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Reduction of A-type Potassium Currents in Dorsal Root Ganglion Neurons ina Rat Model of Functional DyspepsiaShiying Li, Jiande Chen

Background and aims: Although without evidence of organic structural abnormalities, painor discomfort is one of prominent symptoms of functional dyspepsia (FD) and is consideredto reflect visceral hypersensitivity. Abnormal hyper-excitability of primary sensory neuronsplays an important role in neuropathic pain. A-type potassium channels (KA) are criticalfor neuronal excitability and responsible for the resting membrane potential and the repolar-ization and frequency of the action potential. The aim of this study was to investigate possiblechanges in KA channels in dorsal root ganglion (DRG) neurons in a rat model of functionaldyspepsia induced by neonatal gastric irritation. Materials and Methods: Male Sprague-Dawley rat pups at 10-day old received 0.1% iodoacetamide (IA) or vehicle by oral gavagefor 6 days (Gastroenterology 2008;134:2070). At 8 weeks, the rat stomach was surgicallyexposed and a retrograde tracer DiI was injected into 10 sites in the gastric wall. T8-T12dorsal root ganglia (DRG) cells were harvested for the KA current recording using wholecell current and voltage clamp technique. Results: 1) Gastric specific DRG neurons showedan enhanced excitability in IA-treated rats compared to the control with a depolarized restingmembrane potential of 52.8±3.8 mV (vs. 56.2±2.8 mV in control, p<0.05), and a lowercurrent threshold for action potential activation (295±48 Vs186±40pA in control, p<0.05).Fifteen percent of DRGneurons in the IA-treated rats generated spontaneous action potentials.2) The transient KA current was pharmacologically separated from delayed rectify potassiumchannel current based on their different sensitivities to external Tetraethylammonium chloride(TEA). The current density of TEA insensitive KA current was significantly smaller in IA-treated rats (56.8±7.2 Vs 87.6±10.4 pA/pF in control, p<0.05) whereas the cell capacitance,and the steady state activation and inactivation of KA current remained unchanged in IA-treated rats. 3) 4-Aminopyridine, a KA channel specific blocker, significantly depolarizedthe resting membrane potential, lowered the current threshold and changed the phasic firingpattern to the tonic firing pattern in control rats compared to that in IA-treated rats. The4-Aminopyridine sensitive current in IA-treated rats was significantly lower than the control.Conclusions: These data suggested that A-type potassium currents are significantly reducedin the gastric-specific DRG neurons in adult rats with mild neonatal gastric irritation, whichin part contribute to the enhanced DRG neuron excitabilities that leads to the developmentof gastric hyperalgesia.

773

Expression of DNA Damage Response Genes, Gastric Cancer, andAngiodysplasia in Non Human PrimatesHui Liu, Cristina Semino-Mora, Matthew Goldman, Eric Lombardini, D. Scott Merrell,Andre Dubois

H. pylori plays an important role in the development of gastric cancer. We demonstratedthat H. pylori infection plus the dietary nitrosamine carcinogen ENNG synergistically inducedgastric neoplasia and dendritic vascular lesions (Liu, Gastro 2009;137:1367-79). We hypo-thesize that disease occurs due to the combined mutagenic effects of H. pylori and ENNGon the DNA Damage Response Genes with resulting disruption of the dominant checkpointto tight cell cycle control and replication of damaged DNA. Goals: To determine the relationbetween endoscopic and pathologic observations and the expression of the DNA damageresponse genes ATM, MDM2, and p53 as well as the p21 and VEGF-A genes during 8 years

