PLAA levels in stimulated cells with a concomitant increase in PGE2 and PEA 2 activity. The role of PLC and PLD in PG production remains unclear and it is not known whether PLAA could activate PLC and PLD. P!_AA shares homology with melittin, a 26 amino acid peptide, which others and we have shown recently to regulate PLD activity. Methods: Inhibitors to PLC and PLD were tested for their ability to reduce PGE2 production in LPS and melittin- stimulated murine macrophages. PLAA's rote as an intermediate signaling molecule in regulat- ing phospholipase activity was investigated by immunoprecipitation to determine if PI_AA required phosphorylation for activity. Finally, biopsy specimens from patients with various levels of inflammation were analyzed by Western analysis for levels of PI-A2, COX-2, PLC, PLD and PLA2 activity to establish the role of these mediators in clinical forms of inflammation. Results: Our studies with D6O9, a PC-PLC inhibitor, and Propranolol, a PLD inhibitor, indicated significant reduction in PGE2 production in LPS-stimulated RAW cells. Results from immuno- precipitation analysis of PI_AA showed it to be a phosphoylated protein both under stimulated and unstimulated conditions indicating that indeed PLAA is phosphorylatecl, although this phosphorylation is not necessapj for its activity. Melittin, which shares homology with a region of PL.AA increased PGE2 production in stimulated cells primarily through activation of PLD. Biopsy specimens from patients with inflammation showed elevated levels of PLAA, COX-2 and phospholipase activity as compared to non-inflamed patients. Conclusions: Our data indicate that PLC and PLD play a role in inflammation in addition to the widely accepted role of PLA2. Therefore, both PLC and PLD provide new targets, in addition to PLAA, for controlling inflammation in IBD.

980

Enhanced Urinary F2-1soproslanes In Patients With Crohn's Disease Bruno Bonaz, Dept of Gastroenterology, Grenoble France; Jean-Luc Cracowsld, J Beasard, Lab Pharmacology, LSCPA (EA2937), Grenoble France; Catherine Anglade, Dnpt of gastroenterology, Grenoble France; Germain Bessard, Lab Pharmacology, LSCPA (EA2937), Grenoble France; Jacques Fournet, Dept of Gastroenterology, Grenoble France

Background: /n vitro studies have implicated free radical generation in the pathopbysiology of Crohn's disease. However, definitive evidence is still lacking due to the limitations of current indexes of oxidative stress in vivo. A new family of prostaglandin F2 isomers, called 1:2- isoprostanes, produced in rive by free radical peroxidation of arachidonic acid, has recently been described (1). The quantification of iPF2,~-III, an F2-isoprostane isomer, has been sug- gested to be a reliable marker of oxidative injury in vivo (2). However, no data is currently available in Crohn's disease. The objectives of the present study were: a) to investigate urinary iPF2=III formation as an index of lipid peroxidation in patients with Crohn's disease, b) to test whether lipid peroxidation correlates to inflammation. Methods: 23 patients with Crohn's disease either with clinical relapse (n = 12; CDAI>150) or in remission (n = 11; CDAI<150) and 23 healthy controls matched in sex, age and cigarette smoking were studied. Urinary iPF2a-III concentrations were quantified using gas chromatography/electronic impact mode mass spectrometry. Results: iPF2,~-ltl urinary concentrations were 1.8 times higher in patients compared to controls (179 _+ 29 vs 94 + 7 pmol/mmol creatinine respectively; p < 0,01, Mann-Whitney test). There was a non-significant trend towards an increased iPF2a-III urinary concentrations in patients with clinical relapse compared to patients with clinical remission (227 _+ 49 vs 125 _+ 19 respectively; p = 0.09, Mann-Whitney test). Plasma C-reactive protein corretated to iPF2~ctil urinary concentrations (r = 0,46; p < 0,05, Spearman rank correlation test). Conclusions: this study provides evidence of enhanced lipid peroxiclation in patients suffering from Croltn's disease, with a correlation to inflammatory markers, iPFz,x- III urinary concentrations may represent a valuable tool in the follow-up of such patients. (1) Morrow JD et al. Proc Nafl Acad Sci USA 1990; 23: 9383-87. (2) Roberts LJ et al. Free Radic Biol Med. 2000; 28: 505-13.

