Inflamed Gastric Tissues Research Articles

Helicobacter pylori senses bleach (HOCl) as a chemoattractant using a cytosolic chemoreceptor.

The gastric pathogen Helicobacter pylori requires a noncanonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here, we reconstituted a complete chemotransduction signaling complex in vitro with TlpD and the chemotaxis (Che) proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found that H. pylori is resistant to killing by millimolar concentrations of HOCl and responds to HOCl in the micromolar range by increasing its smooth-swimming behavior, leading to chemoattraction to HOCl sources. We show related protein domains from Salmonella enterica and Escherichia coli possess similar reactivity toward HOCl. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy that H. pylori uses to aid in colonizing and persisting in inflamed gastric tissue.

Recruitment of CCR6+ Foxp3+ regulatory gastric infiltrating lymphocytes in Helicobacter pylori gastritis.

Helicobacter pylori (H.pylori) infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. Increased T-cell infiltration is found at sites of H.pylori infection. The CCR6+ subset of CD4+ regulatory T cells (Tregs), a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6+ Tregs are present in H.pylori gastritis, and what their relationship is to disease prognosis, remains to be elucidated. In this study, gastric infiltrating lymphocytes were isolated from endoscopic biopsy specimens of H.pylori gastritis patients and analyzed. We found that in gastric infiltrating lymphocytes, CCR6+ CD4+ CD25high Tregs, which express high levels of CD45RO, are positively associated with more severe inflammation in gastric mucosa during H.pylori infection. Furthermore, the frequency of CCR6+ Tregs in gastric infiltrating lymphocytes, but not CCR6- Tregs, is significantly increased in inflamed gastric tissues, which is inversely correlated with significantly lower expression of IFN-γ+ CD8+ T cells. We also found that the frequency of CCR6+ Tregs is positively correlated with the frequency of CD4+ IFN-γ+ T cells. In addition, the frequency of CCR6+ Tregs, but not that of CCR6- Tregs, is significantly correlated with increased inflammation in H.pylori gastritis. This study demonstrates that immunosuppression in H.pylori gastritis might be related to the activity of CCR6+ Tregs, which could influence disease prognosis.

Hop-family Helicobacter outer membrane adhesins form a novel class of Type5-like secretion proteins with an interrupted β-barrel domain.

The human stomach pathogen Helicobacter pyloriattaches to healthy and inflamed gastric tissue through members of a paralogous family of 'Helicobacter outer membrane proteins' (Hops), including adhesins BabA, SabA, HopQ, LabA and HopZ. Hops share a conserved 25 kDa C-terminal region that is thought to form an autotransporter-like transmembrane domain. Instead, our results show that Hops contain a non-continuous transmembrane domain, composed of seven predicted β-strands at the C-terminus and one at the N-terminus. Folding and outer membrane localization of the C-terminal β-domain critically depends on a predicted transmembrane β-strand within the first 16 N-terminal residues. The N-terminus is shown to reside in the periplasm, and our crystal and small angle X-ray scattering structures for the SabA extracellular domain reveal a conserved coiled-coil stem domain that connects to transmembrane β-strand 1 and 2. Taken together, our data show that Hop adhesins represent a novel outer membrane protein topology encompassing an OmpA-like 8-stranded β-barrel that is interrupted by a 15-108 kDa domain inserted inside the first extracellular loop. The insertion of large, folded domains in an extracellular loop is unprecedented in bacterial outer membrane proteins and is expected to have important consequences on how these proteins reach the cell surface.

Novel urease-negative Helicobacter sp. 'H. enhydrae sp. nov.' isolated from inflamed gastric tissue of southern sea otters.

