Characterization of anti-apoptotic proteins derived from the probiotic Lactobacillus rhamnosus GG

phosphorylation of STAT1 in gastric epithelial cells. This is a direct bacterial effect independent of the cag pathogenicity island and vacuolating cytotoxin activity. H. pylori could inhibit this signal transduction cascade in order to evade host mucosal immune responses.

treated with 100 ng/ml purified flageflin or TNF (verified to be a maximal dose for both agonists). Activation of STATs, p38, and NF-KB was assessed via immunoblotting with phospho-specific antibodies. IL-6 and IL-8 were measured via ELISA. Expression levels of many other genes were measured via our mRNA microarray. Results: Flageflin and TNF appeared to be equipotent inducers of p38 MAPK and NF-KB. However, flageflin induced substantially higher levels of STAT 3 (3-fold) and moderately higher levels of STAT 1 (1.5 fold). FlageUin-induced STAT activation was blocked by inhibition of either p38 or NF-KB and exhibited delayed kinetics (minimum time 2 h vs. 15 rain for NF-KB activation) suggesting it resulted from new protein synthesis, in agreement, pretreatment of epithelia with cycloheximide prevented flagellin-induction of STAT 1 and 3 but did not affect activation of p38 or NF-kB. Expression profiling suggested IL-6 was a good candidate to be mediating this STAT activation. In support of this notion, flagellin induced more than twice as much 1L-6 secretion (155 _+ 2.1 vs 7.2 -+ 2.2 pg/ml for flagellin and TNF respectively) than did TNF even though TNF was a more potent inducer of IL-8 (1.41 _+ 0.17 vs. 1.83 _+ 0.15 vs for flagellin and TNF respectively) Furthermore, neutralizing antibodies to the IL-6 receptor completely blocked activation of STAT 3 (no effect on STAT 1) while control neutralizing antibodies to IL-8 were without effect. Such blockade of the IL-6 receptor significantly attenuated induction of several genes (e.g. iNOS by 40%) but had no effect on the induced expression of TNF nor 11_-8.Conclusion: While both TNF and flagellin potently" activate pro-inflammatory gcne expression m general, flageflin activates a subset of genes not significantly induced by TNF, for example iNOS, in part due to fiageflin-induced IL-6 secretion that results in activation of STAT3.

790 Unique and Divergent Roles for WASP Family Members in Salmonella Entry Tomoyuki Shibata, Fuminao Takeshima, Iron Bhan, Catherine T. Yan, Deanna Nguyen, Anna Lyubimova, Frederick W. Alt, Scott B. Snapper Background & Aims: The enteric pathogen Salmonella invade and survive within epithelial cells in the gastrointestinal tract by usurping a variety of host-encoded signaling pathways To invade cells, Salmonella employs a type lI1 secretion system to inject several bacterial proteins that modulate the host's actin cytoskeleton. Specifically, Salmonella SopE activates Rho-family GTPases (i.e., Cdc42, Rac 1) which in turn activate unknown downstream proteins that facilitate the cytoskeletal reorganization required for invasion. Since activated Cdc42 and Racl are known, in other systems, to stimulate Wiskott-Aldrich syndrome protein (WASP) family members (N-WASP and WAVE respectively) to mediate actin polymerization, we sought to determine whether WASP-family members had an important role in Salmonella invasion. Methods: We isolated murine embryonic fihrohlasts (MEFS) from raid-type (WT), N-WASP-deficient (KO) and WAVE-2 KO embryos as described (Nat.CeiL Biol., 5;897,2001; Yah et al., in prep). We then determined the rate of invasion of Salmonella typhimurium in these cell lines employing a modified gentamicin protection assay and confirmed results using a microscopic assay (Curt. Biol., 12:341,2002). In gentamicin protection assays, following a lhr infection (MOl: 100), extracellular bacteria are killed with gentamicin, and the number of intracellular bacteria determined by plating out lysates on agar plates. To rule out invasion defects that might not be attributed to the targeted protein, similar assays were performed with N-WASP-KO and WAVE-2 KO MEFS that were stably infected with retroviruses expressing N-WASP-GFP and WAVE-2 -GFP respectively. Results and Discussion: We determined that there is a significant decrease in Salmonella entry in WAVE2-KO cells compared with WT cells (>30% reduction, p<0.05). While these data identify a role for WAVE-2 in Salmonella entry, they suggest a predominant WAVE-2 independent invasion pathway. Surprisingly, Salmonella invasion was significantly increased in N-WASP-KO cells (>40% increase, P<0 01). This increase in invasion rate was reduced to WT levels in NWASP KO cells expressing N-WASP ectopically. These data imply that N-WASP may negatively regulate Salmonella invasion. Conclusions: N-WASP and WAVE 2, known downstream mediators of Cdc42 and Racl, have divergent roles in Salmonella entry.