981

Platelet Activation May Initiate Lencocyte-Platelet Aggregation In Inilammatmy Bowel Disease Urooj Azam, St Bartholomew's and the Royal London Sch of Medicine, London United Kingdom; Peter M Irving, St Bartholomew's and the Royal London Sch of Medicine, London United Kingd; Lee Webb, Louise Langmead, Marion G. Macey, David S. Rampton, St Bartholomew's and the Royal London Sch of Medicine, London United Kingdom

Background: We have recently shown that the formation of leucocyte-platelet aggregates is increased in the peripheral blood of patients with IBD (irving, UEGW 2000). Platelets are known to have proinflammatory as well as thrombotic effects and their activation is increased in IBD (Collins, Gastroenterology 1994;106:840-5). Aims: To assess whether platelet and/or leucocyte activation are correlated with, and might therefore induce formation of leucocyte- platelet aggregates in IBD. Methods: 56 patients with IBD (29 Crohn s disease, 27 ulcerative colitis) and 19 healthy controls had venous blood drawn into EDTA and CTAD (citrate, theophylline, dipyridamole and adenosine). Samples were immediately mixed and then analysed by flow cytometry for P-selectin (CD62P for platelet activation), L-selectin (CD62L for neutrophil activation) and CD45/CD42a (leucocyte-platelet aggregates). Platelet activation was also assessed by analysis of mean platelet mass, an inverse composite measure of platelet degranu- lation and volume, using an ADVIA Haematology System (Bayer). Results: We confirmed that leucocyte-platelet aggregation was increased in IBD (median 4.3 (range 1.2-17.3)) compared with controls (3.3 (1.5-7.4), p<O.05). The mean piatelet mass was lower in IBD (1.9 {1.6- 2.2)) than in controls (2.0 (1.7-2.3), p<O.05), but activation of platelets as measured by CD62P and of leucocytes (CD62L) were similar in the two groups. Platelet (CD62P), but not leucocyte (CD62L), activation correlated positively with leucocyte-platelet aggregates in IBD (R0.44, P<O.01). Conclusions: Leucocyte-platelet aggregation is increased in patients with IBD, and the correlation with piatelet activation measured by P-selectin, supports the hypothesis that platelets may contribute to the pathogenesis of the disease by aggregating with circulating neutrophils. The formation of aggregates does not appear to be related to the expression of CD62L on neutrophils.

982

Glycoalkaloids Ingested From Potatoes Adversely Affect intestinal Permeability and Aggravate inflammatory Dowel Disease Bijal Patel, Robert Schatte, Peter Spurns, Richard N. Fedorak, Univ of Alberta, Edmonton Canada Background: Disruption of intestinal epithelial barrier integrity is important in initiation and perpetuation of inflammatory bowel disease (IBD). Glycoalkaloids, e-solanine (S) and chaconine (C), as toxins present in high concentrations in fried potatoes, permeabilize choles- terol containing biomembranes and disrupt intestinal barrier integrity. Coincidentally, the prevalence of IBD is highest in countries where consumption of fried potatoes is prominent. Objective: We examined the effect of potatoe glycoalkaloids on epithelial barrier integrity and intestinal injury. Methods: Project 1:T84 epithelial monolayers were exposed to S (O-50p.M), C (O-20pM), or an S:C mixture (1:1, O-20/~M) and alterations in epithelial monolayer permeability assessed. Project2: To determine whether glycoalkaloids affected mammalian intestinal barrier integrity and function in animals with IBD, to a greater extent than control animals, an S:C mixture (1:1, 5-lOp.M) was exposed to sheets of intestine mounted in Ussing chambers from control and ILlO-deficient mice with geneticlly-induced IBD. Intestinal barrier integrity (3H mannitol flux) and epithelial fluid and electrolyte absorption was determined. Project 3: Finally, the effect of glycoalkaloids on in vivo intestinal function and injury was examined in ILIO deficient (IBD) and control mice, orally fed, for 10 days, amounts of glycoalkaloids that would be consumed in a normal diet. Results: Project 1:In T84 monolayers, glycoalkaloids embedded themselves and permeabilized the epithelial membrane bilayer in a dose dependent fashion with S:C>C>S. Project 2: ln vitro Ussing chamber experiments also illustrated a glycoalkaloid- induced dose-dependent intestinal epithelial barrier disruption, which permitted increased exposure to luminal antigens. In addition, animals with a genetic predisposition to develop IBD had greater glycoalkaloid-induced disruption of their intestinal epithelial barrier than did controls. Project 3: Similarly, in vivo feeding experiments demonstrated that orally ingested S:C caused a more severe mucosal inflammatory response in the intestine of mice with IBD, relative to non-IBD control mice. Conclusion: Intraluminal concentrations of glycoalkaloids, attainable during ingestion of deap-fried potatoes: (1) adversely affects the function of mamma- Ilan intestine, (2) disrupts epithelial barrier integrity, thereby permitting sieving of luminal antigens, and (3) aggravates existing IBD.

