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670 CagA SHP2 Binding in Natural Infections By Western and East Asian Helicobacter pylori Strains
this kinase might regulate acid responses. Aim: To identify the rapid response of urease apoenzyme regulated by HP0244 and its functional consequence. Methods: Wildtype and HP0244 mutants were exposed to pH 4.5 for 30 min and membrane recruitment of urease apoenzyme and accessory genes and urease activity were compared in a highly purified membrane preparation. Since the addition of 50mM NH4Cl results in, first, influx of NH3 producing alkalization and then NH4+ with re-acidification, the role of UreI in transport of NH3 and NH4+ was determined by the effect of 50mM NH4Cl on pHin and membrane potential using BCECF-AM and DiSC3(5) fluorimetry in wildtype and ureI deletion mutants. Protein synthesis was measured by 35S-methionine incorporation. Results: There is UreI dependent recruitment of UreA, UreB and UreE to the membrane at pH 4.5out independent of protein synthesis since it is not affected by chloramphenicol. There is a ~ twofold increase in membrane urease activity and equivalent depletion of cytosolic urease. Deletion of HP0244 abolished recruitment. Membrane potential collapsed at pH 2.5 in HP0244 deletion mutants with 10mM urea but not in the wildtype, accounting for the loss of survival. UreI is able to transport NH3 as evidenced by the decreased rate of alkalization following NH4Cl addition in the presence of methylamine that competes with NH3. UreI also transports NH4+ since cytoplasmic re-acidification and membrane repolarization after NH4Cl addition are abolished in ureI deletion mutants. Conclusions: UreI-dependent membrane recruitment and activation of urease is regulated by the histidine kinase, HP0244. Formation of this pH-dependent complex allows direct access of urea to urease via UreI and efflux of urease-generated NH3 and NH4+ through UreI. This enables rapid periplasmic neutralization and consistent cytoplasmic pH homeostasis. Functioning of this complex appears essential for survival at the pH of the site of H. pylori habitation. HP0244 is a therefore new target for eradication.
Molecular Detection of Bacterial Contamination in Gnotobiotic Rodent Units Christopher D. Packey, Ian M. Carroll, Maureen A. Bower, Susan L. Tonkonogy, R. Balfour Sartor Gnotobiotic rodents facilitate the study of commensal bacteria and their roles in human physiology and pathophysiology. To ensure sterility, animals must be screened frequently for contamination by the traditional approach of culturing and Gram staining of feces. Yet many bacteria are uncultivable, fecal Gram stains can be difficult to interpret, and these methods are labor-intensive and time-consuming. Aims: To develop molecular methods of detecting contamination in gnotobiotic units. Methods: We collected fresh fecal pellets from mice housed in germ free (GF) isolators (N=8), a contaminated ex-GF isolator, an isolator colonized with 2 bacterial species, and a specific pathogen free (SPF) animal room (N=4). DNA from fecal samples was extracted using a phenol/chloroform extraction method combined with physical disruption of bacterial cells. Polymerase chain reactions (PCR) were carried out using templates of fecal DNA and primers that amplify the genes encoding the 16S rDNA from all bacterial groups. The numbers of 16S rDNA gene copies in each DNA sample were then quantified using quantitative Real-Time PCR (qPCR) with the same templates and primers and the appropriate set of standards. Finally, the 16S rDNA gene was amplified from fecal DNA from contaminated ex-GF mice, sequenced, and crossreferenced with a computerized data base to identify the bacterial species. Results: PCR yielded amplicons from isolated fecal DNA from, in descending order of intensity, all SPF mice, dual associated mice, and contaminated ex-GF mice, as well as from mice from one presumed GF isolator. Fecal DNA from mice from the other 7 GF isolators did not yield amplicons. qPCR confirmed that the level of bacteria in the mouse feces from the questionable isolator, (4.0 X 108 copies 16S rDNA gene/μg feces), was 210-fold greater than the levels in the other 7 GF isolators, (1.9 X 106 copies/μg, p < 0.0001), and was comparable to the level in the known contaminated isolator, (3.0 X 108 copies/μg.) The source of DNA in mouse feces from GF isolators detected by qPCR was dead bacteria in autoclaved food. Upon review, both cultures and Gram stains from the isolator that we determined to be contaminated based on PCR and qPCR results had been deemed indeterminate by gnotobiotic facility staff. Sequencing the 16S rDNA gene revealed the contaminating bacterial species in the ex-GF isolator to be Bacillus simplex. Conclusions: We developed molecular methods to screen for contamination in gnotobiotic units that are more reliable and less timeconsuming than current traditional methods and can be used to identify the contaminating species.
669 H. pylori Peptide HP(2-20) Promotes Healing of Injured Gastric Mucosa By Interacting with Formyl-Peptyde Receptors Marco Romano, Nella Prevete, Francesca W. Rossi, Felice Rivellese, Fiamma Salerno, Gabriele Delfino, Bianca Liccardo, Stefania Staibano, Giuseppe D'Argenio, Gianni Marone, Amato De Paulis Background & Aims: Peptide Hp(2-20) derived from the N-terminal sequence of Helicobacter pylori (H. pylori) ribosomal protein L1 possesses a broad-spectrum of anti-microbial activity. In addition to its antimicrobial action, Hp(2-20), through the interaction with formyl-peptide receptors (FPRs), exerts several immunomodulatory properties in eukaryotic cells. It has recently been shown that activation of FPRs facilitates intestinal epithelial cell restitution. We studied whether Hp(2-20) accelerated healing of injured gastric mucosa and assessed the mechanism(s) underlying any such effect. Methods: 1) Gastric epithelial cell proliferation was assessed by flow cytometry and cell migration was determined by chemotaxis assay using a modified Boyden chamber technique, in two gastric cell lines, i.e MKN28 and AGS cells; 2) mRNA expression was assessed by RTPCR; 3) protein expression was determined by Western blot analysis; 4) the effect of Hp(2-20) on mucosal healing was assessed in rats following indomethacin-induced injury both macroscopically and microscopically. Results: 1) FPR-like receptors are expressed in both cell lines, both at the mRNA and protein level; 2) Hp(2-20) significantly stimulates migration and proliferation of gastric epithelial cells; 3) this effect is specifically mediated by the interaction with FPRL1 and FPRL2; 4) Hp(2-20) causes activation of FPRs-related downstream signalling pathways inducing phosphorylation of MAPK, Akt, and STAT3; 5) Hp(2-20) upregulates mRNA expression and stimulates secretion of VEGF; 5) Hp(2-20) significantly accelerates healing of rat gastric mucosa following injury brought about by indomethacin causing an approximately 40% reduction in gastric mucosal injury compared with control, both at the macroscopic and microscopic level. Conclusions: 1) FPRs are expressed in human gastric epithelial cells; 2) H. pyloriderived peptide Hp(2-20) stimulates gastric epithelial cell migration, proliferation and VEGF expression through interaction with FPRL1 and FPRL2 and activates FPRs-related downstream signalling pathways; 3) oral delivery of Hp(2-20) significantly improves gastric mucosal healing following damage brought about by indomethacin in the rat. This study provides further evidence to the complexity of the relationship between H. pylori and its host and opens possible scenarios where a bacterial product may prove of use in the therapy and/or prevention of exogenous injury to the human stomach.