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of observation of Rhesus macaques after H. pylori infection and/or ENNG administration.Methods: Gastritis, cancerous lesions and telangiectasia were scored on a 0 to 3 scale byreview of digital chromogastroscopies performed in 23 H. pylori-negative macaques assignedto either controls (C); ENNG alone (E); inoculation with USU101 H. pylori strain (H), orUSU101 inoculation plus ENNG (EH). Biopsies fixed for histopathology and immunohisto-chemistry (IHC) or flash-frozen for subsequent extraction of RNA were analyzed. Rhesusmacaque microarray, real-time qRT-PCR and in situ hybridization (ISH) [for RNA transcriptexpression] and Western blot analysis and IHC [for protein expression] were determinedat regular intervals. Results: Gastric mucosal neoplastic transformation was present only inEH group and endoscopic telangectasia scores increased only in the E and EH groups startingat 5 years. Microarray analysis demonstrated significant increases in MDM2 expression inE and EH relative to C. These changes were also more pronounced in comparison to theH group (table; *p<0.01). qRT-PCR of RNA from biopsies at 5 years and later confirmedthese changes and showed a significant (p <0.03) increase in MDM2 expression in E andEH groups. IHC demonstrated significant (p <0.01) increases in protein expression of ATM,MDM2, and in tumor protein 53 (TP53), which is an oncogene. In the EH group, theseincreases were localized to the cytoplasm of inflammatory cells of the lamina propria andwithin gastric glands and also in invasive neoplastic cells. ATM, MDM2, and VEGF werealso expressed in the cytoplasm of endothelial cells in E and EH groups. IHC in controlsand early biopsies in H, E, and EH groups was negative.Conclusions: DNA damage responsegene regulation is altered during long-term progression to cancer in an H. pylori infection plusdietary carcinogen human-like model. Normal ATM-induced activation of the checkpoint thatleads to cell cycle arrest appears to be disrupted, thus leading to replication of mutatedgenes and uncontrolled increased mitosis and neoangiogenesis. A better understanding ofthese changes will have important therapeutic implications.

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Helicobacter pylori Induces CagA and Slt-Dependent Gastric Epithelial CellMicroRNAs That Target H,K-ATPase α Subunit 3' Untranslated RegionCharles E. Hammond, Melissa Eustace, Adam J. Smolka

H. pylori infection of human gastric epithelial cells (GEC) represses H,K-ATPase α subunit(HKα) gene transcription through NF-κB p50 homodimer binding to HKα promoter (AmJ Physiol, 294:G795, 2008). The bacterial cagA and slt gene products have been implicatedin HKα repression (Gut, 59:874, 2010), which facilitates gastric H. pylori colonization andmay underlie transient clinical hypochlorhydria. We hypothesized that H. pylori also regulatesH,K-ATPase expression post-transcriptionally by miRNA interaction with 3' untranslatedregion (UTR) HKα mRNA, blocking transcript translation. We amplified the HKα 3' UTRfrom human gastric biopsy cDNA with PCR primers incorporating XbaI restriction sites.The amplicon was ligated into the XbaI site of a pGL3-Luc Control vector, placing the HKα3' UTR downstream of the Luc open reading frame. Sequence and orientation of the ligatedamplicon were verified by DNA sequencing. AGS cells were transfected with HKα 3' UTR-Luc reporter construct and 24 h later infected (MOI=50, 6 h) with strain 7.13 H. pylori.Luciferase activity in cell lysates was measured by luminometry. Compared to pGL3-Lucvector alone, the HKα 3' UTR repressed Luc activity by 70%, and H. pylori infection repressedLuc activity another 19%, consistent with GEC miRNA(s) binding to HKα 3' UTR and up-regulation of an miRNA subset by H. pylori. HKα 3' UTR-Luc reporter repression was moresignificant with H. pylori MOIs of 100, allowing us to question the role of cagA and solublelytic transglycosylase (slt) genes in this repression. Wild-type H. pylori infection repressedHKα 3' UTR-Luc reporter activity by 80%, while cagA or slt-deficient isogenic mutantinfections repressed activity by only 50% and 58% respectively. In silico analysis (TargetScan-Human 5.2) of HKα 3' UTR identified miR-1289 as a highly-conserved potential HKα-regulatory miRNA. We measured AGS cell content of miR-1289 in response to wild-typeH. pylori infection (6 hr, MOI=100) using a custom primer/probe set (SABiosciences) andqRT-PCR; infection caused a 3.6-fold up-regulation of miR-1289 levels. Lastly, AGS cellstransfected with the human HKα 3' UTR-Luc construct were transfected 24 h later with a23-nucleotide oligoribonucleotide mimic of miR-1289. HKα 3' UTR activity was maximallyrepressed (54%) within 30 min of miR-1289 transfection; UTR activity was unchanged bynon-targeting siRNA transfection. Taken together, these data indicate that H. pylori infectionup-regulates GEC miRNAs that target HKα 3' UTR and block translation, and that CagAand Slt activity are implicated in miRNA up-regulation. The sensitivity of HKα mRNA 3'UTR to binding of GEC miRNAs, some of which are up-regulated by H. pylori, suggests anovel regulatory mechanism of gastric acid secretion, and potentially offers new insightsinto molecular mechanisms underlying H. pylori-induced hypochlorhydria.

