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Liver free papers 001–011

001 PLASMA INTERLEUKIN-18 (IL-18) LEVELS IN CHRONIC ALCOHOLIC LIVER DISEASE (ALD) AND ALCOHOLIC HEPATITIS (AH) ARE RELATED TO DISEASE SEVERITY

C.D.J. Evans1, S.E. Robertson2, J.A. Gracie2, I.B. McInnes2, A.J. Morris.

1Department of Gastroenterology, Glasgow Royal Infirmary, UK; 2CRD, Glasgow Royal Infirmary, UK

Aim and Background: IL-18 is a novel pro-inflammatory cytokine of the IL-1 family and is critical in the development of Th-1 responses. Th-1 responses have been implicated in the pathogenesis of ALD. We investigated IL-18 expression in patients with ALD and AH.Gut i):A1–A116

Patients and Methods: We studied inpatients with ALD (n = 48, I/P ALD), outpatients with compensated ALD (n = 13, O/P ALD), and healthy volunteers (n = 15, HVs). The mean age of the I/P ALD (40 males) was 47.7 years and that of the O/P ALD group 55.2 years. Other forms of chronic liver disease were excluded by standard serological tests. IL-18 levels were measured by ELISA (Diaclone). Whole blood cell culture (WBC) +/− lipopolysaccharide (LPS) was performed to assess IL-18 production. A Childs-Pugh score and a Maddrey’s Discriminant Function were calculated for all patients. The results were analysed by Mann-Whitney U test.

Results: Inpatients consisted of five with Childs grade A, 17 B, and 26 C. Eighteen had a MDF < 24, 9 a MDF of 24–32, and 21 a MDF > 32. Plasma IL-18 levels were significantly elevated in the I/P ALD group when compared with O/P ALD and HVs (all units pg/ml), (174 +/− 170, 45 +/− 52, and 32 +/− 83; p < 0.01 and p < 0.001). IL-18 levels in Child’s C inpatients (226 +/− 214) were significantly elevated compared to Child’s A inpatients (27 +/− 38; p < 0.01). Higher plasma IL-18 levels were found in inpatients with moderate/severe AH (MDF > 24) when compared with inpatients with MDF < 24, O/P ALDs, and HVs (224 +/− 203, 93 +/− 89, 45 +/− 83, and 32 +/− 52; p < 0.02, p < 0.002, and p < 0.0001, respectively). Patients with Childs grades B/C showed no incremental rise in IL-18 production following LPS stimulation. A similar result was obtained on analysis of patients with moderate/severe AH (206 +/− 190 to 217 +/− 214).

Conclusions: Plasma IL-18 is elevated in patients with ALD. IL-18 levels are higher in patients with increasing severity of ALD (Childs grade B/C) and AH (MDF > 24). In patients with Childs B/C ALD and AH, LPS stimulation resulted in no incremental rise in IL-18 production, suggesting prestimulation to a ‘maximal’ level in more severe ALD. These results support the role of immune dysregulation in moderate/severe ALD and AH.

002 TREATMENT WITH ANTI-TUMOUR NECROSIS FACTOR α(TNFα) ANTIBODY IMPROVES CLINICAL PARAMETERS AND LOWERS PORTAL PRESSURE IN ALCOHOLIC HEPATITIS

R.P. Mookerjee, S. Sen, N.A. Davies, S.J. Hodges, R. Williams, R. Jalan.

Institute of Hepatology, UC, 69–75 Chenies Mews, London WC1E 6HX, UK

Background: Patients with alcoholic hepatitis (AH) and cirrhosis exhibit an inflammatory state in addition to a hyperdynamic circulation and portal hypertension. TNFα is a key mediator in AH, promoting a cascade of inflammatory activity. In this study we addressed the effects of the chimeric anti-TNFα antibody, infliximab, on clinical parameters and portal haemodynamics, and tested the hypothesis that the development of portal hypertension in AH is associated with inflammation.

Methods: Sixteen patients with biopsy proven AH (mean age 57 ± 4 and discriminant function 52.4 ± 6.8) were treated with the chimeric, monoclonal anti-TNFα antibody, infliximab (5 mg/kg lean body weight). Ten of these patients had assessment of their wedged hepatic venous pressure (WHVP) before, 24 hours, and 28 days after infliximab treatment. Patients’ clinical and biochemical profiles were monitored during the study period.

Results: Significant improvements were noted by 28 days in bilirubin (293 ± 33 to 130 ± 21; p < 0.001), CRP (73 ± 10.9 to 36.1 ± 12.7; p <0.01), white cell count (15.9 ± 2.1 to 10.51 ± 1.5; p < 0.05) and SIRS score (2.6 ± 0.4 to 0.23 ± 0.12; p < 0.01). Three patients died during the study period, with two developing sepsis, both from Staphylococcus aureus. No patient developed renal failure or hepatorenal syndrome, or required intensive care support. WHVP reduced significantly from 38.63 ± 2.39 to 29.14 ± 1.42 mm Hg at 24 hours, and the reduction was sustained at 28 days (27.4 ± 2.82 mm Hg; p < 0.01) with no significant differences in free HVP or CVP. Mean arterial pressure (MAP) increased significantly (71.6 ± 1.72 to 81.1 ± 3.15 mm Hg; p < 0.05 at 28 days).

Conclusion: Anti-TNFα treatment results in a significant reduction in bilirubin, white blood cell count, CRP, and SIRS score, coupled with an apparent improved clinical outcome. This improvement in clinical and inflammatory parameters is accompanied by a highly significant, early, and sustained reduction in HVPG, as well as a significant increase in MAP. We conclude that portal hypertension in AH has a significant inflammatory component that can be partially corrected with anti-TNFα therapy. This provides some insight in to the possible mechanisms of portal hypertension in AH.

003 PREDICTORS OF SHORT AND MEDIUM TERM MORTALITY IN ACUTE ALCOHOLIC HEPATITIS

E.H. Forrest1, C.D. Evans2, L.S. Murray3, A.J. Morris2. 1Department of Gastroenterology, Victoria Infirmary, Glasgow;2 Glasgow Royal Infirmary, Glasgow; 3Department of Medicine and Therapeutics, University of Glasgow, Glasgow

Introduction: The modified Maddrey’s Discriminant Function (DF) is used to identify poor prognosis acute alcoholic hepatitis (AAH). This calculation requires conversion of serum bilirubin from mg/dl to μmol/l and a corrected calculation of prothrombin time (PT) prolongation. The DF is therefore awkward to use in a clinical setting. We aimed to identify variables related to mortality in patients with AAH.

Methods: Clinical and laboratory data was collected from 256 patients with clinical AAH. Stepwise logistic regression identified variables associated with mortality.

Results: Day 1 and 7 variables associated with short term (28 day) and medium term (84 day) mortality are displayed in Table 1.

Abstract 3 Table 1

The correct predictions for each model and the DF are shown in Table 2.

Abstract 3 Table 2

Conclusions: The DF failed to identify the majority of patients who died. The variables we identified significantly improved the identification of patents with poor short and medium term prognosis.

004 WORSENING IF CEREBRAL HYPERAEMIA IN ACUTE LIVER FAILURE (ALF) WITH TERLIPRESSIN

D.L. Shawcross1, R.P. Mookerjee1, P.C. Hayes2, A. Lee2, R. Williams1, R. Jalan1. 1Liver Failure Group, Institute of Hepatology, 69–75 Chenies Mews, London WC1E 6HX, UK; 2Scottish Liver Transplantation Unit, Royal Infirmary of Edinburgh

Background: The role of terlipressin in ALF patients is not clear. Terlipressin acts through the V1 and V2 receptors. V1 receptors, distributed in the systemic circulation, mediate vasoconstriction. V2 receptors, distributed in the cerebral vasculature, in contrast mediate cerebral vasodilatation through a nitric oxide dependent mechanism. It is therefore possible that terlipressin may accentuate cerebral hyperaemia and worsen intracranial pressure (ICP) in ALF. This study evaluated the haemodynamic effect of a small dose of terlipressin in six patients [median age 27 (22–46), four female] with ALF (paracetamol four, acute fatty liver of pregnancy one, non A/non B viral one) and grade IV encephalopathy.

Method: ICP was continuously monitored in five/six patients using a subdural fibreoptic system. Cardiovascular haemodynamics were monitored with pulmonary artery, right atrial, and arterial catheters. Jugular venous oxygen saturation (JVOS) was monitored using a reverse jugular catheter and cerebral blood flow (CBF) was measured using a modified Kety-Schmidt technique, prior to and 1 hour after administration of 0.01 mg/kg of terlipressin intravenously as a single bolus.

Results: There was no significant rise in mean arterial pressure (MAP). ICP increased in all patients [median 15 (range 13–18) to 20 (range 16–23) mmHg; p < 0.02] after 1 hour and returned to baseline values after 3 hours. CBF increased significantly at 1 hour [median 69 (range 48–79) to 81 (range 62–97) ml/100 g/min; p < 0.02. JVOS increased at 1 hour [median 75% (range 67–89) to 87% (range 75–94)].

Conclusions: Administration of even a sub-therapeutic dose of terlipressin increases CBF and consequently ICP, without causing significant change to systemic haemodynamics. Terlipressin may therefore have deleterious consequences through worsening of cerebral hyperaemia and ICP. These data suggest extreme caution and close monitoring if this drug is used in patients with ALF.

005 ISOLATION AND CHARACTERISATION OF HUMAN HEPATIC STEM CELLS IN FETAL LIVER

I.S. Currie1, N.M. Masson1, S.R. Dundas1, J.R. Black1, R.A.L. Bayne2, R.W. Parks1, O.J. Garden1, J.A. Ross (introduced by N. Finlayson)1. 1University of Edinburgh Dept of Clinical and Surgical Sciences, Royal Infirmary of Edinburgh, Lauriston Place, Edinburgh, EH3 9YW; 2MRC Human Reproductive Science Unit, Centre for Reproductive Biology, University of Edinburgh, Little France Crescent, Edinburgh

Introduction: Hepatic stem cells have been described in rat which express the surface antigens CD90 (Thy-1) and CD34. We hypothesised that a similar population may exist in human fetal liver, giving rise to hepatocyte precursors after cytokine treatment in vitro.

Methods: Human fetal liver was subject to collagenase digestion. The cells were labelled with anti-CD90 monoclonal antibody, anti-mouse phycoerythrin conjugate and FITC-conjugated monoclonal anti-CD34 antibody. Fluorescence-activated cell sorting provided CD90+ve, CD90−ve/CD34+ve or CD90+ve/CD34+ve cells, which were cultured for 7 days in the presence and absence of cytokines (stem cell factor, thrombopoietin and Flt3-ligand). Cultures were fixed and immunostained to show biliary tract (cytokeratin 19 (CK19)) and hepatocyte (cytokeratin 18 (CK18), Fibrinogen (FIB)) markers.

Results: Two per cent of the liver cell suspension was CD90+ve, 9% was CD34+ve/CD90−ve, and 2% was CD34+ve/CD90+ve. By day 7 in vitro, CD34+ve cells were absent. By contrast, CD90+ve cells were seen in cultures derived from CD90+ve cells and, surprisingly, from CD34+ve/CD90-ve cells. In cultures prepared from CD34+ve/CD90+ve cells, survival depended on cytokines. Many small, fibrinogen−ve, CK19+ve cells and other slightly larger cells staining for CK18 were noted. In cultures of CD34+ve/CD90−ve cells, we observed cells positive for FIB or CK19. In CD90+ve cell cultures, cytokines had little effect. Occasional cells with hepatocyte morphology, which were positive for FIB and CK19 or CK18, were seen in these cultures alone.

Conclusion: Cultures of purified human hepatic stem cells showed that CD90+ve cells can produce bipotential hepatocyte/ biliary tract (FIB+ CK19+) cells and monopotential hepatocytes (FIB, CK18). Conversely, other stem cell types may produce earlier liver cell precursors.

006 TOLL-LIKE RECEPTOR EXPRESSION IN CIRRHOSIS

S.M. Riordan1, N. Skinner2, A. Nagree1, H. McCallum2, C.J. McIver1, J. Kurtovic1, J.A. Hamilton3, S. Bengmark4, R. Williams4, K. Visvanathan2.

1Gastrointestinal and Liver Unit, The Prince of Wales Hospital, Sydney, Australia; 2Murdoch Children’s Research Institute, Melbourne, Australia; 3Department of Medicine, Royal Melbourne Hospital Melbourne, Australia; 4Institute of Hepatology, University College London, UK

Background: Pro-inflammatory cytokines such as tumour necrosis factor α (TNFα) contribute to liver damage in cirrhosis. Relevant to this is the expression of toll-like receptor (4) and TLR2, critically involved in TNFα production in response to endotoxin and Gram-positive stimuli, respectively, the first studies on which in cirrhosis are reported here. Methods: We measured in 36 cirrhotic patients and 32 controls (a) circulating endotoxin and TNFα levels; (b) peripheral blood mononuclear cell (PBMC) expression of TLR4 and TLR2; and (c) in vitro production of TNFα by PBMCs in response to stimulation by endotoxin or Staphylococcus aureus enterotoxin B (SEB), a Gram-positive microbial antigen. To determine the role of Gram-positive gut flora, TLR2 expression and TNFα production were re-assessed after supplementation for 7 days with a synbiotic regimen (Synbiotic 2000; Medipharm, Sweden) known to increase intestinal levels of Gram-positive bacteria.

Results: Circulating endotoxin and TNFα levels were significantly increased in cirrhotic patients but not correlated. Expression of TLR2, but not TLR4, was significantly up-regulated and correlated significantly with serum TNFα levels. In vitro production of TNFα in response to stimulation with SEB was significantly blunted, in keeping with chronic stimulation by Gram-positive antigens in vivo. Oral supplementation with the synbiotic regimen resulted in further, significant up-regulation of systemic TLR2 expression. Further impairment of TNFα production in response to SEB occurred in most patients.

Conclusions: Upregulation of TLR2 but not TLR4 in cirrhosis implies, contrary to previous assumptions, an important stimulatory role for Gram-positive microbial antigens but not endotoxin. Such Gram-positive antigens may be derived from the gut. Signalling via TLR2 contributes to increased circulating TNF-α levels in cirrhosis.

007 BALANCE OF TIMP-1 AND MMP-2 DETERMINES N-CADHERIN CLEAVAGE IN HEPATIC STELLATE CELL APOPTOSIS

F. Murphy1, J. Waung1, N. Patel1, J Collins2, K. Brew3, H. Nagase4, M.J.P. Arthur1, R.C. Benyon1, J.P. Iredale1. 1Liver Group, Division of Infection, Inflammation and Repair, Southampton University, SO16 6YD, UK; 2Mucosal Immunology, Southampton University; 3The Kennedy Institute, Imperial College, London; 4Department of Biochemistry & Molecular Biology, Miami University School of Medicine, Miami, USA

Introduction: The hepatic stellate (HSC) is known to synthesise the majority of excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC also express matrix degrading metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Whereas during spontaneous recovery from experimental liver fibrosis, there is a fall in the expression of the MMP inhibitor TIMP-1 and an increase in hepatic collagenolytic activity accompanied by HSC apoptosis; in advanced cirrhosis, TIMP-1 expression is maintained, and HSC persist. We have previously demonstrated that TIMP-1 can inhibit apoptosis of HSC by mechanisms involving MMP inhibition. We have studied the role of N-cadherin because it is known to be up regulated during HSC activation and may have a role in determining HSC survival and apoptosis.

Aims: To determine the effect of blockade of N-cadherin binding on HSC; observe the fate of N-cadherin during HSC apoptosis; determine which MMP is involved and its direct effect on HSC.

Results: By Western blot both rat and human HSC express 135kDa N-cadherin. Blockade of N-cadherin promoted apoptosis of HSC. During apoptosis of HSC there is cleavage of N-cadherin into fragments of 20–100kDa in size, which is protected by TIMP-1 and a selective inhibitor of MMP-2, but not inhibitors of MMP-1 or MMP-3 or a non functional mutant T2G TIMP-1. Active MMP-2 directly cleaves N-cadherin in vitro. Active MMP-2 also promotes apoptosis of HSC.

Conclusions: These data suggest that the balance of MMP-2 and TIMP-1 determine HSC survival in hepatic fibrosis via stabilising N-cadherin.

008 IMPROVEMENT IN INDOCYANINE GREEN CLEARANCE FOLLOWING SYNBIOTIC TREATMENT IN CIRRHOSIS

J. Kurtovic1, U. Ruettimann2, H. Adamson2, D. Bihari2, S. Bengmark3, R. Williams3, S.M. Riordan1. 1Gastrointestinal and Liver Unit; 2Department of Intensive Care, The Prince of Wales Hospital and University of New South Wales, Sydney, Australia; 3Institute of Hepatology, University College London, England

Background: Intervention aimed at reducing intestinal levels of endotoxin containing Gram-negative bacteria is reported to improve systemic haemodynamic disturbance in cirrhosis. Any beneficial effect on hepatic blood flow is unknown. This study addressed this issue.

Methods: We studied 15 cirrhotic patients (hepatitis C virus, n = 7; alcohol, n = 6; primary biliary cirrhosis, n = 1; idiopathic, n = 1; Child-Pugh grade A, n = 6; B, n = 5; C, n = 4) and 11 patients with chronic hepatitis (hepatitis C, n = 9; hepatitis B, n = 1; non-alcoholic steatohepatitis, n = 1). Indocyanine green retention at 15 min (ICGR15) following an intravenous bolus of 0.5 mg/kg body weight was assessed as an index of hepatic blood flow, using a non-invasive transcutaneous system (LiMon, Pulsion, Germany). ICGR15 was measured at baseline and following oral supplementation for 7 days with a synbiotic (probiotic and fermentable fibre) preparation including Lactobacillus plantarum 2362, L paracasei subsp paracasei 19, Pediacoccus pentoseceus 5–33:3, and L raffinolactis 32–77:1, designed to increase the intestinal content of Gram-positive bacteria (Synbiotic 2000, Medipharm, Sweden).

Results: ICGR15 was significantly higher in cirrhotic patients (median 38.3, range 5.0–60%) than those with chronic hepatitis (median 5.3, range 1.8–9.7%) (p < 0.0005). Supplementation with the synbiotic regimen was associated with a significant reduction in ICGR15 in the cirrhotic group (p = 0.003). ICGR15 was reduced by a median 17.5% (range 1.4–65%) of baseline values in 14/15 (93%) such patients and increased by 4.1% in the other patient. Treatment led to no significant change in ICGR15 in patients with chronic hepatitis (p = 0.65).

Conclusions: Use of a Gram-positive synbiotic preparation significantly improves ICG clearance in cirrhotic patients, presumably by reversing a disturbance in gut flora occurring in cirrhosis but not chronic hepatitis that adversely affects hepatic blood flow.

009 CHARACTERISATION OF DIFFUSE LIVER DISEASE IN PATIENTS WITH HEPATITIS C USING31P MAGNETIC RESONANCE SPECTROSCOPY (MRS)

A.K.P. Lim1, N. Patel1, G. Hamilton1, J.V. Hajnal1, R. Goldin2, S.D. Taylor-Robinson1. 1Robert Steiner MRI Unit, MRC Clinical Sciences Centre, Imperial College, Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0HS, UK; 2Department of Histopathology, Imperial College, Faculty of Medicine, St. Mary’s Hospital, Praed Street, London W2 1NY, UK

Purpose: Liver biopsy remains the gold standard for characterising and staging diffuse liver disease. This invasive test is associated with significant morbidity and, rarely, mortality. Our aim was to investigate whether a non-invasive technique, in vivo 31P-(MRS) could grade the severity of diffuse liver disease in patients whose liver disease was attributable to hepatitis C (HCV) infection only.

Materials and Methods: Twelve controls and 47 patients with biopsy-proven HCV infection were studied prospectively. Based on their histological fibrosis (F) and necroinflammatory (NI) scores, patients were divided into mild hepatitis, n = 17 (F 2/6, NI 3/18); moderate/severe hepatitis, n = 19 (F 3/6, NI 4/18); and cirrhosis, n = 11 (F = 6). Hepatic 31P MR spectra were obtained using a 1.5 Tesla spectroscopy system (TR: 10 000; TE: 2).

Results: There was a monotonic increase in the mean ± 1 s.e. phosphomonoester (PME) to phosphodiester (PDE) ratios for the control, mild hepatitis, moderate hepatitis and cirrhosis groups: 0.16 ± 0.01, 0.19 ± 0.07, 0.25 ± 0.02, 0.38 ± 0.04, respectively (ANOVA p < 0.001). No other significant spectral changes were observed.

Conclusion:31P MRS cannot diagnose HCV infection, but in patients with proven infection, this test can characterise the severity of liver disease. We have shown that the ratio of PME to PDE resonance in 31P MRS is able to separate mild from moderate hepatitis and these two groups from cirrhosis. The ability to differentiate these populations of patients has therapeutic implications and 31P MRS, in some situations, would not only complement a liver biopsy but could replace it.

010 INVESTIGATION OF HEPATIC BLOOD FLOW USING MICROBUBBLE ENHANCED ULTRASOUND IN THE DIAGNOSIS OF CIRRHOSIS

A. Anderloni1, J. MacQuarrie2, E. Leen3, K. Oein4, W.J. Angerson2, A.J. Morris1. 1Department of Gastroenterology, Glasgow Royal Infirmary; 2University Department of Surgery, Glasgow Royal Infirmary; 3Department of Radiology, Glasgow Royal Infirmary, 4Department of Pathology, Glasgow Royal Infirmary

Background: It has been previously shown that alterations in hepatic haemodynamics can be assessed in cirrhotic patients by measuring the hepatic vein transit time of an ultrasound contrast agent.

Aim: To evaluate the clinical usefulness of a new contrast agent SonoVue, in differentiating between patients with cirrhosis, hepatitis C and normal subjects.

Methods: We studied 13 healthy controls, 14 subjects with biopsy proven hepatitis C and 18 subjects with proven cirrhosis, using a Philips-ATL 5000 scanner with a 3.5 MHZ curvilinear scanhead. An intercostal scan including the hepatic vein (HV), the hepatic artery (HA), and the portal vein (PV) was selected. Colour-Doppler gain was reduced until no signal was displayed and MI was set at 0.8. A bolus injection of 1 ml SonoVue was given. The transit time, defined as the time interval in seconds between the start of the injection and first appearance of colour-Doppler signal in the vessel, was recorded for the HA, HV, and PV. The “Intrahepatic time index”(ITI) was defined as the difference between the HV arrival time and the HA arrival time. Each scan was recorded on SVHS tape for offline review.

Results: Overall differences between groups were significant for all variables (p < 0.001). Pairwise differences between the cirrhotic group and each of the other two groups were all statistically significant (p < 0.05), and those between the hepatitis C group and controls were significant for all variables except HA (p < 0.05). An ITI 9 sec showed a sensitivity of 0.90 in detecting cirrhosis, and specificities of 0.93 and 1.00 in distinguishing cirrhosis from hepatitis and controls respectively. An ITI between 9 and 13 sec showed a sensitivity of 0.64 and specificity of 0.90 in distinguishing hepatitis from the other groups.

Conclusion: ITI is accurate in detecting cirrhosis and may be useful in identifying hepatitis C patients with underlying cirrhosis.

011 NATIONAL NEEDS ASSESSMENT FOR HEPATITIS C

J. Parkes, B. Bennett-Lloyd, P. Roderick, W. Rosenberg. Liver Epidemiology Group, University of Southampton, HCRU Level B South Academic Block, Southampton General Hospital, Tremona Road, Southampton SO16

Background: Hepatitis C virus (HCV) infection is a major health care problem. The publication of the National Strategy for Hepatitis C emphasised the need to scope the current service configuration, workloads, and capacity in the UK in order to implement the improvements in care detailed by the strategy, and to inform future planning of services. BASL and the Liver section of the BSG commissioned this national needs assessment of hepatitis C to gain this knowledge.

Aim: To conduct a national survey to determine the workload and configuration of current services available to manage patients with hepatitis C, and to identify models of good practice.

Methods: A questionnaire survey was conducted on a purposive sample of consultant members of BASL (n = 49), consultants in infectious diseases (n = 42), a 1:5 sample of GUM and GI physicians (n = 47, n = 186) stratified by Health Region. In-depth field interviews and semi-structured telephone interviews were conducted where novel or innovative models of care were identified.

Results: Preliminary data are reported, with full results available for the BSG meeting. Questionnaire response rate was 75%. Forty per cent of respondents provided a complete service from various clinical settings: hepatology (34%), GI (50%),and infectious diseases (16%). Total number of patients managed by the respondents was > 23 000 with upward trend over the past 3 years. Seventy-five per cent of referrals to these specialist service providers came with an established diagnosis of HCV. There is variation in practice for treatment decisions. Eligibility for treatment was in range 25–49% with 50–74% receiving treatment. Twenty-four per cent of specialist service providers have outreach clinics. Identified barriers to management include staffing capacity, patient non-attendance, and funding of treatment.

Conclusions: Detailed quantitative and qualitative data from this survey will inform the future planning of hepatitis C services. Preliminary data suggest significant variation in service delivery.

Endoscopy free papers 012–021

012 COLONOSCOPIC APPEARANCES HELP PREDICT DYSPLASIA RISK IN ULCERATIVE COLITIS

M.D. Rutter, G. Schofield, K.H. Wilkinson, A. Forbes, B.P. Saunders. St Mark’s Hospital, Harrow, UK

Background and aim: Colonoscopic surveillance attempts to reduce the excess cancer risk in ulcerative colitis (UC). We aimed to assess whether colonoscopic appearances help to predict cancer risk.

Methods: A case control study of patients on UC surveillance; 68 patients with dysplasia/colorectal cancer (CRC) and 136 patients without dysplasia/CRC were matched for age at onset of UC, duration, and extent of UC and sex. Medical and colonoscopic reports were reviewed, and data on colonoscopic appearances documented.

Results: 204 patients underwent 1217 colonoscopies. Patients who had one or more macroscopically normal surveillance colonoscopies were at reduced risk of dysplasia/CRC (p = 0.004, OR 0.4 [0.2–0.7]). Patients with one or more segments of severe inflammation at any of their surveillance colonoscopies were at increased risk of dysplasia/ CRC (p < 0.001, OR 4.9 [2.0–12.2]). Other results are shown in the table.

Abstract 10

Conclusion: Colonoscopic features can help predict cancer risk in UC. Severe inflammation, strictures, a shortened colon, and postinflammatory polyps appear to confer significant increased dysplasia risk, whereas a macroscopically normal colonoscopy confers a reduced (although not zero) risk of subsequent dysplasia development. Contrary to a recent report, we did not find that patients with backwash ileitis were at greater risk of developing colorectal cancer.

013 DYSPLASIA IN ULCERATIVE COLITIS IS USUALLY VISIBLE AT COLONOSCOPY

M.D. Rutter, K.H. Wilkinson, B.P. Saunders. Wolfson Unit for Endoscopy, St Mark’s Hospital, Harrow, UK

Background and Aim: During colonoscopic surveillance for dysplasia in longstanding extensive ulcerative colitis (UC), multiple non-targeted “random” biopsies of colonic mucosa are advised, based on historical data suggesting dysplasia may only be detectable microscopically. We aimed to assess what proportion of dysplastic lesions were macroscopically evident at colonoscopy.

Methods: All cases of colonoscopically detected dysplasia in a major UC surveillance programme from 1/1/88 to 1/1/02 were reviewed. Details were obtained from our colonoscopy database, case notes (including colonoscopic photographs), and histology reports. All dysplasia was assumed to be macroscopically invisible unless stated otherwise at the time.

Results: During the study period, 300 patients underwent 2189 colonoscopies. Fifty six patients had one or more biopsies showing colorectal dysplasia. In total, 92 colonoscopies yielded 106 positive biopsies. Eighty one (76%) dysplastic biopsies were from macroscopically visible lesions, and 25 sites of dysplasia (24%) were macroscopically invisible. Thirty three lesions were considered endoscopically and histologically to be tubular adenomas. Excluding these, there were 73 dysplastic biopsies (38 patients, 65 colonoscopies). 48 (66%) of these were from macroscopically visible lesions. Overall, 50 patients (82%) had macroscopically detectable dysplasia during the study period, and six patients with dysplasia (18%) had only macroscopically invisible lesions.

Conclusion: Over 80% of patients with dysplastic lesions in ulcerative colitis will develop a colonoscopically visible lesion. Even after excluding tubular adenomatous lesions, the majority of dysplasia is colonoscopically visible. Colonoscopists should concentrate on careful mucosal scrutiny for dysplastic lesions, rather than relying solely on detection by random biopsies.

014 EUS GUIDED FINE NEEDLE ASPIRATION FOR UNDIAGNOSED HILAR STRICTURES SUSPICIOUS OF CHOLANGIOCARCINOMA

A. Fritscher-Ravens1, P. Swain1, X. Rogiers2, T. Topalidis2, F. Thonke2, N. Soehendra2, D.C. Broering1. 1Department of Gastroenterology, Royal London Hospital, London; 2University Hospital Eppendorf, Hamburg, Germany

Background: Despite improvements of diagnostic modalities differentiation between benign and malignant strictures at the liver hilum remains a challenge. Extensive hepatic resections with intention to cure or modern palliative concepts require preoperative diagnosis, to avoid risk of misdiagnosis and inappropriate surgical treatment for the patients. Preoperative tissue diagnosis, most commonly achieved during ERCP, can be difficult. We used endosonography guided fine needle aspiration (EUS-FNA) for preoperative diagnosis of hilar cholangiocarcinoma.

Methods: Prospective evaluation of 44 patients (31 male, age 37–74, mean age 59) with strictures at the liver hilum diagnosed by computed tomography and/or ERCP. All were highly suspicious of hilar cholangiocarcinoma but had inconclusive brush cytology or biopsy, and subsequently underwent EUS-FNA with a linear echoendoscope and 22 gauge needles.

Results: Adequate material was achieved in 43 out of 44 patients. Cytology revealed chloangiocarcinoma in 26 patients, hepatocellular carcinoma, Hodgkin’s disease, metastasis of colon and ovarian cancer, and neuroendocrine tumour in one each. Twelve patients had benign diseases with chronic fibrosing inflammation in four, inflammation in two, sarcoid like changes in one and primary sclerosing cholangitis (PSC) in five. Four of the benign results were false negatives. Thirty five patients underwent surgery. Patients with benign results and PSC were followed up, patients with Hodgkin’s disease and metastases of other primaries had chemotherapy. In one patient adenocarcinoma was discovered at repeat surgery. No complication occurred. Accuracy, sensitivity, and specificity were 91%, 90%, and 100%, respectively. EUS and EUS-FNA changed preplanned surgical approach in 22/43 patients.

Conclusion: These results suggest that EUS-FNA may be a valuable, less invasive approach for tissue diagnosis of unknown hilar strictures. It proved to be technically feasible without significant risks, when other diagnostic tests were inconclusive and was able to change preplanned management in about half of the patients.

015 RADIATION EXPOSURE TO PERSONNEL PERFORMING ERCP

S. Singhal1, L. Naidu1, D.E. Preece2, A. Vohrah3, D.E. Loft1. 1Department of Gastroenterology, University Hospitals Coventry and Warwickshire NHS Trust; 2Department of Clinical Physics and Bioengineering, University Hospitals Coventry and Warwickshire NHS Trust; 3Department of Radiology, University Hospitals Coventry and Warwickshire NHS Trust

Background: ERCP relies on the use of ionising radiation but the risks to operator and patient associated with radiation exposure (in terms of subsequent biological damage) are unclear. The aim of this prospective study was to estimate the radiation dose received by personnel performing fluoroscopic endoscopic procedures, mainly ERCP.

Methods: Consecutive procedures over a 2 month period were included. The use of thermoluminescent dosemeters to measure radiation exposure to the abdomen, thyroid gland, and hands of the operator permitted an estimation of the annual whole body effective radiation dose (ED) equivalent to be made.

Results: During the study period 66 fluoroscopic endoscopic procedures (61 ERCP) were performed and the estimated annual whole body ED equivalent received by consultant operators ranged between 3.35 and 5.87 mSv. These values are similar to those received by patients undergoing barium studies and equate to an estimated additional lifetime fatal cancer risk between 1 in 7000 and 1 in 3500. Although within the legal safety limits for radiation exposure to personnel, these radiation doses are considerably higher than the levels deemed acceptable for the general public.

Conclusions: We suggest that personnel as well as patients may be exposed to significant levels of radiation during ERCP. We emphasise the need to carefully assess the indication for, and to employ measures that, minimising radiation exposure during any fluoroscopic procedure.

016 COULD CANCER STAGING AND TUMOUR MEASUREMENTS BE IMPROVED BY USING 3D ENDOSCOPIC ULTRASONOGRAPHY?

S. Inglis1, K.V. Ramnarine1, J.N. Plevris2. 1Medical Physics, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH3 9YW; 2Centre for Liver and Digestive Disorders, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH3 9YW

Introduction: Endoscopic ultrasound (EUS) is routinely used to stage upper GI cancers. This technique estimates depth of invasion, but may not provide reliable information on tumour length. 3D EUS could improve staging and treatment monitoring by providing complete datasets, detailing the progression of the tumour along the oesophagus and accurate dimensional measurements.

Aims: To compare (a) the measurement capabilities of 2D and 3D EUS in an oesophageal cancer phantom; and (b) the staging of patients with oesophageal cancer from 2D and 3D EUS.

Methods: Two experienced endoscopists used the Olympus GF-UM200 scope/EU-M30 processor to scan the phantom and patients at 7.5 and 12 MHz. Images were captured at 25 frames/sec. (a) In vitro 3D data were acquired from an EUS anthropomorphic oesophageal cancer phantom with known dimensions. (b) In vivo 3D data were obtained during conventional 2D EUS as the scope was withdrawn. 3D volumes were reconstructed from a series of parallel B-mode images. Dimensional measurements and cancer staging was performed during the routine test. A retrospective comparison was made between the cancer staging and dimensions obtained from 2D EUS and from our 3D EUS system.

Results: Routine 2D length measurements performed on the phantom had a mean error of 9% (max 23%). Identical measurements performed using 3D EUS had a mean error of 1.7% (max 3.7%). 3D EUS could visualise nodes < 5 mm and detected nodes not observed during routine EUS. The 3D system improved the reproducibility of the X, Y, and Z measurements of tumour and nodes with mean intra and inter observer coefficient of variation < 5%. Conclusions: This study has shown that the 3D technique can accurately measure tumour and node dimensions with good reproducibility. Since the main in vitro error was estimating the length of embedded objects, it is likely that 2D EUS cannot estimate tumour and node length as accurately as previously thought. This preliminary work suggests that 3D EUS may increase diagnostic accuracy of node staging and introduce a fifth criterion (length) to diagnose malignant nodes.

017 CHROMOENDOSCOPY WITH METHYLENE BLUE IN BARRETT’S OESOPHAGUS: ADDING INSULT TO INJURY?

J.R. Olliver1, C.P. Wild1, P. Sahay2, S.P.L. Dexter3, L.J. Hardie1. 1Molecular Epidemiology Unit, School of Medicine, University of Leeds, Leeds LS2 9JT; 2Gastroenterology Department, Pontefract General Infirmary, Friarwood Lane, Pontefract WF8 1PL; 3Department of Surgery, School of Medicine, University of Leeds, Leeds LS2 9JT

Chromoendoscopy with methylene blue (MB) is increasingly being applied during endoscopic examination of Barrett’s oesophagus (BO). MB selectively stains specialised intestinal metaplasia as it is specifically absorbed by the goblet cells characterising this tissue. Experimental studies show that photosensitisation of MB with visible light stimulates the formation of singlet oxygen species leading to the generation of single strand breaks and oxidative alterations to guanine residues of which the promutagenic DNA adduct, 8-hydroxydeoxyguanosine (8-OHdG) predominates. We hypothesised that the application of MB to the oesophagus followed by exposure to endoscopic light may stimulate high levels of DNA damage in BO. Biopsies were collected before and after MB exposure from immediately adjacent sites within BO mucosa from 15 patients. Biopsies were subject to DNA damage analysis using the comet assay to detect strand breaks and alkali-labile sites. In addition the enzyme Fapy-DNA glycosylase (FpG) was incorporated into the assay to allow the detection of FpG sensitive sites, including 8-OHdG. For every patient studied, DNA damage levels increased (range 6.0–71.9%) following MB chromoendoscopy. Statistical comparison of matched biopsies revealed a significant difference (p < 0.005) in the DNA damage level before (28.8% (24.6–32.4%), median percentage tail DNA (1st-3rd quartile)) compared to after chromoendoscopy (36.5% (32.1–42.7%)). Following MB chromoendoscopy the percentage of cells with highly damage DNA attributable to FpG sensitive sites, approximately doubled. Barrett’s mucosa is a recognised preneoplastic tissue, which exhibits many genetic and epigenetic alterations. Exposing this and any associated dysplasia to further promutagenic DNA damage, via MB chromoendoscopy warrants caution as it could potentially accelerate the carcinogenic process.

Abstract 12

018 MISSED UPPER GI CANCER: IN THE COMMUNITY OR IN THE HOSPITAL?

S. Yalamarthi, P. Witherspoon, D. McCole, C.D. Auld. Department of Surgery, Dumfries and Galloway Royal Infirmary, Bankend Road, Dumfries DG1 4AP

Aim: To determine the incidence and causes for failure in diagnosis of oesophageal and gastric cancer after referral to a surgical endoscopy unit (1994–2001).

Methods: Since the introduction of open access endoscopy in 1994, over 13 000 patients have been entered into a prospective database. From a consecutive series of oesophageal and gastric cancer (n = 305), the number of patients undergoing an endoscopy within 3 years of diagnosis were identified and the reasons for missed diagnosis documented.

Results: Thirty patients (9.89%) had a minimum of one endoscopy within 3 years of which 20 (67%) occurred within 1 year. Of those patients with a definite missed diagnosis (n = 22), the causes are outlined in the Table.

Abstract 18

In oesophageal cancer (n = 16), the initial diagnosis was oesophagitis or benign stricture in 56%. In gastric cancer (n = 14), the initial diagnosis of gastritis, ulcer, or suspicious lesion was made in 71.4%. In the overall group, > 4 biopsies were taken in 23% at initial endoscopy with 63% at final endoscopy (p = 0.002).

Conclusion: This study emphasises the importance of detecting cancer at an early stage with a low threshold for multiple biopsies of an abnormal finding.

019 PAN-COLONIC INDIGO CARMINE DYE SPRAYING FOR THE DETECTION OF DYSPLASIA IN ULCERATIVE COLITIS

M.D. Rutter, G. Schofield, A.B. Price, I.C. Talbot, B.P. Saunders. St Mark’s Hospital, Harrow, UK

Background and Aim: Patients with longstanding extensive ulcerative colitis (UC) are at increased risk of colorectal cancer. Colonoscopic surveillance is often performed, and relies heavily on multiple non-targeted mucosal biopsies detecting occult dysplasia. Chromoendoscopy can aid detection of subtle mucosal abnormalities. We hypothesised that the routine use of pan-colonic indigo carmine dye spray would improve the macroscopic detection of dysplasia while reducing the dependence on non-targeted biopsies.

Methods: One hundred patients with longstanding extensive UC attending for colonoscopic surveillance underwent “back-to-back” same day colonoscopies. During the first examination, all visible abnormalities were biopsied and quadrantic, non-targeted biopsies were taken every 10 cm throughout the colon and rectum (20–40 biopsies per patient). Indigo carmine dye (0.1%) was used to coat the entire mucosal surface during the second colonoscopic withdrawal, and additional abnormalities detected with dye spray were biopsied.

Results: Extubation times for first and second colonoscopies were 11 and 10 min, respectively. Forty three mucosal abnormalities (from 20 patients) were detected by pre-dye spray targeted biopsies, and following indigo carmine dye-spraying 114 additional abnormalities (in 55 patients) were detected. Pre-dye spray targeted biopsies detected two lesions considered to be dysplasia associated lesions/masses (DALMs) in two patients. Dye spraying detected seven additional dysplastic lesions (2–6 mm) in five patients. All seven were considered histologically and colonoscopically to be sporadic adenomas. No dysplasia was detected in 2904 non-targeted biopsies.

Conclusions: Pan-colonic indigo carmine dye spraying increased the yield of suspicious mucosal areas, but the vast majority of these (107/114) were non-dysplastic. No dysplasia was detected from 2904 non-targeted biopsies. Chromoendoscopy may be more time-effective than taking high numbers of non-targeted biopsies, however most significant abnormalities can be detected by careful mucosal examination and targeted biopsies.

020 IMPROVING COLONOSCOPY COMPLETION RATES: THE IMPACT OF AUDIT

A. Bassi, P. O’Toole. University Hospital Aintree, Liverpool, UK

Introduction: The BSG National Colonoscopy Audit reported that caecal intubation rates in the UK fall far short of the recommended 90%. Addressing this requires both national and local initiatives. The audit process is one tool for producing local service improvement. We report our experience of colonoscopy audit in a large teaching hospital.

Methods: All colonoscopy reports produced between 1 February and 31 July 2000 were reviewed retrospectively. Completion rates and main reasons for failure were recorded for each operator. Procedures were regarded as complete if the ileum was entered or the ileocaecal valve identified, or if scope advancement was prevented by stricture, tumour, or severe colitis. Based on these findings, several changes were implemented. The audit was then repeated using an identical method, for the same six month period in 2001.

Results: During the first six month period 676 colonoscopies were performed. Overall completion rate was 75%. This was influenced by the poor performance of a small number of endoscopists. The commonest reason for failure was poor bowel preparation (35%). Following this, new bowel preparation guidelines were introduced, three endoscopists were retrained and the principal trainer attended the “Training the Trainers” course. During the second audit period there were 729 colonoscopies. Overall completion rate had improved to 89%, with improvement across all subgroups (Table). No perforations occurred in either audit period.

Abstract 20

Discussion: We acknowledge the obvious limitations of using self-reported caecal intubation rates, but believe this study confirms the value of audit as a tool for improving colonoscopy performance.

021 IS THE UK GASTROINTESTINAL ENDOSCOPY TRAINING ADEQUATE? THE TRAINER’S AND TRAINEE’S PERSPECTIVES

T. Mahmood, A. Darzi, D. Boucier-Hayes, S. Bann. Academic Surgical Unit, St Mary’s Hospital NHS Trust, London

Objectives: To measure the training standards in gastrointestinal endoscopy from the perspective of the specialist registrars (SpRs) and the trainers.

Design: Content validated, computer coded, mailed questionnaire.

Setting: Academic Surgical Unit, Imperial College of Medicine, London.

Participants: Randomly selected 100 SpRs and 100 consultants throughout the United Kingdom from attendants of BSG meeting 2000 or members of the society.

Main Outcomes: Level of training already achieved in endoscopy, according to the year of training, compared with the standard expected by the SpRs and the consultant trainers.

Results: There was an unequivocal lack of training in gastroscopy, flexible sigmoidoscopy, and colonoscopy acknowledged by both the trainees and the trainers. (p < 0.000). The SpRs in the final year of training had not achieved level five training (therapeutic endoscopy) in 4% gastroscopy, 15.4% flexible sigmoidoscopy, and 37.5% colonoscopy (p < 0.000 for all).

Conclusions: Almost one third of the SpRs in the final year of training (appointable as a consultant), and their trainers did not feel that appropriate level of training had been achieved in colonoscopy to run independent endoscopy lists. Courses and ongoing objective assessment in endoscopy should be an integral part of training in gastroenterology. Guidelines regarding standards required should be validated. Should we have a system of training and assessment in GI endoscopy with basic, therapeutic, and advanced levels?

Cell/molecular biology free papers 022–030

022 REPLENISHMENT OF THE ENTERIC NERVOUS SYSTEM IN AGANGLIONOSIS

N. Thapar, N. Bondurand, D. Natarajan, V. Pachnis (introduced by I.R. Sanderson). National Institute for Medical Research, Mill Hill, London, UK

In humans aberrant development of the enteric nervous system (ENS) manifest as motility disorders. The commonest is Hirschsprung’s disease. Mutations in a number of genes implicated in ENS development have been identified, eg receptor tyrosine kinase (RET), GDNF, Endothelin 3 (ET3), and Sox 10. Mouse models of aganglionosis share many of these mutations, providing good models for studying ENS development, isolation, and characterisation of ENS progenitor cells (EPCs), and their use to replenish aganglionic bowel. Myenteric plexus from wild type and mutant postnatal mouse guts were dissected out and cell cultures established. After culturing in defined medium large clusters of cells or neurosphere like bodies (NLBs) were isolated. Dissociated NLBs were infected with a retroviral vector containing a fluorescent protein incorporated into dividing cells. Positive cells were selected using a FACS cell sorter and characterised for the properties of stem cells. The effects of various growth factors, eg ET3 and GDNF, were studied. Labelled cells were injected into wild type and mutant gut in organ culture to assess their ability to migrate, colonise the gut, and differentiate into mature ENS cell types. Cultures of cells isolated from postnatal wild type and mutant gut gave rise to NLBs with 30% of sorted cells exhibiting EPC characteristics. At clonal density single cells gave rise to both neurons and glia. In sub-clonal studies such colonies when re-plated at clonal density showed self-renewal and multi-potency. Preliminary results suggest that ET3 and GDNF alter EPC differentiation and proliferation. Injected cells migrate, proliferate, and differentiate into neurons and glia. This is one of the first descriptions and isolation of EPCs from postnatal gut. They have the properties of stem cells and appear to be modulated by ET3 and GDNF. Injection experiments to date are encouraging as a degree of replenishment is observed. We describe for the first time isolation of EPCs from the proximal ganglionic segment of partially aganglionic bowel, opening exciting new prospects for replenishment therapies.

023 DYSREGULATION OF THE MAJOR SQUAMOUS CELL STRESS RESPONSIVE SYSTEM IN BARRETT’S METAPLASIA

H.H. Dalziel, E. Pohler, A.L. Craig, N. Kernohan, D. Hopwood, J.F. Dillon, T.R. Hupp. GI Research Laboratories, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK

The epithelial cells of the oesophagus are routinely exposed to unique environmental pressures, including thermal stress, acid and bile reflux, and dietary carcinogens, which appear to play a role in development of metaplasia, increased selection pressure for early mutation of p53, and therefore a heavy burden on stress-responsive and cell-cycle checkpoint control systems. The human oesophageal epithelium is easily accessible by endoscopy and provides a unique opportunity to integrate biochemical and clinical studies of stress protein responses relevant to understanding mechanisms of initiation of the cancer progression sequence to the intermediate Barrett’s metaplasia. To begin to address these issues, we have performed a comprehensive analysis on the nature of the stress responsive systems in normal squamous epithelium and Barrett’s metaplasia. We have unexpectedly found that the major stress-responsive genes (SEP53, SEP70, and Trans-glutaminase) of normal epithelium represent a novel stress control system generally confined to normal squamous epithelium. Strikingly, these stress responsive proteins are shown to be differentially expressed in Barrett’s epithelium from different patients, thus identifying a likely epigenetic pathway involved in modulating disease progression. Transfection of the SEP53 gene into cells enhances proliferation as judged by colony formation assays. This ability of SEP53 to enhance proliferation is similar to oncogenic mutant p53, indicating that SEP53 functions as a proto-oncogenic survival factor. Together, these data suggest two functions for SEP53 depending upon cell type: (a) in normal epithelium, the ubiquitous expression of SEP53 suggests a role in maintaining normal tissue integrity; and (b) in patients with Barrett’s metaplasia, high levels of expression of SEP53 are likely to result in enhanced cellular proliferation, whereas patients with no SEP53 are likely to have higher rates of cell death. These biochemical studies identified a novel pathway whose dysregulation may play a role in modulating the rates of tissue injury and ultimately assist in developing novel therapies.

024 OESOPHAGEAL CELL LINES SHOW DIFFERENTIAL SUSCEPTIBILITY TO BILE ACID INDUCED APOPTOSIS THAT IS P53 INDEPENDENT

J. Darragh1, P.E. Ross1, J.F. Dillon1, N.M. Kernohan1, P.W. Dettmar2.

1Ninewells Hospital and Medical School, University of Dundee, UK; 2Reckitt Benckiser Healthcare Ltd

Aim: Bile acids are one component of gastric contents that can reflux into the oesophagus in patients with GORD. Bile acids have been shown to induce apoptosis in colonic epithelium and hepatocytes. Similar activity on oesophageal epithelium may contribute to the pathogenesis of GORD. As the effect of bile acids on the oesophagus has not been examined in detail we have investigated the effects of bile acids on apoptosis of established oesophageal cell lines.

Methods: Oesophageal cell lines (OE19, OE21, OE33, and KYSE30), a wild type p53 colon cell line (HCT116), and it’s derived isogenic p53 null cell line were grown in medium containing different biochemically pure bile acids. Apoptosis was identified by cell morphology, the presence of sub G1 DNA fragments by flow cytometry and detection of activated caspase-3 by Western blot.

Results: OE 33 and OE 19 (derived from an adenocarcinoma) exhibited a dose dependent induction of apoptosis in response to deoxycholic acid (DCA) and chenoDCA. The cell lines derived from squamous cancers (OE21 and KYSE30) were resistant to the proapoptotic effect of bile acids. Less hydrophobic bile acids, such as cholic acid and tauroDCA were unable to induce apoptosis. Caspase 3 activation was observed in apoptotic cells, however p53 protein levels remained unaffected. The proapoptotic activity of DCA was p53 independent, both p53 wild type and null isogenic colonic cell lines being equally sensitive.

Conclusion: DCA and chenoDCA induce p53 independent apoptosis in some oesophageal cell lines, although those that exhibit squamous differentiation are resistant. The proapoptotic activity of particular bile acids may contribute to mucosal damage. Our results also suggest that this response may compensate for loss of p53 activity that occurs in oesophageal cancers and that loss of this bile acid induced effect in vivo may favour tumour progression.

025 EXPRESSION ANALYSIS OF THE METAPLASIA DYSPLASIA CARCINOMA SEQUENCE IN BARRETT’S OESOPHAGUS AND ADENOCARCINOMA

I.D. Penman1, V. Smith2, E.F. Shen1, D. Wieand2, T.H. Landon2, N.A.C.S. Wong2, A.M. Lessells1, S. Paterson-Brown1, J.Z. Tang2, T. Wu2, K.J. Hillan2.

1Western General Hospital and Royal Infirmary, Edinburgh, UK; 2Genentech Inc., San Francisco, Calif, USA

Introduction and Aims: The molecular genetic events involved in the metaplasia dysplasia carcinoma (MDC) sequence in Barrett’s oesophagus (CLO) are incompletely understood. We applied microarray expression analysis of endoscopic biopsies to study further these events and to detect novel genes involved in the process.

Methods: Paired biopsies representing progression through Barrett’s oesophagus (CLO), low and high-grade dysplasia (LGD, HGD), adenocarcinoma (Adca), and CLO adjacent to adenocarcinoma (Adca-BO), were taken from patients undergoing surveillance endoscopy. Biopsies were also taken from normal squamous mucosa. cDNA microarrays of 9031 genes were used to identify genes that were expressed in different disease stages. Data for each microarray were normalised to calculate Z scores and expression ratios: genes with Z scores > 1.7 (ratios 2–20) occurring in > 25% of samples from each “stage” were considered to be significantly expressed.

Results: 460 genes satisfied these criteria. The mean number of expressed markers increased with progression from CLO (7.6) through LGD (11.7) to HGD (16.4). The data reveal progressive increases with dysplasia in a variety of markers involved in inflammation (eg IL-1 homologue H1, IL-17 and its receptor, chemokine receptor CXCR4, COX-2), intestinal differentiation (eg myosin MY01A, AGR2) and carcinogenesis (eg c-fos, EGFR, VEGFC), suggestive of a differentiated small intestinal enterocyte lineage, along with increased expression of TCF4. Gene expression profiles in adenocarcinoma also show evidence of Wnt-related expression, similar to colonic carcinoma.

Conclusions: These results define a collection of markers that may assist in identifying patients with higher risk of developing cancer, and highlight multiple novel genes that merit further study in Barrett’s carcinogenesis.

026 INTESTINAL CALCIUM ABSORPTION: STUDIES OF POLYMORPHISMS IN TWO KEY GENES

J.R.F. Walters1, D.A. Van Heel2, M. Khanji1, O. Rhodes-Kendler1, U. Khair1, N.F. Barley1. 1Gastroenterology Section, Faculty of Medicine, Imperial College, London, UK; 2Wellcome Trust Centre for Human Genetics, Oxford, UK

The absorption of dietary calcium by the intestine varies between individuals and those with low absorption are at greater risk of osteoporotic fractures. Only part of the variation is dependent on circulating vitamin D metabolites and the differences in intestinal expression of genes involved in calcium transport remain unexplained. The aim of this study was to investigate whether polymorphisms in two key genes could be implicated in the observed differences.

The expression of the epithelial apical membrane calcium transporter, ECAC2/CAT1 (TRPV6 gene) differs tenfold between individuals. Approximately 1.5 kb of the 5’ flanking region of this gene was sequenced in DNA from 15 subjects, including 13 with known levels of duodenal ECAC2/CAT1 RNA expression. Only one DNA contained a single nucleotide polymorphism (SNP) and this was from a subject with expression in the mid-range. A novel polymorphism in the vitamin D receptor (VDR) gene has recently been associated with differences in bone density in a Japanese population. This SNP (G or A) is in a caudal-related homeobox (CDX2) binding element and has been shown to affect transcription of the VDR gene. It was hypothesised that differences in expression of the VDR in the intestine could then affect the expression of vitamin D dependent genes involved in calcium absorption. In 82 British patients the allelic frequencies of this SNP were G:A = 0.76:0.24, different to those found in Japan. There were relatively few AA homozygotes, and no significant differences could be demonstrated in bone mineral density. The SNP genotypes were not associated with differences in mean levels of expression of duodenal calcium transport genes (ECAC2/CAT1, calbindin-D9k, or PMCA1), although there was evidence of significantly different responsiveness to 1,25-(OH)2D3. In summary, no significant SNP has been found in the promoter of the gene for ECAC2/CAT1, but the effect of the CDX2 binding element SNP in the VDR gene merits further study.

027 ELEVATED PLASMA CONCENTRATIONS OF AMIDATED GASTRIN CAUSE INCREASED CRYPT SURVIVAL IN MURINE INTESTINAL EPITHELIA AFTER γ RADIATION

P.D. Ottewell1, A.J.M. Watson1, T.C. Wang2, A. Varro3, G.J. Dockray3, D.M. Pritchard1. 1Department of Medicine, University of Liverpool, UK; 2University of Massachusetts Medical Center, Worcester, MA, USA; 3Department of Physiology, University of Liverpool, UK

Background and Aims: Amidated gastrin has well characterised mitogenic and morphogenic properties in the stomach, but its role in the distal intestine remains unclear. We have previously demonstrated no differences in the levels of apoptosis and mitosis in small intestinal and colonic epithelia of mice that overexpress amidated gastrin (INS-GAS) compared to their wild-type counterparts, either in the untreated state or 4.5 hours following 8 Gy γ radiation. In order to complete our analysis of the effects of hypergastrinaemia upon the distal intestine in vivo, we have now investigated the effects of elevated plasma concentrations of amidated gastrin upon intestinal crypt survival following γ radiation.

Methods: Mice analysed were adult INS-GAS and their wild-type counterparts (FVB/N). Clonogenic crypt survival was assessed by light microscopy of small intestinal and colonic crypts four days after 10, 12, or 14Gy γ radiation. We confirmed that the differences observed were specifically induced by hypergastrinaemia by analysing INS-GAS mice treated with the specific gastrin/CCKB receptor antagonist YF476 and FVB/N mice treated with omeprazole.

Results: Four days following 12 and 14Gy γ radiation, INS-GAS mice exhibited significantly higher (∼3 fold) small intestinal and colonic crypt survival compared with their wild-type counterparts. INS-GAS mice treated with YF476 showed significantly lower (∼4 fold) small intestinal and colonic crypt survival after 14Gy γ radiation compared with mice receiving vehicle alone. FVB/N mice dosed with omeprazole to induce hypergastrinaemia showed significantly increased (∼4 fold) survival of small intestinal and colonic crypts after 14Gy γ radiation.

Conclusions: (a) Increased small intestinal and colonic crypts survival is observed in mice with elevated plasma concentrations of amidated gastrin following γ radiation. (b) This protective effect of hypergastrinaemia occurs as a result of signalling via the gastrin/CCKB receptor.

028 MONITORING M CELL CONVERSION IN VITRO: THE ROLE OF LYMPHOCYTE EPITHELIAL CELL CONTACT

S.W.J. Cochrane1, D.P. O’Donoghue2, A.W. Baird1. 1University College Dublin; 2St Vincent’s University Hospital Dublin

M cells are of paramount importance in microbial-intestinal cross talk. There is sparse data on human M cells due to their distribution and the lack of specific markers, however, in vitro co-culture models have been described.1,2 The nature of lymphocyte epithelial cell interactions required for M cell transformation as well as the degree of phenotype specific expression in vitro never-the-less remain unclear. We compared co-cultures of polarized Caco-2 cells and Raji B lymphocytes with monocultures of Caco-2 cells in two configurations: one in which there is direct contact between the two cell types (mode 1) and one in which the epithelial cells are exposed to B cell secreted factors only (mode 2). M like cell transformation was assessed by (a) scanning and transmission electron microscopy (EM); (b) apical membrane enzyme activity; (c) translocation of FITC-labelled latex microparticles; and (d) assessing the interaction of the co-cultures with Salmonella typhimurium and Clostridium difficile. EM demonstrated cells with M like morphology in both co-culture models. Down regulation of alkaline phosphatase expression could only be proven in mode 1 (p < 0.005). Microparticle transport was increased in both co-culture configurations compared to caco-2 cell monoculture but was much greater in mode 1 (mode 1 1629+/−453 v 104 +/−11 events, n = 36, p < 0.0001; mode 2 265+/−99 v 121 +/−29 events, n = 34, p = NS). Adherence of S typhimurium to mode 1 was increased compared to caco-2 monoculture > 6-fold (p < 0.005) but that of C. difficile was unchanged. Transformation of absorptive enterocytes to M like cells can be achieved by co-culturing immortalised cell lines of human origin. This transformation is more reproducible if a model using direct contact between epithelial cells and B lymphocytes is employed. Such studies will prove useful in vitro examining the interaction of bacteria which exploit the M cell pathway with polarized epithelia.

  1. Kerneis S, et al. Science 1997;277:949–52.

  2. Gullberg E, et al. Biochem Biophys Res Commun 2000;279:808–813.

029 HELICOBACTER PYLORI REGULATES ID-1 AND ID-3 EXPRESSION, BUT NOT ID-2

B.A. Manzo1, M. Bajaj-Elliott1, J. Atherton2, R. Thomas2, I.R. Sanderson, J.W. Wilson. 1Adult and Paediatric Gastroenterology, Barts and The London, Queen Mary School of Medicine and Dentistry; 2Division of Gastroenterology, University Hospital, Nottingham

H pylori can induce both apoptosis and proliferation of gastric epithelial cells. The balance between these two processes during bacterial infection depends on both host and microbial determinants and underlay the risk of developing cancer. Inhibitor of differentiation (Id/DNA binding) helix-loop-helix proteins are critically related to cell cycle progression, differentiation, and apoptosis. These effects are mediated by inhibiting the DNA binding of basic HLH transcription factors, such as the ubiquitous E47 and tissue-specific factors like myoD. We hypothesised that H pylori could regulate Id expression in gastric epithelial cells following infection. AGS cells (from a poorly differentiated gastric adenocarcinoma) were co-cultured with H pylori 60190 wild type strain (1×108 bacteria/cell) from 2 to 48 hours. RT-PCR analysis revealed down-regulation for Id-1 and Id-3 mRNA levels, which occurred over the first 6 hours of exposure to H pylori. In contrast, Id-2 mRNA levels remained constant. In agreement with transcriptional data, Western blot analysis showed that protein levels were strongly and rapidly downregulated by the bacteria; again Id-2 protein levels were unaltered. Culture of AGS cells in presence of H pylori resulted in the accumulation of cells in the G1 phase of the cell cycle (69% after 24 hours compared with 48% in control cultures), as assessed by FACS analysis. No significant apoptosis was observed. In conclusion, H pylori results in decreased Id-1 and Id-3 expression in AGS cells in vitro, which is associated with arrest of the cells in G1 phase of the cell cycle. These results indicate that H pylori can alter the expression of key regulatory transcription factors controlling gene expression in the cell cycle.

030 DIRECT VISUALISATION OF CELL SHEDDING FROM THE SMALL INTESTINE OF THE LIVING MOUSE

A.J.M. Watson1, S. Chu2, M.H. Montrose2. 1Department of Medicine, University of Liverpool, Royal Liverpool University Hospital; 2Department of Physiology, Indiana University School of Medicine, Indianapolis, USA

Introduction: Epithelial cells arise from stem cells at the base of the crypt and migrate up the crypt/villus axis to the tip of the villus where they are shed. The mechanism of cell shedding is unknown but two theories have been proposed. In the first, the shedding process is initiated by the cell undergoing apoptosis, which causes the cell to detach from the basement membrane and neighbouring cells, and thereby be shed into the lumen. In the second, the shedding process is initiated by neighbouring cells extending processes under the cell to be shed, forming tight junctions that extrude the cell into the lumen. Once detached from the basement membrane the cell undergoes apoptosis as a secondary event.

Methods: Mice were anaesthetised with inactin and loop of bowel brought out and opened. The mouse was placed on the stage of a two-photon microscope so the mucosal surface of the bowel could be observed. Nuclei were visualised with Hoechst 33258 1 mg/ml i.p. Caspase 3 activation was monitored with the cell permeant fluorescent dye 10 μM PhiPhilux (Oncoimmunin).

Results: Optical sections were taken of the upper 30 μm of the mouse villi in vivo. Cell shedding was observed from the epithelial monolayer using time lapse imaging. While the cells were within the monolayer no changes in the morphology of the nuclei was observed. However, once shedding was complete cells in the intestinal lumen had condensed nuclei, suggestive of apoptosis. Cell shedding took place at a rate of 13.2 cells/hour/villus ± 3.5 (n = 4 mice). In a minority of cells caspase 3 was found to be activated at the same time as shedding took place. However, the majority of cells were shed without activation of caspase 3. The average rate of the cell moving out of the monolayer into the lumen was 0.83 μm/min ± 0.06, (n = 53 cells). This is more than twice the rate at which cells migrate up the villus.

Conclusions: The majority of cells are shed without undergoing apoptosis. The observation that cells are shed at a rate greater than the migration rate suggests that cells are actively extruded from the monolayer.

Small bowel free papers 031–036

031 ROLE OF T CELLS IN THE REGULATION OF HUMAN INTESTINAL α DEFENSIN GENE EXPRESSION

W. Dhaliwal, M. Bajaj-Elliott, P. Kelly. Department of Adult and Paediatric Gastroenterology, Barts & The London School of Medicine, London E1 2AD, UK

Background: In a 3 year study of an urban African population, we have explored the relationship of intestinal α defensin expression to tropical enteropathy and intestinal infection. Quantification of human defensins (HD) 5 and 6 mRNA revealed marked variation between individuals and with season. There were strong correlations with changes in mucosal architecture and diarrhoeal incidence. This suggests that environmental determinants elicit changes in mucosal α defensin mRNA.

Aims: In view of the role of T cell activation in the pathogenesis of tropical enteropathy we hypothesise that HD5 and HD6 expression may be modulated by T cells.

Methods: An in vitro organ culture system was developed and small intestinal biopsies from healthy adults undergoing endoscopy were stimulated with staphylococcal enterotoxin B (SEB) over the range 0.1–10 μg/ml for 8 or 24 hours. Biopsies were also cultured for 24 hour with SEB (10 μg/ml) or dexamethasone (10−7M) or both. HD5 and HD6 mRNA was quantified by competitive RT-PCR.

Results: A dose dependent decrease of up to 1.5 log mRNA transcripts/μg total RNA was observed (HD5, p < 0.01; HD6, p = 0.05) with increasing SEB. Dexamethasone abrogated the effect of SEB on mRNA levels, and dexamethasone alone increased mRNA levels to above those of controls (HD5, p = 0.03; HD6, p < 0.05). Preliminary data with pro-inflammatory cytokines also show down-regulation of HD5 and HD6.

Conclusions: These data suggest that T cell activation downregulates human intestinal α defensin expression and that the effects seen in tropical enteropathy are at least partly due to interactions with adaptive immune cells.

032 THE EFFECT OF HYDROXYPROPYLCELLULOSE ON BILE ACID INDUCED WATERY DIARRHOEA

G. Brydon1, R. Ganguly1, S. Ghosh2. 1Western General Hospital, Edinburgh, 2Hammersmith Hospital, London

Introduction: Hydroxypropylcellulose (HPC) is a food additive found in soups and ice creams where it acts as a thickener and emulsifier. Watery diarrhoea caused by bile acid malabsorption may be helped by HPC, which binds bile acids and may thereby reduce the laxative effect of bile acids on the colon.

Patients and Methods: Five patients (two men, three women) with idiopathic bile acid malabsorption (IBAM) and five patients (two men, three women) with quiescent Crohn’s disease with ileal resection were recruited into the study. All suffered from watery diarrhoea and were intolerant of cholestyramine. All underwent a 1 week baseline study period (week 0) when they kept a diary of their bowel habits and also discontinued loperamide or other anti-diarrhoeals. All patients then had HPC in water for 6 weeks (1 gm/day for week 1 to week 4 and 1.5 gm/day for week 5 to week 6). A diary of bowel habits was maintained daily throughout the study. The study was approved by the Lothian Ethics in Research Medicine and Oncology Subcommittee.

Results: HPC forms a viscous solution in water. All patients tolerated the intake of HPC and no untoward side effects were noted. The mean stool frequency per day decreased from 5.2 (SD 2.1) during week 0 to a mean frequency of 2.9 (SD 1.5) per day during week 6. This was a significant reduction in bowel frequency (p = 0.001; paired t test) compared to bowel frequency in the week prior to HPC administration. Nine out of 10 patients also reported a subjective improvement in their stool consistency, urgency, and incontinence.

Conclusion: In this proof of concept study, HPC resulted in a significant reduction in stool frequency in patients with watery diarrhoea due to bile acid malabsorption. It may provide a safe treatment of this condition as an alternative to cholestyramine and it does not bind to concurrently administered medications. Randomised trials are warranted to further establish the role of HPC in watery diarrhoea caused by bile acid malabsorption.

033 SERUM 7α HYDROXY-4-CHOLESTEN-3-ONE CONCENTRATIONS IN DIFFERENT TYPES OF BILE ACID INDUCED DIARRHOEA

W.G. Brydon1, R. Ganguly1, S. Ghosh2. 1Western General Hospital, Edinburgh, UK; 2Imperial College School of Medicine, Hammersmith Hospital, London

Introduction and Aims: Bile acid induced diarrhoea can be of three types: type 1: caused by pathology or resection of terminal ileum; type 2: idiopathic bile acid malabsorption (IBAM) due to increased bile acid pool size or rapid ileal transit, rarely a specific bile acid ileal transport defect; and type 3: non-ileal disease secondary to other conditions such as post-cholecystectomy, IBS, diabetes mellitus, bacterial overgrowth, and chronic pancreatitis. Serum concentration of a hepatic intermediary of bile acid synthesis, 7 α hydroxy-4-cholesten-3-one (7HCO) reflects the rate of bile acid synthesis in man and is elevated in clinical conditions associated with bile acid malabsorption (BAM). Serum 7HCO correlates well with SeHCAT test.