667 Temporal Stability of Faecal Microbiota By Inter Spacer rDNA Bacterial Profiling (IS-pro) Matthijs E. Grasman, Andries E. Budding, Chris J. Mulder, Paul H. Savelkoul, Adriaan A. van Bodegraven Introduction Faecal microbiota is considered to be relatively stable in time within healthy individuals, unlike the markedly fluctuating microbiota in IBD patients, particularly during flare-up. Since most intestinal bacteria are uncultivable, we developed a rapid, highly reproducible molecular method for high-throughput bacterial profiling: IS-pro. Aims To determine short-term and long-term stability of faecal microbiota of healthy individuals by means of IS-pro. Materials and methods Short-term intestinal microbiota variation was assessed with day-to-day faecal samples in one individual over a three-week period. General composition of diet was different between weekdays and weekends. In addition, eight individuals provided two faecal samples with an interval of one month. Long-term variation was assessed in eight healthy individuals who provided two faecal samples with an interval up to two years. Samples were analysed by use of IS-pro. IS-pro is based on two features of the bacterial genome: species-specific length of the inter spacer (IS) region between the 16s and 23s rDNA and phylum-specific labelled primer sequences. With these specific labelled primers, species can be directly sorted into either Firmicutes/Actinobacteria or Bacteroidetes phylum. By colour and size sorting of amplified fragments specific bacterial profiles are created. Results Short-term: mean similarity of day-to-day bacterial profiles was 86% (79-92%). Substantial variation was observed between pre- and post-weekend profiles (70-74% similarity), in particular in species belonging to the Firmicutes phylum (53-59% similarity). Diet seemed to account for this short-term difference. Mean similarity of faecal profiles after one month in eight individuals was 71% (48-82%) Long-term: each of eight individuals showed a unique bacterial profile, sharing few species with other individuals. In cluster analysis, faecal profiles were host-specific, even after an interval between samples up to two years. Intra-individual profiles were relatively stable in time with a mean similarity of 70% (4884%). Conclusion By use of IS-pro, faecal microbiota showed a relatively high day-to-day stability within an individual: 79-92% similarity. Short-term (1 month) and long-term (up to 2 years) stability was equal, both with similarity around 70%. Inter-individual similarity was low. Diet seems to alter faecal bacterial composition. Short-term and long-term differences, although limited and stable, may thwart analysis of disease-specific changes in intestinal microbiota.
670 CagA SHP2 Binding in Natural Infections By Western and East Asian Helicobacter pylori Strains Hiroaki Ogiwara, Mitsushige Sugimoto, Ellen J. Beswick, Victor E. Reyes, David Y. Graham, Yoshio Yamaoka Background and aims: Helicobacter pylori CagA protein tyrosine-phosphorylation site is at EPIYA motifs within the 3' repeat region of the gene. These repeat regions have been classified based on sequences surrounding EPIYA motifs into types A, B, C, and D. EPIYA-A and -B are conserved in both Western and East Asian strains; EPIYA-C is specific for Western strains and EPIYA-D is specific for East Asian strains. CagA transfection experiments reported that East Asian type CagA (e.g., ABD) exhibited stronger SHP-2 binding activity than Western type (e.g., ABC, ABCC). This difference was related to structural differences between EPIYAC and -D motifs and it was hypothesized that SHP-2 binding activity might relate to the high incidence of gastric cancer in East Asia compared to Western countries. We investigated CagA-SHP-2 binding during natural infection experiments using live Western and East Asian H. pylori. Methods: 27 East Asian and 26 Western H. pylori with various numbers of EPIYA motifs were co-cultured with gastric epithelial AGS cells. CagA levels in the cell lysates were measured by immunoblot and phospho-CagA levels and CagA-SHP2 binding by immunoprecipitation. Results: Western and East Asian strains produced similar amounts of CagA protein in AGS cells irrespective of the number of EPIYA motifs. The amount of H. pylori attached to the AGS cells was also equal irrespective of the origin of strains and the number of EPIYA motifs. In contrast, significantly higher total CagA protein levels translocated into AGS cells from Western strains compared to East Asian strains. Phospho-CagA and SHP-2 bound CagA levels showed similar patterns to total CagA protein levels and the levels directly correlated with the number of EPIYA-C motifs among Western strains. Importantly, translocated CagA protein levels were significantly higher in ABC than in ABD type H. pylori
668 Regulation of Urease Membrane Recruitment and Activation By the Cytoplasmic Histidine Kinase, HP0244 of Helicobacter pylori: A Rapid Response to Acidity George Sachs, Yi Wen, Jing Feng, Siddharth Singh, Elizabeth A. Marcus, David R. Scott Background: The neutralophile, H. pylori, is the only known pathogen colonizing the normal human stomach. It uses unique acid acclimation rather than acid tolerant/resistance mechanisms. Important for this acclimation is very high UreA/UreB apoenzyme content and urea access to urease via the H+-gated urea channel, UreI. Since the pH of its gastric habitat can fall rapidly from pH ~5.0 to pH < 3.0 (Scott et al PNAS 2007), a rapid reaction to high acidity is needed. Deletion of the cytoplasmic histidine kinase, HP0244, abolishes survival at pH 2.5 with 10 mM urea in contrast to wildtype (Wen et al J Bact 2008) suggesting that
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strains. Phospho-CagA and SHP-2 bound CagA levels were similar among ABC and ABD strains and significantly lower than with ABCC and ABCCC strains. Conclusions: Transfection prevents assessment of critical bacterial-host cell interactions by the type IV secretion system and hypotheses derived from transfection experiments must be confirmed using natural infection. The use of natural infections failed to confirm the hypothesis of higher SHP-2 binding of East Asian type CagA reported in CagA transfection experiments (i.e., CagA-SHP-2 binding during natural infection with Western strains was greater than with East Asian strains).