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Deacetylation of Helicobacter pylori Peptidoglycan Attenuates NOD1Activation and Promotes Colonization of the StomachGiovanni Suarez, Judith Romero-Gallo, M. Blanca Piazuelo, Dawn Israel, Barbara G.Schneider, Pelayo Correa, Richard M. Peek

H. pylori colonizes the human stomach for decades and one consequence of microbial-induced gastritis is gastric adenocarcinoma. A cancer-linked H. pylori locus is the cag patho-genicity island (cagPAI), which translocates peptidoglycan (PGN) and CagA into host cells.Nod1 is an intracellular innate immune receptor that senses PGN derived from Gram-negative bacteria and responds by activating NF-κB, resulting in inflammation-mediatedbacterial clearance; however chronic pathogens can evade Nod1-mediated clearance byaltering PGN structure. We previously demonstrated that a rodent-adapted derivative (7.13)of a human H. pylori cagPAI+ strain (B128) rapidly induces gastric cancer in Mongoliangerbils. Using 2D-DIGE and mass spectrometry, we subsequently identified a peptidoglycan

deacetylase, PgdA, which exists as different isoforms in strain 7.13 vs. B128; therefore, wesought to define the role of PgdA in Nod1-dependent activation of NF-κB and inflammationthat develops in response to H. pylori. Expression of pgdA in strain 7.13 was dependentupon bacterial density, and peaked during late exponential growth phase. Co-culture ofwild-type (WT) strain 7.13 or its △pgdA isogenic mutant with AGS gastric epithelial cells,or HEK293T epithelial cells expressing a NF-κB reporter, revealed that pdgA inactivationreciprocally increased NF-κB activation (AGS: △pgdA 1.5 fold over WT; HEK293T: △pgdA1.3 fold over WT; p<0.01), indicating that deacetylation of PGN by PgdA attenuates Nod1activation. We confirmed these results using a gerbil model of acute infection. Colonizationefficiency for gerbils challenged with the 7.13 △pgdA mutant was 50% compared to 100%for the WT strain 2 weeks post-inoculation. Animals infected with the mutant strain hadsignificantly lower colonization densities (1.5-2 log CFU lower) but more severe inflammationcompared with animals infected with the WT H. pylori strain. We then extended thesestudies into a unique collection of H. pylori clinical strains harvested from persons residingin areas with high or low rates of gastric cancer in Colombia. H. pylori strains inducedvarying levels of NF-κB activation; however, when histopathology scores and levels of NF-κB activation induced by the corresponding H. pylori strain were examined, there was asignificant relationship between the severity of gastritis and levels of NF-κB activation amongthe low risk (p<0.0001), but not the high risk strains (p=0.1957), indicating that NF-κBactivation and inflammation are uncoupled among H. pylori-infected persons at high riskfor gastric cancer. Collectively, these results suggest that PGN deacetylation may play animportant role in modulating the host inflammatory response to H. pylori, allowing thebacteria to persist long term in the gastric niche.

