proximal and distal 1/2 of the gut. The proximal gut was perfused with pH 7.0 phosphate buffer at 2 ml/min while the distal gut was perfused with 60mM oleate at 2 ml/min to tngger the ileal brake response. Buffer was perfused into both compartments as the buffer control, intestinal transit across the proximal gut was measured by the cumulative % recovery of a radioactive marker out of the midgut fistula during the last 30 ram of the 90-mni perfusion (data = mean _+ SE). H3016 (kappa opioid antagonist)(Bachem) or saline was administered iv at 4 l.tg/kg over 90 toni. Results: We found that 1) % recovery was reduced from 72.8 -+ 3.8% for buffer to 31.9 _+ 6.1% for fat (p<O.005) as the ileal brake response and 2) The kappa opioid receptor antagonist reversed the ileal brake so that % recovery increased from 31.9 _+ 6.1% to 69.6 + 7.2% (p< 0.005). Conclusion: Slowing of intestinal transit by fat depends on an opioid pathway acting on kappa receptors.

311

Surgical Resection of the Small Intestine Acutely Disrupts ICC Populations and Electrical Rhythmicity Hiroe Yanagida, Kenton M Sanders, Scan M Ward

Surgical manipulation of the gastrointestinal tract, including intestinal resection and anasto- mosis, leads to motility disorders. The aims of the present investigation were to i) determine the mechanisms underlying the loss of intestinal motility in a murine model of intestinal reconstruction, and ii) to follow the recovery of intestinal motility after surgery. Two cm segments of ileum were removed from 20 mice without interfenng with blood supply. The intestine was reconstructed using fine suture thread. Following surgery, the structure and activity of the ileums, 0-5 cm oral and aboral to the site of resection, were examined (5-24 hr) using intracellular recording techniques, isometric force measurements and Kit immuno- hisiochemistry (using ACK2, an antibody that specifically labels pacemaking cells in the GI tract) Five hours after surgery there was an acute loss of electncal slow waves and phasic contractions of the circular muscle layer. Loss of activity decreased as a function of distance, 0ral and aboral to the anastomosis. Activity was only 50% of control 3 cm from the surgery site. Tissues in the affected region were poorly responsive to carbachol (10-6M) and transmural nerve stimulation. Kit immunohistochemistry revealed that loss of motility was associated ~ t h disruption in ICC networks at the level of the myenteric plexus (1C-MY). Lesions in IC-MY decreased with distance from the site of surgery. Slow waves and mechanical acttvity partially recovered at the site of anastomosis after 24 hrs and appeared normal at 2 centimeters Slow waves and mechanical activity recovered more rapidly when tissues were incubated in the iNOS inhibitor (L-NIL, 5x10-5M). Loss of motihty following surgery ts associated with an acute disruption of ICC networks and slow wave activity and that this can partially recover within a short time period. Supported by DK 41315.

312

Fructose Intolerance: A Randomized, Double Blind, Dose Response Study In Healthy Humans Leslie Anderson, Joan Kempf, Phyllis Stumbo, Satish Rao