673 FHL2 Promotes Epithelial Mesenchymal Transition in Colon Cancer Through Modulating the Composition of E-Cadherin/β-Catenin Complex Wenjing Zhang, Jide Wang, Bing Zou, Juan Ma, Qing Gu, Liang Qiao, Hui Y. Lan, Benjamin C.Y. Wong Aims: We aim to evaluate the role of FHL2 in the regulation of epithelial mesenchymal transition (EMT). Methods: The expression of FHL2 by colon cancer cells was detected by immunohistochemistry. The effect of FHL2 on cell adhesion, invasion and migration was evaluated by various functional assays. FHL2 expression was altered by either over-expression by generating stable transfectants or suppression by siRNA. The response of FHL2 to TGFβ was investigated too. Expression of vimentin, MMP9 and E-cadherin was detected by RTPCR, real-time PCR and western blot. The transactivity of β-catenin was determined by both the luciferase-reporter system and the detection of its downstream genes. The composition of E-cadherin/β-catenin complex was visualized under fluorescent microscopy. The mechanism for FHL2 to down-regulate E-cadherin was defined by investigating the interaction between FHL2 and the negative regulator of E-cadherin, Snail. Results: FHL2 was overexpressed in colon cancer cells that could penetrate through the basement membrane in colon cancer tissues. FHL2-siRNA inhibited while FHL2 over-expression promoted invasion capacity of cancer cells. FHL2 expression was inducible by TGF-β and FHL2 mediated TGFβ induced vimentin expression. Over-expression of FHL2 increased while FHL2-siRNA suppressed the expressions of vimentin and MMP-9. Furthermore, FHL2 siRNA suppressed the transactivity of β-catenin and inhibited the expressions of its downstream genes including survivin and cyclin D1 through modulating the phosphorylation of β-catenin. FHL2 siRNA and truncated mutation increased E-cadherin expression and the presentation of membraneassociated E-cadherin/β-catenin complexes. FHL2 protein physically interacted with the transcriptional factor snail. FHL2 increased snail level through suppressing its cytosolic translocation and degradation. Site-directed mutation of snail-binding elements within Ecadherin promoters abrogated the effect of FHL2 in suppressing E-cadherin transcription. Conclusion: FHL2 promotes EMT of colon cancer cells through modulating the organization of E-cadherin/β-catenin complex; thus, facilitate the invasion or metastasis of colon cancer.
671 Increased L-Arginine Availability Enhances Helicobacter pylori Colonization in the Chronic Mouse Model Daniel P. Barry, Mohammad Asim, Nuruddeen D. Lewis, Thibaut de Sablet, M. Blanca Piazuelo, Rupesh Chaturvedi, Keith T. Wilson Introduction: The host response to Helicobacter pylori (Hp) infection involves activation of macrophages and upregulation of both arginase II and inducible nitric oxide synthase (iNOS), two enzymes that use L-arginine (L-Arg). Uptake of this amino acid is facilitated by cationic amino acid transporter 2 (CAT2). L-Arg is also an essential nutrient for the bacterium used to generate urea, and its utilization contributes to the resistance to killing by macrophages by preventing availability for host iNOS. Our aim was to determine if colonization levels could be altered by knockout of CAT2 or arginine supplementation in mice. Methods: C57BL/6 wild-type or congenic CAT2-/- mice were orally infected three times with Hp SS1. Some mice were administered supplemental L-Arg in their drinking water (1% w/v). After 4 months mice were sacrificed and stomachs were dissected. Bacterial colonization was assessed by serial dilution and plating of homogenized tissue and chronic (lymphocytic) and acute (granulocytic) gastritis was quantified by microscopic examination of stained sections. Results: Hp colonization was significantly increased by more than a log order from 5.93 ± 0.16 in wild-type mice to 7.16 ± 0.14 log(CFU/g) in CAT2-/- mice (p < 0.0001, n = 20-25). In contrast, there was no observed difference in gastritis between wild-type and knockout groups; specifically, there was an increase in histologic gastritis score induced by Hp infection in wild-type mice from 1.0 ± 0.3 in controls (n = 17) to 4.2 ± 0.4 in Hpinfected (n = 41; p < 0.001) and from 0.8 ± 0.3 in CAT2-/- controls (n = 15) to 3.5 ± 0.4 in Hp-infected CAT2-/- mice (p < 0.001, n = 32). A strong negative correlation was observed between gastritis score and colonization in CAT2-/- mice (r2 = 0.462, p = 0.001, n = 20). In contrast this correlation was not significant in the wild-type mice (r2 = 0.096, p = 0.115, n = 28). L-Arg supplementation enhanced Hp colonization in WT mice increasing the load from 5.53 ± 0.26 to 6.52 ± 0.12 log(CFU/g), (p < 0.01, n = 7-8 per group). In contrast, in the CAT2-/- mice L-Arg administration had no effect on the colonization density (n = 8 per group). In this experiment, L-Arg treatment increased gastritis from 3.2 ± 0.9 in wild-type mice to 5.6 ± 1.4 in the CAT2-/- mice (p < 0.05), suggesting that activation of CAT2independent pathways may be pathogenic. Conclusions: Increasing availability of L-Arg to Hp either by knockout of the mouse arginine transporter CAT2 or by L-Arg supplementation of mice leads to significantly increased Hp colonization. This indicates that L-Arg utilization by the host is part of innate immune defense, by preventing L-Arg use by Hp.