Methods: Serum HCO was measured on fasting blood sample using HPLC. Standard 7HCO was a kind gift from Professor I. Bjorkhem, Karolinska Institute, Stockholm. Results were calculated as: test peak area X concentration of standard/standard peak area (ng/ml). Serum 7HCO concentrations > 35 ng/ml were considered to be abnormally raised. A total of 190 patients with watery diarrhoea had serum 7HCO assayed as well as diarrhoea investigated with radiology, endoscopy, celiac serology, pancreatic function tests, and glucose hydrogen breath test.

Results: Thirty one out of thirty two patients with ileal Crohn’s disease (including 4 resections) had abnormally high 7HCO (type 1 BAM). The median concentration was 158 ng/ml (range 37–590 ng/ml). Twenty six patients with IBAM (type 2 BAM) had a significantly lower (p < 0.00001) 7HCO concentration (median 67.5; range 39–303 ng/ml) than patients with type 1 BAM. Of patients with IBS, post-cholecystectomy, bacterial overgrowth, and chronic pancreatitis, 16/132 had abnormally elevated 7HCO (type 3 BAM). However, the concentration of 7HCO in type 3 BAM (median 46; range 36–71 ng/ml) was significantly lower than type 2 BAM (p < 0.01).

Conclusion: The thee types of BAM stratify according to serum 7HCO type 1 BAM have the highest concentration, and type 3 the lowest concentration. IBAM (type 2) have intermediate values of 7HCO with some overlap with type 1 and type 3.

034 IS THE RISK OF ADULT COELIAC DISEASE CAUSALLY RELATED TO CIGARETTE EXPOSURE?

S. Suman1, E.J. Williams1, P.W. Thomas2, S.L. Surgenor1, J.A. Snook1

1Gastroenterology Unit, Poole Hospital NHS Trust, Dorset, UK; 2Research and Development Support Unit, Bournemouth University, Dorset, UK

Introduction and Aim: Previous studies have shown an association between cigarette smoking and the risk of development of adult coeliac disease (CD), but it has yet to be established whether this relationship is causal. The aim of this study was to assess causality using the Bradford-Hill criteria, specifically seeking evidence of a biological gradient.

Method: Matched case control study using a questionnaire to establish a detailed smoking history for 138 incident cases of adult CD and 276 age and sex matched controls. Subjects were categorised according to various measures of the duration and intensity of active cigarette exposure prior to diagnosis of the matched case. Conditional logistic regression was used to calculate odds ratios and linear trends.

Result: At the time of diagnosis, 10% of cases and 30% of controls were current smokers (odds ratio 0.21, 95% CI 0.11–0.40 for CD in current v never-smokers). The odds of developing CD fell significantly with both increasing total lifetime exposure and exposure to cigarettes over the 15 years prior to diagnosis. However, the strongest relationship was with the number of cigarettes smoked per day at the time of diagnosis (odds ratio 0.15, CI 0.06–0.37, for CD in current heavy v never-smokers). All linear trends were highly statistically significant, and controlling the data for standard of living did not alter the findings.

Conclusion: This study strengthens the case for a causal relationship between smoking and CD by demonstrating a strong, temporally appropriate and dose dependent effect, thus meeting the Bradford-Hill criteria. This suggests that cigarette smoking truly protects against the development of adult CD.

035 COMPLICATIONS OF COELIAC DISEASE – HOW COMMON AND CAN THEY BE PREVENTED?

H. Gillett1, H. Drummond1, C. Goddard2, A. Shand3, J. Satsangi1. 1University of Edinburgh; 2West Lothian NHS Trust; 3Lothian Universities NHS Trust

Introduction: Coeliac disease (CD) is a common condition with wide clinical variation in both severity and type of symptoms. Many complications have been reported to occur but the protective effect of gluten free diet (GFD) remains controversial. The aim of this study was to provide data on the prevalence of complications and assess the effect of strict GFD in a large population with CD.

Methods: Consecutive patients attending the coeliac clinic at the Western General Hospital, Edinburgh, were invited to participate. Data were collected from case notes and from questionnaires and entered onto a secure database.

Results: Clinical data were obtained from 270 patients, and questionnaires completed by 199. Age at diagnosis was 0.2–88 years (median 41.5 years) with sex ratio of 1M:2.6F. Duration of follow up was 1 month to 68 years (median 6 years). Ninety one per cent reported symptoms of various severity and 9% were asymptomatic, even in retrospect. At diagnosis 75/199 (38%) were anaemic, improving to 30/225 (13%) with treatment; 16% were vitamin B12 deficient at diagnosis; 53% were iron deficient at diagnosis; and 7/32 (22%) had osteoporosis of the hip and 10/24 (42%) of the spine at diagnosis, improving to 15/130 (12%) (p = 0.006) and 34/109 (31%) (p = 0.012) with treatment. Those who described strict compliance to GFD on the questionnaire had significantly higher T scores on DEXA scan of the neck of femur compared with those who took gluten regularly (p = 0.05). T scores of the spine did not reach statistical significance. Epilepsy occurred in nine patients and was not more common in those less strict with GFD, but in 7/9 the diagnosis of epilepsy was made before that of CD. Low birth weight (LBW) babies (< 2.5 kg) occurred in 5/23 pregnancies in those regularly taking gluten and 6/146 pregnancies in those strict with diet (p = 0.005). Osteoporosis and anaemia did not differ in the symptomatic group, but LBW babies did (11/221 pregnancies v 5/24 (p = 0.011)).

Summary: Complications occur commonly in coeliac disease. The risk of osteoporosis of the hip and LBW babies may be prevented by strict GFD.

036 LACTOSE MALABSORPTION IN THE ELDERLY

J. Kurtovic, S.M. Riordan. Gastrointestinal and Liver Unit, The Prince of Wales Hospital and University of New South Wales, Sydney, Australia

Background: Limited data suggest a high prevalence of lactose malabsorption (LM) in the elderly. The mechanism is uncertain. A significant increase in enterocyte apoptosis raises the possibility that a subtle reduction in mucosal absorptive area, undetectable by standard structural analyses, may be responsible, especially if compensatory enterocyte proliferation is inadequate. Assessment of intestinal absorption of mannitol, taken up transcellularly and hence an index of absorptive area, and intestinal permeability, reflected by urinary lactulose/mannitol ratios, may resolve this issue.

Methods: We studied 20 asymptomatic elderly subjects (median age 76, range 65–94 years) and 20 controls (median age 29, range 21–35 years) with no history of gastrointestinal disease or use of drugs that disturb gut permeability. All were hydrogen producers following ingestion of lactulose. A 50 g lactose breath hydrogen test was performed after dietary preparation and an 8 hour fast. Breath hydrogen was measured by gas chromatography. Lactulose/mannitol ratios were measured by gas-liquid chromatography in urine collected over 6 hours after ingestion of 10 g lactulose and 5 g mannitol. Urinary creatinine levels were measured so that differences in renal function did not confound interpretation of urinary mannitol levels.

Results: LM, defined by a rise in breath hydrogen > 20 ppm above baseline, was present in 10/20 (50%) elderly subjects and 0/20 controls. Urinary mannitol/creatinine ratios did not differ significantly in elderly and control subjects (median 111.1, range 17.6–149 v median 92.8, range 45.9–140.8; p = 0.62) or in elderly subjects with and without LM (median 111.1, range 35.6–149 v median 106.7, range 17.6–138; p = 1.0). Urinary lactulose/mannitol ratios were significantly increased in the elderly (median 0.018, range 0.007–0.063) compared to controls (median 0.011, range 0.007–0.025; p = 0.003), but did not differ significantly in elderly subjects with and without LM (median 0.022, range 0.007–0.063 v median 0.014, range 0.011–0.057; p = 0.19).

Conclusions: The prevalence of LM in the asymptomatic elderly is high and cannot be explained by any reduction in small intestinal absorptive area. LM in this group is unrelated to disturbed intestinal permeability.

Abstract 25

Nutrition free papers 037–041

037 EFFECT OF FLAVOURING ON ISOTONIC SOLUTIONS FOR SHORT BOWEL SYNDROME

J. Williams1, J. Dart1, D. Van Heel2, S.P.L. Travis2. 1Department of Dietetics, John Radcliffe Hospital, Oxford, UK; 2Department of Gastroenterology, John Radcliffe Hospital, Oxford, UK

Background: Isotonic oral rehydration solutions (IORS) facilitate nutritional management of short bowel syndrome (SBS) and ileostomy dysfunction by reducing stoma output and improving hydration. Maximal intestinal Na+ and water absorption occurs at [Na+] 90–120 mmol/L and is facilitated by glucose. Patient compliance with IORS is poor but can be improved by the addition of flavourings such as fruit juice or squash. Amounts are unspecified and the effect of adding flavouring on the solution biochemistry has not been established.

Aim: To determine the effect on [Na+], [glucose] and osmolality, after the addition of fruit juice or squash as flavouring components to IORS.

Method: John Radcliffe Hospital (JRH) prepared IORS and 15 commercial sports/energy drinks were analysed for [Na+], [glucose], and osmolality by standard biochemical techniques. The JRH IORS prepared solutions were: (a) 200 ml no added sugar (NAS) squash + 800 ml H2O + 1 metric teaspoon NaCl; (b) 200 ml regular squash + 800 ml H2O + 1 metric teaspoon NaCl; (c) 750 ml fruit juice + 250 ml H2O + 1 metric teaspoon NaCl; and (d) 1000 ml Lucozade Sport and 1 metric teaspoon NaCl. A variety of sports/energy drinks were analysed.

Results: See Table.

Abstract 37

Conclusion: None of the commercial solutions had adequate [Na+] (range < 20–30 mmol/L); they were on average hyperosmolar (range 335–1031 mOsm; median 595 mOsm) and had variable, mostly high glucose concentrations (range 10.6–655.7 mmol/L; median 190 mmol/L). Flavouring with fruit juice or squash made IORS hypertonic, therefore rendering them less suitable in managing SBS. All JRH IORS contained adequate [Na+] (> 90 mmol/L) but the addition of 200 ml NAS squash was the only one to remain isoosmolar after flavouring.

038 PERCUTANEOUS ENDOSCOPIC GASTROSTOMY SITES INFECTED BY METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS: IMPACT ON OUTCOME

I. Mainie, A. Loughrey, J. Watson, T.C.K. Tham. Divisions of Gastroenterology and Bacteriology, Ulster Hospital, Belfast, N. Ireland

Background: Methicillin resistant S aureus infection (MRSA) appears to be associated with an increased incidence of colonisation of percutaneous endoscopic gastrostomy (PEG) sites.

Aim: We investigated the impact of prior MRSA colonisation on the incidence of symptomatic PEG site wound infection and mortality.

Method: Consecutive patients who had PEG tubes inserted recently were identified and their notes studied retrospectively. Presence or absence of MRSA prior to PEG placement was noted. After PEG placement, patients suspected of having wound infection had swabs taken from the site (no routine swabs taken). Mortality within 30 days of PEG placement was determined. Significant wound infection was defined as those requiring antibiotic treatment.

Results: Eighty three patients underwent PEG placement; 23 (28%) of these patients had known MRSA infection prior to PEG placement. Of these, 13 (57%) developed symptomatic MRSA colonisation of the PEG site. The remainder of the patients, 60 (73%), had no known prior MRSA infection. In these patients, 9 (15%) developed symptomatic MRSA colonisation of the PEG site. Thus, there was a significantly higher risk of MRSA infection of the PEG site if patients had prior MRSA infection (p = 0.00013). Only 4 patients with MRSA colonisation of the PEG site required treatment with antibiotics. Thus the incidence of significant wound infection was 5% of total undergoing PEG placement or 18% of those with MRSA colonisation. The mortality (none related to the PEG tube) of those with symptomatic MRSA infection of the PEG site was 9% (2 out of 22), although the mortality from non MRSA infected PEGs was 20% (12 out of 61). There was no significant difference, (p = 0.25).

Conclusion: Patients with prior MRSA infection had a significantly higher risk of developing symptomatic MRSA infection of the PEG site. However, there was still a significant risk (15%) for patients with no known prior MRSA infection developing MRSA infection of the PEG site. MRSA infection of the PEG site did not seem to affect mortality.

039 SYNBIOTICS IN IRRITABLE BOWEL SYNDROME: A DOUBLE BLIND PROSPECTIVE RANDOMISED CONTROLLED TRIAL

P. Barker, C.E. McNaught, A.D.G. Anderson, C.J. Mitchell, J. MacFie.

Combined Gastroenterology Research Group, Scarborough Hospital, Woodlands Drive, Scarborough YO12 6QL, UK

Introduction: Alterations in resident GI microflora have been implicated in the aetiology of irritable bowel syndrome (IBS) and this provides a theoretical rationale for the use of probiotics in these patients. The aim of this study was to investigate the effects of synbiotics (pre- and probiotics) in this group.

Methods: Patients fulfilling the Rome 2 criteria were randomised to receive a synbiotic preparation (oligofructose/Lactobacillus acidophilus, Bifidobacterium Bb12) or placebo for an 8 week period. Frequency of stool and abdominal pain were charted during weeks 0 (pretreatment), and 8, along with linear analogue (100 mm) measures of abdominal pain, flatulence, bloating, and Hospital Anxiety and Depression (HAD) scores. Data from weeks 0 and 8 were treated as “paired,” and the difference between the data points calculated for each patient. Non-parametric tests were then applied.

Results: A total of 34 patients were randomised (19 placebo, 15 synbiotic). The groups were similar with regards to age (45 ± 17 years placebo, 50 ±13 years synbiotic) and sex distribution (m:f = 4:15 placebo, 7:8 synbiotic). Both groups of patients showed significant improvement in pain frequency (median reduction three episodes per week), p < 0.05 and severity (median difference 12 mm on analogue score), p < 0.02. Both groups showed significant improvement in HAD score (median improvement 1.5 points), p < 0.05. There were no significant differences between synbiotic and placebo groups in any measured parameter (abdominal pain, flatulence, bloating, frequency of stool, or HAD score).

Conclusion: In this group of IBS patients there is no evidence of benefit from 8 weeks of synbiotic treatment. Both synbiotic and placebo groups showed significant clinical improvement, which is likely to represent a “placebo effect.”

040 FATTY ACID COMPOSITION AND FUNCTION OF MONONUCLEAR CELLS IN CROHN’S DISEASE

T. Trebble, M.A.Stroud, N.K. Arden, A. Ballinger, D.R. Fine, M.A. Mullee, S.A. Wootton. Southampton University Hospital Trust, Southampton SO14 6GB

Inflammation in Crohn’s disease (CD) is mediated by mononuclear cells and is associated with increased production of TNF α and prostaglandin E2 (PGE2). In active CD, dietary supplementation with fish oil results in anti-inflammatory effects associated with increases in eicosapentaenoic acid (EPA) and reductions in arachidonic acid (AA) concentrations in plasma. However, in the unsupplemented state the relationship between habitual dietary fat intake, EPA, and AA incorporation into mononuclear cells and TNF α/PGE2 production is uncertain. The fatty acid composition and synthetic function of peripheral blood mononuclear cells (PBMC) were determined in 53 CD patients, stratified into active and inactive disease groups by Crohn’s Disease Activity Index, and age and sex-matched healthy controls. There were no differences between CD and controls in dietary fat or total energy intake. Lower concentrations of AA were noted in active CD (20.2%) v controls (24.0%) (p < 0.001) and inactive CD (20.2%) v controls (23.8%) (p < 0.001). Higher concentrations of EPA were noted in active CD (0.7%) and inactive CD (0.8%) v controls (0.3% for each control group; p = 0.001). α linolenic acid, the precursor of EPA, was increased in active CD v controls (p = 0.003) but no differences were noted in concentrations of the AA precursor, linoleic acid. Trends towards a reduction in PGE2 production were noted in inactive CD v controls, but no differences in production of TNF α were seen. CD is associated with alterations in the availability of AA and EPA in both active and inactive states. This cannot be simply explained by altered dietary intake but may be an adaptive or pathological response to the disease process. The anti-inflammatory response to dietary fish oil may reflect a pharmacological effect, and not the correction of a deficiency of EPA. The response of mononuclear cell composition and function to dietary fish oil warrants further investigation.

041 SCREENING FOR MALNUTRITION: IMPACT OF THE MALNUTRITION UNIVERSAL SCREENING TOOL (MUST)

A. Jaafar1, C. Hawkyard1, K. Lapworth2, B. Davidson3, J. Ledger4, J. Wilkins4, J. Mansfield1, N.P. Thompson1. 1Department of Medicine, Freeman Hospital & Royal Victoria Infirmary, Newcastle-upon-Tyne; 2Audit Department, Freeman Hospital & Royal Victoria Infirmary, Newcastle-upon-Tyne; 3Dietetic Department, Freeman Hospital & Royal Victoria Infirmary, Newcastle-upon-Tyne; 4Department of Surgery, Freeman Hospital & Royal Victoria Infirmary, Newcastle-upon-Tyne

Introduction: Malnutrition is commonly found in hospital in-patients, with a frequency of up to 40%. Intervention studies suggest this is an independent and reversible prognostic factor. The MUST is a validated, simple tool enabling systematic identification of malnutrition.

Aims: To establish whether patients were being screened for malnutrition before the introduction of a specific screening tool; then to assess the practicality and effect of introducing the MUST.

Methods: All patients on eight wards (general medical, general surgery, care of the elderly, and orthopaedic) at two teaching hospitals were surveyed at one time-point. Patients’ medical and nursing notes and bedside charts were examined. Demographic, nutritional status and dietetic referral details were recorded. The audit was repeated 2 months after the MUST was introduced to these eight wards.

Results: In the first audit 172 patients were surveyed; mean age 68 years, 90 men, 111 medical/61 surgical, mean length of stay (LOS) 17 days. Sixty four (37%) had weights recorded, 6 (3%) had a BMI recorded, and 120 (70%) had comment about appetite made; 55 (31%) patients were referred to the dietetic service, 40% within 2 days of admission. In the second audit 173 patients were surveyed; mean age 66 years, 96 men, 99 medical/74 surgical, mean LOS 13 days; 103 (60%) had weights recorded, 125 (72%) had comment about appetite made; 62 (36%) had a BMI recorded (p < 0.001 compared to previously).Forty six (26%) patients were referred to the dietetic service, 44% within 2 days of admission. The MUST tool was used in 70 (40%) patients, in these patients the BMI was recorded in 89% patients and the dietetic referral rate was 56% compared with 11% where the MUST was not used (p < 0.001).

Conclusions: Prior to the introduction of a specific screening tool patients’ weight and BMI were rarely recorded. A pilot introduction of the MUST tool was partially successful and when used increased appropriate dietetic referral rate significantly.

Inflammatory bowel disease free papers 042–052

042 COMPLEX GENETIC INTERACTIONS REVEALED IN AN IBD GENOME SCAN STRATIFIED BY CROHN’S DISEASE ASSOCIATED VARIANTS

D.A. van Heel, B.M. Dechairo, G. Dawson, D.P.B. McGovern, K. Negoro, A.H. Carey, L.R. Cardon, I. MacKay, D.P. Jewell, N.J. Lench. Wellcome Trust Centre for Human Genetics and Gastroenterology Unit, University of Oxford and Oxagen Ltd, Abingdon, UK

Background and Aims: Genetic studies in inflammatory bowel disease (IBD) have identified multiple susceptibility loci. The significance of these findings depends on verification in independent cohorts. Genetic variants associated with Crohn’s disease have now been identified on chromosomes 5 (IBD5 risk haplotype) and 16 (IBD1 locus, CARD15/NOD2 mutations). These variants now allow stratification of linkage analyses, which will improve the ability to identify other loci, and allow assessment of potential complex interactions between genetic factors. Such gene–gene interactions have been shown to occur in animal models of IBD.

Methods: We performed a genome-wide scan of 228 IBD families. Multipoint linkage was assessed using MERLIN for IBD (288 affected relative pairs), Crohns disease (CD, 137 pairs) and ulcerative colitis (UC, 95 pairs) phenotypes. CD analyses were further stratified by common CARD15/NOD2 mutations and the IBD5 haplotype.

Results: We confirmed loci on chromosomes 3q (CD, LOD 2.1; p = 0.0009), 6p (IBD, LOD 2.2; p = 0.0008) and Xp (CD, LOD 2.0; p = 0.001). Linkage (CD, LOD 2.2; p = 0.0007) was observed 25cM q telomeric to CARD15 in CARD15 negative CD affected. The chromosome 19q locus, also demonstrated in a Canadian CD population, showed significant genetic heterogeneity with CARD15 (significance test p = 0.002, LOD 2.9; p = 0.0001 in CARD15 negative CD) and epistasis with the IBD5 haplotype (significance test p = 0.02, LOD 2.4; p = 0.0005 in CD IBD5 carriers).

Conclusions: Stratification of a genome scan by Crohn’s disease associated variants demonstrates the complex genetic basis to IBD, with genetic heterogeneity and epistatic interactions between loci. Our data support previous suggestions of a second chromosome 16 locus.

043 MORTALITY CAUSED BY INFLAMMATORY BOWEL DISEASE IN THE UK: A TWO YEAR SURVEY

S. Dolwani, A.B. Hawthorne. Department of Gastroenterology, University Hospital of Wales, Cardiff, UK (conducted via the BSG-blue card surveillance scheme)

Background and Aims: Previous studies in the United Kingdom have either been regional if population based or entirely based on statistics from the Registrar General’s office. We aimed to ascertain the non-cancer mortality in persons < 75 years of age ascribed to inflammatory bowel disease via a notification system throughout the UK conducted through all members of the BSG from Jan 2000 to Dec 2001. We then compared this data with that from the Office for National Statistics (ONS).

Methods: All deaths in patients with IBD were notified to the BSG central audit office. Further details and death certificate diagnosis were then requested from the notifying clinician. Subgroup analysis was done according to diagnosis, age, sex, cause of death, duration, site, extent, and operative mortality. These data were then compared with that from the ONS in England and Wales, Scotland, and Northern Ireland.

Results: One hundred and thirty two patients were recorded as having died due to IBD during this period, of which 108 were < 75 years. Of these, 61 had Crohn’s disease, 44 had ulcerative colitis (UC), and 3 had indeterminate colitis. Twenty six patients (24%) died within a year after a diagnosis was made; 36 (33.3%) died within 2 years of diagnosis; 7 (6.5%) had death attributed to medical therapy; and 51 (47.2%) had surgery prior to death. The median time to death after surgery was 7 days. When analysed according to immediate cause (IA on death cert), 23 patients (21.3%) had septicaemia, 18 patients (16.7%) colonic perforation, 9 patients (8.3%) multi-organ failure, 8 (7.4%) thromboembolic events (PE), and a similar percentage had pneumonia. There were more female deaths (35 v 20) in Crohn’s compared to UC. Patients aged < 35 years accounted for 25% of all deaths. During the same period the ONS reported 128 deaths due to UC and 240 to Crohn’s disease (which would include cancer related IBD mortality).

Conclusions: Younger patients and those within the first year from diagnosis, as well as those undergoing surgery, account for a significant proportion of all deaths due to IBD in the UK.

044 COMBINING CLINICAL RISK FACTORS AND GENOTYPE TO PREDICT POSTOPERATIVE RECURRENCE OF CROHN’S DISEASE

E. Johns1, T. Ahmad1, R. Hubbard2, D.P. Jewell1, S.P.L. Travis1. 1Gastroenterology Unit, Oxford; 2University Dept Statistics, Oxford

Introduction: There is a need to be able to identify patients at highest risk of relapse after surgery for Crohn’s disease, so that preventative treatment can be targeted at those most likely to benefit. Combining clinical risk factors and genotype to predict risk has not previously been explored.

Methods: All 105 patients who had had surgery for ileocaecal Crohn’s disease at the John Radcliffe Hospital, Oxford, on whom DNA had been collected and follow up data obtained were selected. Three endpoints were defined: duration of steroid free remission (EP1), time to clinical relapse (EP2), and time to surgical relapse (EP3). Forty one clinical and 57 genetic variables were recorded. Multivariate analysis was performed using the Accelerated Life Model, before decision tree analysis to identify prognostic groups. Owing to the large number of variables, genotypic analysis focused on three mutations of the NOD2 gene and 10 other genes in the HLA region on chromosome 6.

Results: Mean follow up duration was 175 months (range 6–840). Median time (range) to EP1 was 56 (0–540) months; to EP2, 74 (0–810) months; and to EP3, 120 (3–810) months. Perianal disease at index surgery and retinoid X receptor beta 1 genotype were the only variables associated with shorter times (all p < 0.0004) and heat shock protein 1 genotype with longer times to all three endpoints (p = 0.0145, p < 0.0001, p < 0.0001). Smoking and IkB14 genotype predicted a shorter duration of steroid free remission and time to further surgery, while age > 40 year, male sex, and NOD3020 genotype predicted a longer duration. Decision tree analysis only predicted steroid free remission: if transmembrane receptor notch 4 genotype positive (n = 80), 60% require steroids within 5 years, compared with 24% if notch 4 negative.

Conclusions: Smoking and perianal disease are confirmed as predictors of a higher relapse rate after surgery. Genotypes associated with a higher relapse rate are different to those currently associated with disease causation. Patients who are notch 4 negative have a 76% chance of prolonged (> 5 years) steroid free remission after surgery. Combining clinical and genotypic factors did not enhance the ability to predict the risk of relapse after ileocaecal resection for Crohn’s disease.

045 INFLAMMATORY BOWEL DISEASE AND THE RISK OF FRACTURE

T.P. van Staa1,2,3, C. Cooper3, C.S.J. Probert4, H.G.M. Leufkens1, M.K. Javaid3, N. Arden3. 1Utrecht Institute for Pharmaceutical Sciences, The Netherlands; 2Procter & Gamble Pharmaceuticals, UK; 3Medical Research Council, UK; 4University of Bristol, UK

Patients with inflammatory bowel disease (IBD) have an increased risk of low bone mass, the pathogenesis of which is multifactorial. There are limited data on fracture. We therefore conducted a primary care based case-control study to determine the risk and major risk factors of fracture in IBD patients. 231 778 patients with a fracture and 231 778 age and sex matched controls were recruited from the General Practice Research Database. The database has been previously demonstrated to be a representative sample of the general population of England and Wales. Adjusted odds ratios (OR) were estimated from conditional logistic regression. The mean age of cases and controls was 51 years and 52.5% were women. A history of IBD was found in 1134 fracture cases, compared with 896 of the controls (adjusted OR 1.21; 95% CI 1.10 to 1.32). The OR was 1.72 (1.13–2.61) for vertebral fracture and 1.59 (1.14 to 2.23) for hip fracture. The risk of fracture was greater in patients with a history of Crohn’s disease (OR 1.32 (1.13–1.53)) than in patients with ulcerative colitis (OR 1.13 (1.02–1.27)). The risk of fracture in patients with a history of IBD was significantly related to disease severity as assessed by the number of symptoms (OR in IBD patients without symptoms, 1.02 (0.90–1.17); 1 symptom, 1.66 (1.41–1.96); and ≥2 symptoms, 1.74 (1.43–12.12)). Similarly, severity assessed by medication demonstrated increasing fracture risks compared with untreated patients: aminosalicylates use 1.32 (1.14–1.54), oral corticosteroids 1.46 (1.18–1.82), and steroid sparing agents 1.86 (1.15–3.02). IBD patients with a history of bowel surgery did not have an increased risk of fracture (OR 1.03 (0.84–1.28)). Use of oral corticosteroids increased with severity of disease; after adjustment for oral corticosteroid use the risk of hip fracture remained elevated (OR 1.46 (1.04–2.04)). In conclusion, IBD patients have a higher risk of fractures, which is due to a combination of disease activity and oral corticosteroid use.

046 AZATHIOPRINE AND SMOKING STATUS IN CROHN’S DISEASE PREDICT RESPONSE TO INFLIXIMAB

I.D.R. Arnott, J. Satsangi. Gastrointestinal Unit, University Department of Medical Sciences, Western General Hospital, Edinburgh

Introduction: Up to 75% of patients given infliximab for Crohn’s disease (CD) will respond. A number of clinical parameters that may predict response have been proposed but none has been reproduced in an independent cohort. We aimed to identify clinical and laboratory markers of response in patients receiving infliximab at our institution.

Methods: Seventy four well characterised CD patients (42 females, median age 34 years (IQR 23–42.5)) were assessed. Full clinical data were collected prospectively and blood was taken for inflammatory markers, ASCA and ANCA. Eleven had ileal disease, 25 colonic disease, 28 ileocolonic disease and 10 had recurrence following ilesotomy. Sixteen had received azathioprine for > 3 months and 5 methotrexate. Single infusion infliximab (5 mg/kg) was given for luminal disease (60 patients) and 3 infusions for fistulating disease (14 patients). Disease activity was assessed by the Harvey-Bradshaw index and a response was defined as a reduction of four or more points or a 50% reduction in draining fistula.

Results: Those who smoked greater that 5 cigarettes per day for > 6 months were less likely to respond to infliximab than those who did not (11/21 v 39/47, χ2 = 7.0, p = 0.008, OR 0.22 (CI 0.08 to 0.41)). Patients on azathioprine for longer that three months at infusion were more likely to respond (15/16 v 35/52, χ2 = 4.4, p = 0.036, OR 7.31 (CI 3.34 to 10.31)). There was a trend towards better response rates in those with colonic disease (χ2 = 2.7, p = 0.09) and worse response rates in those with perianal disease (χ2=3.7, p = 0.056). Age at diagnosis, previous surgery, sex, pre-infusion disease activity, CRP, and ASCA/ANCA status did not influence response rates in our cohort.

Conclusion: Smoking has a strong adverse effect on response rates to infliximab. Furthermore, patients established on azathioprine for greater than 3 months had greater response rates. Every effort should be made to discourage CD patients from smoking. Ninety two per cent of patients established on azathioprine prior to treatment responded: this has clear implications for clinical practice.

047 MUTATION IN THE ITPA GENE PREDICTS INTOLERANCE TO AZATHIOPRINE

A. Marinaki, A. Ansari, M. Arenas, S. Sumi, E.M. Shobowale-Bakre, C.L. Lewis, I. Woodman, J. Duley, J.D. Sanderson. Departments of Gastroenterology, Genetics and Chemical Pathology, Guy’s and St Thomas’ Hospitals, London SE1 9RT, UK

Background and Aims: Withdrawal of azathioprine due to adverse drug reactions occurs in up to 20% of patients. Polymorphism in the TPMT gene predicts intolerance in a small proportion of these patients. Thus, the pharmacogenetic basis of side effects is unexplained in most patients. Inosine triphosphate pyrophosphatase (ITPase) deficiency occurs at polymorphic frequencies in Caucasian populations and results in accumulation of inosine triphosphate (ITP) in red cells. We recently reported the genetic basis of ITPase deficiency, a 94C>A missense mutation (Pro32 to Thr) resulting in complete deficiency in homozygotes and < 25% activity in heterozygotes. 6-Mercaptopurine (6-MP) is activated through a thio-IMP intermediate and we predicted that in ITPase deficient patients treated with AZA, the metabolite thio-ITP would accumulate, resulting in toxicity.

Methods: ITPase genotype, TPMT phenotype and TPMT genotype in 64 IBD patients with adverse drug reactions to AZA treatment were compared with 71 patients who did not experience side effects to therapy. Odds ratios were calculated using a dominant model.

Results: Overall, the ITPase 94C>A mutation was significantly associated with adverse drug reactions (OR 4.239, CI 1.562 to 11.504; p = 0.0033). Variant TPMT genotypes were not significantly associated with adverse drug reactions overall but did predict side effects in a subset of 14 patients with nausea and vomiting (OR 5.079, CI 1.325 to 19.465; p = 0.0 239). Conversely, ITPase 94C>A genotype was significantly associated with flu-like symptoms in 11 patients (OR 6.190, CI 1.400 to 27.371; p = 0.0251) and rash in 6 patients (OR 10.833, CI 1.780 to 65.938; p = 0.0190). Myelosuppression, pancreatitis, and hepatitis were not predicted by ITPase or TPMT genotype in this study.

Conclusion: The ITPase 94C>A mutation predicts intolerance to AZA and may be particularly associated with the “flu-like” adverse effects. Thioguanine would be an alternative therapy in these patients.

048 AZATHIOPRINE ACTIVE METABOLITE CONCENTRATIONS IN INFLAMMATORY BOWEL DISEASE

S. Wright1, D.S. Sanders2, L. Lennard1, A.J. Lobo1. 1University of Sheffield, Section of Medicine and Pharmacology, The Royal Hallamshire Hospital, Sheffield S10 2JF, UK; 2Department of Gastroenterology, The Royal Hallamshire Hospital, Sheffield S10 2JF, UK

Introduction: Azathioprine is well established in the treatment of inflammatory bowel disease (IBD). Thioguanine nucleotides (TGNs) are active azathioprine metabolites, elevated TGNs are associated with myelosuppression.

Aim: To investigate TGN metabolite concentrations in a large cohort of IBD patients.

Method: IBD patients on constant dose azathioprine were studied over a 2 year period. Blood samples (10 mL) were taken for the monitoring of blood counts and the measurement of red cell (RBC) TGN metabolite concentrations at each clinic visit.

Results: 159 patients (88 men, 71 women) were recruited into the study. 156 had multiple metabolite assays. In 111 patients (3 to 14 assays, median 6) TGN concentrations varied by < 50%, this group was defined as steady-state. 104 patients remained in remission, 7 developed active disease. Twenty patients (13%) on constant azathioprine dosage showed evidence of non-compliance (eg nil metabolites). Twenty-five had dosage adjustments, 20 due to active IBD, 1 with concurrent rheumatoid arthritis and 4 dose reductions (2 in prolonged IBD remission, 2 with neutropenia). TGN concentrations in the n = 104 steady state group ranged from 70 to 717 pmol/8×108 RBCs (median 236) compared with 70 to 517 (median 175) in the 27 IBD patients with active disease (median difference between remission and active disease = 44 pmol, p <0.05). The two neutropenic patients (azathioprine 1.96 and 1.78×109/L) had TGN concentrations of 452 and 376 pmol. The azathioprine dose for the 104 patients in remission ranged from 0.4 to 2.8 mg/kg (median 1.8), no different from that in those with active disease (0.6 to 2.5 mg/kg). There was no correlation between azathioprine dose and TGN concentrations (rs=0.2).

Conclusion: IBD patients with active disease have significantly lower TGN concentrations than those patients in remission.

049 CO-LOCALISATION OF ESCHERICHI COLI WITH MACROPHAGES IN LAMINA PROPRIA IN PATIENTS WITH ACTIVE INFLAMMATORY BOWEL DISEASE (IBD)

N. Rayment1, M. Mylonaki2, B. Hudspith1, J. Brostoff1, D.S. Rampton2.

1Infection and Immunity Group, Department of Life Sciences, King’s College London; 2Academic Department of Adult and Paediatric Gastroenterology, Barts and the London, Queen Mary’s School of Medicine & Dentistry, London

Background: Gut bacterial flora are thought to play a pathogenic role in IBD. We and others have previously reported the presence of E coli, but not other commensals, in the lamina propria of patients with active IBD (Gut 2002;50(Suppl II) A29; Scand J Gastroenterol 2002;37:1034–41). We have now investigated the possibility that these organisms may contribute to the pathogenesis of IBD by interaction with lamina proprial macrophages and dendritic cells.

Methods: Snap frozen rectal biopsies were taken at routine colonoscopy from patients with ulcerative colitis (UC, n = 35), Crohn’s (CD, n = 6), and controls with normal colorectal mucosa (n = 25). Fluorescent in situ hybridisation was used to identify E coli, bifidobacteria, lactobacillus, and bacteroides using RNA probes. Cellular co-localisation of bacteria was sought immunohistochemically using the phenotypic markers CD68 (macrophages) and CD1a (dendritic cells).

Results: In controls and patients with inactive IBD, all four bacterial species were confined to the mucosal surface. However, individual organisms and clusters of E coli (but not other species) were seen also in the lamina propria of 7/9 patients with active UC and 4/6 those with active CD (p < 0.0004 from controls and inactive IBD). In patients with active IBD, E coli were found adjacent to (n = 7) and/or internalised by CD68+ macrophages (n = 4). E coli were not co-located with CD1a+ dendritic cells in any patients (p < 0.00006 from macrophages).

Conclusion: Their close apposition to, and in some cases internalisation within lamina proprial macrophages in patients with active IBD, suggests that E coli could contribute to the pathogenesis of the disease.

050 CROHN’S DISEASE MUCOSA-ASSOCIATED E COLI INDUCE IL-8 RELEASE FROM INTESTINAL EPITHELIAL CELLS AND ADHERE VIA CARBOHYDRATE DEPENDENT MECHANISMS

H.M. Martin1, B.J. Campbell1, C.A. Hart2, H. Williams2, M. Nayar1, J-.F. Colombel3, A. Darfeuille-Michaud4, J.M. Rhodes1.

1Department of Medicine, University of Liverpool, UK; 2Department of Medical Microbiology, University of Liverpool, UK; 3Faculte de Pharmacie, Clermont-Ferrand, France; 4CHRU, Lille, France

Introduction: There is general consensus that intestinal inflammation in IBD is caused by an abnormal response to the intestinal microflora. It is our hypothesis that the altered mucosal glycosylation seen in IBD could allow mucosal recruitment of otherwise non-pathogenic bacteria and thus cause inflammation.

Methods: Mucosa-associated bacteria were isolated from colonoscopic biopsies from CD (n = 14), UC (n = 18), and control patients (IBS and sporadic polyps, n = 28) after removal of surface mucus with dithiothrietol. CD ileal-associated E coli were isolated by our French collaborators. Bacteria identified as E coli were screened for possession of known pathogenicity and adhesin genes, agglutination of a panel of human red blood cells, attachment and invasion of, and release of pro-inflammatory cytokines from intestinal cell-lines.

Results: 79% (11/14) of CD patients were positive for mucosa-associated bacteria compared with 39% (11/28) of control patients (p = 0.017) and 38% (7/18) of UC patients. Haemagglutinating E coli were identified in 39% (5/14) of CD patients compared with 4% (1/28) of controls (p = 0.01). Agglutination in all cases was inhibited by both soluble plantain fibre and bovine submaxillary mucin (BSM) but not following mild acid hydrolysis of BSM to remove sialic acid/fucose. A range of other carbohydrates and glycoconjugates, including ovine submaxillary mucin were non-inhibitory. The agglutinating E coli all possessed at least one adhesin gene but lacked conventional virulence genes of pathogenic E coli. Of the CD ileal-associated E coli, one strain, LF10, was shown, using both haemagglutination and PCR, to possess an adhesin specific for M blood group antigen. All agglutinating E coli were shown to adhere to both HT29 and I407 cell lines, inducing release of the pro-inflammatory cytokine IL-8 up to 4-fold above baseline levels (p < 0.01), but not IL-1β.

Conclusions: CD is associated with an increased prevalence of mucosally associated E coli, capable of attaching to intestinal cell lines and inducing a pro-inflammatory response. Soluble plantain fibre inhibits E coli attachment and deserves study as a potential prebiotic therapy for Crohn’s disease.

051 COLONIC INFLAMMATION IS A RISK FACTOR FOR DYSPLASIA IN ULCERATIVE COLITIS

M.D. Rutter, K.H. Wilkinson, G. Schofield, S. Rumbles, B.P. Saunders, A. Forbes. St Mark’s Hospital, Harrow, UK

Background and Aim: The cancer risk in patients with longstanding extensive ulcerative colitis (UC) is highly variable. We aimed to study potential factors that might predict cancer risk in UC.

Methods: Case control study of 204 patients on biennial colonoscopic surveillance of extensive UC. 68 cases with dysplasia or CRC and 136 controls without dysplasia/CRC were matched for sex, age at UC onset, disease duration, and extent. Data were obtained from colonoscopy and pathology reports, case notes, and prospective patient survey. Segmental colonoscopic and microscopic inflammation was recorded using a simple score (0, normal; 1, quiescent/chronic inflammation; 2,3, and 4, mild, moderate, and severe active inflammation). Each surveillance colonoscopy for every patient was scored. The mean value was used for analysis.

Results: Univariate analysis is shown in the table. On multivariate analysis, only the microscopy score remained significant (p < 0.001).

Abstract 51

Conclusion: These data suggest that colonic inflammation is an important risk factor for dysplasia/CRC development. Endoscopic and histological grading at surveillance colonoscopy could allow better risk stratification for surveillance programmes.

052 5-AMINOSALICYLIC ACIDS AND THE RISK OF RENAL TOXICITY: A LARGE BRITISH EPIDEMIOLOGICAL STUDY

T.P. van Staa1,2, S.P.L. Travis3, H.G.M. Leufkens1, R.F. Logan4.

1Utrecht Institute for Pharmaceutical Sciences, The Netherlands; 2Procter & Gamble Pharmaceuticals, Egham, UK; 3John Radcliffe Hospital, Oxford, UK, 4University of Nottingham, Nottingham, UK

The main objective of this study was to evaluate the risk of renal toxicity in patients using aminosalicylates (5-ASA). The medical records of GPs in the UK (from the General Practice Research Database) were used to estimate the incidence rates of renal toxicity of adult patients with either a record of inflammatory bowel disease (IBD) or prescription for 5-ASA (eg mesalazine or sulfasalazine) and that of control patients. Each case of renal toxicity was subsequently matched by age, sex, practice, and calendar time to one patient without renal toxicity. 37 984 adult patients with a record of IBD or 5-ASA prescription and a similar number of control patients were included. In the patients without a history of arthropathy, we found that IBD patients using 5-ASA had an increased risk of renal toxicity: the overall incidence was about 1 case per 1000 person-years of treatment (double compared to non-users). The case control analysis revealed the risk of renal disease was related to indicators of IBD severity. It was also increased in current and recent users (ie their last prescription in the 3 to 12 months before the index date) of 5-ASA. Compared to non-users, the odds ratio (OR) for renal events was 1.80 (95% CI 1.22 to 2.60) in current 5-ASA users. This excess risk markedly reduced in current users after adjustment for concomitant disease and drug use (adjusted OR 1.22 [0.69–2.16]). For recent users, the crude OR was 3.96 (2.20–7.13) and adjusted OR 2.80 (1.33–5.91). Users of mesalazine and sulfasalazine had comparable risks (crude ORs 1.66 [0.94–2.96] and 1.93 [1.18–3.14], and adjusted ORs 1.05 [0.47–2.31] and 1.34 [0.68–2.62], respectively). There was no relationship between 5-ASA dose and risk of renal disease. Numbers were too small to compare individual 5-ASA compounds. Users of 5-ASA had an increased risk of renal disease, which may be influenced by the underlying disease severity. There were no differences in risk of renal disease between mesalazine and sulfasalazine.

Neurogastroenterology/motility free papers 053–058

053 A CLINICAL INVESTIGATION OF THE EFFECTS OF TEGASEROD TREATMENT REGIMEN ON THE SYMPTOMS OF IBS-C IN TEGASEROD RESPONSIVE PATIENTS

K.D. Bardhan, A. Forbes, C.L. Marsden. District General Hospital, Rotherham, S60 2UD, St Mark’s Hospital, Harrow, HA1 3UJ; Novartis Pharmaceuticals UK Ltd, GU16 5SG

Background and Aims: Tegaserod is effective in relieving the symptoms of IBS with constipation (IBS-C), including abdominal discomfort or pain and bloating. However, the post-withdrawal characteristics and optimal long term treatment strategy still remain to be defined. The aim of this study was to investigate the effects of withdrawing tegaserod, as compared to maintaining patients on continuous treatment.

Methods: A randomised, open label, parallel group, multi-centre trial. 519 patients (≥18 years) diagnosed with IBS-C (457 females, 62 males) were treated with tegaserod (6mg twice daily) for 4 weeks. Responders were randomised (1:1) to either continue on tegaserod at the same dose, or to withdraw from treatment for 8 weeks. The absence or recurrence of IBS-C symptoms was measured at 4 weekly intervals, as derived from the patient’s weekly Overall Relief Assessment (ORA).

Results: 274/410 (68%) patients who completed the initial 4 weeks of treatment were responders (experienced relief of symptoms for ≥2 weeks). The remaining patients had little or no benefit (n = 131, 32.3%), or were not assessed (n = 5). At the end of the 8 week comparator phase, 90/104 (87%) of those maintained on tegaserod continued in symptomatic remission, judged by ORA scores at the end of week 12, compared with only 61/105 (58%) in whom therapy was withdrawn (p < 0.0001). Adverse events (AEs) were mild or moderate severity. The most frequent AEs in the treatment-continuation arm were diarrhoea (8/130, 6%), reflecting the promotile effect of the drug, and headache (15/130, 12%). In the treatment withdrawal arm no patients reported diarrhoea and 6% (9/141) reported headaches.

Conclusions: Based on weekly ORA scores, when patients who are responsive to tegaserod were maintained on treatment for 12 weeks, the recurrence of IBS-C symptoms was significantly reduced. Tegaserod treatment was well tolerated.

054 BIOFEEDBACK, NOT LAXATIVES, IMPROVES SYMPTOMS, TRANSIT AND AUTONOMIC TONE IN FUNCTIONAL CONSTIPATION

C.D.R. Murray, A.V. Emmanuel, M.A. Kamm. St Mark’s Hospital, Northwick Park, Harrow, Middlesex HA1 3UJ, UK

Background: Behavioural therapy biofeedback (BF), is an established treatment for patients with functional constipation, allowing avoidance of laxatives in successfully treated patients. Improvement with BF is known to be associated with enhanced autonomic input to the hindgut, as measured by laser Doppler flowmetry (LDF) of rectal mucosal blood flow. It is unknown whether laxatives affect autonomic tone and whole gut transit (WGT).

Methods: Forty-nine consecutive consenting female patients were randomised to receive either BF (n = 27, mean age 46 years, range 23–75) or bisacodyl, 5 to 10 mg as required (n = 22, 41 years, 22–81). Outcome with treatment was subjectively reported. Physiological studies were made blind to knowledge of treatment group and outcome. Physiological data comprised WGT and LDF measurement, and was assessed before, and at the end of treatment. WGT was performed using radio-opaque markers, slow transit defined as excess retention of one or more of the three marker sets used. On the same day as WGT studies, LDF of rectal mucosa was performed.

Results: Subjectively, 19/27 (70%) BF and 6/22 (27%) laxative patients reported improvement, p < 0.01. Nineteen BF patients had slow transit initially, and this normalised in 9 (47%), while 11 laxative treated patients had slow transit, normalised in 1 (9%), p < 0.05. The number of retained markers significantly reduced with BF (31±3 to 22±3, p = 0.002) associated with increased LDF (157±7 to 192±7, p < 0.001). There was no change in either transit (27±5 to 24±5, p = 0.3) or LDF (167±8 to 168±7, p = 0.8). There was a significant negative correlation between WGT and LDF in the BF treated patients (r=−0.51, p < 0.01) but no such correlation in the laxative group (r=−0.17, p > 0.1).

Discussion: Biofeedback is effective in improving symptoms, whole gut transit, and autonomic tone. Bisacodyl improved symptoms in a minority of patients and did not alter transit or autonomic tone. Acceleration of transit was associated with improved autonomic tone in BF treated patients only.

055 KETAMINE, AN NMDA RECEPTOR ANTAGONIST PREVENTS THE INDUCTION OF CENTRAL SENSITISATION IN A HUMAN MODEL OF VISCERAL PAIN HYPERSENSITIVITY

R.P. Willert1, A.R. Hobson1, C.J. Woolf2, D.G. Thompson1, Q. Aziz1. 1Hope Hospital, University of Manchester, UK; 2Massachusetts General Hospital and Harvard Medical School, Boston, USA

Introduction: Recent studies indicate that proximal oesophageal (PO) pain hypersensitivity to distal oesophageal acid infusion occurs due to increased neuronal excitability, ie central sensitisation (CS). In somatic tissues the induction of CS in the spinal cord is dependent on the N-methyl-D-aspartate (NMDA) receptor, however, the role of the NMDA receptor in mediating visceral hypersensitivity is unknown.

Aim: To determine if the NMDA receptor antagonist ketamine attenuates CS in a model of human oesophageal hypersensitivity.

Methods: 14 healthy subjects (7 male, age 18–43 years) were studied in a randomised two way double blind placebo controlled crossover study. Pain thresholds (PT) to electrical stimulation were determined in the PO and foot, and then either ketamine (0.075 mg/kg) or placebo (0.9% NaCl) was given as an intravenous bolus. A 30 min infusion of 0.15 M acid was then given in the distal oesophagus together with an intravenous infusion of ketamine (0.005 mg/kg) or placebo (0.9% NaCl) for 30 min. PT in the PO and foot were then repeatedly tested for 120 min post-infusion. PO pH and an attention task was performed throughout.

Results: In all but one subject (excluded from analysis) the pH remained above 5 in the PO during each study. Ketamine attenuated the reduction in PT in the PO in response to acid in the distal oesophagus (Area under curve (AUC): 16.9±3.9 and 12.3±2.6 for ketamine and placebo, respectively, p < 0.002). Ketamine did not affect PT in the foot compared with placebo (AUC 10.8±4.7 and 11.2±5.2, p = 0.5). Ketamine reduced attention scores during the infusion compared with baseline (Mean 54.4±2.6 and 46.1±4.6, respectively, p < 0.001) but this had ceased 30 min post-infusion (mean 54.9±2.8 and 54.4±2.6, p = 0.27).

Conclusion: The attenuation of PO hypersensitivity by ketamine suggests that the NMDA receptor contributes to the generation of CS in visceral pain. Therefore, NMDA receptor antagonists may have a role in the management of visceral pain hypersensitivity states.

056 POSTPRANDIAL HYPERSEROTONINAEMIA AND DELAYED GASTRIC EMPTYING: NEW INSIGHTS INTO POSTPRANDIAL SYMPTOMS IN UNTREATED COELIAC DISEASE

N.S. Coleman1, G.K.T. Holmes2, S.P. Dunlop1, L. Marciani3, P.A. Gowland3, G. Singh4, C.A. Marsden4, R.C. Spiller1. 1School of Medical and Surgical Sciences; 2University of Nottingham and Derbyshire Royal Infirmary; 3Magnetic Resonance Centre; 4School of Biomedical Sciences

Background: Anorexia and nausea are common but unexplained features in coeliac disease. We have recently shown that a 5-HT3 agonist delays gastric emptying and induces nausea in healthy subjects. Untreated coeliacs have marked duodenal enterochromaffin cell hyperplasia and we hypothesised that the postprandial dyspepsia and delayed gastric emptying frequently seen in these patients is due to excess serotonin release following a meal.

Methods: Untreated coeliacs (n = 13) and controls (n = 12) received a 500 kcal meal and blood samples were collected during fasting and for 3 hours postprandially. Serotonin was measured in platelet poor plasma using HPLC-EC. The presence of postprandial dyspepsia was assessed using a questionnaire. On a separate occasion gastric volumes and motility were determined by echoplanar magnetic resonance imaging.

Results: (Mean ±SE) Serotonin rose significantly after the meal, coeliac patients showing a significantly greater postprandial AUC over 3 hours (4386±622) compared with controls (1756±295 nmol/L.min, p = 0.001). Peak 5HT levels were also significantly higher in coeliacs (114±26 v 59±27 nmol/L, p < 0.01) and occurred sooner after the meal than in controls (71±16 v 133±9 min, p < 0.01) Coeliacs with significant postprandial dyspepsia (n = 7) had higher postprandial 5HT release than those without symptoms (n = 6) (5264±556 v 33611089 nmol/L.min) this difference was not significant (p = 0.13) Gastric emptying in coeliacs was significantly delayed (72±11 v 40± 13 min, p = 0.01) but this did not correlate with serotonin levels (r = 0.29).

Conclusion: Coeliac disease is associated with markedly elevated postprandial serotonin release, postprandial dyspepsia, and delayed gastric emptying. Whether these are causally linked remains to be determined by intervention studies using 5HT antagonists.

057 SMALL INTESTINAL INFLAMMATION IS ASSOCIATED WITH INCREASED ENTEROENDOCRINE CELL NUMBERS AND HYPOPHAGIA

F.C. Leslie1, J.R. McDermott2, S. Kazmi1, R. Grencis2, D.G. Thompson1, J.T. McLaughlin1. 1GI Science, Hope Hospital; 2School of Biological Sciences, Manchester University

The mechanisms by which small intestinal infection and inflammation produce symptoms such as anorexia are poorly understood. Upregulation of enteroendocrine (EEC) cell function is a plausible mechanism, because regulatory peptides/amines, for example CCK and 5HT, regulate food intake. Trichinella spiralis infection in mice is a well described model of small intestinal inflammation, and has been proposed as an animal model of postinfective functional GI disorders. Effects on feeding and EEC have not been assessed.

Methods: Male NIH mice were infected with 300 T spiralis larvae by oral gavage. Food intake was measured and compared with a control group of naive mice. On days 6, 9, 13, and 20 postinfection (PI), duodena were fixed and immunostained for 5HT and CCK. Results are expressed as numbers of positive staining EEC per 15 crypt/villus units. To assess the contribution of the mast cell infiltration seen in this model, an anti c-Kit antibody (Ack-2) was administered i.p. to attenuate mast cell responses. A control group received nonspecific IgG.

Results: Six days PI, increased numbers of CCK-positive EEC cells were seen (9.0±1.3) compared to naive mice (6.7±0.7, p = 0.03). This peaked from day 9 (15.3±1.7, p < 0.01) to day 13 (15.0±0.6, p < 0.01) but improved by day 20 (8.5±0.9, p = ns), when inflammation has resolved. Similar changes were seen with 5HT-positive EEC cells, although not significantly increased until day 9 (19.8±2.6, p < 0.01) and day 13 (17.0±0.9, p = < 0.01). This again resolved by day 20 (7.2±1.5, p = ns). Food intake dropped from 4.5±0.1 g/mouse/day in naive mice to 3.4±0.5 at day 7, to 2.1±0.8 at day 9, and to 3.1±0.2 at day 11 (all p < 0.05). This normalised by day 14. Studies following Ack-2 showed that hypophagia and increased EEC persist despite effective mast cell attenuation.

Conclusions: Acute small intestinal infection is associated with an increase in EEC cell numbers that coincides with, or contributes to, marked hypophagia. Infiltration by mast cells is not responsible for driving these responses. This represents a potentially important host response to intestinal infection. Both CCK and 5HT influence motility: this may facilitate parasite clearance. CCK may also protect via reduced bacterial translocation and increased IgA secretion.

Supported by Wellcome Trust (FCL) and DDF (JMcL).

058 DECREASED POST-PRANDIAL 5 HT IN CONSTIPATION PREDOMINANT IRRITABLE BOWEL SYNDROME (C-IBS)

S.P. Dunlop, N.S. Coleman, P.E. Blackshaw, A.C. Perkins, G. Singh, C.A. Marsden, R.C. Spiller. Divisions of Gastroenterology, Medical Physics and Neurosciences, University Hospital, Nottingham

Introduction: 5 HT (5-hydroxytryptamine), an important stimulator of intestinal motility and secretions, is stored predominantly within enterochromaffin cells (EC) of the GI tract, which are increased in post-infectious IBS (PI-IBS). Recent reports have associated diarrhoea-predominant IBS with increased 5HT release but whether reduced release plays a role in causing constipation has not been examined.

Aims: To determine the relationship between postprandial plasma serotonin release and colonic transit in C-IBS compared with PI-IBS and healthy controls.

Methods: 15 C-IBS, 15 PI-IBS with diarrhoea predominant symptoms, and 15 healthy volunteers recruited through advertisement, underwent serial plasma serotonin measurement after a standard 520 kcal meal for 3 hours. Blood was taken through an 18G cannula into a syringe containing citrate, adenosine, dipyrimadole, and theophylline to inhibit platelet activation. Platelet poor plasma (PPP) and platelet rich plasma (PRP) was assessed for 5 HT using HPLC with electrochemical detection. Colonic transit was measured using marker pellets.

Results: Mean (SEM) colonic transit was prolonged in C-IBS (49.4±3.8 h) compared with PI-IBS (26.7±4.5) and controls (34.1±4.5) (p = 0.002). The area under the curve (AUC) of PPP serotonin from immediately after the meal to 180 min was lower in C-IBS (2593±309 nmol/L.min) compared with PI-IBS (5623±721) and controls (4822±598) (p < 0.001). Although PI-IBS showed increased AUC compared with controls, these differences were not significant owing to substantial variability. There was a negative correlation between AUC and transit in all subjects (r = 0.354, p = 0.02). Platelet 5HT was greater in C-IBS (652±56.1) compared with PI-IBS (PRP = 484.1±40.7 ng/109 platelets, p = 0.039) but not significantly different from controls (598.5±36.4).

Conclusions: Reduced 5 HT release may be an important contributor to slow intestinal transit in C-IBS.

Gastroduodenal free papers 059–067

059 WHEN IS PROPHYLACTIC SURGERY FOR DUODENAL ADENOMATOSIS IN FAP JUSTIFYABLE?

M.C. Gallagher, R.K.S. Phillips. The Polyposis Registry, St Mark’s Hospital, Harrow, Middlesex, UK

Background: Despite > 90% of patients with familial adenomatous polyposis having duodenal adenomas, only 5% develop cancer. This risk rises to more than 30% in patients with more advanced disease in whom prophylactic surgery is often advised.1–3

Aims: The outcome of prophylactic pylorus preserving pancreatoduodenal resection performed for Spigelman Stage IV duodenal polyposis detected in an endoscopic surveillance programme is presented.

Methods: FAP patients entered into the surveillance programme at St Mark’s Hospital were included in this study. Endoscopy is performed with a side viewing duodenoscope to a set protocol. Data were collected prospectively on the Polyposis Register, and the case notes of patients undergoing prophylactic surgery reviewed retrospectively.

Results: 419 FAP patients have entered the endoscopic surveillance programme since 1989. Between 1994 and 2002, 15 patients with advanced duodenal polyposis (six male, average age 54 years and 4 months) were referred for PPPDR. Six suffered major postoperative complications (40%). Although the pathology of the resected specimen revealed less severe changes in two patients, five with Stage IV disease showed adenocarcinoma, all ampullary. One patient (with benign histology) died from a pulmonary embolus shortly after hospital discharge. Four of the five patients with adenocarcinoma have died (10–36 months postoperatively) and one further patient has died form a brain tumour. The remainder are alive at a mean of 36 months (2–103).

Conclusion: Surprise invasive adenocarcinoma was already present in 33% of patients. Survival once cancer has developed is poor. Prophylactic surgery may be most appropriate for individuals with large ampullary polyps. Advising resection for earlier stage disease is limited by the complication rate.

  1. Spigelman, et al. Lancet, 1989;2:783–5.

  2. Groves, et al. Gut 2002;505:636–41

  3. Nugent, et al. J R Soc Med 1994;87:704–6.

060 A SYSTEMATIC REVIEW AND ECONOMIC ANALYSIS OF THE COST-EFFECTIVENESS OF PROTON PUMP INHIBITOR THERAPY IN NON-ULCER DYSPEPSIA

P. Moayyedi1,2, B. Delaney1. 1Gastroenterology Unit, City Hospital, Birmingham, UK; 2Dept of Academic Primary Care and General Practice, Birmingham University, Birmingham B15 2TT

Introduction: Proton pump inhibitor (PPI) therapy is an established treatment for gastro-oesophageal reflux disease, but their efficacy in non-ulcer dyspepsia (NUD) is controversial. Randomised controlled trials (RCTs) have given conflicting results so we have conducted a rigorous Cochrane systematic review of the literature and evaluated the cost effectiveness of this strategy.

Methods: The Cochrane Controlled Trials Register, Medline, EMBASE, and CINAHL electronic databases were searched for RCTs evaluating PPIs in NUD. Experts in the field and pharmaceutical companies were contacted for information on any unpublished RCTs. A single investigator reviewed papers generated from this search according to predefined eligibility and validity criteria. Outcomes were dichotomised into dyspepsia minimal/resolved versus same/worse. The results were entered into a Markov model (Tree Age version 4.0) that compared costs and effects of PPI with antacid therapy over a 12 month period from a health service perspective. Full dose PPI was assumed to cost 22.75, low dose PPI 12.43, antacid 2.48/month, and a GP visit 18. Monte Carlo simulations of 5000 patients were performed using the 95% CI of the meta-analysis data to give stochastic estimates of cost effectiveness.

Results: Seven RCTs evaluating 3031 patients for 2–8 weeks were eligible for the review. PPI therapy was significantly superior to placebo in treating NUD (relative risk (RR) of remaining dyspeptic = 0.86; 95% CI = 0.80–0.93, p < 0.0001; random effects model). There was significant heterogeneity between the studies. The number needed to treat to cure one case of dyspepsia = 9 (95% CI = 6 to 26). Six RCTs evaluating 2032 patients compared high versus low dose PPI with no statistical difference in curing NUD symptoms (RR = 0.98; 95% CI = 0.92 to 1.05). The Markov model suggested full dose PPI cost 60/month free from dyspepsia (95% CI = 42–112) while half dose PPI cost 29/month free of dyspepsia (95% CI = 19–55).

Conclusions: PPI is modestly effective in NUD and the economic model suggests low dose PPI is cost-effective in NUD.

061 PLASMA GHRELIN FOLLOWING CURE OF HELICOBACTER PYLORI

D. Freshwater, H.S. Randeva, P. O’Hare, C.U. Nwokolo. University Hospitals Coventry and Warwickshire NHS Trust and Biomedical Research Institute, University of Warwick, UK

Background: In the Western world, the incidence of oesophageal adenocarcinoma has increased over the past 30 years, coinciding with a decrease in the prevalence of H pylori. Trends of increasing oesophageal adenocarcinoma can be linked causally to increasing gastroesophageal reflux disease (GORD), which can be linked to an increasingly obese population. However, there is no plausible biological mechanism of association between H pylori, obesity, and GORD. Ghrelin, a peptide produced in the stomach that regulates appetite, food intake, and body composition, is studied in H pylori positive asymptomatic subjects.

Methods: Plasma ghrelin, leptin, and gastrin were measured for 6 h after an overnight fast, before and after cure of H pylori in 10 subjects; 24 h intragastric acidity was also assessed.

Results: After cure median (95% CIs) integrated plasma ghrelin increased from 1160.5 (765.5–1451) pg/ml/h to 1910.4 (1675.6–2395.6) pg/ml/h (p = 0.002 Wilcoxon Rank Sum Test), a 75% increase. This was associated with a 14% increase in 24 h intragastric acidity (p = 0.006). There was a significant positive correlation between plasma ghrelin and intragastric acidity (rs 0.44, p = 0.05, Spearman rank correlation) and no significant change in leptin or gastrin.

Conclusions: After H pylori cure, plasma ghrelin increases profoundly in asymptomatic subjects. This could lead to increased appetite and weight gain and contribute to the increasing obesity seen in Western populations where H pylori prevalence is low. This plausible biological mechanism links H pylori, through increasing obesity and GORD, to the increase in oesophageal adenocarcinoma observed in the Western world.

062 PCR DETECTION OF CAG PATHOGENICITY ISLAND GENES DOES NOT ACCURATELY PREDICT FUNCTIONALITY OF CAG ENCODED PROTEINS

R.H. Argent1, M. Kidd2, R.J. Owen3, M.C. Limb1, J.C. Atherton1. 1Division of Gastroenterology and Institute of Infections and Immunity, University of Nottingham, UK; 2School of Medicine, Yale University, New Haven, CT, USA; 3Laboratory of Enteric Pathogens, PHLS, London, UK

H pylori associated disease is more common in patients infected with CagA+ strains. CagA is one of 31 proteins encoded on the cag pathogenicity island (PaI). CagA itself is translocated into host cells via a syringe like structure comprising other cag encoded proteins. Here it becomes tyrosine phosphorylated and causes proliferative cell signalling and cytoskeletal changes. Interaction of the cag syringe itself with epithelial cells results in induction of proinflammatory cytokines, particularly interleukin-8 (IL-8). One or both of cagA phosphorylation and cag induced IL-8 secretion are likely responsible for the increased disease risk from CagA+ strains. Thus, we aimed to assess whether PCR detection of selected cag PaI genes could predict whether H pylori strains could induce epithelial cells to phosphorylate cagA and/or secrete IL-8.

Methods/Results:H pylori strains were obtained from 26 patients from South Africa. The presence of cagA, cagE, cagM, cagT, and cag6–7 genes was determined by PCR analysis. cagA was present in all 26 strains, and 16 had all the tested cag PaI genes and so were predicted to encode an intact PaI. Possession and phosphorylation of cagA was determined by use of a cell free system and by co-culturing H pylori with AGS cells at 37° for 6 h, followed by lysis of the cells, and western blotting. Of the 26 strains, 20 induced cagA phosphorylation within AGS cells, and all of these strains induced the secretion of IL-8. Of the remaining 6 strains none induced IL-8 secretion. The degree of cagA phosphorylation induced and the amount of translocated cagA were not related to the level of IL-8 secretion induced. Six strains that possessed cagA did not produce cagA protein. Four strains predicted to lack a complete cag PaI by PCR analysis induced both cagA phosphorylation and IL-8 production, and one strain predicted to possess an intact PaI by PCR did not induce cagA phosphorylation or IL-8 production.

Conclusion: PCR analysis of selected cag PaI genes cannot reliably predict whether the PaI is functional, either in terms of cagA translocation and phosphorylation or IL-8 induction in epithelial cells. PCR analysis is therefore unlikely to be as accurate as these phenotypic assays in measuring strain virulence.

063 EVOLUTION OF THE HELICOBACTER PYLORI VACA TOXIN BY RECOMBINATION IN VIVO

F. Aviles1, D.P. Letley1, G. Gonzales2, J. Torres2, J.C.Atherton1.

1Division of Gastroenterology and Institute of Infections and Immunity, University of Nottingham, UK; 2IMSS, Mexico City, Mexico

Introduction: The H pylori vacuolating cytotoxin, VacA, varies between strains as a result of genetic recombination. The three main types have different toxicity (s1/m1>s1/m2>s2/m2) and this results in different disease associations. We aimed to show whether VacA can evolve to alter its toxicity within an individual stomach.

Methods: Single H pylori colonies were cultured from gastric biopsies from a Mexican patient with duodenal ulcer and typed for the toxin gene, vacA, by allele specific PCR. The relationship between isolates was determined by two PCR based genomic fingerprinting methods, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), and for two isolates of different vacA type by partial sequencing of the housekeeping genes, A/G adenine glycosylase (mutY) and GTPase (yphC). For these two isolates, vacA was sequenced and vacuolating activity was determined on the gastric cell line, AGS.

Results: Among 11 individual isolates cultured, we found 4 of vacA type s1/m1 and 7 of type s1/m2. RAPD and AFLP analysis showed that all isolates were closely related. Nucleotide sequencing of mutY and yphC confirmed a clonal origin. The vacA sequence of candidate vacA s1/m1 and s1/m2 clones showed differences in two vacA segments of 439 and 378 base pairs, respectively. Outside these regions, vacA nucleotide sequences were identical. The vacA s1/m1 clone induced AGS cell vacuolation whereas the s1/m2 clone induced no vacuolation.

Conclusion/Discussion: We have demonstrated evolution, by homologous recombination, of the toxin gene, vacA, and hence of toxin phenotype within an individual stomach. From our vacA sequence data, it appears most likely that the less pathogenic strain is the result of this evolution process. Evolution of VacA in vivo might represents an adaptation process for survival in the changing gastric environment. Changing H pylori virulence within an individual stomach has important implications for pathogenicity and for vaccine development.

064 A SYSTEMATIC REVIEW OF HELICOBACTER PYLORI ERADICATION THERAPY IN DUODENAL AND GASTRIC ULCER HEALING AND MAINTENANCE

A. Ford1, B. Delaney2, P. Moayyedi3. 1Leeds General Infirmary, Great George Street, UK; 2Leeds and University of Birmingham, Birmingham, UK

Introduction:H pylori eradication (HE) therapy is regarded as the treatment of choice for both duodenal (DU) and gastric ulcer (GU). Surprisingly there have been few systematic reviews on the efficacy of this approach both for acute healing and maintenance.