Fructose intolerance affects 30-70% of patients with unexplained gastrointestinal symptoms. However, the optimal method for testing this problem is unclear. Our aim was to perform a dose response study in heahhy subjects to assess fructose malabsorption METHODS: In a double-blind study, twenty healthy subjects (M/F = 10/10, age range 18-70 years) were randomized to receive ] 5g of fructose in 150ml of water, 25g of fructose in 250ml of water, 50g of fructose in 500ml of water, or 50g of fructose in 150ml of water (33% solution) on four separate days at weekly intervals. Breath samples were collected for hydrogen and methane at baseline and at 30 minute intervals. If the subjects experienced any symptom(s) it was recorded and the severity scored on a visual analog scale. The data was compared using repeated measures anova. An abnormal study was defined as a rise of ) 5 ppm of H2 or CH4 or both over the baseline value and a rise that was sustained over three consecutive breath samples. RESULTS: The mean baseline for H2 was 5ppm, and for CH4 was 0.37ppm. Six subjects had methanogenic flora and their mean baseline was 16.7ppm A rise in breath H: or CH4 always occurred within three hours. We found that no subject tested positive ~th the 15/150 solution, whereas two(lO%) tested positive with the 25/250 solution [max peak H2 conc= 67ppm] but without symptoms. In contrast, 12(60%) had an elevation of H: [max peak H2 cone = 76ppm], and three(15%) had a rise in both H2 and CH, with the 50/]50 solution. Among these, five(42%) experienced symptoms including belching, bloat- ing, gas, pain, diarrhea, and headaches. Also, with the 50/500 solution, 16(80%) subjects tested positive [max peak H2 conc= 62ppml. Out of these, 14(88%) had elevated H2 and two(12%) had elevated CH4 or both gases and nine(56%) experienced symptoms. The mean area under the curve for H2 and CH4 was 605ppm (15/150), 1415ppm (25/250), 4976ppm (50/150), and 5596ppm (50/500) respectively. CONCLUSIONS: All heathly subjects could absorb 15g fructose. Although 90% of subjects could absorb 25g fructose, the two (10%) who malabsorbed had no symptoms. In contrast, a 50g fructose concentration, given either as 33~ or as 10% solution was malabsorbed by 60%-80%, and produced symptoms in about 50% of subjects. Because almost all healthy subjects can absorb 25g fructose, and many malabsorb (with symptoms) at a higher dose, 25g seems to be the optimal dose for testing patients with suspected fructose intolerance. Breath sampling at 30 minute intervals for three hours should detect most malabsorbers.

349

Nodl-Dependent Proinflammatory Responses to Helicobacter pylori Infection in Gastric Epithelial Cells Jerome Viala, Catherine Chaput, Ivo G. Boneca, Stephen E. Girardin, Anthony P. Moran, Michel R Huerre, Philippe J. Sansonetti, Agnes Labigne, John Bertin, Dana J. Philpott, R~cfiard L. Ferrero

Helicobacter pylon bacteria that harbour a cag pathogenicity island (cagPAI) induce NF-KB activation and IL-8 synthesis in gastric epithelial cells. Nevertheless, neither the host cell receptor, nor the bacterial effector molecule(s) involved in this process have been elucidated.

The aim of the study was to clarify the mechanism by which epithelial cells respond to extracellular H. pylori infection. The reported role of Nod1, an intracellular receptor with similarities to plant resistance proteins, in epithelial cell signalling to pathogens led us to investigate the involvement of this molecule in host sensing of H. pylofi. Epithelial cell signalling to H. pylori was investigated by measuring IgK-luc reporter activity in HEK293 cells, which express endogenous levels of Nodl. Consistent with the findings for AGS gastric epithelial cells, only those isolates harbouring a functional cagPAI induced luciferase reporter activity in transfected HEK293 cells. Moreover, whereas cagM-deficient mutants no longer induced NF-/CB activation, a cagA mutant was unaffected in its ability to activate this transcription factor. Co-transfection of HEK293 cells with increasing concentrations of a dominant negative Nod1 construct abrogated the effect of cagPAI+ H. pylori strains on Ig/C-luc activity. Since Nodl was recently shown to specifically respond to Gram-negative peptidoglycan that had been delivered intracellularly, we tested HEK293 cells for their ability to respond to purified H. pylori peptidtglycan. Peptidoglycan from H. pylori, but not LPS, was effective in inducing NF-KB activation in these cells. Moreover, an H. pylori mutant lacking lyric transglycosylase activity, responsible for peptidoglycan turnover and release in bacteria, was affected in its capacity to induce NF-KB activation in epithelial cells. Consistent with the in vitro data, Nodl-deficient mice were significantly more susceptible to infection with a cagPAI H. pylori isolate, when compared to wild-type animals (6.4 vs 5.1 and 5.7 vs 3.3 logCFU/g, at 7 and 30 days post-inoculation, respectively; P = 0.008 and P = 0.009). Conversely, no significant differences were noted at either time-point for a cagPA I- Helicobacter sp (H. fells). In conclusion, Nod1 in host epithelial cells has been shown to specifically recognise and respond to peptidoglycan released by cagPAI + H. py Iori bacteria. Nod1 is thus likely to playan important role in innate immune responses to H. pyloriinfection.