674 Interleukin-1β Induces COX-2 in Colonic Fibroblasts: Roles of Paracrine Amphiregulin and Autocrine PGE2 Reba Mustafi, Hongyan Zhu, Apameh Pezeshk, Hardik B. Shah, Zhiqun Song, Loren J. Joseph, Maria Sibilia, Alessandro Fichera, Marc Bissonnette Background: Epidermal growth factor receptors (EGFR) are over-expressed in colon cancer. These receptors are expressed in malignant colonocytes and tumor fibroblasts and regulate cyclooxygenase-2 (Cox-2) levels. Cox-2 is the rate limiting enzyme for biosynthesis of prostaglandin E2 (PGE2) that in turn stabilizes Cox-2 mRNA. Interleukin 1β (IL-1β) is also increased in colonic tumors and induces Cox-2 in colonocytes and fibroblasts. To begin to dissect the role of EGFR in colon cancer cells on IL-1β signals in fibroblasts, we examined human colonic fibroblasts co-cultured with human colon cancer cells that expressed an inducible dominant negative EGFR (DN-EGFR). To delineate the role of fibroblast EGFR on IL-1β stimulation of these cells we also examined EGFR sufficient (+) and EGFR null () murine embryonic fibroblasts (MEF). Methods: Caco-2 colon cancer cells were stably transfected with doxycycline-regulated expression vector that codes for dominant negative EGFR (DN-EGFR). This construct lacks the cytoplasmic domain and silences EGFR signals. CCD-18Co human colonic fibroblasts were plated in monoculture or co-cultured on transwells with DN-EGFR Caco-2 cells. EGFR ligands were quantified by real time PCR and PGE2 assayed by enzyme immunoassay. Cox-2 expression was measured by Western blotting. For MEF studies, cells were treated with IL-1β or vehicle and cytokines assayed by murine cytokine arrays. Results: When active EGFR was expressed in Caco-2 cells under co-culture conditions, IL-1β significantly up-regulated the EGFR ligand amphiregulin in Caco-2 cells. This cytokine also concomitantly increased Cox-2 levels and PGE2 secretion in CCD-18Co cells. When EGFR was silenced by DN-EGFR, however, increases in Caco-2 amphiregulin and fibroblast Cox-2 and PGE2 were reduced by >60% (p<0.05). In MEF monoculture experiments, IL-1β increased RANTES, TIMP-1 and TNF-α by 2-3-fold in MEF+ compared to MEF- cells (p<0.05) as assessed by cytokine arrays. Summary: Enhanced Cox-2 expression in human colonic fibroblasts requires EGFR signals in co-cultured colon cancer cells. Cox2 increases in fibroblasts induced by IL-1β are likely paracrine mediated by Caco-2-derived amphiregulin and autocrine mediated by fibroblast PGE2 that stabilizes Cox-2 mRNA. MEF studies revealed that fibroblast EGFR co-regulates several IL-1β induced pro-inflammatory cytokines. Conclusions: Thus, EGFR signals in both colon cancer cells and fibroblasts are likely required for the full spectrum of cytokine responses that contribute to colonic tumorigenesis.
672 Need for H. pylori Adhesins in Colonization of the Primate Stomach Hui Liu, Cristina Semino-Mora, D. Scott Merrell, Steven Mog, Thomas Borén, Andre Dubois H. pylori adherence to gastric epithelial cells is critical for gastric colonization and life-long persistence in the primate stomach. Adherence depends on the ABO/Leb blood group antigen binding BabA adhesin and on the sialic acid binding SabA adhesin that binds to the host's sialyl-Lewis glycosphingolipid. To explore the respective roles of these adhesins in gastric colonization, we inoculated H. pylori negative rhesus monkeys with 107 CFU of either wild type (WT; N=6) H. pylori J99, or babA::cam sabA::kan double mutant (DM), babA::cam, or sabA::kan single mutants (SM) (Mahdavi et al., Science 2002) given at 9-month successive intervals. Videogastroscopies were performed at regular intervals for nine months after inoculation and biopsies were cultured, fixed for histology or flash-frozen at -80C. Fluorescence in situ hybridization (FISH) was used to detect sabA, and babA expression in WT and mutant strains and in the gastric mucosa of monkeys before and after inoculation. RNA expression of H. pylori-specific 16S rRNA (copies/100 ng total RNA) were determined using absolute real-time quantitative RT-PCR (TaqMan). Relative quantification of infection was established by comparison to a standard curve of cloned 16S rRNA and normalization to monkey 18S rRNA. All monkeys inoculated with WT bacteria were persistently infected whereas cultures of all DM and SM post-inoculation biopsies were negative. The DM strain was detected by RT-PCR in only two of the five monkeys at 7 days post inoculation but not at any later time point. In keeping with this, gastritis scores and IL-1β and IL-8 expression were lower than in WT-infected controls. Since some monkey show natural immunity to H. pylori infection, we recently reinoculated the 5 animals that had failed inoculation with SM and DM strains with a GFP-labeled WT J99 strain to ensure these animals were not naturally resistant to infection. Dual FISH demonstrated merged fluorescence of H. pylori 16S rRNA and GFP in the biopsies of three of the five monkeys and H. pylori positivity of these three monkeys was also confirmed by real-time RT-PCR. Additionally, we were able to culture and characterize the infecting strain from one of the monkeys after inoculation, thus demonstrating that the lack of colonization of DM and SM was not due to natural immunity. Further follow-up is ongoing, but these current observations indicate that the expression of SabA and BabA adhesins are necessary for successful establishment of colonization and persistent H. pylori infection in primates.