Methods: The Cochrane Controlled Trials Register, Medline, EMBASE, and CINAHL electronic databases were searched for RCTs evaluating predefined HE therapies in DU and GU. Comparison therapies were ulcer healing drugs (UHD) or placebo/no therapy. Experts in the field and pharmaceutical companies were contacted for information on any unpublished RCTs. Articles were included on predefined eligibility and validity criteria.

Results: 82 articles were reviewed, 57 were eligible, and data was extractable in 52 papers. The results are given in the table in terms of relative risk (RR) of ulcer unhealed/relapsed. Where statistically significant heterogeneity (p < 0.2) existed (denoted by *) a random effects model was used (see Table).

Abstract 64

The pooled relapse rate for DU recurrence in those allocated to no treatment over 12–52 weeks was 60% compared with 14% in the HE group. The corresponding figures for GU patients were 45% and 12%. Meta-regression was performed to evaluate factors that might explain the heterogeneity in the DU and GU maintenance meta-analyses. Eradication rate was not a significant factor in this model with only concealment of allocation an independent predictor of trial outcome.

Conclusions: HE is an effective therapy to prevent DU and GU recurrence although the efficacy of this may be overestimated and there is variability in study results not explained by efficacy of eradication. HE has additional benefit to UHD in healing DU (but not GU) patients.

065 GASTRIC EPITHELIAL ANTI-MICROBIAL PEPTIDES—HISTOLOGICAL CORRELATION AND INFLUENCE OF SITE, ULCERATION, AND LOW DOSE ASPIRIN

A.S. Taha1, E. Faccenda2, W.J. Angerson3, R.W. Kelly3. 1Crosshouse Hospital, Kilmarnock; 2University Department of Surgery, Glasgow, Scotland, UK; 3Medical Research Council Reproductive Sciences Centre, Edinburgh, UK

Background and Aims: Recent in vitro and animal model studies have identified a number of epithelial peptides with antimicrobial and potentially gastroprotective activities. To investigate their relevance to human peptic ulcer disease, we assessed gastric epithelial secretory leukocyte proteinase inhibitor (SLPI) and human beta defensins, HBD1, HBD2, and HBD5 in a range of peptic disorders.

Methods: Gastric biopsies were taken from 52 patients, median age of 55 years, including 31 males and 8 smokers. Expression of SLPI, HBD1, HBD2, and HBD5 mRNA was determined using real time quantitative PCR. Histological assessment was carried out on biopsies taken from the same anatomical regions. The activity of gastritis was graded on a 0–3 scale, 0 being normal and 3 severe. Specimens carried code numbers for blind assessment.

Results: The gastric antrum had a median SLPI level of 0.93 and HBD1 of 0.42, compared with 0.13 (p = 0.001) and 0.08 (p = 0.002) ,respectively, in the gastric body. The antral histological scores correlated positively with HBD2 expression (r = 0.69; p < 0.001) and negatively with HBD1 (r = −0.47; p = 0.006), particularly in the absence of aspirin. Patients with H pylori gastritis, gastric, or duodenal ulcers had lower expression of HBD1 and greater expression of HBD2, and this pattern was only partially reversed by H pylori eradication. The intake of low dose aspirin, 75 mg daily, by 16 patients had no influence on epithelial peptides of those free of H pylori infection. However, in the infected group, aspirin was associated with lower expression of SLPI (0.33 v 0.58; p = 0.029) and higher expression of HBD5 (42.3 v 3.25; p = 0.039).

Conclusions: The expression of some endogenous epithelial antimicrobial peptides is influenced by the anatomical site, the grade of gastritis, H pylori, and peptic ulceration. It can also be modulated by low dose aspirin particularly in subjects infected with H pylori.

066 HELICOBACTER PYLORI INFECTION IS ASSOCIATED WITH UPREGULATION OF MATRILYSIN (MMP-7) IN GASTRIC EPITHELIAL CELLS IN VIVO

J.R. Bebb1, F. Aviles1, N. Hand2, A. Zaitoun2, J.C. Atherton1. 1Div Gastro/ Inst Infect Immun, University Hospital, Nottingham, UK; 2Dept Histopath, University Hospital, Nottingham, UK

Background: MMP-7 (matrilysin) is a member of the metalloproteinase family of enzymes, which are important in normal and pathological remodelling of epithelial matrix interactions. It is upregulated in gastric cancer. We have previously shown that H pylori co-culture induces upregulation of MMP-7 in epithelial cells in vitro at both the protein and RNA level, and that this is more marked for strains with an intact cag pathogenicity island. We now examined whether this occurs in vivo.

Methods: Gastric biopsies were taken at endoscopy from H pylori infected (n = 17) and uninfected (n = 16) patients, and MMP-7 expression examined by immunohistochemistry (paraffin-embedded sections), ELISA (R&D systems), and Real Time PCR. H pylori strains were cultured and PCR-typed for cagA, cagE, and vacA. For immunohistochemistry, slides were examined by two blinded observers and staining intensity was graded 0–4. Results were compared by Mann Whitney U test (immunohist) and t test (ELISA, Real Time PCR).

Results: Epithelial cells from H pylori infected patients stained more intensely for MMP-7 than uninfected patients both in antrum and corpus. This was most marked superficially in the antrum (median Hp+ 2, Hp− 1, p < 0.05) and in the proliferative zone in the corpus (median Hp+ 3, Hp− 2, p < 0.05). There was also a significant increase in MMP-7 staining inflammatory cells in infected patients. ELISA confirmed these results with a threefold increase in infected patients (Hp+ 0.182 v Hp− 0.059, p = 0.009). Real Time PCR demonstrated upregulation of MMP-7 at RNA level (antrum Hp+ 0.038 v Hp− 0.008, p = 0.007). It was not possible to correlate changes with strain type due to small numbers of cag-negative strains.

Conclusion: This study demonstrates increased expression of MMP-7 in vivo in nonmalignant gastric epithelial cells colonised by H pylori. We speculate that upregulation of MMP-7 in H pylori gastritis may play a role in H pylori induced gastric carcinogenesis.

067 THE EFFECT OF NAPROXEN ON GENES ASSOCIATED WITH DNA DAMAGE AND REPAIR IN THE STOMACH OF HEALTHY VOLUNTEERS BY MICROARRAY ANALYSIS

J.A. Smith1, A. Rose2, M.W. James1, J.R. Bebb1, C.T. Atherton1, N. Bailey-Flitter1, A. Zaitoun1, R.A. Jones2, N.P. Shankley2, C.J. Hawkey1. 1Division of Gastroenterology, University Hospital, Nottingham, UK; 2Johnson & Johnson Pharmaceutical Research & Development, La Jolla, CA, USA

Introduction: Microarray analysis provides the opportunity to study large numbers of drug—gene interactions. Here we report some of the effects of the non steroidal anti-inflammatory drug (NSAID) naproxen on the transcription of genes in the human gastric mucosa.

Methods: Four (2m, 2f) healthy volunteers received naproxen 500 mg twice daily. for 2 days. Four control subjects (2m, 2f) received no active drug. Antral biopsies (4) were taken by endoscopy before and following (3, 12, and 48 h) treatment. Total RNA was extracted from 2 of the biopsies with RNeasy™ mini kits and 2 were used for histology. The expression of ∼8000 genes were assessed by cDNA microarray. After normalisation the expression of 1258 genes were significantly altered by naproxen as judged by ANOVA (p < 0.05). Data in brackets are maximum percent increase compared to control.

Results: Scrutiny of the microarray data identified increased transcription of genes coding for the following proteins at 3 h: caspase 1 (29%) and 7 (21%), E2F dimmerization partner 2 (18%); at 12 h: caspase 3 (35%), poly (ADP-ribose) synthase (33%), NFkB (14.5%), methyl GpG binding domain protein 4 (33%), APC (20κ), DNA protein kinase (43%), damage specific DNA binding protein 2 (24%), cell division cycle 25A (17%), checkpoint kinase 2 (47%), cyclin dependent kinase (CDK) 6 (20%), cyclin H (33%); and at 48 h: caspase 9 (31%), xeroderma pigmentosa group A (22%), mothers against decapentaplegic homologue 6 (30%), CDK 7(40%), CDK associated protein 1 (37%). Histological assessment found no inflammatory changes in the gastric mucosa correlated to drug treatment.

Conclusion: These transcriptional changes suggest naproxen not only causes apoptosis but also DNA double-strand breaks, stimulates DNA repair responses and checkpoint cell cycle arrest. This preliminary analysis of an in vivo human experiment is consistent with the in vitro findings of others. The data also allow identification of previously unrecognised changes in gene expression that may be related to the anticancer properties of NSAIDs.

Pancreatic free papers 068–069

068 SECRETIN MRCP DERIVED FUNCTION IN CHRONIC PANCREATITIS

A. Gillams, S. Pereira, W. Lees. Department of Medical Imaging and Gastroenterology, The Middlesex Hospital, Mortimer Street, London, W1T 3AA

Introduction: We have previously reported the use of secretin MRCP to quantify pancreatic function. We have now studied a group of normal patients and patients with chronic pancreatitis.

Methods: 62 patients referred with suspected or known pancreatic pathology underwent SSFSE MRCP both before and after 0.1 ml/kg IV secretin. The sequence with identical positioning and receiver gain was performed prior to secretin and at 2 min intervals post-secretin to a mean of 7 min. All patients also underwent parenchymal pancreatic imaging with a coronal trufisp and axial T1W sequence. The pancreatic flow rate was calculated as the change in signal intensity over time within a ROI that encompassed not only the pancreas but the small bowel. A voxel containing 100% water was used to correct for variations in receiver gain between individuals. 63 patients had concurrent CT scans. Using the Cambridge classification patients were divided into mild, moderate, and severe chronic pancreatitis.

Results: There were 31 patients with a normal pancreas. The mean flow rate was 8.0 ml/min +/− 2.6. 31 patients had evidence of chronic pancreatitis, 6 mild, 6 moderate, and 19 severe chronic pancreatitis. The mean flow rates were; mild pancreatitis 6.9 +/− 1.7, moderate 6.0 +/− 1.7, and severe 5.9 +/− 2.9. This was statistically significant from the normal group.

Conclusion: Significantly different flow rates were found in patients with severe chronic pancreatitis compared with normals. Further, there appeared to be trend down through the different levels of severity of chronic pancreatitis. Changes in flow rates in a given individual should provide useful information and a trend down on sequential measurements may prove to be an early indicator for pancreatic disease.

069 SITE AND SEQUENCE OF INITIATION OF INTRACELLULAR ENZYME ACTIVATION IN EXPERIMENTAL ACUTE PANCREATITIS

M.G.T. Raraty, J.P. Neoptolemos, O.H. Petersen, R. Sutton. Departments of Surgery & Physiology, University of Liverpool, UK

Intracellular activation of both trypsin and cathepsin B is known to occur early in the course of acute pancreatitis. This study aimed to determine the time course and subcellular location of such activation and its relationship to abnormal Ca2+ signalling. Isolated mouse pancreatic acinar cells were loaded with 10 μM IPR-CMAC (a fluorescent substrate specific for trypsin) and/or (FR)2-R110 (specific for cathepsin B), together with fura-2 for measurement of cytosolic Ca2+ ([Ca2+]i) and were then perifused with stimuli. Enzyme activation was visualised by confocal microscopy, and changes in cellular morphology by electron microscopy. Cells exposed to 10nM CCK or 2 μM thapsigargin showed a rapid rise in [Ca2+]i followed by a modest sustained elevation. Within 300 seconds of application of the stimulus, fluorescence appeared within multiple discrete rounded compartments approximately 1 μm in diameter within the granular pole of the cell. These gradually enlarged with continued stimulation and became less distinct. By 60 min, typical vacuoles were apparent on electron microscopy. The fluorescence from both enzyme substrates developed within spatially indistinguishable compartments at the apical pole of the cell but trypsinogen activation preceded cathepsin B activation by a mean of 85 ± 34s. Trypsinogen activation occurred more rapidly and reached a plateau 200 seconds earlier than that from cathepsin B. Attenuation of the abnormal [Ca2+]i signals with the Ca2+ chelating agent BAPTA prevented both trypsinogen activation and vacuolisation. These findings build on our previous findings (PNAS 2000;97:13126–31) and confirm the crucial role of abnormal cytosolic Ca2+ signals in the initiation of intracellular enzyme activation. Trypsinogen activation occurs within zymogen granules, which then become vacuoles. These results are consistent with the central role trypsinogen activation is thought to play in acute pancreatitis with results that are subsequently amplified by cathepsin B.

Oesophageal free papers 070–075

070 ENDOSCOPIC GASTROPLICATION FOR THE TREATMENT OF PAEDIATRIC GASTRO-OESOPHAGEAL REFLUX DISEASE

M.A. Thomson1, N. Afsal1, R. D’Souza2, A. Fritscher-Ravens2, P. Swain2.

1Centre for Paediatric Gastroenterology, Royal Free Hospital, London; 2Royal London Hospital, London

The aim of this work is to assess the safety and efficacy of endoscopic gastroplication (EG) (BARD Endocinch© device) for the treatment of GORD in children and adolescents. 20 patients (3 cerebral palsy, 8 male, median (range) age 12.8 years (6.1–17.7), weight 46 kg (16.5–75)) with symptoms of GORD dependent/refractory wrt PPIs for > 12 months underwent EG. Median follow up: 15 months. Exclusion criteria were age > 17 years, dysphagia, obesity (BMI) > 99th centile, previous upper GI surgery, and hiatus hernia > 2 cm. Pre-procedure assessment included symptom scoring, upper GI endoscopy, barium upper GI series, oesophageal manometry (in 4), liquid/solid phase gastric scintiscan, 24 hour oesophageal pH, and completed reflux quality of life (QOLRAD) questionnaire. Post-procedure symptomatology, QOL and adverse events were assessed at 1, 3, 6, and 12 months. Repeat 24 h pH was performed at 2 months (16 patients). All occurred under general anaesthesia as is standard practice in paediatric endoscopy. The median duration of the procedure for 3 plications (3 pairs of sutures) was 65 min. The median heartburn symptom score (daily heartburn frequency x severity (0–10)) was 24 pre-procedure and 0 at six weeks and 6 months. Total, and all sub-parameters, of QOLRAD showed sustained improvement for the follow up period. Total QOLRAD (best 175) increased from a median of 83 (70–142) to 157 (93–175) (p < 0.001). All pH parameters improved significantly. Median reflux index from 14.8% (0.9–67.9) to 2.7% (1.0–6.9) (p < 0.001). Median pH Demeester score from 67.1 (6.1–68.3) to 11.6 (5.0–31.2) (p < 0.001). 19/20 patients had a normal pH profile at 2 months and 19/20 did not require any further PPI use at any stage (median PPI dose pre-EG 0.89 (0.3–2.42) mg/kg/day). One patient had localised gastric bleeding requiring red cell transfusion that settled spontaneously and no other adverse events were seen. This is the first study reporting paediatric experience with an endoscopic anti-reflux procedure and this shows it to be a safe and effective method of managing GORD in the paediatric age group.

071 LIFE THREATENING AND FATAL COMPLICATIONS IN FUNDOPLICATION: ANALYSIS OF 11 974 PATIENTS

J.A. Salo, T.K. Rantanen, J.T. Sipponen. Section of General Thoracic and Oesophageal Surgery, Helsinki University Central Hospital, Helsinki, Finland

The number of fundoplications has increased since the introduction of the laparoscopic technique. Usually excellent reports from specialised centres are reported and no comprehensive nationwide studies of dangerous complications of fundoplication have been made. We have analysed all the serious and fatal complications after both open and laparoscopic fundoplication in all primary fundoplications performed between 1.1.1987 and 31.12.2000. Altogether 11 974 fundoplications were performed in Finland (population ca. 5 million), (5975 open and 5999 laparoscopic). Patients undergoing open fundoplication had more operative risk factors. According to the Central Statistical Office, Patient Insurance Association and National Research and Development Centre for Welfare and Health there were altogether 65 (0.54%) fatal or life-threatening complications; 40 (0.67%) in laparoscopic and 25 (0.42%) in open (p = n.s.). The complications were severe infections caused by perforation of the oesophagus or fundus, intestinal perforation/intra-abdominal abscess, several re-operations due to haemorrhage requiring massive transfusion, total oesophageal obstruction leading to total parenteral nutrition with/without re-operation, incarceration of the intestine after hiatal repair, and lung embolism. The total mortality was 15 (0.13%). Five patients died in laparoscopic (mortality 0.08%) and 10 in open fundoplication (mortality 0.17%, p = n.s.). Laparoscopic fundoplication had 35 (0.58%) non-fatal life-threatening complications, which is significantly more (p < 0.01) than in open 15 (0.25%). In conclusion the current mortality of fundoplication is relatively low (0.13%), but still, almost every 200 patient suffers from life-threatening and/or fatal complications. There is no significant difference in the mortality between laparoscopic and open fundoplication, but laparoscopic fundoplication is associated with significantly more life-threatening complications. These facts must be considered both when planning operations and when informing patients.

072 IS CHEWING GUM EFFECTIVE IN REDUCING POSTPRANDIAL REFLUX IN THE OESOPHAGUS?

R. Moazzez1, A. Anggiansah2, D. Bartlett2.

1Department of Conservative Dentistry (GKT), St Thomas’ Hospital, London, UK; 2Oesophageal Laboratory, St Thomas’ Hospital, London, UK

Introduction: Saliva is reported to have an important role in preventing damage by gastro-oesophageal reflux (GOR). Chewing gum stimulates the flow of saliva and initiates primary peristalsis. Consequently peristalsis clears the volume of the refluxate and then the saliva dilutes and neutralises the remaining acid in the oesophagus. However, this novel idea needs further investigation.

Aim: This study was designed to assess the effect of chewing gum on postprandial GOR. The objective was to compare postprandial pH on the same patient on two occasions by keeping the experimental conditions identical.

Method: A standard refluxogenic meal was devised with 60% fat using a computer program (comp.eat). 21 subjects with symptoms of GOR were chosen. Each subject had standard manometry followed by the insertion of the pH catheter. Subjects were given the refluxogenic meal on the first and the second day for lunch having starved for 4 h prior to eating the meal on both occasions. They were randomly selected to chew a piece of gum for half an hour after eating the meal on either the first or the second day. pH was measured for 2 h during the postprandial period on both occasions under the same conditions. Percentage time pH below 4 was compared for the two postprandial periods.

Results: The mean (sd) and median (IQ range) values for the percentage time pH below 4 during the postprandial period without chewing gum were 9.2(8.9) and 5.8(2–13.5) respectively and with chewing gum 4.7(5.4) and 3.6(0.6–6.8). This difference was statistically significant (p = 0.005).

Discussion: Chewing gum significantly reduced postprandial reflux in these symptomatic patients who acted as their own controls. This study allowed the assessment of the role of chewing gum as the differential factor on the reduction of postprandial GOR.

Conclusion: Chewing a piece of gum for half an hour after a refluxogenic meal significantly reduced acid exposure in the oesophagus during the postprandial period in these subjects.

073 HIGH GRADE DYSPLASIA IN BARRETT’S OESOPHAGUS: SUCCESSFUL ABLATION BY PHOTODYNAMIC THERAPY WITH ALA REQUIRES INTENSIVE THERAPY

N.F. Jamieson, S. Thorpe, A. Mosse, S.G. Bown, L.B. Lovat. National Medical Laser Centre, Royal Free and University College London Medical School, London, UK

Background: Oesophagectomy is considered first line therapy for high grade dysplasia (HGD) in Barrett’s oesophagus but has considerable morbidity and mortality. Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) offers a minimally-invasive method for mucosal ablation of HGD. No published studies have attempted to optimise treatment conditions. Our aim was to confirm safety and determine the effect of light dose and treatment regime on outcome.

Methods: With ethical approval, 22 patients with HGD (Vienna category 4) were treated from 1998. HGD was confirmed and carcinoma excluded by multiple biopsies, CT and EUS. Oesophagectomy was inappropriate because of co-morbidity or patient refusal. Patients received 60 mg/kg ALA orally (DUSA Pharmaceuticals Inc.) At endoscopy 4–6 h later, laser light (wavelength 633 nm) was delivered by diffuser fibre through an oesophageal balloon or silicone bolster at a dose of 500, 750 or 1000J/cm diffuser fibre. Follow up was by endoscopy and multiple biopsies after 1,3,6,9 and 12 months. PDT was repeated up to three times if HGD persisted.

Results: Overall, 12/22 (55%) patients are clear of HGD, after a median follow up of 18 months. However, 9/12 (75%) patients treated with 1000 J are clear v 3/10 with lower light-doses (500–750 J) (p < 0.02). Two 1000 J treatments scheduled 4 weeks apart appears optimal: 8/9 (89%) clear of HGD at a median follow-up 7 months. HGD eradication was more likely when associated with a shorter Barrett’s segment: median length 4 cm in successful treatments, 6 cm in failures (p < 0.02). There have been no deaths or oesophageal strictures. In a total follow-up of 40 patient-years, 4 HGD progressed to carcinoma but are clear of carcinoma after surgery (2), chemo-DXT (1) or PDT with a more potent agent (1).

Conclusions: Photodynamic therapy using ALA is a safe initial approach to HGD in Barrett’s oesophagus. Intensive therapy with two 1000 J/cm treatments 4 weeks apart appears the most effective treatment regime.

074 RISK OF OESOPHAGEAL CANCER IN BARRETT’S OESOPHAGUS AND IN GASTRO-OESOPHAGEAL REFLUX

M. Solaymani-Dodaran1, C. Coupland2, R.F.A. Logan1. 1Division of Epidemiology and Public Health, University Hospital, Nottingham; 2Division of General Practice, University Hospital, Nottingham

Introduction: Barrett’s oesophagus is recognised to be a pre-malignant condition and the incidence of oesophageal adenocarcinoma (OAC) in people with Barrett’s is much higher than general population. Nevertheless, it has been suggested that gastro-oesophageal reflux per se is a more important determinant of risk. The current study was set up to explore the risk of oesophageal cancer (OC) and specifically OAC in Barrett’s using a total of 166 228 person-years follow-up experience from General Practice Research Database (GPRD).

Methods: The 27 813 study subjects constitute four groups: Barrett’s oesophagus group (1677), oesophagitis group (6392), simple reflux group (6328), and normal group (13 416). The last three groups were 4/1, 4/1, and 8/1 matched to the Barrett group according to their GP practice, date of birth, and sex, respectively. All prevalent cases of OC, including those diagnosed before or in the first year after the diagnosis of their condition, were excluded from the analysis. Hazard ratios were calculated using Cox-proportional hazard regression analysis. Using the England and Wales cancer registry data, the incidence of non-AC cancer of oesophagus and subsequently the estimated number of observed AC were worked out in study groups and the relative rate were calculated by standardised incidence ratio method.

Results: A total of 137 oesophageal cancer cases were identified in study subjects of which 80 cases were excluded as being prevalent. 57 were entered into the analysis. The results have been presented in the table.

Abstract 74

Conclusion: Barrett’s oesophagus increases oesophageal cancer risk about 20 times and oesophageal adenocarcinoma about 50 times in comparison to general population. There is only a modest increase in risk of oesophageal cancer in patients with reflux but no record of Barrett’s. Given that the person-years follow-up in Barrett’s group is about twice the largest previous study lower relative risk estimates further emphasises the present publication bias in the estimates of risk of adenocarcinoma in Barrett’s oesophagus.

075 IN BARRETT’S OESOPHAGUS, ACID REFLUX GENERATES HIGH CONCENTRATIONS OF NITRIC OXIDE DERIVED FROM DIETARY NITRATE

H. Suzuki, K. Iijima, E. Henry, K. McElroy, G. Scobie, K.E.L. McColl.

Department of Medicine and Therapeutics, Western Infirmary, Glasgow, UK

Background: The lumen of the oesophagus has a high nitrite concentration due to the enterosalivary recirculation of dietary nitrate. On entering the acidic stomach, the nitrite is converted to nitric oxide, which may contribute to mutagenesis at the cardia.

Aim: To study luminal nitrite chemistry in patients with Barrett’s oesophagus.

Methods: Using microdialysis probes, we studied nitrite concentration in saliva, proximal oesophagus within the Barrett’s segment, and proximal and distal stomach of 10 Barrett’s patients before and following nitrate (2 mmol) administration. Results were compared with 17 healthy controls. In a subgroup of Barrett’s patients, we simultaneously monitored oesophageal pH and nitric oxide concentration.

Results: In the healthy controls, a high nitrite concentration was present in saliva (mean fasting = 37 μM, after nitrate = 203 μM) and throughout the length of the oesophagus (fasting = 29 μM, after nitrite = 181 μM) but fell by 75% on entering the acidic gastric cardia. The fall in nitrite was associated with a high cardia concentration of nitric oxide (fasting = 2.4 μM, after nitrite = 7.5 μM). In the Barrett’s patients, similarly high concentrations of nitrite were present in saliva and in the proximal oesophagus but the nitrite concentration fell by 98% on entering the Barrett’s segment exposed to acid reflux. Simultaneous recording of pH and nitric oxide concentration within the Barrett’s segment indicated that the reflux of acidic gastric juice into the Barrett’s segment resulted in the immediate generation of high concentrations of nitric oxide within the oesophageal lumen (fasting = 13 μM, after nitrate = 20 μM).

Conclusions: The lumen of the normal oesophagus contains high concentrations of nitrite derived from the enterosalivary recirculation of dietary nitrate. The reflux of acidic gastric juice into the distal oesophagus immediately converts the nitrite to nitric oxide. The high concentrations of nitric oxide generated in this way may contribute to the mutagenesis and neoplasia associated with gastroesophageal reflux.

Plenary free papers 076–079

076 DOES SIZE MATTER, OR, WHERE SHOULD OESOPHAGO-GASTRIC CANCER BE MANAGED?

A.M. Thompson, K.P. Park. For the Scottish Audit of Gastric and Oesophageal Cancer, Department of Surgery and Molecular Oncology, Ninewells Hospital, Dundee DD1 9SY, UK

The Scottish Audit of Gastric and Oesophageal Cancer prospectively collected population based data on 3293 cancers diagnosed over a 2 year period with a minimum 1 year follow up. One aim of the project was to examine whether hospital size was related to outcome. The 53 contributing hospitals were divided by caseload, with small hospitals having significantly less delay in diagnosis than larger institutions (p = 0.001). However, this did not significantly improve subsequent survival (see Table 1).

Abstract 76 Table 1

There was no statistically significant difference in postoperative mortality for either gastric or oesophageal cancer by hospital size (Table 2). Overall survival was 32% at 1 year (54% for surgical patients) and 17% at 2 years (33% for surgical patients). The factors adversely affecting survival by multivariate analysis in the surgical patients were American Society of Anaesthesiologists grade 4 (Hazard Ratio 1.35, CIs 1.00 to 1.81, p = 0.047) or 5 (HR 2.46, CI 1.42 to 4.27, p = 0.001) and margin involvement by tumour (HR 1.97 CI 1.32 to 2.96, p = 0.001). Conversely, non-smokers (HR 0.78, CI 0.64 to 0.94, p = 0.01), patients with junctional cancers (HR 0.7, CI 0.51 to 0.95, p = 0.021), and those with a history of H pylori infection (HR 0.79, CI 0.63 to 0.99, p = 0.04) did better. Following univariate, case mix adjusted and multivariate analysis, there was no difference in survival according to the size of the hospital of presentation or hospital of surgery. In combination with data emerging from elsewhere, the guidance on the organisation of oesophago-gastric cancer services in the UK should be reviewed.

Abstract 76 Table 2

077 DECREASED BONE MINERAL DENSITY AT DIAGNOSIS IN PAEDIATRIC INFLAMMATORY BOWEL DISEASE

N.M.Croft, J.Ho, O.A. Akanle, M.O. Savage, I.R. Sanderson. Departments of Adult and Paediatric Gastroenterology and Paediatric Endocrinology, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London, UK

Introduction: Significantly reduced bone mineral density (BMD) has been reported in cross sectional studies of children with established inflammatory bowel disease (IBD). In this study we have examined the bone mineral density within 3 months of diagnosis in children with IBD.

Methods: Children with IBD underwent dual-energy x ray absorptiometry (DEXA) of the whole body (BMD WB) and lumbar spine (BMD LS) within 3 months from diagnosis. The data were matched for age, sex, and race and expressed as standard deviation scores (SDS). Bone age was recorded (Tanner-Whitehouse) and the BMD adjusted accordingly. CD and UC/IC group data were compared using the Mann-Whitney U test. Osteopenia is defined as BMD SDS between −1.0 and −2.5, osteoporosis < −2.5.

Results: 21 children (8 male and 13 female), median age 12.8 years, have been studied. 14 had Crohn’s disease (CD), 6 ulcerative colitis (UC), and 1 indeterminate colitis (IC). Bone age was delayed (> 2 years) in 1 and advanced (> 2 years) in 2 children. Median body mass index Z score was −1.08, significantly lower in CD v UC (−0.97 v 1.0). Height for age Z score was −0.06. BMD was reduced both in the whole body (median SDS −1.08) and lumbar spine (−1.38). There was a trend to these being worse in the CD group (BMD WB: CD −1.35 v UC/IC −0.46, p = 0.2; BMD LS, −1.95 v −1.0, p = 0.5). BMD WB SDS was < −1 in 52% of the 21 subjects (osteopenia in 39% and osteoporosis in 13%), and for BMD LS was 66% (osteopenia 42%, osteoporosis 24%). Using BMD not corrected for bone age for the whole body 25% had a BMD score < −1 (osteopenia 25%, osteoporosis 0%) and lumbar spine 38% (osteopenia 28% and osteoporosis in 10%). There was no significantly increased frequency in these abnormalities in the CD group over the UC group.

Conclusions: These data demonstrate reduced bone mineral density at diagnosis in a high proportion of children with IBD. As peak BMD is not reached until early adulthood evidence based strategies to manage low BMD must be clarified for the paediatric population.

078 ANATOMICAL CLUSTERING OF TYPE II FLAT AND DEPRESSED COLORECTAL LESIONS: RELATIONSHIP BETWEEN DYSPLASIA, NEOPLASIA, AND MORPHOLOGY SUBTYPE

D.P. Hurlstone1, A.J. Shorthouse2, I. Adam2, S. Brown2, S.S. Cross3, C. Korulla1, D. Davies1, A.J. Lobo1.

1Department of Gastroenterology, Royal Hallamshire Hospital, Sheffield; 2Department of Surgery, Royal Hallamshire Hospital, Sheffield; 3Academic Unit of Pathology

Introduction: Current data suggest that more than 30% of all adenomatous colorectal lesions are of flat morphology (Japanese Research Society Classification type II - JRSC). Reporting of anatomical preponderance has however been inconsistent in Western cohorts. Malignant potential in this group is significant. The optimal approach to diagnosis, management and eventual screening in the UK will be influenced by anatomical distribution.

Aim: To evaluate the anatomical distribution of JRSC type II colorectal lesions in the UK and establish if any association exists between site and dysplastic/neoplastic transformation.

Subjects and Methods: Total colonoscopy was performed on 600 consecutive patients, by a single endoscopist using the Olympus CF240Z endoscope from 01/02 to 09/02. 0.5% indigo carmine was used to facilitate detection. Anatomical site was recorded for each lesion identified. Morphology was documented using the JRSC system. Histological analysis was obtained on all specimens by cold biopsy, endoscopic mucosal resection or en-block resection.

Results: See Table. For lesions with sd or beyond, 78% were located in the right colon, 14.6% left, and 6.5% rectum. 32/41 (92.6%) of lesions with sd or beyond had a depressed component morphologically.

Abstract 78

Conclusions: Flat lesions with areas of depression are associated with sd and show a right-hemi-colonic preponderance. Total colonoscopy is required for adequate detection of these lesions (95% would not be detected using flexible sigmoidoscopy alone).

079 HEPATIC INFLAMMATION IS AN IMPORTANT DETERMINANT OF PORTAL PRESSURE IN ALCOHOLIC LIVER DISEASE

R.P. Mookerjee, S.J. Hodges, N.A. Davies, R. Williams, R. Jalan. Institute of Hepatology, UCL, 69–75 Chenies Mews, London WC1E 6HX, UK

Background: A hyperdynamic circulation and elevated portal pressure are established features of cirrhosis. Furthermore, in patients with alcoholic cirrhosis with superadded inflammation, defined on biopsy as alcoholic hepatitis (AH), an inflammatory cytokine cascade perpetuates further injury. We hypothesise that this additional hepatic inflammation in these patients is central to further deterioration in their portal haemodynamics.

Methods: 10 patients with cirrhosis and superadded inflammation on biopsy, fulfilling histological criteria for AH, (5 male; age 49.5 [34–69]; discriminant function [DF] 36.8 ± 9.6) were compared with 13 patients with only alcoholic cirrhosis (12 male; age 51 [40–62]; DF 17.4 ± 5.5). Cardiovascular haemodynamics (cardiac output [CO] and systemic vascular resistance [SVR] using a Swan-Ganz catheter, and mean arterial pressure) and wedged hepatic venous pressure gradient [HVPG] were assessed in both groups using standard techniques, during routine clinical assessment, in accordance with local ethics approval. Clinical and biochemical profiles were also assessed. IL-6 and IL-8 plasma levels were assayed via commercially available ELISAs.

Results: The patients fulfilling AH biopsy criteria had significantly elevated white blood cell counts (13.7±2.7 v 7.8±0.9; p < 0.05), C-reactive protein (36.0±5 v 18.9±3.8; p < 0.05) and systemic inflammatory response syndrome scores (1.4±0.3 v 0.2±0.1; p < 0.01) compared with patients with alcoholic cirrhosis alone (AC). Significant differences in haemodynamics were noted between the 2 groups, following nonparametric analysis (Mann-Whitney, *p < 0.05; **p < 0.01). The measured pro-inflammatory cytokines, IL-6 and IL-8, also showed substantial increases in the AH group compared to AC (p < 0.05 in each case).

Conclusions: This study demonstrates a significant difference in portal and systemic haemodynamics between patients with alcoholic cirrhosis and those with additional hepatic inflammation, highlighting the important contribution of inflammation to the development of portal hypertension in these patients and the potential benefit to portal pressure from future anti-inflammatory treatments.

Gastrointestinal physiology 080–083

080 DOES BETHANECHOL PROVOCATION TESTING (BPT) PREDICT SYMPTOM RELIEF AFTER CHOLECYSTECTOMY FOR ACALCULOUS BILIARY PAIN?

A. Smythe, A.W. Majeed, R. Ahmed, M. Fitzhenry, A.G. Johnson. Division of Clinical Sciences South, University of Sheffield, UK

Introduction and Aims: We have previously shown that cholecystokinin provocation testing does not predict symptomatic benefit from cholecystectomy in patients with acalculous biliary pain (ABP). Bethanechol is a muscarinic agent, which stimulates the gall bladder in vitro. The aim of this study was to evaluate the value of BPT in patients with acalculous biliary pain as a predictor of symptom relief after cholecystectomy.

Methods: 51 patients with ABP were given saline and bethanechol (given s/c in a dose 0.50 μg/kg) to provoke biliary pain (blinded). If pain was reproduced with bethanechol, patients were classed as BPT+ve. If pain was not reproduced with either saline or bethanechol, patients were classed as BPT−ve. If pain was reproduced with saline, bethanechol was not given and patients were analysed separately. Percentage gall bladder emptying was monitored with serial ultrasonography. All patients were offered cholecystectomy irrespective of the outcome of the BPT and symptoms were re-assessed at 6 months.

Results: 20 patients were BPT+ve and 24 patients were BPT−ve. Percentage gall bladder emptying (mean±SD) was similar in both groups (BPT+ve: 30%±16%, BPT−ve: 32±16%, p = 0.7). 17/20 BPT+ve patients underwent cholecystectomy of whom 9 (53%) remained symptomatic 6 months after operation. 11/24 BPT−ve patients underwent cholecystectomy and 6 (54%) remained symptomatic. 2/20 BPT+ patients did not undergo cholecystectomy and were subsequently lost to follow up. 9/24 BPT patients did not undergo cholecystectomy, of these 7 patients had spontaneous reduction in symptoms at 6 months. Of 7 patients positive for saline, five underwent cholecystectomy and two remained symptomatic.

Conclusion: Bethanechol provocation of gall bladder symptoms in patients with ABP does not predict symptom relief at 6 months after cholecystectomy.

081 OESOPHAGEAL MANOMETRY IN CHILDREN WITH FEEDING DIFFICULTIES

S.K. Arthur, D.F. Evans, F. Smith, E. Yazaki, N. Meadows. Barts and the London School of Medicine and Dentistry

23 Children aged between 11 months and 15.5 years were seen in our clinic within the period October 01 2001–2002. They presented with feeding difficulties that required oesophageal manometry, among other diagnostic investigations. The commonest symptoms were dysphagia for solids, regurgitation, and vomiting, in order of frequency. Lower Oesophageal Sphincter (LOS) pressure and body motility were measured using a standard eight channel, water perfused adult catheter (3.9 mm diameter) or a 2.3 mm catheter purpose designed for paediatric use (Mediplast). LOS pressure and oesophageal body motility were measured using a Flexilog 4000 recording system. Nine out of 23 patients had a hypertensive LOS (pressure > 26 mm Hg). These patients with hypertensive LOS but without achalasia often had regurgitation and vomiting as major presenting symptoms. Only 2 out of 23 patients had achalasia and only one of the two had a hypertensive LOS. Non-specific motility disorder (7/23), in which there were few peristaltic waves with a majority of simultaneous contractions, was the commonest cause of dysphagia. Of significance was a subgroup (4/23) with a partially relaxing LOS (residual pressure 6–11 mm Hg) but who did not have achalasia. This group had varied symptoms such as vomiting, regurgitation, and dysphagia for solids. A large hiatus hernia (7 cm) was the only finding in an adolescent girl with unexplained vomiting, normal oesophageal body motility, and “normal” OGD.

In summary, children with feeding problems who present with regurgitation, vomiting, and dysphagia for solids are most likely to have a hypertensive LOS or non-specific oesophageal dysmotility rather than achalasia. Unexplained vomiting with normal LOS function and body motility may be due to hiatus hernia, which could be missed during OGD. In conclusion, oesophageal manometry is an essential diagnostic tool in the investigation of upper gastrointestinal manifestations. From our experience, it is best suited to children aged over six years, as in younger children, manometry can be difficult because of non-compliance, and other diagnostic tests for detecting oesophageal dysmotility may be preferred.

082 ANTIREFLUX SURGERY IN BARRETT’S OESOPHAGUS: SYMPTOMATIC VERSUS PHYSIOLOGICAL OUTCOME

J.M. O’ Riordan1, P.J. Byrne1, N. Ravi1, P.W.N. Keeling2, J.V. Reynolds1.

1University Department of Surgery, St James’ Hospital, Dublin 8, Ireland; 2University Department of Medicine, St James’ Hospital, Dublin 8, Ireland

Background: The appropriate management of Barrett’s disease remains controversial. We report the long term outcome of antireflux surgery in Barrett’s patients in a tertiary referral centre.

Methods: Between 1985 and 2001, 67 patients with Barrett’s oesophagus underwent Nissen Fundoplication. Follow up via questionnaire (n = 58), endoscopy/histology (n = 57) and 24 h pH monitoring (n = 41) were examined.

Results: At a median follow up of 59 months, 95% (55/58) of patients considered the surgery either excellent/good. The median DeMeester score reduced from 69.5(2–309) before to 6.1(0–222) after surgery (p < 0.001). Lower oesophageal sphincter pressure increased from 7(3–27) mm Hg before to 15(3–40) mm Hg afterwards (p < 0.001). Six patients (10%) had developed recurrent symptoms. 17/41(41%) patients had abnormal post-operative DeMeester scores (> 15) of whom 11 had a reduction in their DeMeester scores but were still outside the normal range. Six out of 8 patients with preoperative dysplasia showed evidence of regression. Dysplasia developed after surgery in 2 patients. Two patients developed adenocarcinoma within Barrett’s 4 and 7 years postoperatively, respectively. These 4 patients had abnormal postoperative acid scores.

Conclusions: Nissen Fundoplication provides excellent long lasting relief of symptoms in Barrett’s patients. Postoperative 24 h pH studies establish that acid reflux has been abolished. Abnormal postoperative studies may be associated with dysplasia and adenocarcinoma in the long term and emphasises the need for long term follow up.

083 REFLUX EXPOSURE UNITS—;A NEW WAY OF EVALUATING OESOPHAGEAL PH MEASUREMENTS?

K.R. Haylet1, P. Vales2, S.H. Lee3, R.F. McCloy4. 1Medical Engineering, 2GI Investigation Unit; 3Department of Radiology;4University Department of Surgery, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL

Background and Aims: Although considered the “gold standard” for assessing gastro-oesophageal acid reflux, evidence suggests a poor correlation between pH measurements and the degree of oesophagitis seen during endoscopy. The current pH features examined, described as exposure, focus on the time the measured pH falls below 4. Importantly, these measures do not take into account distension of the oesophageal lumen. This study examined the diameter of the oesophageal lumen, its effect on mucosal folds and the effect that these may have on acid exposure at the mucosa.

Methods: The diameters of the oesophageal lumen of 25 patients under investigation for oesophageal disorders were studied using a video barium swallow. The widest luminal diameter was measured for each subject for a series of five controlled swallows and the results critically examined with regard to the usual measure of acid exposure.

Results: The results showed the lumen diameter varies widely from 0.9 to 3.8 cm. No difference in maximum luminal diameter was found between successful and failed swallows (student t, p > 0.05). In addition, results from endoscopy show oesophagitis often occur at the tips of the mucosal folds creating linear longitudinal patterns of erthyma. Changes in lumen diameters have also been previously shown to affect the different degrees of mucosal folding. These results suggest that individual patients may have varying areas of mucosa exposed to luminal content. As a result of these considerations a new Acid Exposure Unit was developed to take into account not only the time of exposure but also pH and the area of mucosa exposed.

Conclusion: In conclusion the range of luminal diameters measured and the developed exposure units highlight that the current measures of oesophageal pH exposure, which do not include the area of mucosa exposed, may explain why only a poor correlation is found between pH measurements and the degree of oesophagitis seen during endoscopy.

Endoscopy free papers 084–097

084 INFORMED CONSENT FOR ENDOSCOPY: AN OBSERVATIONAL STUDY

A.J. Brooks, J. Fotheringham, J. Gane, D.S. Sanders, M.E. McAlindon.

Dept of Gastroenterology, Royal Hallamshire Hospital, Sheffield Teaching Hospitals Trust, UK

Background: The amount and type of information that patients require about risk in order to consent to endoscopic procedures has never been established.

Aims: To evaluate the amount of information patients undergoing endoscopic procedures require regarding risk.

Methods: Patients were given written and verbal information about the nature, incidence, and consequences of complications, then asked how common each complication would have to be before requiring information for informed consent.

Results: 150 colonoscopy and 150 gastroscopy patients were studied. 24–32% of gastroscopy and 31–43% of colonoscopy patients wanted to be informed of one or more complications irrespective of frequency. 19% of gastroscopy and 14% of colonoscopy patients wanted to know about all possible complications. This was unrelated to the severity of the complication. Among gastroscopy patients, 17–25% did not want to be informed of one or more complications no matter how serious or frequent they were, and again, this was unrelated to severity. In the colonoscopy patients only 2–17% did not want to be informed of one or more complications. Significantly fewer [0.7%] colonoscopy patients required no information about any complications, compared to gastroscopy patients (10%, p < 0.001). In both groups many more required information about serious complications than minor complications when the complications occurred with a risk rate < 1/1000 (p < 0.001).

Conclusions: Up to a fifth of patients want to be informed of all possible complications. Consenting for colonoscopy requires greater risk disclosure. Patient’s threshold for information on major complications is much lower than for minor ones. Serious complications likely to occur in >1/1000 cases should be disclosed to all patients.

085 ASSESSING RESECTION MARGINS USING HIGH MAGNIFICATION CHROMOSCOPY FOR “EN-BLOCK” COLORECTAL ENDOSCOPIC MUCOSAL RESECTION OF FLAT LESIONS: A USEFUL TOOL FOR PREDICTING REMNANT TISSUE

D.P. Hurlstone1, A.J. Shorthouse2, I. Adam2, S. Brown2, S.S. Cross3, C. Korulla1, R. Davies1, A.J.I. Lobo1.

1Department of Gastroenterology; 2Department of Surgery, 3Academic Unit of Pathology Royal Hallamshire Hospital, Sheffield

Introduction: EMR in parallel with high resolution chromoscopic colonoscopy (HRCC) may permit safer and more accurate resection practice. Residual tumour or a disrupted, incomplete resection interface can be difficult to detect with conventional endoscopic techniques.

Aim: To evaluate the efficacy of HRCC in predicting complete horizontal and vertical resection margins for flat lesions less than 10 mm diameter.

Subjects and Methods: Total colonoscopy was performed on 600 consecutive patients using the Olympus CF240Z endoscope from 01/02 to 09/02 by a single endoscopist. 196 lesions were evaluated using HRCC pre- and post-EMR, using locally applied 0.5% indigo carmine (IC). The crypt appearance of all lesions was graded according to the modified Kudo class. Those demonstrable of a type V(n) or V(a)/IIIs were excluded due to the high risk of submucosal-massive invasion, and lesions failing to “lift” symmetrically at EMR. Lesions with a Kudo I crypt exclusively were additionally excluded (hyperplastic). Resection was performed en-block with submucosal lift using 0.5% IC and an Olympus barbed snare. Resection margins were then reassessed with a second dye spray around the circumferential horizontal and vertical border. Type I /II crypt was used as evidence of complete resection endoscopically. Histological confirmation was obtained on all specimens.

Results: See Table. Percentage agreement between HRCC and actual histological verification of vertical and horizontal clearance was calculated using the Kappa statistic, κ = 0.78 (excellent agreement, 95% CIs).

Abstract 79

Conclusion: Prospective evaluation of resection margins using HRCC proves it is a useful tool for predicting excision completeness post-EMR.

Abstract 85

086 IN VIVO ANTICIPATION OF SUBMUCOSAL INVASION IN FLAT AND DEPRESSED COLORECTAL LESIONS: CLINICAL IMPLICATIONS OF SUBTYPE ANALYSIS OF THE KUDO TYPE V PIT PATTERN USING HIGH RESOLUTION CHROMOSCOPIC COLONOSCOPY

D.P. Hurlstone1, A.J. Shorthouse2, I. Adam2, S. Brown2, S.S. Cross3, A.J. Lobo1. 1Gastroenterology, Royal Hallamshire Hospital, Sheffield; 2Academic Department of Surgery, Royal Hallamshire Hospital, Sheffield; 3Academic Department of Pathology, Royal Hallamshire Hospital, Sheffield

Background: Focal submucosal invasive colorectal cancers (localised to the upper third of the submucosa, ie sm1) can be managed by endoscopic mucosal resection (EMR) as local lymph node metastasis (LLNM) are rare. Morphologically, these lesions are usually flat, depressed, or mixed – Japanese Research Society Classification (JRSC) IIa/b/c. In deeper vertical submucosal invasion (ie sm2/3) LLNM rates exceed 10–15%. EMR within this group can be complicated with perforation and non-curative resection. It is thus essential to differentiate accurately focal sm1 disease from submucosal sm2/3 disease.

Aim: To evaluate the relationship between the Kudo type Vn (A–C) crypt pattern and submucosal invasion depth for flat type (JRSC type II) colorectal lesions.

Methods: 600 consecutive patients were colonoscoped, by a single endoscopist using the Olympus CF240Z, from 01/02 to 09/02. Kudo type V pits were identified using 0.5% crystal violet (CV) applied directly to the lesion with a steel tipped catheter. Type V pits were graded into class Va or Vn as per the modified Kudo class. Vn pits were subsequently subtyped as grades A,B,C according to Vn appearance as described by Tanaka. Morphology was documented using the JRSC. Histological analysis was performed on all lesions.

Results: 47 JRSC class II lesions had a type V(n) pit pattern (see Table).

Abstract 86

Conclusions: High resolution chromoscopy is a predictor of invasive depth. The safety of EMR can be greatly enhanced using this technique.

087 SINGLE LUMEN ACCESS ANASTOMOSIS DEVICE FOR FLEXIBLE ENDOSCOPY

A. Fritscher-Ravens1, A.C. Mosse1, D. Mukherjee1, T. Mills1, P.O. Park2, C.P. Swain1. 1Department of Gastroenterology, Royal London Hospital; 2General Hospital Vaxjoe, Sweden

Introduction: Forming anastomoses between two lumens at flexible endoscopy might relieve obstruction and reduce the need for traumatic surgery. Current methods require access to both lumens.

Aim: to develop methods of forming anastomosis at flexible endoscopy when access to only one lumen is feasible, such as gastrojejunostomy in the presence of obstructing pancreatic cancer.

Methods: A large channel echo-endoscope was used to image small intestine. A modified needle was passed from the accessible lumen into the target hollow organ. Out of four anastomosis devices designed one was chosen for extensive animal experiments. It was formed using two short 7F catheter segments attached together by a thread, which can be pushed over a wire into the target non-accessible lumen. They are released by withdrawing the guidewire to form a cross shape. These can form a cruciate anastomosis when compressed by a spring against a plate from the accessible side creating ischaemia.

Results: These devices were tested on the bench in postmortem tissue and in live porcine tissue. In 18 pigs anastomoses were formed between small intestine and stomach and gall bladder and stomach. There were no complications. The size of the anastomoses were 3–8 mm. They underwent balloon dilatation and were patent 3–4 weeks after they had been formed at postmortem. In 8 of them an 11 mm gastroscope could pass through.

Conclusion: It is feasible to form anastomoses at flexible endoscopy when access is limited to a single side.

088 PROSPECTIVE COMPARISON OF SECRETIN STIMULATED MRCP WITH SPHINCTER OF ODDI MANOMETRY IN THE DIAGNOSIS OF SPHINCTER OF ODDI DYSFUNCTION

S.P. Pereira1, A.R. Gillams2, A.W.R. Hatfield1. 1Department of Gastroenterology; 2Department of Gastroenterology; 3Department of Radiology, The Middlesex Hospital, UCL Hospitals NHS Trust, London, UK

Background: Sphincter of Oddi manometry (SOM) is the gold standard for the diagnosis of sphincter of Oddi dysfunction (SOD) and predicts response to sphincterotomy, but it is invasive and associated with complications.

Aim: To evaluate the role of secretin stimulated magnetic resonance cholangiopancreatiography (ssMRCP) in predicting the results of ERCP+SOM in patients with clinically suspected type II (pancreaticobiliary type pain + duct dilatation or abnormal liver biochemistry/recurrent pancreatitis) or type III (pain alone) SOD.

Methods: 43 patients (35F, 8M; mean age 46 years, range 27–69 years) referred for SOM from 28 hospitals were studied. MRCP was performed at baseline and at 1, 3, 5, and 7 min after IV secretin (0.1 ml/kg). Five 10–15 mm coronal images were obtained in a single breath hold using a heavily T2 weighted, fat suppressed fast spin-echo sequence on a Siemens 1.5T MR system. All MRCP studies were reported by one radiologist who was blinded to the SOM findings. SOM was performed using a standard 5F wire-guided triple lumen water perfused manometry catheter, and SOD diagnosed when the mean basal sphincter pressure was > 40 mm Hg (sustained for > 30 sec and observed in both leads).

Results: 23 patients (53%) had manometrically-proven SOD: 16 type II (8 biliary, 7 pancreatic, 1 both) and 7 type III. ssMRCP was abnormal in 10 of the 16 patients (63%) with type II SOD, but in none of the 7 with type III SOD. 14 patients had normal ERCP+SOM studies, in whom ssMRCP was also normal, and a further 6 patients with normal SOM were found to have morphological changes of moderately severe chronic pancreatitis on both ERCP and ssMRCP.

Conclusions: ssMRCP is insensitive in predicting abnormal manometry in patients with suspected type III SOD, but correlates well with ERCP+SOM in detecting structural disease of the sphincter or pancreas.

089 SINGLE CENTRE EXPERIENCE OF ENDOSCOPIC THERAPY FOR PRIMARY SCLEROSING CHOLANGITIS (PSC), 1984–2000

G.J.M. Webster, J. Wittmann, A.R. Hatfield, S.P. Pereira.

The Middlesex Hospital, UCL Hospitals NHS Trust, London, UK

Background: In patients with PSC, the optimal management of the minority who develop dominant biliary strictures remains uncertain. Several studies suggest a survival benefit of endoscopic dilatation +/− stent insertion compared with expectant management alone, but they have generally reported only short term follow up.

Aim: To review the endoscopic management of PSC in a tertiary referral centre, and to assess the impact of intervention on outcome.

Methods: Cases were identified via endoscopy, pathology and clinical databases, from 1984–2000. The numbers of ERCPs, interventions, length of endoscopic follow up, and outcome, were compared in patients with and without dominant strictures.

Results: Of 161 patients identified with PSC, 131 (85M, 46F; mean age 55 years, range 14–86 years) underwent 382 ERCPs during the study period (3.4% of 11 326 ERCPs). Interventions and endoscopic follow up in the patients are shown in the table.

Abstract 89

Of stricture interventions performed, stent insertion alone was performed in > 65% of cases, dilatation alone in < 25% of cases.

Discussion: This single centre experience indicates that patients with dominant strictures due to PSC can be successfully managed long term with endoscopic therapy. Ongoing prospective follow-up will allow the effects of biliary interventions on clinical outcome to be assessed in detail.

090 A PROSPECTIVE SINGLE BLIND STUDY COMPARING CAPSULE ENTEROSCOPY WITH PUSH ENTEROSCOPY AND SMALL BOWEL ENEMA IN OBSCURE GASTROINTESTINAL BLEEDING

P. Georgopoulos, F.W. Poon, A. Anderloni, J.F. Mackenzie, A.J. Morris.

Glasgow Royal Infirmary, Glasgow, Scotland, UK

Background: Obscure GI bleeding remains a clinical problem in up to 10% of patients with anaemia with the small bowel as the most possible source of bleeding. Wireless capsule endoscopy has not been evaluated in comparison with the established methods of small bowel examination, enteroscopy, and small bowel enema in anaemic patients.

Aim: To compare prospectively the diagnostic yield of capsule enteroscopy with push enteroscopy and small bowel enema.

Method: Patients with obscure GI bleeding with negative upper GI endoscopy and colonoscopy were studied. Each examination was performed and reported independently by physician or radiologist.

Results: Preliminary data from the first 9 enrolled patients are presented. Mean age was 63.5 years (range 58–79) and male to female ratio was 1:2. Comorbitity existed in 5/9 (55.5%) patients with polycystic kidneys (1), previous gastric operation for peptic disease (2), right hemicolectomy (1), and cardiac valve replacement (2). Push enteroscopy discovered a definite bleeding source in 4/9 patients and a possible in one. Capsule enteroscopy found a definite bleeding source in 7/9 patients and a possible cause in one. Six of them had a definite lesion beyond the reachable area of the enteroscope. Other findings not related with bleeding source were found in one patient in each method. None of the patients had abnormal small bowel enema findings. The diagnostic yield of push enteroscopy was 44.4% in comparison to capsule enteroscopy, which was 77.7%. Additional information within the small intestine was obtained using the capsule enteroscopy in 66.6% of the patients.

Conclusion: This prospective single blind study shows for first time that capsule enteroscopy detects more lesions than push enteroscopy or small bowel enema in patients with obscure GI bleeding. It can also diagnose intestinal bleeding source beyond the reach of push enteroscopy in a substantial larger number of patients.

091 EVALUATING MRC AGAINST ERCP IN THE ASSESSMENT OF BILE DUCT STONES

K. Shafiq, E. Thomas, B. Brett, C. Jamieson. James Paget Hospital, Gorleston, Norfolk

Aim: To evaluate magnetic resonance cholangiography (MRC) in the detection of bile duct stones.

Methods: The study was performed over 2 years. Patients presenting with biliary symptoms who were assessed as having a low to medium probability of bile duct stones according to predefined criteria had an MRC. In general patients who had positive MRC had an endoscopic retrograde cholangiogram (ERCP), while patients who had a negative MRC did not have further biliary imaging. MRC and ERCP data were gathered prospectively. At completion of the study the patients who had both procedures were compared for presence of stones on each modality. A rigorous retrospective case notes review was carried out for all those who had a negative MRC to spot subsequent admissions related to probable biliary disease, biliary symptoms in new referral or follow up clinic letters and mortality.

Results: 229, 133, and 27 patients had ERCP, MRC, and both, respectively. MRCP was positive in 30. ERCP was scheduled for these patients and performed in 24. In the remainder ERCP was not performed for the following reasons; 2 declined, 2 cancelled, 1 ERCP failed, and 1 had surgery. The positive predictive value of MRC was 91.7%. Three patients with a negative MRC had a subsequent ERCP (for clinical indications) and all were negative. The number of patients with a negative MRC later admitted with biliary disease was 6. Five had a cholecystectomy for gall stones, (3 emergency, 2 elective) and 1 declined to have follow up. One had pancreatitis while awaiting surgery. None of the patients who underwent surgery had bile stones at operation. Five patients have had persistent or recurrent symptoms; 1 was found to have pancreas divisum and 4 patients continue to have upper abdominal pain which appears musculoskeletal after further investigation. Three patients have died due to unrelated causes. The complications of ERCP were pancreatitis in 4 cases out of 229 (2 mild, 2 moderate), a minor bleed in 1 case (no transfusion required). There were no complications in the MRC group.

Conclusion: This study demonstrates that MRC has a high positive predictive value in the assessment of the presence of bile duct stones. Patients having a negative MRC rarely develop subsequent symptoms in relation to bile duct stones.

092 PATIENT TOLERABILITY OF VIRTUAL COLONOSCOPY IN COMPARISON TO CONVENTIONAL COLONOSCOPY AND BARIUM ENEMA

S. Moreea1, A. Singhal1, C.G. Beckett1, C.L. Kay2. 1Department of Gastroenterology; 2Department of Radiology, Bradford Royal Infirmary, Bradford BD9 6RJ

Aim: To evaluate the discomfort associated with bowel cleansing and the degree of discomfort/abdominal pain in patients having a virtual colonoscopy (VC), conventional colonoscopy (CC) and barium enema (BE) and to determine patient preference between VC and CC.

Methods: Patients referred for CC were recruited to have a VC prior to their CC. A separate group of patients having a BE or CC alone were also recruited. Using a 100 point visual analogue scale, patients were asked to rate the following after the individual procedure: discomfort caused by the bowel preparation (Kleanprep for VC/CC and picosulphate for BE), discomfort and abdominal pain during the procedure. They were asked about the lack of respect during the procedure, whether they would have the procedure again, their preferred procedure, and the length of time they were prepared to wait to have their preferred procedure. On the visual analogue scale 0 meant no discomfort/pain and 100 was severe discomfort/pain. Conscious sedation was used for CC.

Results: 33 patients had both a VC and CC, 32 patients had CC alone (total number of CCs: 65) and 61 patients had a BE. Discomfort of bowel cleansing was rated for VC/CC: 49/100, and for BE: 22/100. Mean procedure discomfort was VC: 34/100, CC: 25/100, and BE: 30/100. Mean procedure pain was VC: 37/100, CC: 22/100, and BE: 23/100. Lack of respect was reported in 9% of VCs, 4.6% of CCs, and none of the BEs. 9%, 12%, and 11% of patients would not have a repeat VC, CC, and BE, respectively. 27/33 patients in the VC/CC arm of the study returned a 24 h questionnaire: 11 preferred CC and 13 preferred VC and 3 had no preference. 54% of patients were prepared to wait for 4 weeks or more to have their preferred procedure.

Conclusion: Bowel cleansing for VC/CC was more uncomfortable than the actual procedure and that for BE (picosulphate) was less uncomfortable than that of VC/CC (Kleanprep). Patients reported more pain with VC than with either CC or BE. The greater relative comfort of CC v VC may be partly due to the effect of conscious sedation but other factors may be involved as unsedated patients reported less pain with BE v VC. Patients would wait 4 weeks for their preferred test.

093 THE “TWO WEEK RULE” (2WR) DIRECTIVE : GOOD OR BAD POLITICS FOR PATIENTS?

A. Douglass, A. Agarwal, R.G. Wilson, P.A. Cann. Endoscopy Centre, James Cook University Hospital, Middlesbrough

Introduction: The 2wr directive for GI malignancies, suspected by GPs, purports to enhance and accelerate diagnosis and care. The imperative is to “see” the patient within 2 weeks, whereas the relevant first key clinical step is to confirm or refute the diagnosis for all potential cases. This is optimised by fast tracking to definitive investigation—usually endoscopy. If resources do not allow for this within 2 weeks, urgent clinic slots are taken up mostly just to explain the need for that test. No evidence base shows this approach as helpful overall and perhaps it disadvantages a significant proportion of patients.

Aims and Objectives: We determined the potential number of 2wr referrals out of all colorectal referrals received (catchment 300 K). We also established the proportion of actual colorectal cancer (CRC) cases that would not have fulfilled the national 2wr criteria.

Methods: All (medical and surgical—clinic and open access) referrals to our unit in an 8 week period and all CRC cases diagnosed in a 1 year period were reviewed.

Results: Referral audit: 773 referrals total (86 excluded—insufficient data in referral). 248 fulfilled the 2wr criteria and were referred via open access colonoscopy (87) and clinic (161), representing 48% and 32% of all such referrals, respectively. Only 8% were actually referred by 2wr—a further 18% were assigned other degrees of urgency by the GP. 248 in 8 weeks is ∼30/week. If 20 min is allowed per 2wr clinic slot, this amounts to 10 h/week even before diagnosis is organised for most. (The impact for upper GI cases could be similar). CRC Audit: 178 CRC cases total, 46 presented acutely. 33 (25% of non-acute) did not fulfil 2wr criteria (Dukes A and B 53%), the remainder did (A and B 52%).

Conclusions: Many CRC cases do not fulfil 2wr referral criteria but have similar staging. If the 2wr was strictly adhered to in primary care, these face potential disadvantage in timely attention. Furthermore, clinic time spent on achieving 2wr targets might be better spent on improving or providing fast track definitive investigation, managing diagnosed cancer cases or the majority of our patients without cancer.

094 PROSPECTIVE COMPARISON OF ENDOSCOPIC ULTRASOUND GUIDED 22G FINE NEEDLE ASPIRATION (EUS-FNA) WITH 19G TRUCUT NEEDLE BIOPSY (EUS-TNB)

S.P. Pereira, M. Falzon, G. Kocjan, M. Novelli. Departments of Gastroenterology and Pathology, The Middlesex Hospital, UCL Hospitals NHS Trust, London, UK

Background: EUS-FNA of mediastinal and gastrointestinal mass lesions is sensitive for diagnosing malignancy and has a low complication rate. However, EUS-FNA ideally requires an on-site cytopathologist to ensure adequate sampling, and the diagnosis of certain neoplasms is difficult based on cytology alone. A 19G trucut needle (Quick-Core, Wilson-Cook) has been recently developed for EUS, but there are no prospective data on its safety or accuracy.

Aim: To compare the sens/spec of EUS-FNA with EUS-TNB in patients with suspected mediastinal or gastrointestinal mass lesions.

Methods: Over a 10 week period, 12 patients (10M, 2F; mean age 68 year, range 43–82 year) underwent combined 22G EUS-FNA (median 3 passes, range 1–4) and 19G EUS-TNB (2 passes, range 1–3) of suspected mediastinal (n = 2) or pancreaticobiliary mass lesions (transgastric approach in 4, transduodenal in 6). Pathologic samples were assessed independently by two pathologists, neither of whom attended the EUS. Patients were contacted 24–48 h later to assess complications.

Results: The median size of the lesions biopsied was 2 cm (range 0.6–7 cm). The final diagnoses were benign disease in 6 and malignancy in 6 (adenocarcinoma in 4, lymphoma in 1, gastrinoma in 1). In two patients, EUS-TNB allowed additional characterisation of the tumour not provided by cytology. There were two false negative results for EUS-FNA (1 ‘highly suspicious’, 1 inadequate), and two needle failures during attempted transduodenal EUS-TNB, but the accuracy of the combined tests in detecting malignancy was 100%. One patient reported mild abdominal discomfort for < 24 h after the procedure.

Conclusions: Combined EUS-FNA and EUS-TNB appears to be safe and to have a high diagnostic yield. EUS-TNB from the second part of the duodenum is associated with a high technical failure rate. Large prospective studies are needed to determine in what situations EUS-TNB may improve on the accuracy of EUS-FNA.

095 AGE THRESHOLD FOR ENDOSCOPY AND RISK OF MISSING UPPER GI MALIGNANCY: DATA FROM THE SCOTTISH AUDIT OF GASTRIC AND OESOPHAGEAL CANCER

C.A. Salmon1, K.G.M. Park2, T. Rapson3, P.S. Phull1. 1Gastro-intestinal and Liver Service; 2Department of Surgery, Aberdeen Royal Infirmary, Foresterhill, Aberdeen; 3ISD, Common Services Agency for NHS Scotland, Edinburgh

Introduction: BSG suggest that it may be possible to increase the age threshold for endoscopy from 45 to 55 years in patients with uncomplicated dyspepsia. Patients with ‘alarm’ symptoms would still undergo an urgent endoscopy. However, there is only limited evidence quantifying the risk of missing upper GI malignancy in the 45–55 year old patients who would no longer warrant an endoscopy. The aim of this study was to assess the numbers of patients under 55 years of age with upper GI malignancy but without ‘alarm’ symptoms.

Methods: The Scottish Audit of Gastric and Oesophageal Cancer collected data prospectively for all upper GI malignancies diagnosed in Scotland between July 1997 and July 1999. The presenting symptoms of all the patients under the age of 55 years were analysed. ‘Alarm’ symptoms were defined as dysphagia, weight loss, GI bleeding, anaemia, vomiting, history of gastric surgery, and history of peptic ulcer disease.

Results: Of the 3293 patients with upper GI malignancy, 290 patients were under 55 years of age. Seventy-three (25%) patients had gastric cancer, 55 (19%) had OG junction cancer, and 162 (56%) had oesophageal cancer. Twenty-one (7.2%) patients had no alarm symptoms; 5 patients were under 40 years (1.7%), 9 patients were under 45 years (3.1%), and 15 patients were under 50 years (5%). Thirteen of the 73 (17.8%) patients with gastric cancer had no alarm symptoms compared to 4 of 55 (7.2%) patients with OG junction cancer and 4 of the 162 (2.4%) patients with oesophageal cancer.

Conclusion: Increasing the age threshold for endoscopy from 45 years to 55 years in Scotland would double the number of patients with missed upper GI malignancy. This would predominantly affect patients with gastric cancer.

096 OUTPATIENT ENDOSCOPY FOR PATIENTS PRESENTING WITH MINOR UPPER GI BLEEDING

G. Mulholland, J.A.H. Forrest. Stobhill Hospital, Glasgow, UK

Introduction and Aim: Acute upper GI bleeding (UGIB) accounts for approximately 8% of acute medical admissions and such patients are routinely admitted for observation and diagnostic endoscopy. This study aimed to assess the safety and outcome of rapid access outpatient endoscopy for patients presenting with minor upper GI bleeding (Rockall score zero).

Study Design: Over a 3 year period patients presenting with UGIB who had a pre-endoscopy Rockall score of zero were identified in A&E.They were discharged home with an appointment for an OP endoscopy.