793

Probiotic Inhibition of the Entry of Enteroinvasive E.coli into Human Intestinal Epithelial Cells Involves both Rho-Dependent and -Independent Pathways Kashyap Tnvedi, Kim E. Barrett, Silvia C. Resta-Lenert Background: lntracelMar actin dynamics are modulated by MAP kinases and Rho family GTPases. We have previously shown that enteroinvasive E.coli (E1EC) induces dramatic changes in the actin cytoskeleton and tight junctions upon adhesion and entry in intestinal epithelial cells, and that these effects can be prevented by treatment with probiotics We hypothesized that probiotics and commensals may inhibit pathogen entry by modulating MAP kinase and/or GTPase function. Methods: HT29/cI19A human intestinal epithelial cell monolayers were exposed to EIEC for 3 h in the presence or absence of probiotics, S.thermophF lus (ST) and Lacidophflus (LAy, and with or without ERK 1,2, p38, or Rho-associated coiledcral-contaiinng kinase (ROCK) inhibitors (PD98059, SB203580, and Y27632). MAP kinase and ROCK inhibition had no effect on cell viability (by dye exclusion assay). Cells were harvested and tested for EIEC invasion by CFU counts and for protein levels by Western blot. Results: Probiotic pretreatment inhibited EIEC entry and associated redistribution of ocdudin, actinin and ZO-1. Treatment of cells with PD98059 (2.6_+0.4 vs. 5.4_+0.2 vs. 8.7_+0.6 CFU/ml, EIEC+ST/LA vs. EIEC+STAA+ PD98059 vs EIEC; n = 4 , p<0.01) or SB203580 ( 1.3 _+0.2 vs. 5.5 _+0.3 vs. 8.2 _+0.2 CFU/ml, EIEC + ST/LA vs. EIEC + ST/LA + SB203580 vs. E1EC ; n = 4, p
791 Flagenin Is the Major EHEC 0157:H7 Virulence Factor Required for Upregnlated Expression of Proinflammatory Signals by Intestinal Epithelial Cells Yukiko Miyamoto, Mitsutoshi Iimura, James B. Kaper, Alfredo G. Tortes, Ludger Johannes, Martin F. Kaguoff EHEC O157:H7 attach to human colon epithehal cells (IECs), produce shiga toxin (Stx), and cause mucosal inflammation and hemorrhagic colitis. Unlike Salmonella and Shigella, EHEC do not generally invade IECs. Prior studies have proposed that Stx is a major bacterial virulence factor responsible for upregulating epithelial cell proinflammatory cytokine responses thought to initiate mucosal inflammation. The aim of this study was to determine the major EHEC virulence factor(s) responsible for upregulating human IEC proinflammatory signals. Methods: Human IEC lines were infected with wild type EHEC O157:H7 strain 8624, or isogeinc St:< negative (six-), or flagellin negative (fliC-) mutants of 86-24. Human intestinal mucosal biopsies were immunostained for toll-like receptor 5 (TLR5), the receptor for bacterial flagellin, and were stained for Gb3, the receptor for Stx, with Cy3 coupled Six B subunit. TLR5 expression by human IECs was determined by flow cytometry. TLR5 expression on polarized IEC monolayers was assessed by confocal microscopy NF-KB activation was assessed by RelA/p65 immunostaining. IL-8 secretion was assayed by ELISA. Results: fliC- EHEC 0157:H7 stimulated little to no epithelial cell IL-8, whereas both wild type and stx- EHEC O 157:H7 were potent inducers of IL-8. TLR5 was expressed by IEC's (e.g. Caco-2, HCA-7) with an apical, basolateral and cytoplasmic distribution pattern. Flagellin activated TLR signaling in IECs through NF-KB and MAP kinase pathways. Consistent with EHEC binding to the apical IEC membrane, apical exposure to flagellin was a more potent stimulus of IL-8 production than basolateral addition. Like the cell lines, human intestinal epithelium in vivo expressed TLR5. However, in contrast to human colon cancer cell lines, Gb3 was not expressed by normal or inflamed human intestinal epithelium in vivo, or by epithelium in colon cancers, although Gb3 was abundantly expressed by endothelial cells lining blood vessels in normal and inflamed human intestinal mucosa. Conclusion: Flagellin, and not Stx, is the major bacterial virulence factor responsible for upregulated expression of epithelial cell proinflammatory cytokines thought to signal a neutrophil influx after infection with EHEC O157:H7. In contrast, Six likely translocates to the lamma propria where it can bind Gb3 on endothelial cells and miuate vascular damage with ensuing hemorrhagic colitis. Supported by NIH grants DK58960 & DK35108.