993

Effects oi 61ecocodiceids on Circulating Concentrations el Soluble Intercellular Adhesion Moleonle-1 (ICAM-1) in Ulcerative Colitis. Die H. Nielsen, Dept of Gastroenterology C, Hurley Univ Hosp, Herlev Denmark; Ben Vainer, Dept of Medicine M, Glostrup Hosp, Glostrup Denmark

Background: Glucocorticoids (e.g. prednisolone) are cornerstones in the treatment of acute exacerbations of ulcerative colitis (UC). Their mode of action remians unclear, but influence on the leukocyte/endothelial interaction appears to be part of their functional mechanism, although only little evidence suggest a direct inhibition of the expression of specialized cell adhesion molecules. During exacerbations of UC, a soluble form of intercellular adhesion molecule-1 (slCAM-1) is present in the circulation, and it has been hypothesized that slCAM- 1 might exert anti-inflammatory properties through competitive binding to its leukocyte recep- tors. Previous studies have shown that prednisolone specifically decreases the ICAM-1- mediated migration of neutrophils. Thus, the aim of the present study was to evaluate slCAM- 1 concentrations during prednisolone treatment of UC patients. Methods: Prednisolone (40 mg) was prescribed to 15 UC patients with active disease scored by a semi-quantitative scale. At inclusion and after two weeks of prednisolone treatment, plasma slCAM-1 levels were measured with an ELISA technique. Results: The concentrations of slCAM-1 was significantly decreased during the treatment period from median 256.2 ng/ml (interquartile range: 239.7- 321.0 og/ml) at Day 0 to median 220.4 ng/ml (196.0-276.3 ng/ml) at Day 14 (p<O.01). The decrease in slCAM-1 concentrations correlated with an improvement of disease activity, assessed both clinically and endoscopically (rs=O.8; p<O.O03). Conclusion: Although the observations indicate that plasma slCAM-1 is an inactive by-product of local inflammation occurring through shedding from the endothelial membrane, it was shown that plasma slCAM- 1 might be used as a biochemical disease activity marker in the individual UC patients.

984

Felminant Jejuno-Ileitis Induced By Ablation Of Enteric Gila In Adult Transgonic Mice Toby G. Bush, Univ of Nevada Sch of Medicine, Reno, NV; Tor C. Savidge, Harvard Medical Sch, Boston, MA; Tom C. Freeman, Sanger Ctr, Cambridge United Kingdom; Hilary J. Cox, Brain Repair Ctr, Cambridge United Kingdom; Elizabeth A. Campbell, Sanger Ctr, Cambridge United Kingdom; Michael V. Sofroniew, UCLA Sch of Medicine, Los Angeles, CA Enteric gila form a large and widespread network of cells at all levels of the gastrointestinal (GI) tract. To investigate the roles of such glial cells, we targeted their ablation genetically. METHODS: We created transgenic mice expressing herpes simplex virus thymidine kinase (HSV-tk) from the mouse glial fibrillary acidic protein (GFAP) promoter. Cells that express HSV-tk are rendered selectively vulnerable to the anti-viral agent ganciclovir (GCV). GCV was administered by sub-cutaneous mini-osmotic pump (lOOmg/kg/day). RESULTS: GCV delivery was invariably fatal within 19 days. RT-PCR screening of major organs revealed expression of both GFAP and HSV-tk in the brain, gut, heart, lung, liver, kidney, adrenal gland and spleen. Histopathological analysis, however, revealed major changes were confined to the small intestine, which showed mucosal degeneration, capillary dilation, erythrostasis, granulocytic inflammatory infiltration and haemorrhagic necrosis. There was never any evidence of stasis of gut contents; in all cases normally formed stool pellets were found in the colon. Pathological changes correlated with a loss of GFAP and HSV-tk mRNA from the small intestine and not other organs. Morphometrical analysis demonstrated perturbation of the crypt-villus architecture and mucosal hyperplasla with a 50% increase in crypt dept. Villus atrophy was not pronounced, but villi exhibited a range of cellular abnormalities. There was an accompanying

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