Results: 64 patients were referred from A&E; 42 M and 21 F, mean age 37.8 (range 19 –79). 42 endoscopies were performed, there being 20 DNAs and 2 inappropriate referrals (vomiting alone). 51 patients presented with haematemesis, 4 with melaena, and 7 with both. 8 patients had taken aspirin or an NSAID and 24 excess alcohol. Of the 20 DNAs 14 had misused alcohol. At endoscopy 14 patients had oesophagitis,8 a peptic ulcer, 4 gastritis, 4 duodenitis, and 1 oesophageal varicies. 7 patients had dual pathology and 18 had a normal endoscopy. H pylori was positive in 16 of 34 patients tested. Average Hb (n = 24) was 14.3 (range 11.3–16.8). Average interval between presentation and endoscopy was 14 days (range 1–77). No patient had recurrennt bleeding.

Conclusion: The study showed it to be safe practice to discharge patients from A&E with minor UGIB and a pre endoscopy Rockall score of zero and to perform an OP endoscopy. The high DNA rate (31%) reflected the significant number of patients who misused alcohol. 57% of those attending for endoscopy had significant pathology; oesophagitis being the most common abnormality. The number of A&E referrals with UGIB for OP endoscopy was significantly less than expected; probably because SHOs were unhappy to discharge such patients home.

097 A PROSPECTIVE RANDOMISED COMPARISON OF 10 FR VERSUS 7 FR BIPOLAR ELECTROCOAGULATION CATHETER IN COMBINATION WITH ADRENALINE INJECTION IN THE ENDOSCOPIC TREATMENT OF BLEEDING PEPTIC ULCERS

G.A. Paspatis, I. Charoniti, N. Papanikolaou.

Department of Gastroenterology, Benizelion General Hospital, Heraklion-Crete, Greece

Background and Aims: Our study sought to compare the efficacy of bipolar electrocoagulation (gold probe) with 10 Fr (Group A) versus 7 Fr (Group B) catheter following adrenaline injection in the treatment of bleeding peptic ulcers. To the best of our knowledge, this is the only prospective, randomised study in humans.

Methods: 77 consecutive patients with endoscopic evidence of peptic ulcer with active bleeding or a non-bleeding visible vessel were randomly assigned to one of the above protocols. 39 patients (31 males, 8 females, mean age 62 years) were included in group A and 38 (28 males, 10 females, mean age 61 years) in group B.

Results: The initial haemostasis rate, rebleeding rate, duration of hospital stay, volume of blood transfused, number of operations needed, and number of deaths were not significantly different between the two groups. The mean number of electrocoagulations and the subsequent mean duration of electrocoagulations were significantly higher in group B patients (7.0±3.8, 14.1±7.6 seconds, respectively) compared with those of group A (4.6±2.6, 9.3±5.3 seconds, respectively) (p < 0.01). Multivariate stepwise logistic regression analysis revealed that among sex, age, location of bleeding, ulcer size, endoscopic severity of bleeding, and the size of the gold probes, the endoscopic severity of the bleeding, the small size of the gold probe, and the increased ulcer size were the only factors significantly associated with an increased number of electrocoagulations (χ2 = 31.1, p < 0.01, χ2 = 23.9, p < 0.01 and χ2 = 13.4, p < 0.01, respectively).

Conclusions: Our data suggest that the use of the large size gold probe was significantly associated with a decreased number of electrocoagulations resulting in the reduction of electrocuagulation duration.

Colorectal free papers 098–107

098 SHOULD DIGITAL RECTAL EXAMINATION STILL BE PART OF THE PHYSICAL EXAMINATION OF PATIENTS WITH COLONIC SYMPTOMS?

L. Langmead, D.B. Jones, P.H. Katelaris. Gastroenterology Department, University of Sydney, Concord Hospital, Sydney, Australia

Background: It is an unchallenged axiom that digital rectal examination (DRE) is an essential part of the physical examination of patients with colonic symptoms. However, there is no evidence demonstrating that routine unsedated, unprepared DRE at the initial consultation is useful in patients in whom colonoscopy is indicated.

Aims: To assess the value of DRE in patients presenting to gastroenterologists with colonic symptoms.

Methods: Consecutive patients presenting to gastroenterologists with colonic symptoms, in whom a colonoscopy was later performed, were studied. Patients with perianal pain or tenesmus and those in whom DRE was done for indications other than routine were excluded. Endoscopic findings were evaluated. Rectal cancers were recorded as distal (within 7 cm of the anal verge and potentially palpable on unsedated DRE) or proximal (beyond 7 cm).

Results: In 4834 patients undergoing colonoscopy, 166 cancers were diagnosed, of which 68 (41%) were rectal cancer (23 distal, 45 proximal). Anaemia was a more frequent indication in those with colonic cancers (p < 0.001). A qualitative questionnaire revealed unanimous patient and doctor preference for DRE at the time of colonoscopy rather than unsedated, unprepared DRE. In a presumptive analysis of usefulness, a 75% sensitivity of DRE for distal cancers was assumed. 280 DREs, therefore, would be required to detect one distal cancer in this cohort, with the outcome of this being a possibly reduced waiting time to colonoscopy.

Conclusion: Routine DRE was not sensitive for the diagnosis of colorectal cancer. It was unpopular with patients and doctors and would not alter management in more than 0.4% of patients. In those with a positive DRE, the maximum benefit would be to shorten the interval to colonoscopy. In practice, valuable time spent performing DRE may be better spent in the endoscopy suite reducing colonoscopy waiting times.

Abstract 99

099 DO CALIUM ANTAGONISTS PROTECT AGAINST PERFORATED COLONIC DIVERTICULAR DISEASE? A CASE-CONTROL STUDY

C.R. Morris1, I.M. Harvey1, W.S.L. Stebbings2, C.T.M. Speakman2, H.J. Kennedy2, A.R. Hart1. 1School of Medicine, Health Policy & Practice, University of East Anglia NR4 7TJ, UK; 2Norfolk & Norwich University Hospital NHS Trust NR4 7UY, UK

Background: Perforated colonic diverticular disease (PCDD) may result from a combination of high intracolonic pressures, secondary to excessive colonic segmentation, and impairment of the mucosal barrier. Calcium channel blockers and antimuscarinic drugs, which reduce colonic contractility and tone, could potentially protect against perforation. The aim of this study was to test this hypothesis using a case-control design.

Methods: All cases of acute PCDD were identified over a 5 year period in two hospitals in Norfolk, UK. Each case was matched for age, sex, and date of admission to two controls groups: (a) patients undergoing cataract surgery and (b) patients with basal cell carcinoma. Data on drug use prior to hospital admission were obtained from medical and nursing records and compared between cases and controls.

Results: 120 cases of PCDD were identified and matched to 240 controls in each group. A statistically significant protective association was seen between calcium channel blocker use and PCDD using both control groups (see table). No association was found with antimuscrainic drugs.

Abstract 103

Conclusions: This study has shown for the first time that a protective association exists between calcium channel blockers and PCDD. Further studies are required to confirm this association but calcium channel blockers may represent a potential preventive therapy in PCDD.

100 PATIENT SUITABILITY FOR NURSE LED FOLLOW UP: A PROSPECTIVE AUDIT

R. Christer, E.V. Robinson, E. Raey, A.F. Horgan (introduced by N. Thompson). Department of Colorectal Surgery, Freeman Hospital, Newcastle-upon-Tyne Hospitals NHS Trust, Newcastle-upon-Tyne, NE7 7DN, UK

Government’s targets, including the 2 week cancer rule, derived from the New NHS: modern and dependable (1997) document have resulted in increased pressure on already stretched resources within the NHS. Managing increasing referrals within a shorter timeframe has therefore become more difficult. Following endoscopic investigations, the waiting time for outpatient review appointments for patients with colorectal conditions within many UK trusts is up to 3 months. This delay has an impact on the patients’ experience, quality, and access to colorectal services. A prospective audit of 122 patients attending outpatient review appointments, identified the method of referral and the reason for review. This was broken down further as patients were allocated as to whether it would have been suitable for them to be reviewed by a nurse, either via nurse led clinic or by telephone. Patient satisfaction of current practice was assessed by means of a questionnaire.

78/122 suitable for nurse led follow up; 40 telephone follow up, 33 nurse led clinic; 49 patients discharged for hospital care following review appointment.

We have therefore shown that the majority of patients (64%) referred with colorectal symptoms are suitable for nurse led follow up. We therefore intend to set up a nurse led follow up service that ultimately will provide a more efficient colorectal service, with improved utilisation of resources.

101 PRIMARY RESTORATIVE PROCTOCOLECTOMY FOR FAMILIAL ADENOMATOUS POLYPOSIS

M.C. Gallagher, R.K.S. Phillips. The Polyposis Registry, St Mark’s Hospital, Harrow, Middlesex

Introduction: Some surgeons favour colectomy and ileoanal pouch (RPC) as prophylactic surgery in familial adenomatous polyposis (FAP), citing the absent risk of rectal cancer and avoidance of further surgery. However, the disadvantages of early postoperative complications, particularly sepsis, late pouch dysfunction, and reduced fertility should all be considered.

Aims: To investigate the long term outcome in a cohort of FAP patients undergoing primary RPC.

Methods: All patients undergoing prophylactic colectomy between January 1980 and December 2001 were identified from a single polyposis register. Cases where the primary operation was colectomy with construction of an ileoanal pouch were included, case notes and the registry database being examined to determine outcome.

Results: Primary RPC was performed in 59 of 285 patients (21%). Follow up was complete for 58 patients (98%) and for an average of 8 years and 8 months (6 months to 20 years and 7 months). There were 34 males and 25 females, average age at surgery 32 years and 1 month (range 10 years 10 months). 20 patients (35%) suffered at least one early postoperative complication including pelvic sepsis (9), anastomotic leak (3), prolonged small bowel obstruction (4), postoperative haemorrhage (2), pulmonary complications (4), early pouchitis (1), and wound breakdown (1). Five patients required subsequent pouch excision, one for carcinoma, three for pouch evacuation problems and one for chronic pelvic sepsis; all had suffered early postoperative complications. In only one case was revision surgery successful, the remainder being managed with an end ileostomy. Four patients died during follow up, three of these from colorectal carcinoma.

Conclusion: Pouch surgery is not without complications. Failed pouch surgery has resulted in an ileostomy in 4% of patients. The choice of primary surgery for FAP remains difficult.

102 SCREENING FOR IRON DEFICIENCY AMONG SUBJECTS AGED 55–74 YEARS IN PRIMARY CARE: A PILOT STUDY

M.C. Allison, A.G.K. Edwards, S. Paling, M.D. Penney. Department of Primary Care, University of Wales College of Medicine, Cardiff, UK; Departments of Gastroenterology and Pathology, Royal Gwent Hospital, Newport

Background: Most patients with proximal colonic cancer have iron deficiency anaemia at presentation. Therefore, measurement of serum iron and stores could prove a potential adjunct to faecal occult blood testing (FOBT) in screening and early detection of colorectal neoplasia.

Methods: From a general practice serving 6677 in November 2000 we identified 1331 subjects aged 55–74 years. Of these 86 were excluded (comorbidity, previous colorectal cancer or inflammatory bowel disease and/or already undergoing endoscopic surveillance and/or investigation of established iron deficiency during the previous 3 years). Thus, 1240 subjects were mailed to explain the study and invite them to the surgery. After obtaining consent, blood was taken for serum iron, iron saturation, and ferritin. Those with iron saturation < 10% and/or ferritin < 15 μg/l were invited to see their GP for history and physical examination, plus FOBT, blood count and coeliac serology, and offered hospital referral for consultation, gastroscopy and colonoscopy.

Results: There were 23 subjects who had recently died or moved away. Of the remaining 1217 subjects 570 (47%) have so far come forward for venepuncture. Iron deficiency was detected in 26 of these (5%), of whom 24 agreed to hospital referral and 18 consented to endoscopic investigation. One had a large sessile adenoma requiring right hemicolectomy and another had 5 pedunculated sigmoid polyps. One patient was found to have coeliac disease. Erosive oesophagitis, gastric erosions, and angiodysplasia were each found in two patients. Just before the study 2 patients from the practice (aged 56 and 73) were diagnosed with iron deficiency anaemia due to colorectal cancer, both of whom would have been detected by the study had it started 3 months sooner.

Conclusions: Screening middle aged and older subjects for iron deficiency in primary care is feasible, and is accepted by 47%, which compares favourably with studies using FOBT. Clinically significant pathology in addition to colorectal neoplasia can be detected. Larger studies of population screening in parallel with FOBT are indicated.

103 THE PREVALENCE OF IRON DEFICIENCY AMONG PATIENTS PRESENTING WITH COLORECTAL CANCER

A.L. Beale, M.D. Penney, M.C. Allison. Departments of Adult Medicine and Chemical Pathology, Royal Gwent Hospital, Newport, UK

Background: Retrospective studies suggest that up to half of patients with colorectal cancer (CRC) have iron deficiency anaemia at presentation. Iron deficiency (ID) is likely to precede anaemia by several months, hence measurement of serum iron and ferritin could have a role in screening and in early diagnosis of CRC.

Aim: To examine prospectively the prevalence of ID among patients presenting with colorectal cancer.

Methods: All new patients with CRC presenting to our unit over a 12 month period were invited to participate. Blood was taken at diagnosis or prior to surgery. Criteria for anaemia were Hb < 12.5 g/dl for men and 11.5 g/dl for women with coexistent ID. ID was defined as serum ferritin < 15 μg/l and/or iron saturation < 14%. Right-sided lesions were defined as those proximal to the splenic flexure. We excluded patients with recurrent cancer or co-existing inflammatory bowel disease.

Results: During the study period 157 patients presented with new onset CRC, of whom we were able to study 130. Established iron deficiency anaemia was present in 54 (42%) at presentation and a further 23 (18%) were found to be iron deficient without anaemia. 53 patients (40%) had normal iron status at diagnosis. Iron deficiency was more common among patients with right-sided CRC than those with left-sided and rectal tumours (χ2 = 13, p < 0.001).

Conclusions: Four-fifths of patients presenting with CRC proximal to the splenic flexure are iron deficient at presentation. Measurement of iron status could assist in the early diagnosis of CRC and might also have a role in selecting healthy subjects for screening by colonoscopy.

104 DISTRIBUTION OF COLORECTAL CANCERS AND ADENOMAS IN A SCREENED POPULATION

D.A.L. Macafee1, J.H. Scholefield1, D.K. Whynes2. 1Department of GI Surgery, E Floor, West Block, Queens Medical Centre, Nottingham, NG7 2UH, UK; 2Department of Economics, University of Nottingham, University Park, Nottingham, UK

The Nottingham trial of faecal occult blood (FOBT) screening, randomised 152 850 individuals between the ages of 45 and 74, with the identification of 1551 cancers and 1497 individuals with at least one adenoma, within the screened group. The distribution of colorectal cancers was 37% (576) in the rectum, 31% (484) in the left colon and 32% (491) proximal to or including the splenic flexure (right-sided lesions). Rectal cancers among males remained high (43%) compared with females (29%), while 39% (261) of female cancers were right-sided. Of those who had adenomas detected by colonoscopy, 80% (1201) had only one adenoma. Considering only the distribution of the index polyp of each individual, 91% (1358) were found distal to the splenic flexure. This is the first time a screening study has identified a right-sided shift in colorectal cancer distribution. This trend has been noted by other recent papers but not within a screening programme. This shift has important implications for any screening modality if a national screening programme is to be considered.

105 TRUNCATING AND MISSENSE GERM LINE MUTATIONS IN MYH ARE A FREQUENT CAUSE OF A SPECIFIC MULTIPLE COLORECTAL ADENOMA PHENOTYPE

O.M. Sieber1, L. Lipton1,2,3, S. Jones4, K. Heinimann5, E. Barclay1, S.V. Hodgson3, J.P. Cheadle4, T.F. Orntoft6, L.A. Aaltonen7, J.R. Sampson4, I.P.M. Tomlinson1, H.J.W. Thomas2. 1The Molecular and Population Genetics Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK; 2The Cancer Research UK Colorectal Unit, St Mark’s Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, UK; 3The Department of Clinical Genetics, Guy’s Hospital, London SE1 9RT, UK; 4The Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK; 5The Research Group Human Genetics, Division of Medical Genetics, University Clinics, 4031 Basel, Switzerland; 6The Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, DK 8200 Aarhus N, Denmark; 7The Department of Medical Genetics, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland

Background: Compound heterozygosity for missense variants of the base excision repair (BER) gene MYH has been linked to inheritance of multiple colorectal adenomas or carcinoma in a single UK family. Colorectal adenomas from affected individuals displayed an excess of somatic G:C→T:A transversions in the adenomatous polyposis coli (APC) gene, consistent with a BER defect.

Methods: 157 unrelated UK patients with multiple (3 to 115) colorectal adenomas were screened for germ line mutations in MYH and subsets were screened for the related BER genes, MTH1 and OGG1. Adenomas from patients harbouring pathogenic MYH germ line mutations were tested for somatic APC mutations and loss of heterozygosity at MYH. Clinicopathological and molecular data were compared between patients with and without MYH mutations.

Results: Fourteen (9%) patients harboured germ line MYH variants, with 8 (5%) having biallelic, pathogenic mutations. Both nonsense and protein truncating MYH mutations were found. Missense variants Y165C and G382D were the most frequent alterations observed. Patients with biallelic MYH mutations had more polyps than carriers of single mutations or MYH wild-type patients (medians, 55 v 3 v 7, respectively; p < 0.01). Four out of 28 (29%) patients with 15 to 115 adenomas had biallelic MYH mutations. All somatic APC mutations identified in such patients were the expected somatic G:C→T:A transversions. No clearly pathogenic MTH1 or OGG1 germ line mutations were identified.

Conclusions: The data provide strong evidence to show that individuals with two allelic MYH mutations are predisposed to a multiple adenoma phenotype. Progression to colorectal carcinoma occurs in some cases.

106 AETIOLOGY OF COLORECTAL CANCER AND RELEVANCE OF MONOGENIC INHERITANCE

M. Ponz de Leon, P. Benatti, M. Pedroni, F. Borghi, A. Scarselli, C. Di Gregorio, L. Losi, G. Rossi, L, Roncucci. Departments of Internal Medicine and Morphological Sciences, Università di Modena e Reggio Emilia, Pathology Unit, Ospedale di Carpi (MO), Italy

Although many factors contribute to the development of colorectal tumours, the only clearly identified aetiological factors include inheritance (Lynch syndrome (HNPCC) and familial polyposis (FAP)), inflammatory bowel diseases (ulcerative colitis and Crohn’s disease, IBD), papillomavirus and AIDS (HIV).

Purpose: To find our what proportion of colorectal malignancies can be attributed to each of these specific factors.

Patients and Methods: Data of a colorectal cancer registry have been analysed, over a period of 15 years, during which nearly 2500 cases were recorded. In patients with clinical suspicion of hereditary tumours, microsatellite instability was assessed, and in positive families constitutional mutations of the main mismatch repair genes (hMSH2, hMLH1, hMSH6) were evaluated by single strand conformation polymorphism and sequencing.

Results: IBD, FAP, and AIDS were rare causes of colorectal cancer (3, 3 and 1 cases, respectively). Anal squamous carcinoma (attributed to papillomavirus infection) developed in 27 patients (1.0%). In 58 patients (from 34 families) a clinical diagnosis of HNPCC could be established (2.4% of the total). Altogether, cases with a know aetiology (N = 92) accounted for only 3.7% of all patients. Microsatellite instability was found in 15 HNPCC families, while germ line mutations in one of the mismatch repair genes were detected in 6 families (12 patients, 0.5% of the total). Microsatellite positive families, regardless the mutational status, were clinically similar, thus suggesting an involvement of the mismatch repair system even when mutations were not detected.

Conclusion: The study suggests that the aetiology of colorectal neoplasms remains elusive in the large majority of cases. Among specific causes, HNPCC represents by far the most frequent. However, by using a population based approach, constitutional mutations of the main genes responsible for HNPCC can be detected in only 20% of the cases.

107 INVESTIGATING THE ROLE FOR NORADRENALINE IN NEUROGENIC RESPONSES OF THE SHEEP INTERNAL ANAL SPHINCTER

A.G. Acheson1, J.H. Scholefield1, V.G. Wilson2. 1Department of Surgery; 2School of Biomedical Sciences, Queen’s Medical Centre, Nottingham, NG7 2UH, UK

Introduction: Nitric oxide (NO) is the principal inhibitory neurotransmitter in the internal anal sphincter (IAS) of humans and sheep with noradrenaline (NA) responsible for causing neurogenic contractions via α adrenoceptors following inhibition of NO synthase. Previous work using isolated IAS tissue from sheep has surprisingly shown that phentolamine, an α adrenoceptor antagonist, fails to enhance neurogenic relaxations under control conditions. This study aims to further investigate the nature of neurogenic responses of the sheep IAS.

Methods: Using an established sheep IAS model, strips of internal sphincter were placed in isolated organ baths. The neurogenic responses to 1 Hz and 10 Hz stimulation (300 mA pulse strength, 0.3 ms pulse width, 30 s every 180 s) in the presence and absence of 3 μM phentolamine were tested.

Results: There was no significant difference in the magnitude of the neurogenic relaxations observed following stimulation at 1 Hz or 10 Hz and the addition of 3 μM phentolamine failed to enhance these responses at either frequency. The time taken for the neurogenic relaxations to return to 50% of the initial tone was significantly longer with 1 Hz (34.6 ± 0.6 sec) compared with 10 Hz (25.8 ± 1.9 sec, n = 24, p < 0.05). Phentolamine had no effect on the duration of these responses at 1 Hz but significantly prolonged the responses at 10 Hz (32.1 ± 1.5 sec, n = 24, p < 0.05).

Conclusion: Based on the effects of phentolamine, NA is released at 10 Hz but not 1 Hz. NA does not alter the magnitude of the neurogenic relaxations, but limits the time course of the response. Phentolamine, an α adrenoceptor antagonist, increased the time course of the neurogenic relaxations and therefore preserved the effects of neuronal NO. These findings raise the possibility that topical α adrenoceptor antagonists may be useful, either with or without a direct acting relaxant, in instances of excessive adrenergic tone (eg anal fissures).

Neoplasia free papers 108–119

108 CCK2 RECEPTOR AS A THERAPEUTIC TARGET FOR NEUROENDOCRINE TUMOURS

M. Stubbs, K. Khan, K. Savage, M. McStay, A.P. Dhillon, M.E. Caplin.

Royal Free and University College Medical School, Rowland Hill Street, London, NW3 2PF, UK

Aim: To investigate the role of gastrin and the CCK2 gastrin receptor in the proliferation of neuroendocrine tumour cell lines.

Methods: CRI-G1, NCI-H727, RIN 5F, and SHP 77 neuroendocrine tumour cells were studied. CCK2 receptor was detected in cell lysates by immunoblotting using an antibody (antiGRE1) raised against the C-terminal sequence of the receptor. For uptake studies, cells were cultured in chamber well slides. The cells were incubated with either gastrin 7 coupled to rhodol green dye or antiGRE1 labelled with Alexa Fluor 546 dye for 1 h at 37°C. After fixation, cells were viewed under a fluorescence microscope. Cells exposed to the antiGRE1 antibody were subsequently counterstained for apoptosis using the TUNEL method (ApopTag FITC kit). Proliferation studies were performed by incubating cells alone or with CCK2 antagonist PD 135 at 10–4 M for 96 h followed by measurement of cell number using the MTT assay.

Results: Immunoreactivity to CCK2 was detected in all cell lines. A 122kD band was seen at in all 4 cell lines with an additional 186 kD band seen only in the CRI G1 and RIN 5F cells. Uptake of rhodol green labelled gastrin 7 and of Alexa Fluor 546 labelled antiGRE1 antibody was seen in all 4 cell lines with the fluorescence being most prominent in the SHP 77 cells. ApopTag FITC staining revealed a coincidence of antibody uptake and apoptosis in the latter cell line. PD135 at a concentration of 10–4 M gave inhibition of proliferation in all 4 cell lines (27–92% reduction compared to controls).

Conclusions: We present evidence for the existence of CCK2 receptor in NET cell lines. We have demonstrated uptake of gastrin peptide and antibody by tumour cells and a role for CCK2 in proliferation. These results suggests that the gastrin pathway could be a potential target for treatment in neuroendocrine tumours.

109 ADHERENT E COLI FROM COLON CANCER TISSUE INCLUDE ISOLATES CAPABLE OF INDUCING IL-8 RELEASE AND COX2 EXPRESSION

H.M. Martin1, B.J. Campbell1, M. Nayar1, H. Williams2, C.A. Hart2, J.M. Rhodes1. 1Department of Medicine, University of Liverpool, UK; 2Department of Medical Microbiology, University of Liverpool, UK

Introduction: A previous study by Swidsinski et al1demonstrated the presence of intracellular E coli in both the carcinoma and in the macroscopically normal tissue of colon cancer patients. It is our hypothesis that the altered mucosal glycoconjugates seen in colon cancer and pre-cancerous polyps may function as receptors for adhesions of otherwise non-pathogenic bacteria, promoting mucosal recruitment and intestinal inflammation.

Methods: Mucosally associated and intraepithelial bacteria from both malignant and macroscopically normal colonic mucosa of patients with colon cancer (n = 21) were isolated after removal of surface mucus followed by the gentamicin protection assay. Bacteria identified as E coli were screened for agglutination of human red blood cells and characterised for genotype/phenotype using PCR. E coli were also assessed for attachment/invasion to intestinal cells and for their ability to induce release pro-inflammatory cytokines.

Results: 71% (15/21) of tumour tissue specimens were positive for mucosa associated bacteria compared with 57% (12/21) of macroscopically normal tissue. However, we were unable to confirm previous reports of intra-epithelial E coli in the unaffected mucosa of colon cancer patients. 38% (8/21) of tumour specimens were positive for agglutinating E coli compared to 24% (5/21) of distant normal specimens. Using a panel of glycoconjugates, all agglutinating E coli were inhibited by soluble plantain fibre and bovine submaxillary mucin. PCR showed that all agglutinating E coli possessed at least one adhesin gene but no known virulence genes. E coli isolated from 3/8 cancer patients possessed the cytotoxic necrotising factor 1 gene, encoding a toxin known to induce COX2 expression in fibroblasts. All agglutinating E coli attached to both HT29 and I407 cell lines, with invasion only in I407 cells. All agglutinating E coli induced IL-8 release, up to 5 times above basal levels (p < 0.01).

Conclusions: These results support the hypothesis that altered mucosal glycosylation in colon cancer causes alterations in mucosa associated flora, leading to recruitment of non-pathogenic bacteria, which may then result in inflammation. This could be relevant to the progression from dysplastic polyps to invasive cancer. Soluble plantain fibre inhibits adherence of these E coli and further studies are warranted to assess its possible role in tumour prevention.

  1. Swidsinski et al. Gastroenterol 1998;115:281–6.

110 EXPRESSION OF MACROPHAGE MIGRATION INHIBITORY FACTOR IN INTESTINAL ADENOMAS

J.M. Wilson1, N. Scott2, P.J. Guillou3, A.F. Markham1, P.L. Coletta1, M.A. Hull1. 1Molecular Medicine Unit, 2Department of Histopathology, 3Department of Surgery, University of Leeds, St James’s University Hospital, Leeds LS9 7TF, UK

Background: The cytokine macrophage migration inhibitory factor (MIF) is expressed in normal human colorectal mucosa and promotes tumour growth in several experimental cancer models.

Aims: The aim of this study was to investigate MIF expression and activity in human sporadic colorectal adenomas and in intestinal adenomas from the ApcMin/+ mouse model of familial adenomatous polyposis.

Methods: Immunohistochemistry was performed on archival formalin-fixed, paraffin-embedded human sporadic colorectal adenomas (n = 56), and on small and large intestine of ApcMin/+ mice and wild-type littermates. Human MIF protein levels were analysed in fresh paired colorectal adenoma and normal mucosal samples by ELISA (n = 16) and by Western blot analysis (n = 8). A p-hydroxyphenylpyruvate (HPP) tautomerase assay was used to measure specific MIF activity in paired fresh tissue (n = 20).

Results: In human colorectal tissue, immunohistochemistry and Western blot analysis both demonstrated increased MIF protein levels in adenomas compared with paired normal colorectal mucosa. MIF was localised to both epithelial (particularly at the apical membrane) and stromal cells in adenomas. The ELISA demonstrated a mean 1.9-fold increase (95% CI 1.1 to 2.7) in immunoreactive MIF in adenomas compared with paired normal mucosa. The HPP tautomerase assay revealed a mean 1.5-fold increase (95% CI 1.2 to 1.7) in MIF activity in adenomas. Immunohistochemical analysis of ApcMin/+ mouse intestine revealed a similar pattern of protein localisation in adenomas and histologically normal mucosa, with prominent MIF protein expression by dysplastic epithelial cells in adenomas.

Conclusions: MIF protein levels are increased in human sporadic colorectal adenomas and in intestinal adenomas from the ApcMin/+ mouse. The precise role of MIF in epithelial and stromal cell compartments of adenomas during the early stages of intestinal tumorigenesis is currently being investigated.

111 TOP DOWN OR BOTTOM UP MORPHOGENESIS OF COLORECTAL ADENOMAS: COMPETING MANAGEMENT STRUCTURES IN THE ORIGINS OF COLORECTAL NEOPLASIA

S. Preston1,2, R. Wong3, M. Brittan2, N. Direkze3, M. Novelli4, I.P. Tomlinson2, R. Poulsom2, C. Lee2, R. Goodlad2, N. Mandir2, W. Bodmer2, N. Wright1,2. 1Department of Histopathology, Barts and the London Hospital, London; 2Cancer Research UK; 3Department of Gastroenterology, University of Hong Kong; 4Department of Histopathology, University College and the Royal Free Medical School, London, UK

Introduction: The adenoma-carcinoma sequence is well established. Understanding the molecular pathology of the adenoma is therefore important. There is great controversy within the field. The Vogelstein group champion the “top down” theory: colorectal adenomas arise and grow across the mucosal surface, and down into the crypts, whereas other studies, including our own, propose ‘bottom up’ spread.

Methods: Serial sections of 40 small (< 3 mm) sporadic colorectal adenomas were stained with H&E, and for MIB-1 and β catenin. 11 early adenomas were Feulgen stained and microdissected. In addition, we examined the flat mucosa of 3 patients who had undergone colectomies for familial adenomatous polyposis (FAP) and also specimens from an XO/XY individual with FAP, the latter using in situ hybridisation for the Y chromosome.

Results: In the earliest sporadic adenomas there were crypts entirely filled with adenomatous epithelium, which showed proliferative activity and nuclear localisation of catenin. There was a sharp cut off between crypt epithelial cells showing nuclear β catenin and surface cells with no membrane staining. In slightly larger lesions, adenomatous spread from above was seen. Microdissected adenomas showed multiple fission events, with proliferation equally distributed throughout. In FAP tissue, numerous isolated monocryptal adenomas, which were clonal in origin, were seen. Examination of adenomas in the XO/XY individual showed no instances of XY or XO adenomatous epithelium growing down into crypts of the other genotype.

Conclusions: Both sporadic and FAP adenomas start as a unicryptal adenomas, and grow initially by crypt fission—a “bottom up” pattern. Later, in sporadic adenomas, there is evidence of growth down into adjacent crypts—“top-down”.

112 THE PREVENTION OF COLORECTAL CANCER BY COLONOSCOPIC SURVEILLANCE IN INDIVIDUALS WITH A FAMILY HISTORY OF COLORECTAL CANCER

I. Dove-Edwin1, P. Sasieni2, J. Adams2, H.J.W. Thomas1. 1Family Cancer Clinic, Cancer Research UK Colorectal Cancer Unit, St Mark’s Hospital, Middlesex HA1 3UJ, UK; 2Department of Mathematics, Statistics and Epidemiology, Cancer Research UK, Lincoln’s Inn Fields, London WC2A 3PX, UK

Background: Surveillance by colonoscopy has been recommended for individuals with a significant family history of colorectal cancer. We report results of 2983 surveillance colonoscopies in 1519 individuals.

Methods: Families were classified into four groups: hereditary nonpolyposis colorectal cancer (HNPCC = Amsterdam Criteria positive), and those with 1, 2, or 3 affected first-degree relatives. Colonoscopy was offered 5 yearly or 3 yearly if an adenoma was detected. The frequencies of advanced neoplasia (high risk adenoma or cancer) were analysed by age, extent of family history, and findings on previous colonoscopies. Colorectal cancer incidence and mortality during over 11 000 person-years of follow up were compared to those expected in the absence of surveillance.

Results: Advanced neoplasia was most frequent in HNPCC (6.5% on initial colonoscopy). In non-HNPCC it was infrequent (1.2%) under age 45 (even when a relative had developed cancer under age 45 years) and on follow up if advanced neoplasia was absent initially (1.3%). After adjusting for the relative risk based on family history and age, there was a highly significant reduction in colorectal cancer incidence (92% in non-HNPCC, 58% in HNPCC) and mortality (91% in non-HNPCC, 80% in HNPCC).

Conclusions: HNPCC family members require surveillance with short intervals. Those with a lesser family history do not require surveillance under age 45 years, and if advanced neoplasia is absent on initial colonoscopy, surveillance intervals may be lengthened. Colonoscopic surveillance reduces the risk of colorectal cancer in those with a strong family history.

113 LOSS OF TRANSFORMING GROWTH FACTOR-α REDUCES TUMOUR INITIATION BUT DOES NOT AFFECT TUMOUR PROMOTION

O. Bashir, R.A. Goodlad. Imperial Cancer Research Fund, Histopathology Unit, 44 Lincoln’s Inn Fields, WC2A 3PX, London, UK

Background: TGF-α may be the main ligand for the EGF receptor in the gut and thus plays an important role in maintaining the integrity of the gastrointestinal tract. TGF-α null mice show some changes in the morphometry of the colon and also have an increased susceptibility to colitis. TGF-α expression is increased in humans with adenomas and autocrine stimulation of the EGF-receptor by TGF-α has been linked to the growth of adenoma cell lines.

Aims: To determine if TGF-α knock out lead to altered susceptibility to carcinogen induced colonic tumours.

Methods: TGF-α null mice and appropriate wild type mice were either injected with saline or dimethylhydrazine (DMH) for 16 weeks. Two weeks after the last injection they were given BrdU and vincristine and then autopsied. Polyp number, cell proliferation, and crypt fission were then assayed.

Results: Small bowel weight was significantly greater in the TGF-α null mice (p < 0.001), but colon weight was not changed. DHM had no effect on small bowel weight but significantly increased colon weight and cell proliferation (p < 0.001). There was a small but non-significant reduction in polyp number (7.1±03 v 6.1±0.6 WT/KO) but the number of aberrant crypt foci was reduced from 97.5±7.4 to 53.2±4.1 (p < 0.001) in the null mice.

Discussion: TGF-α knock out mice had half the number of aberrant crypt foci of the wild type mice, but polyp number was not altered, suggesting that TGF-α is involved in the initiation rather than the promotional stages of carcinogenesis.

114 SEVERITY OF DYSPLASIA IN COLONIC POLYPS AND NOT LESION SIZE IS DIFFERENTIALLY ASSOCIATED WITH ELEVATED FAECAL CALPROTECTIN—A COMPARATIVE STUDY WITH FAECAL α1 ANTITRYPSIN

D. Watts1, S. Campbell2, N. Anderson1, W.G. Brydon1, J. Satsangi1, S. Ghosh3. 1Western General Hospital, Edinburgh, UK; 2John Radcliffe Hospital, Oxford, UK; 3Hammersmith Hospital, London, UK

Introduction: Levels of both calprotectin(Cal) and α1antitrypsin(α1at) have been shown previously to be elevated in colonic neoplastic disease. There are, however, little data on either their comparative performance as markers of neoplasia or their discriminatory function as markers of dysplastic change within polyps.

Aim: To assess the sensitivity of faecal Cal and α1at levels in patients with severely dysplastic colonic polyps.

Method and Results: A prospective study of 137 patients (male:female 69:73,median age 68 years, range 24–86) undergoing colonoscopy was made. 44 patients underwent a normal colonoscopy with a median faecal calprotectin of 4 μg/g (range: 1–367) and α1at 44 μg/g (2–168). 28 patients had one or more large (> 1 cm) polyps (2 right colon, 21 left colon and 5 multiple polyps) but neither Cal (13 μg/g, 1–397, p = 0.1) nor α1at (51 μg/g, 9–296, p = 0.6) was significantly raised. 8 patients had one or more estimated small (< 1 cm) polyps at colonoscopy (2 right colon, 5 left colon and 1 both sides of colon); again neither faecal Cal (median 2 μg/g, 1–15 μg/g, p = 0.5) nor α1at levels (44 μg/g, p = 0.39) were significantly higher than the normals. 11 adenocarcinomas (10 left colon, 1 right colon) and 31 adenomas (24 left colon, 5 right colon and 1 both sides) were reported. Significant differences in Cal values were found between adenocarcinoma (44 μg/g, 12–397) and adenoma (3.5 μg/g (1–94 μg/g) p = 0.0002) and metaplasia (n = 16, median 5.5 μg/g (1–45 μg/g) p = 0.0009). α1at was not significantly higher in carcinomas compared to either severely dysplastic adenomas (71 v 46 μg/g, p = 0.53), or the normal group (71 v 45 μg/g, p = 0.38). In the severely dysplastic adenomas (n = 15), significantly higher Cal (8 μg/g, 1–48) was found than in those with only little dysplasia (n = 17, 1 μg/g, 1–94, p < 0.05). α1at was no different in either group (p = 0.33). Using ROC analysis, at a Cal level of 8 μg/g, sensitivities for carcinomas, 100%, severely dysplastic adenomas, 54%, and adenomas with little dysplasia, 25%, were achieved.

Conclusion: Faecal calprotectin and not faecal α1at is significantly higher in those patients with adenocarcinoma or severely dysplastic adenomas by contrast to adenomas with little dysplasia, irrespective of polyp size or location.

115 THE ACCURACY OF EUS, HCT AND PET IN RE-STAGING OESOPHAGEAL CANCER FOLLOWING CHEMOTHERAPY

J. Meenan, S. Rankin, G. Marx, P. Harper. Departments of Gastroenterology, Radiology, Oncology, and Surgery, Guy’s and St Thomas’ Hospital, London, UK

The best approach to re-staging oesophageal cancer following chemotherapy is unknown. Potential limitations exist for all imaging modalities: hCT and EUS cannot differentiate between viable tumour and inflammation, whereas although PET scanning may detect remnant malignancy, it lacks spatial resolution. The purpose of this study was to compare the accuracy of CT, EUS, and PET scanning in the assessment of oesophageal tumour response to chemotherapy using pathological correlation. Initial results are presented.

12 oesophageal cancer cases underwent CT, EUS, and FDG-PET before and after 3 cycles of ECF chemotherapy. The follow up FDG-PET was performed at least 4 weeks after the end of the 5FU and 8 weeks after the E/C. Quantitative PET data using standard uptake values (SUV) were measured. Results were compared with surgical pathological findings. Baseline EUS gave initial T stage as T4 in 2, T3 in 9 and T2 in 1 patient. Ten patients were staged as N1 and 2 as N0. CT and EUS concurred in all the patients for nodal status with CT understaging the T stage in 3 patients. Initial PET identified all the tumours but was unable to T stage accurately and identified only 5 of the N1 patients.

10 patients underwent oesophagectomy. The two remaining cases did not undergo surgery due to early death and disease progression. Of the 10 surgical patients, only 2 had responded. CT identified one of the responders correctly, overstaged the other and understaged 3/8 of the non-responders. EUS significantly overstaged one of the responders (1/2) and understaged 4/7 of the non-responders, particularly the N stage. Quantitative PET measurement of the reduction in tumour uptake was significantly different between the responders (-77% ±6%) and the non-responders (-39% ±22%), but FDG-PET underestimated nodal disease in 4 patients. Qualitative assessment alone using FDG-PET was unreliable as 2 non-responders were labelled responders.

Neither CT nor EUS could accurately predict response following chemotherapy. FDG-PET could differentiate responders from non-responders based on the reduction in uptake values and was better at identifying liver metastases than CT. No single imaging modality conferred, however, adequate accuracy in assessing oesophageal tumour response to chemotherapy.

116 HGF/MET INDUCES DOWNREGULATION OF E-CADHERIN AND INCREASES TCF/BETA-CATENIN SIGNALING IN OESOPHAGEAL ADENOCARCINOMA

M.R. Anderson1, M. Campbell1, J.A.Z. Jankowski2. 1University of Birmingham, UK; 2Digestive Diseases Centre, Leicester, UK

Background: The development of oesophageal adenocarcinoma is characterised by progression along the Barrett’s metaplasia–dysplasia–carcinoma sequence. The HGF receptor, Met, shows increased expression along this sequence and patients with carcinomas that overexpress Met exhibit poorer short term survival. The perturbation of cadherin/catenin complexes has been shown in oesophageal adenocarcinoma with downregulation of E-cadherin a common finding. We sought to investigate a link between Met activation and cadherin/catenin biology.

Aims: To investigate the effect of Met activation on E-cadherin expression and on Beta-catenin nuclear signaling in oesophageal cells.

Methods: Two cell lines that express Met (OE33, SEG1) and a cell line that does not (TE7) were incubated with HGF at doses ranging from 1n g/ml to 500 ng/ml. At set time points from 30 min to 24 h, mRNA and protein were harvested. Real time PCR was used to assess levels of E-cadherin mRNA. Western blot was used to assess levels of E-cadherin protein. Levels of nuclear TCF/Beta-catenin signaling were assessed following transient transfection with a TCF/luciferase reporter construct. An ELISA was used to measure levels of HGF in the culture media at different time points to detect any endogenous synthesis of HGF by the cell lines themselves.

Results: OE33 and SEG1 showed a 37% and 69% reduction in E-cadherin mRNA following 30 min stimulation with HGF at 100 ng/ml (p < 0.01). This minimum dose was reduced by altering the amount of calf serum supplemented in the culture media. Reduced E-cadherin protein expression was seen. OE33 showed a two-fold increase in the relative levels of luciferase activity following HGF stimulation (p < 0.01). TE7 (which lacks the Met receptor) showed no response.

Conclusions: Met activation induces downregulation of E-cadherin and increases nuclear TCF/Beta-catenin signaling. Thus Met activation may impair cell adhesion and play a role in regulating gene transcription.

117 SURVEY OF CURRENT CLINICAL PRACTICE IN THE DIAGNOSIS, MANAGEMENT AND SURVEILLANCE OF BARRETT’S METAPLASIA: A UK NATIONAL SURVEY

S. Ishaq, E. Harper, J. Brown, P. Moayyedi, T. Wicks, P. Watson, H. Barr, S. Attwood, R. Harrison, J. Jankowski. Division of Medical Sciences, University of Birmingham, Edgbaston and Digestive Disease Centre, Leicester, UK

Introduction: Barrett’s metaplasia (BM) is the precursor lesion for oesophageal adenocarcinoma, a tumour with a rising incidence and a uniformly poor prognosis. In spite of the recognition of this fact and the almost exponential increase in clinical and basic science research, much uncertainty remains as to the optimum management of Barrett’s metaplasia in terms of screening, surveillance, and treatment.

Methods: In order to assess the current situation in the UK an anonymous questionnaire was sent to members of the BSG asking for details of their current practice.

Results: Of 401 questionnaires sent, 228 were returned (57%), indicating wide differences in practice. Of the responders, 50% routinely consider screening in patients with gastro-oesophageal reflux disease, 1/3 for all patients, but 2/3 only for those with long standing reflux disease. Sixty-five percent will diagnose BM using histological criteria by the presence of intestinal metaplasia anywhere in oesophagus. Although the majority (90%) consider surveying patients with known BM using a 4-quadrant protocol, there is considerable variation in the frequency and implementation. Endoscopy intervals also vary in patients with low grade dysplasia. Although there is greater agreement in management of patients with high grade dysplasia (HGD), with the majority considering surgery for suitable patients, there was considerable variation in access to pathological expertise and utilisation of this expertise in cases of HGD. The majority supported the idea of clinical trials in BM, particularly into the value of surveillance.

Conclusion: This survey indicates a wide variation in the management of BM in the UK. This is likely to reflect the lack of good evidence to support one protocol over another and in certain cases a lack of clinical resources, particularly in pathology. There is a clear need for a more robust evidence base on which to base management and direct resources by a large randomised multicentre intervention and surveillance study.

118 MANAGEMENT OF HIGH GRADE DYSPLASIA (HGD) IN BARRETT’S OESOPHAGUS: A RETROSPECTIVE 10 YEAR REVIEW

T. Thomas, C.J. Richards, J. de Caestecker, R.J. Robinson. The Digestive Diseases Centre, University Hospitals of Leicester

Background: Management of HGD in Barrett’s remains controversial.

Aim: To review management and outcome of HGD from three hospitals in one district.

Methods: Case record study. Patients with dysplasia identified from pathology database. All HGD verified by at least two upper GI pathologists and those with coincident adenocarcinoma (AC) with HGD excluded.

Results: 99 cases of dysplasia identified; 36 had HGD arising in Barrett’s mucosa, M/F 31/5, mean age 67 (range 34–95). 9/36 (25%) were identified during Barrett’s surveillence. 7 had no further interventions due to age and comorbidity. On repeat endoscopy and biopsy of the remaining 29 (usually within 3 months), 9 had intramucosal AC and are not further considered. Of the remaining 20, 16 had persisting HGD and 4 became and remained negative for dysplasia (mean follow up 17.2 months). Of the 16 with persisting HGD, 8 had oesophagectomy of which 6 had AC on operative histology. At a mean follow up of 18.7 months, 6 patients were disease free. There were no immediate (30 day) postoperative deaths; 3 died (2–47 months after surgery), 2 of unrelated causes and one of metastatic AC. 5 were treated with argon ablation of which 2 had nodules and/or ulcers in the Barrett’s segment. In 4/5 the Barrett’s segment was ablated; absence of HGD was confirmed histologically in 2 (both originally had HGD in “flat” mucosa) who remained disease free (mean FU 10 months); 2/5 developed metastatic AC of whom 1 has died. The final 3/16 continued to have surveillance: 2 developed AC (I death) and 1 died of an unrelated cause. In summary: 15 of 36 (42%) of patients with biopsy proven HGD, have underlying AC either on resection or on early rebiopsy. A further 4 developed AC during surveillance (median 17 months, range 4–64). A small proportion of HGD regress (4/36–11%).

Conclusions: Oesophagectomy remains the treatment of choice in HGD because a high proportion have coincident AC. Argon ablation can be effective in a proportion of those unfit for surgery but may be less successful if the Barrett’s segment contains nodules/ulcers.

119 NOVEL P53 MUTATIONS BUT LACK OF A MUTATIONAL FINGERPRINT IN HUMAN INTRAHEPATIC CHOLANGIOCARCINOMA

S.A. Khan1, S.D. Taylor-Robinson1, P.L. Carmichael2, N. Habib3, N. Lemoine4, H.C. Thomas1. 1Liver Unit, St Mary’s Campus; 2Department of Biological Chemistry, South Kensington Campus, Faculty of Medicine, Imperial College; 3Department of Surgery; 4Molecular Oncology Unit, Hammersmith Campus, Faculty of Medicine, Imperial College

Background: Cholangiocarcinoma (CCa) may arise from cholangiocyte DNA damage due to genotoxic compounds in bile. We have previously shown that human biliary tissue is exposed to genotoxic agents, as evidenced by the presence of DNA adducts. The correlation of DNA lesions along a target gene with a known “mutational signature” induced by an environmental carcinogen is a means of linking cause and effect in human cancer. The tumour suppressor gene p53 is known to have “hotspots” for particular chemical carcinogens. Previous studies of p53 mutation in CCa have focused on exons 5–8, potentially missing gene alterations at other sites. This study examined the link between environmental carcinogens and CCa, by analysing CCa DNA for complete p53 mutational signatures.

Methods: Entire p53 cDNA (all exons) from 31 intrahepatic CCa patients were screened by single strand conformational polymorphism. Exons exhibiting aberrant bands were fully sequenced. Mutations were compared to known p53 mutations in CCa, and to mutations induced by environmental mutagens, as described in p53 databases.

Results and Conclusions: Five non-silent p53 mutations were found, including an exon 5 missense and a nonsense mutation, both previously reported. Three frameshifts (2 deletions and 1 insertion) in exons 4, 5, and 6, and two intron mutations were discovered, none of which have been previously reported. No predominant mutational signature was seen. This may be due to host or environmental differences in study populations, lack of data outside exons 5–8, bias in mutation reporting or because non-coding regions or other genes are involved. Further epidemiological and molecular studies are required to establish risk factors and elucidate genotoxic and host mechanisms in cholangiocarcinoma.

Abstract 120

Plenary posters 120–149

120 FLAT AND DEPRESSED COLORECTAL LESIONS HAVE A HIGH MALIGNANT POTENTIAL IN THE UK: A PROSPECTIVE STUDY USING HIGH RESOLUTION CHROMOSCOPIC COLONOSCOPY

D.P. Hurlstone1, A.J. Shorthouse2, I. Adam2, S. Brown2, S.S. Cross3, C. Korulla1, R. Davies1, A.J. Lobo1. 1Department of Gastroenterology, 2Department of Surgery, 3Department of Pathology, Royal Hallamshire Hospital, Sheffield.

Introduction: Flat and depressed colorectal (CR) lesions are well described in Japanese and now western European cohorts. Studies assessing their malignant potential have shown high variability. Detection using conventional colonoscopy can be difficult. High resolution chromoscopic colonoscopy (HRCC) may facilitate detection of such lesions and allow histological characteristics to be predicted in vivo.

Aim: To evaluate the efficacy of HRCC in predicting the malignant potential of flat CR lesions.

Subjects and Methods: Total colonoscopy was performed on 600 consecutive patients, by a single endoscopist using the Olympus CF240Z endoscope from 01/02 to 09/02. Locally applied 0.5% indigo carmine (IC) was used to facilitate crypt pattern appearance of all flat lesions, graded according to the modified Kudo class. Morphology was documented using the Japanese Research Society Classification (JRSC). Endoscopic Mucosal Resection (EMR) was performed on all lesions unless > 20mm diameter, had evidence of a type V(n)/V(a)/IIIs crypt pattern, or those demonstrable of asymmetrical “lift”. Histological analysis was obtained on all specimens. Lesions excluded from EMR were cold biopsied and marked with an intramucosal tattoo or endoclip.

Results: See table.

Conclusions: Flat depressed CR lesions exhibit a high malignant potential in the UK. HRCC is a useful tool when predicting histological type in vivo.

121 ENDOSCOPING PATIENTS WITH UNCOMPLICATED DYSPEPSIA OVER AGE 55 HAS MINIMAL IMPACT ON MORTALITY FROM UPPER GASTROINTESTINAL CANCER

A.C. Casburn-Jones, D. Gillen, K.E.L. McColl. Western Infirmary, Glasgow, Scotland, UK

Most guidelines recommend endoscoping patients over 55 years of age with uncomplicated dyspepsia. This rationale is based on the assumption that an upper gastrointestinal (GI) cancer may be detected at a curable stage in such patients.

Aim: To determine the proportion of upper GI cancers that present with uncomplicated dyspepsia in patients ≥ 55 years and determine the proportion of these upper GI cancers that are curable.

Method: We reviewed the case notes of patients within a catchment area of the North Glasgow Trust, population of approximately 300 000, who were ≥ 55 years age, diagnosed with an upper GI cancer between October 1995 and December 1998 inclusive. These patients were identified by the West of Scotland Cancer Registry. Presenting symptoms were taken to be those recorded as present at the time of referral for initial investigation.

Results: Of the 106 cancer cases identified only 23.5% (24) had dyspepsia (complicated or uncomplicated) as their predominant symptom. Only 8.5% (9) of patients presented with uncomplicated dyspepsia. Of those 9 patients presenting with uncomplicated dyspepsia and found to have upper GI cancer, 6 were found to have lymph node metastases and/or extensive metastases at the time of diagnosis. Each of these 6 patients died from their cancer between 3 and 26 months from diagnosis. One patient had no record of lymph node spread but died 55 days after diagnosis. Of the 2 patients presenting with simple dyspepsia without evidence of lymph node spread, 1 died 57 days post-diagnosis from post-gastrectomy complications. Only 1 patient presenting with simple dyspepsia and found to have cancer remains alive at 5 years follow up.

Conclusion: A policy of endoscoping patients ≥ 55 years with simple dyspepsia will reduce death from upper GI cancers by less than 1% in our population.

122 CAN AN INTENSIVE, STRUCTURED TRAINING WEEK IMPROVE COLONOSCOPY PERFORMANCE?

S. Thomas-Gibson, C.J. Thapar, G. Schofield, M.D. Rutter, N. Suzuki, C.B. Williams, B.P. Saunders. Wolfson Unit for Endoscopy, St Mark’s Hospital, London

Introduction: We developed a structured colonoscopy training course aimed at improving technique of trainees with intermediate colonoscopy skills.

Methods: 12 specialist registrars (8 surgeons, 4 physicians) attended for a week of comprehensive colonoscopy training and assessment in 3 main areas: core knowledge; two teaching videos; and a CD Rom on colonoscopy/basic polypectomy technique were watched by each trainee. Knowledge was assessed using 2 purpose designed, multiple choice question (MCQ) papers shown to be of equal difficulty. Simulator (hand skills): a mean of 24 (16–31) computer simulator cases were performed. A standardised simulated test case (SSTC) was used to score performance. Clinical skills were taught during 5 training lists by two experts. A mean number of 21 (14–26) clinical cases were performed by each trainee throughout the week. Structured subjective scores using 100 point visual analogue scales were made by both trainers. All assessments were made at the start and end of the week.

Results: Eight had performed < 200 cases previously, 4 had performed more than this. The MCQ score significantly increased: mean score 54.8% v 64% (n = 8; p = 0.008). Structured subjective scoring demonstrated an improvement in clinical skill: overall subjective pre- and post-scores 59.6 v 70.8 (p = 0.0004). Mean total time taken to complete the SSTC improved significantly from 1631 secs pre-training v 1163 secs post-training (p = 0.02). There were no perforations or haemorrhages during the SSTCs and only one vasovagal episode.

Discussion: We believe that training must be shown to be effective in achieving its goals. We have demonstrated that a week of structured intensive training can result in an improvement in colonoscopy clinical skills and core knowledge in moderately experienced trainees.

123 ENDOSCOPIC MANAGEMENT OF POSTOPERATIVE BILIARY TRAUMA IN A DISTRICT GENERAL HOSPITAL.

L.P Maiden, P. Hurley, A. Theodossi. Mayday University Hospital, Thornton Heath, Surrey, UK

Introduction: The role of endoscopic intervention in the management of biliary trauma following cholecystectomy and hepatobiliary surgery in tertiary referral centres has been reported previously. This paper reports the practice of a district general hospital in the management of such cases over a 13 year period.

Patients and Methods: Between June 1989 and June 2002, 24 patients (15 female) underwent an endoscopic retrograde cholangio-pancreatogram (ERCP) following suspected biliary trauma sustained during hepatobiliary surgery. Mean age was 54 years (range 29–79). Symptoms were of right upper quadrant pain (n = 16), fever (n = 1), jaundice (n = 3), or none (n = 4).

Results: At ERCP 14 patients were found to have a leak in the biliary tree and underwent a papillary sphincterotomy and single stent insertion (mean duration = 92 days). 7 had strictures: 2 patients with Bismuth type I strictures required stenting only once and 5 patients with type II or III strictures required multiple stent changes (mean = 3). Symptoms resolved when the stents were finally removed. Three patients had both a biliary leak and a stricture (Bismuth type I in all cases). All 3 required multiple stent changes. 2 out of the 24 (8%) required subsequent surgical intervention in a tertiary referral centre. One patient with a leak and stricture was referred for hepaticojejunostomy 2 years after index ERCP due to a permanent stricture. A patient with 2 strictures was referred for surgery 11 days after ERCP due to continuing symptoms. Complications noted were stent migration (n = 2), ascending cholangitis (n = 1), and acute pancreatitis (n = 2).

Conclusion: Postoperative complications of biliary surgery may be managed by therapeutic ERCP effectively and safely in the district general hospital setting. Bile duct leaks and uncomplicated Bismuth type I strictures may both be treated effectively by a single stent for 3 months. Type II and III strictures may require a limited number of stent changes. Patients with multiple strictures or a combination of leak and a stricture may be more difficult to treat endoscopically.

124 ONE STOP INVESTIGATION OF IRON DEFICIENCY ANAEMIA. THE WAY AHEAD?

R. Beale, R. Pinder, A. Hall, M. Rogers (introduced by D. Walls). Department of Surgery, Pinderfields Hospital, Aberford Road, Wakefield WF1 4DG, UK

Adults with iron deficiency anaemia require GI tract investigation. Traditionally gastroscopy (FOG) and barium enema (DCBE) are used, with colonoscopy (FOC) if DCBE is normal. Primary FOC is not used because caecal intubation rates may be as low as 57%1 and it is costly. We investigate iron deficiency anaemia with FOG and FOC at one visit. Over 3 years, 64 consecutive patients with iron deficiency anaemia underwent same day FOG and FOC. Preparation was 4 litres kleenprep and fasting from midnight. All received supplemental nasal oxygen monitored by pulse oximetry. All received pharyngeal local anaesthesia and midazolam for FOG, with fentanyl and hyoscine for FOC. No patients required reversal of sedation. There were no perforations or significant haemorrhage. Two patients required admission overnight. In two patients FOG revealed a gastric carcinoma and FOC was omitted. FOC was incomplete in 7 but in 5 of these obstructing pathology prevented completion (true completion 60/62, 97%).

Findings: See table.

Abstract 124

Investigation of iron deficiency anaemia by FOG and FOC at one sitting is safe and effective. Significant pathology was found in 17 (27%) patients and 21 (33%) patients were returned to their general practitioner after the examination. This cohort of patients are at high risk of having malignant disease and need expeditious investigation. This approach allows prompt, complete investigation and discharge of many patients directly to primary care with the reassurance that no significant abnormality has been found.

  1. Bowles C.J.A., Leicester R., Swarbrick E. et al. Intercollegiate-BSG National Colonoscopy Audit: methods used to identify the caecum and caecal intubation rate. Gut 2001;48(Suppl 1); A10.

125 PERFORMANCE AT SCREENING FLEXIBLE SIGMOIDOSCOPY: A NEW VIDEO ASSESSMENT TOOL

S. Thomas-Gibson1, W. Atkin2, M.D. Rutter1, N. Suzuki1, M.E. Vance1, M.S. Quraishy1, D. Swain1, A. Nicholls1, B.P. Saunders1. 1Wolfson Unit for Endoscopy, St Mark’s Hospital, London; 2Department of Imperial Cancer Research Fund, St Mark’s Hospital, London

Background: Adenoma detection rates (ADR) at screening flexible sigmoidoscopy (FS) are known to be variable. No existing objective method for scoring endoscopy performance exists.

Aims: To develop a video assessment tool that evaluates performance in FS for potential screening purposes, the main objective being confidence that no lesions are missed during an examination (exam).

Methods: Eight experiments using video footage from a selection of 40 000 cases from the UK Flexible Sigmoidoscopy Screening Trial were performed. 5 experienced endoscopists independently scored, on average 5 exams from different endoscopists with varying ADRs. All experiments tested inter-observer variability (IOV), experiments 7 and 8 tested correlation of scores with ADR (validity) by looking at several cases from an individual endoscopist back-to-back. Cases were selected and extubations edited together by independent researchers. Variables assessed were: Bowel mucosa NOT visualised; Confidence NO lesion missed; Time spent viewing mucosa; re-exam of poorly viewed areas; Suctioning of fluid; Luminal distension; lower rectal exam; and overall quality of exam. Experiments 1–3 used Visual Analogue Scales (VAS), replaced in experiments 4–8 (due to high IOV) by grades A-E (A = excellent, B = good, C = needs improving, D = not good enough, E = unacceptable).

Results: The A-E system produced less variability and more validity in predicting overall quality of exam. In experiments 7 and 8 the endoscopist with highest ADR was ranked as most technically proficient. In experiment 8 there was 100% agreement in ‘Overall’ score given for 2 of 3 endoscopists when 5 cases from each were viewed back-to-back. For the third, all scorers agreed that the endoscopist needed to improve their technique (Grades C or D).

Conclusions: An objective performance score for screening FS based on video footage is possible. There is less inter-observer variability when 5 cases from an individual endoscopist are viewed back-to-back and then scored. Further validation is being performed in a larger sample.

126 THE EFFECTS OF LONG TERM CHRONIC HELICOBACTER PYLORI INFECTION ON GASTRIC GROWTH FACTOR EXPRESSION IN THE MONGOLIAN GERBIL

A.H.T. Jeremy1, M.A. Aboshkiwa1, M.F. Dixon2, P.A. Robinson1, J.E. Crabtree1. Molecular 1Medicine Unit, St James’s Hospital, Leeds, UK; 2Dept Pathology, The General Infirmary, Leeds, UK

Introduction: Chronic H pylori infection in the Mongolian gerbil has been demonstrated to result in gastric cancer. To gain a greater understanding of the mechanisms involved, this study investigated the effects of infection on gastric expression of growth factors.

Methods: Male Mongolian gerbils were orally infected three times with H pylori SS1 strain. Infected gerbils (n = 20) and controls (n = 17) were sacrificed at 36 and 62 weeks post-infection (p.i). Gastric mucosa was snap frozen for analysis of growth factor transcripts by RT-PCR. Cross-species PCR and sequencing was used to identify gerbil transcripts for heparin-binding epidermal growth factor (HB-EGF) and transforming growth factor alpha (TGFα). The ratio of growth factors to β-actin was determined by computer image analysis. In addition, HB-EGF localisation was detected by immunohistochemistry.

Results: All infected gerbils had chronic gastritis, with more severe pathology and corpus atrophy observed in the 62 week infected animals. Uninfected controls exhibited an age-related increase in transcripts of both HB-EGF (36 week 1.1 ± 0.19 v 62 week 2.1 ± 0.17, p < 0.01) and TGFα (36 week 1.57 ± 0.195 v 9 62 week 2.28 ± 0.15, p < 0.02). H pylori infection was associated with significantly (p < 0.01) decreased TGFα transcripts at 62 weeks p.i. In infected gerbils HB-EGF also increased with age, but levels were not significantly different to controls. Immunolocalisation showed HB-EGF was present in parietal cells in controls. In contrast, in 62 week H pylori infected gerbils with corpus atrophy HB-EGF was localised in gastric epithelial cells.

Conclusions: In the gerbil there are age related increases in gastric growth factor expression. Gastric TGFα but not HB-EGF, was decreased in long term chronic H pylori infection. In H pylori induced corpus atrophy, the presence of HB-EGF in the gastric epithelial cells may play an important role in epithelial hyperproliferation.

127 IDENTIFICATION OF RISK FACTORS FOR THE DEVELOPMENT OF OESOPHAGO-GASTRIC CANCERS IN SCOTLAND

E. Fernandes, A.G.K. Li, H. Rashid, K.G.M. Park on behalf of the SAGOC Steering Group Aberdeen Royal Infirmary, Foresterhills, Aberdeen, UK

Background: The early detection and treatment of oesophageal and gastric cancer offers the only chance of an improved outcome. However, the relatively low incidence of these tumours renders screening programmes uneconomical and unpractical.

Aim: To target groups of patient who are at increased risk of developing gastric and oesophageal cancer and identify risks factor for this disease.

Methods: Data from the Scottish Audit of Gastric and Oesophageal Cancer (SAGOC) were prospectively collected form 3293 patients with oesophageal and gastric cancer from 1997 to 1999. χ2 analysis was performed between the risk factors for each tumour type.

Results: The population study included cancers of the oesophagus (45%), gastro-oesophageal junction (16%), and stomach (39%). Of the oesophageal tumours, 51% were oesophageal andenocarcinomas (OAC) and 39% squameous cell carcinomas (SCC). Significantly high rates of tobacco and alcohol consumption were seen in patients with SCCs. 14% of patients with OAC had pre-existing Barrett’s oesophagus, which was present in 4.3% of the tumours of the OG junction, 0.9% of SCCs and 0.9% of gastric tumours. A history of GOR was present in 16% of the total study group. Conversely, 27% of patients with OAC had history of GOR compared with 20% of junctional tumours, 13% of SCCs, and 12% of gastric cancers.

Conclusion: These data indicate the relative importance of known risk factors in the development of upper gastrointestinal cancers in Scotland. While each of these may be proportionally common within the general population, a targeted screening algorithm based upon a combination of such factors could be developed to direct investigation of patients.

128 HEPATIC MANIFESTATIONS OF THE ANTIPHOSPHOLIPID SYNDROME

N. Zakaria, J. Wendon, J. Devlin. King’s College Hospital, London, UK

Background and Aims: Antiphospholipid syndrome (APS) is increasingly recognised to have a wide range of clinical manifestations, although involvement from this disorder within the liver or its vasculature is poorly identified.

Patients and Methods: 49 consecutive patients with a liver disorder investigated between 1994 and 2002 who fulfilled the diagnostic criteria for APS were studied. Patients were followed up for a median of 10 months (range 6–94 months).

Results: Of the 49 cases, 21 were male, 28 female. Median age at presentation was 42 (range 17–67). 29 patients were north European (60%), 5 (10%) Mediterranean, 8 (16%) Afro-Caribbean, and 7 (14%) Asian. 36 patients had primary APS (80%), 7 had APS associated with SLE (14%) and 3 associated with other autoimmune diseases (6%). 3 patients had “equivocal” APS. A diagnosis of APS had been made in only 2 of 49 (4%) symptomatic cases prior to referral. A wide range of clinical and pathological findings involving the liver parenchyma and its vascular compartments were identified. 6 (12%) had either thrombosis or arteritis of the hepatic arterial tree. Porto-mesenteric thrombosis was present in 13 cases (27%) and nodular regenerative hyperplasia in 7 (14%). In 9 patients, Budd Chiari syndrome (6), veno-occlusive disease (2) or congestion secondary to right ventricular failure (1) was diagnosed. Autoimmune hepatitis was diagnosed in 9 patients. Associated non-hepatic clinical manifestations included vasculitis (10 (20%)), thromboses (8 (16%)) and unexplained myocardial dysfunction (5 (10%)). Anticardiolipin antibodies were detected in 48 of 49 (98%) and Lupus anticoagulant in 14 of 36 (39%) of the patients tested. Complement C4 levels were low in 22 of 33 (67%) patients tested. 7 of 49 (14%) had SLE. 11 underwent transplantation for APS related liver disease (Budd-Chiari syndrome 3, hepatic artery thrombosis 3, NRH 3, autoimmune hepatitis 1, veno-occlusive disease 1). 4 of the 49 patients died.

Conclusion: The antiphospholipid syndrome has multiple hepatological manifestations and should be actively excluded in arterial, vasculitic and venous vaso-occlusive complications. This newly identified association with autoimmune hepatitis has rarely been reported.

129 TRANSJUGULAR LIVER BIOPSY: SAFETY, ADEQUACY AND CLINICAL IMPACT IN A NON-TRANSPLANT CENTRE

A.M. Elsharkawy, A.S. Austin, S.D. Ryder. Division of Gastroenterology, University Hospital, Queens Medical Centre, Nottingham, NG7 2UH, UK

Introduction: Histological examination of liver tissue is the gold standard for diagnosing and staging liver disease. We assessed the safety, efficacy, and clinical impact of transjugular liver biopsy (TJBx) in a non-transplant centre.

Methods: 180 consecutive biopsies were performed using an automated transjugular cutting biopsy needle with a maximum core length of 25 mm and diameter of 2.2 mm. Mean patient age was 54.2 ± 13.8 (17–89) of whom 114 were male.