794 Characterization of Anti-Apoptotic Proteins Derived from The Probiotic Lactobacillus Rhamnosus GG Fang Yan, Guinn Wilson, Sutha K. John, D.-Brent Polk Background: Probiotics are living microorganisms that exert beneficial efl~:cts on host health such as enhancing immunity, preventing and treating diarrhea, infection and intestinal inflammation. We reported that Lactobacilhs GG (LGG) is a model probiotic agent, which prevents cytokine-induced apoptosis m mouse and human colon epithelial cell models through activation of anti-apoptotic Akt and inhibition of pro-apoptotic p38. Furthermore, factors recovered from LGG culture supernatant (LGG-s) activate Akt and prevent apoptosis. To further investigate the mechanisms of communication between LGG and epithelial ceils, the purpose of this study is to characterize proteins recovered from LGG culture broth and their effect on intestinal cell survival and proliferation. Methods: LGG (ATCC 53103) was grown m MRS broth (37~ LGG-s was generated by cemrifuging and filtering (0.2 mm) LGG culture broth. The filtrate was concentrated using Centricon Pins-20 (5-100 kDa). Proteins in LGG-s were separated by SDS-gels and eluted from the gel slices using ElectroEluter, purified and concentrated by column chromatography and renatured in RPMI media. LGG-s or purified proteins were prepared for heat-denaturing (hd, boiling, 1 h) or protease digestion (trypsin or proteinase K). LGG, LGG-s or purified proteins were used to treat young adult mouse colon (YAMC) or HT29 cells in the presence or absence of tumor necrosis factor (TNF, 100 ng/ml) alone or the "cytokine cocktail" combination of TNF (100 ng/ml), iL-lc~ (10 ng/ml) and y-IFN (100 ng/ml). Apoptosis was determined using TUNEL and DAPI and proliferation determined by colorimetric assay using MTS tetrazolium. Cellular lysates were prepared to determine Akt activation by Western blot analysis. Results: Two proteins with molecular weight of ~ 80 kDa (p80) and 42 kDa (p42) are purified from LGG-s. Both of these proteins stinmlate Akt activation in a concentration-dependent manner in YAMC cells. Furthermore, these two proteins are heat-labile, and protease-sensinve for Akt activation. In addition, both p80 and p42 inhibit "cytokme cocktail" induced apoptosts in HT29 cells. Furthermore, YAMC cell proliferation ~s stimulated by LGG-s or the purified proteins. Conclusion: Our findings suggest that p80 and p42 purified from LGG-s mediate