A total of 31 sea otters Enhydra lutris nereis found dead or moribund (and then euthanized) were necropsied in California, USA. Stomach biopsies were collected and transected with equal portions frozen or placed in formalin and analyzed histologically and screened for Helicobacter spp. in gastric tissue. Helicobacter spp. were isolated from 9 sea otters (29%); 58% (18 of 31) animals were positive for helicobacter by PCR. The Helicobacter sp. was catalase- and oxidase-positive and urease-negative. By electron microscopy, the Helicobacter sp. had lateral and polar sheathed flagella and had a slightly curved rod morphology. 16S and 23S rRNA sequence analyses of all 'H. enhydrae' isolates had similar sequences, which clustered as a novel Helicobacter sp. closely related to H. mustelae (96-97%). The genome sequence of isolate MIT 01-6242 was assembled into a single ~1.6 Mb long contig with a 40.8% G+C content. The annotated genome contained 1699 protein-coding sequences and 43 RNAs, including 65 genes homologous to known Helicobacter spp. and Campylobacter spp. virulence factors. Histological changes in the gastric tissues extended from mild cystic degeneration of gastric glands to severe mucosal erosions and ulcers. Silver stains of infected tissues demonstrated slightly curved bacterial rods at the periphery of the gastric ulcers and on the epithelial surface of glands. The underlying mucosa and submucosa were infiltrated by low numbers of neutrophils, macrophages, and lymphocytes, with occasional lymphoid aggregates and well-defined lymphoid follicles. This is the second novel Helicobacter sp., which we have named 'H. enhydrae', isolated from inflamed stomachs of mustelids, the first being H. mustelae from a ferret.

Modulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by Helicobacter pylori in immune pathogenesis of gastric mucosal damage.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphomas. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Enhanced gastric epithelial cell apoptosis during H. pylori infection was suggested to play an important role in the pathogenesis of chronic gastritis and gastric pathology. In addition to directly triggering apoptosis, H. pylori induces sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in gastric epithelial cells. Human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death-receptor signaling. The induction of TRAIL sensitivity by H. pylori is dependent upon the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex through downregulation of cellular FLICE-inhibitory protein. Moreover, H. pylori infection induces infiltration of T lymphocytes and triggers inflammation to augment apoptosis. In H. pylori infection, significant increases in CCR6+ CD3+ Tcell infiltration in the gastric mucosa was observed, and the CCR6 ligand, CCL20 chemokine, was selectively expressed in inflamed gastric tissues. These mechanisms initiate chemokine-mediated T lymphocyte trafficking into inflamed epithelium and induce mucosal injury during Helicobacter infection. This article will review recent findings on the interactions of H. pylori with host-epithelial signaling pathways and events involved in the initiation of gastric pathology, including gastric inflammation and mucosal damage.

Abstract 5366: Mutation signature of TP53 gene in H. pylori-associated inflamed gastric mucosa during gastric carcinogenesis

Abstract Helicobacter pylori (H. pylori) infection and the resultant chronic gastric inflammation is an important etiologic factor for gastric cancer development. To explore the genetic basis of gastric cancer that develops in inflamed gastric mucosa, we investigated genetic aberrations that latently accumulate in non-tumorous gastric epithelium with H. pylori infection. Whole exome sequencing on gastric cancer, non-cancerous gastritis tissues and peripheral lymphocytes of 5 patients revealed that somatic mutations accumulated in various genes in inflamed gastric tissues. Additional deep sequencing analyses of selected tumor-related genes in 34 gastritis mucosa samples from patients with, or without, gastric cancer revealed non-synonymous low-abundance mutations in TP53 in 15 cases (44.1%) and ARID1A in 5 cases (14.7%). The mutation signature of whole exome sequences in gastric cancer genomes as well as low-abundance TP53 mutations latently accumulated in H. pylori-inflamed gastritis mucosa were predominantly biased to C:G>T:A transitions in the context of GpCpX motifs, the typical footprints of cytidine deamination triggered by activation-induced cytidine deaminase (AID). In vivo studies with a human TP53 knock-in mouse model revealed that constitutive AID expression in the gastric mucosa caused the emergence of human TP53 mutations at the amino acid positions identical to those detected in human gastric cancers. These findings provide novel evidence that somatic mutations latently accumulated in various genes in inflamed gastric mucosa with H. pylori infection, and cytidine deaminase activity might provide a putative genetic basis for the increased susceptibility to gastric carcinogenesis in patients with H. pylori infection. Citation Format: Takahiro Shimizu, Hiroyuki Marusawa, Tsutomu Chiba. Mutation signature of TP53 gene in H. pylori-associated inflamed gastric mucosa during gastric carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2014-5366