Results: Indications for TJBx were platelet count < 100 × 109/l (49%), INR > 1.4 (41%), bilirubin > 100 μmol/l (35%), tense ascites (23%) or other (17%). 44% had two or more indications. Minor transient complications occurred in 11 cases only (5 hypotension, 4 pain, 2 fever). Biopsy sample was adequate for histological diagnosis in 93%, inadequate in 4%, and technically unsuccessful in 2%. Mean biopsy size was 14.8 ± 0.6 (5–35) mm. Adequacy and length did not differ between cirrhotic and non-cirrhotic biopsies. In 63% of cases, alcoholic liver disease was the presumed diagnosis before biopsy. TJBx had a clinical impact in 78%; changed diagnosis in 38% (drug induced liver injury 11, haemachromatosis 8, normal liver 6, cryptogenic cirrhosis 5, other 39); and changed stage in 40%. The likelihood of a change in diagnosis was greater in the non-cirrhotic 21/64 compared with the cirrhotic 35/116 (Chi test; p = 0.001). In addition, a change in diagnosis was less common in those with a presumptive diagnosis of alcoholic liver disease 25/113 compared with those without 44/67 (Chi test; p < 0.001).

Conclusions: Transjugular liver biopsy is a safe procedure in high risk patients in a non-transplant centre. It provides adequate tissue samples in the vast majority irrespective of the presence or absence of cirrhosis. It has a clinical impact in 78% leading to an unsuspected diagnosis in 38%.

130 UP-REGULATION OF TOLL-LIKE RECEPTOR EXPRESSION IN CHRONIC HEPATITIS C: CORRELATION WITH CIRCULATING PRO-INFLAMMATORY CYTOKINE LEVELS AND HEPATIC NECRO-INFLAMMATORY ACTIVITY

K. Visvanathan1, N. Skinner1, J. Kurtovic2, A. Nagree2, C.J. McIver2, R. Williams3, S.M. Riordan2. 1Murdoch Children’s Research Institute, Melbourne, Australia; 2Gastrointestinal and Liver Unit, The Prince of Wales Hospital and University of New South Wales, Sydney, Australia; 3Institute of Hepatology, University College London, UK

Background: Innate immunity to microbial pathogens, leading to the production of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, occurs as a result of activation of toll-like receptors (TLRs). Expression of TLRs has not been investigated in chronic hepatitis C (CHC). This is important as TNF-α may contribute to liver damage in this disorder.

Methods: We studied 16 hepatitis C virus RNA-positive patients (genotype 1, n = 11; 2, n = 2; 3, n = 3) with CHC (median Ishak histological activity score 4, range 2–7; median stage 2, range 1–4) and raised serum ALT levels (median 109, range 55–350 U/L; normal 15–45 U/L) and 32 healthy controls. TLR2 and TLR4 expression on CD14+ve peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry. Serum TNF-α levels were measured by ELISA (R&D Systems, USA).

Results: TLR2 expression on PBMCs (expressed as a ratio to results in controls) was significantly increased in patients with CHC (median 1.18, range 0.77–2.41, p = 0.001). TLR4 expression was also significantly increased in this group (median 1.25, range 0.94–1.66, p < 0.0005). Serum TNF-α levels were significantly higher in CHC patients (median 2.3, range 0.8–9.3 pg/mL) than controls (median 1.9, range 0.8–3.4 pg/mL) and were correlated significantly with PBMC expression of TLR2 (r = 0.58, p < 0.0005) but not TLR4 (r = 0.01, p = 0.96). In CHC patients, serum ALT levels were correlated significantly with TLR2 expression (r = 0.74, p = 0.001) and TNF-α levels (r = 0.53, p = 0.05).

Conclusions: Up regulation of PBMC expression of TLR2 and TLR4 occurs in CHC. Cell signalling via TLR2, in particular, may contribute to both increased circulating TNF-α levels and liver damage in this disorder. These findings provide novel insight into the pathogenesis of CHC.

131 IMPAIRED TOLL-LIKE RECEPTOR EXPRESSION IN CHRONIC HEPATITIS B

K. Visvanathan1, N. Skinner1, J. Kurtovic2, A. Nagree2, S. Locarnini3, R. Williams4, S.M. Riordan2. 1Murdoch Children’s Research Institute, Melbourne, Australia; 2Gastrointestinal and Liver Unit, The Prince of Wales Hospital and University of New South Wales, Sydney, Australia; 3Victorian Infectious Diseases Reference Laboratory and University of Melbourne, Melbourne, Australia; 4Institute of Hepatology, University College London, UK

Background: Mechanisms by which hepatitis B virus (HBV) establishes persistent infection remain unclear. In particular, expression of toll-like receptors (TLRs), increasingly recognised as critically involved in the innate immune response to bacterial and viral pathogens, has not been investigated.

Methods: Eighteen non-cirrhotic patients with chronic hepatitis B and on-going viral replication (HBV DNA > 200 000 genomes/mL, n = 12 and 200–10 000 genomes/mL, n = 6; Cobas Amplicor HBV MonitorTM Test, USA) and 32 healthy control subjects were studied. TLR2 and TLR4 expression on CD14+ve peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry using anti-CD14 (Becton Dickinson) and anti-TLR2 and anti-TLR4 (eBioscience, USA) monoclonal antibodies. TLR expression was re-assessed in 5 patients in whom HBV DNA fell from > 200 000 to < 200 genomes/mL following treatment with lamivudine. In vitro TLR2 expression by PBMCs was measured in 5 control subjects at baseline and following stimulation for 20 h by partially purified recombinant HBV.

Results: TLR2 expression (expressed as a ratio to control results) was significantly reduced in chronic hepatitis B patients with HBV DNA > 200 000 genomes/mL (median: 0.63; range: 0.05–1.52) compared with controls (p = 0.001) and those with HBV DNA 200–10 000 genomes/mL (median: 0.98; range: 0.94–1.17, p = 0.04). TLR4 expression did not differ significantly between the 3 groups. TLR2 expression normalised in each of the 5 lamivudine-treated chronic hepatitis B patients in whom HBV DNA became undetectable. In vitro expression of TLR2 fell in a concentration dependent manner following exposure to recombinant HBV.

Conclusions: HBV downregulates expression of TLR2 on PBMCs. This virus induced defect in innate immunity may contribute to the development of persistent infection.

132 MOLECULAR PROFILES OF PANCREATIC ADENOCARCINOMA

T. Crnogorac-Jurcevic, N.R. Lemoine. Cancer Research UK Molecular Oncology Unit, Imperial College, Hammersmith Hospital, DuCane Road, London, UK

In order to expand our understanding of the molecular changes underlying the complex pathology of pancreatic malignancy, global gene expression profiling of pancreatic adenocarcinoma compared to normal pancreatic tissue was performed. Human cDNA arrays comprising 9932 elements were interrogated with fluorescently labelled normal and adenocarcinoma samples using a reference control design, so that a pool of normal pancreatic tissues was always labelled with direct incorporation of Cy3 dCTP, while test samples (nine tumours, three normal pancreata and three cell lines) were always labelled with Cy5 dCTP. The enrichment with tumour cells was performed by manual trimming of the frozen blocks controlled by frequent microscopical examination of H&E stained sections. This resulted in enrichment of the specimens to at least 80% of tumour cells. The data were analysed for differential gene expression, which was confirmed by serial analysis of gene expression (SAGE), digital differential display (DDD) analysis and immunohistochemistry for selected cases. The array data were filtered to produce a total of 75 genes significantly upregulated or downregulated in pancreatic adenocarcinoma. Two of those showing the highest differential were members of the S100 family of Ca binding proteins (S100P and S100A6) and were selected for additional studies by immunohistochemistry. As neither of them is expressed in normal pancreatic tissues, they could represent potential markers for pancreatic carcinogenesis.

133 P53 DEPENDENT DIFFERENTIAL EXPRESSION ANALYSIS OF ETOPOSIDE INDUCED STRESS IN THE MURINE SMALL INTESTINE

A. Davies, P.D. Ottewell, A.V. McNamara, A.J.M. Watson, J.R. Jenkins. Henry Wellcome Laboratory for Molecular and Cellular Gastroenterology, Department of Medicine, University of Liverpool, UK

Background and Aims: Etoposide (VP16) induced stress in the mouse small intestine displays a dose and time dependent apoptotic response. Apoptosis only occurs in the stem cell compartment of the crypts and is Bax and p21 independent, but p53 dependent. Apoptosis is absent in the villi. We exploited the differential response to VP16 along crypt-villus axis to identify novel p53 dependent genes that are either pro-apoptotic or protective against cell death.

Methods: cDNA array analysis was performed in the p53 +/+ and −/− mouse total epithelium of the small intestine (10 mg/kg VP16 exposure for 4.5 h). Data were analysed using GeneSpring software (Silicon Genetics), which generated a list of >2-fold p53 dependent gene expression changes. Laser capture microdissection (LCM) was used to isolate pure crypt and pure villus epithelial cell populations, and real time PCR (QPCR) performed to detect differential gene expression along the crypt-villus axis. Protein expression was analysed by Western blot and immunohistochemistry techniques.

Results: Presenilin 2 (PS2) and baculoviral IAP repeat 3 (BIRC3) were identified as VP16 induced p53 dependent genes by array analysis. Western blot analysis of total epithelium showed an upregulation of PS2 and BIRC3 protein levels in a p53-dependent manner. QPCR analysis of LCM isolated p53-wildtype crypts and villi revealed a differential gene expression of PS2 along the crypt-villus axis: a decrease of gene expression in the crypts, but an increase in the villus. Our initial histopathological observations support these data. BIRC3 expression was downregulated in both the crypt and villus.

Conclusions: BIRC3 does not appear to regulate apoptosis in intestinal epithelium. The differential mRNA expression of PS2 along the crypt-villus axis may indicate a pro-apoptotic role for low PS2 expression in the crypts, while providing apoptotic protection in the villus following VP16-induced stress.

134 DYSREGUALTION OF MYC NETWORK PROTEINS IN BARRETT’S METAPLASIA

C. Tselepis, N. Sharma, R. Hardy. Division of Medical Sciences, University of Birmingham, Birmingham and Royal Infirmary of Edinburgh, Lauriston Place, Edinburgh, UK

Barrett’s metaplasia is a premalignant lesion predisposing to oesophageal adenocarcinoma. Patients with Barrett’s metaplasia have an approximate 0.5–2% annual risk of developing adenocarcinoma, 30–125 times the risk seen in the general population. Barrett’s metaplasia is associated with severe gastro-oesophageal reflux disease and there is increasing evidence that the reflux of bile acids is likely to be important in malignant development. We have previously demonstrated that c-Myc is upregulated in the malignant progression of Barrett’s metaplasia, and using in vitro cell models shown that bile acids can induce c-Myc. However, because c-Myc alone is unable to transactivate genes associated with mitogenic and oncogenic functions we sought to characterise the other essential c-Myc network proteins, namely Mad-1 and Max in this malignant process.

By use of microarrays on RNA extracted from Barrett’s metaplasia (n = 4) we have identified the c-Myc antagonist Mad-1 as downregulated. These data were verified by quantitative real time PCR and have shown that in 19/20 Barrett’s metaplasia samples there was Mad-1 repression. By Western blotting we were able to demonstrate that repression also occurred at the level of protein. Furthermore, Mad-1 expression was also modulated in cell culture experiments. Thus our data would suggest that in the malignant progression of this disease an imbalance in the c-Myc regulatory system exists, with c-Myc being over-expressed while the c-Myc antagonist Mad-1 repressed. Furthermore, with increasing evidence that components of the refluxate can modulate these proteins this may provide a useful tool for identifying Barrett’s patients at greatest risk of developing adenocarcinoma, leading to an impact on surveillance programmes and patient healthcare.

135 LOW BAT-26 AND BAT-25 POLYMORPHISM (PM) RATES IN A UK POPULATION SAMPLE: ROUTINE COLORECTAL CANCER (CRC) ANALYSIS FOR MSI IS POSSIBLE

M. Lockett, W. Smale., K. Pack, W. Atkin. Cancer Research UK CRC Unit, St Mark’s Hospital, Harrow, Middlesex, UK

Background: Microsatellite instability (MSI) is a change in length of a simple repeat sequence (microsatellite (MS)) in tumour DNA compared to normal. It is a non-specific marker of a mismatch repair defect. It occurs in hereditary non-polyposis CRC (HNPCC) and in a proportion of sporadic CRCs. MSI-CRCs have distinctive clinicopathological features, behaviour, and response to chemotherapy. A panel of five MS markers is recommended for analysis of MSI in tumours, but this requires normal DNA for comparison. The use of mononucleotide markers alone (eg Bat-26) has been suggested as a simple screen for MSI without the need for normal DNA. Germline PMs in mononucleotide markers occur, and without normal DNA, may be misinterpreted as MSI. The PM rates vary according to the population studied (13–18% in African-Americans). The PM rates in the UK are not known.

Aim: To determine rate of germline Bat-26 and Bat-25 PMs in a sample of the UK population.

Methods: A population based sample of germline DNAs (prepared from blood) from 789 individuals from one centre of the UK flexible sigmoidoscopy screening trial was analysed. Blood DNA was amplified at Bat-25 and Bat-26 loci by polymerase chain reaction (PCR) using fluorescent labelled primers. Products were electrophoresed on 5% denaturing polyacrylamide gels on an ABI 377 sequencer and analysed using Genescan and Genotyper software. Two independent observers read the traces. Any DNAs thought to be polymorphic were re-PCRed to confirm.

Results: Results were available in 787. Four (0.5%) were polymorphic at Bat-25, 1 (0.1%) at Bat-26 and 0 at both loci.

Conclusions: There is a low rate of Bat-25 and Bat-26 PMs in this UK sample. If MSI analysis was conducted on tumour DNA without matched normal tissue, about 0.6% would be incorrectly classified as MSI-positive. Bat-26 is the better marker to screen tumours for MSI as it has the lowest PM rate. MSI-positive tumours should be confirmed by comparison to normal DNA, although analysis for MSI at both Bat-25 and Bat-26 is probably sufficient.

136 RELATIONSHIP BETWEEN HELICOBACTER PYLORIE AND COX2 EXPRESSION IN PATIENTS WITH GORD

G.F. Abouda, J.F. Dillon. Department of Molecular and Cellular Pathology, Ninewells Hospital, University of Dundee, UK

Background: The factors involved in the progression of GORD to Barrett’s oesophagus, and then to oesphageal adenocarcinoma are still unclear, however, gastric and bile reflux, cytokines (IL-1B, TNF-α), and inflammatory mediators eg PGs are known to play a role. COX2 activity has been shown to effect cell turnover in oesphageal cancer. Previous studies have also shown that COX2 is induced by H pylori in the stomach.

Aim of Work: To study COX2 expression in patients with reflux symptoms and Barrett’s oesophagus, and correlate this with their helicobacter status.

Method: 53 patients with reflux symptoms, 35 Barrett’s patients and 20 non-reflux control patients were recruited. For purpose of analysis we divided the reflux group into GORD and NERD, 33 and 20 respectively. During an upper GI endoscopy, biopsies were taken from the oesophagus, the gastric body and antrum of each patient. CLO test, ELISA and, histopathology were used to assess the H pylori status. COX2 estimation was carried out by ELISA on the oesophageal and antral biopsies.

Results: COX2 was expressed more in Barrett’s (mean value 412 ng/ul) than in GORD patients (mean value of 312 ng/ul) (p < 0.005) this difference was more evident in patients with dysplasia. COX2 expression was higher in GORD patients than NERD (mean value of 245 ng/ul) (p < 0.005), whereas control group expressed minimal levels of COX2 (mean value of 187.5 ng/ul). When comparing these results with H pylori status, COX2 expression was greater in oesphageal biopsies for both reflux and Barrett’s in H pylori +ve (370 ng/ul, and 450 ng/ul, respectively) than H pylori –ve patients (237 ng/ul and 392 ng/ul, repectivley). In H pylori +ve patients COX2 was expressed at a greater level in the oesphageal biopsies compared with antral biopsies (450 ng/ul vs 240ng/ul), for H pylori –ve patients this trend was less marked.

Conclusion: COX2 is over expressed in the oesophagus, in GORD and Barretts this is exaggerated further by H pylori infection in the stomach, whatever benefit H pylori may have on initiation of GORD it may be detrimental to progression to dysplasia.

137 DYSPHAGIA AND THE 2 WEEK RULE: A HARD ACT TO SWALLOW?

D.L. Morris, A. Ainsworth, C. Leckenby, P.B. McIntyre, S.M. Greenfield. Queen Elizabeth II Hospital, Howlands, Welwyn Garden City, Herts, UK

Background: Guidelines from the DOH guarantee a specialist sees everyone with suspected cancer within 2 weeks of urgent GP referral. Symptom guidelines for suspected upper GI cancers were distributed.

Aims: To evaluate the symptom guidelines for identifying oesophageal cancer. To assess delays in the patient pathway by comparing time from urgent referral to first hospital appointment (endoscopy or outpatients), before and after the guidelines were introduced, and waiting times to first endoscopy in those with benign and malignant dysphagia.

Methods: Patients with oesophageal cancer were identified from PAS and endoscopy, for the 1 year before and after the guidelines were introduced. A sample of age/sex matched patients with benign dysphagia was identified for comparison. Case notes were reviewed.

Results: 44 subjects with oesophageal cancer were identified, 21 before and 23 after guidelines introduced. 8 had ‘curative’ surgery. Symptom guidelines would have missed 3/23 GP referrals presenting with anaemia, haematemesis and weight loss without dyspepsia. Since guidelines, referrals to endoscopy with dysphagia (excluding outpatient visits) increased from 12 to 13 per month, but the benign:malignant ratio reduced from 6.4: to 4.6:1, suggesting better case selection. Since the guidelines, the mean waiting time to first consultant episode for urgent referrals found to have oesophageal cancer has increased from 19 (range 2–46) to 23 days (3–56), the percentage seen within 14 days dropping from 56 to 46%. The wait to first endoscopy has remained stable (33 days range 2–120). Non-urgent referrals with cancer also showed an increase in wait to first consultant episode from mean 27 (4–50) to 36 days (23–46), the percentage seen within 14 days dropping from 50 to 0%. The wait to first endoscopy has increased from mean 27 (4–50) to 65 (3–107) days. Waiting times have also increased for benign dysphagia.

Conclusions: Identifying delays in the patient pathway may improve the service to patients. However, introducing the 2 week rule is unlikely to have a significant effect on outcome, and may delay diagnosis in those with early, asymptomatic disease, who are not referred urgently.

138 SALIVARY FLOW RATE AND BUFFERING CAPACITY: A COMPARISON BETWEEN PATIENTS WITH REFLUX SYMPTOMS AND CONTROLS

R. Moazzez1, A. Anggiansah2, D. Bartlett1. 1Department of Conservative Dentistry (GKT); 2Oesophageal Laboratory, St Thomas’ Hospital, London, UK

Introduction: Heartburn and regurgitation are known to be reliable indicators of gastro-oesophageal reflux (GOR). Saliva has been shown to prevent GOR damage, by diluting and buffering the gastric acid and also by initiating primary peristalsis.

Aim: The aim was to assess salivary flow rate and buffering capacity of stimulated saliva in patients complaining of GOR symptoms compared with a group of controls without GOR symptoms.

Method: 47 patients (mean age 44) referred with GOR symptoms and 30 age matched controls (mean age 41) were studied. Patients and controls were starved for 4 h. Saliva was stimulated by chewing a piece of paraffin wax. Salivary flow rate was calculated as time taken to collect 3 ml of saliva. The salivary buffering capacity was assessed by mixing 1 ml of saliva with a standard buffer solution to analyse the final pH. All saliva was collected mid-morning. All patients then proceeded to have manometry and ambulatory 24 h pH tests.

Results: 57.6% of patients were diagnosed with reflux disease. 65.9% had symptoms associated with reflux and 56% had a symptom index (percentage of symptom associated with reflux) > 50%. The median and range values for salivary buffering capacity were significantly lower for patients than controls (p = 0.0004). There was no significant difference in the salivary flow rate between patients and controls.

Conclusions: Pathological reflux and symptoms associated with GOR were common in this group of patients. The causes of GOR disease and symptoms are multifactorial, however, the poor buffering capacity of saliva may be one of the important factors for the presence of GOR symptoms and disease.

Abstract 138

139 NEUROENDOCRINE TUMOURS OF GUT, LIVER, AND PANCREAS: OVERALL SURVIVAL IN A LARGE COHORT

A.H.G. Davies, A.J. Stangou, N. Jervis, S.J.B. Aylwin, M. Buxton-Thomas, J.K. Ramage. Institute of Liver Studies (Carcinoid Clinic), King’s College Hospital, London; Department of Gastroenterology and Hepatology, North Hampshire Hospital, Basingstoke

Introduction: Neuroendocrine tumours constitute a heterogeneous group of neoplasms, which originate from neuroendocrine cells in the gut, pancreatic islet cells, respiratory epithelium, thyroid, and pituitary glands. They are rare tumours, hence series tend to be small from individual centres. These are slow growing tumours for which many expensive therapies exist. It is important to assess background survival rates to compare to treated groups.

Aim: To determine the 3, 5, and 10 year survival in all the patients followed up in the carcinoid clinics at King’s College and North Hampshire Hospitals.

Method: We carried out a retrospective analysis of the notes and computer database of the carcinoid and neuroendocrine tumour clinics at both hospitals.

Results: There were 212 patients on the database. 49 were excluded due to incomplete data. 163 were analysed with an average age of 54.61 years. There were 79 (48.5%) male and 84 (51.5%) female. There were 19 (11.7%) pancreatic islet cell neuroendocrine tumours, 7 (4.3%) fore gut, 66 (40.5%) midgut, 3 (1.8%) hindgut, 9 (5.5%) lung, 52 (31.9%) unknown primary, and 7 (4.35) from various other sites. The peak age of diagnosis was 50–59 years. The 1, 3, 5, and 10 year survival were 97.97%, 82.5%, 64.5%, and 33.72%, respectively. The mean survival was 5.46 years. The 5 year survival for lung tumours was 100% and for midgut 84%. The worst prognosis was for tumours of unknown origin with 45%, 5 year survival.

Conclusion: Neuroendocrine tumours have a good overall prognosis compared to other gastrointestinal malignancies. Those with lung and midgut primaries showed the best prognosis, whereas those with an undiagnosed primary the worst. Trials of new therapies are rarely randomised but survival data need to be compared with these figures to demonstrate efficacy.

140 THE CO-EXPRESSION OF CYTOKINES AND P-CADHERIN IN INFLAMED BARRETT’S METAPLASIA

E.Cobby1, R. Bryan1, M. Campbell1, R. Harrison1, J. Jankowski 2. 1Epithelial Laboratory, University of Birmingham, UK; 2Digestive Diseases Centre, Leicester, UK

Introduction: Barrett’s metaplasia is a known premalignant lesion associated with a chronic inflammatory cell infiltrate. Recently work from our and other groups have identified some of the important inflammatory cytokines expressed, but their association along the metaplasia-dysplasia-adenocarcinoma sequence has not been fully elucidated. In particular it has been shown that the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) may downregulate E-cadherin (the prime epithelial cell-cell adhesion molecule) expression with consequential effects on differentiation and migration. However, ulceration in established Barrett’s metaplasia is very rare and it has been postulated that upregulation of other cell-cell adhesion molecules may occur to prevent loss of tissue integrity.

Aim: To assess the co-expression of cytokines and P-cadherin (the cadherin most associated with proliferating and migrating cells) along the metaplasia-dysplasia-adenocarcinoma sequence.

Methods and Results: We assessed expression of P-cadherin and interferon gamma (IFNγ) by routine immunocytochemistry and Western blotting on 10 patients from each of the following tissue types: normal oesophagus; reflux oesophagitis; Barrett’s metaplasia; Barrett’s dysplasia; oesophageal adenocarcinoma; lymph node adenocarcinoma. P-cadherin expression was upregulated along the metaplasia-dysplasia-adenocarcinoma sequence being expressed in predominantly proliferating cells. Similarly IFNγ was upregulated in the same tissues and was significantly correlated with P-cadherin expression. IFNγ was expressed in both the inflammatory and epithelial cells.

Conclusions: This data lends further support to the notion that cytokines may regulate cadherin expression in the remodelling of epithelial cells arising in the background of chronic mucosal inflammation. In addition this correlation is currently being tested in functional assays in order to assess the mechanism of cytokine regulation of cadherin transcription and translation.

141 THE PREVALENCE OF UNRECOGNISED ADULT COELIAC DISEASE IN PATIENTS WITH REDUCED BONE MINERAL DENSITY: A CASE FINDING APPROACH

D.S. Sanders, D. Patel, F.B. Khan, R.H. Westbrook, C.V. Webber, A. Milford Ward, A.J. Lobo, E.V. McCloskey. Department of Gastroenterology, Royal Hallamshire Hospital, Sheffield, UK

Introduction: Patients with coeliac disease (CD) may have reduced bone mineral density (BMD). The value of case finding for CD in patients presenting with reduced BMD has not been fully assessed.

Aim: The aim of this study was to determine the prevalence of CD in patients with reduced BMD, verified by Dual energy x ray absorptiometry (DXA).

Methods: All patients attending for a DXA scan (at the Metabolic Bone Unit of the Royal Hallamshire Hospital) were investigated for CD using immunoglobulins, IgG/IgA anti-gliadin antibodies (AGA), and endomysial antibody (EMA). Patients were questioned about any possible coeliac associated diseases or gastrointestinal (GI) symptoms (for example anaemia). Any patient with a positive IgA AGA or a positive EMA or only IgG AGA in the presence of IgA deficiency was offered a small bowel biopsy to confirm the diagnosis of CD.

Results: 978 patients were recruited from January 01 to December 02. Direct questioning revealed that all patients with unrecognised CD had subtle (GI) symptoms or a previous history of anaemia. Excluding patients without these symptoms would give a prevalence of 3.9% for osteoporosis (5/127) and 2.6% for osteopaenia (5/191).

Conclusion: A case finding approach will allow recognition of undiagnosed adult CD in patients with DXA proven reduced BMD. This may avoid delays in diagnosis of CD, a potentially treatable cause of reduced BMD. Initial selection of patients who should be investigated for CD could be based on questioning about GI symptoms or anaemia.

142 SEX HORMONAL STATUS AFFECTS MEAL STIMULATED PLASMA 5-HYDROXYTRYPTAMINE (5-HT) LEVELS IN FEMALE PATIENTS WITH DIARRHOEA PREDOMINANT IRRITABLE BOWEL SYNDROME (D-IBS)

W. Atkinson1, L.A. Houghton1, P.J. Whorwell1, P. Whitaker2. 1Department of Medicine, University Hospital of South Manchester, UK; 2Chem. Pathology, Leicester Royal Infirmary, UK

We have previously shown that 5-HT may play a role in the post-prandial exacerbation of symptoms seen in female patients with d-IBS. As both progesterone (P) and oestrogen (O) can influence the 5-HT system, the aim of this study was to compare plasma 5-HT concentrations in female patients with d-IBS (Rome II) with high and low levels of P/O. Platelet depleted plasma (PDP) 5-HT concentration was therefore assessed for 2 h (60 min intervals) under fasting conditions and 4 h (30 min intervals) after a standard carbohydrate meal (457 kcal) in 39 patients (aged 19–52 yrs) with high P/O (studied either during the luteal phase of the menstrual cycle or while taking combined P/O oral contraception) and in 11 aged matched patients (aged 22–45 years) with low P/O levels (menses). 5-HT concentration was measured by reverse-phase high performance liquid chromatography with fluorimetric detection. In addition, symptomatology (0–4; 4 = severe) was assessed throughout the study.

Results: Under fasting conditions there was no difference in PDP 5-HT concentration between patients with a high (5.41 ng/ml, (mean)) and low (4.98 ng/ml; difference high low (95%CI), 0.43 ng/ml (−0.69,1.55) ng/ml; p = 0.443) P/O status. Following meal ingestion, however, patients with high P/O levels have a significantly higher PDP 5-HT peak (13.99 ng/ml; (geometric mean) than those with low P/O levels (8.57 ng/ml; ratio high:low, 1.63 (1.02,2.62); p = 0.043). This difference was particularly evident in patients reporting post-prandial symptoms (patients with high P/O levels (n = 31): 15.60 ng/ml v patients with low P/O levels (n = 11): 8.57 ng/ml; ratio high:low, 1.82 (1.12, 2.96); p = 0.017) and was not related to a difference in symptom severity between the two groups (patients with high P/O levels: 0.70 (mean) v low P/E levels: 0.58; difference high-low, 0.12 (−0.28, 0.53); p = 0.547.

Conclusions: Female sex hormones may play a role in the gastrointestinal tract 5-HT response to a meal in female patients with d-IBS.

143 CROHN’S DISEASE AND THE NOD2 GENE: A ROLE FOR PANETH CELLS

S.G. Lala1, Y. Ogura2, S.K. Hor1, M. Abeya1, C. Osborne1, S. Davies1, O. Ogunbiyi1 G. Nuńez2, S. Keshav1. 1Department of Medicine, Royal Free & University College Medical School, London, UK; 2Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA

Background and Aims: Mutations in the NOD2 gene predisposing to Crohn’s disease (CD) are predominantly associated with terminal ileal disease. The mechanisms by which these mutations cause ileitis are unknown, as NOD2 is most highly expressed by circulating peripheral blood monocytes, which are ubiquitously distributed throughout the body. Paneth cells are specialised intestinal epithelial cells critically important in enteric antibacterial defence, which respond to bacterial lipopolysaccharide (LPS) through undefined pathways. They are also most numerous in the terminal ileum, and we therefore determined if Paneth cells as well as tissue macrophages in the terminal ileum expressed the NOD2 gene.

Methods: In situ hybridisation and immunohistochemistry were used to determine RNA and protein expression in tissue sections, and quantitative real time RT-PCR was used to measure gene expression in intestinal epithelial cell lines and peripheral blood mononuclear cells.

Results: Paneth cells and monocytes express NOD2 mRNA and protein in CD affected and normal terminal ileum. In Crohn’s ileitis, monocytes but not macrophages located within the lamina propria and granulomas, express NOD2 protein and RNA. Paneth cells, monocytes, and tissue macrophages also express tumour necrosis factor alpha (TNFα) mRNA in CD. In vitro, a decrease in NOD2 expression is seen in monocytes differentiating into macrophages. Caco-2 cells, which display some features of Paneth cell differentiation, constitutively express more NOD2 mRNA than monocyte derived macrophages.

Conclusions: NOD2 expressing-Paneth cells could play a critical and hitherto unrecognised role in the pathogenesis of terminal ileal CD, while the role of the NOD2 gene in mononuclear cells probably depends critically on their differentiation state.

144 HIGHER FREQUENCY OF ZERO TPMT DEFICIENCY: A REVIEW OF TPMT REQUESTS TO A NATIONAL REFERENCE LABORATORY

A. Yim1, A. Holmes2, A. Ansari1, M. Arenas2, E.M. Shobowale2, A. Marinaki2, J.A. Duley2, J.D. Sanderson1. 1Department of Gastroenterology, 2Department of Chemical Pathology, Guy’s & St. Thomas’ Hospitals, London SE1 9RT, UK

Background and Aims: Detection of a zero TPMT activity prior to commencing azathioprine (AZA) avoids the risk of severe and potentially fatal myelosuppression. Zero TPMT activity has been estimated as 1:300 in a Caucasian population. The aim of this study was to define the distribution of TPMT activity among patients referred from throughout the UK to a National Purine Reference laboratory.

Methods: TPMT data were obtained from the first assay in 1990 to present. Where available, data on AZA treatment and referring centre were recorded.

Results: TPMT demand has risen exponentially since 1990. 500/year were perfomed in 1998 and 7500 so far in 2002. Requests from gastroenterologists have increased from 34% to 51% in the same period. From an analysis of 5000 consecutive requests, the frequency for zero TPMT activity was 1:210–220 (0.45%). This figure remained 0.45% when only those who had not started AZA treatment were analysed (n = 1747). The mean and median were 32.3 units. The intermediate activity (between 10–25 units) frequency was 16%. This is also higher than previously reported (11%) although it is known that not all intermediates are carriers. TPMT activity was distributed non-normally, with two “shoulders” to the main curve. At the lower end of TPMT activity (10–20 units), the heterozygote deficient group form a distinct group. At the high end of activity there is reproducible shoulder that maybe a separate genetic entity.

Conclusions: TPMT requests have increased exponentially throughout the UK, especially from gastroenterologists. Zero TPMT frequency is probably higher than predicted from previous studies. This has important cost implications for those seeking financial justification for checking TPMT prior to commencing AZA. At 1 in 220, it would cost £8800 to prevent each episode of potentially fatal myelotoxicity.

145 ASSOCIATION BETWEEN P47PHOX PSEUDOGENES AND INFLAMMATORY BOWEL DISEASE

M. Harbord, A. Hankin, R. Day, S.L. Bloom. Rayne Institute, University College London, London WC1E 6JJ, UK

Chronic bowel inflammation is a feature of chronic granulomatous disease, caused by deficiency in components of the neutrophil and macrophage NADPH oxidase. NCF1, which encodes one such component (p47phox), is found within a region of significant linkage to inflammatory bowel disease (IBD) on chromosome 7. We hypothesised that NCF1 haplo-insufficiency was a susceptibility factor for IBD. We adapted a Genescan method to identify NCF1 heterozygotes, who were expected to have a high p47phox pseudogene (ξNCF1) to NCF1 ratio (ΔGT:GTGT), in 138 patients with IBD and 37 controls. We were surprised to find an excess of IBD patients with a ΔGT:GTGT ratio of approximately 1:1, which was greater in CD patients (CD 22.4%; control 8.1%; Fisher’s exact test p < 0.05, odds ratio 3.3), than in those with ulcerative colitis (UC) (UC 14.1%; p = 0.28). Co-incidentally1, it has been shown that a 1:1 ratio occurs following DNA exchange by recombination or conceivably gene conversion between NCF1 and ξNCF1, to produce a gene hybrid (Type II ξNCF1). The presence of Type II ξNCF1 means that the ΔGT:GTGT ratio cannot be used to assess NCF1 heterozygosity, so we were unable to confirm our original hypothesis. The functional significance of Type II ξNCF1 remained unknown.

Therefore, we assessed the effect of the ΔGT:GTGT ratio on cellular migration into an acute inflammatory cantharidin blister. In patients with a ratio < 1.2 (n = 10; 5 CD, 3 normal subjects), there was a significant increase in blister cell number compared to those patients with a ratio > 1.2 (n = 62; 28 CD, 26 normal subjects), comprising 5.6 +/- 1.5 × 106/mL (mean +/- standard error) and 2.6 +/- 0.4 × 106/ml respectively (Mann-Whitney test p = 0.03). This applied to both neutrophils (p = 0.06) and macrophages (p = 0.19), which were quantified in 46 subjects, including 8 with a ratio < 1.2. It is therefore conceivable that the presence of Type II ξNCF1 increases the inflammatory response and so predisposes to IBD, particularly CD.

  1. Heyworth PG, Noack D, Cross AR. Blood 2002;100:1845–51.

146 CHARACTERISATION OF MUCOSAL ADDRESSIN CELL ADHESION MOLECULE (MAdCAM-1) IN INFLAMMATORY BOWEL DISEASE

A. Ala, M. Stubbs, S. Coward, D. Brown, H.J. Hodgson. Centre for Hepatology, Royal Free Campus, Royal Free & University College Medical School, Rowland Hill Street, London NW3 2PF, UK

Introduction: MAdCAM-1 is an endothelial cell adhesion glycoprotein that regulates α4β7 lymphocyte trafficking. It is upregulated in active inflammatory bowel disease (IBD). No satisfactory in vitro models of MAdCAM-1 expression have been described. In vitro, brain endothelium (bEnd3) is reported to express MAdCAM-1 in response to cytokines. In this study, we used a mouse lymph node endothelial cell line (SVEC4–10), which expresses functional levels of MAdCAM-1 in response to TNF-α and is a more realistic model of high endothelial venules.

Methods: Cells were exposed to TNF-α (20 ng/L) for 0–72 h and incubated with anti-MAdCam-1 antibody, followed by FITC anti-Rat IgG. For quantification of fluoresence, 5 fields were selected for each time point and imaged using a Nikon Coolpix 990 digital camera. Using imaging software the total amount of fluorescence was measured for each field. Mean values were calculated for each time point.

Results: Unstimulated SVEC cells exhibited no staining. After TNF-α there was marked increase in MAdCAM-1 immunostaining especially at cell junctions. Surface expression of MAdCAM-1 increased to 48 h and remained elevated at 72 h. Quantitation of fluorescence revealed that MAdCAM-1 expression was increased by 74% (9 h), 187% (23 h), 443% (48 h) and 505% (72 h). The elevation of MAdCAM-1 at 24 h was confirmed using microwell plate immunofluoresence of cultured SVEC cells. TNF-α treated cells expressed a 326% increase in MAdCAM-1 compared with unstimulated cells. MAdCAM-1 expression in normal and inflamed tissues of the gut was also characterised using immunostaining and RT PCR, showing upregulation in active IBD.

Conclusion: Our studies demonstrate that MAdCAM-1 is upregulated by TNF-α on SVEC 4–10 cells and in inflamed gut tissues. This will provide the basis for investigating the expression and modulation of MAdCAM-1 both in vitro and in human disease.

147 TWO WEEK REFERRALS FOR COLORECTAL CANCER: DOES IT DELAY FINAL DIAGNOSIS?

S. Chaudhri, E. Reay, S. Nair, L. Jones, A. Nath, S. Gonsalves, H. Houng, A.F. Horgan (introduced by N. Thompson). Department of Colorectal Surgery, Freeman Hospital, Newcastle-upon-Tyne

The two week cancer referral rule was introduced to minimise the delay in diagnosis of cancers and introduce a fast track pathway for management of these patients. It has had significant implications for colorectal surgical practice leading to increased pressure on clinic and endoscopy lists. All patients referred with symptoms suggestive of colorectal cancer over a 6 month period were studied. Patients were seen at the outpatient clinic, flexible sigmoidoscopy clinic, or a direct access rectal bleeding clinic (DARC). In addition all referrals sent via the two week guidelines for colorectal cancer during the same period were audited. Methods of referral and utilisation of investigations were analysed, including the waiting times, investigations performed, and outcome. Information was analysed for 709 patients. Of those patients referred via the two week rule (243), 80% were first seen at OPD and 97% underwent complete colonic investigation. Colorectal cancer was diagnosed in 6% of these patients, and all but one cancer would have been within range of a flexible sigmoidoscope. The cancer pick up rate was 6% and median time to histological diagnosis of cancer was 63 days (range 34–155). Referrals to the DARC (210) during the same period led to a diagnosis of CRC in 4.8%, with a median time to diagnosis of (x) days. Of 100 patients with colorectal cancer, 90% would have met the eligibility criteria for 14 day referral. Despite compliance with the two week rule, the diagnosis of colorectal cancer in this group of patients is delayed compared with patients where referral are made directly to flexible sigmoidoscopy clinics. Given that 97% of these patients require complete visualisation of their colon it may be more appropriate to reorganise colorectal services so as to enable a greater number of patients to be assessed directly by means of endoscopic examination.

148 MUTATIONS OF THE HMSH6 GENE AS A POSSIBLE CAUSE OF HEREDITARY NONPOLYPOSIS COLORECTAL CANCER

M. Ponz de Leon, A. Scarselli, P. Benatti, L. Roncucci, G. Ponti, L. Losi, M. Pedroni, F. Borghi, M. Menigatti, C. Di Gregorio.

Departments of Internal Medicine and Morphological Sciences, Università di Modena e Reggio Emilia; Pathology Unit, Ospedale di Carpi (MO), Italy

The large majority of mutations causing hereditary nonpolyposis colorectal cancer (HNPCC) occurs in hMLH1 or hMSH2 genes, but recent observations have shown that other genes can be involved. The aim of this study was to evaluate the role of hMSH6 gene in HNPCC, through an immunohistochemical approach. 28 colorectal cancer patients with clinical diagnosis of HNPCC or suspected HNPCC were selected. Immunohistochemical studies of hMSH6, hMLH1, and hMSH2 were carried out in paraffin embedded tumour samples. Immunoperoxidase staining was carried out with the NEX-ES, Automatic System. Monoclonal antibodies to hMLH1 and hMSH2 proteins were used at 1:40 dilution; monoclonal antibody to hMSH6 protein was used at 1:2000 dilution. Each tumour sample was tested for microsatellite instability (MSI). Immunohistochemical expression of hMSH6 lacked in 7 of 28 tumours. In 4 cases, absence of hMSH2 expression was associated. The patients (mean age 56.6 years) were all affected by right-sided colon cancer, frequently mucinous and MSI+. Three patients were from HNPCC families fulfilling Amsterdam Criteria I or II. In 2 cases Muir-Torre syndrome (MTS) could be diagnosed, whereas the other 2 were suspected HNPCC. In almost all the pedigrees, an excess of extracolonic tumours was observed: interestingly, in both MTS patients, colorectal cancers and sebaceous dermatologic lesions showed the same immunohistochemical pattern. The complete gene sequencing was carried out in 3 of 7 patients: two hMSH6 and one hMSH2 frameshift mutations leading to a stop codon with a truncated protein were detected. In conclusion, hMSH6 mutations could characterise a subset of families with hereditary colorectal cancer. Altered hMSH6 expression (even if often associated with lack of hMSH2 protein), MSI+, proximal localisation, later age at diagnosis, and the association with extracolonic tumours, seem to be features of suspicion for the presence of hMSH6 mutations.

149 2300 WEEKS OF HOME PARENTERAL NUTRITION IN A DISTRICT GENERAL HOSPITAL

D.A. Freshwater, A. Saadeddin, B.J.M. Jones. Dudley Group of Hospitals NHS Trust

Background: Home Parenteral Nutrition (HPN) is accepted in the treatment of intestinal failure but is mostly restricted to a few large specialist centres in the UK.

Methods: Adult HPN patients at a single district general hospital (Dudley Group of Hospitals NHS Trust) were analysed by indications, complications and outcome.

Results: 2310 patient weeks of HPN were provided to 23 patients, aged 18–80 years with intestinal failure (Crohn’s disease 8, small bowel infarction 5, other GI disease 3, radiation enteritis 2, colonic carcinoma 2, stricture 2, cancer small bowel 1, ulcerative colitis 1, pseudo-obstruction 1, gastric cancer 1, volvulus 1) N.B. some patients had more than one underlying diagnosis. 28% were subsequently able to discontinue HPN. 46% of patients died while on HPN but cause of death was not related to HPN. 82% had a Karnowsky Index of 60 (generally self-caring) or greater. HPN complications for 22 of the 23 patients were 14 confirmed line infections with a total of 33 suspected line infections being recorded, 10 line occlusions and 6 line breakages. There was a proven catheter sepsis rate of 1 catheter sepsis per 3.17 years with an overall suspected catheter sepsis rate of 1 suspected infection per 1.35 patient years. The overall proven line complication rate was 1 complication per 1.48 patient years. One patient was excluded from complication analysis as he was unable to grasp aseptic technique.

Conclusions: HPN can be practised at DGH level achieving complication rates broadly comparable to large specialist centres, and this lends weight to the argument of using a “hub and spoke” model to widen provision of HPN beyond large specialist centres.

Oesophagus posters 150–175

150 A NEW CONCEPT: FUNCTIONAL OESOPHAGEAL MAPPING

B.S.F. Stacey, P. Patel.

Southampton General Hospital, Tremona Rd, Southampton SO16 6YD, UK

Introduction: Advances in computer software and acquired image quality have meant that new and more sensitive means of interpreting scintigraphy data are possible. Described here is the creation of a three dimensional construction of the function of the oesophagus throughout its length, with time as the fourth variable. This is not an anatomical model. It provides a visual image indicating the site of hold up or delayed transit over the length of the oesophagus and is quantifiable—ie the ratio of the diameter of the cylinder in two scans reflects the change in rate of transit.

Methods: The distance travelled by the mode of the scintigraphic count on each acquisition frame is measured by density analysis. This is performed using Scion Image—an image density analysis programme from ther NIH. This series is then plotted against time to produce a curve. An equation for the graph can be generated by computer. The differential of this curve gives its gradient and this, superimposed along the length of a cylinder, gives a functional map of the oesophagus. The model shown is a normal subject with physiological holdup at the striated/smooth muscle junction.

Summary: This technique can be used to assess dysmotility and stricture and gives quantitative data for follow up scans.

151 PREVALENCE OF HELICOBACTER PYLORI VIRULENCE FACTORS IN PATIENTS WITH REFLUX OESOPHAGITIS AND BARRETT’S OESOPHAGUS

G.F. Abouda, J.C. Cotton, J.F. Dillon. Department of Molecular and Cellular Pathology, Ninewells Hospital, University of Dundee, UK

Background:Helicobacter pylori (Hp) is a microaerophilic spiral rod, which is associated with gastritis, duodenitis and gastric carcinoma. Its role in GORD is unclear, recent studies have suggested a protective role of a virulent strain against the development of GORD.

Aim of work: To evaluate the prevalence of this virulence factor in patients with oesophagitis and Barrett’s oesophagus.

Method: 67 patients with reflux oesopahgitis, 60 patients with Barrett’s oesophagus, and 25 non reflux patients (control group) underwent upper GIT endoscopy. 4 biopsies were taken from each patient, 2 from the oesophagus, 1 from the body of the stomach, and 1 from the antrum. Clo test, ELISA for Hp IgG, Western Blot for Cag, Vac, and HSP 60 of Hp, and histopathological grading of the severity, was performed on each patient.

Results: 21(31.3%) of reflux patients were CLO positive, 18(30%) of Barrett’s patients were Clo positive, and 5 (20%) of the control group were CLO positive. The Cag and Vac strain was +ve in 22 patients with reflux and 15 patients with Barrett’s. HSP 60 was +ve in 25 (37%) patients with reflux oesophagitis and 19 patients with Barrett’s. 4 patients exhibited high grade dysplasia, and were negative for all strains except HSP60. IgG ELISA was positive in 35 (52.2%) of the patients with reflux and in 23(38%) of the Barrett’s patients. The non reflux group (control group) were all Cag, Vac, and HSP 60 negative. When combining the histological grading and serological tests, patients were divided into active, past or never, according to their status of Hp infection. 22 of the GORD group and 18 of Barrett’s patients were actively infected, of which 87% of GORD patients and 57% of Barrett’s patients were positive for Cag, Vac, and HSP60.

Conclusion: 70% of patients presenting with symptoms of reflux or Barrett’s are Hp negative, of those 34% had a history of previous eradication treatment. 80% of control patients were Hp negative. In the Hp positive group, the virulent strains, in particular HSP 60, seem to predominate and appear to be associated with dysplasia.

152 LENGTH OF BARRETT’S OESOPHAGUS SEGMENT: DEMOGRAPHIC ASSOCIATIONS AND CANCER RISK

P.A.C. Gatenby1, C.P.J. Caygill1, A. Charlett2, R. Fitzgerald1, A. Watson1.

1On behalf of UK National Barrett’s Oesophagus Registry (UKBOR), University Department of Surgery, Royal Free Hospital, London NW3 2PF, UK; 2PHLS Statistics Unit, London NW9 5EQ, UK

Introduction: Studies have suggested a higher incidence of adenocarcinoma (AC) in longer Barrett’s oesophagus (BO) segments, but this has not been stratified. Although AC has been described in short BO segments ≤3cm (SSB), its incidence is controversial. The influence of age, gender, smoking, alcohol, and BMI on the development of BO has been studied in small series, but not their influence on segment length.

Methods: Medical records of 1000 BO patients from 5 hospitals registered with UKBOR were examined. Data were extracted on age, gender, BMI, tobacco, and alcohol use, and length of BO segment at BO diagnosis. Data on AC development were also abstracted. Segment lengths were categorised as SSB, >3 ≤6cm and >6cm. The relationships between demographic parameters and segment length, and segment length and AC development were determined, both for overall cancer risk and true incident cancers (occurring >1 year after BO diagnosis).

Results: Histology and segment length were available in 625 records. There was a small, non-significant increase in BO length with age, but no correlation between gender, BMI, tobacco and alcohol consumption and segment length. The distribution of the 28 overall and 9 incident ACs according to segment length is shown in the table.

Abstract 152

Conclusions: The risk of both overall and incident cancers is greater for SSB than for segments >3 ≤6cm in length, but the greatest risk is for length >6cm (Pearson χ2 p=0.02). Whilst demographic factors have previously shown an influence on the risks of developing BO, there is little correlation with the length of segment which develops.

153 NOVEL MECHANISM OF NITROSATIVE STRESS FROM DIETARY NITRATE RELEVANT TO GASTROESOPHAGEAL JUNCTION CANCER

K. Iijima1, J. Grant, K. McElroy, S. Anderson, V. Fyfe, S. Paterson, T. Preston, K.E.L. McColl.

1Dept of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan; Dept of Medicine & Therapeutics, Western Infirmary, Glasgow, UK

Abstract: High concentrations of nitric oxide are generated at the gastro-oesophageal (GO) junction due to the reduction of salivary nitrite to nitric oxide by acidic gastric juice containing ascorbic acid. Salivary nitrite is derived from the enterosalivary recirculation of dietary nitrate.

Aims: To determine whether nitric oxide generated in the above way will exert nitrosative stress on the adjacent epithelium.

Methods: A benchtop model was constructed reproducing the chemistry occurring at the GO junction and incorporating an epithelial compartment maintained at pH 7.4 separated from the lumen by a thin hydrophobic barrier. The secondary amine morpholine was added to each compartment and N-nitrosomorpholine formation at 15 min measured.

Results: Adding 100μM nitrite to the acidic (pH 1.5) luminal compartment in the absence of ascorbic acid generated 6.2±2.0 (mean±SE) N-nitrosomorpholine in that compartment and 2.2±0.1μM in the epithelial compartment. When 100μM nitrite was added to the acidic luminal compartment (pH 1.5) containing ascorbic acid, all the nitrite was immediately converted to nitric oxide and no N-nitrosomorpholine was formed within that compartment. However, the nitric oxide rapidly diffused into the adjacent epithelial compartment (pH 7.4) where it generated very high concentrations of N-nitrosomorpholine (137±5.6μM). The addition of ascorbic acid or glutathione to the epithelial compartment only reduced this nitric oxide induced nitrosation within the epithelial compartment by 40%.

Conclusion: Ascorbic acid in gastric juice prevents acid-catalysed nitrosation within the gastric lumen. However, in doing so it generates nitric oxide which exerts a far higher nitrosative stress on the adjacent epithelium. This mechanism may be relevant to the aetiology of mutagenesis at the GO junction.

154 THE ADVANTAGES OF HIGH RESOLUTION OESOPHAGEAL MANOMETRY (HRM) IN CLINICAL PRACTICE

M. Fox1, G. Hebbard2, J. Brasseur3, W. Schwizer1 (introduced by A. Harris).

1University Hospital Zürich, Switzerland; 2Royal Melbourne Hospital, Australia; 3Pennsylvannia State University, USA

Background: Conventional manometry (CM) with 5–8 oesophageal pressure channels and a lower oesophageal sphincter (LOS) sleeve sensor is limited by poor resolution. HRM techniques with 21–32 pressure channels may increase diagnostic accuracy.

Aim: To analyse quantitatively whether HRM improves the assessment of motility compared to CM, and to determine qualitatively the situations in which HRM provides clinically important information not obtained by conventional investigation including CM.

Method: Control subjects and patients with dysphagia underwent HRM. 95 records were reviewed independently by two blinded physicians using both limited CM (pull-through plus 6 recording sites) and HRM analysis (all 32 sites). Further HRM records from dysphagic patients with non-diagnostic endoscopy and radiology were compared to the limited CM analysis to identify the additional information leading to positive diagnosis provided by HRM.

Findings: Receiver-operating curve (ROC) analysis revealed for HRM at a sensitivity level of 90% a specificity of 100%, whereas for CM a specificity of 89% was associated with a sensitivity of 70%. Qualitative analysis demonstrated that HRM provided positive diagnoses in cases where conventional tests including CM were non-diagnostic. Advantages included: (i) detection of localised disturbances of peristalsis (eg sub-mucosal leiomyoma); (ii) ability to locate and follow LOS movement during esophageal spasm without loss of detail (eg vigorous achalasia with pseudo-relaxation); and (iii) detailed analyses of LOS pressure topography (eg dysfunction post-fundoplication).

Conclusion: HRM is more accurate than CM and identifies clinically important peristaltic and LOS dysfunction not detected by conventional investigations including CM.

155 QUALITY OF LIFE ASSESSMENT IN PATIENTS UNDERGOING ENDOSCOPIC MUCOSAL ABLATION FOR DYSPLASTIC BARRETT’S OESOPHAGUS

K. Ragunath, N. Krasner, V.S. Raman, M.T. Haqqani, W.Y. Cheung

University Hospital Aintree, Liverpool, University of Wales, Swansea, UK

Background: A number of thermal and non-thermal modalities have been applied in an effort to eradicate dysplasia and reverse the Barrett’s epithelium. Quality of life (QOL) assessment is an important outcome measure, since these interventions result in procedure related morbidity.

Aim: To assess QOL measurement in a cohort of patients with dysplastic Barrett’s oesophagus undergoing two different endoscopic mucosal ablation modalities as part of a randomised clinical trial.

Methods: Twenty-nine patients with dysplastic Barrett’s oesophagus underwent mucosal ablation: Photodynamic therapy (PDT) with Photofrin was performed in one session-13 patients and Argon Plasma Coagulation (APC) was performed in 1-6 (mean 3) sessions-16 patients. Treatment and disease specific QOL questionnaire was constructed based on previous validated Gastrointestinal QOL tool. A 10-item questionnaire was constructed with 5 responses in each question. Most desirable option: 5 points, and least desirable option: 1 point (maximum: 50, minimum: 10). Patients were asked to fill the questionnaire immediately before the procedure and 2 weeks after the procedure (in the case of APC after the first session). The scores before and after treatment were compared using the paired t test and the difference between the two treatment groups was analysed using the unpaired t test.

Results: All but 3 patients in the APC group completed the study. In the PDT group the mean pre-treatment QOL score was 45 and post treatment QOL score was 39, p=0.0002 (95% CI 3.54–8.76). In the APC group the mean pre-treatment score was 39 and post-treatment score was 33, p=0.0008 (95% CI 2.80–8.27). There was no significant difference in QOL pre and post-treatment between the two groups.

Conclusion: QOL is significantly affected in patients undergoing endoscopic mucosal ablation therapy for at least 2 weeks following the procedure. Future studies comparing various endoscopic mucosal ablation modalities should assess QOL as well, when comparing the outcomes of the endoscopic intervention.

156 EXPRESSION OF THE CDX2 HOMEOBOX PROTEIN IN BARRETT’S METAPLASIA (BM)

N. Kapoor1, M. Hulbert2, M. Haqquani2, L.G. Yu3, K. Bodger1,3.

1Aintree Centre for Gastroenterology; 2Dept of Pathology, University Hospital Aintree, Liverpool, UK; 3Dept of Medicine, University of Liverpool, UK

Background: Cdx2 is an intestinal transcription factor that is a key regulator of development and homeostasis of intestinal epithelium. In the adult, Cdx2 expression is confined to the small and large intestine. Neo-expression occurs in gastric intestinal metaplasia and ectopic gastric expression of Cdx2 leads to an intestinalised mucosa in transgenic mice. Preliminary reports suggest that Cdx2 is expressed in BM. We aimed to examine Cdx2 expression within various histological sub-types of BM and during malignant progression.

Methods: With informed consent, 68 oesophageal surveillance biopsies from 52 patients with BM were studied. Formalin-fixed, paraffin-embedded sections were subject to immunohistochemistry using an anti-Cdx2 mAb (BioGenex) and an automated avidin-biotin-based system. Control sections comprised normal colon and duodenum. Both H&E and mucin staining (Gomori’s aldehyde fushcin) were employed in serial sections to identify and type foci of metaplastic epithelium. Nuclear immunostaining was scored semi-quantitatively (0-absent; 1-focally absent or weak; 2-strongly positive). We also screened confluent TE7 cells (oesophageal adenocarcinoma) for Cdx2 expression by immunoblotting of cell lysates (CACO-2 cells were employed as positive control).

Results: All examples of squamous mucosa, cardiac/fundic-type glands and deep oesophageal glands were negative for Cdx2 expression. Staining intensity within intestinal-type mucosa and dysplasia is shown in the table. No Cdx2 expression was identified in immunoblots of TE7 adenocarcinoma cells.

Conclusions: Neo-expression of Cdx2 is almost invariable in SIM but does not occur in gastric-type variants of Barrett’s oesophagus. Down-regulation of Cdx2 may occur during oesophageal malignant progression, consistent with a tumour suppressor function. The molecular mechanisms regulating Cdx2 expression during oesophageal carcinogenesis require further study.

Abstract 156

Abstract 157

157 EFFICACY AND SAFETY OF PALLIATIVE STENTING FOR MALIGNANT DYSPHAGIA: A REVIEW OF 120 PATIENTS

S. Subramanian, K.R. Hine.

Princess Royal Hospital, Haywards Heath RH16 4EX, UK

Background: Oesophageal carcinoma is a largely incurable disease. The main aim of palliation is to restore oesophageal patency. Current oesophageal endoprostheses comprise semirigid plastic stents and self-expandable metal stents (SEMS). In recent years, prospective randomised controlled trials have demonstrated a higher efficacy and a lower complication and mortality rate (0 v 15.8%) for SEMS v plastic stents.

Aims: We reviewed our experience with the efficacy, safety and complications with stents placed for palliation of malignant dysphagia.

Materials and Methods: The case notes of all patients that had a stent inserted between 1985 and 2002 were reviewed. Data including demographics, type of stent, dysphagia score prior to after implantation of stent, complications and survival were recorded on a database.

Results: A total of 120 stents were implanted during the study period. We used the conventional Atkinson plastic prosthesis and four different types of SEMS: Z-stent (N=22), In-stent (N=21), Uncovered wall stent (N=5) and Choo stent (N=4). Complications were divided into immediate (<1 week) and delayed (>1 week), following stent insertion. Immediate complications for all stents in our series included chest pain – 1(0.8%), failure to deploy stent – 1 (0.8%), perforation – 8 (6.7%), stent displacement – 4 (3.3%) and death – 1 (0.8%), resulting from the procedure. Delayed complications for all stents included stent displacement – 10 (8.3%), recurrent dysphagia – 10 (8.3%), food impaction – 3 (2.5%), broncho-oesophageal fistula formation – 4 (3.3%), bleeding – 5 (4.2%) and tumour ingrowth/ overgrowth – 4 (3.3%).

Conclusions: Both immediate and delayed complication rates were higher in the plastic endoprostheses group. The complication rates are similar to previously reported series. In conclusion, SEMS are a safer and efficacious mode of palliation for oesophageal carcinoma. However, there is no added survival benefit in this group.

158 BARRETT’S OESOPHAGUS–IS SURVEILLANCE REQUIRED?

S. Gupta, C. Fernandez, A. Arnaout, A. Theodossi, M.A. Mendall.

Department of Gastroenterology and Pathology, Mayday University Hospital, Croydon, Surrey, UK

Introduction: Controversy exists about the utility of a screening endoscopy programme and the value of proton pump inhibitors (PPI) in preventing the progression from metaplasia to dysplasia to cancer in Barrett’s oesophagus (BO). Earlier studies to determine the risk of oesophageal adenocarcinoma in BO were conducted largely before the era of widespread usage of PPIs. We present a long term follow up study of subjects with BO, a high proportion of whom were on PPIs.

Aims and Methods: The aim was to determine the efficacy of screening to detect oesophageal cancer in patients with BO in the context of high rates of PPI usage. All cases of BO diagnosed at our hospital from 1990 to 1998 were identified from a histopathology database. Case notes were retrieved and a database prepared. Patients who died during follow up had the cause of death ascertained from the Department of Public Health, Registry of Births and Deaths and Office of National Statistics. PPI usage was recorded at each follow up visit.

Results: A total of 308 patients were identified of whom 35 patients did not have intestinal metaplasia. 19 files could not be traced. Of the 273 subjects with intestinal metaplasia, 179 (65.6%) were male. Total follow up till October 31 2000 was 1145 patient years (1110 years with the untraceable files excluded). Mean age of diagnosis was 66.7±14.1 years (range 21–94 years). 136/156 (87.2%) patients on first follow up endoscopy, 74/79 (93.7%) patients on second follow up endoscopy and 28/32 (87.5%) patients on third follow up endoscopy were on PPIs. 12 (4%) oesophageal/cardia adenocarcinomas were detected of which 9 were detected on first endoscopy. 3 cases were suspicious of at initial endoscopy. One of these was confirmed as adenocarcinoma 1 month and the other two 3 months later. Two of the 11 patients had high grade dysplasia. One of these was referred for treatment and is still alive while the other died of an unrelated cancer. 84 patients died during the period of observation. Of these 10 died of oesophageal/gastric cancer.

Conclusion: In the era of PPI therapy, rate of malignant transformation is low and it questions the need for a intensive surveillance endoscopy programme.

Abstract 159

PPR per Meals per Quarters (q)

159 WHEN SHOULD ANTACIDS BE INGESTED AFTER MEALS?

R. Anggiansah, A. Chandra, A. Anggiansah, W.J. Owen.

Dept of Surgery, St Thomas’ Hospital, London, UK

Introduction: Post-prandial reflux (PPR) is a significant component of gastro-oesophageal reflux (GOR) in health and disease. PPR is generally analysed within 2 hours after meals. Various studies have attempted to modify PPR to determine the effectiveness of intervention. However, there is little documentation regarding when maximal PPR occurs.

Aim: This study examined the PPR profile of pathological GOR patients.

Patients and Methods: A sample of 55 patients (32 males, mean age 50.3 years) was chosen randomly from those with pH-documented GOR disease who had attended the unit in 2002. During 24 hr pH monitoring (Synectics), patients were advised to go about their normal activities and had their usual meals but avoiding food or drink with pH <5. GOR was defined as pH <4. The two-hour PPR periods were divided into 30 min quarters. Kruskal-Wallis and Mann-Whitney statistical tests were used.

Results: There were no significant differences in PPR comparing meals (p=0.92). However, there were significant differences in PPR quarters for each meal (p≤0.03). The greatest acid exposure after breakfast was in the 2nd quarter, which was significantly higher than the 1st (p=0.003) and the 4th quarters (p=0.002). After lunch and dinner, the highest GOR occurred in the 3rd quarter, which in both was significantly higher than the 1st quarter (p≤0.03) only.

Conclusion: PPR is a major source of GOR and in this group of patients up to 74.4% of the post-prandial period demonstrated acid exposure. Maximal PPR occurs in the 2nd or 3rd quarter of the post-prandial period. For maximal effect patients with pathological GOR should ingest antacids half an hour after meals.

Abstract 162

160 COST-EFFECTIVENESS ANALYSIS OF PROTON PUMP INHIBITOR STRATEGIES IN THE MAINTENANCE OF HEALED REFLUX OESOPHAGITIS

J.M. Plumb, S.J. Edwards (introduced by P.D.I. Richardson).

AstraZeneca UK Ltd, 600 Capability Green, Luton LU1 3LU, UK

Objective: To assess the cost-effectiveness of combined high and low dose PPI strategies using UK licensed dose for the maintenance of healed reflux oesophagitis over 12 months from the perspective of the UK NHS.

Methods: A decision analysis model was constructed to depict the sequential management of the maintenance of healed reflux oesophagitis, based on a systematic review of remission rate data at 6 and 12 months. Patients relapsing at 6 months followed a healing strategy identified in a survey of UK general practitioners and gastroenterologists. Treatment strategies, in terms of high and low dose PPIs prescribed, were based on UK prescribing patterns. Resource units were multiplied by national published resource unit costs at 2000/01 prices. Sensitivity analyses were conducted to assess the robustness of the model.

Outcome Measure: The measure of clinical effectiveness was, “the proportion of patients relapse-free for 12 months”. Relapse was defined as endoscopic evidence of oesophagitis and/or symptomatic relapse.

Results: Esomeprazole dominates all other PPI strategies (ie it is more effective and less costly). In terms of cost alone, the mean cost per patient treated for the esomeprazole strategy is numerically less than the lansoprazole strategy but would be considered cost-neutral (£276.61 v £279.98). However, the additional effectiveness of the esomeprazole strategy compared to the lansoprazole strategy (0.763 v 0.738) results in a lower mean cost per relapse-free patient for 12 months (£362.53 v £379.38). A sensitivity analysis indicated that the results were relatively robust to changes in key model parameters.

Conclusion: Esomeprazole is the most cost-effective strategy for the maintenance of healed reflux oesophagitis over 12 months.

161 IMPAIRMENT OF TGFβ SIGNALLING IN BARRETT’S ASSOCIATED OESOPHAGEAL ADENOCARCINOMA: ROLE OF SMAD4

B.A. Onwuegbusi, R.C. Fitzgerald.

Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge CB2 2XZ, UK

Introduction: The accumulation of somatic mutations during the Barrett’s metaplasia-dysplasia-adenocarcinoma sequence leads to uncontrolled epithelial cell proliferation. Since transforming growth factor β (TGFβ) is a potent anti-proliferative agent, and signalling mutations frequently occur in gastrointestinal malignancies, we hypothesise that alterations in this pathway may be important in oesophageal adenocarcinoma development.

Methods: Western blot, RT-PCR and immunohistochemistry were used to analyse the expression of TGFβ receptors I and II, and Smad2, 3 and 4 in oesophageal samples from normal squamous mucosa (n=20), Barrett’s oesophagus (BE) without dysplasia (n=20), BE with low-grade dysplasia (n=10), BE with high-grade dysplasia (n=10) and BE adenocarcinoma (n=20). TGFβ responsiveness, TGFβ receptor and Smad expression of a panel of oesophageal cell lines were also determined, and mutational analysis was performed by PCR and sequencing.

Results: There was a significant decrease in the mRNA expression of Smad2 (p<0.001), Smad3 (p<0.001) and Smad4 (p<0.002) in BE samples, and in high-grade dysplasia samples (Smad2 p<0.001; Smad3 p<0.01; Smad4 p<0.001) when compared to squamous epithelium. Smad4 mRNA was also significantly decreased in adenocarcinoma samples compared to squamous epithelium (p<0.05). A shift in protein mobility was seen for Smad4 in 25% of adenocarcinoma samples analysed by Western blot. OE21 cells (squamous carcinoma), OE33 and SEG-1 (BE adenocarcinoma) were responsive to TGFβ. BIC-1 and TE7 (BE adenocarcinoma) were unresponsive to TGFβ, as were KYSE-30 (squamous carcinoma). BIC-1 did not express Smad4 mRNA and protein, due to a base pair substitution in exon 9 of the Smad4 gene. TGFβ receptor and Smad mRNA and protein were expressed in these cells, and no mutations could be identified in exon 3, 5, or 7 of the TβRII gene.

Conclusion: The TGFβ signalling pathway is impaired in Barrett’s associated adenocarcinoma and the alteration of Smad4 expression may be an important factor in neoplastic progression.

162 COMPARING VOLUME CLEARANCE AND ACID CLEARANCE IN OESOPHAGEAL ACID REFLUX EVENTS

A. Chandra, R. Anggiansah, A. Anggiansah, W.J. Owen.

Department of Surgery, Guy’s & St. Thomas’ Hospital, London

Introduction: Manometry can detect peristalsis while pH sensors can show acid clearance. Flow and volume clearance can be detected by changes in impedance pairs of electrodes. This ambulatory study uses pressure and impedance to determine on a temporal basis the motility patterns during a reflux event, in order to investigate the role of peristalsis in acid and volume clearance.

Subjects and Methods: Recordings from 3 control and 13 patients (symptoms of gastro-oesophageal reflux) were analysed. All underwent 24-hour ambulatory oesophageal study. The combined catheter consisted of 4 pressure transducers at 5, 10, 15, and 28 cm proximal to the manometrically defined lower oesophageal sphincter (LOS). Two impedance electrode pairs were placed at 5 and 10 cm. A pH sensor on a separately bound catheter was sited at 5cm. A RE occurred when distal oesophageal pH was <4, (>4s), Acid clearance was defined by a return of pH >4. Pharyngeal activity determined primary or secondary activity. This was divided into peristalsis and others (e.g. simultaneous, non-transmitted, reverse, isolated). An impedance RE was defined by a 33% decrease in baseline values (>2s), when pH <4. Subsequent volume clearance was defined as a return to impedance baseline.