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675 Chromatin Remodeling Tumor Suppressor Inhibitor of Growth 2 (P33ING2) As a Novel Mediator of Transforming Growth Factor-β-Dependent Responses Hiromi Kataoka, Takashi Joh, Karl Riabowol, Shirin Bonni Background: Members of the Inhibitor of Growth (ING) family of chromatin modifying proteins (ING1-ING5) have emerged as critical regulators of gene expression and cellular responses, suggesting that the ING may impinge on specific signal transduction pathway. Frequent down-regulation of tumor suppressor ING1 and ING2 expression in esophageal, stomach and colorectal carcinomas has been reported. On the other hand, dysfunction of the transforming growth factor β (TGF-β) signaling pathways have been implicated in cancer, but its precise mechanism has not been fully elucidated. As the relation between ING2 and growth factors and their associated signal cascades remains poorly understood, we investigated the role of ING2 in TGF-β signaling pathway. Methods: MvlLu, HepG2, 293T and SNU16 cell lines were used. Dual Luciferase assay were performed by using effecter plasmids (ING1a, ING1b, ING1c, ING2, ING3, TGF-β receptor I) with reporter plasmid [plasminogen activator inhibitor 1 (PAI-1), Smad binding elements]. Recombinant TGF-β1 (rTGF-β1) and
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Molecular Detection of Bacterial Contamination in Gnotobiotic Rodent Units Christopher D. Packey, Ian M. Carroll, Maureen A. Bower, Susan L. Tonkonogy, R. Balfour Sartor Gnotobiotic rodents facilitate the study of commensal bacteria and their roles in human physiology and pathophysiology. To ensure sterility, animals must be screened frequently for contamination by the traditional approach of culturing and Gram staining of feces. Yet many bacteria are uncultivable, fecal Gram stains can be difficult to interpret, and these methods are labor-intensive and time-consuming. Aims: To develop molecular methods of detecting contamination in gnotobiotic units. Methods: We collected fresh fecal pellets from mice housed in germ free (GF) isolators (N=8), a contaminated ex-GF isolator, an isolator colonized with 2 bacterial species, and a specific pathogen free (SPF) animal room (N=4). DNA from fecal samples was extracted using a phenol/chloroform extraction method combined with physical disruption of bacterial cells. Polymerase chain reactions (PCR) were carried out using templates of fecal DNA and primers that amplify the genes encoding the 16S rDNA from all bacterial groups. The numbers of 16S rDNA gene copies in each DNA sample were then quantified using quantitative Real-Time PCR (qPCR) with the same templates and primers and the appropriate set of standards. Finally, the 16S rDNA gene was amplified from fecal DNA from contaminated ex-GF mice, sequenced, and crossreferenced with a computerized data base to identify the bacterial species. Results: PCR yielded amplicons from isolated fecal DNA from, in descending order of intensity, all SPF mice, dual associated mice, and contaminated ex-GF mice, as well as from mice from one presumed GF isolator. Fecal DNA from mice from the other 7 GF isolators did not yield amplicons. qPCR confirmed that the level of bacteria in the mouse feces from the questionable isolator, (4.0 X 108 copies 16S rDNA gene/μg feces), was 210-fold greater than the levels in the other 7 GF isolators, (1.9 X 106 copies/μg, p < 0.0001), and was comparable to the level in the known contaminated isolator, (3.0 X 108 copies/μg.) The source of DNA in mouse feces from GF isolators detected by qPCR was dead bacteria in autoclaved food. Upon review, both cultures and Gram stains from the isolator that we determined to be contaminated based on PCR and qPCR results had been deemed indeterminate by gnotobiotic facility staff. Sequencing the 16S rDNA gene revealed the contaminating bacterial species in the ex-GF isolator to be Bacillus simplex. Conclusions: We developed molecular methods to screen for contamination in gnotobiotic units that are more reliable and less timeconsuming than current traditional methods and can be used to identify the contaminating species.
669 H. pylori Peptide HP(2-20) Promotes Healing of Injured Gastric Mucosa By Interacting with Formyl-Peptyde Receptors Marco Romano, Nella Prevete, Francesca W. Rossi, Felice Rivellese, Fiamma Salerno, Gabriele Delfino, Bianca Liccardo, Stefania Staibano, Giuseppe D'Argenio, Gianni Marone, Amato De Paulis Background & Aims: Peptide Hp(2-20) derived from the N-terminal sequence of Helicobacter pylori (H. pylori) ribosomal protein L1 possesses a broad-spectrum of anti-microbial activity. In addition to its antimicrobial action, Hp(2-20), through the interaction with formyl-peptide receptors (FPRs), exerts several immunomodulatory properties in eukaryotic cells. It has recently been shown that activation of FPRs facilitates intestinal epithelial cell restitution. We studied whether Hp(2-20) accelerated healing of injured gastric mucosa and assessed the mechanism(s) underlying any such effect. Methods: 1) Gastric epithelial cell proliferation was assessed by flow cytometry and cell migration was determined by chemotaxis assay using a modified Boyden chamber technique, in two gastric cell lines, i.e MKN28 and AGS cells; 2) mRNA expression was assessed by RTPCR; 3) protein expression was determined by Western blot analysis; 4) the effect of Hp(2-20) on mucosal healing was assessed in rats following indomethacin-induced injury both macroscopically and microscopically. Results: 1) FPR-like receptors are expressed in both cell lines, both at the mRNA and protein level; 2) Hp(2-20) significantly stimulates migration and proliferation of gastric epithelial cells; 3) this effect is specifically mediated by the interaction with FPRL1 and FPRL2; 4) Hp(2-20) causes activation of FPRs-related downstream signalling pathways inducing phosphorylation of MAPK, Akt, and STAT3; 5) Hp(2-20) upregulates mRNA expression and stimulates secretion of VEGF; 5) Hp(2-20) significantly accelerates healing of rat gastric mucosa following injury brought about by indomethacin causing an approximately 40% reduction in gastric mucosal injury compared with control, both at the macroscopic and microscopic level. Conclusions: 1) FPRs are expressed in human gastric epithelial cells; 2) H. pyloriderived peptide Hp(2-20) stimulates gastric epithelial cell migration, proliferation and VEGF expression through interaction with FPRL1 and FPRL2 and activates FPRs-related downstream signalling pathways; 3) oral delivery of Hp(2-20) significantly improves gastric mucosal healing following damage brought about by indomethacin in the rat. This study provides further evidence to the complexity of the relationship between H. pylori and its host and opens possible scenarios where a bacterial product may prove of use in the therapy and/or prevention of exogenous injury to the human stomach.