792 STAT Signaling Underlies Differences Between Flagellin-Induced and TNFInduced Epithelial Gene Expression Yimin Yu, Hui Zeng, Scan Lyons, Adam Carlson, Didier Merlin, Andrew S. Neish, Shanthi V. Sitaraman, Andrew T. Gewirtz Background: We have shown that, although both bacterial flagellin and the cytokine TNF can be categorized as being potent activators of gut epithelial pro-inflammatory gene expression, there are distinct differences in the specific patterns of gene expression induced by these agonists. The goal of this study was to characterize differences in the signaling mechanisms that underlie these differences by examining the 3 major signal transduction pathways that regulate pro-inflammatory gene expression, namely; 1) NF-KB, 2) MAPK, and 3) STAT (Signal Transducer and Activator of Transcription). Methods: T84 Model epithelia were

AGA Abstracts

A-106

intestinal cell proliferation and survival. This response appears to be through activation of cell growth regulating signal, Akt.

E~Brneprazole40 mg mep'azole 20 mg Change in upper GI mean symptom =core Study A p value vs placebo Study B p value vs placebo

795 t Esomeprazole Improves Quality of Life in Patients with Upper GI Symptoms Associated with Long-Term NSAID Therapy Chris J. Hawkey, Neville D. Yeomans, Roger Jones, James M. Scheiman, Goran hagstrom, Martha Lamm, Jorgen Naesda], Ingela Wiklund

-2.41 <0.0001 -2.19 <0.0001

-1.63 -1.58

797

Purpose: Upper gastrointestinal (G1) symptoms such as abdominal pain and heartburn occur in up to 40% of NSAID users, which significantly impacts on their health-related quality of life (HRQL). This muhicenter, double-blind study compared the effects of esomeprazole and placebo on HRQL in a non-ulcer population with upper Gl symptoms associated with continuous NSAID use. Methods: 608 patients with upper Gi symptoms of at least moderate seventy associated with continuous daily NSAID treatment were randomized to receive esomeprazole 20 mg (n = i95) or 40 mg (n = 206), or placebo (n = 207), once daily for up to 4 weeks. Patients were excluded if they had gastric or duodenal ulcers, were Helicobacter pylori-positive or had erosive esophagitis. HRQL assessments were made at baseline and the last study visit using the Quality of Life in Reflux and Dyspepsia (QOLRAD) instrument, the Gastrointestinal Symptom Rating Scale (GSRS) and the Short Form-36 (SF-36). Results: On the SF-36, the study population scored significantly lower on all dimensions than reference populations of healthy individuals or patients with GERD, and on the dimensions of mental health, role emotional and general health compared with a reference population of patients with arthritis. Esomeprazole significantly improved HRQL on the QOLRAD (increased scores) and GSRS dimensions (decreased scores) a priori specified (Table 1). In addition, both doses of esomeprazole produced greater improvements compared with placebo in the vitality and physical/social functioning QOLRAD dimensions and the diarrhea and constipation GSRS dimensions. Conclusion: Treatment with esomeprazole 40 mg or 20 mg equally improves important aspects of HRQL in patients with upper G[ symptoms associated with long-term NSAID use. Data from the SF-36 confirm that in such patients, in addition to the impact of ~fie underlying condition requiring NSAID therapy, NSAID-induced upper GI symptoms contribute to further worsening of HRQL