Accumulation of Somatic Mutations in TP53 in Gastric Epithelium With Helicobacter pylori Infection

Helicobacter pylori infection is a risk factor for gastric cancer. To explore the genetic basis of gastric cancer that develops in inflamed gastric mucosa, we investigated genetic aberrations that latently accumulate in nontumorous gastric epithelium with H pylori infection. We performed whole-exome sequencing of gastric tumors, noncancerous tissues with gastritis, and peripheral lymphocytes from 5 patients. We performed additional deep-sequencing analyses of selected tumor-related genes using 34 gastritis mucosal samples from patients withor without gastric cancer. We also performed deep sequencing analyses of gastric mucosal tissues from mice that express transgenic human TP53 and constitutively express activation-induced cytidine deaminase (AICDA or AID) (human TP53 knock-in/AID-transgenic mice). Whole-exome sequencing revealed that somatic mutations accumulated in various genes in inflamed gastric tissues. Additional deep-sequencing analyses of tissues from regions of gastritis confirmed nonsynonymous low-abundance mutations in TP53 in 15 cases (44.1%) and ARID1A in 5 cases (14.7%). The mutations that accumulated in gastric mucosal tissues with H pylori-induced gastritis, as well as gastric tumors, were predominantly C:G>T:A transitions in GpCpX motifs-a marker of cytidine deamination induced by AID. Constitutive expression of AID in the gastric mucosa of mice led to mutations in the human TP53, at amino acid coding positions identical to those detected in human gastric cancers. Studies of gastric tumors and tissues from humans and mice indicate that somatic mutations accumulate in various genes in gastric mucosal tissues with H pylori infection. Increased cytidine deaminase activity in these tissues appears to promote the accumulation of these mutations and might promote gastric carcinogenesis in patients with H pylori infection.

Regulation of Helicobacter pylori adherence by gene conversion

Genetic diversification of Helicobacter pylori adhesin genes may allow adaptation of adherence properties to facilitate persistence despite host defences. The sabA gene encodes an adhesin that binds sialyl-Lewis antigens on inflamed gastric tissue. We found variability in the copy number and locus of the sabA gene and the closely related sabB and omp27 genes due to gene conversion among 51 North American paediatric H. pylori strains. We determined that sabB to sabA gene conversion is predominantly the result of intra-genomic recombination and RecA, RecG and AddA influence the rate at which it occurs. Although all clinical strains had at least one sabA gene copy, sabA and sabB were lost due to gene conversion at similar rates in vitro, suggesting host selection to maintain the sabA gene. sabA gene duplication resulted in increased SabA protein production and increased adherence to sialyl-Lewis antigens and mouse gastric tissue. In conclusion, gene conversion is a mechanism for H. pylori to regulate sabA expression level and adherence.