Results: There were a total of 775 REs. Of these 527 (68%) had an associated impedance event. Of these, primary peristalsis has a significantly larger role (p=0.0001) in volume clearance 276/527 (52%) than in acid clearance 322/775 (42%). There were no statistical differences between controls or patients.

Conclusions: Ambulatory impedance has a high concordance with reflux events (68%). This study shows that primary peristalsis has a significantly larger role in volume clearance than in acid clearance.

163 IMMEDIATE AND EARLY GENE RESPONSE TO IN VITRO ACID EXPOSURE IN A BARRETT’S ADENOCARCINOMA CELL LINE

C. Morgan, W. Alazawi, P. Sirieix, T. Freeman1, N. Coleman, R. Fitzgerald.

Cancer Cell Unit, Research Centre, Hutchison/ MRC, Hills Road, Cambridge, UK; 1MRC, Human Genome Mapping Project, Hinxton, Cambridge, UK

Introduction: Acid, a principal component of refluxate, may contribute to the neoplastic progression of Barrett’s oesophagus. Previously published data have demonstrated that brief acid exposure in vivo and in vitro increases cell proliferation. The mechanisms underlying the hyperproliferative response are not well elucidated but may include alterations in Na/H exchanger activity and MAPK signalling pathways.

Aim: To ascertain the effects of acid exposure on gene expression in a Barrett’s adenocarcinoma cell line (SEG-1) using expression micro arrays and RT-PCR.

Methods: SEG-1 cells were grown to 60% confluency and exposed to either acidified DMEM at pH 3.5 (0.1M HCl) or pH 7.4 (control) for 20 minutes followed by neutralisation of the medium for up to 10 hours. Total RNA was extracted before acid exposure and over a 10 hour time course (0.5, 2, 4, 6, 8, and 10h) and hybridised to an Affymetrix human U133A oligonucleotide array. Data were analysed using the Affymetrix statistical expression algorithms. Only alterations in gene expression >2, <-2 were taken as significant and a subset of interest were validated by RT-PCR.

Results: An up-regulation of genes associated with proliferation (PCNA, FGFR3 and VEGFC) and a down-regulation of genes associated with apoptosis (caspase-9, GADD45A) were shown throughout all time points. DUSP2 and 8, which are involved in the inactivation of the MAPK pathway were down regulated throughout the time course. At specific time points the following cell cycle regulatory genes were significantly altered: E2F (up at 0.5h), Rb binding protein 2 homolog 1(down at 0.5, 2h), Cyclin E2 (up at 2, 6, 8h) Cyclin E1 (up at 4, 6h) and Cyclins D1 and A1 (up at 8h). Cyclo-oxygenases were unaffected.

Conclusion: Suppression of apoptosis via the p53 pathway, stimulation of proliferation via the MAPK pathway and alterations in cell cycle components may be involved in the proliferative response to an acid pulse. This study provides candidate genes for further studies on cell responses to acid.

164 OPTIMISATION OF AN ENDOSCOPIC BIOPSY PROTOCOL IN LONG SEGMENT BARRETT’S OESOPHAGUS: EVIDENCE FROM 125 CONSECUTIVE CASES

I. Perry, R.F. Harrison, R.T. Bryan, W. Haddadin, P. Taniere, N.A. Shepherd, J.A.Z. Jankowski.

University Hospital Birmingham; Digestive Diseases Centre, Leicester; Histopathology Unit Gloucester Royal Hospital

Background: Intestinal Metaplasia within a Columnar Lined Oesophagus (Barrett’s Oesophagus) is believed to be pre-malignant. At present it is not clear how best to diagnose this condition reliably when encountered at endoscopy.

Objectives: We aimed to assess the frequency and distribution of intestinal metaplasia in 125 consecutive patients with endoscopically observed long segment Barrett’s metaplasia to determine the optimal protocol for OGD and biopsy. In addition we assessed whether additional PAS histological staining would improve the diagnostic yield.

Methods: A total of 1646 individual biopsies from 296 endoscopies performed on 125 patients were examined.

Results: Intestinal metaplasia was demonstrated in 80/125 (64%) patients and 150/296 (51%) endoscopies, although only 557/1646 (34%) individual biopsies contained foci of intestinal metaplasia. Of 296 endoscopies, 166 contained cardiac mucosa, 158 fundic mucosa, 64 glandular mucosa of no special type and 111 squamous mucosa. The highest rate of intestinal metaplasia detection occurred when 8 biopsies per endoscopy were taken. However, there was a notable increase in the pick-up rate of intestinal metaplasia moving from 4 biopsies per endoscopy to 5 biopsies per endoscopy. Performing multiple endoscopies for individual patients resulted in only a modest increase in detection of intestinal metaplasia, with no new cases of intestinal metaplasia being diagnosed after the third endoscopy. While PAS staining identified only an extra 5.4% of new cases of intestinal metaplasia, it identified many more positive biopsies in those who were already known to have intestinal metaplasia.

Conclusions: We recommend taking between 5 and 8 biopsies per endoscopy to reliably determine if a patient has intestinal metaplasia. Conventional H&E staining seems to be the mainstay of diagnosis.

165 ENDOSCOPIC DILATATION OF OESOPHAGEAL STRICTURES IN EPIDERMOLYSIS BULLOSA

S.H.C. Anderson, L. Doig, J. Meenan.

Department of Gastroenterology, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK

Background: Epidermolysis bullosa (EB) is a rare inherited disorder of the stratified squamous epithelium, characterised by blistering and scarring following minor trauma. Patients with the most severe forms, particularly dystrophic EB, develop bullae, inflammation, and scarring of the oesophagus following the ingestion of solid food. This results in dysphagia in the first two decades of life, severely compromising the ability to eat. There is no specific medical treatment for the disease and maintaining an adequate nutritional intake is often a central aim.

Methods: We report the results of 53 adults and children with EB and oesophageal strictures who were treated with endoscopic balloon dilatation. The procedure was performed using propofol anaesthesia and “through the scope” balloons.

Results: The median age at the time of index endoscopy was 16 years (range 3 to 61 years). A median of 2 barium studies (range 0 to 16) were performed per patient, identifying strictures at a median of 20 cm from the incisors (range 15 to 29 cm). 75% of patients had a single stricture (median =1, range 1 to 6 strictures). Recurrent strictures tended to occur in the same position. The total number of dilatations performed was 182—the median number of dilatations per patient was 2, over a mean follow up period of 3.5 years. The median interval between dilatations was 18 months. The median balloon size used was 45 Fr (range 42–56 Fr). All but three patients had an improvement in the dysphagia score. The median change in weight following the procedure was an increase of 2.6 kg (p<0.0001) over a median 29 days. Apart from self-limiting odynophagia in 3 patients, there were no other post-procedural complications and no oesophageal perforations.

Conclusion: Endoscopic balloon dilatation is a safe and effective treatment for the oesophageal strictures of EB, producing a long-term relief of dysphagia and an improvement in the nutritional status in the majority of patients.

166 HOW BEST TO ASSESS SYMPTOMATIC OUTCOMES OF THERAPY IN GASTRO-OESOPHAGEAL REFLUX DISEASE TRIALS: A SYSTEMATIC REVIEW

N. Sharma1, C. Donnellan2, C. Preston2, B. Delaney3, G. Duckett1, P. Moayyedi 1, 2, 3.

1Gastroenterology Unit, City Hospital; 2Centre for Digestive Diseases, Leeds General Infirmary, Leeds, UK; 3Dept. of Academic Primary Care and General Practice, Birmingham University, Birmingham, UK

Introduction: Traditionally drug trials have used oesophagitis healing as the main outcome of treatment success and the performance of oesophagitis scales have been well characterised. Symptoms are also an important outcome for clinical trials to measure. The optimal symptoms to assess however have not been well characterised. We conducted a systematic review to assess the symptoms that have been evaluated and how well these correlate with oesophagitis healing and relapse.

Methods: The Cochrane Controlled Trials Register, Medline, EMBASE, and CINAHL electronic databases were searched for RCTs evaluating drug therapies in oesophagitis. Experts in the field and pharmaceutical companies were contacted for information on any unpublished RCTs. Articles were included on predefined eligibility and validity criteria. Data were extracted on scales used, method of collecting data, duration of assessment, individual and global symptoms assessed, and whether improvement or absence of symptoms was the main outcome measure, types of symptoms assessed, frequency and severity of symptoms. The proportion of patients with a successful outcome according to these different symptom measures was compared with the proportion of oesophagitis healed/relapsed after therapy. The results are primarily evaluated in the form of L’Abbé plots.

Results: 324 papers were evaluated and data were extractable from 143 eligible trials. L’Abbé plots suggested no or minimal symptoms correlated well with oesophagitis healing and relapse whereas symptom “improvement” overestimated treatment effects. Trials that measured symptoms over a fixed period of time correlated better with oesophagitis healing and relapse than those that did not state the time period. Heartburn was the most important symptom to measure to predict oesophagitis healing but L’Abbé plots suggested additional information may be obtained from regurgitation and dysphagia.

Conclusions: This systematic review provides a comprehensive summary of the symptom outcome measures that have been used in trials and indicates how these might be improved.

167 RISK OF EXTRA-OESOPHAGEAL MALIGNANCIES IN BARRETT’S OESOPHAGUS AND IN GASTRO-OESOPHAGEAL REFLUX

M. Solaymani-Dodaran1, C. Coupland2, R.F.A. Logan1.

1Division of Epidemiology and Public Health; 2Division of General Practice, University Hospital, Nottingham, UK

Introduction: The relationship between Barrett’s oesophagus and extra-oesophageal malignancies (EOM) has been a matter of controversy. Some researchers have pointed out that the incidence of colon cancer is higher in Barrett patients while others have found no such association. The current study explores the relationship between Barrett’s oesophagus and EOM in general and colon cancer in particular in 27 813 subjects in General Practice Research Database (GPRD).

Methods: The mean follow-up experience was about 6 years and the study subjects constitute four groups: Barrett’s oesophagus group (1677), oesophagitis group (6392), simple reflux group (6328), and normal group (13 416). Respectively the last three groups were 4/1, 4/1, and 8/1 matched to the Barrett group according to their GP practice, date of birth and sex. All malignancies occurring before or in the first year after the diagnosis of Barrett’s, oesophagitis, or reflux were regarded as prevalent and excluded from the analysis. Risk of occurrence of an unrelated condition such as cataract was also explored for comparison. Number of visits to the GP was defined as a date on which there are both medical and drug records available and its confounding effects were controlled. Hazard ratios were calculated using Cox-proportional hazard regression analysis.

Results: A total of 2167 EOM, 260 colorectal cancer, and 1918 cataract diagnoses were identified in study subjects of which 1119, 113, and 853 cases were excluded respectively as being prevalent. The hazard ratios have been presented in the table after adjusting for age, sex, and number of visits to the GP.

Conclusion: The risk of colorectal cancer was not specifically higher in any of these groups. There were small increases in risk of EOM in the Barrett’s oesophagus, oesophagitis and reflux groups in comparison to the general population. The explanation for these increases is unclear but they may be mediated by smoking if smoking rates are increased in Barrett.

Abstract 167

168 GENDER DIFFERENCES IN THE EPIDEMIOLOGY OF GORD

J.P. Cotton, M. Lopez, S. McLeod, J.A. Todd1, D.A. Johnston, P.W. Dettmar, J.F. Dillon.

Department of Digestive Diseases and Clinical Nutrition, Ninewells Hospital, Dundee, UK; 1Leicester Royal Infirmary, Reckitt Benkiser, Hull.

Introduction: GORD affects up to 30% of the population, it is associated with an increased risk of oesophageal adenocarcinoma, a disease that is on the increase. Modern lifestyle has been attributed to the changing epidemiology of oesophageal cancer. We aimed to describe the epidemiology of reflux disease and its complications.

Methods: Consecutive consenting patients with symptoms of GORD were recruited from an endoscopy clinic. Demographic data, social habits, and symptom scoring were collected by the administration of a questionnaire, and an upper GI endoscopy was performed. Oesophagitis was graded using the LA grading system.

Results: 1165 subjects were recruited, 615 with no endoscopic features of oesophagitis (EN, 52.7%), 318 with evidence of erosive oesophagitis (EO 27.4%) (LA Grades A 43%, B 33%, C 16%, D 8%) and 232 with Barrett’s oesophagus (BO 19.9%). The proportion of male patients was greater in BO subjects (62.5%) than those with EO (52.5%) and EN (35.6%). When stratified for age male subjects with EN, EO, and BO were on average 5 years younger than their female counterparts (0.002), and that subjects with BO were 7 years older than those subjects with EN or EO (0.002), there was no statistically significant difference in age between those with EN and EO. Although males, regardless of diagnosis, on average drink more alcohol (mean 7.7 units/week, female 1.4 units per week, 0.001) males and females with BO do not drink more than their counterparts with EN or EO (P > 0.05). There was no difference in pack years smoked between diagnosis and gender. There was no difference in the body mass index between gender and diagnosis.

Conclusions: Male subjects are more likely to develop BO, and they are likely to develop the disease at an earlier age. Tobacco consumption and obesity are not associated with and increased risk of developing BO. Alternatively women may have some protection against developing BO until later in life. Other factors such as diet or the nature of the refluxate may be important in the gender differences.

169 MINI–CHROMOSOME MAINTENANCE PROTEINS PROVIDE A NOVEL METHOD FOR DETECTING PATIENTS AT RISK OF DEVELOPING ADENOCARCINOMA IN BARRETT’S OESOPHAGUS

P.S. Sirieix, M. O’Donovan1, J. Brown, N. Coleman, R.C. Fitzgerald.

MRC-Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge, CB2 2XZ, UK; 1Department of Histopathology, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK

Introduction: Oesophageal adenocarcinoma incidence is increasing rapidly. Endoscopic screening for the population at risk is not feasible and surveillance of patients with known Barrett’s oesophagus (BE) is prone to sampling bias and the subjective interpretation of dysplasia.

Aims: To determine whether a novel marker of cell cycle entry, mini-chromosome maintenance (MCM) protein predicted cancer risk and whether this could be used in combination with a surface sampling method.

Methods: Archival specimens (30 squamous oesophagus (SE), 62 BE +/- dysplasia, 16 adenocarcinoma (AC)) were stained for Mcm2. In addition, 9 patients with 3-13 years follow up who developed AC were compared with 18 controls matched for age and length of follow up who did not progress. Endoscopic cytological brushings were taken from a prospective cohort (61 SE, 90 BE +/- dysplasia and 11 AC) and scored blind as Mcm2 positive or negative.

Results: Correlation between Mcm2 surface expression and dysplasia (p<0.05 for biopsies and brushings). Since ∼40% of non-dysplastic BE samples were Mcm2 positive we tested whether surface expression predicted progression. 13/14 (93%) cancer patient biopsies, prior to any dysplasia diagnosis, were Mcm2 positive on the surface compared with 13/67 (19%) biopsies from matched controls.

Conclusion: Surface expression of Mcm2 can be used to detect the population at risk of developing high grade dysplasia and AC. A combined brushing technique with Mcm staining has the potential to be exploited as a non-endoscopic screening test.

Abstract 169

170 PHOTODYNAMIC THERAPY USING MTHPC FOR EARLY BARRETT’S–ASSOCIATED OESOPHAGEAL CANCERS

N.F. Jamieson, S. Thorpe, A. Mosse, S.G. Bown, L.B. Lovat.

National Medical Laser Centre, Royal Free and University College London Medical School, London, UK

Background: Oesophageal adenocarcinoma is rising in incidence and is strongly associated with Barrett’s oesophagus. Oesophagectomy is the standard treatment for early carcinoma but has considerable morbidity and mortality. Photodynamic therapy (PDT) is a non-thermal minimally invasive technique for mucosal ablation. Meso-tetrahydroxy-phenylchlorin (mTHPC, Foscan) is a potent photosensitiser which has been used with PDT in small series with squamous-cell oesophageal cancer.

Aims: To assess efficacy and safety of ablating high-grade dysplasia (HGD) or early oesophageal adenocarcinoma by PDT using mTHPC.

Methods: 5 patients with dysplasia (Vienna 4) within nodules and 11 with early adenocarcinoma (Vienna 5) staged by CT and EUS as T2N0 or less were included: T1(6), T1/2(3), T2(2). Oesophagectomy was inappropriate because of patient refusal or co-morbid pathology. Patients were photosensitised intravenously with mTHPC (0.15mg/kg). Three days later, at endoscopy, red light (wavelength 652nm) was delivered to the tumour by bare-tipped fibre or diffuser fibre within a windowed silicone bolster. Further PDT was given within 48 hours if needed. Patients were followed up by endoscopy and multiple biopsies (1,3,6,9,12 months) and EUS (3 monthly).

Results: 16 patients have been treated since Sept 1995. One patient died before initial follow-up unrelated to PDT and is excluded from analysis. At a median follow up of 17 months (range 9–73 months) 8/11 (72.7%) cancer patients are clear of carcinoma. All 4 patients with HGD are clear of dysplasia (median 13 months). There were 2 serious complications—an aorto-oesophageal fistula from which the patient died (an early case, later recognised as due to excessive light dose), and a tracheo-oesophageal fistula, successfully treated by covered oesophageal stent. Of 3 carcinomas not responding to PDT, there is no evidence of residual carcinoma after subsequent chemoradiotherapy (2) or surgery (1).

Conclusions: PDT with mTHPC offers an effective therapy for early oesophageal adenocarcinoma where surgery is not appropriate.

171 BILE ACIDS: DO THEY PLAY A ROLE IN BARRETT’S OESOPHAGUS?

F.R. D’souza1, G.J. Jenkins1, E.M. Parry1, J.N. Baxter1,2, J.M. Parry1.

1Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK; 2Department of Surgery, Morriston Hospital, Swansea SA6 6NL, UK

Background/Aims: The incidence of adenocarcinoma of the oesophagus has rapidly increased over the past few decades. Virtually all adenocarcinomas arise in Barrett’s oesophagus (BE), the exact aetiology of which is still unknown. There is increasing evidence that bile acids play a significant role in the pathogenesis of BE. Studies have shown that patients with BE have increased bile reflux compared to patients with uncomplicated gastro-oesophageal reflux. Effects of bile acids on oesophageal cell lines have not been previously demonstrated. Our study aimed at the evaluation of genotoxic potential of physiological concentrations of bile acids on oesophageal cell lines.

Methods: OE33 cells were purchased from ECACC (European Collection of Cell Cultures). Six different bile acids, namely Taurocholic acid (TCA), Glycocholic acid (GCA), Taurodeoxycholic acid (TDCA), Glycodeoxycholic acid (GDCA), Deoxycholic acid (DCA) and Cholic acid (CA) were used in near physiological concentrations ranging from 50μmol–1mmol. The Cytokinesis Blocked In-vitro Micronucleus Assay, a well accepted genotoxicity assay, was used to assess both structural and numerical chromosomal damage. Kinetochore labelling was performed to distinguish aneugens from clastogens. Apoptosis and necrosis was measured based on the morphological criteria.

Results: All the bile acids induced cytotoxicity in a dose dependent manner. The un-conjugated bile acids (DCA & CA) were more cytotoxic than the conjugated bile acids (TCA, GCA, TDCA, & GDCA). None of the bile acids except DCA showed any significant induction of micronuclei. DCA induced a statistically significant increase in the frequency of micronucleus in comparison with normal control (42 v 14 micronuclei/2000 cells, p=0.0029, Fisher exact test). DCA was predominantly clastogenic in action as revealed by the kinetochore labelling (66% kinetochore positive and 34% kinetochore negative). DCA also induced significant apoptosis (21 v 3 /500 cells, P<0.001). DCA induced apoptosis was maximum at concentrations between 100–200μmol.

Conclusion: Our studies showed that all bile acids were cytotoxic and that DCA in particular can induce DNA damage and apoptosis. Although DCA is found in a very low concentration in patients with BE, the fact that bile acids can become trapped and accumulate within mucosal cells (up to eight times the luminal concentration) makes even small concentrations significant. Our study shows that bile acids could play a major role in the development of oesophageal injury and its subsequent progression to dysplasia and oesophageal adenocarcinoma.

172 COX-2 EXPRESSION IS AN INDICATOR OF POOR SURVIVAL IN PATIENTS UNDERGOING RESECTION FOR OESOPHAGEAL ADENOCARCINOMA

P. Bhandari, B. Stacey, A.C. Bateman1, P. Patel.

Depts of Gastroenterology and 1Cellular Pathology, Southampton University Hospitals, Tremona Road, Southampton SO16 6YD, UK

Introduction: The clinical relevance of cox-2 overexpression in oesophageal adenocarcinoma is uncertain.

Aim: To test the hypothesis that cox-2 overexpression in oesophageal adenocarcinoma is associated with decreased patient survival.

Methods: Oesophageal resection specimens were obtained from a retrospective sample of patients who underwent intentionally curative resection for oesophageal adenocarcinoma (1990–96). Paraffin sections were used for immunohistochemical staining with a monoclonal antibody for cox-2 (Caymen chemicals) at a dilution of 1/100. Cox-2 expression was assessed using a semi-quantitative intensity-proportion scoring system (H-score i.e. intensity (0–3) × proportion of positive cells (%); possible scores from 0–300). Statistical analysis was performed using the Mann–Whitney U test and regression analysis.

Results: Specimens were identified from 79 patients with full pathological and clinical follow up data (minimum five years).

There was a negative association between cox-2 expression and survival (p=0.03).

Conclusion: Cox-2 overexpression is a marker of poor survival in patients with oesophageal adenocarcinoma. Differences in survival between groups with high and low cox-2 expression were not accounted for by variations in tumour differentiation or nodal stage.

Abstract 172

173 M2–PK EXPRESSION IN THE PROGRESSION OF BARRETT’S OESOPHAGUS TO ADENOCARCINOMA

K. Koss1, R.F. Harrison2, J. Jankowski3.

1Queen Elizabeth Hospital, Birmingham, UK; 2Department of Pathology, The Medical School University of Birmingham, UK; 3Leicester Royal Infirmary, Leicester/Warwick Medical School, UK

Background: During tumour formation, the tissue-specific isoenzymes of pyruvate kinase (PK), such as L-PK in liver and M1-PK in muscle and brain are lost and M2-PK is expressed. The dimeric form of M2-PK is specifically expressed by a wide range of different tumour cells, including those of the gastrointestinal tract, pancreas, kidney, breast, lung, and prostate. This study aimes to characterize the expression of M2-PK in the progression of Barrett’s oesophagus to adenocarcinoma.

Material and Methods: Oesophageal biopsies from 113 patients: 17 reflux oesophagitis, 37 intestinal metaplasia of the oesophagus, 21 Barrett’s high grade dysplasia and 38 Barrett’s adenocarcinoma were stained and semiquantified using monoclonal mouse anti-human antibodies against dimeric M2-PK. Staining was assessed with regard to location and intensity and the proportion of cells staining.

Results: All cases of reflux oesophagitis showed positive staining in the basal layers of the squamous epithelium, where the cells are immature and proliferating. All cases of Barrett’s metaplasia stained positively but staining was very variable from <30% of cells to 100% of cells. The majority of cases of Barrett’s dysplasia showed widespread positive staining in dysplastic cells, but also some negative areas. All adenocarcinomas cases were strongly positive for M2-PK antibody.

Conclusion: The results of this study have shown inappropriate expression of M2-PK in neoplastic Barrett’s mucosa. There was an increase of M2-PK expression as the Barrett’s metaplasia-dysplasia-adenocarcinoma sequence progressed.

174 THE USE OF THREE MODALITY SEQUENTIAL STAGING IN OESOPHAGEAL CANCER

P.C. Leeder, T.C.B. Dehn.

Royal Berkshire Hospital, Reading, Berkshire, UK

Aim: Following recent government recommendations regarding the management of patients with oesophageal cancer, it was useful to evaluate the process of sequential staging for patients with oesophageal cancer.

Methods: Patients admitted with a diagnosis of oesophageal cancer between 1996 and 2001 were included in the study. Data were collected prospectively. All patients considered for surgery embarked on sequential staging with computerised tomography (CT), followed by endoluminal ultrasound (introduced at the end of 1998), then laparoscopy.

Results: A total of 244 patients were admitted over the six-year period. Seventy-three patients (30%) were turned down for surgery because of high operative risk, while seven (3%) refused the offer of surgery. The remaining 164 patients went on to have staging CT. Thirty-eight patients were refused surgery because of metastatic spread seen on CT. Of 46 patients undergoing endoluminal ultrasound, nine were refused surgery because of signs of widespread disease. Thirty-five of 118 patients (30%) were denied surgery on the grounds of findings at staging laparoscopy. A total of 85 patients underwent resection (35%). Thirty-five patients were given pre-operative chemotherapy. In two patients attempted resection was abandoned because of local spread. Patients who did not undergo surgery were referred for either radical chemoradiotherapy (ten) or palliative treatment.

Conclusions: It is important to select those patients who are most likely to benefit from surgery and to avoid incomplete resection. Three modality, sequential staging is an invaluable tool in this process of patient selection for oesophageal resection.

175 BARRETT’S OESOPHAGUS IS AN INDEPENDENT RISK FACTOR IN TUMOURS OF THE UPPER GASTROINTESTINAL TRACT

A.G.K. Li, E. Fernandes, J. Baird, K.G.M. Park, on behalf of SAGOC Steering Group.

Aberdeen Royal Infirmary, Foresterhill, Aberdeen, UK

Background: Epidemiological studies suggest that Barrett’s specialised intestinal metaplasia (SIM) is a pre-malignant precursor for adeno-carcinomas of the oesophagus (OAC) and oesophago-gastric junction (OGJ). Surveillance programmes are currently based on the stringent endoscopic biopsy of all patients with SIM to identify “at risk” individuals.

Aim: To determine whether Barrett’s SIM is an independent prognostic factor in patients with carcinoma of the oesophagus and stomach.

Methods: The Scottish audit of Gastric and Oesophageal Cancer (SAGOC) analysed survival amongst patients with respect to possible contributory factors including Barrett’s oesophagus. Univariate and multivariate analyses was performed to account for compounding factors and hazard ratios (HR) for 1 year survival were derived from this.

Results: A history of pre-existing Barrett’s oesophagus was present in 14% of OACs, 4.3% OGJ tumours, 0.9% squamous cell carcinoma, and 0.9% of gastric tumours. In patients undergoing resectional surgery, Barrett’s SIM was found in 44% of OACs, compared with 17% of OGJ tumours. Pathological data for the presence of SIM were not recorded in up to 44% and 55% respectively. Adjusted hazard ratios were as follows: 1 year survival for all patients with and without pre-existing Barrett’s was 31% and 51.5%, respectively, and for those undergoing potential curative resection, was 52.7% and 72.6%, respectively.

Conclusion: Survival odds are greater in patients with Barrett’s than those without. This is increased in patients undergoing surgery with intent to cure and is independent of tumour stage and grade. However, given the prevalence of known pre-existing Barrett’s in this cohort, large scale surveillance programmes would benefit only a small number of individuals with tumours of the oesophagus and oesophago-gastric junction.

Abstract 175

Gastrointestinal physiology posters 176–180

176 SENSITIVITY TO ACID REFLUX IN BARRETT’S OESOPHAGUS IS RELATED TO IMPAIRED OESOPHAGEAL MOTILITY

J.M. O’Riordan, P.J. Byrne, E.D. Mulligan, P.W.N. Keeling1, Reynolds JV. University Depts of Surgery and1Medicine at St James’s Hospital, Dublin 8, Ireland

Background: Patients with Barrett’s oesophagus have increased acid and duodenogastric reflux compared to non Barrett’s patients. Impaired sensitivity to acid infusion and distension has also been described, but the relationship between oesophageal motility and symptomatology is poorly documented.

Methods: 74 patients with Barrett’s oesophagus were compared with 216 GORD patients with abnormal acid scores and 50 symptomatic patients who had normal acid exposure. All patients had oesophageal manometry and 24 hour pH monitoring. 36 Barrett’s patients also had 24 hour bile monitoring. Symptoms were assessed by event marker and the Symptom Index (SI) was calculated. Analysis was based on the patient’s motility status.

Results: Barrett’s patients with normal motility had significantly less symptoms than GORD patients for similar acid exposure (p<0.01). Barrett’s patients with abnormal motility had higher acid exposure than those with normal motility (p<0.01), but the SI values for this group were not significantly different from the GORD patients. Bile reflux in Barrett’s oesophagus did not appear to be a significant factor in patient’s symptoms.

Conclusions: Symptoms in Barrett’s oesophagus are less than GORD patients and are related to oesophageal motility. Normal motility in Barrett’s oesophagus is associated with the poorest sensitivity and the presence of increased acid exposure is required in order to achieve sensitivity levels comparable with GORD patients.

177 IS THERE A RELATIONSHIP BETWEEN PH MEASUREMENTS AND SYMPTOMS? A PROBABILITY BASED ANALYSIS USING NEURAL NETWORKS

K.R. Haylett1, P. Vales2, R.F. McCloy. 1Medical Engineering,2GI Investigation Unit,3University Department of Surgery, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK

Background and Aims: Ambulatory pH studies are widely accepted as the “Gold Standard” for detecting acid reflux within the oesophagus. However, many difficulties with analyses still exist. Patients frequently have evidence of reflux, on the day of the test, but no correlation with symptoms can be clinically found. This has led some investigators to suggest that more than one pH study would have to be done to identify the reflux pattern for each individual. The aim of this study was to carry out cluster-based analyses on a large database of ambulatory pH investigations and to compare the results of those patients with a clinical correlation of symptoms with acid reflux with those where no such correlation was found.

Methods: A self-organising neural network was used to partition the data from 900 clinical investigations into clusters. First, similar clusters between the two groups were found, eg investigations with reflux with or without correlating symptoms. Then the numbers of cases within the similar clusters were compared to give an estimate of the probability of correlating reflux with symptoms.

Results: The results show that investigations with reflux can be divided into four classes. Those patients with the highest values of reflux fell into a class indicating that there is up to a 78% probability of reflux correlating with symptoms on the day of the investigation. While those patients with the lowest levels of reflux have a 49% probability of symptoms correlating with reflux.

Discussion: The developed neural network allows the data from any study to be placed into the most appropriate class. This enables the probability of the symptoms being related to the observed reflux to be estimated despite no correlation being found on the day. This study suggests that, by using historical data to form the basis of the network and applying these techniques, a series of repeat tests are not necessary to assess the individual patient.

178 ATTITUDES OF BSG MEMBERS TO THE DIAGNOSIS AND MANAGEMENT OF BILIARY DYSKINESIA

E.W. Seward, M.J. Guinane, D.F. Evans, C.A. Ainley. Wingate Institute, 26 Ashfield Street, London E1 2AJ, UK

Introduction: Sphincter of Oddi dysfunction (SOD) and acalculous cholecystitis (AC) are contentious causes of biliary pain that require specialised investigative techniques. While data exist to support their diagnosis and treatment, many gastroenterologists doubt their existence.

Aim: to survey the attitudes of British gastroenterologists towards these functional diagnoses.

Methods: We contacted all BSG full members listed in the 2002 handbook.

Results: 1225 questionnaires were sent, of which 535 (43.7%) were returned. Of these, 355 (66.4%) were medical gastroenterologists, 118 (22.1%) were surgeons, and 62 (11.6%) were “other”. Respondents were asked what diagnoses they would consider in “unexplained” abdominal pain.

Conclusions: Of those BSG members who returned questionnaires, the majority would consider a diagnosis of SOD in previously unexplained abdominal pain. Standard diagnostic criteria for SOD are accepted although a significant minority would not accept pain alone as a reason to consider SOD. Cholecystectomy is largely accepted for AC, particularly amongst those surgeons performing the procedure.

Abstract 178, Table 1

Numbers of respondents that would consider a diagnosis of SOD, AC, or both AC and SOD

Abstract 178, Table 2

SOD diagnostic criteria accepted by respondents

Abstract 178, Table 3

Numbers who would recommend cholecystectomy for AC

179 THE ASSOCIATION BETWEEN ELECTROGASTROGRAPHY AND GASTRIC EMPTYING IN NON-ULCER DYSPEPSIA

P. Wong, M. Harbord, M. Jacovicic, O. Epstein. Centre for Gastroenterology, Royal Free Hospital, London NW3 2QG, UK

Background: In non ulcer dyspepsia (NUD), radionuclide imaging and electrogastrography (EGG) are used to assess gastric emptying (GE) and gastric pacemaker (slow wave) activity respectively.

Hypothesis: In NUD, gastric dysrythmias predispose to gastric dysmotility.

Aim: (1) To correlate GE and EGG findings in patients with NUD. (2) To correlate GE and EGG with the dominant symptom complex.

Methods: A GE study and pre- and post-prandial EGG were performed in 51 patients (34 female; mean age 40 years) with NUD (chronic epigastric symptoms, normal endoscopy, negative Helicobacter pylori and no other cause for symptoms). GE was reported as normal, rapid, or delayed and EGGs as normal (>70% slow waves, with mean frequency of 3 cycles/minute), tachygastria or bradygastria. The symptoms were epigastric pain (n=27); nausea with or without vomiting (N&V) (n=15); epigastric bloating (n=3); nausea and bloating (n=3); and reflux and belching (n=3). Linear regression was used to assess any association between age, gender and symptoms.

Results: (1) Both GE and EGG were normal in 12 patients (24%). GE was abnormal in 33 patients (65%) of whom 21 had delayed emptying. The EGG was abnormal in 18 patients (36%), with tachygastria reported in 13. In these 18 patients, the pre-prandial EGG was abnormal in 15, the post-prandial EGG abnormal in 5, whereas GE was normal in 6 (all tachygastrias). (2) GE was abnormal in 16 patients (59%) with pain (delayed in 10), in 11 (73%) with N&V (delayed in 6), in the 3 patients with nausea and bloating (delayed in 2), and delayed in the 3 patients with bloating. GE was normal in the patients with reflux. (3) EGG was abnormal in 12 patients (44%) with pain (tachygastria in 9), in 5 patients (33%) with N&V (bradygastria in 4), and in 1 patient with bloating. There was no corellation between gender, age, GE, EGG, and symptoms.

Conclusions: Abnormality of GE or gastric slow wave activity occurs in up to 76% of NUD patients. There appears to be no association between abnormal GE and EGG. Abnormal GE or EGG does not correlate with the dominant symptom complex although 73% of patients with N&V had abnormal GE. Abnormal EGG usually normalises after a meal. A further study is required to determine whether delayed GE (but normal EGG) predisposes to bloating.

180 SMALL BOWEL MANOMETRY ON CHILDREN WITH SUSPECTED SMALL INTESTINAL MOTILITY DISORDER

E. Yazaki, N. Meadows, F. Smith, K. Wiles , M. Hirata, S.K. Arthur, D.F. Evans. Adult and Paediatric Gastroenterology, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, UK

Background: Small bowel manometry (SBM) has widely been used to investigate adult patients with gastrointestinal motility disorder. However, the use of this technique to paediatrics is uncommon. The aim of this study was to evaluate feasibility of SBM on paediatric patients with suspected small intestinal motility disorder.

Methods: We have performed SBM on 14 paediatric patients (age range 2–10 years) who had symptom(s) including recurrent abdominal pain (n = 10), nausea (n = 5), vomiting (n = 7), abdominal distension (n = 2) and constipation (n = 2). A 3-channel manometry catheter was introduced nasally and placed in the proximal small bowel under fluoroscopic control. Ambulatory recordings were made for 24 hours using a portable recorder. Recorded data were analysed using dedicated software.

Results: Four had normal motility when applied to the normal ranges in adults. At least one of the following abnormalities was found in eight patients; possible neuropathy findings including excess of motor activity (n = 3), prolonged Phase III (n = 2), Phase III-like prolonged bursts (n = 3), non-propagated Phase III (n = 2), retrograde propagation of Phase III (n = 2), and low-amplitude Phase III (n = 1) suggesting possible myopathy. Intubation failed in two patients due to no migrations of the catheter into the small intestine.

Summary: In this study a high proportion of the patients had abnormal small intestinal motility and this suggests that SBM can be used to evaluate those conditions. Normal values during infant and childhood development need establishing before adopting the technique for clinical diagnosis.

Cell/molecular biology posters 181–213

181 SODIUM BUTYRATE MEDIATED SP3 ACETYLATION REPRESSES HIGFBP-3 EXPRESSION IN INTESTINAL EPITHELIAL CELLS

N.R. White, P. Mulligan, P.J. King I.R. Sanderson. Department of Adult and Paediatric Gastroenterology, St Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK; Department of Endocrinology, St Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK

Introduction: Butyrate induces histone acetylation, but this does not easily explain the down-regulation of hIGFBP-3 by butyrate, as histone acetylation is associated with expansion of the nucleosome. Furthermore, the down regulation occurs in the absence of de novo protein synthesis, excluding the induction of a repressor as a possible mechanism. However, promoter analysis revealed the presence of a butyrate responsive element that included binding sites for p300 and Sp1/Sp3.

Methods: We examined the acetylation and activity of Sp3 in hIGFBP-3 expression.

Results: Transfection of Caco-2 cells with E1A, an inhibitor of p300 acetyltransferase activity, reversed the butyrate induced repression of hIGFBP-3. Sp3 is a known repressor of gene activation. Its potential for acetylation has recently been reported. We hypothesized that butyrate might increase the acetylation of Sp3. We, therefore, performed EMSA and supershift studies on nuclear extracts from butyrate treated and non-treated Caco-2 cells with the hIGFBP-3 promoter. We also studied nuclear extracts by Western blotting analysis and immunoprecipitation. 5mM butyrate retarded the Sp3-specific band in EMSAs. This band was further supershifted by an anti-acetyl lysine-specific antibody. Immunoprecipitation indicated an increase in the amount of the acetylated form of Sp3, in the presence of NaB, whereas the total amount of Sp3 protein was unchanged. Western blot analysis revealed the acetylation of an isoform of Sp3 in the presence of 5mM NaB. Transfection with E1A abrogated the repression by NaB as evidenced by semi-quantitative PCR and Western blot analysis.

Conclusions: Our evidence supports the hypothesis that butyrate down-regulates hIGFBP-3 by butyrate by the acetylation of the repressor Sp3 and likely involves p300. We have shown for the first time that butyrate affects the acetylation status of a non-histone DNA-binding protein.

182 CHARACTERISATION OF BINDING OF CLOSTRIDIUM DIFFICILE TOXIN A TO MEMBRANE PREPARATIONS OF HUMAN COLONIC EPITHELIAL CELLS

N.A. Ali, Y.R. Mahida. Division of Gastroenterology, University Hospital, Queen’s Medical Centre, Nottingham, UK

Introduction: Clinical presentation after C difficile infection can range from asymptomatic carriage to pseudomembranous colitis. C difficile induces diarrhoea and colonic inflammation via secreted toxins A and B. To induce disease, the toxins have to first interact with colonic epithelial cells. We have investigated the binding of C difficile toxin A to membrane preparations of primary human colonic epithelial cells.

Methods: Primary colonic epithelial cells from different individuals (A to D) were isolated by treatment with EDTA and used for membrane preparations (confirmed by electron microscopy). Caco2 cell membrane preparations were also obtained (and shown to be enriched for sucrase-isomaltase and alkaline phosphatase). Purified C difficile toxin A was labelled with tritium [3H] using the Bolten-Hunter reagent N-succinimidyl [2,3-3H] propionate. Binding studies were carried out using purified epithelial membrane preparations and [3H]-toxin A. Specific binding was calculated by subtracting non-specific binding in the presence of 1000-fold excess unlabelled toxin A.

Results: Biological activity of [3H]-toxin A was confirmed using Vero cells. In studies using membrane preparations from different individuals, binding of [3H]-toxin A varied markedly (binding per mcg protein, expressed as % of binding to Caco2 membrane preps: A - 25.7%, B-32.8%, C-184.9% and D–0.8%). Using pooled samples of membrane preparations, specific binding of [3H]-toxin A occurred at 4°C, 19 °C, and 37 °C. The binding achieved saturation in the presence of increasing concentrations of [3H]-toxin A, and reached equilibrium within 60 min at 4°C.

Conclusions: The specificity of [3H]-toxin A binding to membrane preparations of colonic epithelial cells and the ability to saturate this binding suggests involvement of receptor-ligand interaction. Variation in the binding of [3H]-toxin A to membrane preparations from different individuals may explain the different types of clinical presentation in patients infected with toxigenic C difficile.

183 BONE MARROW DERIVED CELLS CONTRIBUTE TO A POPULATION OF FIBROBLASTS AND MYOFIBROBLASTS WHICH ENGRAFT TO MULTIPLE SITES, INCLUDING THE GASTROINTESTINAL TRACT

N.C. Direkze, S. Forbes, M. Brittan, T. Hunt, R. Jeffery, S.L. Preston, R. Poulsom, K. Hodivala-Dilke, M. Alison, N.A. Wright. Cancer Research UK, Imperial College London, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London, UK

Background: Our previous work has shown that bone marrow can make important contributions to the myofibroblast population in the lamina propria after bone marrow transplantation (Gut 2002;50:752–7). Here we show that the bone marrow can also contribute to fibroblast populations in sites of injury, and in fact, the engraftment of cells destined to be myofibroblasts from the bone marrow appears to be a systemic phenomenon not confined to gastrointestinal tissues.

Methods: C57/black female mice were irradiated with a total of 12 Gray to ablate the bone marrow followed immediately by I.V. injection of male wild type whole bone marrow. The mice received 2 doses of paracetamol at 5 and 8 weeks post transplantation at a dose of 400mg/kg I.P. Tissue sections were examined using in situ hybridisation to detect the Y-chromosome and immunohistochemistry for intermediate filaments and cytoskeletal elements.

Results: Examination of the intestine and stomach showed numerous myofibroblasts of bone marrow origin distributed throughout the gut wall. In addition, areas of fibrosis contained numerous Y chromosome positive cells with a fibroblastic phenotype. Myofibroblast engraftment was also demonstrated in the skin, adrenal capsule, lung, and kidney.

Conclusions: We conclude that bone marrow provides a circulating population of cells which can contribute to myofibroblasts in healing tissues. Our data also suggest that these cells can adopt a fibroblastic phenotype and contribute to fibrosis. We therefore hypothesise that circulating bone marrow-derived precursors are available which are able to colonise injured tissues and give rise to myofibroblasts and then fibroblasts. These conclusions have important connotations for tissue repair in gastrointestinal and other tissues.

184 INTERPHASE FLUORESCENCE IN SITU HYBRIDISATION (FISH) TO DETECT CHROMOSOMAL ABNORMALITIES IN GASTRIC CANCER PROGRESSION

L. Williams1, J.G. Williams1, A.P. Griffiths2, T. Brown2, S.H. Doak3, G.J.S. Jenkins3, E.M. Parry3, J.M. Parry3. 1Neath General Hospital, Neath, UK;2Morriston Hospital, Swansea, UK;3School of Biological Sciences, University of Wales Swansea, UK

Introduction: Gastric cancer is the second commonest fatal cancer worldwide and Helicobacter pylori has been causally linked with cancer progression. Gastric cancer presentation is at the advanced stage, and survival rates are poor. As such, interest in the causative agents and genetic changes responsible for gastric carcinogenesis has increased. Advanced gastric cancer shows widespread chromosomal rearrangement of chromosomes 20, 8, and 17(p53). In addition, chromosome 4 has previously been implicated in Barrett’s oesophagus.

Aim: To develop FISH to look at chromosomal abnormalities (using 4, 8, 17, 20) in gastric tissue and to correlate the chromosomal abnormalities with histological diagnosis, patient characteristics, H pylori presence and subtype.

Methods: Patients were enrolled, gastric biopsies were taken for histology and for PCR to detect H pylori (and Cag A status). Gastric/oesophageal cytology brushes were taken and FISH performed with centromeric probes 20, 8 and 4, and a locus specific probe for p53. In addition, surgical resections of dysplastic/cancer patients were obtained for FISH analsysis.

Results: Oesophageal cells show little chromosomal instability, whilst histologically normal gastric cells do show genetic instability, possibly due to the adverse environment of the stomach. Histologically abnormal gastric cells show significantly more chromosome abnormalities with all probes (p<0.0001), when compared to oesophageal cells or normal gastric cells. This demonstrates the increasing genetic nstability in the histological progression of gastric cancer. Specifically, p53 gene deletion, chromosome 8 and 4 amplifications and chromosome 20 instability is detected.

In the surgically resected specimens, a range of histological subtypes were noted (normal, intestinal metaplasia, dysplasia, adenocarcinoma). The same chromosomal abnormalities seen in the premalignant cells (above), were seen in these cancer cells, but the incidence of these abnormalities was greater.

Conclusion: Cytology brush/FISH analysis allows adequate numbers of gastric cells to be collected and analysed. This approach allows cytogenetic screening of gastric cancer progression to be performed. Importantly, the chromosomal instability detected in pre-malignant gastric tissues increased during the histological progression to cancer. This suggests that these specific chromosomal alterations are important in cancer progression.

Work in progress: PCR for H pylori in the gastric biopsies collected from enrolled patients to correlate the chromosome data with H pylori presence and subtype (cag A).

185 PROSTAGLANDIN E2 RECEPTOR (EP RECEPTOR) EXPRESSION IN PRE-MALIGNANT AND MALIGNANT GASTRIC MUCOSA

A. Wood, A.B. Ballinger, G.V. Smith.

St Barts and the London, Queen Mary’s School of Medicine, London, UK

Prostaglandin E2 (PGE2) is significantly over-expressed in gastric adenocarcinoma and as a result of H pylori (Hp) infection. This is associated with over expression of cyclooxygenase 2. The effects of PGE2 are mediated by both nuclear and cell surface receptors. The PGE2 cell surface (EP) receptors activate several intracellular homeostatic mechanisms and have been implicated in cell cycling. The expression of these receptors in gastric carcinogenesis is poorly understood.

Methods: Paraffin embedded sections of normal gastric mucosa, Hp gastritis, mucosal atrophy, intestinal metaplasia (IM), dysplasia and cancer were analysed immunohistochemically for the expression of the four subtypes of EP receptor. Polyclonal antibodies raised against these receptors were utilised. Controls comprised oesophageal and duodenal biopsies. Negative controls were analysed by substitution of the primary antibody with diluent or its pre-incubation with a specific control peptide for each receptor. Two observers scored each slide. Median scores were compared with a Mann–Whitney analysis.

Results: See table.

Abstract 141

Abstract 185

Discussion: Significant variations in EP receptor expression are seen in gastric pathology. EP4 receptors appear to be up-regulated in inflammatory conditions associated with Hp infection, whereas EP2 and EP3 are over-expressed in premalignant and malignant tissue. These patterns of expression, at the time of increased PGE2 synthesis, may provide a mechanism for increased cell cycling and proliferation and play a significant role in inflammation and neoplasia.

186 THE ROLE OF PPARγ IN THE ANTI-COLORECTAL CANCER ACTIVITY OF INDOMETHACIN

G. Hawcroft, S.H. Gardner, M.A. Hull.

Molecular Medicine Unit, University of Leeds, St James’s University Hospital, Leeds LS9 7TF, UK

We have previously shown that indomethacin inhibits proliferation and induces apoptosis of several human colorectal cancer (CRC) cell lines in vitro. The anti-proliferative effects of indomethacin (100–600 μM) were associated with a decrease in expression of β-catenin and the β-catenin/TCF target gene cyclin D1 (CD1), in a COX-2-independent manner. However, mutation of the TCF-binding site in the CD1 promoter did not abolish down-regulation of CD1 by indomethacin, implying that other cis- and/or trans-acting elements must also mediate the effects of indomethacin on CD1 expression. It has previously been demonstrated that indomethacin can activate peroxisome proliferator-activated receptor γ (PPARγ) in adipocytes and that growth arrest by PPARγ ligands can occur via PPARγ-dependent repression of CD1. Therefore, the aim of this study was to investigate whether growth arrest and down-regulation of CD1 expression by indomethacin occurs via a mechanism involving PPARγ activation in human CRC cells.

SW480 and HCT116 human CRC cells expressed PPARγ mRNA and protein. PPARγ function was tested using the thiazolidinedione (TZD) PPARγ ligand troglitazone (10–50 μM) and a PPAR response element-luciferase (PPRE-tk-luc) reporter gene. Both SW480 and HCT116 cells contained functional PPARγ that was also activated by indomethacin (HCT116 > SW480). The functional relevance of PPARγ activation for the anti-proliferative effects of indomethacin was tested by transient transfection of dominant-negative PPARγ (dnPPARγ) in HCT116 cells. dnPPARγ inhibited TZD-induced PPRE-tk-luc activity. However, dnPPARγ expression did not inhibit indomethacin-induced down-regulation of CD1.

Despite the fact that indomethacin directly activates PPARγ in human CRC cells, PPARγ activation does not explain the anti-proliferative activity of indomethacin (including down-regulation of CD1) in human CRC cells and does not represent a COX-independent mechanism of the anti-CRC activity of the NSAID indomethacin.

187 INDOMETHACIN ALTERS HELICOBACTER PYLORI INDUCED EFFECTS ON GASTRIC EPITHELIAL CELLS IN VITRO

M.W. James, R.H. Argent, R.J. Thomas, C.J. Hawkey, J.C. Atherton.

Division of Gastroenterology, University Hospital, Nottingham, UK

Introduction:Helicobacter pylori infection is a major risk factor for distal gastric adenocarcinoma. Pathogenicity determinants include production of an active form of vacuolating cytotoxin (VacA) and possession of the cag pathogenicity island (cag PaI). Toxigenic strains cause marked epithelial cell vacuolation in vitro whilst cag-dependent effects include rearrangement of the actin cytoskeleton to form a “hummingbird” phenotype. NSAID ingestion is associated with a reduced incidence of gastric adenocarcinoma, although mechanisms are unclear. We tested the hypothesis that NSAIDs modulate H. pylori-induced effects on epithelial cells, in particular effects induced by VacA and the cag PaI.

Methods: AGS cells (1–2 × 105 cells/ml) were co-cultured with H pylori strain 60190 (cag+, vacA s1/m1) or its VacA- or CagE- isogenic mutants at a bacteria:cell ratio of 0.02–70:1. Indomethacin (a non-selective NSAID) or vehicle control was added at a concentration of 0.01–100μM. After overnight incubation with 5% CO2 at 37°C, vacuolated and hummingbird cells were counted directly using phase contrast microscopy. Statistical analysis was by ANOVA and Student’s paired t test.

Results: Co-culture with H pylori strain 60190, but not its VacA- mutant, increased AGS cell vacuolation from 0.8 ± 0.8 to 25.4 ± 6.5%. Indomethacin had no significant effect in the absence of H pylori, but inhibited H pylori-induced vacuolation in a dose-dependent manner (100μM: by 56 ± 14%, p=0.02; 1μM: by 42 ± 15 %, p=0.05; 0.01μM: by 26 ± 13%, p=0.26). Co-culture with H pylori strain 60190, but not its CagE- mutant increased AGS hummingbirds from 0 to 6.2 ± 1.7%. Indomethacin had no effect in the absence of H pylori. In the presence of H pylori, indomethacin at 100μM surprisingly increased hummingbirds by 240 ± 98%, p=0.05.

Conclusion: Indomethacin reduces H pylori-induced vacuolation, but in contrast, at high concentrations, increases H pylori-induced cytoskeletal changes. We speculate that these actions contribute to NSAID-induced reduction in H pylori-associated gastric cancer risk.

188 EFFECTS OF SULINDAC SULPHIDE ON MITOCHONDRIAL OXYGEN CONSUMPTION AND MEMBRANE POTENTIAL

M.J. Garle, B. Middleton, C.J. Hawkey.

Division of Gastroenterology and School of Biomedical Sciences, Queens Medical Centre, Medical School, University of Nottingham, NG7 2UH, UK

Introduction: Sulindac (active metabolite: sulindac sulphide (SS)), is useful for the chemoprevention of colorectal cancer. Sulindac derivatives are active against COX-negative cancers by promoting apoptosis indicating that mechanisms unrelated to prostaglandin synthesis are involved. We have shown that SS inhibits β-oxidation of long chain fatty acids. To explore precise mechanisms, we measured ADP-stimulated oxygen consumption, proton transport (mitochondrial membrane potential (MMP)), and peroxide generation with different metabolic fuels: octanoate (OCT, C8 fatty acid), palmitoylcarnitine (PC, C16 fatty acid), pyruvate (PYR, glycolytic intermediate) and succinate (SUC, TCA cycle intermediate).

Methods: Rat liver mitochondria were prepared by differential centrifugation and ADP-stimulated oxygen uptake was measured using a Clark oxygen electrode. MMP and peroxide generation was determined in HCT-116 cells using JC-1 accumulation and dichlorofluorescin oxidation respectively.

Results: SS (100μM) inhibited octanoate and succinate, but stimulated pyruvate, oxidation (see table). It enhanced MMP by 86% but suppressed peroxide generation.

Abstract 188

Conclusions: SS selectively inhibits fatty acid and succinate oxidation. Our data imply flavoproteins, eg succinate dehydrogenase, and medium chain acyl CoA dehydrogenase as possible targets. Suppression of peroxide formation may indicate an additional antioxidant action of SS.

189 EXPRESSION OF THE CYTOPROTECTIVE TREFOIL PROTEIN TFF2 IN HUMAN GASTRIC AND DUODENAL BIOPSIES

C.E. Johns, J.L. Newton, B.R. Westley, F.E.B. May.

University of Newcastle, UK

Introduction: TFF2 is the second member of the trefoil family of peptides. These small proteins are found in epithelial surfaces and contribute to mucosal repair and protection. It has been suggested that the glycosylated form of TFF2 is more active in promoting ulcer healing in rats. Immunohistochemical studies have demonstrated TFF2 in the antral glands of the stomach and the Brunner’s glands of the duodenum. The relative amounts in these areas have not been quantified previously in man.

Methodology: Seven patients attending for routine oesophagogastroduodenoscopy gave written informed consent. In each subject gastric biopsies were taken from within 2 cm of the pylorus and duodenal biopsies from each of the first, second and third parts of the duodenum. Separate parallel biopsies were taken for immunohistochemistry. The amount of TFF2 in the biopsies was measured by quantitative Western transfer analysis.

Results: High glycosylated TFF2 levels were found in the gastric antrum and first part of the duodenum. There was no difference between these areas (means both 3.4 ng per μg total protein; p=0.99 ci –4.2,4.1). TFF2 was present in D2 in all subjects, and 4 had detectable TFF2 in D3 (mean 0.2 ng per μg protein). The proportion of total TFF2 that is non-glycosylated was greater in the duodenum than in the stomach, (means 7.3% and 3.1% respectively; p=0.03, ci –7.9, −0.44). The profile of TFF2 glycosylation in the duodenum was different from that in the stomach.

Discussion: The presence of TFF2 in D3, beyond the pancreatic duct, where Brunner’s glands are thought to be absent, indicates that Brunner’s glands may not be the only source of TFF2 in the human duodenum, and that TFF2 is active in D3. The higher proportion of non-glycosylated TFF2 in the duodenum suggests this peptide may have a different function here.

190 THE EXPRESSION OF MUCIN GLYCOPROTEINS AND TREFOIL PEPTIDES IN THE HUMAN FETUS

D. Carroll1, P. Soothill3, C. Sergi4, R.D. Spicer1, A. Corfield2.

Departments of 1Paediatric Surgery and 4Paediatric Pathology, Bristol Children’s Hospital, 2Department of Biochemistry, University of Bristol; 3Professor Department of Fetal Medicine, Bristol University

Introduction: Mucin glycoproteins are important constituents of the supramucosal barrier. Trefoil peptides are fundamental to the maintenance of epithelial integrity. Immaturity of the gastrointestinal tract is associated with a number of pathological conditions. We sought to examine the timing of expression of the gene products of these proteins within the developing gastrointestinal tract.

Methods: fetal gastrointestinal tract was collected with maternal consent and prior approval of the regional ethics committee from 9 fetuses from 8–13 weeks gestation. Tissue was immediately fixed in formalin for 24h and embedded in paraffin. Sections were subjected to in situ hybridisation and immunohistochemistry. Sections were then examined by two independent observers under light microscopy to determine the cellular and subcellular pattern of gene and protein expression in the developing gastrointestinal tract. Data analysis was performed using independent t test in SPSS for Windows v.10.

Results: Mucin gene expression was not detected before 9+3 weeks gestation in the fetal gastrointestinal tract by either in situ hybridisation or immunohistochemistry. From 10 weeks gestation mucin gene expression adopted a pattern similar to that seen in normal neonatal tissue. Trefoil factor 3 product was detected in primitive secretory cells of the small and large intestine from 10 weeks gestation onward.

Conclusions: Mucin glycoproteins and trefoil peptides are present in detectable levels in the gastrointestinal tract from late in the first trimester. Deficiencies in post-translational modification or quantitative changes in expression may account for presumed deficiencies in the integrity of the supramucosal barrier.

191 PEPTIDE TARGETING OF CCK-2 RECEPTOR IN TUMOUR CELL LINES

M. Stubbs, K. Khan, S.A. Watson, S. Grimes, D. Michaeli, K. Savage, M. McStay, A.P. Dhillon, M.E. Caplin.

Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK

Background: Gastrin binds preferentially to the CCK2 receptor. We have previously demonstrated expression of the CCK2 receptor in a number of hepatopancreaticobilary cancers and uptake of gastrin analogue peptides by CCK2 expressing tumour cell lines. The study also demonstrated dimerization of the peptides in solution.

Aim: To compare the uptake of dimer and monomer forms of gastrin analogue peptides in hepatic and pancreatic cancer cell lines.

Methods: 4 derivatives of GRTL-1 were labelled using Alexa Fluor 488 dye (Molecular Probes, USA); untreated GRTL-1, GRTL-1 with the cysteine sulfhydryl group protected with N-ethylmaleimide (GRTL-1-NEM), a GRTL-1 dimer with a bismaleimidohexane linker ((GRTL-1)2 BMH) and a GRTL-1 dimer with a bismaleimidotetraethyleneglycol spacer linker ((GRTL-1)2 BM(PEO)4). The labelled peptides were exposed to HepG2 (human hepatocyte carcinoma), AR42J (rat pancreatic adenocarcinoma) and PLC/PRF/5 (human liver hepatoma) cells at a concentration of 20 μg/ml for 1 hour at 37°C. Cells were fixed with buffered formaldehyde and examined under a fluorescence microscope.

Results: (GRTL-1)2 BMH (AR42J and PLC/PRF/5) and GRTL-1 (HepG2) gave the highest proportion of cells with visible uptake of labelled analogue. High uptake was also seen with (GRTL-1)2 BM(PEO)4 but the fluorescence intensity was slightly less intense than for GRTL-1 or (GRTL-1)2 BMH. The level of GRTL-1-NEM uptake was much lower.

Conclusion: Dimeric forms of GRTL-1 show more endocytosis than the monomeric form. GRTL-1 with unsubstituted cysteine undergoes significant dimerization in aqueous solution, so the high uptake seen with this form is probably due to the dimer. Dimeric GRTL-1 therefore shows greater potential for intracellular delivery of potential therapeutic agents to CCK2 positive tumours.

192 HELICOBACTER PYLORI INFECTION IN MONGOLIAN GERBILS INDUCES A TH1 GASTRIC MUCOSAL RESPONSE

M.A. Aboshkiwa1, A.H.T. Jeremy1, M. Court1, M.F. Dixon2, P.A. Robinson1, J.E. Crabtree1.

1Molecular Medicine Unit, St James’s University Hospital, UK; 2 Department of Pathology, The General Infirmary, Leeds, UK

Introduction: Chronic H pylori infection in Mongolian gerbils has been demonstrated to result in gastric cancer. The gastric mucosal inflammatory and immune response to infection has not been characterised. The aims of this study were to identify transcripts for gerbil cytokines and to examine the gastric cytokine responses to chronic H pylori infection.

Methods: Female Mongolian gerbils were orally challenged three times with H pylori SS1 strain. Infected animals (n = 15) plus controls (n = 17) were sacrificed at 4, 12, and 36 weeks post-infection (p.i.). Infection was confirmed histologically and by culture. Gastric mucosa was snap frozen for analysis of cytokine transcripts by RT-PCR. Cross-species PCR and sequencing was used to identify gerbil transcripts for IFNγ, IL-12p40, IL-10 and TGFβ. The ratio of cytokines to β-actin was determined by computer image analysis.

Results: Gastric IFNγ transcripts in H pylori infected gerbils were significantly increased at 12 (p<0.05) and 36 weeks (p<0.01) p.i. compared to uninfected controls. (IFNγ:β-actin ratio 12 weeks 0.85 v 0.057; 36 weeks 1.51 v 0.16). IL-12p40 transcripts in H pylori infected gerbils were significantly increased (p<0.05) at 36 weeks p.i. compared to controls (1.512 v 0.512). In contrast, no differences in IL-10 or TGFβ transcripts were observed with H pylori infection, although an age-related increase in TGFβ transcripts was evident in both infected and control gerbils.

Conclusions: Long term chronic infection with H pylori in the Mongolian gerbil is associated with upregulation of the Th1 cytokines IFN-γ and IL-12p40 in the gastric mucosa. The lack of upregulation of key immunomodulatory cytokines IL-10 and TGF-β in H pylori infection may contribute to the severe pathology associated with infection in the Mongolian gerbil.

193 SPECIFIC SERUM ANTIBODIES ARE INVOLVED IN H PYLORI-INDUCED PLATELET AGGREGATION

P.A. Corcoran, S.W. Kerrigan, D. Cox, J.C. Atherton, D.J. Fitzgerald, F.E. Murray, M.F. Byrne.

Clinical Pharmacology/Gastroenterology, RCSI/Beaumont Hospital, Dublin, Ireland and Duke University Medical Center, NC, USA

Background: Clinical studies have suggested an association between cardiovascular disease and infection with H pylori. We have described strain specific H pylori-induced platelet aggregation that involves platelet glycoprotein Ib and von Willebrand factor (vWf). We examined the role of H pylori antibodies in this aggregation.

Methods and Results: Addition of pooled immunoglobin (Ig) and fibrinogen along with H pylori strain 60190 (coated in vWf) to washed platelets led to platelet aggregation (60±4%, n=3). Antibodies involved were specific to H pylori as bacteria incubated in pooled Ig and subsequently washed could support platelet aggregation (49±7%, n=3). To confirm this, pooled Ig was incubated with H pylori or Streptococcus sanguis. Bacteria were removed by centrifugation and the depleted Ig used in aggregation assays. H pylori-depleted Ig showed greatly reduced platelet aggregation to H pylori (13±9%, n=3, p<0.01) while the S sanguis-depleted Ig was not affected (54±3%, n=3, p=NS). All 20 plasma donors were tested for H pylori seropositivity using a H pylori ELISA. All 4/4 of the positive volunteers consistently aggregated in response to H pylori while only 2/15 of the H pylori negative donors aggregated consistently (p=0.039, Fisher’s Exact Test). The relative risk of H pylori positivity for aggregation was 7.5 (CI 2.0-27.3).

Conclusions:H pylori-induced platelet aggregation requires binding of H pylori-specific IgG. This pro-aggregatory phenotype is not unique to H pylori and has been described with S sanguis and P gingivalis. Exposure to oral bacteria such as S sanguis can lead to infective endocarditis and, in the case of P gingivalis, to enhancement of atherosclerosis in animal models. Repeated exposure to pro-aggregatory bacteria such as certain strains of H pylori may not cause coronary artery disease but could accelerate the progression of the disease. Local platelet effects may also contribute to the pathogenesis of H pylori-associated peptic ulcer disease.

194 STIMULATION OF MATRIX METALLOPROTEINASE-7 (MMP-7) BY HELICOBACTER PYLORI IN GASTRIC EPITHELIAL CELLS: ROLE IN EPITHELIAL CELL MIGRATION

A. Varro, L.E. Wroblewski, A. Pagliocca, 1D.M. Pritchard, P-J.M. Noble, 2C.A. Hart.

Physiological Laboratory, 1Departments of Medicine and 2Medical Microbiology, University of Liverpool, Liverpool, UK

Background and Aims: Epithelial cell responses to bacterial infection include induction of MMP-7. We have examined the cellular mechanisms of H pylori stimulation of MMP-7 in gastric cells, and the consequences for gastric epithelial cell migration.

Methods: Gastric biopsies of H pylori positive and negative subjects were probed for MMP-7 by Western blot. Biopsies were cultured for up to 72hr and MMP-7 localised by immunocytochemistry; cell migration in these cultures was examined by time lapse videomicroscopy. Cellular signalling mechanisms were examined using a MMP-7-promoter/luciferase (-luc) reporter vector transfected into AGS cells.

Results: In H pylori positive patients there was increased MMP-7 in both corpus and antrum. MMP-7 was localised to mucus and chief (but not parietal or endocrine) cells in cultured gastric epithelial cells, and was identified in lamellipodia at the advancing edge of migrating cells. The spreading of gastric glands was significantly increased in H pylori positive cultures compared with control and migration was inhibited by MMP-7 antisense oligonucleotides. In addition, H pylori stimulated AGS cell migration and invasion and this was inhibited by an MMP-7 antibody. In AGS cells, H pylori induced expression of MMP-7-luc and this was significantly inhibited by co-transfection with dominant negative (DN-) forms of Rho and Rac, and by inhibitors of NFκ-B (BAY11-7082) and MEK (PD98059). A constitutively active (CA-) form of Rho also stimulated MMP-7-luc, and this was inhibited by BAY 11-7082, PD98059 and DN-Jun; in contrast, stimulation of MMP-7-luc by CA-Rac was inhibited by BAY but not DN-Jun.

Conclusions: 1. MMP-7 is increased in both corpus and antrum in response to H pylori. 2. MMP-7 plays a role in H pylori stimulated cell migration. 3. H pylori acts via RhoA, Rac, AP-1 and NFκB to stimulate MMP-7 expression.

195 INTERACTION BETWEEN THE HELICOBACTER PYLORI VIRULENCE FACTORS CAGA AND VACA

R.J. Thomas, R.H. Argent, D.P. Letley, K.R. Hardie, J.C. Atherton.

Div. Gastroenterology and Inst. Infections and Immunity, Univ. Nottingham, UK

Introduction:H pylori strains possessing the cag Pathogenicity Island (cag PaI) or expressing active vacuolating cytotoxin (vacA) are associated with increased risk of peptic ulceration and gastric adenocarcinoma. Most strains have both or neither of these virulence factors. This is unlikely to be due to genetic linkage as H pylori recombine freely and the loci are distant on the chromosome. We hypothesised that the association was due to a gain of function from possessing both factors.

Methods: In two toxigenic cag+ strain backgrounds, 60190 and 84-183, we constructed separate isogenic mutants null for vacA, and for each of two genes encoded on the cag PaI, cagA and cagE. These and wild type strains were passaged in parallel to avoid differential laboratory adaption. For all strains, we assayed vacA activity on cultured epithelial cells by direct counting and by neutral red uptake (NRU) assay; cag-induced pro-inflammatory activity by interleukin-8 (IL-8) ELISA on supernatant from co-cultured epithelial cells; and cagA phosphorylation in epithelial cells by immunoblot with anti-phosphotyrosine antibodies. Results (mean±SD) were compared using t tests.