Non-selective NSAIDs and COX-2 Selective Inhibitors: GI Risk and Use of Proton Pump Inhibitor Prophylaxis Joshua J. Ofman, Enkhe Badamgarav, Kevin Knight, James M. Henning, Katrine Wallace, John Wong, Michelle Dylan, Loren Laine Background: NSAIDs are associated with serious complications such as ulcer bleeding and perforation. In high-risk patients, PPI co-therxpy or use of COX-2 selective inhibitors (coxibs) may reduce GI complications. Objective: To assess use of PPIs and subsequent occurrence of G1 complications in patients initiating non-selective NSAIDs or coxibs. Methods: The Medstat MarketScan database of 4.6 covered million fives was used to identify enrollees aged 18 or older with coverage from 6/1/1998 to 12/31/2001. Coxib and non-selectwe NSA1D initiators were those with at least 3 prescription fills within 180 days. Subjects 65 or older with ulcer, G1 hemorrhage, or anticoagulant use were defined as high risk. PPI use within 15 days of initiating coxibs/NSAIDs was considered prophylactic. Proportions with GI complications (evidence of ulcer, GI hemorrhage and perforation) in the year following initiation were calculated. Results: 45.7% of coxib initiators and 22.6% of non-selective NSAID initiators were at high risk. Prophylactic PPI use was more common among coxib initiators (difference = 6.5%, 95% CI 5.8 %-7.1% (high risk pts) and 6.5%, 95% CI 6.1%6.9% (others)). Occurrence of GI complications was greater in coxib initiators (difference = 1.1%, 95 % CI 0.5%-1.6% (high risk pts) and 1.3 %, 95% CI 1.0%-1.5% (others))(Table 1). Conclusion: A higher proportion of coxib initiators were at risk for GI complications, and GI complications were more common among coxib initiators than those starting nonselective NSA1Ds. While a relatively small proportion of patients initiating coxibs/NSAIDs received PPIs, their use was more common in coxib initiators, even those with lower G1 risk. Outcomes studies are needed to justify combined use of coxibs and PPIs and to explore the association between NSAID type and G1 complications.

Table 1. Mean changes from baseline in QOLRAD and GSRS scores at last visit. Esemeprazole 40 rag Esomeprazole20 nlg QOLP~D EmoGcoaldl~rses 1.451" 1.54" Sleep dysfunction 1.43T 1.54T Food/drink problems 1,56T 1.76" GSR8 Reflux -t.69" -1.87" Abdominal pain -1.25~t -1.48t Indigestion -0.99 -1,26T *p~.O001 vs placebo; 1p~0.01 vs placebo; $p<0.05 vs placebo

-2.12 0.0003 -2.08 0,0002

Placebo

Placebo

Table I

1.00 1.09 1,18

Group

N

Coxib GI risk No GI risk Non-selective NSAID GI dek No GI dsk

-1.09 -1.05 -0.95

69,464 31,725 37,739 37,100 8,395 28,705

N (%) with Prophylactic PPI use 7,347 (10.6) 3,680 (11.6) 3,667 (9.7) 1,705 (4.8) 525 (6.3) 1,180 (4.1)

N (%) with GI complica. .b~ona 3,153 (4.5) 1,832 (5.8) 1,321 (3.5) 1,068 (2.9) 405 (4.8) 663 (2.3)

796

798

Esomeprazole Provides Effective Control of NSA1D-Associated Upper GI Symptoms in Patients Continuing to Take NSAIDs Neville D. Yeomans, Chris J. Hawkey, Roger Jones, James M. Scheiman, Goran I.angstrom, Martha Lamm, Jorgen Naesdal, Bolennart Eriksson

Proton Pump Inhibitor Use in Patients with Indications for Low-Dose Aspirin Who Start COX-2 Selective Inhibitors or Nonselective NSAIDs Joshua J. Ofman, Kevin Knight, Enkhe Badamgarav, James M. Henning, Katrine Wallace, John Wong, Michelle Dylan, Loren Laine

