacter pylori infection

A hallmark of infection by the pathogen Helicobacter pylori, which colonizes the human gastric epithelium, is the simultaneous activation of the classical and alternative nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, underlying inflammation and cell survival. Here, we report that the classical NF-κB target gene product A20 contributes to the negative regulation of alternative NF-κB signaling in gastric epithelial cells infected by H. pylori. Mechanistically, the de novo synthesized A20 protein interacts with tumor necrosis factor receptor-associated factor-interacting protein with forkhead-associated domain (TIFA) and thereby interferes with the association of TIFA with the NIK regulatory complex. We also show that alternative NF-κB activity contributes to the up-regulation of anti-apoptotic genes, such as baculoviral IAP repeat containing 2 (BIRC2), BIRC3 and B-cell lymphoma 2-related protein A1 (BCL2A1) in gastric epithelial cells. Furthermore, the observed over-expression of RelB in human gastric biopsies with type B gastritis and RelB-dependent suppression of apoptotic cell death emphasize an important role of the alternative NF-κB pathway in H. pylori infection.

Optical diagnosis of gastric tissue biopsies with Mueller microscopy and statistical analysis

We investigate a possibility of producing the quantitative optical metrics to characterize the evolution of gastric tissue from healthy conditions via inflammation to cancer by using Mueller microscopy of gastric biopsies, regression model and statistical analysis of the predicted images. For this purpose the unstained sections of human gastric tissue biopsies at different pathological conditions were measured with the custom-built Mueller microscope. Polynomial regression model was built using the maps of transmitted intensity, retardance, dichroism and depolarization to generate the predicted images. The statistical analysis of predicted images of gastric tissue sections with multi-curve fit suggests that Mueller microscopy combined with data regression and statistical analysis is an effective approach for quantitative assessment of the degree of inflammation in gastric tissue biopsies with a high potential in clinical applications.

Lacticaseibacillus casei Strain T21 Attenuates Clostridioides difficile Infection in a Murine Model Through Reduction of Inflammation and Gut Dysbiosis With Decreased Toxin Lethality and Enhanced Mucin Production.

Clostridioides difficile is a major cause of diarrhea in patients with antibiotic administration. Lacticaseibacillus casei T21, isolated from a human gastric biopsy, was tested in a murine C. difficile infection (CDI) model and colonic epithelial cells (Caco-2 and HT-29). Daily administration of L. casei T21 [1 × 108 colony forming units (CFU)/dose] for 4 days starting at 1 day before C. difficile challenge attenuated CDI as demonstrated by a reduction in mortality rate, weight loss, diarrhea, gut leakage, gut dysbiosis, intestinal pathology changes, and levels of pro-inflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α, macrophage inflammatory protein 2 (MIP-2), and keratinocyte chemoattractant (KC)] in the intestinal tissue and serum. Conditioned media from L. casei T21 exerted biological activities that fight against C. difficile as demonstrated in colonic epithelial cells by the following: (i) suppression of gene expression and production of IL-8, an important chemokine involved in C. difficile pathogenesis, (ii) reduction in the expression of SLC11A1 (solute carrier family 11 member 1) and HuR (human antigen R), important genes for the lethality of C. difficile toxin B, (iii) augmentation of intestinal integrity, and (iv) up-regulation of MUC2, a mucosal protective gene. These results supported the therapeutic potential of L. casei T21 for CDI and the need for further study on the intervention capabilities of CDI.

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.

APRIL-producing eosinophils are involved in gastric MALT lymphomagenesis induced by Helicobacter sp infection

The roles of the inflammatory response and production of a proliferation-inducing ligand (APRIL) cytokine in gastric mucosa-associated lymphoid tissue (MALT) lymphomagenesis induced by Helicobacter species infection are not clearly understood. We characterized the gastric mucosal inflammatory response associated with gastric MALT lymphoma (GML) and identified APRIL-producing cells in two model systems: an APRIL transgenic mouse model of GML induced by Helicobacter infection (Tg-hAPRIL) and human gastric biopsy samples from Helicobacter pylori-infected GML patients. In the mouse model, polarization of T helper 1 (tbet), T helper 2 (gata3), and regulatory T cell (foxp3) responses was evaluated by quantitative PCR. In humans, a significant increase in april gene expression was observed in GML compared to gastritis. APRIL-producing cells were eosinophilic polynuclear cells located within lymphoid infiltrates, and tumoral B lymphocytes were targeted by APRIL. Together, the results of this study demonstrate that the Treg-balanced inflammatory environment is important for gastric lymphomagenesis induced by Helicobacter species, and suggest the pro-tumorigenic potential of APRIL-producing eosinophils.