667 Temporal Stability of Faecal Microbiota By Inter Spacer rDNA Bacterial Profiling (IS-pro) Matthijs E. Grasman, Andries E. Budding, Chris J. Mulder, Paul H. Savelkoul, Adriaan A. van Bodegraven Introduction Faecal microbiota is considered to be relatively stable in time within healthy individuals, unlike the markedly fluctuating microbiota in IBD patients, particularly during flare-up. Since most intestinal bacteria are uncultivable, we developed a rapid, highly reproducible molecular method for high-throughput bacterial profiling: IS-pro. Aims To determine short-term and long-term stability of faecal microbiota of healthy individuals by means of IS-pro. Materials and methods Short-term intestinal microbiota variation was assessed with day-to-day faecal samples in one individual over a three-week period. General composition of diet was different between weekdays and weekends. In addition, eight individuals provided two faecal samples with an interval of one month. Long-term variation was assessed in eight healthy individuals who provided two faecal samples with an interval up to two years. Samples were analysed by use of IS-pro. IS-pro is based on two features of the bacterial genome: species-specific length of the inter spacer (IS) region between the 16s and 23s rDNA and phylum-specific labelled primer sequences. With these specific labelled primers, species can be directly sorted into either Firmicutes/Actinobacteria or Bacteroidetes phylum. By colour and size sorting of amplified fragments specific bacterial profiles are created. Results Short-term: mean similarity of day-to-day bacterial profiles was 86% (79-92%). Substantial variation was observed between pre- and post-weekend profiles (70-74% similarity), in particular in species belonging to the Firmicutes phylum (53-59% similarity). Diet seemed to account for this short-term difference. Mean similarity of faecal profiles after one month in eight individuals was 71% (48-82%) Long-term: each of eight individuals showed a unique bacterial profile, sharing few species with other individuals. In cluster analysis, faecal profiles were host-specific, even after an interval between samples up to two years. Intra-individual profiles were relatively stable in time with a mean similarity of 70% (4884%). Conclusion By use of IS-pro, faecal microbiota showed a relatively high day-to-day stability within an individual: 79-92% similarity. Short-term (1 month) and long-term (up to 2 years) stability was equal, both with similarity around 70%. Inter-individual similarity was low. Diet seems to alter faecal bacterial composition. Short-term and long-term differences, although limited and stable, may thwart analysis of disease-specific changes in intestinal microbiota.
670 CagA SHP2 Binding in Natural Infections By Western and East Asian Helicobacter pylori Strains Hiroaki Ogiwara, Mitsushige Sugimoto, Ellen J. Beswick, Victor E. Reyes, David Y. Graham, Yoshio Yamaoka Background and aims: Helicobacter pylori CagA protein tyrosine-phosphorylation site is at EPIYA motifs within the 3' repeat region of the gene. These repeat regions have been classified based on sequences surrounding EPIYA motifs into types A, B, C, and D. EPIYA-A and -B are conserved in both Western and East Asian strains; EPIYA-C is specific for Western strains and EPIYA-D is specific for East Asian strains. CagA transfection experiments reported that East Asian type CagA (e.g., ABD) exhibited stronger SHP-2 binding activity than Western type (e.g., ABC, ABCC). This difference was related to structural differences between EPIYAC and -D motifs and it was hypothesized that SHP-2 binding activity might relate to the high incidence of gastric cancer in East Asia compared to Western countries. We investigated CagA-SHP-2 binding during natural infection experiments using live Western and East Asian H. pylori. Methods: 27 East Asian and 26 Western H. pylori with various numbers of EPIYA motifs were co-cultured with gastric epithelial AGS cells. CagA levels in the cell lysates were measured by immunoblot and phospho-CagA levels and CagA-SHP2 binding by immunoprecipitation. Results: Western and East Asian strains produced similar amounts of CagA protein in AGS cells irrespective of the number of EPIYA motifs. The amount of H. pylori attached to the AGS cells was also equal irrespective of the origin of strains and the number of EPIYA motifs. In contrast, significantly higher total CagA protein levels translocated into AGS cells from Western strains compared to East Asian strains. Phospho-CagA and SHP-2 bound CagA levels showed similar patterns to total CagA protein levels and the levels directly correlated with the number of EPIYA-C motifs among Western strains. Importantly, translocated CagA protein levels were significantly higher in ABC than in ABD type H. pylori
668 Regulation of Urease Membrane Recruitment and Activation By the Cytoplasmic Histidine Kinase, HP0244 of Helicobacter pylori: A Rapid Response to Acidity George Sachs, Yi Wen, Jing Feng, Siddharth Singh, Elizabeth A. Marcus, David R. Scott Background: The neutralophile, H. pylori, is the only known pathogen colonizing the normal human stomach. It uses unique acid acclimation rather than acid tolerant/resistance mechanisms. Important for this acclimation is very high UreA/UreB apoenzyme content and urea access to urease via the H+-gated urea channel, UreI. Since the pH of its gastric habitat can fall rapidly from pH ~5.0 to pH < 3.0 (Scott et al PNAS 2007), a rapid reaction to high acidity is needed. Deletion of the cytoplasmic histidine kinase, HP0244, abolishes survival at pH 2.5 with 10 mM urea in contrast to wildtype (Wen et al J Bact 2008) suggesting that
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strains. Phospho-CagA and SHP-2 bound CagA levels were similar among ABC and ABD strains and significantly lower than with ABCC and ABCCC strains. Conclusions: Transfection prevents assessment of critical bacterial-host cell interactions by the type IV secretion system and hypotheses derived from transfection experiments must be confirmed using natural infection. The use of natural infections failed to confirm the hypothesis of higher SHP-2 binding of East Asian type CagA reported in CagA transfection experiments (i.e., CagA-SHP-2 binding during natural infection with Western strains was greater than with East Asian strains).