Gastric Mucosal Calcinosis in a Patient with Abdominal Pain

Purpose: Background: Calcification of the gastric mucosa (gastric mucosal calcinosis) is a rare condition and is usually found incidentally on upper endoscopy. It is most frequently associated with conditions which cause hyperphosphatemia and hypercalcemia. In patients with chronic renal disease, it may be representative of impending systemic calcinosis. We herein report a case of gastric mucosal calcinosis in an otherwise healthy patient with abdominal pain. To our knowledge, such a presentation has not been described. Case Description: A 33 year-old female with no significant past medical history was referred for outpatient evaluation of worsening post-prandial epigastric pain of several months duration. She had intermittent headaches for which she used intermittent Excedrin®. Her only other medication was an oral contraceptive pill daily; no calcium-containing products. Physical Examination was notable for minimal epigastric tenderness but otherwise was unremarkable. Recent hepatic panel and serum chemistries (including calcium and phosphate) were normal. Upper endoscopy was significant for diffuse linear whitish plaques in the gastric antrum which did not wash off. Biopsy of these areas revealed gastric mucosal calcification and marked reactive changes. No Helicobacter pylori, inflammation, or metaplasia were seen. Stains for Congo red and PAS were additionally negative. Discussion: Gastric mucosal calcinosis is an extremely uncommon condition with a reported incidence of less than 0.1%. It has been described in association with chronic renal disease, hyperparathyroidism, gastric cancer, solid organ transplant recipients, and aluminum-containing antacids and sucralfate. Gastric mucosal calcinosis has been classified as metastatic, dystrophic, or idiopathic. In metastatic calcification, elevated serum calcium and phosphate lead to calcium salt deposition in normal gastric mucosa. Other organs including the lungs, kidneys and heart can also be affected. Multiple myeloma, tumor lysis syndrome, hyperparathyroidism, end-stage renal disease, and hypervitaminosis D are associated with this pattern. In dystrophic calcification, serum electrolytes are normal and calcium salt deposition occurs in damaged or inflamed gastric tissue. Peptic ulcer disease and malignancy are examples of associated conditions. The precise significance of gastric mucosal calcinosis in association with other diseases is unclear. Our patient was placed on omeprazole 20 mg daily and reported improved symptoms at follow-up.

Interplay between Helicobacter pylori and immune cells in immune pathogenesis of gastric inflammation and mucosal pathology

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. Recent studies have shown that apoptosis of gastric epithelial cells is increased during H. pylori infection. Apoptosis induced by microbial infections are factors implicated in the pathogenesis of H. pylori infection. The enhanced gastric epithelial cell apoptosis in H. pylori infection has been suggested to play an important role in the pathogenesis of chronic gastritis and gastric pathology. In addition to directly triggering apoptosis, H. pylori induces sensitivity to tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in gastric epithelial cells via modulation of TRAIL apoptosis signaling. Moreover, H. pylori infection induces infiltration of T lymphocytes and triggers inflammation to augment apoptosis. In H. pylori infection, there was significantly increased CCR6(+)CD3(+ )T-cell infiltration in the gastric mucosa, and the CCR6 ligand, CCL20 chemokine, was selectively expressed in inflamed gastric tissues. These results implicate that the interaction between CCL20 and CCR6 may play a role in recruiting T cells to the sites of inflammation in the gastric mucosa during Helicobacter infection. Through these mechanisms, chemokine-mediated T lymphocyte trafficking into inflamed epithelium is initiated and the mucosal injury in Helicobacter infection is induced. This article will review the recent novel findings on the interactions of H. pylori with diverse host epithelial signaling pathways and events involved in the initiation of gastric pathology, including gastric inflammation, mucosal damage and development of MALT lymphomas.

Helicobacter pylori colonization of the human gastric epithelium: A bug's first step is a novel target for us

After Helicobacter pylori enters the stomach, three steps are vital for infection: (i) establishing colonization; (ii) evading host immunity; and (iii) invading gastric mucosa; the last step is what is associated with diverse outcomes. Urease activity and motility mediated by the flagella of H. pylori are important in harboring colonies beneath the gastric mucus in niches adjacent to the epithelium. Several putative adhesins attach the organism to the gastric epithelium and prompt the succeeding processes for evading host immunity and invading the mucosa. Successful colonization is thus the leading and critical step. From another point of view, this can be a novel target to control this common and important infection. This review summarizes the putative adhesins that influence the evasion of host immunity, and how these could determine different clinico-pathologic outcomes. The putative adhesins include the interplay between bacterial and host Lewis antigens (type I: Le(a) and Le(b); type II: Le(x) and Le(y)), the dominant pathway between BabA and Le(b), the SabA adhesin binding to sialylated Le(x) that is upregulated in inflamed gastric tissue or those with weak-Le(b), the CagL apparatus to adapt with the alpha5beta1 integrin to mediate a type IV secretory system for CagA translocation into the epithelium; and other outer membrane proteins as HopZ, AlpA/AlpB, or OipA, without known corresponding receptors. This review implicates the adhesins vital for bugs that could be alternatively provided as novel targets for us to overcome the colonization.