350

Helicobacter pylori activates Akt via transactivation of the EGF receptor in AGS gastric epithelial cells Sarah Keates, Andrew C. Keates, Stavros Sougloultzis, Richard M. Peek Jr., Ciaran P. Kelly

Background: Attachment of the bacterial pathogen Helicobacter pylori to the gastric epithe- fium results in the activation of numerous signaling pathways. Recently, we reported that H. pylori was able mediate transactivation of the EGF receptor in AGS gastric epithelial cells, and that this was an upstream event leading to both ERK1/2 phosphorylation and the upregulation of interleukin-8 protein production and gene expression. We now report that H. pylori are able to activate the protooncogene Akt / protein kinase B via transactivation of the EGF receptor. Method: AGS cells were infected with type i(cag +) and type 2(cag-)strains of H. pylori and isogenic mutants. Akt activation by H. pylori was determined by Western blot analysis using a phospho-specific antibody. P13 kinase involvement was investigated using Wortman- nin and LY294002 (10p.M), and EGF receptor kinase activation was inhibited using tyrphos- tin AG1478 (II~M). Results: Infection of AGS cells with type 1 strains of H. pylon led to phosphorylation of Akt (Ser473) by 30 minutes, which was maximal at 2 hours. A type 2 strain of H. pylori and a picB- isogenic mutant of H. pylon demonstrated an impaired ability to cause Akt phosphorylation. H. pylori mediated Akt phosphorylation, was completely prevented using the PI 3 kinase inhibitors LY294002 and Wortmarmin, and reduced with the EGE receptor kinase inhibitor tyrphostin AG1478. Conclusions: We find that: 1) H. pylori infection of AGS gastric epithelial cells results in the activation of the protooncogene Akt, 2) that an intact type IV secretion apparatus is required for full activation of Akt 3) and that it is mediated via activation of PI 3 kinase and EGF receptor transactivation. As Akt plays a pivitol role in apoptosis, cell proliferation, and angiogenesis, its activation by H. pylon could potentially be a slguificant event in gastric carcinogenesis.

351

Degradation of P27 ~pi by Helicobacter Pylori in Gastric Epithelial Cells Is Proteasome-Dependent But Independent of Ubiquitination or Mitogen-Activated Protein Kinase (MAPK) Phosphorylation Hidetnshi Eguchi, Steven E. Moss

Background: The expression of p27, a cyclin-dependent kinase inhibitor of the G] to S cell cycle phase transition, is down-regulated in gastric cancer. We reported that p27 expression in gastric epithelial cells is decreased by Helicobacter pflori through increased p27 protein degradation, but the responsible mechanisms are not known. Post-translational modification of p27, including ubiquitin-proteasome or MAPK-dependent degradation, may play impor- tant roles in the regulation of p27. We therefore investigated how H. pylon increases p27 degradation in gastric epithelml cells. Methods: 5 clinical strains of H. pylofi and isogenic mutants (cagA, vacA or cagE-disrupted) of the wild type strain 60190 were co-cuhured with AGS or MKN28 gastric epithelial cells at bacterial to epithelial cell ratios of 0:1 to 1000:1 for 0-24 hrs. Cell cycle distribution, p27 mRNA and protein levels, p27 degradation and the effect of proteasome inhibition by MG-132 or MAPK inhibition by PD98059 or SB203580 were evaluated. In vitro ubiquitination of exogenous p27, CDK2 activity and proteasome activity were also determined. Results: H. pylori decreased the expression of p27 protein in both cell lines via increased p27 protein degradation, dose and time-dependently, p27 degradation was abolished by proteasome inhibition, but not changed by inhibition of ERK1/ 2 or p38 MAPK's. Threonine-187-phosphorylated-p27, skp2 (p27's ubiquitni hgase), p27 ubiquitination, global proteasomal function and CDK2 activity were all reduced by H. pylori. The reduction in p27 was dependent on adherence of live H. pylori to gastric cells but was not related to H. pylori's cagA or cagE genes. Clinical strains lacking the cag island showed a small decrease in their abihty to decrease p27 but iceA and vacA allelotypes played no role in this decrease. Summary: H. pylori increases the degradation of p27 in gastric epithelial cells through a proteasome-dependent, but ubiquitin-independent pathway. ERK1/2 and p38 MAPK pathways are not involved in this degradation, cag island genes but not cagA may play a minor role. Thus H. pylori decreases the level of p27 protein through a novel degradative pathway that may link H. pylori to cell cycle dysregulatinn and carcinogenesis.

A-43 AGA Abstracts