673 FHL2 Promotes Epithelial Mesenchymal Transition in Colon Cancer Through Modulating the Composition of E-Cadherin/β-Catenin Complex Wenjing Zhang, Jide Wang, Bing Zou, Juan Ma, Qing Gu, Liang Qiao, Hui Y. Lan, Benjamin C.Y. Wong Aims: We aim to evaluate the role of FHL2 in the regulation of epithelial mesenchymal transition (EMT). Methods: The expression of FHL2 by colon cancer cells was detected by immunohistochemistry. The effect of FHL2 on cell adhesion, invasion and migration was evaluated by various functional assays. FHL2 expression was altered by either over-expression by generating stable transfectants or suppression by siRNA. The response of FHL2 to TGFβ was investigated too. Expression of vimentin, MMP9 and E-cadherin was detected by RTPCR, real-time PCR and western blot. The transactivity of β-catenin was determined by both the luciferase-reporter system and the detection of its downstream genes. The composition of E-cadherin/β-catenin complex was visualized under fluorescent microscopy. The mechanism for FHL2 to down-regulate E-cadherin was defined by investigating the interaction between FHL2 and the negative regulator of E-cadherin, Snail. Results: FHL2 was overexpressed in colon cancer cells that could penetrate through the basement membrane in colon cancer tissues. FHL2-siRNA inhibited while FHL2 over-expression promoted invasion capacity of cancer cells. FHL2 expression was inducible by TGF-β and FHL2 mediated TGFβ induced vimentin expression. Over-expression of FHL2 increased while FHL2-siRNA suppressed the expressions of vimentin and MMP-9. Furthermore, FHL2 siRNA suppressed the transactivity of β-catenin and inhibited the expressions of its downstream genes including survivin and cyclin D1 through modulating the phosphorylation of β-catenin. FHL2 siRNA and truncated mutation increased E-cadherin expression and the presentation of membraneassociated E-cadherin/β-catenin complexes. FHL2 protein physically interacted with the transcriptional factor snail. FHL2 increased snail level through suppressing its cytosolic translocation and degradation. Site-directed mutation of snail-binding elements within Ecadherin promoters abrogated the effect of FHL2 in suppressing E-cadherin transcription. Conclusion: FHL2 promotes EMT of colon cancer cells through modulating the organization of E-cadherin/β-catenin complex; thus, facilitate the invasion or metastasis of colon cancer.
671 Increased L-Arginine Availability Enhances Helicobacter pylori Colonization in the Chronic Mouse Model Daniel P. Barry, Mohammad Asim, Nuruddeen D. Lewis, Thibaut de Sablet, M. Blanca Piazuelo, Rupesh Chaturvedi, Keith T. Wilson Introduction: The host response to Helicobacter pylori (Hp) infection involves activation of macrophages and upregulation of both arginase II and inducible nitric oxide synthase (iNOS), two enzymes that use L-arginine (L-Arg). Uptake of this amino acid is facilitated by cationic amino acid transporter 2 (CAT2). L-Arg is also an essential nutrient for the bacterium used to generate urea, and its utilization contributes to the resistance to killing by macrophages by preventing availability for host iNOS. Our aim was to determine if colonization levels could be altered by knockout of CAT2 or arginine supplementation in mice. Methods: C57BL/6 wild-type or congenic CAT2-/- mice were orally infected three times with Hp SS1. Some mice were administered supplemental L-Arg in their drinking water (1% w/v). After 4 months mice were sacrificed and stomachs were dissected. Bacterial colonization was assessed by serial dilution and plating of homogenized tissue and chronic (lymphocytic) and acute (granulocytic) gastritis was quantified by microscopic examination of stained sections. Results: Hp colonization was significantly increased by more than a log order from 5.93 ± 0.16 in wild-type mice to 7.16 ± 0.14 log(CFU/g) in CAT2-/- mice (p < 0.0001, n = 20-25). In contrast, there was no observed difference in gastritis between wild-type and knockout groups; specifically, there was an increase in histologic gastritis score induced by Hp infection in wild-type mice from 1.0 ± 0.3 in controls (n = 17) to 4.2 ± 0.4 in Hpinfected (n = 41; p < 0.001) and from 0.8 ± 0.3 in CAT2-/- controls (n = 15) to 3.5 ± 0.4 in Hp-infected CAT2-/- mice (p < 0.001, n = 32). A strong negative correlation was observed between gastritis score and colonization in CAT2-/- mice (r2 = 0.462, p = 0.001, n = 20). In contrast this correlation was not significant in the wild-type mice (r2 = 0.096, p = 0.115, n = 28). L-Arg supplementation enhanced Hp colonization in WT mice increasing the load from 5.53 ± 0.26 to 6.52 ± 0.12 log(CFU/g), (p < 0.01, n = 7-8 per group). In contrast, in the CAT2-/- mice L-Arg administration had no effect on the colonization density (n = 8 per group). In this experiment, L-Arg treatment increased gastritis from 3.2 ± 0.9 in wild-type mice to 5.6 ± 1.4 in the CAT2-/- mice (p < 0.05), suggesting that activation of CAT2independent pathways may be pathogenic. Conclusions: Increasing availability of L-Arg to Hp either by knockout of the mouse arginine transporter CAT2 or by L-Arg supplementation of mice leads to significantly increased Hp colonization. This indicates that L-Arg utilization by the host is part of innate immune defense, by preventing L-Arg use by Hp.