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Epidermal Growth Factor Receptor (EGFR) Activation by Helicobacter pylori isMediated by cag-Dependent and Independent MechanismsJohanna C. Sierra, Fang Yan, Judith Romero-Gallo, M. Blanca Piazuelo, RupeshChaturvedi, Keith T. Wilson, Richard M. Peek, D. Brent Polk

Helicobacter pylori colonizes human gastric mucosa and induces an inflammatory responsethat can progress to gastric atrophy, intestinalmetaplasia, dysplasia and ultimately adenocarci-noma. EGFR signaling has been implicated in carcinogenesis and we previously demonstratedthat H. pylori transactivates EGFR in gastric epithelial cells, which inhibits H. pylori-inducedapoptosis. Since infection with H. pylori strains containing the cag pathogenicity island (cagPAI), encoding a type IV secretion system (T4SS), has been associated with increasedproliferation without a concomitant increase in apoptosis, we investigated the role of bacterialconstituents associated with the T4SS in EGFR activation. Gastric epithelial cells were infectedwith the H. pylori cag+ wild-type strain PMSS1 or its △cagE isogenic mutant at varyingtime-points and phosphorylation status of EGFR was assessed by Western blot analysis.Wild-type H. pylori activated EGFR in a biphasic manner characterized by a transient increasein EGFR phosphorylation as early as 30 minutes post infection (1.8-fold increase overuninfected) followed by a second activation peak 120 min (2.8-fold increase) after infection.In contrast, EGFR activation was delayed until 2 hours (2.2-fold increase) post-infectionwith the △cagE mutant, yet activation was sustained up to 4 hours (2.2-fold increase)following infection. Based on these results, we examined EGFR activation in a mouse modelof infection by using C57Bl/6 WT and EGFRwa5 mice that express a dominant negativeEGFR. WT and EGFRwa5 mice were infected with wild-type strain PMSS1 or its △cagEmutant for 4 weeks. H. pylori colonization levels were increased in both WT (p<0.001) andEGFRwa5 (p<0.05) mice infected with the △cagE mutant compared to mice infected withwild-type PMSS1. In Vivo-adapted wild-type H. pylori colonies recovered from infectedanimals retained the ability to translocate CagA into gastric epithelial cells in an In Vitroassay. Inflammation and proliferation were significantly increased in WT (inflammationp<0.01, proliferation p<0.05) and EGFRwa5 (inflammation p<0.01, proliferation p<0.05)mice infected with H. pylori wild-type strain PMSS1 compared to uninfected controls, andamong WT mice, the H. pylori wild-type strain induced more inflammation than the △cagEmutant (p<0.001). However, increased levels of inflammation (p<0.05) and cell proliferation(p<0.05) developed in EGFRwa5 mice infected with the △cagE mutant H. pylori straincompared to WT mice infected with the △cagE mutant. These results suggest that theinability of the △cagE mutant to induce inflammation in WT mice is dependent upon afunctional EGFR signaling axis. These data also demonstrate that, in addition to cag PAI-encoded virulence factors, there is also a cag PAI independent mechanism of EGFR activationthat develops in response to H. pylori.

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Identification of Dominant Immunoregulatory Sequences in H. pylori GenomeWhich Exert Anti-Inflammatory Effects on Intestinal Inflammation in Mice:Therapeutic Implications in Chronic Inflammatory DiseasesStephanie Y. Owyang, Christopher C. Owyang, Min Zhang, Yundong Sun, John Y. Kao