Purpose: An estimated 15-40% of NSAID users experience upper GI symptoms such as abdominal pain and heartburn, which significantly impacts on their quality of life. Recent studies have demonstrated that symptomatic patients have an increased risk to develop upper GI mucosal damage, bleeding or perforation. Two studies were performed to compare esomeprazole with placebo in treating upper G1symptoms associated with continuous NSAID use in a non-ulcer population. Patients & Methods: Patients, aged 18 years or over, were required to have a chronic condition requiring stable, continuous daily NSAID treatment for at least 7 months. Endoscopy was performed to ensure that patients had no gastroduodenal ulcers, no erosive esophagitis and were Helicobacter pylori-negative. Following a 7-11 day run-m, 608 and 556 patients with moderate or worse upper GI symptoms (measured on a 7-point scale, from 0=none to 6 = very severe) on at least 3 days of the run-in period were entered into two randomized, double-blind, parallel-group trials (studies A and B). Patients received esomeprazole 40 rag, 20 mg or placebo orally, once daily for 4 weeks. The primary endpoint was the mean change in the upper G[ symptom score from the last 7 days of the run-in to the last 7 days in the study. A difference of 0.5 on this scale between active treatment and placebo is considered clinically significant Relief of symptoms was defined as "any 7 consecutive days with a diary assessment of 'none' or 'minimal', but with up to 2 days rated as 'mild' during the 7day period" Results: In the ITT populations of 594 and 552 patients, both doses of esomeprazole were significantly more effective than placebo in relieving upper GI symptoms (table). Median time to relief of symptoms was 10 and 11 days with esomeprazole 40 mg and 11 and 10 days in each study with esomeprazole 20 mg, compared with 17 and 21 days with placebo (p=0.0053 and p = 0 0002 for esomeprazole 40 mg vs placebo and p=0.0137 and p
Background: Many NSAID users take low-dose aspirin. Concomitant use of other NSAIDs in such patients significantly increases risk of GI bleeding. Use of gastroprotective therapy among aspirin users who initiate NSAID therapy has not been well explored. Objective: To examine use of proton pump inhibitor (PPI) therapy in patients likely to be on aspirin treated with cox-2 selective inhihitors (coxibs) or non-selective NSAIDs. Methods: The Medstat MarketScan database of 4.6 covered million lives was used to identify enrollees aged 18 or older with coverage from 6/1/1998 to 12/31/2001. Coxib and non-selective NSA1D initiators were defined as those with at least 3 prescription fills within 180 days. Coxib initiators were divided into those with (i.e. switchers) and without non-selective NSAID use in the previous year. High risk for cardiac events (evidence of coronary artery disease, diabetes, or >10% 10-year risk for a cardiac event by Framingham criteria) was considered a proxy for aspirin use. PP1 use within 15 days of initiating coxibs/NSAIDs was considered prophylactic. Results: Prophylactic PPI use was more common in those with vs. without indications for low-dose aspirin (p<0.001) in both the coxib and non-selective NSAID initiator groups. Prophylactic PPI use was also more prevalent among coxib initiators than NSAID initiators (p<0.001) for high and low cardiac risk patients. Conclusion: Coxib initiators were more likely to meet criteria for aspirin use and receive prophylactic PPIs than those starting non-selective NSAIDs. Even in patients likely to be taking NSAIDs plus aspirin, with attendant risk of GI bleeding, prophylactic use of PPI was infrequent. Reasons for limited use of PPIs in patients on NSAIDs and aspirin merit exploration.

Group Coxib with no prior NSAID use Coxib with prior NSAID use Naproxen Non.ulectlve NSAIDs

Ao107

High ask for cardiac events N

Prophylactic PPI use

Prophylactic PPI use

N (%)

Low dek for cardiac events N

36,472

11,848

1,503 (13)

24,624

2,410 (10)

32,992

10,059

1.199 (12)

22,933

2,235 (10)

8,341 28,759

1,777 6,309

92 (5) 441 (7)

6,564 22,450

242 (4) 930 (4)

N

N (%)

AGA Abstracts