Association Between Expression of Interleukin-32 Gene and Various Helicobacter pylori Virulence Factors in Human Infected Gastric Biopsy

Helicobacter pylori (H. pylori) is a spiral bacterium that infects the human gastric mucosa. Various clinical aspects of the infection may mirror distinctive forms of cytokine expression. It correlates with immune cell penetration to the gastric mucosa with numerous cytokines production and gastric inflammation. IL-1 and IL-8 directly contribute to H. pylori effected gastritis. IL-32 is a pro-inflammatory cytokine categorized by the training of Immune cell activation, which has a vital role in human immunity. H. pylori virulence and danger factors are critical in gastritis, such as the outer inflammatory protein (OipA) and the cytotoxin associated gene a (cagA). We aimed to study the IL-32 mRNA expression in H. pylori-positive and negative patients as well as its relation with bacterial cagA, oipA, and severity of gastritis. Endoscopic biopsies were taken from the antrum of 60 H. pylori-infected patients and 62 uninfected individuals. Mucosal IL-32 mRNA expression was assessed by real-time PCR. With PCR, the H. pylori virulence factors were evaluated. Showed that the mRNA expression of IL-32 levels was significantly lower in biopsies of H. pylori-uninfected patients compared to positive individuals (P=0.01). A straight communication between virulence factor up, cagA, and heightening in IL-32 mRNA expression (P<0.001) was observed. Furthermore. IL-32 mRNA expression levels were approximately equal in both chronic and active gastritis (P=0.1). IL-32 may have a critical role in different situations like inflammation and the severity of inflammatory changes in the gastric mucosa.

NEIL2 plays a critical role in limiting inflammation and preserving genomic integrity in H. pylori‐infected gastric epithelial cells

The accumulation of Helicobacter pylori infection‐ induced DNA damage in gastric epithelial cells is a risk factor for developing gastric cancer; however, the underlying mechanisms remain poorly understood. Here we report that suppression of the DNA repair enzyme NEIL2 is one such mechanism via which H. pylori infection may fuel the accumulation of DNA damage during the initiation and progression of gastric cancers (GCs). We found that NEIL2 was significantly downregulated upon H. pylori infection both in vitro and in human gastric biopsies. The H. pylori infection‐induced downregulation of NEIL2 is specific, as Campylobacter jejuni has no such effect. Using gastric organoids isolated from the murine and human stomach in co‐culture studies with live microbes, mimicking the infected stomach lining, we found that H. pylori infection was associated with proinflammatory cytokines release; this response was more pronounced in Neil2 knock out (KO) mouse cells compared to wild type (WT) cells suggesting that NEIL2 suppresses inflammation under physiological conditions. In vivo studies in mice showed that despite a lower bacterial load, as determined by UreB expression, DNA damage was significantly higher in Neil2 KO mice compared to WT mice. Interestingly, after infection with H. pylori, Neil2 KO mice has higher inflammation and epithelial cell damage compared to WT cells. Taken together, our data suggest that downregulation of NEIL2 is a plausible way of how H. pylori infection derails DNA damage repair mechanisms and initiates GCs.Support or Funding InformationThis work was supported, in whole or in part, by National Institute of Health Grants: DK107585 and DK099275 (to SD); R01 NS073976 (to TH).

Genomic Diversity, Virulence, and Antimicrobial Resistance of <i>Helicobacter suis</i> Isolated from Human Stomachs

Background: Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases, such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. The aim of this study is to elucidate the pathogenicity of H. suis by genetic characterization and through animals infected with H. suis isolated from human stomachs. Methods: H. suis from human gastric biopsies was isolated as previously described in pigs, with modifications. Mice were orally infected with H. suis for 4 months, and then pathogenesis was analyzed. Whole-genome analysis of H. suis isolates was performed by short-read and long-read sequencing, and their genomes were subjected to comparative analysis. Findings: H. suis was successfully isolated from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings in all patients. H. suis infection in mice using clinical isolates elicited gastric and systemic inflammatory responses, and H. suis could be isolated from the stomachs of infected mice. Therefore, according to Koch’s postulates, these findings demonstrate that H. suis is a human gastric pathogen. Genomic analysis revealed high similarity between isolates from humans and pigs, suggesting possible horizonal transmission of H. suis. Interpretation: This is the first study to directly demonstrate virulence-associated features of H. suis infection using isolates obtained from human stomachs. Since H. suis would have been missed by routine diagnostic tests for Helicobacter pylori, large-scale epidemiological research on H. suis is needed to identify causal relationships with gastric diseases and develop novel diagnostic methods. Funding: MEXT/JSPS KAKENHI Grant numbers JP19H03474 for (HM) and JP20K08365 for (KT), and AMED Grant Numbers JP20fk0108148 (ER) and JP20fk0108133 (MS). Conflict of Interest: ER has received research funding from GlaxoSmithKline, outside the submitted work. All other authors declare no competing interests. Ethical Approval: Ethical approvals were obtained from all participating hospitals and the National Institute of Infectious Diseases (NIID). Written informed consent was obtained from all participants.