Results:cagA mutants had increased vacuolating activity compared with cagA+ parent strains, both by direct counting and by NRU assay (60190, 0.085±0.0073 adjusted OD units v 0.131±0.017, p=0.0001). cagE mutants had similar effects (60190, 0.085±0.0073 v 0.151±0.018, p=0.00005). As expected, the vacA mutants lacked vacuolating activity. In contrast, vacA mutants were similar to vacA+ parent strains in inducing IL-8 secretion from epithelial cells and in inducing cagA phosphorylation. As expected, the cagE mutant but not the cagA mutant had significantly reduced IL-8 stimulating activity (60190, 2145±140.89 pg/ml vs cagE, 662.5±90.85 p=0.000002) and the cagE mutant did not induce cagA phosphorylation in epithelial cells. Similar results were obtained in the strain 84-183 background.

Conclusion: CagA significantly downregulates vacA-induced epithelial cell vacuolation but vacA does not affect cagA or cagE-induced effects. We speculate that toxigenic H pylori strains depend on cagA signalling to control epithelial cell damage and prolong the epithelial cell interaction which is beneficial to H pylori.

196 THE IMPORTANCE AND GENETIC DETERMINANTS OF DIFFERENCES IN VACA TRANSCRIPTION BETWEEN HELICOBACTER PYLORI STRAINS

G.L. Narayanan, D.P. Letley, F. Aviles, J.R. Bebb, K.R. Hardie, J.C. Atherton.

Division of Gastroenterology and Institute of Infections and Immunity, University of Nottingham, Nottingham, UK

Introduction: Levels of vacuolating cytotoxin gene (vacA) transcription vary among laboratory strains of H pylori. We aimed to study the importance of vacA transcriptional differences among wild type strains and explore whether specific genetic determinants within vacA explained these differences.

Methods:H pylori single colonies were isolated from gastric biopsy specimens from 8 patients and their vacA type determined by allele-specific PCR. Total RNA was prepared for each isolate and for the control strains 60190 (Tox+; vacA s1/m1) and Tx30a (Tox; s2/m2). Level of vacA transcription was determined by densitometric analysis of RNA dot blots. Level of VacA protein production was determined by densitometric analysis of immunoblots of 24 hour broth culture supernatants. For each isolate, nucleotide sequences were determined for the region between vacA and its 5’ gene, cysS (the cysS-vacA intergenic region) and for 122 nucleotides of the 5’ end of vacA. Finally, the vacuolating activity of each strain was determined by cell culture assay.

Results:vacA transcription varied 6 fold among the clinical isolates. VacA RNA levels correlated closely with protein levels suggesting that variation in vacA production resulted mainly from variation in transcription. Level of vacA transcription was not related to vacA genotype (genotypes of wild type isolates were: 2 s1/m1; 4 s1/m2; 1 s2/m2; 1 s2/m1) nor was it associated with disease state or specific gastric histological features. Analysis of the cysS–vacA intergenic region revealed 70% identity across all 10 strains (including control strains 60190 and Tx30a). The –10 and –35 promoter regions were well conserved. Identity within the vacA 5’ untranslated region was 77%. Despite high variation within the cysS-vacA intergenic region, none of the genetic differences were associated with vacA transcription level. A potential stem-loop structure at the +4 site was conserved between all strains.

Conclusion: Level of vacA transcription appears to be the main determinant of vacA production. This is independent of vacA genotype and has no consistent association with nucleotide sequence differences in the vacA promoter region. Interestingly, a conserved, potential stem-loop region was identified which may regulate vacA expression through mRNA stability, and we are investigating this.

197 GLUCOCORTICOID (GC) ACCESS AND ACTION IN THE RAT COLON: EXPRESSION AND REGULATION OF MULTIDRUG RESISTANT GENE 1A (MDR1A), GLUCOCORTICOID RECEPTOR (GR), MINERALOCORTICOID RECEPTOR (MR) AND 11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE 2 (11βHSD2)

F.M. Moodie, J. Noble, J. Satsangi, J. Seckl.

GI and Mol. Endocrinology Units, Western General Hospital, Edinburgh University, UK

Background: Recent studies suggest that altered tissue sensitivity to glucocorticoids may underlie steroid insensitivity in patients with Ulcerative Colitis (UC). The expression of mdr1a, GR, MR and 11βHSD2 (a key enzyme in the inactivation of circulating glucocorticosteroids-GCs) along a healthy colon and the effect of altered levels of GCs on these key targets has not been studied in detail.

Methods: Wistar rats were either adrenalectomised (ADX), sham operated or had no surgery, and injected daily for one week with either dexamethasone (dex) (200ug/kg) or vehicle (2.5% et/OH). Rats were sacrificed, colons removed, sectioned and snap-frozen. Radioactive in situ hybridisation was used to analyse mRNA distribution of the above genes in colonic epithelial cells (ECs).

Results: The expression of mdr1a mRNA was increased in the proximal colon compared to the distal colon (p<0.001), especially within the upper crypts. Dex treatment decreased mdr1a mRNA in all ECs. After ADX, mdr1a expression was increased in the upper crypt ECs (p<0.001). Both GR and MR increased after ADX (p<0.001), although dex treatment only reduced GR mRNA levels (p<0.05). 11βHSD2 mRNA was highest in ECs in the upper crypts compared to the base of the crypts (p<0.01), however in the distal colon all ECs expressed high levels. After ADX 11βHSD2 mRNA increased both within cells in the crypt axis and along the colon.

Conclusion: Expression of mdr1a and 11βHSD2 varies within the crypt axis and along the colon, in contrast to GR and MR. In particular GC excess down-regulation of GR may reduce steroid sensitivity, but this is set against dex-induced reduction in MDR and 11βHSD2 which favours GC. GCs influence colonic expression of these genes, and may directly influence the efficacy of steroids in the treatment of UC.

Abstract 199

198 FATTY ACIDS ACT DIRECTLY ON INTRACELLULAR CALCIUM STORES TO RELEASE CALCIUM IN THE ENTEROENDOCRINE CELL LINE, STC-1

T. Hira T1,2, R.M. Case2, D.G. Thompson3, J.T. McLaughlin3.

1Graduate School of Agriculture, Hokkaido University, Japan; 2School of Biological Science & 3Gastrointestinal Sciences, Hope Hospital, University of Manchester, UK

Background and Aims: Nutrient-sensing mechanisms in the gut epithelium are poorly characterized. Fatty acids with chain length =12 carbon atoms increase intracellular Ca2+ levels to stimulate cholecystokinin (CCK) release from enteroendocrine cells. Using the CCK-producing enteroendocrine cell line, STC-1, and monitoring intracellular Ca2+, we investigated (a) whether classical intracellular pathways transduce the fatty acid signal, or (b) whether fatty acids act directly on intracellular stores to release Ca2+.

Methods: STC-1 cells were loaded with Ca2+ sensitive fluorescent dyes, and the intracellular Ca2+ level was measured ratiometrically using a fluorescence-microscope imaging system. (a) Intact cells (Fura-2-loaded cytoplasm) were exposed to fatty acid solutions (C8, C10, C12, C18:1) under several conditions, e.g. removal of extracellular Ca2+, or pretreatment with drugs that block candidate signal transduction pathways. (b) To examine direct effects of fatty acids on the intracellular Ca2+ store, Ca2+ release was measured in cells permeabilised using Streptolysin O. In permeabilised cells, the calcium store compartment was loaded with the lower affinity dye, Mag-fura-2.

Results: (a) Fatty acids (C12 and C18:1, but not C8 or C10) induced a dose-dependent increase in the cytosolic Ca2+ level, occurring in the presence or absence of extracellular Ca2+. Various G protein blockers, phospholipase inhibitors, protein kinase inhibitors, IP3 receptor antagonists and ryanodine receptor antagonists all failed to abolish fatty acid-induced Ca2+ responses. (b) In permeabilised cells, C12 induced release of stored Ca2+ in a dose-dependent manner, as did the physiological Ca2+ releaser IP3. The fatty acid chain length specificity in permeabilised cells was unchanged from intact cells.

Conclusions: Fatty acids (= C12) induce Ca2+ release from intracellular Ca2+ stores, independently of the presence of extracellular Ca2+. The data strongly suggest that fatty acids can act directly on the intracellular Ca2+ store to release Ca2+, by a novel nutrient sensing mechanism, operating independently of the major transcellular signal pathways.

Supported by the DDF Senior Fellowship (JMcL), and the Japan Society for the Promotion of Science (TH).

199 C DIFFICILE TOXIN A INDUCES RAPID AND SELECTIVE MONOCYTE CELL DEATH

K. Solomon1, R.A. Robins2, Y.R. Mahida1.

Divisions of 1Gastroenterology & 2Immunology University Hospital, Nottingham, UK

Introduction:C difficile causes an intense inflammatory colitis through the actions of secreted toxins A and B. Responses of immune cells to C difficile toxins appear to be pivotal in the progression of the disease. We have investigated the effects of toxin A on peripheral blood mononuclear cells (PBMNCs).

Methods: PBMNCs and purified monocytes were exposed to 1–1000 ng/ml of purified toxin A. Cells were studied by flow cytometry (FACS), Hoechst staining and electron microscopy (EM).

Results: In PMNCs, proportions of CD3 events (T cells) and CD19 events (B cells) did not change significantly in response to toxin A for up to 72 h. By contrast, CD14 events (monocytes) were lost in a dose and time dependent fashion (table). Hoechst staining of purified monocytes exposed to >10 ng/ml toxin A showed many cells with nuclear fragmentation characteristic of apoptotic cells. DNA fragmentation was confirmed by analysis of propidium iodide-stained monocytes exposed to high conc. of toxin A. EM of toxin A-exposed purified monocytes confirmed the presence of numerous apoptotic cells, but also of some cells showing features of necrotic cell death.

Conclusion: 1. Within 24 h exposure to high concentrations of C difficile toxin A, cell death is induced in peripheral blood monocytes but not T and B cells. 2. Toxin A-induced monocyte cell death occurs by apoptosis although in some, features of necrotic cell death were also seen. 3. Early loss of monocytes would impair host innate and adaptive immune responses to C difficile toxins.

200 PROGASTRIN STIMULATES MURINE COLONIC MITOSIS AFTER DNA DAMAGE VIA THE CYCLIN D-CDK4 COMPLEX

P.D. Ottewell1, A.J.M. Watson1, T.C. Wang2, G.J. Dockray3, D.M. Pritchard1.

Departments of 1Medicine and 3Physiology, University of Liverpool, UK; 2University of Massachusetts Medical Center, Worcester, MA, USA

Background and Aims: Transgenic mice which overexpress human progastrin (hGAS) are more susceptible to the induction of colonic aberrant crypt foci and adenomas by the chemical carcinogen azoxymethane than wild-type mice (FVB/N) and mice which overexpress amidated gastrin (INS-GAS). We have previously shown that 4.5h following DNA damage by 8Gy γ-radiation, colonic mitosis persists at significantly higher levels in hGAS compared with FVB/N or INS-GAS mice. We have now investigated the molecular mechanisms responsible for this continued mitosis.

Methods: Mice used were 10–12 week old hGAS and FVB/N. 4.5h following 8Gy γ-radiation colonic epithelial cells were harvested using a modified Weiser technique. Expression of cell cycle regulatory genes were assessed using a GEArrayTM pathway specific gene expression profiling system (SuperArray.com). Protein expression of p21WAF1/CIP1, p27KIP1, cdk6, cdk4 and cyclin D1 were analysed by Western blotting.

Results: Gene array analysis showed increased abundance of cdk4, cdk6 and cyclin D1 mRNA in hGAS colonic epithelial cells compared to FVB/N 4.5h following 8Gy γ-radiation. Western blots confirmed that the abundance of cdk4 protein was significantly higher in hGAS colonic epithelium compared to FVB/N and did not change following γ-radiation. In FVB/N mice, cyclin D1 protein expression was decreased in colonic epithelial cells 4.5h following 8Gy γ-radiation. However, no change was observed in cyclin D1 protein expression in hGAS colonic epithelial cells after this treatment. No significant differences were observed between hGAS and FVB/N mice in the levels of p27KIP1, p21WAF-1/CIP1 and cdk6 proteins in colonic epithelia.

Conclusion: (1) Progastrin overexpression results in increased expression of cdk4 in murine colonic epithelium. (2) The persistent colonic epithelial mitosis found in hGAS mice after γ-radiation is due to continued expression of cyclin D1.

201 ANALYSIS OF THE CC CHEMOKINE RECEPTOR CCR5 δ32 MUTATION IN BEHCETS’S DISEASE

X. Yang, T. Ahmad, F. Gogus, G. Wallace, W. Madanat, C.A. Kanawati, H. Yazici, S.E. Marshall, D.P. Jewell.

1Dept. of Gastroenterology, University of Oxford, Oxford, UK; 2Dept of Rheumatology, Cerrahpasa Medical Faculty, Istanbul, Turkey; 3Department of Ophthalmology, St Thomas Hospital, London; 4The Jordan Hospital, Amman, Jordan; 5St John Ophthalmic Hospital, East Jerusalem, Israel; 6Dept of Immunology, Wright-Fleming Institute, Imperial College, St Mary’s Hospital, London

Background: Behcet’s disease (BD) is a chronic multi-system inflammatory disorder characterized by recurrent oral and genital inflammation, uveitis and pathergy. Reports of an association between HIV-I infection and BD suggest that retroviruses may play a role in the pathogenesis of this disease. Chemokine receptor CCR5 acts as a co-receptor permitting entry of the HIV-1 virus into CD4-positive T cells. Here we investigate whether a 32bp deletion of this gene (CCR5 δ32), implicated in susceptibility to, and progression of, HIV disease, is associated with BD.

Methods: We studied 350 BD patients and 559 healthy ethnically matched controls recruited from 3 ethnic groups (Turkish: 109 BD, 96 controls; Palestinian 100 BD, 98 controls; UK white Caucasian: 131 BD, 365 controls). Genotyping for the CCR5 δ32 deletion was perfomed using PCR-SSP. Data were stratified by the presence of HLA-B*51, an allele previously shown to be associated with BD in the 3 ethnic groups.

Results: The CCR5 δ32 allele was significantly more common in UK white Caucasians than in either the Turkish or Palestinian cohorts (control allele frequency 14.2%, 4.2% and 0.5% respectively: P<1×10-8). No association was found with the CCR5 δ32 allele and BD in any of the three cohorts, even when stratified by the BD associated allele HLA-B*51.

Conclusions: The CCR5 δ32 allele is common in UK white Caucasians but rare in both Turkish and Palestinian populations. The CCR5 δ32 allele is not associated with Behcet’s disease in white UK Caucasians, Turkish, or Palestinian populations.

202 FUNCTIONAL CANNABINOID RECEPTOR EXPRESSION IN NORMAL HUMAN COLONIC EPITHELIUM

K.L. Wright1, N. Rooney2, J. Tate3, M.A. Feeney1,4, D.A. Robertson4, M.J. Welham1, S.G. Ward1.

1Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK; Departments of 2Pathology, 3Surgery, and 4Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK

Background and Aims: We investigated the presence of functional cannabinoid receptors in normal human colon and purified normal human colonic epithelial cells and human colonic epithelial cell lines, HT29 and CaCo.

Methods: The location of both the CB1 and CB2 receptors in normal human colonic biopsies was determined by immunohistochemistry. Purified normal human colonic epithelial cells from resected tissue and human colonic epithelial cell lines were treated with both synthetic and endogenous cannabinoids in vitro. Phosphorylation of the p42/44 mitogen-activated protein kinases (MAPKs) and glycogen synthase kinase (GSK)-3α/β was tested by Western immunoblotting.

Results: CB1 receptor expression was evident in colonic epithelium as well as smooth muscle and the submucosal myenteric plexus. The signal was particularly strong on the apical membrane of the epithelial cells. CB2 receptor expression was expressed on plasma cells and macrophages in the lamina propria. Both purified primary colonic epithelial cells and related cell lines responded to cannabinoids by activation of the p42/44 MAPK pathway and phosphorylation of GSK3α/β.

Conclusions: Given the accepted role of colonic epithelium in immune host responses and the inflammatory bowel diseases, the presence of functional cannabinoid receptors in normal human colon implies a role for the endocannabinoid system in normal gastrointestinal physiology, which may impact on mucosal immunity and modulate inflammation.

203 IMMUNITY TO CRYPTOSPORIDIUM PARVUM: A ROLE FOR IL-4 IN THE EARLY ADAPTIVE IMMUNE RESPONSE

S.A.C. McDonald, M. Bajaj-Elliott, P.T. Smith, A.B. Ballinger, V. McDonald.

Adult and Paediatric Gastroenterology, Barts and the London Queen Mary School of Medicine, London, UK

Introduction: Immunological control of Cryptosporidium parvum infection is dependent on a Th1 response with interferon-γ production. However, in a neonatal murine infection model we have shown that the Th2 cytokine IL-4 is also involved as BALB/c IL-4 knockout (KO) mice develop more intense oocyst shedding at the peak of infection (Day 7) than wild-type mice. Paradoxically, at this time, no increase in IL-4 mRNA could be detected in the intestines of wild-type mice. The aim of this study was to characterise further IL-4 involvement in the early priming of immunity to C parvum.

Methods: Neonatal BALB/c wild-type or SCID mice (which lack T and B cells) were infected at 4 or 7 days of age with a cervine isolate of C parvum and infections were subsequently measured microscopically from acid-fast stained smears of colonic contents. Murine recombinant IL-4 (1.0 and 0.75μg) was injected sc prior to infection and 6 h later. Rat anti-IL-4 IgG monoclonal antibody 11B11 (100μg) was injected prior to infection or 4 days post-infection.

Results: Injection of BALB/c mice with IL-4 decreased the level of C parvum reproduction on day 7 by a factor of 4.6. Administration of anti-IL-4 to BALB/c mice prior to infection—but not 4 days post-infection—increased susceptibility to infection. To determine if an IL-4 dependent innate mechanism was at work, SCID mice were given anti-IL-4 prior to infection, but this did not increase parasite reproduction.

Conclusion: IL-4 may act early in infection to boost the adaptive immune response to the intracellular mucosal parasite, C parvum.

204 TUMOUR NECROSIS FACTOR ALPHA MEDIATES RESISTANCE TO CRYPTOSPORIDIUM PARVUM INFECTION IN ENTEROCYTE CELL LINES BUT IS NOT REQUIRED FOR PROTECTION IN A MOUSE MODEL OF INFECTION

I.S. Lean, S.A.C. McDonald, V. McDonald (introduced by A.B. Ballinger).

Barts and the London Queen Mary’s School of Medicine and Dentistry, UK

Introduction: It has been suggested that TNF-α may play a role in immunity to infection by the intracellular parasite C parvum. Increased mucosal expression of this pro-inflammatory cytokine has been noted in the intestine of infected mice and humans whilst exogenous TNFα has been shown to reduce oocyst shedding in susceptible mouse models. However, its exact mechanisms of action and its importance in infection have yet to be established.

Methods: The human enterocyte cell lines HT 29, Caco2 and the mouse enterocyte cell line CMT 93 were pre-incubated for 24 h with TNFα prior to infection with C parvum. Cells were incubated for a further 24 h then fixed and stained in Giemsa and the number of parasites estimated per 50 high power fields. Using previously established in vitro models of infection, the possible mechanisms of action involved were also studied. Finally, C57BL/6 TNFα deficient neonatal mice were infected with C parvum and the outcome of disease measured.

Results: TNFα inhibited the development of C parvum in all 3 enterocyte cell lines. In HT 29 cells TNFα led to a 29 (+/-10)% decrease in parasite numbers in concentrations as low as 0.04 ng/ml. One mechanism identified was the inhibition of invasion. Tryptophan depletion and alteration of intracellular iron did not appear to be involved. In the murine model of infection however, no significant difference was noted in either the course of infection or parasite burden between controls and mice lacking the functional gene for TNFα.

Conclusion: We have demonstrated that TNFα inhibits C parvum development in enterocytes. We have also identified, for the first time, a possible mechanism of its action. Although TNFα appears to have an inhibitory effect in vitro, it does not appear to be essential for recovery against infection in an in vivo mouse model.

205 HEPATOCYTES BEARING THE PHILADELPHIA CHROMOSOME REVEAL TRANSDIFFERENTIATION OF BONE MARROW TO LIVER WITHOUT TRANSPLANTATION

R. Poulsom1, V. Campbell2, R. Jeffery1, T. Hunt1, M.R. Alison 2, A. Quaglia3, P.A. Dhillon3, N.A. Wright1,4.

1Histopathology Unit, Cancer Research UK, London; 2Imperial College, London; 3 The Royal Free Hospital, London; 4Department of Histopathology, Barts and the London, Queen Mary’s College of Medicine and Dentistry, London, UK

Introduction: Bone marrow cells can cross lineage boundaries and transdifferentiate into a wide variety of cell types. Such plasticity is seen after transplantation, if histocompatibility or genetic markers are used. Chronic granulocytic (myeloid) leukaemia (CML) is a neoplastic proliferation of an early haematopoietic stem cell that can differentiate into most haematopoietic lineages; ∼95% of CML cases have a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22); the abnormal chromosome 22 is known as the Philadelphia (Ph) chromosome. We sought to discover if Ph-positive bone marrow cells could transdifferentiate into hepatocytes in vivo.

Methods: We examined liver biopsies of 4 cases of CML (cytogenetics confirmed that 3 cases were Ph-positive and one had a different translocation) and a biopsy from an unrelated pathological case. The presence of normal or fused genes was visualised by fluorescence and confocal microscopy using directly-labelled DNA Fluorescence In Situ Hybridisation (FISH) probes. One thousand morphologically identified hepatocytes were examined from each case.

Results: In the two Ph-negative cases most hepatocytes had two separate orange signals and two separate green signals; no fusion of orange and green signals (producing an overlapping yellow signal) indicative of the BCR-ABL fusion gene was seen. In contrast, in the liver biopsies of the 3 Ph-positive cases of CML, the hybrid fusion gene was observed in 0.6, 1.0 and 1.3% of hepatocyte nuclei. Ph-positive hepatocytes were not morphologically abnormal.

Conclusions: Our study supports the concept that bone marrow cells transdifferentiate into hepatocytes in the human liver without the trauma and associated stresses of transplantation. The presence of the Philadelphia chromosome appears not to impede this lineage switch.

206 TOPOISOMERASE II ASSOCIATED PROTEINS IN COLON CANCER CELLS.

C.R. Barker1, S. Rackstraw1, J. Hamlett2, S.R. Pennington2, A.J.M. Watson1, J.R. Jenkins1.

Departments of 1Medicine and Anatomy and 2Cell Biology, University of Liverpool, UK

Background and Aims: Topoisomerase II is an essential enzyme required for the viability of all cells. It plays important roles in DNA replication, recombination, chromosome segregation, mRNA expression, and maintenance of the nuclear scaffold. In humans there are two isoforms of the topoisomerase II enzyme designated α and β. Numerous topoisomerase II inhibitors are used in the treatment of acute cancers for remission induction, as salvage therapy and conditioning therapy for bone marrow transplantation. However, these chemotherapeutic drugs are highly toxic and cells are developing drug resistance. Therefore, it is of great importance to investigate the effect of current topoisomerase II inhibitors upon cells with regard to protein-protein interactions with topoisomerase II. These interacting proteins represent potential chemotherapeutic targets.

Methods: Immunoprecipitations using anti-human topoisomerase II rabbit polyclonal antibodies were performed on proteins extracted from a pair of human colon cancer cell lines, HCT116 (WT, p53 KO, isogenic), following treatment with etoposide (VP16), a topoisomerase II inhibitor. Samples were subjected to 1 and 2 dimensional electrophoresis and associated proteins identified by MALDI-TOF mass spectrometry and confirmed by counter-immunoprecipitations. Newly identified interactions were then disrupted using chemical inhibitors in growth inhibition assays. The effect of the drugs, when used in combination with topoisomerase II inhibitors was also tested.

Results: Mass spectrometry and subsequent counter-immunoprecipitations identified heat shock protein 90 (HSP90) to be specifically associated with topoisomerase II. The use of VP16, a topoisomerase II poison, and geldanamycin (GA), an HSP90 inhibitor, when used in combination produced synergistic growth inhibition at concentrations where there is little or no effect when they are used individually.

Conclusions: (1) There is a direct physical interaction between topoisomerase II and HSP90. (2) Upon treatment with VP16 and GA in combination a syngeristic effect upon growth inhibition is observed. (3) This is independent of p53 status. (4) These data demonstrate the possibility of a new combination therapy for the treatment of colon cancer.

207 THE MATURATION AND CYTOKINE EXPRESSION OF HUMAN DENDRITIC CELLS ARE DIFFERENTIALLY MODULATED BY PROBIOTIC BACTERIA

A.L. Hart1,2, M.A. Kamm2, S.C. Knight1, A.J. Stagg1.

1APRG, Imperial College; 2St Mark’s Hospital, Harrow, UK

Introduction: Dendritic cells (DC) are antigen-presenting cells present throughout the gastrointestinal tract that interact with bacteria and determine the subsequent T cell response. Probiotic bacteria are effective in the treatment of some inflammatory bowel diseases. Probiotic bacteria modulate immune function, but the effect of these bacteria on DC is unclear. This study aimed to determine whether probiotic bacteria influence DC phenotype and cytokine production.

Methods: DC were identified by multi-colour flow cytometry as an HLA-DR+ lineage- (CD3-, CD14-, CD16-, CD19-, CD34-, CD56-) population in blood. Cell wall fractions of the 8 probiotic bacteria present in the probiotic combination VSL#3 in addition to Streptococcus faecium, Escherichia coli Nissle strain and lipopolysaccharide (LPS) were prepared by sonication and centrifugation and cultured with whole blood. Changes in maturation and co-stimulatory markers (CD80, CD86, CD83 and CD40) and cytokine production (IL-12 and IL-10) were analysed by flow cytometry.

Results: The combination of organisms in VSL#3 downregulated IL-12 and upregulated IL-10 production by DC in a dose-dependent manner. All the individual components downregulated IL-12 production, but only the bifidobacteria strains enhanced IL-10 production. All the bacteria upregulated all the co-stimulatory and maturation markers with the exception of the bifidobacteria strains which downregulated CD80 expression. An anti-IL-10 antibody did not prevent this effect. LPS had opposing effects to VSL#3, enhancing IL-12 and decreasing IL-10 production. Co-culturing VSL#3 with LPS indicated cross-regulation of the DC cytokine response. The enhanced IL-12 seen with LPS alone was reduced in the presence of VSL#3 but IL-10 production seen with VSL#3 alone was maintained in the presence of LPS.

Conclusions: Probiotic bacteria differentially activate DC in vitro in a manner that may favour an anti-inflammatory T cell response.

208 EFFECT OF COX-2 INHIBITION ON SPECIFIC IMMUNE RESPONSES TO COLON CARCINOMA CELLS

A.J. Walmesley1, S.E. Christmas2, A.J.M. Watson1.

1Department of Medicine and 2Department of Immunology, University of Liverpool, UK

Background: We have previously demonstrated effective suicide gene therapy of MC26 colorectal cancer cells in vivo using the HSV-TK/GCV model system in mice. This therapy achieves successful tumour regression, possibly involving the immune response. This may be enhanced by inhibition of the cyclooxygenase-2 (COX-2) enzyme, which produces potentially immunosuppressive prostaglandins in vivo (e.g. PGE2). COX-2 is also overexpressed in colon tumours, suggesting it has a role in colon carcinogenesis.

Hypothesis: Selective COX-2 inhibition will augment the specific host immune response to colon carcinoma cells.

Methods: The effect of a COX-2 inhibitor was studied on murine T-cell proliferation in response to mitogen and tumour cells. Splenocytes were isolated from control mice and mice that had undergone tumour induction with MC26 cells and regression following HSV-TK suicide gene therapy without tumour recurrence. The erythrocytes were lysed and the splenocytes were treated with either Con-A (2μg/ml) or irradiated MC26 cells (1:100 ratio of irradiated MC26: splenocytes), and the COX-2 inhibitor Rofecoxib. Proliferation was measured by 3H-thymidine incorporation after 3 or 7 days.

Results: Rofecoxib increased splenocyte proliferation in a mitogenic response over 3 days by 50% at concentrations above 1μM, which are known to inhibit PGE2 production by >90% in vivo. Irradiated MC26 cells induced a 60% increase in control splenocyte proliferation over 7 days, enhanced to 100% by 1μM Rofecoxib. Splenocytes from treated mice showed a 300% increase in proliferation in response to irradiated MC26 cells also over 7 days, amplified to 450% by 1μM Rofecoxib.

Conclusions: A specific immune response is generated to tumour cells in mice that have undergone HSV-TK suicide gene therapy. COX-2 inhibition enhances this effect, possibly by inhibition of PGE2 production.

209 THE IDENTIFICATION OF P53 MUTATIONS IN METAPLASTIC BARRETT’S TISSUES: A PROGNOSTIC MARKER?

G.J.S. Jenkins, S.H. Doak, A.P. Griffiths, J.M. Parry, J.N. Baxter.

Swansea Clinical School, University of Wales Swansea, Swansea, UK

We have analysed multiple biopsies from over 40 Barrett’s patients with a range of pre-malignant conditions, for the presence of p53 mutations. We have employed a molecular technique, the Restriction Site Mutation (RSM) assay to detect the presence of low-frequency p53 mutations. The advantage of employing the RSM methodology in this study is that microdissection or flow cytometry of the tissue is not required to enrich the mutant cell population. Instead, the RSM methodology selects mutated sequences at the DNA level. Hence p53 mutations are detectable, even if the cells carrying such mutations are present within an excess of normal cells (sensitivity 1 mutant sequence amongst 10 000 normal sequences).

Our results have shown that approximately 30% of our metaplastic and Low Grade Dysplastic Barrett’s patients carry p53 mutations. This figure increases to 40% with High Grade Dysplastic patients. These p53 mutations were mainly located in exon 7 of the p53 gene (specifically at codon 248) and resemble the type of mutation (GC to AT) previously shown by ourselves to be induced by oxidative DNA damage. This strengthens the argument that inflammatory mediated release of Reactive Oxygen Species (ROS) may play a role in tumour development in Barrett’s oesophagus. Interestingly, our study has identified a subset of Barrett’s patients carrying early p53 mutations, before clonal expansion has occurred. The key question we would like answered is—do these patients exhibit an increased rate of adenocarcinoma development compared to the patients without p53 mutations? If so, then the early detection of p53 mutations may represent a useful prognostic marker in Barrett’s oesophagus. Only long term follow up of this cohort of patients will answer this question. Through concurrently studying the chromosomal instability of these same Barrett’s patients, we have shown a lack of correlation between p53 mutation and chromosomal instability. This contradicts suggestions that the p53 protein is essential for maintaining chromosomal integrity. We are currently examining the status of other (p53 regulated) proteins in these samples to determine if the loss of p53 related proteins leads to chromosomal instability.

210 FIBROBLAST-DERIVED MATRIX METALLOPROTEINASES ACTIVATE A POTENT NEUTROPHIL CHEMOATTRACTANT FROM INTESTINAL EPITHELIAL CELLS

L. Kruidenier1, T.T. MacDonald2, I.R. Sanderson1.

1Department of Adult and Paediatric Gastroenterology, Barts and The London, Queen Mary School of Medicine and Dentistry, London; 2Division of Infection, Inflammation, and Repair, School of Medicine, University of Southampton, UK

The upregulation of matrix metalloproteinases (MMPs) in the inflamed gut has mainly been associated with mucosal degradation and ulceration. However, their in vitro capacity to specifically cleave inflammatory mediators indicates that MMPs may also have a profound immunoregulatory impact.

In this study, we assessed whether MMPs proteolytically modify intestinal epithelial chemokine signalling. Fully differentiated CaCo-2 cells were grown on filters, stimulated with IL-1β, and exposed basolaterally to nanomolar concentrations of activated MMP-3. Chemotaxis assays of the conditioned media revealed that MMP-3 dose-dependently induced the neutrophil, but not monocyte, chemoattractant capacity of CaCo-2 cells. A similar response was obtained when these cells were co-cultured with IL-1β-stimulated colonic fibroblasts (CCD-18co), which expressed various MMPs, including MMP-3, -10, and -12. The addition of doxycyclin, a broad-spectrum MMP inhibitor, disrupted the CaCo-2 chemotactic response. The principal mediator of these protease-related effects was identified as the potent neutrophil chemokine neutrophil activating peptide 2 (NAP-2, CXCL7), a cleavage product of biologically inactive platelet basic protein (PBP). Antibodies against NAP-2 greatly inhibited the MMP-induced chemotactic response, and PBP mRNA and protein was detected in stimulated CaCo-2, but not in CCD-18co cells.

Our data suggest that fibroblast-derived MMPs proteolytically activate the neutrophil chemokine NAP-2 from the intestinal epithelium, adding a novel dimension to MMP function and to our understanding of the pathogenesis of intestinal inflammation.

211 PRELIMINARY ASSESSMENT OF P53 AND RELATED GENES AS THERAPIES FOR OESOPHAGEAL CANCER

G.F. Abouda, E. Pohler, J.F. Dillon.

Department of Molecular and cellular Pathology, Ninewells Hospital, University of Dundee, UK

Background: Oesophageal cancer is a disease with a dreadful prognosis and a rapidly rising incidence. New therapies for treatment of this cancer need to be evaluated and in this area gene therapy may have a role to play.

Aims: Using liposomes as a transfer agent, to assess the expression of the p53 gene (human and mouse wild type and mutants) in oesophageal cancer cell lines, estimating the degree of apoptosis in these cell lines. In addition, to determine the effect the mouse Scottin gene, a p53 inducible pro-apoptotic gene, on cell survival.

Method: Two squamous cell carcinoma cell lines (OE21 and KYSE30), and two adenocarcinoma cell lines (OE33 and OE19) were transfected with human and mouse wild type p53 constructs. Following 48 hours incubation, cells were analysed by Western blotting (to examine expression of p53) and FACScan (to detect apoptosis). Cells were also transfected with mutant forms of both the human and mouse p53 genes, and the effects of these on the cell survival compared to the wild types. Finally, cells were transfected with the mouse Scottin gene and the consequences of its expression determined.

Results: p53 expression was found to be higher in OE33 and OE21 cells by Western blotting than in KYSE30 and OE19. FACScan analysis revealed high levels of apoptosis were achieved in OE21 and OE 33 cells following wild type p53 expression. Apoptosis was also observed in KYSE30 cells but to a lesser degree. In all three of these cell lines, this effect was more pronounced using the mouse p53 gene compared to the human p53. The levels of apoptosis observed were increased further in OE21, OE33 and KYSE30 cells following expression of the mutant forms of both human and mouse p53. However, highest degrees of apoptosis were obtained in these cell lines with expression of the Scottin gene. No apoptosis was seen following expression of any of the constructs in OE19 cells.

Conclusion: We have succeeded in inducing apoptosis in three different oesophageal cancer cell lines following introduction of wild type p53 genes. This effect was enhanced using mutant forms of p53 and was greatest following expression of the Scottin gene. Having established a successful gene transfer model for induction of apoptosis in cell lines, it would be of great interest to test this on human oesophageal biopsies to explore its potential as a future gene therapy.

212 BREAST MILK LACTOFERRIN REGULATES GENE EXPRESSION BY BINDING TO EXTRACELLULAR PRO-INFLAMMATORY CPG DNA SEQUENCES, BUT NOT NUCLEAR DNA

P. Mulligan1, N.R.J. White1, G. Monteleone2, P. Wang1, J.W. Wilson1, Y. Ohtsuka3, I.R. Sanderson1.

1Department of Adult and Paediatric Gastroenterology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Dominion House, 59 Bartholomew Close, London EC1A 7BE, UK; 2Unita di Gastroenterologia, Dipartimento di Medicina Interna, Universita di Roma Tor, Vergata, Roma, Italy; 3Department of Pediatrics, Juntendo University, Tokyo, Japan

We hypothesised that lactoferrin (LF) alters the expression of immune genes in the infant intestine by binding to DNA. We examined both the direct and indirect effects on immune gene regulation of its binding to DNA. Two biologically relevant compartments were studied: binding to pro-inflammatory, bacterial DNA sequences (CpG motifs) in the extracellular environment, and binding to gene promoters in the cell nucleus. LF inhibited CpG motif-induced NF-κB activation and IL-8 and IL-12 gene transcription in B-cells at LF concentrations greater than 0.5 μM. However, the intestinal epithelial cell lines, Caco-2 and HT-29, were unresponsive to CpG motifs under a variety of conditions. Using three independent techniques, we demonstrated that LF does not regulate reporter gene transcription, nor does it bind specifically to putative DNA response elements. We conclusively showed no LF localisation into enterocyte and lymphocyte nuclei by tagging the LF with green fluorescent protein. Purification of intact, DNA-binding LF from the urine of preterm infants has been previously described, indicating significant survival and transcytosis or leakage of LF across the infant intestine. This suggests that lymphocytes in the lamina propria and Peyer’s patches of the infant intestine are exposed to high concentrations of LF. We conclude that DNA binding by LF extracellularly, but not in the nucleus, is an important mechanism of immune gene regulation. Such a mechanism is likely to function in B-cells exposed to bacterial DNA in the lamina propria and Peyer’s patches of the breast-fed infant.

213 THE EFFECT OF SIROLIMUS ON THE NEUTROPHIL OXIDATIVE BURST

I. Gee, A. Trull, S. Charman, G.J. Alexander.

Departments of Medicine & Biochemistry, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ; MRC biostatistics unit, Cambridge

Introduction: Sirolimus is an immunosuppressive drug that is being used increasingly in transplant recipients. It has been observed that some patients develop bacterial sepsis during treatment.

Methods: We have developed a physiological in vitro model to investigate the effects of therapeutic concentrations of sirolimus on the neutrophil oxidative burst (NOB), a mechanism by which immunity to bacterial and fungal infection may be impaired. Whole blood from 24 healthy subjects was equilibrated with 0, 1, 5, 10 and 50μg/L sirolimus or 60mg/L propofol (a known inhibitor of neutrophil function) for 2 hours at 37°C. The cells were washed and the neutrophils stimulated with phorbol myristate acetate (PMA). NOB was measured by flow cytometry using the fluorescent marker dichlorofluorescein.

Results: A significant mean inhibition of NOB (95 % C.I. of mean % inhibition did not overlap zero) was found with 50mg/L sirolimus (mean 6.3; C.I. 1.5, 11.1 %) and 60mg/L propofol (mean 5.1; C.I. 0.4, 9.8 %). 10mg/L sirolimus also inhibited N.O.B. (mean 4.6; C.I. –1.3, 10.6; NS) but inhibition was <1.5% at lower concentrations. Repeated measures ANOVA confirmed a linear relationship between sirolimus concentrations and inhibition of NOB (P=0.01). The two hour incubation period had no effect on red blood cell integrity or white cell viability in vitro.

Conclusion: Sirolimus had a dose-dependent inhibitory effect on NOB but this was not significant at low therapeutic concentrations. This may partly explain the predisposition to sepsis in patients receiving sirolimus. The 2 hour exposure of blood cells to sirolimus in these studies probably underestimates the in vivo effects of sirolimus and we are currently investigating the effect of chronic oral dosing with sirolimus on NOB

Inflammatory bowel disease posters: 214–258

214 MICROBIAL MANNAN SUPPRESSES NEUTROPHIL AND MONOCYTE RESPIRATORY BURST AND ENHANCES MYELOPEROXIDASE RELEASE: POSSIBLE MECHANISMS FOR GRANULOMATOUS INFLAMMATION IN CROHN’S DISEASE

C.M. Mpofu, J.M. Rhodes, B.J. Campbell, S.W. Edwards1.

Department of Medicine and 1School of Biological Sciences, University of Liverpool, Liverpool L69 3GA, UK

Background: Crohn’s disease (CD) patients commonly have serum antibodies to baker’s yeast (Saccharomyces cerevisiae). The epitope for this antibody is oligomannan, which is present in bacterial and yeast cell walls. CD-like intestinal lesions occur in chronic granulomatous disease, a condition caused by a defect in phagocyte function. We previously reported that oligomannan inhibits the neutrophil respiratory burst in a dose-dependent manner, with a maximal effect at 1mg/ml. We have now assessed the effect of oligomannan on the respiratory burst in peripheral blood monocytes and have also assessed its effect on myeloperoxidase release from neutrophils.

Methods: Neutrophils were purified from heparinised venous blood by a one-step centrifugation method using Neutrophil Isolation Medium. Monocytes were isolated by Ficoll Hypaque separation followed by adherence to plastic wells. The effect of S cerivesiae oligomannan on phorbol ester (PMA) induced-respiratory burst of peripheral blood monocytes was measured by isoluminol-amplified chemiluminescence. Oligomannan dose of 1mg/ml was used. Granulocyte-macrophage colony stimulating factor (GMCSF) primed neutrophils in the presence/absence of oligomannan were treated with cytochalasin B (CB) and fMLP to stimulate secretion of myeloperoxidase which was then quantified by immunoblotting.

Results: Oligomannan (1mg/ml) inhibited peak PMA-stimulated isoluminol-amplified chemiluminescence in peripheral blood monocytes by (mean ± SD) 52.8% ± 6.0. Isoluminol largely measures extracellular superoxide secretion, which is dependent on NADPH oxidase activity. Oligomannan (1mg/ml) increased myeloperoxidase secretion by (mean ± SD) 74% ± 8.4 in fMLP/CB stimulated, primed neutrophils. Secreted myeloperoxidase has been shown to cause local tissue damage that is independent of the phagocyte respiratory burst.

Conclusion:S cerivesiae oligomannan causes inhibition of the PMA-induced respiratory burst in phagocytic cells but also causes increased secretion of MPO. These results further support the hypothesis that microbial cell wall oligomannan is capable of causing an intramucosal defect in phagocyte function, leading to the granulomatous inflammation that typifies CD.

215 FAECAL CALPROTECTIN AS AN AID TO DIAGNOSIS IN INTESTINAL INFLAMMATION

S. Dolwani1, J. Wassell2, M. Metzner1, H. Losty2, A. Yong3, B.W. Lawrie3, A.B.Hawthorne1.

1Departments of Gastroenterology, 2Clinical Biochemistry & 3Radiology, University Hospital of Wales, Cardiff, UK

Background/aims: The neutrophil derived protein Calprotectin has previously been found to be raised in intestinal inflammatory conditions. We aimed to evaluate the discriminant value of a stool calprotectin assay in predicting the likelihood of an abnormal result on a Barium follow through examination (BaFT) in patients being investigated for abdominal pain and or diarrhoea.

Patients and Methods: Patients being investigated for abdominal pain and or diarrhoea and undergoing a BaFT as part of their workup (n=65) provided a one off stool sample for estimation of calprotectin level. This was compared with patients with known active Crohn’s disease (positive controls), normal healthy volunteers & patients with irritable bowel syndrome (IBS) as negative controls. The biochemist performing the assay was blinded to all clinical details. Other clinical and laboratory indices such as ESR, CRP, and CDAI were assessed concomitantly.

Results: The median level of calprotectin in the active Crohn’s group (n=23) was 226.5 μg/g of stool compared to a median of 17.3 in the group with IBS (n=27) and 10.9 in normal healthy controls (n=24).

A sensitivity of 94%, specificity of 68.7% & negative predictive value of 97% for the stool calprotectin assay. Of the 6 patients with IBD & a normal barium follow through, 5 had colonic Crohn’s disease & 1 had ulcerative colitis on further investigation.

Conclusion: Patients being investigated in a gastroenterology clinic for diarrhoea and or abdominal pain do not need small bowel radiology to rule out Crohn’s disease if their stool calprotectin level is <60 μg/g.

Abstract 215

216 INNATE IMMUNOGENETICS AND INFLAMMATORY BOWEL DISEASE (IBD)

D.P.B. McGovern, D.A. van Heel, K. Negoro, T. Ahmad, D.P. Jewell.

Wellcome Trust Centre for Human Genetics and Gastroenterology Unit, The University of Oxford, Oxford, UK

Background: The identification of NOD2(CARD15) as a susceptibility gene for Crohn’s disease (CD) confirms the role of innate immunity in IBD. More recently a haplotype at IBD5 (Ch 5) has shown association with CD.

Aims: To identify and test innate immunity single nucleotide polymorphisms (SNPs) for association with IBD.

Methods: Interesting (positional and functional) SNPs were identified from databases or by direct sequencing. A two-stage approach was adopted to overcome problems of multiple testing. Stage 1: Case-control analysis (191 CD, 247 ulcerative colitis (UC) and 240 controls (HC)). Stage 2: Positive results from stage 1 to be confirmed by the transmission-disequilibrium test (TDT) (556 IBD (294 CD and 252 UC) trios). All results were stratified by phenotype and NOD2/IBD5 genotype.

Results:Case-control analysis(all % allele frequency (p value)): Toll-like receptor (TLR) 2, 3 intronic SNPs and 1 microsatellite marker, no IBD association. TLR2 ‘bacterial hyporesponsive’ SNP (R753Q), HC 4.0, IBD 2.5 (0.13), UC 2.4 (0.12) and CD 2.6 (0.25). Fulminant UC requiring surgery (130 cases) 4.3%, ‘mild’ UC not requiring surgery (135 cases) 0.4% (0.0032)(OR 0.08). TLR4 SNP (bronchial hypo-responsiveness) HC 4.4, CD 3.1 (0.34) and UC (4.9 (0.71). Two novel MAL (TIRAP) SNPs, 4 novel Triggering receptor expressed on myeloid cells -1 (TREM-1) SNPs, TLR9 SNP, 4 P2X7 SNPs, and lactoferrin SNP, all - no association seen with any IBD geno- or phenotype. TDT analysis(transmissions/non-transmissions): TLR4 SNP (A896G): IBD 64/44 (0.054), UC 11/11, CD 36/23 (0.098), CD NOD2 negative 28/18 (0.14).

Conclusions: These innate immunity SNPs are not significantly associated with IBD. The colectomy, non-colectomy TLR2 association suggests that intact innate immunity is needed for the development of fulminant UC. Studies of other SNPs in the innate immune system and their role in IBD are warranted.

Abstract 218

217 A DISTINCT SUBSET OF CHEMOKINES, INDUCED IN COLONIC EPITHELIUM BY IL-1β, DOMINATES THE MUCOSAL CHEMOKINE RESPONSE IN ULCERATIVE COLITIS

J. Puleston1, M. Cooper2, S. Murch1, S. Makh2, P. Ashwood1, F. Torrente1, A. Bingham2, H. Green2, P. Moss2, A. Dhillon1, R. Gelinas2, R. Pounder1, A. Platt2.

1Royal Free Hospital, London, NW3 2QG, UK; 2Celltech R&D Ltd, Cambridge, CB1 6GS, UK

Purpose: Inflammatory bowel disease (IBD) is characterised by intense mucosal recruitment of activated leukocytes. As chemokines determine inflammatory leukocyte recruitment and retention, we compared expression of the entire chemokine family within colonic mucosa from IBD patients and uninflamed controls.

Methods: A microarray, representing every member of this superfamily and their cognate receptors, was hybridised with probes derived from colonoscopic biopsies. The array levels were correlated with histopathological inflammatory scores and expression of their cognate receptors by quantitative PCR and immunohistochemistry. Flow cytometry was performed on mucosally-derived colonic cells. Caco-2 and keratinocyte cell lines were stimulated with IL-1β and TNF-α, and analysed using the same microarray.

Results: A distinct subset of chemokines, consisting of CXCLs 1–3 and 8 and CCL20, was upregulated in active colonic IBD, compared to uninflamed areas or tissue from controls. This corresponded to histopathological scores. Increased expression of their cognate receptors, CXCR1, CXCR2, and CCR6, was confirmed by quantitative PCR and immunohistochemistry. Flow cytometric revealed an increase of CCL20 expression on epithelial cells in IBD specimens, particularly in severe disease. An identical chemokine response was induced in Caco-2 cells by stimulation with IL-1β, but not TNF-α. By contrast, IL-1β and TNF-α were synergistic in keratinocytes.

Conclusion: IL-1β appears to be the pivotal mediator of an epithelial response that dominates the mucosal chemokine environment in ulcerative colitis. These data suggest several new therapeutic targets for IBD, as well as identifying a previously unrecognised co-ordinated epithelial chemokine response.

218 NOD2/CARD15 MUTATIONS IN THE EXTRAINTESTINAL MANIFESTATIONS (EIMS) OF INFLAMMATORY BOWEL DISEASE

T.R. Orchard1,2, T. Ahmad2, D.P. Jewell2.

1St Mary’s Hospital, London, UK; 2Gastroenterology Unit, University of Oxford, UK

Introduction: There has been much interest in the role of the innate immune system in inflammatory rheumatological conditions. CARD15 plays an important role in triggering an immune response to bacterial LPS, and polymorphisms in this gene are associated with Crohn’s disease (CD). We have previously shown a genetic predisposition to EIMs, and have demonstrated abnormalities in the humoral immune response to enterobacteria in patients with arthritis associated with IBD. This study was performed to investigate whether polymorphisms in the CARD15 gene are associated with the presence of EIMs in IBD.

Methods: DNA was obtained from 104 patients who had suffered Type 1 or Type 2 peripheral arthritis, erythema nodosum (EN) or uveitis (60 Crohn’s disease and 44 ulcerative colitis). Genotyping for the 3 common polymorphisms of CARD15 (Arg702Trp, Gly908Arg, and Leu1007fsinsC) was undertaken by SSP PCR, and allele frequencies were compared with 354 healthy controls and 244 CD patients.

Results: Allele frequencies (%) are shown in the table below:

EIMs were significantly associated with CARD15 polymorphisms, when compared to controls, particularly type 1 arthritis and EN. Further analysis was undertaken by IBD diagnosis, and the association between CARD15 polymorphisms and EIMs was seen in both UC and Crohn’s patients.

Discussion: CARD15 polymorphisms may be important in determining the EIMs of IBD, suggesting that bacteria may trigger them. Further studies are required to confirm these associations, and to investigate the interaction with previously described HLA associations.

Abstract 219, Table 1

Allelic frequencies for CD, UC, and HC. Results are expressed as percentages and comparisons are between CD and HC.

Abstract 219, Table 2

Genotype frequencies for CD, UC and HC (%). Genotypic status did not correlate with any phenotypic characteristics.

219 MUTATIONS OF TOLL-LIKE RECEPTOR 4 AND CD14 GENES ARE NOT ASSOCIATED WITH SUSCEPTIBILITY OR DISEASE BEHAVIOUR IN INFLAMMATORY BOWEL DISEASE.

E.R. Nimmo, I.D.R. Arnott, H.E. Drummond, E. MacKinlay, J. Morecroft, J. Hutchinson, J. Satsangi.

Gastrointestinal Unit, University Department of Medical Sciences, Western General Hospital, Edinburgh, UK

Introduction: Following the identification of the NOD2 gene in Crohn’s disease (CD), much interest has been focused on other aspects of the innate immune system. CD14 acts as a receptor for bacterial LPS. Toll-like receptor 4 (TLR4) is involved in signal transduction of the LPS/CD14/MD-2 complex with resulting activation of NFκB. We aimed to assess the frequency of recently described germline mutations of the CD14 and TLR4 genes in a cohort of Scottish patients with CD.

Methods: Genomic DNA was extracted from venous blood of 489 patients with inflammatory bowel disease (IBD) (249 CD and 240 UC) and 182 healthy controls (HC). PCR based genotyping was carried out using allele specific primers designed to identify mutations of the TLR4 (A299G) and CD14 genes (T-159C).

Results: Results are displayed in tables 1 and 2.

Conclusion: Although these are plausible candidate genes, the mutations analysed in the TLR4 and CD14 do not appear implicated in IBD. Detailed assessment of other allelic variants of these and other functional/ positional candidate genes are in progress.

220 SEQUENCE VARIATION IN THE CARD15 (NOD2) GENE AND SUSCEPTIBILITY TO CROHN’S DISEASE

K. King1, C. Onnie1, M. Mirza1, S. Fisher1, A. Cuthbert1, S. Sanderson2 A. Forbes3, J. Mansfield4, C. Lewis1, C. Mathew1.

1Division of Medical and Molecular Genetics and Department of Medicine, Guy’s King’s and 2St Thomas’ School of Medicine, London; 3St Mark’s Hospital, Harrow, UK; 4Department of Gastroenterology, School of Clinical and Medical Sciences, University of Newcastle, UK

Crohn’s disease (CD), a subtype of inflammatory bowel disease (IBD), is a complex disorder, with both genetic and environmental aetiology. Three sequence variants in the CARD15 gene have recently been shown to be associated with susceptibility to CD. There is also evidence of an excess of rare CARD15 variants in CD, but whether these are true disease susceptibility alleles (DSAs) is unknown. Clarification of the status of rare variants would facilitate a more accurate assessment of the extent of the contribution of CARD15 to Crohn’s disease, and of the likely genetic model for the effect of CARD15.

In order to investigate the contribution of rare variants to CD we have selected 100 Crohn’s patients who are heterozygous for one of the three associated DSAs (R702W, G908R, or 1007fs) for comprehensive mutation screening. The coding sequence of CARD15 was screened by denaturing HPLC for mutations to identify both common polymorphisms and rare variants. 21 patients had one or more additional rare CARD15 mutations, 10 of which were non-synonymous. One of these was a novel nonsense mutation that would produce a truncated protein (R896X), and four (R235C, S431L, A612T, R1019G) were amino acid substitutions that were predicted to alter the function of the protein. The finding that only a minority of patients who are heterozygous for common disease susceptibility alleles in CARD15 have a second mutation is consistent with the gene dosage model for CARD15 in Crohn’s disease.

221 GENETIC EVIDENCE FOR INTERACTION OF THE 5Q31 CYTOKINE LOCUS AND THE CARD15 GENE IN CROHN’S DISEASE

M.M. Mirza1, S.A. Fisher1, K. King1, A.P. Cuthbert1, J. Hampe2, J. Sanderson3, J. Mansfield4, P. Donaldson4, A. Macpherson5, A. Forbes6, S. Schreiber2, C.M. Lewis1, C.G. Mathew1.

1Dept of Medical & Molecular Genetics, GKT School of Medicine, Guy’s Hospital, London, UK; 2Dept of General Internal Medicine, Christian-Albrechts-Universität, Kiel, Germany; 3Gastroenterology Dept, St Thomas’s Hospital, London, UK; 4Dept of Gastroenterology & Hepatology, University of Newcastle upon Tyne, Royal Victoria Infirmary, UK; 5Inst. of Experimental Immunology, Universitätsspital, Zurich, Switzerland; 6St Mark’s Hospital, Northwick Park, Middx, UK

A common haplotype containing 11 single nucleotide polymorphisms (SNPs) and spanning 250kb in the cytokine gene cluster on chromosome 5q31 has recently been reported to be strongly associated with Crohn’s disease (CD) in Canadian families. We analysed 2 SNPs from this haplotype (C2063G and C2198G) in 267 individuals, and found them to be in strong linkage disequilibrium (D′=0.91). The C2063G SNP was then genotyped in Northern European IBD families which contained a total of 511 offspring affected with CD and 320 with ulcerative colitis (UC). Excess transmission of the 2063G allele was observed in CD (p=0.011) but not in UC. Genotyping of C2063G in an independent set of unrelated British cases with CD (n = 684) and UC (n = 388) and in 701 British controls showed that the 2063G allele was present at a significantly higher frequency in CD cases than in controls (p=0.008), but was not increased in UC. However, the increase in disease risk was small (odds ratio 1.49 for homozygotes, 95% CI: 1.11–2.0). The disease risk haplotype frequency was significantly elevated in 943 CD patients who also carried mutations in the CD susceptibility gene CARD15 (p=0.0018). Kaplan-Meier survival analysis of age of disease onset showed a significantly earlier onset in homozygotes for the 5q31 risk haplotype (p=0.0019). These findings suggest that genetic variants at the 5q31 (IBD5) locus may hasten the onset of Crohn disease, and co-operate with CARD15 in disease causation.

222 PHOSPHOINOSITIDE SIGNALLING IN INFLAMMATION AND COLORECTAL CANCER

C.L.E. Osborne, O. Ogunbiyi, S. Keshav.

Departments of Medicine and Surgery, Royal Free and University College Medical School, London, UK

Background and Aims: Phosphoinositide-3-kinase gamma (PI-3-Kγ) is a lipid kinase activated by G-protein coupled receptors (GPCR) to regulate such cellular functions as chemotaxis, cell proliferation and apoptosis. Transgenic mice that lack PI-3-Kγ are immune suppressed and develop colorectal cancer (CRC), and mice lacking the G-protein subunit Gαi2, that may interact with PI-3-Kγ develop ulcerative colitis (UC) and CRC. Patients with UC have a 30% lifetime risk of CRC and UC associated CRC (UC-CRC) develops by a distinct pattern of genetic changes to sporadic CRC. We investigated the expression of PI-3-Kγ in colonic tissue from patients with UC, UC-CRC and CRC.

Methods: PI(3)Kγ expression was investigated in normal colon, UC, CRC and UC-CRC by immunohistochemistry and in situ hybridisation. Using RT-PCR and western blotting, PI-3-Kγ expression was determined in normal colonic mucosa, colonic epithelial cells, and the colon cancer cell line (Caco2). Methylation specific PCR was performed on Caco2 cell, sporadic CRC and UC-CRC DNA to determine if silencing of PI-3-Kγ was associated with hypermethylation.

Results: PI-3-Kγ expression was demonstrated in leukocytes, colonic epithelial cells and Caco2 cells. All UC specimens expressed PI-3-Kγ protein at higher levels than normal tissue, and expression was lost in the majority of UC-CRC. PI-3-Kγ mRNA was detected in normal tissue, CRC and UC, but was absent in UC-CRC. In Caco2 cells treated with stimulated lymphocyte conditioned medium, expression of PI-3-Kγ was increased, and as the gene is unmethylated in these cells, it is probably upregulated by inflammation, and subsequent loss of expression may be mediated by hypermethylation.

Conclusion: Loss of expression of PI(3)Kγ is seen more frequently in UC-CRC than sporadic CRC and our data suggest that it plays a critical role in intestinal epithelial cell function in the context of inflammation, where its expression is increased. We hypothesise that subsequent loss of PI-3-Kγ expression, possibly through hypermethylation, promotes neoplastic transformation in the inflamed colonic epithelium.

223 ANTI-INFLAMMATORY EFFECT OF N-ACETYLCYSTEINE (NAC) IN A MODEL OF COLITIS

O.G. Azooz, A. De Silva, D.S. Rampton, A.B. Ballinger.

Dept. of Adult & Paediatric Gastroenterology, Barts & The London, Queen Mary’s School of Medicine & Dentistry, London, UK

Introduction: Oxidative stress is a potential mechanism in the pathogenesis of inflammatory bowel disease (IBD) where reactive oxygen species (ROS) are produced in excess by the intestinal mucosa. N-acetylcysteine (NAC) is a synthetic thiol compound which has antioxidant properties and suppresses production of NK-κB, and proinflammatory cytokines including TNF-α.

Aims: The aim of this study was to assess the effect of NAC on the severity of colonic inflammation in a rat model of colitis.

Methods: Colitis was induced in Wistar rats by intrarectal administration of 2,4,6 trinitrobenzenesulphonic acid (TNBS) and rats treated with i.p. NAC (50 or 200 mg/kg/day) or saline control (8/group). 16 healthy controls were treated with either NAC (50 mg/kg/day) or saline. Animals were sacrificed 5 days after induction of colitis. Severity of intestinal inflammation was assessed macroscopically, and by measurement of colonic tissue myeloperoxidase (MPO) and water content. ROS were measured by chemiluminescence (CLS).

Results: TNBS induced distal colitis with ulceration, increased tissue MPO concentration and water content, and CLS (table). NAC reduced all these measures of intestinal inflammation except water content. There was no significant difference between 200 and 50 mg/kg of NAC.

Conclusion: NAC has anti-inflammatory effects in the acute phase of TNBS-induced colitis. This treatment may be worth further assessment in the treatment of human IBD.

224 SENESCENCE RATES AND TELOMERE SHORTENING IN CULTURED RECTAL FIBROBLASTS IN ULCERATIVE COLITIS

K.M. Getliffe, D.M. Aldulaimi, C.U. Nwokolo.

University Hospitals Coventry and Warwickshire NHS Trust and Dept of Biological Sciences, University of Warwick, UK

Introduction: Human somatic cells have a finite lifespan and cease to divide after a certain number of cell divisions, a phenomenon known as replicative senescence. One mechanism believed to trigger this process is continuous shortening of chromosomal ends (telomeres), with each cell division until a critical length is reached at which cell replication ceases. In cancerous and normal proliferating cells (including the epithelial cells of the bowel) this mechanism can be bypassed by telomerase, which adds telomeric repeats to the ends of chromosomes to maintain their length and thereby prevent senescence. A telomerase knockout mouse suffers ulceration and atrophy of the bowel. In ulcerative colitis (UC), the colonic mucosa is deficient in telomerase and epithelial cells in inflamed areas have short telomeres. Furthermore, in UC patients we have observed a decrease in lymphocyte telomerase activity and others have found lymphocyte chromosomal abnormalities.

Aim: To determine whether these observations are caused simply by the increased cell turnover inherent in the inflammatory process or whether UC patients have a global telomere maintenance defect which contributes to the disease.

Methods: Rectal fibroblast cultures were generated from 9 UC patients and 9 age-matched non-UC controls; their rates of senescence and telomere lengths were assessed throughout their life span in culture. Senescence rates were measured by counting the percentage of cells staining for the proliferation marker Ki67 at each passage and telomere lengths were measured using a standard TRF measurement southern blotting assay.

Results: Preliminary results have identified little difference between the rates of senescence in UC and non-UC fibroblasts, suggesting that telomere/telomerase dysfunction is not global but may be a downstream effect of inflammation in this disease. However, this study is ongoing and further cultures are in the process of being analysed.

Abstract 223

225 CIRCULATING TUMOUR NECROSIS FACTOR ALPHA CONCENTRATIONS AND PERIPHERAL CD4+ T CELL CHEMOKINE RECEPTOR EXPRESSION IN PATIENTS WITH PYODERMA GANGRENOSUM

T.N. Brooklyn, A.M. Williams, C.S. Probert.

Bristol Royal Infirmary, Bristol, UK

Background: Pyoderma gangrenosum (PG) is recognised as an extraintestinal manifestation of inflammatory bowel disease (IBD). Recent reports of PG responding to infliximab suggest that tumour necrosis factor alpha (TNFá) may be important, although in our hands the response of PG to infliximab has been variable. Subsets of CD4+ T cells are functionally polarised according to their cytokine profile and one of the ways of identifying these groups of cells is to examine the chemokine receptors they express. Little is known of the cytokines involved in the development of PG and it is not known whether the disease fits a T-helper type 1 (Th1) or Th2 pattern.

Aims: To investigate the hypothesis that, if PG responds to infliximab, TNFα must be important in the pathogenesis of the disease and it is also likely that PG would display a Th1 phenotype.

Patients: Ten patients with PG, half of whom also had underlying IBD, were compared with seven healthy controls.

Methods: The concentration of circulating TNFα was determined using enzyme linked immunoadsorbent assay (ELISA). The expression of the chemokine receptors CCR4 and CXCR3 on CD4+ T cells was determined by flow cytometry. The results were compared using the Mann-Whitney U test.

Results: The plasma concentrations of TNFα in the PG patients were found to be significantly higher than in the healthy individuals (p=0.002). The expression of CCR4 on CD4+ T cells was significantly higher in the PG patients (p=0.048), but there was no difference in the expression of CXCR3.

Conclusions: Plasma concentrations of TNFα in patients with PG are significantly greater than those found in normal individuals, but not as high as those found in active IBD. Patients with PG preferentially express CCR4 not CXCR3 on peripheral CD4+ T cells, suggesting that PG may be a Th2 disease. These findings may explain the variable clinical response to infliximab.

226 EFFECTS OF NICOTINE ON SPONTANEOUS AND LPS-INDUCED NF-κB ACTIVATION AND APOPTOSIS IN COLORECTAL CELLS: RELEVANCE TO INFLAMMATORY BOWEL DISEASE?

M.C. Aldhous, L.A. Stark1, M.G. Dunlop1, J. Satsangi.

Gastrointestinal Research Laboratory, University of Edinburgh Department of Medical Sciences, and 1MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland UK

Background: Complex genetic, environmental, and immunological interactions are involved in the pathogenesis of Crohn’s disease (CD). Mutations within the NOD-2 gene are present on up to 50% of patients with CD and clearly influence disease susceptibility and behaviour. Early functional data implicate NOD-2 as an intracellular sensor of bacterial lipopolysaccharide (LPS) which directly influences NF-κB activation. Cigarette smoking is the best defined environmental risk factor for CD. While nicotine therapy has had limited success in ulcerative colitis, it is not clear whether nicotine itself causes the increased severity of CD, nor whether nicotine supplements could be used to aid patients stop smoking.

Methods and Results: We have examined the effect of nicotine on NF-κB activation in SW480 colorectal cancer cells. Immunohistochemistry for NF-κB (p65) expression showed a dose-dependent increase in NF-κB activation after 30 minutes with nicotine alone, which was reversed by addition of LPS. Cells stimulated overnight with nicotine and/or LPS, showed a dose-dependent decrease in apoptosis with nicotine alone, which was modified by LPS. In kinetic studies using western blot analysis, nicotine induced degradation of the NF-κB inhibitor protein, I-κB, within 1 hour and levels returned to baseline by 5 hours. Pre-incubation with nicotine for 1–5 hours inhibited I-κB degradation in response to LPS.

Conclusions: These data indicate that nicotine directly influences NF-κB activity and suggest that nicotine may modify cellular responses to LPS. The consequent effects on cytokine production and apoptosis may help explain the effects of smoking in CD and have implications for the use of nicotine in smoking cessation therapy.

227 METAPLASTIC PANETH CELLS IN ULCERATIVE COLITIS AND COLORECTAL CANCER

C.L.E. Osborne, A. Khan, O. Ogunbiyi, S. Keshav.

Royal Free & University College Medical School, London, UK

Background and Aims: Paneth cell metaplasia (PCM) in the colon of patients with inflammatory bowel disease (IBD) represents a distinct form of the host inflammatory response. Paneth cells secrete an array of antimicrobial proteins including lysozyme, defensins, and type IIa secretory phospholipase A2, (PLA2-IIa) which lyses bacterial cell membranes, and is also secreted into the circulation as part of the acute phase response. PLA2-IIa levels are higher in the colonic mucosa of patients with severe colitis who are at greater risk of colorectal cancer (CRC), and the PLA2-IIa gene is a modifier of neoplasia in the Min mouse model of familial adenomatous polyposis. We therefore investigated PLA2-IIa expression in UC, CRC, and UC-associated CRC, to determine if expression was higher in UC-associated CRC compared to sporadic CRC.

Methods: PCM and PLA2-IIa protein expression in paraffin embedded sections of colon from UC, sporadically occurring CRC and UC associated CRC by indirect immunohistochemistry. We compared PLA2-IIa expression with positive (anti-CD45 antibody) and negative controls (secondary antibody only), and used western blot analysis on fresh tissue from paired samples of UC and UC associated CRC, sporadic CRC and normal colon, to confirm the immunohistochemical data.

Results: PCM and PLA2-IIa expression were detected in 67% of UC samples, but not in any normal colon. PCM was detected in 30% of UC associated CRC, compared to 6.3% of sporadically occurring CRC. In the paired tissue samples, PLA2-IIa expression was clearly demonstrated by western blotting in both UC specimens and in one UC associated CRC, but was not in sporadic CRC or normal tissue.

Conclusion: PCM in colonic mucosa is easily and reliably identified by the presence PLA2-IIa protein in Paneth cell granules. PCM can be used as an indicator of disease activity in UC and is also a marker of UC associated CRC, where it may play a role in pathogenesis, which remains to be determined.

228 DEVELOPMENT OF HUMAN ANTI-MURINE ANTIBODIES IN PATIENTS FOLLOWING TREATMENT WITH INFLIXIMAB

L.P. Maiden, G.J. Lawson, A.W. Harris.