Background: Recently we showed that HP DNA has the ability to downregulate the productionof proinflammatory cytokines by bone marrow-derived dentritic cells (BMDCs) In Vitro(Luther et al, Gut 2011). The aim of this study is to identify the dominant immunoregulatoryoligodeoxynucleotides (ODNs) in the HP genome and test their ability to suppress pro-inflammatory cytokines (IL-12 and IL-6) secreted by BMDCs. In addition, we examined theIn Vivo effects of HP-ODNs on reducing intestinal inflammation using a water avoidancestress (WAS) mouse model. Methods: omplete genomes for six different strains of HP (G27,Shi470, HPAG1, P12, 26695, and J99) were obtained from the NCBI website and frequenciesof all 256 octomers with the ending in GGGG (G-rich sequences) were calculated usingMacVector. Results: Among these, three specific G-rich octomers stood out (TTTAGGGG[U1], AAAAGGGG [U2], and TTTTGGGG [U3]), occurring more than 15-fold over theexpected frequency in all HP genomes. The high occurrence of G-rich octomers was observedacross all strains of HP, but not found in other gram-negative bacteria genomes, indicatingthis observed distribution is unique to HP. We next examined the ability of U1, U2, andU3 to inhibit IL-12 release from BMDCs obtained fromC57B/6mice. The immunostimulatorysequence (ISS), TGACGTTC (P1) (5ug/mL) caused a 62±8 fold increase above baselineproduction of IL-12. Addition of U1, U2, and U3 reduced IL-12 production to 40±9, 38±5,

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and 20+/-8 fold increase above baseline, respectively (P<0.05), indicating that these may bepotent immunoregulatory sequences (IRS). To investigate the In Vivo significance of our InVitro observations, we examined the In Vivo effects of U3 on intestinal inflammation usinga WAS mouse model. WAS 1h per day for 10 days induced small intestinal inflammationcharacterized patchy crypt disruption and lymphocytic infiltration in the lamina propria ofthe ileum. This was accompanied by an increase in stool frequency from 15±2 pellets/dayto 42±5 pellets per day (P<0.05). Ileum obtained fromWAS mice secreted a 3-4 fold increasefrom baseline in IL-12 and IL-6. These proinflammatory cytokines were normalized in micetreated with U3 octomer (50ug via gavage every other day). Conclusion: We have identifieda number of previously unrecognized IRS with a high occurrence across the HP genomeand showed that they are able to suppress IL-12 and IL-6 inflammatory response fromBMDC. Our In Vivo studies demonstrated the feasibility of delivering one such octomer inrodents to reduce intestinal inflammation. These findings may have therapeutic significanceas this method may be exploited in the future to treat patients with chronic inflammatorydisorders. * Recipients of AGA-Broad Foundation Student Research Fellowship Award

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Attenuation of the Macrophage Inflammatory Response to Helicobacter pyloriis Mediated by p38 MAPK-Dependent Induction of Heme Oxygenase-1Alain P. Gobert, Thomas G. Verriere, Thibaut de Sablet, Richard M. Peek, RupeshChaturvedi, Keith T. Wilson

Background: Myeloid cells recruited to the gastric mucosa during Helicobacter pylori (Hp)infection express numerous mediators of the innate immune response, including TNF-α,iNOS, and IL-10, which have been directly implicated in modulation of defense against Hpleading to persistent inflammation within the gastric mucosa. Therefore, dissecting thecrosstalk that exists between Hp and macrophages is essential for understanding Hp-induceddiseases. Heme oxygenase-1 (HO-1) is an inducible enzyme that possesses numerous anti-inflammatory properties. We have recently shown that Hp inhibits HO-1 expression ingastric epithelial cells. Thus, our aim was to analyze the effect of Hp on the expression ofHO-1 in macrophages and its role in the innate immune response. Methods: We used themacrophage cell line RAW264.7 and the Hp strains SS1, PMSS1, 60190 and 7.13, aswell as cagA, cagE, slt, ureA, and oipA isogenic mutants of 7.13. Cells were treated withpharmacological inhibitors of various signaling pathways or with chromium-mesoporphyrin(CrMP; an HO-1 inhibitor), and then infected with Hp. In some studies, cells were transfectedwith siRNA directed against ho-1 (encoding for HO-1) or lmnA (encoding for lamin A, usedas control). Expression levels of the genes encoding TNF-α, iNOS, and IL-10 were analyzedby real-time PCR. Detection of HO-1 was performed byWestern blotting.Results: Expressionof the ho-1 gene and levels of HO-1 protein were induced by 25- and 16-fold by Hp strain7.13 when compared to uninfected cells, respectively; similar levels of induction of HO-1occurred with the 3 other Hp strains. This induction was significantly inhibited by 72 ±5% (p<0.01) when infected cells were pretreated with the p38 inhibitor (SB203580), butnot with inhibitors of NF-κB, ERK1/2, JNK, or PI3K. When Hp 7.13 strains lacking cagA,oipA, or ureA were used, there were significant reductions of HO-1 induction compared tothe WT (by 75, 75, and 84%, respectively), or to the slt- and cagE- strains. We then analyzedthe role of HO-1 in the innate response to Hp in macrophages. Hp-stimulated mRNA levelsof the pro-inflammatory mediators, TNF-α and iNOS, were further increased by 2.2 ±0.2- and 3.1 ± 0.4-fold in macrophages treated with CrMP; conversely, expression ofimmunoregulatory IL-10 was decreased by 3.2 ± 0.5-fold in cells treatedwith CrMP. Similarly,mRNA levels of TNF-α and iNOS were increased, while those of IL-10 were decreased, inRAW264.7 cells transfected with ho-1 siRNA prior to Hp infection.Conclusions:Hp upregul-ates HO-1 in macrophages in a p38-dependent manner. Moreover, multiple Hp virulencefactors, including CagA, OipA and UreA, are involved in this induction. Activation of HO-1 attenuates the pro-inflammatory response to Hp in macrophages, and therefore couldrepresent a strategy by which the bacterium escapes antimicrobial host defenses.