Molecular detection of Helicobacter pylori and clarithromycin resistance in gastric biopsies: a prospective evaluation of RIDA®GENE Helicobacter pylori assay

ABSTRACT Background: Empirical treatment of Helicobacter pylori (HP) depends on the local prevalence of clarithromycin resistance but data are lacking and culturing of HP is time-consuming. We evaluated RIDA®GENE Helicobacter pylori assay (r-biopharm), a quantitative PCR assay for detecting HP and clarithromycin resistance mutations in gastric biopsies. Material/methods: Gastric biopsies were obtained from each of 436 consecutive patients referred for gastroscopic investigation and results of qPCR were compared to culture and immunohistochemical staining (IHCS). Results: Of 436 samples, 47 were positive for HP by PCR (11%), 42 by culture (9.7%) and 44 by IHCS (10%). Compared to culture, sensitivity and specificity of the qPCR were 100% and 99%, respectively, and 96% and 99% compared to IHCS. The sensitivity and specificity for clarithromycin resistance detection were 92% and 97%, respectively. Conclusions: RIDA®GENE Helicobacter pylori assay reliably and rapidly detects HP and its resistance to clarithromycin in human gastric biopsies.

Defense against Reactive Oxygen Species during Gastric Wound Repair is Mediated by the Cystine‐Glutamate xCT Transporter in Response to Injury

Background/HypothesisCluster‐of‐differentiation gene 44 (CD44) is an alternatively spliced cell surface protein. CD44 variant isoform 9 (CD44v9) has been shown to mediate gastric repair, but the precise mechanism is unknown. Reactive Oxygen Species (ROS) driven p38/MAPK signaling leads to increased caveolin‐1, and re‐direction of β‐catenin from the nucleus to the membrane where it decreases fundamental events contributing to repair such as cellular proliferation and migration. CD44v9 stabilizes the cystine‐glutamate transporter, xCT, which provides defense against ROS. Thus, we hypothesized that CD44v9 stabilizes the xCT transporter to potentiate defense against ROS to establish an environment conducive to efficient wound repair.MethodsAcetic acid injury was induced in C57BL/6 (BL6) mice followed by vehicle, sulfasalazine (SSZ, an xCT inhibitor) or glutathione inhibitor (GSHinh). Acetic acid gastric injuries were induced in aged mice (>8 months) and animals were treated with p38 inhibitor. Mice were analyzed between days 1 to 30 post‐injury. A novel scratch‐wound assay generated from organoids derived from human stomachs and transferred to a gastric epithelial cell monolayer (huGEM) was treated with vehicle, SSZ or GSHinh.Results1) CD44v9 and xCT co‐expressing cells emerge at the ulcer margin: CD44v9/xCT co‐expressing cells emerged at the ulcer margin within 3‐days post‐injury. Compared to control mice, animals treated with SSZ or GSHinh showed impaired wound healing and loss of normal epithelial regeneration in response to injury. Western blots showed loss of activated β‐catenin, increased expression of caveolin‐1 that coincided with loss in repair in SSZ‐treated mice. FACS sorted CD44v9+ve cells from the ulcer margin expressed markers of EMT. 2) SSZ and GSHinh inhibited wound healing in huGEM scratch wound assays: HuGEM cultures exhibited a cellular composition reflective of primary gastric tissue that included the expression of parietal, chief, and mucous neck and pit cells. CD44v9/xCT co‐expressing cells emerged at the wound edge in huGEM cultures in response to a scratch wound. HuGEM cultures treated with SSZ or GSHinh exhibited significantly delayed wound closure. 3) p38 inhibitor rescues wound healing in vivo in the stomachs of aged mice: In human gastric biopsies, CD44v9, xCT, and activated β‐catenin was significantly decreased in elderly patients. Aged mice treated with p38 inhibitor displayed increased cellular proliferation, CD44v9 expression, and rescued repair when compared to aged vehicle controls.ConclusionCD44v9 and xCT emerge during gastric regeneration which may potentiate defense against ROS to allow for effective healing.Support or Funding InformationR01 DK083402‐06A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genotyping and antibiotic resistance properties of Helicobacter pylori strains isolated from human and animal gastric biopsies.