674 Interleukin-1β Induces COX-2 in Colonic Fibroblasts: Roles of Paracrine Amphiregulin and Autocrine PGE2 Reba Mustafi, Hongyan Zhu, Apameh Pezeshk, Hardik B. Shah, Zhiqun Song, Loren J. Joseph, Maria Sibilia, Alessandro Fichera, Marc Bissonnette Background: Epidermal growth factor receptors (EGFR) are over-expressed in colon cancer. These receptors are expressed in malignant colonocytes and tumor fibroblasts and regulate cyclooxygenase-2 (Cox-2) levels. Cox-2 is the rate limiting enzyme for biosynthesis of prostaglandin E2 (PGE2) that in turn stabilizes Cox-2 mRNA. Interleukin 1β (IL-1β) is also increased in colonic tumors and induces Cox-2 in colonocytes and fibroblasts. To begin to dissect the role of EGFR in colon cancer cells on IL-1β signals in fibroblasts, we examined human colonic fibroblasts co-cultured with human colon cancer cells that expressed an inducible dominant negative EGFR (DN-EGFR). To delineate the role of fibroblast EGFR on IL-1β stimulation of these cells we also examined EGFR sufficient (+) and EGFR null () murine embryonic fibroblasts (MEF). Methods: Caco-2 colon cancer cells were stably transfected with doxycycline-regulated expression vector that codes for dominant negative EGFR (DN-EGFR). This construct lacks the cytoplasmic domain and silences EGFR signals. CCD-18Co human colonic fibroblasts were plated in monoculture or co-cultured on transwells with DN-EGFR Caco-2 cells. EGFR ligands were quantified by real time PCR and PGE2 assayed by enzyme immunoassay. Cox-2 expression was measured by Western blotting. For MEF studies, cells were treated with IL-1β or vehicle and cytokines assayed by murine cytokine arrays. Results: When active EGFR was expressed in Caco-2 cells under co-culture conditions, IL-1β significantly up-regulated the EGFR ligand amphiregulin in Caco-2 cells. This cytokine also concomitantly increased Cox-2 levels and PGE2 secretion in CCD-18Co cells. When EGFR was silenced by DN-EGFR, however, increases in Caco-2 amphiregulin and fibroblast Cox-2 and PGE2 were reduced by >60% (p<0.05). In MEF monoculture experiments, IL-1β increased RANTES, TIMP-1 and TNF-α by 2-3-fold in MEF+ compared to MEF- cells (p<0.05) as assessed by cytokine arrays. Summary: Enhanced Cox-2 expression in human colonic fibroblasts requires EGFR signals in co-cultured colon cancer cells. Cox2 increases in fibroblasts induced by IL-1β are likely paracrine mediated by Caco-2-derived amphiregulin and autocrine mediated by fibroblast PGE2 that stabilizes Cox-2 mRNA. MEF studies revealed that fibroblast EGFR co-regulates several IL-1β induced pro-inflammatory cytokines. Conclusions: Thus, EGFR signals in both colon cancer cells and fibroblasts are likely required for the full spectrum of cytokine responses that contribute to colonic tumorigenesis.
672 Need for H. pylori Adhesins in Colonization of the Primate Stomach Hui Liu, Cristina Semino-Mora, D. Scott Merrell, Steven Mog, Thomas Borén, Andre Dubois H. pylori adherence to gastric epithelial cells is critical for gastric colonization and life-long persistence in the primate stomach. Adherence depends on the ABO/Leb blood group antigen binding BabA adhesin and on the sialic acid binding SabA adhesin that binds to the host's sialyl-Lewis glycosphingolipid. To explore the respective roles of these adhesins in gastric colonization, we inoculated H. pylori negative rhesus monkeys with 107 CFU of either wild type (WT; N=6) H. pylori J99, or babA::cam sabA::kan double mutant (DM), babA::cam, or sabA::kan single mutants (SM) (Mahdavi et al., Science 2002) given at 9-month successive intervals. Videogastroscopies were performed at regular intervals for nine months after inoculation and biopsies were cultured, fixed for histology or flash-frozen at -80C. Fluorescence in situ hybridization (FISH) was used to detect sabA, and babA expression in WT and mutant strains and in the gastric mucosa of monkeys before and after inoculation. RNA expression of H. pylori-specific 16S rRNA (copies/100 ng total RNA) were determined using absolute real-time quantitative RT-PCR (TaqMan). Relative quantification of infection was established by comparison to a standard curve of cloned 16S rRNA and normalization to monkey 18S rRNA. All monkeys inoculated with WT bacteria were persistently infected whereas cultures of all DM and SM post-inoculation biopsies were negative. The DM strain was detected by RT-PCR in only two of the five monkeys at 7 days post inoculation but not at any later time point. In keeping with this, gastritis scores and IL-1β and IL-8 expression were lower than in WT-infected controls. Since some monkey show natural immunity to H. pylori infection, we recently reinoculated the 5 animals that had failed inoculation with SM and DM strains with a GFP-labeled WT J99 strain to ensure these animals were not naturally resistant to infection. Dual FISH demonstrated merged fluorescence of H. pylori 16S rRNA and GFP in the biopsies of three of the five monkeys and H. pylori positivity of these three monkeys was also confirmed by real-time RT-PCR. Additionally, we were able to culture and characterize the infecting strain from one of the monkeys after inoculation, thus demonstrating that the lack of colonization of DM and SM was not due to natural immunity. Further follow-up is ongoing, but these current observations indicate that the expression of SabA and BabA adhesins are necessary for successful establishment of colonization and persistent H. pylori infection in primates.
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675 Chromatin Remodeling Tumor Suppressor Inhibitor of Growth 2 (P33ING2) As a Novel Mediator of Transforming Growth Factor-β-Dependent Responses Hiromi Kataoka, Takashi Joh, Karl Riabowol, Shirin Bonni Background: Members of the Inhibitor of Growth (ING) family of chromatin modifying proteins (ING1-ING5) have emerged as critical regulators of gene expression and cellular responses, suggesting that the ING may impinge on specific signal transduction pathway. Frequent down-regulation of tumor suppressor ING1 and ING2 expression in esophageal, stomach and colorectal carcinomas has been reported. On the other hand, dysfunction of the transforming growth factor β (TGF-β) signaling pathways have been implicated in cancer, but its precise mechanism has not been fully elucidated. As the relation between ING2 and growth factors and their associated signal cascades remains poorly understood, we investigated the role of ING2 in TGF-β signaling pathway. Methods: MvlLu, HepG2, 293T and SNU16 cell lines were used. Dual Luciferase assay were performed by using effecter plasmids (ING1a, ING1b, ING1c, ING2, ING3, TGF-β receptor I) with reporter plasmid [plasminogen activator inhibitor 1 (PAI-1), Smad binding elements]. Recombinant TGF-β1 (rTGF-β1) and
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