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A HNF4A MicroRNA-Inflammatory Circuit is Involved in the Pathogenesis ofUlcerative Colitis (UC)Tiziana Palumbo, Christos Polytarchou, Maria Hatziapostolou, Charalabos Pothoulakis,Dimitrios Iliopoulos

Background & Aims: MicroRNAs are small non-coding RNAs involved in the pathogenesisof different diseases. However their functional role in the development of UC remains tobe determined. Here, we describe for the first time the involvement of miR-24 and itsdownstream target HNF4A (hepatocyte nuclear factor 4 alpha) in UC pathogenesis. Methods:MicroRNA microarray analysis was performed in intestinal tissues derived from 37 activeUC, 31 active Crohn's disease (CD), and 26 control subjects. MiR-24 was overexpressed(50nM) in non-transformed human NCM356 colonic epithelial cells and HNF4A luciferaseactivity and mRNA levels were assessed 48h later by a luciferase assay and real-time PCR,respectively. STAT3 phosphorylation status (Tyr705) was assessed by ELISA, 48h posttransfection of an HNF4A expression vector in DLD-1 colon epithelial cancer cells. MiR-24and HNF4A mRNA levels were assessed by real-time PCR in 48 active UC tissues and 10dextran sulfate salt (DSS)-treated mice for 5 days. A chemically-modified antisense-mic-roRNA-24 (10mg/kg) was administered intracolonically in mice which were subsequentlytreated with DSS (5% for 5d). Mice were sacrificed, RNA and protein were extracted fromtheir colons and HNF4A mRNA levels and STAT3 phosphorylation were assessed by real-time PCR and western blot analyses, respectively. Results: We have identified 18 microRNAsto be deregulated (10 up-regulated and 8 down-regulated) in UC patients relative to controlsubjects. We found that miR-24 was the top (9.4-fold) up-regulated microRNA in UC, butnot CD, patients relative to controls. Bioinformatics analysis revealed that miR-24 has abinding site in the 3'UTR of HNF4A. MiR-24 overexpression suppressed 70% HNF4Aluciferase activity and 5.2-fold HNF4A mRNA levels in NCM356 cells. On the other hand,miR-24 suppression led to increased HNF4A protein levels. Furthermore, miR-24 wasoverexpressed while HNF4A was down-regulated in 36/48 UC tissues, suggesting the diseaserelevance of this interaction. In addition, HNF4A inhibition activated the IL6-STAT3 pathwayin NCM356 cells. Restoration of HNF4A expression suppressed STAT3 phosphorylation

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