Genomic features of the Helicobacter pylori strain PMSS1 and its virulence attributes as deduced from its in vivo colonisation patterns.

The human gastric pathogen Helicobacter pylori occurs in two basic variants, either exhibiting a functional cagPAI-encoded type-4-secretion-system (T4SS) or not. Only a few cagPAI-positive strains have been successfully adapted for long-term infection of mice, including the pre-mouse Sydney strain 1 (PMSS1). Here we confirm that PMSS1 induces gastric inflammation and neutrophil infiltration in mice, progressing to intestinal metaplasia. Complete genome analysis of PMSS1 revealed 1,423 coding sequences, encompassing the cagPAI gene cluster and, unusually, the location of the cytotoxin-associated gene A (cagA) approximately 15 kb downstream of the island. PMSS1 harbours three genetically exchangeable loci that are occupied by the hopQ coding sequences. HopQ represents a critical co-factor required for the translocation of CagA into the host cell and activation of NF-κB via the T4SS. Long-term colonisation of mice led to an impairment of cagPAI functionality. One of the bacterial clones re-isolated at four months post-infection revealed a mutation in the cagPAI gene cagW, resulting in a frame shift mutation, which prevented CagA translocation, possibly due to an impairment of T4SS function. Rescue of the mutant cagW re-established CagA translocation. Our data reveal intriguing insights into the adaptive abilities of PMSS1, suggesting functional modulation of the H. pylori cagPAI virulence attribute.

Helicobacter pylori Depletes Cholesterol in Gastric Glands to Prevent Interferon Gamma Signaling and Escape the Inflammatory Response.

BACKGROUND & AIMS: Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-α-glucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. METHODS: MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-ß-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1-/- mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. RESULTS: In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human ß-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1-/- mice were colonized by PMSS1 to a greater extent than control mice. CONCLUSIONS: H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell-based vaccines against H pylori.

tRNA Lys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs.

Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNA(Lys3) short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNA(Lys3) transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNA(Lys3) expression cassettes are also effective in vivo. Usefulness of tRNA(Lys3) antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing.

Full of sound and fury, signifying something: the impact of autonomic arousal on EGM gambling.

An experiment was conducted to observe the influence of autonomic arousal on subsequent gambling behavior. Thirty-seven male and 32 female regular Electronic Gaming Machine (EGM) players were recruited through newspaper advertisements. Participants were randomly assigned to either: (1) a control condition, or (2) an experimental condition that introduced a loud white-noise event (80 db) at fixed 120 s intervals throughout the 5-min EGM gambling session. Galvanic Skin Response (GSR) measurements showed that the manipulation was successful in elevating autonomic arousal. The results showed differences in behavioral response to the manipulation based on prior experience with gambling problems. Persons with many gambling problems had lower average bet-sizes in the white-noise condition compared to the control, while those with few or no problems had higher average bet-sizes. The results suggest that arousal may provide different signals to gamblers with few versus many problems. Gamblers with many problems may interpret their arousal as a sign that they will soon lose money, while gamblers with few or no problems may associate feelings of arousal exclusively with winning.

The Four Es 1-year later: a tool for predicting the development of gambling problems.

The Four Es is a 40-item scale measuring psychological risk for the development of problem gambling behavior. One-year follow-up interviews (n = 395) from a previously reported phone survey in Queensland, Australia (n = 2,577) (Rockloff & Dyer, 2006) tested the ability of the Four Es instrument to prospectively identify persons who would later develop gambling problems. Two groups of participants were selected for the 1-year follow-up interviews, including (1) persons who had gambling problems, high-risk alcohol abuse problems, and/or substance abuse problems (abuse group); and (2) a random selection of other persons from the original survey (random group). The results indicated that the "Excess" trait, which measures impulsive behavior, was predictive of relative increases in gambling problems for both groups over the 1-year period. Additionally, the Four Es questionnaire showed good psychometric properties in the surveys, with a test-retest reliability of r = .70 and a Cronbach's alpha reliability of alpha = .90 and .92 in the original and follow-up interviews, respectively.

An experiment on the social facilitation of gambling behavior.

Research and theory regarding the social facilitation effect generates the expectation that the presence of other gamblers (or co-actors) in a gaming venue is likely to intensify individual gambling behavior and magnify losses. Fifty male and 66 female participants (116 total) played a computer-simulated electronic gaming machine with a fixed winning sequence, followed by an indefinite losing sequence. Measures of the intensity of gambling behavior included the final payout (a direct measure of losses), average bet-size, number of trials played, and the speed of play. Some participants received false feedback from the computer designed to suggest that other gamers in adjacent rooms were playing and sometimes winning at the same game. Persons who received both sight and sound information, including winning bells and instant messages regarding the wins of other (fake) players, placed more bets and lost more money compared to the other conditions with less information.

The four Es of problem gambling: a psychological measure of risk.

A focus group of Reno area Gamblers Anonymous members identified four psychological traits contributing to risk for problem gambling, including: Escape, Esteem, Excess and Excitement. A panel of four experts authored 240 Likert-type items to measure these traits. By design, none of the items explicitly referred to gambling activities. Study 1 narrowed the field of useful items by employing a quasi-experimental design which compared the answers of Reno area Gamblers Anonymous members (N = 39) to a control sample (N = 34). Study 2 submitted successful items, plus new items authored with the knowledge gained from Study 1, to validation in a random sample telephone survey across Queensland, Australia (N=2577). The final 40 item Four Es scale (4Es) was reliable (alpha=.90); predicted gambling problems as measured by the Canadian Problem Gambling Index of Severity (PGSI, Ferris & Wynne (2001). The Canadian Problem Gambling Index: Final Report: Canadian Centre on Substance Abuse); and distinguished problem gamblers from persons with alcohol abuse problems. The new scale can provide a basis for further study in harm minimization, treatment, and theory development.