Chronische Entzündungen – Rolle am Tumorgeschehen

Die Erkenntnis, dass ein Zusammenhang zwischen chronischen Entzündungen und einem erhöhten Krebsrisiko besteht, wurde bereits vor mehr als 150 Jahren vom Berliner Mediziner Rudolph Virchow beschrieben. Heute wird vermutet, dass ca. 10-15 % aller humanen Krebserkrankungen auf chronische Entzündungsprozesse zurückzuführen sind, wobei die Ursachen sowohl auf humanpathogene Keime (z. B. Helicobacter pylori) als auch auf anorganische/organische Substanzen/Faktoren (Asbestfasern, Magensäure, Zigarettenrauch) zurückgeführt werden können. Chronisch entzündetes Gewebe stellt ein potenziell transformierendes Milieu dar, da es reich an Wachstumsfaktoren, Zytokinen und Chemokinen ist, welche die proliferatorische Aktivität von Zielzellen triggern, sowie an radikalischen Sauerstoff-/Stickstoffverbindungen, die bei hoher Konzentration Schädigungen der DNS bewirken. Durch die Faktor vermittelte bzw. inhärente Zellteilungsaktivität der Zielzellen, kombiniert mit mutagen wirkenden radikalischen Sauerstoff-/Stickstoffverbindungen, kann es zur Akkumulation von chromosomalen Aberrationen und nachfolgend zur malignen Transformation der Zielzellen kommen. Entgegen der alten Lehrmeinung, dass Krebs seinen Ursprung in somatischen Zellen hat, ist heute bekannt, dass die Ursprungszellen für maligne Transformationen Stammzellen (Gewebestammzellen bzw. rekrutierte Knochenmarkstammzellen) bzw. deren Progenitorzellen sind, aus denen die tumorinitiierenden Krebsstammzellen hervorgehen. So konnte z. B. anhand eines etablierten Mausmodells zur Analyse des durch H. felis induzierten Magenadenokarzinoms gezeigt werden, dass das neoplastische Gewebe aus rekrutierten Knochenmarkstammzellen hervorgegangen ist. Neben ihrer Rolle in der Karzinogenese sind chronische Entzündungsreaktionen darüber hinaus maßgeblich an der Progression von Tumorerkrankungen beteiligt. Fatal hierbei ist die symbiotische Beziehung zwischen tumorassoziierten Makrophagen (TAM) und Tumorzellen. TAM sind an sämtlichen Prozessen beteiligt, die eine erhöhte Malignität der Tumorerkrankung nach sich ziehen. Hierzu zählen die Neoangiogenese, die Proliferation sowie die Invasion und Metastasierung von Tumorzellen wie auch die Suppression von zytotoxischen T-Zellen.

Upregulation of CCL20 and Recruitment of CCR6+Gastric Infiltrating Lymphocytes inHelicobacter pyloriGastritis

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. There is increased T-cell infiltration at the site of infection with H. pylori. CCR6, a specific beta-chemokine receptor for CCL20 (MIP-3alpha/LARC/exodus), has recently been reported to mediate lymphocyte homeostasis and immune responses in mucosal tissue, and it may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation. In this study, we investigated the role of CCR6 and its ligand, CCL20, in inducing an inflammatory response in the gastric mucosa during H. pylori infection. Gastric infiltrating T lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed for the expression of the CCR6 chemokine receptor. Our results demonstrated that there was significantly increased CCR6 expression in CD3(+) T cells infiltrating the gastric mucosa, and the CCR6 ligand, the CCL20 chemokine, was selectively expressed in inflamed gastric tissues. The production of CCL20 was upregulated in response to H. pylori in gastric epithelial cells when there was stimulation by the proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha. Furthermore, recombinant CCL20 induced lymphocyte chemotaxis migration in fresh gastric T cells ex vivo, indicating that the gastric T cells could migrate toward inflammatory sites via CCR6/CCL20 interaction. Our results suggest that the interaction between CCL20 and CCR6 may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation in Helicobacter infection.

Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism

Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-kappaB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.