PurposeThe present study was done to assess the prevalence rate, antibiotic resistance pattern and genotyping status of the Helicobacter pylori strains isolated from human and animal gastric biopsy samples.Patients and methodsA total of 1,150 gastric biopsy samples were randomly collected from humans (children and adults) and animals (cows, sheep and goats). All samples were subjected to culture, urease test and histopathologic examination. H. pylori isolates were also confirmed using the 16S rRNA gene PCR-amplification. Antibiotic resistance pattern was assessed by the disk diffusion method. Distribution of different genotypes was studied by PCR.ResultsThe prevalence of H. pylori in gastric biopsy samples which were studied using urease test, culture and histological examination were 57.04%, 55.40% and 60.80%, respectively. Samples that were collected from adult humans (78%) and sheep (70%) had the highest prevalence of H. pylori strains, while those of goats (0.6%) and cows (4%) had the lowest. Findings of the culture method were confirmed using PCR-based amplification of 16S rRNA. Distribution of H. pylori among the gastric ulcers, duodenal ulcers, chronic gastritis gastric cancer and chronic cancer samples were 10.40%, 15.70%, 96.50%, 0.60% and 3.14%, respectively. H. pylori strains harbored the highest prevalence of resistance against ampicillin (74.4%), clarithromycin (63.4%), trimethoprim (61.5%) and metronidazole (61.5%). The most commonly detected genotypes among the H. pylori strains isolated from different types of biopsy samples were cagA (84.79%), vacA m2 (55.95%), vacA s1a (49.84%), cagE (48.58%), iceA1 (47.02%) and iceA2 (47.02%).ConclusionHigh prevalence of antibiotic resistance and virulent genotypes indicates an important public health issue. Similarities in antibiotic resistance and genotyping pattern of H. pylori strains isolated from humans and animals may show their similar routes of infection.

Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway

BackgroundHelicobacter pylori (H. pylori) delivers oncoprotein CagA into gastric epithelial cells via the T4SS and drives activation of multiple oncogenic signalling pathways. YAP, a core effector of the Hippo tumour suppressor pathway, is frequently overexpressed in human cancers, suggesting its potential tumor-promoting role. Although CagA is a casual factor in H. pylori induced gastric carcinogenesis, the link between CagA and YAP pathway has not been identified. In this work, we investigated the regulation of oncogenic YAP pathway by H. pylori CagA.MethodsExpression of YAP and E-cadherin protein in human gastric biopsies were assessed by immunohistochemistry. H. pylori PMSS1 cagA− isogenic mutant strains were generated. Gastric epithelial cells were co-cultured with H. pylori wild-type cagA+ strains or isogenic mutants and were also treated by recombinant CagA expression. Immunofluorescence was performed for YAP localization. Immunoblot and quantitative PCR were performed for examining levels of YAP, downstream effectors and markers of epithelial-mesenchymal transition. Verteporfin and siRNA silencing were used to inhibit YAP activity.ResultsYAP is significantly upregulated in human gastric carcinogenesis. We generated PMSS1 CagA isogenic mutant strains with chloramphenicol resistance successfully. Our analysis indicated that H. pylori infection induced YAP and downstream effectors in gastric epithelial cells. Importantly, knockout of CagA in 7.13 and PMSS1 strains reduced the expression of YAP by H. pylori infection. Moreover, Inhibition of YAP suppressed H. pylori infection-induced Epithelial-mesenchymal transition (EMT).ConclusionOur results indicated that H. pylori CagA as a pathogenic protein promotes oncogenic YAP pathway, which contributes to EMT and gastric tumorigenesis. This study provided a novel mechanistic insight into why cagA+H. pylori infection is associated with a higher risk for the development of gastric cancer.

Interaction between Helicobacter pylori and human gastric mucosa revisited by electron microscopy: still something new to debate?

Helicobacter pylori (Hp) is a pathogen which causes gastric diseases from gastritis to ulcer, cancer, and MALT Lymphoma. The higher grade of virulence belongs to strains harbouring CagA oncoprotein. Hp cag Pathogenicity Island encoded Type IV Secretion System (T4SS) is supposed to form needle-like structures (pili) on Hp surface that injects cagA into the host cell cytoplasm at basolateral membrane level. Other structures such as membranous appendages have also been described on Hp surface membrane. These data were obtained from cultured cells. The aim of the present, preliminary, retrospective, study was to investigate the morphological expression of T4SS also in human gastric biopsies and revisit the morphological aspects of bacteria and host interaction. We reviewed our previous morphological findings on over 300 scanning electron micrographs of 109 gastric bioptic specimens. 1) Needle-like structures (Pili) and membranous appendages have been found at the Hp gastric cell surface interface; 2) Hp polar flagella merge to apical mucous cell membrane; 3) some gastric cell microvilli became taller and projected towards bacteria; 4) in lining epithelium basal membrane holes in which bacteria penetrate are clearly visible. Hp T4SS associated structures have also been observed in human biopsies. Pili could also take nutrients from gastric mucous cells (iron); the polar flagella could anchor Hp tightly to mucous cells; the tall microvilli projected to Hp could represent defence or affinity.