Kent and Sussex Hospital Tunbridge Wells, Kent TN4 8AT, UK

Introduction: Infliximab is a 75% human and 25% murine antibody directed against tumour necrosis factor-alpha. Human anti-mouse antibody (HAMA) directed against the murine component of this antibody could develop in patients given infliximab. The estimated prevalence of HAMAs varies widely from <1–80%. Many biochemical immuno-assays involve the use of mouse antibodies, eg. thyroid function tests (TFTs). If present, HAMAs could interfere with these assays giving rise to misleading results with clinical consequences. The aim of this pilot study was to assess whether patients receiving infliximab for Crohn’s disease or Rheumatoid arthritis subsequently developed abnormal TFTs. If so, this could be due to the development of HAMAs as a result of exposure to infliximab.

Method: Patients who had received infliximab at the Kent and Sussex or Homeopathic Hospitals as treatment for Crohn’s disease or Rheumatoid arthritis between March 2000 and August 2001 subsequently had their TFTs assessed using the standard hospital assay. If any results were abnormal, the assay could be confirmed using “blocking agents” which filter out the HAMAs by binding to them.

Results: 19 patients (15 female; mean age=50 years, range 21–70), with either Crohn’s disease (n=3) or Rheumatoid arthritis (n=16), received infliximab (mean dose=3 infusions at 5mg/kg, range 1–7 infusions). TFTs were assessed between 3 and 15 months (mean=7 months) after the last infusion. One patient had abnormal TFTs. This patient was already known to be hypothyroid and was regularly taking thyroxine. No patients required repeat assays with blocking agents.

Conclusion: This pilot study has failed to show that HAMAs develop following, at least, a single infusion of infliximab. HAMAs may persist for up to 30 months after exposure. HAMAs may have developed but subsequently disappeared prior to the TFT assay, given that 3 patients had assays up to 15 months after their last infusions. Larger studies are needed to confirm these findings as the prevalence of HAMAs may be less than 1%.

229 A REPORT ON THE CLINICAL EFFICACY AND STEROID SPARING EFFECT OF GRANULOCYTE AND MONOCYTE ADSORPTIVE APHERESIS IN PATIENTS WITH CORTICOSTEROID DEPENDENT ULCERATIVE COLITIS

H. Hanai1, K. Takeuchi1, T Iida1, K. Tozawa1, T. Tanaka1, A. Saniabadi2, F. Watanabe3, Y. Maruyama3, I. Matsushita4, M. Yamada5, K. Kikuchi6 (introduced by B. Danesh).

1Dept of Medicine, Hamamatsu University; 2Japan Immunoresearch Laboratories; 3Fujueda General Hospital; 4Seirei General Hospital; 5Hamamatsu Medical Centre; 6National Tosei Hospital, Hamamatsu, Japan

Frequent relapse during steroid tapering is common in many patients with ulcerative colitis (UC) who initially respond to intensive medication including a corticosteroid. Such cases are said to have steroid-dependent (SD) UC. Further, increased granulocyte and monocyte counts, activation and prolonged survival time is a feature of mucosal inflammation in active UC. Likewise, faecal calprotectin (a neutrophil protein) level parallels intestinal inflammation and predicts UC relapse. We thought that granulocyte and monocyte apheresis (GMA) to reduce the circulating level of these leucocytes might suppress relapse during the steroid tapering in SD patients. Forty six patients with SD UC, mean age 38±14 yr, CAI (clinical activity index) 9.2, DAI (disease activity index) 8.6 were given 10 GMA sessions, one session/week for 10 consecutive weeks, by using a 335 mL capacity column filled with 220g cellulose acetate beads of 2 mm in diameter as the column adsorptive carriers (Adacolumn). The carriers selectively adsorb granulocytes and monocytes. Duration of one GMA sessions was 60 minutes, flow rate 30mL/minute. At week 12, CAI was 1.7 DAI 2.8 (n=46) and 39 of 46 patients were in remission. The mean dose of prednisolone, 18 mg/patient/day at entry was reduced to 11mg at week 12 and to 4mg at week 20. Further, at week 20, 42 patients were in remission, 2 had improved, and 2 had relapsed. The treatment was well tolerated and no serious side effects were observed. In conclusion, GMA appeared to be an effective adjunct to standard drug therapy of active UC by promoting remission and suppressing relapse during steroid tapering. It seems that reduction of circulating level of activated granulocytes alleviates inflammation and contributes to remission. However, the full efficacy of GMA is unlikely to be due to reduction of granulocytes per se. Results from other studies together with basic research indicate a marked decline in inflammatory cytokine generation and reduced CXCR3 expression by peripheral blood leucocytes post Adacolumn apheresis.

230 A DOUBLE BLIND, PLACEBO CONTROLLED RANDOMISED TRIAL OF CURCUMIN EXTRACT IN THE TREATMENT OF STEROID DEPENDENT INFLAMMATORY BOWEL DISEASE

R.J. Atkinson, J.O. Hunter.

Department of Gastroenterology, Addenbrookes Hospital, Cambridge CB2 2QQ, UK

Introduction: Nuclear factor kappa B (NFkB) is a pro-inflammatory transcription factor which plays an integral part in the pathogenesis of inflammatory bowel disease (IBD). Curcumin has anti-inflammatory properties through prevention of NFkB activation and is effective in the TNBS model of colitis. We present the results of a trial of Curcuma extract (PYM 50014) as a steroid sparing agent in patients with IBD.

Methods: 27 patients with steroid dependent colitis were entered into the 16 week study. All patients were in remission on 5–30mg prednisolone per day, were 6 weeks from their last clinical relapse and were not on any other steroid sparing agent (e.g. azathioprine). Disease activity was assessed by either Ulcerative Colitis Index (UCI) or Crohn’s disease activity Index (CDAI), standard laboratory parameters (C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet count, albumin) and flexible sigmoidoscopy. Randomisation to PYM 50014 (3×220mg b.i.d) or placebo was at 2 weeks and steroid reduction began 2 weeks later. Patients were assessed every two weeks and their prednisolone reduced as long as they remained in remission. Primary end points were remission at 16 weeks, or relapse, when the minimum effective dose of prednisolone was calculated.

Results: 13 patients received PYM 50014 (8 UC, 5 CD) and 14 placebo (8 UC, 6 CD). The groups were matched for sex, duration of disease and disease activity, although the PYM 50014 group tended to be younger (36.9 ± 10.35; 52.3 ± 12.72 years). 5 (38%) patients in the PYM 50014 and 6 (43%) in the placebo group 16 week treatment period. The average total steroid reduction was 45.2% on PYM50014 and 56.8% on placebo. There were no significant differences between the groups, whether or not patients were analysed according to disease type.

Discussion: Whilst apparently safe, PYM 50014 does not appear to enable steroid dependant patients to reduce their steroid requirements. There may be a role for PYM 50014 in milder disease and further evaluation may be warranted.

231 RE-INTRODUCTION OF AZATHIOPRINE IN PREVIOUSLY INTOLERANT PATIENTS

C.J. Green, A.S. Mee.

Royal Berkshire Hospital, Reading

Introduction: Azathioprine is widely used as a steroid sparing agent in the management of inflammatory bowel disease. However, patient intolerance, which occurs in 9.3%1 limit its use.

Methods: We conducted an observational study assessing the success of the slow re-introduction of azathioprine in previously intolerant patients. Patients who had been unable to take full dose azathioprine (1.5–2.0mg/kg) due to side effects primarily flu like illness, myalgia, and headaches were re-commenced on azathioprine at sub therapeutic doses (10–25mg). Their dose was increased in a stepwise fashion, increasing by 25mg every two weeks until they reached full dose (100mg).

Results: Azathioprine was successfully reintroduced, achieving therapeutic dose in six patients (approximately 40% of the population in whom it was tried). Three patients had ulcerative colitis and three had Crohns disease. The male to female ratio was 2:4. All had initially stopped azathioprine due to a severe flu like illness with headache and myalgia. All were successfully restarted using the above regime. All tolerated the drug and have remained on it for a minimum of seven months at the time of writing. All had TPMT (thiopurine methyl transferase) levels on treatment within the normal range.

Discussion: Azathioprine is a useful drug in the treatment of chronic inflammatory bowel disease. However, there is a significant side effect profile leading to morbidity as stated above. We show good observational evidence that previously intolerant patients can achieve useful dosing of this drug using a step up approach. Our anecdotal experience suggests that approximately 40% of intolerant patients can be managed in this way and therefore, possibly avoid surgery.

1 Sandborn W, Sutherland L, Pearson D, et al. Azathioprine or 6-mercaptopurine for induction of remission in Crohn’s disease (Cochrane Review). The Cochrane Library, Issue 2, 2002. Oxford: Update Software.

232 PARENTERAL METHOTREXATE IN REFACTORY CROHN’S DISEASE: EXPERIENCE IN A DISTRICT GENERAL HOSPITAL

L.P. Maiden, A.W. Harris.

Kent and Sussex Hospital, Tunbridge Wells, Kent TN4 8AT, UK

Background: Methotrexate (MTX) is of proven benefit in treating and maintaining remission in active Crohn’s disease. We report our experience in the use of this immunomodulatory agent over a 24 month period in those patients with refractory Crohn’s disease, demonstrating its useful role in a district general hospital.

Methods: MTX was offered to 13 patients (9 female) with refractory Crohn’s disease. All 13 commenced treatment following the regimen reported by Feagan et al at a dose of 25mg intramuscularly (im) once a week for 16 weeks and continued at 15mg im once a week thereafter. Corticosteroids and aminosalicylates were the only concurrent treatment. Efficacy was assessed by steroid withdrawal and remission rates. Adverse events were recorded at each visit.

Results: Mean age was 46 years, (range 31–69). Mean length of treatment was 38 weeks, (range 5–105). Total mean dose was 700mg, (range 125mg–1735mg). 8 patients (62%) achieved clinical remission. 5 patients were unable to complete 16 weeks of treatment: 2 withdrew due to intolerance from nausea; 2 required surgery for continuing disease; 1 commenced warfarin and was thus unable to receive further intramuscular injections after achieving clinical remission at 14 weeks. Of the 6 who completed 16 weeks of treatment, 4 have continued maintenance at 15mg im weekly and 2 have discontinued treatment (1 wished to become pregnant and the other had few symptoms and wanted to discontinue). 1 patient required further treatment with corticosteroids following a clinical relapse and 1 required 4 weeks of MTX at 25mg/week to control symptoms of a relapse before returning to the maintenance dose. Adverse events reported were mucositis (1) and nausea (3).

Conclusions: MTX is a well tolerated alternative treatment in refractory Crohn’s disease, which is safe and practical to administer in a district general hospital. The results of our experience are in keeping with those of 2 large, double-blind, randomised trials.

233 DA-6034, A NEW THERAPEUTIC AGENT OF INFLAMMATORY BOWEL DISEASE

C.Y. Shin, M. Son, I.K. Lee, M.Y. Jung, D.S. Kim, S.H. Kim, M. Yoo, I.S. Song1, W.B. Kim.

Research Laboratories, Dong-A Pharm. Co. Ltd., Korea;1Department of Internal Medicine, Seoul National University, College of Medicine, Korea

DA-6034, 7-carboxymethyloxy-3’, 4’, 5-trimethoxy flavones, is a synthetic flavonoid known to possess anti-inflammatory activity. In this study, we evaluated the oral therapeutic effect of DA-6034 in four experimental animal models of inflammatory bowel disease (IBD). In acetic acid (4%) induced IBD rat models, prednisolone (1 mg/kg), sulfasalazine (100 mg/kg) and DA-6034 (3 mg/kg) significantly reduced the severity of colitis. Especially, DA-6034 (3 mg/kg) showed more potent effect than other drugs in macroscopic lesion score. In trinitrobenzene sulfonic acid (TNBS) induced IBD rat models, prednisolone (3.0mg/kg) or DA-6034 (3.0mg/kg) inhibited the colonic mucosal damage. DA-6034 (3.0mg/kg) significantly decreased the prostaglandin E2 levels, leukotriene B4 levels and myeloperoxidase activity at 7 day as compared with control. In Dextran sulfate sodium (DSS)-induced colitis mice, DA-6034 reduced the colitis index-diarrhea incidence, body weight, mortality, and intestinal gross lesion. In HLA-B27 transgenic rats, DA-6034 (3 mg/kg) and prednisolone (0.5 mg/kg) were treated orally twice daily for 6 weeks. The HLA-B27 transgenic rats showed only mild colitis, compared with the chemical-induced colitis models. DA-6034 ameliorated the loose stool and decreased microscopic damage, which is the important indicator of this model.

In conclusion, DA-6034 attenuated the macroscopic and histological damages of the colon in all four experimental models of IBD, which suggest that DA-6034 could be a promising drug in the treatment of IBD and we will set out clinical Phase I study of DA-6034 in next year.

234 ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION OF DA-6034, A NEW INFLAMMATORY BOWEL DISEASE TREATMENT IN RATS

H.J. Shim1*, J.M. Jang1, K.J. Park1, D.G. Kim1, H.S. Lee2, M.W. Son1, D.S. Kim1, S.H. Kim1, M.H. Yoo1, W.B. Kim1.

1Research Labororatories, Dong-A Pharm. Co. Ltd1; 2Wonkwang University, College of Pharmacy, Korea

The absorption, distribution, metabolism, and excretion of DA-6034, which is being developed for the treatment of inflammatory bowel disease as an oral dosage form, were investigated using fluorescence-HPLC and LC/MS/MS method. The detection limit was 0.5 ng/ml in plasma. After intravenous administration of DA-6034, 10 mg/kg to rats, the plasma concentrations of DA-6034 declined polyexponentially with the mean terminal half-life of 3.42 hr. Total body clearance and renal clearance were 26.3 and 9.48 ml/min/kg, respectively and fraction of dose excreted in urine for 24 hr was 35.7%. The tissue to plasma ratios in S.I., L.I. and kidney were large, indicating high affinity of DA-6034 to rat GI tract tissues. After oral administration of DA-6034, 10 mg/kg to the rats, the absolute bioavailabilities were only 1.34%. Percents of dose remaining in the gastrointestinal tract were 55.2% of dose at 24 hr after oral administration of DA-6034. It was considered that the superior effect in experimental animal models of inflammatory bowel disease after oral administration of the drug was due to the local action of DA-6034. No metabolites of DA-6034 were produced in the rat liver microsome without and with NADPH generating system. Glucuronide- and sulfate-conjugation were not involved in DA-6034 metabolism. And DA-6034 was not a substrate of human CYP3A4, therefore, clinically significant drug interactions are not expected.

235 THE NUTRICEUTICAL BOVINE COLOSTRUM FOR THE TREATMENT OF DEXTRAN SULFATE SODIUM INDUCED COLITIS IN RATS

A.J. FitzGerald, T. Marchbank, J. Boyle, S. Ghosh, R.J. Playford.

Dept of Gastroenterology, Imperial College Faculty of Medicine, Hammersmith Hospital, Ducane Road, London W12 0NN, UK

Bovine colostrum is a rich source of nutrients, growth factors and antibodies and is marketed as a health food supplement. There is increasing evidence that it may be useful for the specific treatment of gastrointestinal disease

Aims: To examine the effects of a commercially available formulation of colostrum in a rodent model of experimental colitis.

Methods: Colitis was induced with 4% dextran sulphate sodium (DSS). Rats were pre-treated with a gavage of either 20 or 60 mg/kg of colostrum before induction of colitis and daily afterwards until killed at 7 days. An addition group was used as controls (no colitis). Disease activity was assessed daily by combining scores of weight lost, stool consistence and bleeding. Colonic tissue was measured and assessed microscopically by histological scoring of inflammation.

Results: Treatment with colostrum resulted in a reduction in the disease activity index and intestinal inflammation. Disease activity fell by 11% in the 20 mg/kg group and 34% in the 60 mg/kg group while inflammation by 60% in the 20 mg/kg group (p<0.05) and by 85% in the 60 mg/kg group (p<0.01), compared with the saline gavage control.

Conclusion: These preliminary results show improvement in colonic inflammation after administration of colostrum gavage in the rat DSS colitis model.

236 THIOPURINES, BUT NOT CORTICOSTEROIDS OR 5-ASA, REDUCE THE NUMBER OF PLATELET-LEUCOCYTE AGGREGATES IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

P.M. Irving1, M.G. Macey2, U. Shah2, L. Webb2, F. L. Langmead1, D.S. Rampton1.

1Adult & Paediatric Gastroenterology, Barts & The London, Queen Mary’s School of Medicine and Dentistry, UK; 2Department of Haematology, Barts & The London NHS Trust, London, UK.

Background: We have previously shown that formation of platelet-leucocyte aggregates (PLAs) is increased in patients with IBD (UEGW 2000) and correlates with platelet activation (BSG 2001).

Aims: To see if drugs commonly used to treat IBD affect PLA formation, platelet and neutrophil activation.

Methods: Of 66 patients with IBD (31 Crohn’s, 35 ulcerative colitis),17 patients were on thiopurines (11 azathioprine, 2 6-mercaptopurine), 17 prednisolone or hydrocortisone and 53 5-ASAs. Venous blood was drawn into EDTA/CTAD anticoagulants. Samples were immediately mixed and then analysed by flow cytometry for P-selectin (CD62P for platelet activation), L-selectin (CD62L for neutrophil activation) and CD42a/CD45 (platelet-leucocyte aggregates).

Results: Patients taking thiopurines had fewer PLAs (median (IQR)) (0.26 (0.20–0.38) × 109/L, n=17) than those that were not (0.37 (0.25–0.60), n=49, P<0.05). The number of platelets expressing P-selectin was similar in both groups (thiopurines 3.1 (1.4–6.0) × 109/L, no thiopurines 3.7 (1.9–8.7) × 109/L) as was expression of L-selectin on neutrophils (thiopurines 802 (793–814), no thiopurines 792 (780–808)). There was no difference in any of these variables between patients taking and not taking corticosteroids or 5-ASAs.

Conclusions: Thiopurines, but not corticosteroids or 5-ASAs, decrease the number of platelet-leucocyte aggregates in patients with IBD; none of these agents appeared to affect platelet and neutrophil activation. It is conceivable that inhibition of platelet-leucocyte aggregate formation could contribute to the therapeutic efficacy of thiopurines in IBD.

237 TREATMENT OF ZERO AND INTERMEDIATE TPMT PATIENTS WITH A TAILORED DOSE OF AZATHIOPRINE

A. Ansari, M. Escudier, A. Marinaki, A. Yim, J. Hirst, J. Duley, J. Sanderson.

Departments of Gastroenterolgy, Oral Medicine and Chemical Pathology, Guy’s & St. Thomas’ Hospitals, London SE1 9RT, UK

Background: Deficiency of thiopurine methyl transferase (TPMT) is associated with a high risk of adverse effects on azathioprine (AZA) therapy. Conventionally, AZA is avoided in patients with zero TPMT (1in 300) and those with heterozygous TPMT deficiency (1 in 10).

Aims: We have studied the outcome of TPMT deficient patients receiving a tailored low dose of AZA.

Methods: According to a department protocol, patients with zero TPMT were offered treatment with 5% usual dose, heterozygotes with 1mg/kg AZA. Thioguanine nucleotide (TGN) levels were assayed in a proportion of cases. In addition, a questionnaire was sent to physicians regarding the outcome of patients referred for TPMT assay from outside the author’s institution.

Results: Two patients with zero TPMT (One CD, one UC) responded to 5% dosing with a prompt and full clinical remission (HBI / Truelove and Witts score). Both have remained in steroid-free remission for two years. TGN levels were very high yet both remained free of myelotoxicity. 26 heterozygotes 12 CD, 7 UC, 2 autoimmune hepatitis, and 5 with oral ulceration were treated. 20 (77%) patients responded to treatment, 1 did not respond and 5 withdrew treatment as a consequence of side effects (19%). TGN levels were variable and did not correlate with response or side effects.

Of 92 questionnaires sent, 51 replies were obtained. Of these, only 15 patients started azathioprine at 1mg/kg. 11/15 (73%) responded to treatment, 2 failed, and 2 withdrew due to side effects. In 36/51 (71%) the requesting physician decided not to use azathioprine due to the low TPMT level.

Conclusions: Physicians generally avoid AZA in patients with TPMT deficiency. However, the results of this study show that heterozygous patients respond well to low dose AZA. Furthermore, zero TPMT patients can be successfully treated with a very low dose of AZA.

238 GENETIC DETERMINANTS OF THE THIOPURINE METHYLTRANSFERASE INTERMEDIATE ACTIVITY PHENOTYPE

M. Arenas1, A. Marinaki1, C.M. Lewis2, E.M. Shobowale-Bakre1, E. Escuredo1, L. Fairbanks1, A. Ansari3, J.D. Sanderson3, J.A. Duley1.

1Purine Research, Guy’s and St Thomas’ Hospital, London; 2Division of Genetics and Development, GKT School of Medicine, King’s College London; 3Department of Gastroenterology, Guy’s and St Thomas’ Hospital, London

Background and aims: Polymorphism in the TPMT gene open reading frame (ORF) is associated with reduced TPMT activity. Variable number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the TPMT gene have been previously reported to modulate the TPMT activity phenotype. The aim of this study was to investigate the relationship between TPMT activity and ORF and VNTR genotypes.

Methods: Groups of patients were selected with non-overlapping TPMT activities from the intermediate activity range (4–8 U, 108 patients) and normal range (12–15 U, 53 patients) and were genotyped for VNTR type, and TPMT*3A, TPMT*3C, and TPMT*2 ORF mutations. To investigate the influence of the number of VNTR repeat units on TPMT activity, the number of Type A, B, or total repeat units summed for both alleles in the ORF heterozygous, wild type intermediate and normal activity groups and were compared in an analysis of variance.

Results: 41% of patients from the intermediate TPMT activity group were heterozygous for a TPMT ORF mutation. Marked linkage disequilibrium was noted between VNTR*6b - TPMT*3A (D′=1), VNTR*4b - TPMT*3C (Dprime;=0.67), and VNTR*6a - TPMT*1 (D′=1) alleles. For Type A and total repeats, significant differences (p<0.05) in the median number of repeats were found between the heterozygous intermediate activity group and the wild type intermediate and normal activity groups. There was no significant difference in the median Type A, B, or total repeat number between the two wild type groups.

Conculsions: These results suggest that TPMT gene VNTRs do not significantly modulate TPMT enzyme activity.

239 XANTHINE OXIDASE/DEHYDROGENASE ACTIVITY IN IBD PATIENTS RECEIVING AZATHIOPRINE

A. Ansari1, A. De Sica2, J.A. Duley3, E.M. Shobowale-Bakre3, L. Fairbanks3, A. Marinaki3, M. Aslam3, J. Hirst1, C. Smith2, J.D. Sanderson1.

Departments of 1Gastroenterology, 2Dermatology & 3Chemical Pathology, Guy’s and St Thomas’ Hospitals, London SE1 9RT, UK

Catabolism of azathioprine (AZA) occurs via two main pathways: methylation by thiopurine methyltransferase (TPMT) and oxidation by xanthine oxidase/dehydrogenase (XOD). TPMT activity is genetically polymorphic, while XOD deficiency (xanthinuria) is a rare disorder. However, XOD is present in intestinal mucosa and might therefore vary in patients with inflammatory bowel disease (IBD) resulting in altered AZA metabolism, adverse effects and efficacy.

We measured 6-thiouric acid (6TU, end-product of XOD oxidation) in 24-hour urines from 31 IBD (27 CD, 4 UC) patients receiving AZA for more than 2 weeks. As non-IBD controls, 9 patients receiving AZA for eczema or bullous pemphigoid were also studied.

Results: The overall mean oxidation of AZA to 6TU was 11% (95%CI:3.1–26.6) of the dose. In 16 IBD patients not on 5-ASA (which inhibits TPMT in vitro) mean 6-TU excretion did not differ significantly from dermatology controls (9.4% (3.3–16.7) v 8.6% (3.1–15)) For the 15 IBD patients receiving 5ASA drugs, the mean 6TU excretion was (14.2%; 6.326.6%) significantly higher than the IBD patients not on 5ASA or in dermatology controls (p<0.05, Tukey-Kramer test). There was no association between AZA oxidation and IBD disease location or severity of disease (CDAI). Oxidation was not raised in 4 of the IBD patients who had intermediate TPMT deficiency. In contrast, an additional IBD patient with zero TPMT, on low dose AZA, excreted 90% of the dose as 6TU. As expected, there was a mild association between 6TU excreted on a creatinine basis with AZA dose (mg/kg). Interestingly, the study also revealed several non-compliers, who were excluded from the study.

Conclusions: Only about 10% of an AZA dose is oxidised, presumably via XOD: most of the drug is methylated. AZA diversion into oxidation is increased significantly by 5-ASA drugs, but not affected by gut inflammation.

240 POLYMORPHISM IN THE MTHFR GENE AFFECTS TPMT ACTIVITY

A. Ansari1, A. Marinaki2, M. Arenas2, E.M. Shobowale-Bakre2, C.L. Lewis3, J. Duley2, J.D. Sanderson1.

Departments of 1Gastroenterology, 2Chemical Pathology, and 3Genetics, Guy’s & St. Thomas’ Hospitals. London SE1 9RT, UK

Background: Polymorphisms in the thiopurine methyl transferase (TPMT) gene only explain a proportion of side effects on azathioprine (AZA). S-adenosylmethionine (SAM) is the methyl donor for methylation by TPMT and is converted to S-adenosyl-homocysteine (SAH). The adenosyl moiety of SAH is cleaved and homocysteine can be remethylated to methionine in a folate dependent pathway. A common polymorphism in the methylene tetrahydrofolate reductase (MTHFR) gene, the 677C>T variant is associated with decreased MTHFR enzyme activity and thus may indirectly affect TPMT activity.

Aim: To investigate whether MTHFR gene polymorphism influences erythrocyte TPMT activity and might be associated with adverse drug reactions to azathioprine (AZA).

Methods: MTFHR TT genotype frequency was assessed in 52 patients with wild type TPMT genotype but intermediate TPMT activity (4–8U) and compared to 55 patients with normal TPMT activity and genotype (in the range 12–15U). MTHFR genotype frequencies were then compared between 57 IBD patients with adverse drug reactions to AZA therapy and 71 patients tolerant to AZA.

Results: MTHFR TT genotype was significantly underrepresented in the normal compared to the intermediate activity group (7.3% v 23% (p=0.0296). Despite this, MTHFR genotype was not significantly associated with an increase in overall side effects to AZA. The MTHFR 677C>T allele was less frequent amongst those with hepatitis and pancreatitis on AZA (4/16(25%) v 35/71(49%) but this association did not reach statistical significance (p=0.6).

Conclusions: MTFHR genotype influences TPMT activity such that wildtype MTHFR genotype is associated with high methylation capacity. High TPMT activity has been suggested as a cause of pancreatic and hepatic side effects but could not be confirmed in this study. Larger studies are needed to explore this relationship further.

241 ADENOMATOUS POLYPS ARE RARE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

A. Dixon1, P. Wurm1, R. Robinson1, A. Hart2.

1Department of Gastroenterology, University Hospitals of Leicester, UK; 2School of Medicine, University of East Anglia, Norwich, UK

Objective: The prevalence of adenomatous colorectal polyps in inflammatory bowel disease (IBD) is unknown, although recent reports suggest they might be rare in patients with ulcerative colitis (UC). Previous work has suggested 5-ASA compounds may protect against colorectal cancer. The aim of this study was to compare the prevalence of left sided adenomas in patients with IBD with a local age matched control population.

Method: We reviewed the medical notes of 106 patients with histologically proven IBD, aged 55–64 years at the time of endoscopy for the presence of left sided colonic adenomas. Patients were identified through our endoscopy/histology databases. The prevalence of adenomas was compared with a healthy control population in Leicestershire aged 55–64 years who participated in a screening programme for colorectal cancer with flexible sigmoidoscopy.

Results: Of 106 patients (61 male, 45 female, mean age 59 years), 80 suffered from UC, 20 from Crohn’s disease and 6 from indeterminate colitis. All patients had undergone at least a flexible sigmoidoscopy and 75 had a colonoscopy. Adenomas were found in 3 patients with ulcerative colitis compared to 67 of 749 controls (2.8% v 8.9%, chi2 =4.6, p=0.03). Of the 3 patients that had polyps, one patient with a 15 year history of UC had a 8mm rectal tubular adenoma, a second patient had a 4 year history of proctitis had a 2mm rectal adenoma and the third patient had, at diagnosis, a 2 cm tubulovillous adenoma. Dysplasia associated with colitis was seen in a further 4 (3.2%) patients. Pseudopolyps were found in 6 (5.6%) patients and metaplastic polyps in 2 (1.8%) patients. 75 (71%) of the IBD patients were maintained on a 5-ASA compound.

Conclusions: The results suggest that adenomatous polyps are rare in patients aged 55–64 years with IBD compared to a control population. This supports the hypothesis that lesions other than polyps are important for the development of colorectal cancer in patients with IBD and it also supports evidence that the 5-ASA compounds could protect against adenoma formation.

Abstract 244

242 WHAT DO PATIENTS THINK OF COLONOSCOPIC SURVEILLANCE IN ULCERATIVE COLITIS?

M.D. Rutter, B.P. Saunders.

St Mark’s Hospital, Harrow, UK

Background: Colonoscopic surveillance for cancer in ulcerative colitis (UC) has been performed for 30 years, yet there are few data on patients’ quality of life (QOL) or views of cancer surveillance.

Method: At our hospital, patients with over 8 years of extensive UC are counselled about their increased risk of colorectal cancer (CRC) & offered surveillance. A 58 question survey was sent to all patients on surveillance. Information was gathered on demographics, general health (EQ-5D), & views on surveillance, cancer risk, & surgery.

Results: 332 questionnaires were sent. 1 patient had died, & 2 had moved. Of the remaining 329, 276 responded (84%). Median responder age was 55 (range 26–84; 164 male, 112 female). QOL on surveillance was high, but lower than the general population (EQ-5D visual analog scale mean 77.5, c.f. population mean 82.5; p<0.0001). There was no significant reduction in QOL with age. 84% felt the right amount of surveillance information was given, 16% too little, none too much. 92% felt explanations were easy to understand, 3% difficult, & 5% could not recall an explanation. 35% sought additional information. 98% felt surveillance important to them. 80% felt the surveillance interval about right, 11% too long, & 9% too short. 84% were worried about CRC. 96% felt reassured by surveillance, 4% felt it increased their anxieties. 2% believed surveillance completely removed cancer risk, 68% greatly reduced it, 24% moderately reduced, & 6% slightly reduced the risk. On the timing of surgery, 7% stated they would choose surgery after 10 years of colitis, 65% on dysplasia detection, 24% on CRC detection, & 4% said they would not have surgery even on CRC diagnosis.

Conclusion: Responders maintain a good quality of life on surveillance. 98% feel surveillance is important to them, although 11% hypothetically opted for a management strategy other than colonoscopic surveillance (surgery at 10 years, or no surgery even on CRC diagnosis). Most patients believe surveillance greatly reduces their chance of dying of CRC. Although most patients are content with the current information provided, 1/3 sought other information, and no patient felt they had received too much. Ongoing discussion with patients is necessary to reiterate the surveillance rationale & to clarify their wishes.

243 HOW EFFECTIVE IS CANCER SURVEILLANCE IN ULCERATIVE COLITIS?

M.D. Rutter, K.H. Wilkinson, S. Rumbles, A. Forbes, B.P. Saunders.

St. Mark’s Hospital, Harrow, UK

Background & Aim: Patients with longstanding extensive ulcerative colitis (UC) are at increased risk of colorectal cancer (CRC). We assessed the value of a long-running colonoscopic surveillance programme.

Methods: Patients on biennial colonoscopic surveillance (from 1975 onwards) with histologically proven UC of at least 8 years duration, macroscopically extending proximal to the splenic flexure were included. All patients were followed up to 1/1/01. Data were obtained from our prospective UC surveillance database, medical records & reports, GPs, other hospitals & the Office of National Statistics.

Results: 600 patients underwent 2627 colonoscopies & 5080 years of surveillance. 68 patients (11%) had dysplasia or cancer detected during surveillance. Initial dysplasia was low-grade (46), high-grade (10), & CRC (12). 89 patients underwent surgery (86 colectomies, 3 partial resections). Fourteen operations were for a pre-operative diagnosis of CRC, 16 for HGD (6 cancers in specimens), 14 for LGD (3 cancers in specimens), & one for an adenoma. 44 operations were for symptoms (5 cancers in specimens). In addition, 2 patients died of CRC without surgery. In total, 30 patients developed CRC within the surveillance programme. Of the 25 surveillance-detected CRCs, 15 (60%) were Dukes’ A or B. A further 16 patients had colectomies for dysplasia, & had dysplasia confirmed in the colectomy specimen. Thus 31 patients benefited from surveillance. This equates to one benefit per 19 patients, or one benefit per 164 surveillance years. 13 patients presented with Dukes’ C or disseminated malignancy within the surveillance programme.

Conclusion: The current colonoscopic surveillance programme benefits approximately 3/4 of patients who are likely to develop life-threatening cancers. However, it requires considerable effort and expense over a prolonged period of time. A significant minority of patients still present with advanced cancer despite surveillance. Improved criteria for surveillance are required.

244 THE INCIDENCE OF CROHN’S DISEASE IN DERBY 1970–2000.

T.R. Card1, G.K.T. Holmes2.

1Division of Epidemiology and Public Health, University Hospital, Nottingham, UK; 2Derbyshire Royal Infirmary, UK

Background and aims: There is good evidence to suggest that the incidence of Crohn’s disease has increased in western nations including the UK since the mid 20th century.1 Whether this rise is ongoing however is less clear and has been the subject of debate and disagreement for some years now. We have examined changes in Crohn’s disease incidence over 30 years in Derby.

Methods: All diagnoses of Crohn’s disease made at Derby’s two DGHs were collected via the use of hospital activity reports, histopathology records and the personal records of gastroenterologists. Diagnoses were validated via hospital notes. Subjects resident outside the city of Derby were excluded. Diagnoses were counted by quinquennia. Official estimates of Derby’s population were obtained for the same time periods and used to calculate incidence rates.

Results: In total 326 persons living in Derby were diagnosed with Crohn’s disease between 1970 and 2000. Of these 131 were male 195 were female. The table shows incidence rates for each quinquennium overall and by site.

Conclusions: This study supports the findings of other recent work suggesting that the incidence of Crohn’s disease has ceased to rise[2]. An apparent peak in incidence occurred between 1976 and 1990 which predominantly consists of disease involving the colon. The small numbers of cases prevent firm conclusions as to any change in distribution but it is interesting to speculate upon the role of changing diagnostic tests.

  1. Logan RF. Gut 1998;42:309–11.

  2. Yapp TR, et al. Eur J Gastroenterol Hepatol 2000;12:907–11.

245 EVIDENCE OF NORTH-SOUTH GRADIENT IN INCIDENCE OF JUVENILE-ONSET IBD IN SCOTLAND

M.C. Aldhous, E.L. Armitage, H.E. Drummond, J. Satsangi.

Gastrointestinal Unit, University Department of Medical Sciences, Western General Hospital, Edinburgh, UK

Introduction: The north-south gradient of IBD incidence was first described in Europe but the recent EC-IBD study suggests that the difference is narrowing. We and others have shown a high and rising Scottish incidence of IBD in the <16 age range, and using our unique dataset now aim to assess its variation with latitude.

Methods: The Scottish hospital-discharges-linked database was used to identify 933 patients less than 19 years old who were ICD coded as having had IBD between 1981 and 1995. All case records were reviewed, diagnoses confirmed and postcode at symptom onset noted. Incident cases (580) were those with symptom onset up to 16 years of age within the period 1 January 1981 to 31 December 1995. Incidence rates were calculated per 100 000 population per year for each of the 15 Scottish postcode districts and were standardised to the 1991 census population. The latitude of the principal town in each postcode district was used.

Results: The highest sex standardised incidence was found in the Shetland isles (7.62, latitude 60°, 9’N), this was followed by Orkney/NE Scotland (6.64, latitude 58°, 59’) The lowest incidence occurred in the Motherwell district (2.36, latitude 55°48’N). The r2 value for the correlation between latitude and incidence is 0.71.

Conclusion: The high incidence of IBD in NE Scotland is consistent with Kyles’ previous data, in addition we have shown a marked north south gradient in the incidence of juvenile-onset IBD in Scotland. The mechanism underlying this geographical pattern remains uncertain.

246 NEUROLOGY OF INFLAMMATORY BOWEL DISEASE

A. Shetty1, N. Tubridy2, P. Rudge3, R. Vega1, D. Polymeros2, F. Schon2, A. Forbes1.

1Dept. of Gastroenterology, St Mark’s Hospital, Harrow, HA1 3UJ, UK; 2Dept. of Neurology, Atkinson Morley’s Hospital, Wimbledon, SW20, UK; 3Dept. of Neurology, Northwick Park Hospital, Harrow, HA1 3UJ, UK

Background: The literature on the extra-intestinal complications of inflammatory bowel disease (IBD) has always included reference to the neurological problems that occur in patients with these conditions. These patients tend to be more prone to stroke and sensorineural deafness but there is only a small literature on associations with other neurological diseases occuring concurrently with IBD.

Aim: To find if there is any association between inflammatory bowel disease (IBD) and other neurological conditions.

Patients and Methods: We studied prospectively 888 patients attending a single IBD clinic and as hospital controls used 700 patients who attended for open access endoscopy at 2 hospital units. Patients were asked to complete a questionnaire. Subjects for whom there was a high suspicion of neurological disease from the questionnaire were then contacted by telephone by the neurological team to determine more accurately the nature of the problem or the reason for any of the neurological investigations, as were a similar number of negative responders.

Results: Of the IBD patients, 7 (0.8%) had clinically definite multiple sclerosis (MS) compared to none in the control group (p<0.025). One further IBD patient has clinically probable MS. Peripheral neuropathy and epilepsy were also more common in our IBD group, 12 (1.4%) v 4 (0.6%) and 14 (1.6%) v 5 (0.7%) respectively, but this was not statistically significant (p<0.2). Stroke (1.6% v 2.4%), myasthenia gravis (0.1% v 0.2%), deafness (8.4% v 11.1%) and migraine 18.4% v 17%) were equally represented in the IBD group and hospital controls respectively.

Discussion: We conclude that there is a true association between multiple sclerosis and IBD. We also found an increased prevalence of peripheral neuropathy and epilepsy. Further work is ongoing to look at possible immunological and genetic similarities between MS and IBD.

247 PATIENTS WITH INFLAMMATORY BOWEL DISEASE TAKE AS MUCH COMPLEMENTARY/ALTERNATIVE MEDICINE AS PATIENTS WITH FUNCTIONAL BOWEL DISEASE

S.C. Kong, C.Y. Pocock, D.P. Hurlstone, M.G. Bramble, M.E. McAlindon, D.S. Sanders.

Department of Gastroenterology, Royal Hallamshire Hospital, Sheffield.

Introduction: In recent years, the public awareness and usage of complementary/alternative medicine (CAM) has increased. The use of CAM (for example, aloe vera) has previously been described in adult patients with functional bowel disease.

Aim: To assess the incidence of CAM usage by gastroenterology patients at a single university centre.

Method: Patients attending luminal gastroenterology clinics (from June 2002) completed a 30-point, structured questionnaire assessing their use of CAM. A gastroenterologist verified the patient diagnosis.

Results: There were 790 patients recruited. The 3 main diagnostic categories were inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and other gastrointestinal diseases (GID: for example colonic polyps). Demographics and CAM usage in each group are shown in the table below.

Abstract 247

Chi-squared analysis demonstrated significant differences between the IBS versus other GI group (p<0.0001) and IBD versus other GI group (p<0.0001). Multivariate logistical analysis of the whole cohort showed significant demographic differences for sex: female versus male (p<0.05). 64% of all CAM users were women. For every 1-year increase in age there was a 1% reduction in the use of CAM (p<0.03). 87% of all CAM users (253/291) felt they derived some symptomatic benefit.

Conclusion: The percentage of CAM users amongst patients with IBD is similar to those with a functional diagnosis. Patients with IBD are using CAM in addition to conventional treatment. Awareness of this will prevent adverse CAM and conventional drug interactions

248 CAUSES OF MORTALITY IN CROHN’S DISEASE

A. Agrawal, S. Kennedy, A.I. Morris, A. Ellis, K. Leiper, J.M. Rhodes.

Royal Liverpool University Hospital, Liverpool L7 8XP, UK

Introduction: It is recognised that life expectancy in Crohn’s disease is slightly reduced (Jess et al, Gastroenterology 2002;122:1808) but few studies have addressed causes of mortality.

Methods: All patients attending our centre have been logged onto a centralised database since 1996. This includes 676 patients with Crohn’s disease with a median age of 43 years, range 17–93 years. During the study period (Jan 1996–June 2002) 32 of these patients died with a median age of 57, range 18–89. Case notes were available for 30 of these patients but contained incomplete data in 4; the remaining 26 were analysed with regards to cause of death.

Results: Eleven (42%) of the deaths were attributable to Crohn’s disease. Apart from post-operative deaths and Crohn’s related malignancy (small intestinal cancer 1, rectal cancer 1, rectal lymphoma 1) these included amyloidosis and renal failure (1), sepsis (2), right atrial thrombus complicating TPN (1) and malnutrition in an elderly patient (1). The median age for those whose death was related to Crohn’s disease was 48 years (range 36–83) and for those that died of other causes was 72.5 years (range 32–89). All 8 non-malignant Crohn’s related deaths were in patients receiving systemic corticosteroids compared to 39% of those who died of other causes. Seven (64%) patients who died of Crohn’s disease were smokers at the time of death compared to 40% of those who died due to unrelated causes.

Conclusion: Crohn’s disease is still associated with a considerable mortality. Further study is justified to assess whether use of systemic corticosteroids may contribute to this mortality.

Abstract 248

249 ANTIBODIES TO ASCA IN CROHN’S DISEASE ARE ASSOCIATED WITH ILEAL DISEASE BUT NOT NOD2 MUTATIONS IN A SCOTTISH COHORT

L. Walker, M.C. Aldhous, J. Satsangi.

Gastrointestinal Laboratory, University of Edinburgh Department of Medical Sciences, Western General Hospital, Edinburgh, UK

Background: Crohn’s disease (CD) and ulcerative colitis (UC) can be difficult to differentiate clinically. Anti-Saccharomyces cerevisiae antibodies (ASCAs) have been proposed as diagnostic tools for CD. Mutations in the NOD2 gene engender susceptibility to CD but not UC. We investigated the possible relationship between NOD2 mutations, disease phenotype and ASCAs in CD, and whether ASCAs were related to antibodies to other fungal proteins.

Methods: Serum from 308 patients (150 CD, 73 UC, 7 with indeterminate colitis (IC)) and 78 healthy controls (HC) were assayed for ASCA antibodies (IgA+IgG) using the Medizym ELISA kit. Antibodies (IgA, IgG) to other fungal proteins (Fusarium species ATC20334, MP) were measured in the same samples using an in-house ELISA assay. NOD2 mutations in these patients had been identified previously using PCR. The antibody responses were compared with the known NOD2 genotype and disease phenotype of these patients.

Results: ASCA was present in 56% of CD, 16% of UC, 43% of IC, and 8% of HCs. ASCA+ve status was a predictor for CD (OR 10.6 (95% CI 1.13–3.75)) and could discriminate CD from all IBD patients (OR 5.5 (95%CI 0.86–6.46)). Sensitivity of ASCA for CD was 56%, specificity was 89% and positive predictive value was 83%. ASCA was associated with ileal/ileocolonic rather than colonic disease (p<0.001), but not with NOD2 mutations. There was no association between ASCA and antibodies to MP (IgA or IgG). MP IgA titres were higher in CD than HC (p<0.01), while MP IgG titres were associated with ileal, but not ileocolonic nor colonic disease.

Conclusions: ASCA was found to be a specific marker for CD and associated with disease involving the ileum, but not NOD2 mutations. MP antibody titres were higher in CD patients and independently associated with ileal disease alone. These results help to further the understanding of CD pathogenesis.

250 INCIDENCE AND PHENOTYPIC ASSOCIATIONS OF NOVEL ANTIBODY MARKERS IN CROHN’S DISEASE

I.D.R. Arnott1, C.J. Landers2, S.R. Targan2, J. Satsangi1.

1Gastrointestinal Unit, University Department of Medical Sciences, Western General Hospital, Edinburgh, UK; 2IBD Research, Davis Building, Cedars-Sinai Medical Centre, Los Angeles, USA

Introduction: The diagnostic value and pathogenic importance of serological markers in patients with inflammatory bowel disease remain under evaluation. Antibodies directed against Saccharomyces cerevisiae (ASCA) and the perinuclear component of neutrophils (pANCA) have been the most widely studied and may be of diagnostic importance. OmpC and I2, antibodies directed against luminal bacterial components (E coli and pseudomonas respectively), have recently been described in Crohn’s disease (CD). The data reported in the Los Angeles population suggest a selective loss of tolerance to microbial antigens in CD. Phenotypic associations of these antibodies are unknown. We aimed to assess the frequency of ASCA, pANCA, OmpC, and I2 in an independent CD cohort and establish phenotypic associations.

Methods: 141 well-characterised CD patients (76 females, median age 39years (17–88)) and 78 healthy controls (HC) were studied. 36 had ileal disease, 58 had colonic disease and 57 had both ileal and colonic disease. 42 had associated perianal disease. 73 had undergone previous surgery. ELISA assayed ASCA, ANCA, OmpC, and I2. The pattern of ANCA positivity was assessed by immunoflourescence.

Results: All antibodies were more prevalent in CD than HC (ASCA 39% v 4%, ANCA 14% v 1%, I2 52% v 10%, OmpC 37% v 1%, all p<0.001). A positive serum ASCA was associated with ileal disease (76% v 42%, p<0.001) and previous surgical resection (73% v 37%, p<0.001) where as pANCA was a marker for colonic disease (63% v 30%, p<0.05). OmpC and I2 were associated with increasing age, increasing disease duration and the need for intestinal resection (p<0.001). OmpC is also associated with ileal disease (p<0.01).

Conclusion: We have confirmed the previous association of ASCA and ileal disease and ANCA and colonic disease. OmpC is also associated with ileal disease and OmpC and I2 are associated with increasing disease duration.

251 PREVALENCE AND MANAGEMENT OF INFLAMMATORY BOWEL DISEASE: DATA FROM PRIMARY CARE RECORDS, INCLUDING 5-ASA PRESCRIBING AND COMPLIANCE

M.A. Stone1, J.F. Mayberry2, R.B. Baker1.

Department of General Practice and PHC, 1University of Leicester and 2Leicester General Hospital, Leicester, UK

Background: Data from general practice (GP) records in North Tees (Rubin et al, 2000) suggested a higher prevalence of inflammatory bowel disease (IBD) than previous estimates from hospital data. Regular prescribing of 5-aminosalicylic acid (5-ASA) therapy can reduce the risk of colorectal cancer in patients with ulcerative colitis (UC).

Aims: To estimate IBD prevalence from GP records in the Trent region of central England and describe the management of patients with this condition, including data regarding 5-ASA prescribing and compliance in UC.

Methods: 15 general practices recruited through the Trent Focus Collaborative Research Network provided data on confirmed cases of IBD using a standardised data collection form.

Results: 344 patients with IBD were identified from a combined GP list size of 86 801, suggesting a prevalence of 396 per 100 000 (95% confidence interval 356 to 440), much higher than previous estimates from secondary care and similar to results from North Tees. Approximately one third of patients with IBD were considered by GPs to be solely under their care and only 59% of IBD patients had actually been seen as hospital inpatients or outpatients during the previous year. Only 57% of patients with UC (including proctitis) had been prescribed a 5-ASA during the previous 6 months and good compliance with prescribing and taking 5-ASA medication was suggested from the prescribing records of only 42% of these patients (23% all UC patients).

Conclusions: Data collection from hospital records may miss some IBD patients. GPs may play an important role in caring for patients with IBD; they should therefore receive good education regarding IBD management, including the importance of 5-ASA therapy in UC.

  1. Rubin GP et al. Inflammatory bowel disease: epidemiology and management in an English general practice population. Aliment Pharmacol Ther 2000;14:1553–9.

252 COMPARISON OF IN VITRO CHARACTERISTICS OF TWO UK MESALAZINE 400MG PRODUCTS

M. Newton1, A. Mwalupindi2, (introduced by C.S. Probert).

1School of Pharmacy, University of London; 2Procter & Gamble Pharmaceuticals, Norwich, US

Background: The release profile of mesalazine from different aminosalicylates preparations are matched to the site and extent of an individual patient’s inflammatory bowel disease. A new enteric coated mesalazine 400mg tablet (Ipocol) has been launched in the UK. Analytical assessment of Asacol and Ipocol was undertaken for comparative purposes.

Methodology: Three methods were used: scanning electron microscopy for coating thickness; near infrared absorption for coating thickness and tablet composition (evaluated by principal component analysis, (PCA)); and in vitro disintegration with dissolution testing to evaluate 5-ASA release, using the standard methodology for Asacol as found in the UK marketing authorisation application. Both Asacol 400mg tablets and Ipocol 400mg tablets were purchased commercially and assessed under conditions of good laboratory practice.

Results: Scanning electron microscopy showed the mean thickness of the enteric coating of Ipocol ranged from 51 to 57μm. Ipocol had a significantly thinner coating over the dome (50–66μm) (p=0.0002) and edges (38-47μm) (p<0.0001) than Asacol. Near infrared absorption, analysed by PCA, showed Asacol and Ipocol to have unique spectral signatures. PCA plots are influenced by coating thickness, formulation and tablet shape. In vitro dissolution at pH 6.4 showed a mean release of 5-ASA of < 1% from Asacol tablets v 27% from Ipocol tablets. At pH 7.2, the 5-ASA release profiles of the two products were significantly different at all time points (p<0.0001).

Conclusions: The two formulations exhibit significantly different coating characteristics and in vitro dissolution profiles. Since they are not pharmaceutically equivalent this raises concerns about their substitutability.

253 THREE YEARS OF PATIENT-LED THERAPY IN ULCERATIVE COLITIS WITH A SINGLE AMINOSALICYLATE, BALSALAZIDE: A LONG TERM OBSERVATIONAL STUDY

J.R.B. Green1, C.H.J. Swan1, J.A. Gibson2, G.D. Kerr3, E.T. Swarbrick4, P.C. Thornton5.

1City General Hospital, Stoke on Trent; 2Staffordshire General Infirmary, Stafford; 3Royal Shrewsbury Hospital; 4New Cross Hospital, Wolverhampton; 5Biorex Laboratories, Enfield, Middlesex

Patient-led therapy in ulcerative colitis (UC) may have significant advantages for patients but this has never been formally observed in long term practice. Use of a single therapy effective in acute relapse and maintenance of remission (in different doses) could simplify long term management by enabling patients to choose an appropriate dose.

Balsalazide, an aminosalicylate prodrug, is effective and well tolerated at all doses for both these indications in UC. To assess the practicability, safety and efficacy of patient-led dosing, two groups of patients in remission from UC (52 in long-term stable remission (SR) and 76 in recent remission (RR)) were prospectively followed in a 3 year open, non-comparative study receiving balsalazide in a variable dose determined by the patient. Symptoms, mucosal inflammation, general well-being and adverse events were assessed every 12–14 weeks and laboratory assessments made every 6 months.

The average daily dose in both groups was 3g (range 1.5–6g). A total of 23 SR patients (45%) had relapsed by 3 years (median time to relapse 36 months) compared with 45 RR patients (59%) (median time to relapse 22 months) with fewer patients relapsing each successive year in both groups. Clinical scores for SR patients were consistent over the study, while RR patients showed a slight improvement. Both groups needed fewer dose increases over time. Time since last relapse was significantly associated with relapse during the first year of treatment (p<0.033) for SR patients. No adverse medication-related haematological or biochemical changes were recorded.

We conclude that long term patient-led maintenance therapy with balsalazide appears to be well tolerated, safe and effective for patients with UC.

254 PREDICTING OUTCOME AND RISK ASSESSMENT IN SEVERE ULCERATIVE COLITIS

G.T. Ho1, C. Mowat2, C.J.R. Goddard3, J.M. Fennell1, N.B. Shah1, R.P. Prescott4, J. Satsangi1.

1Western General Hospital, Edinburgh; 2Ninewells Hospital, Dundee, 3 St. John’s Hospital, Livingston, 4Department of Statistics and Public Health, University of Edinburgh, UK

Backgrounds/Aims: The rate of failure of medical therapy in severe ulcerative colitis (UC) remains high (30–40%). We have identified predictor factors associated with poor outcome in patients presenting with severe attacks of UC and devised a numerical scoring system that categorises patients by risks.

Methods: All patients admitted with severe UC (defined by the modified Truelove and Witts criteria) in three gastroenterology units in the Lothian region between January 1995 and March 2002. Fifty-six clinical and demographic parameters were analysed. Predictive factors were identified using multiple logistic regression and risk score was formulated using statistical modelling. Outcomes were categorised to non-responders (colectomy during hospitalisation) and responders.

Results: Of 167 patients admitted, 68(40%) failed to respond to medical therapy. Mean stool frequency, albumin, and colonic dilatation within the first three days of treatment were identified as independent predictors of outcome. A numerical score was formulated using these variables (table 1). Patients with scores of 0–1, 2–3 and >4 had a rate of medical therapy failure of 12%, 43%, and 85% respectively. 42%, 34%, and 25% of patients fell into each respective category (table 1).

Abstract 254

Risk score for categories according to coefficients from logistic regression modelling

Conclusion: Using this novel scoring system, we can clearly stratify patients to low, intermediate and high risk groups of non-response.

255 HEALTH-RELATED QUALITY-OF-LIFE (HR-QOL) IN INFLAMMATORY BOWEL DISEASE (IBD): SURVEY OF A PREVALENCE COHORT

A. Bassi, E. Brown, K. Bodger.

Aintree Centre of Gastroenterology; University Hospital Aintree, Liverpool, UK

Introduction: Studies of HR-QoL aim to assess the influence of disease on patients’ lives by seeking patient-perceived valuations of health across discrete domains including physical and psychosocial function. The aim of the present study was to measure HR-QoL in an unselected cohort of IBD patients and to correlate with demographics, disease type, extent and severity.

Methods Subjects: We identified a 6-month prevalence cohort of 479 IBD patients receiving any form of care for IBD at our centre by cross referencing multiple hospital databases (out-patient letters, in-patient coding, diagnostic reports). Disease type, extent and severity (using a published grading system) were abstracted from casenotes.

Postal survey: Self-administered questionnaire, the UK-IBDQ (modified to include an item about interference with household or recreational activities). The IBD-Q has 5 domains (Social; Emotional; Bowel 1; Bowel 2 & Systemic). Data analysed by linear regression.

Results: 233 (48.6%) responded (CD n=87, UC n=122, indeterminate colitis n=24). Data incomplete in 12. Mean age (sex): CD 48.5 yrs (28 male); UC 48 yrs (61 male). Table summarises total QoL scores (% optimal function). Total scores for UC were higher than CD (76% v 70%, p<0.001) and lowest domain scores were in the Systemic domain (CD 55%; UC 64%). Emotional (p=0.011) and Bowel (p=0.03) scores were significantly lower in younger patients. Mean lost days from household / recreational activities in 6 months: CD 21 d; UC 12 d.

Conclusions: Disease severity is the main determinant of overall QoL in IBD with greatest impact being on systemic well-being. QoL scores were lower in CD than UC overall and CD sufferers had greater loss of “social” days. Younger age is associated with lower ratings for emotional functioning.

Abstract 255

256 SOFT STOOLS, SOFT BONES AND SOFT EVIDENCE?—WHAT INFLAMMATORY BOWEL DISEASE PATIENTS THINK OF OSTEOPOROSIS MEDICATION

D.G. Oliver, P.N. Trewby.

Darlington Memorial Hospital, Darlington DL3 6HX, UK

Background: The British Society of Gastroenterology guidelines for osteoporosis in inflammatory bowel disease imply the screening of a large proportion of inflammatory bowel disease patients and treatment with antiresorptive medication if necessary. Despite this, only a small percentage of patients will benefit from fracture prevention as a direct result of taking this medication.

Aims: To explore inflammatory bowel disease patients’ attitudes towards taking medication for osteoporosis in the light of present best evidence for fracture reduction with such medication.

Methods: Questionnaires were sent to all patients on an inflammatory bowel disease database at a district general hospital in North East England. The main outcome measure was the lower limit of absolute risk reduction of fracture below which subjects were not prepared to take a hypothetical osteoporosis drug for three years. All responders were followed up with a telephone interview.

Results: 121 (45%) patients responded. The median value for the lower limit of absolute risk reduction of fracture acceptable to the study population was 50%. There was no significant correlation between the value expressed by the patient and their age, number of previous fractures or current or previous steroid use. Patients who had suffered a fracture within the previous two years showed a significantly lower threshold to taking osteoporosis medication than those with no previous fracture. Patients exhibited wide variations in their attitudes towards taking the preventive treatment with 20% being concerned about possible side effects. 85% wished to have a discussion about the expected benefits of any new preventive drug prior to starting treatment.

Conclusions: Inflammatory bowel disease patients may not be prepared to take osteoporosis medication if they were aware of its true likelihood of benefit. Gastroenterologists should consider their patients’ attitudes as well as the medical criteria before deciding to treat for osteoporosis.

257 OSTEOPOROSIS IN CROHN’S DISEASE IS NOT DETERMINED BY VITAMIN D RECEPTOR AND INTERLEUKIN-6 GENOTYPE

A. Sutherland-Craggs, J.C. Mansfield, P.T. Donaldson, A. Daly, R. Francis R, N.P. Thompson.

Dept. of Gastroenterology, University of Newcastle upon Tyne, and Dept. of Gastroenterology, Freeman Hospital, Newcastle, UK

Background: Osteoporosis is a common and important complication of Crohn’s disease. The risk of osteoporosis is known to be related to body mass index, use of corticosteroid therapy and disease activity. These factors do not fully account for the variation between patients, genetic factors may also be important.

Aim: To determine whether bone density in Crohn’s disease is related to polymorphisms which influence bone density in other conditions: the vitamin D receptor (VDR) TaqI (Exon 9), the VDR Start codon polymorphism FokI (Exon 2) and the Interleukin-6 NlaIII –174 G/C SNPs. The VDR TaqI genotype has been found to be related to osteoporosis in postmenopausal women, and bone loss in rheumatoid arthritis. The VDR FokI genotype and the IL-6 polymorphism have been found to relate to bone mineral density in other populations.

Methods: 255 patients had their bone density assessed by DEXA scanning at the lumbar spine and hip. Osteoporosis was defined by WHO criteria as a T(30) score worse than –2.5 at any site, osteopenia as a T(30) score between –1 and –2.5. DNA was genotyped for VDR SNPs, TaqI and FokI, and the IL-6 G/C SNP.

Results: There are no significant differences in genotype, or allele frequency between the groups or compared to healthy controls.

Conclusion: VDR TaqI and FokI SNPs, and IL-6 G/C SNP do not appear to contribute to the incidence of osteoporosis in Crohn’s disease.

Abstract 257

258 DIFFERENTIATION OF EUROPEAN “ASACOLS”

M. Newton1, A. Mwalupindi2, A. Forbes3.

1School of Pharmacy, University of London; 2Procter & Gamble Pharmaceuticals, Norwich, US; 3St Mark’s, Harrow, UK

Background: The original developers of “Asacol” in Europe were Tillots Laboratories. The subsequent out-licensing of the product across Europe has given rise to potential of variations in tablets. If the companies change excipients &/or manufacturing processes, this can affect the release profile. This in turn influences availability of the product locally in the intestinal mucosa & the amount of product absorbed systemically & hence the products’ overall profile. Analytical assessment of different European “Asacols” (400mg tabs) was undertaken to evaluate tablet characteristics. Tablets were sourced from France (Fivasa), Germany (Asacolitin), Italy (Asacol It), the Netherlands (Asacol NL) & the UK (Asacol UK).

Methodology: Three assessment tools were used in the analysis; scanning electron microscopy (SEM) for coating thickness; near infra red (NIR) absorption for coating thickness & tablet composition, (which was evaluated by principal component analysis (PCA)); & in vitro disintegration and dissolution testing to characterise 5-ASA release. Batches of each product were purchased commercially & assessed under good laboratory practice.

Results: PCA of NIR absorption demonstrated clear differences in individual spectra. These spectra are influenced by coating thickness, formulation & tablet shape. SEM results further showed a range of coating thicknesses. Asacol (It) had the thinnest (39–62μm) & Asacol (NL) the thickest (64–129μm) coating. Since the coating is critical to the release mechanism of ‘Asacol’ the variations shown both by NIR & SEM analysis would be expected to impact the in vitro dissolution profile of 5-ASA release. Differences between the products were identified in terms of the initial release pH & overall profile.

Conclusion: The currently available “Asacol” 400mg tablets across Europe differ from each other in vitro with respect to the pH at which 5-ASA release started and their overall release profile at pH 7.2. Their interchangeability should therefore be viewed with caution until in vivo results are available to demonstrate their equivalence.

Endoscopy posters 259–285

259 APPROPRIATENESS OF UPPER GASTROINTESTINAL ENDOSCOPY: A HOSPITAL BASED RETROSPECTIVE STUDY

S. Aroori1, B. Little1, P.D. Carey1, J.A.A. Archbold2. 1Belfast City Hospital; 2Downe Hospital

Aim: To evaluate whether upper gastrointestinal endoscopy (UGE) is used appropriately according to the British (BSG) and American Society of Gastrointestinal Endoscopy (ASGE) guidelines in a hospital setting and whether there is any relationship between appropriateness of UGE and the presence of lesions detectable by endoscopy.

Materials and Methods: Indications and endoscopic findings for 525 consecutive UGE performed in 500 patients, between Jan 1995 and Dec 2000 in Downe Hospital, retrospectively evaluated using BSG and ASGE guidelines to determine appropriateness of referrals. The endoscopic findings that had direct therapeutic or prognostic consequences were classified as positive; the other were classified as negative.

Results: There were 237 male and 263 female patients with mean age 57.3 years (range: 17–94). In all, 62.9% of UGEs were positive, 37.1% were negative, and 22.7% and 26.7% were inappropriate according to BSG and ASGE guidelines, respectively (p = 0.024). The probability of finding a positive endoscopy was significantly higher in UGE rated as appropriate on the basis of both BSG (p < 0.0001) and ASGE guidelines (p = 0.002). Endoscopies rated as inappropriate according to BSG and ASGE guidelines showed a positive finding in 46.2% and 52.1% of cases, respectively. On multivariate analysis, the positive findings are directly related to age (p = 0.034), male gender (p = 0.006) and inversely related to upper abdominal pain (p < 0.0001) and nausea and vomiting (p = 0.0001). Inpatient referrals showed more positive findings compared with outpatient and open access referrals (p = 0.01).

Conclusions: UGE is frequently used for inappropriate indication. The actual usefulness of appropriateness criteria as proposed by BSG and ASGE is questionable: as their strict observance could lead to missing a large number of significant positive findings.

260 A STUDY TO VALIDATE THE COLONOSCOPY SIMULATOR: IT IS USEFULLY DISCRIMINATORY FOR MORE THAN ONE MEASURABLE OUTCOMES

T. Mahmood, A. Darzi, S. Bann, V. Datta.

ASU, Imperial College and St Mary’s Hospital NHS Trust, London, UK

Objectives: To investigate the relationship between clinical experience and performance with regard to colonoscopic procedures performed on the HT Immersion Medical Colonoscopy Simulator. The hypothesis is that the performance of the novice, intermediate, and experienced operator is different on simulators just as on real patients. Thus validating HT Immersion Medical Colonoscopy Simulator.

Method: 25 postgraduate doctors were divided into three groups according to their level of colonoscopic experience. Candidates at random performed colonoscopy on module 3 or 4 of the HT Immersion Medical Colonoscopy Simulator. Candidates in the first group called “novice”, had each performed less than 10 colonoscopies and included 4 PRHOs, 5 SpRs, and 2 Consultants. This group completed 80 episodes. The second group, called “intermediate”, had each performed 11–100 colonoscopies and included 5 SpRs and 2 Research fellows. This group completed 65 episodes. Members of the third group, called “experienced”, had each performed more than 101 colonoscopies and included 1 SpR and 6 Consultants. This group completed 45 episodes. A result of 3600 seconds (1 h) was used to denote perforation.

Results: The experienced were shown to perform better than the intermediate group, who in turn performed better than the novice. The assessment was made on multiple factors, including time taken to complete the test, percentage of the colonic mucosa visualised, incidence of colonic perforations, and path length used. The results were statistically highly significant for all these factors, p < 0.000 except in path length used.

Conclusions: Operators who differ in terms of their clinical experience and technical ability also differ in their performance of simulated colonoscopy. Thus the simulator technology has been shown to be a powerful discriminator of manipulative skills in colonoscopy. The experienced perform better than the intermediate than the novice on the simulator, thus validating HT Immersion Colonoscopy Simulator.

261 A STUDY OF THE LEARNING CURVE FOR A COLONOSCOPY SIMULATOR WITHOUT ANY FEEDBACK: NO FEEDBACK, NO LEARNING OF THE MACHINE ITSELF

T. Mahmood, A. Darzi, S. Bann, V. Datta.

ASU, Imperial College and St Mary’s Hospital, London, UK

Objectives: To investigate the learning curve for colonoscopic procedures performed on the HT Immersion Medical Colonoscopy Simulator without any feedback. The hypothesis is that no learning curve exists without a feedback. In other words, the student does not learn the simulation machine itself, instead of the colonoscopy technique that it is primarily meant to teach.

Method: 22 postgraduate doctors were recruited, including 5 research fellows, 4 PRHOs, 6 SpRs and 7 consultants. Candidates were asked to perform colonoscopy on the HT Immersion Medical Colonoscopy Simulator. Modules 3 and 4 were used at random. In total, each candidate was asked to perform five consecutive virtual colonoscopies on the same module. These five episodes were collectively referred to as one trial. A time result of 3600 seconds (1 h) was used to denote perforation. No guidance or feedback was given to candidates before, during or after each procedure.

Results: 23 candidates recorded five attempts or episodes each, meaning one trial each, on only one module of the colonoscopy simulator, (Total 115 episodes over 23 trials). Another 15 people recorded five attempts, meaning one trial each, on two modules of the colonoscopy simulator (Total 150 episodes over 30 trials). Therefore the total episodes recorded were 265, over 53 trials. There was no improvement in performance on the simulator from first attempt to the fifth in absence of feedback. The outcomes measured included time taken to complete the test, percentage of the mucosa visualised, depth of the instrument inserted, and the path length used. The results were statistically very significant p < 0.000 for all outcomes.

Conclusions: This study has demonstrated that in absence of feedback, it is not possible to improve performance on the HT Immersion Colonoscopy Simulator. Thus there is no learning curve for the machine itself. The information from this study is vital for using the simulators in training and assessment because any learning curve would be of procedure only and not the machine.

262 THE COLONOSCOPY SIMULATOR AS A TEACHING TOOL

T. Mahmood, A. Darzi, S. Bann, V. Datta.

ASU, Imperial College and St Mary’s Hospital, London

Objectives: The aim of this study was to investigate the relationship between teaching in the form of structured feedback and performance with regard to colonoscopic procedures performed on the HT Immersion Medical Colonoscopy Simulator. The study employed clinically relevant outcome measures as proxies of learning. In contrast to work conducted on more primitive simulators, this study is not reliant upon the time taken to complete a task as the only surrogate of competency. The hypothesis is that learning colonoscopy on the simulator is affected by a structured feedback.

Method: 10 postgraduate doctors, including 5 SpRs, 4 research fellows, and 1 PRHO, who had previously performed at least five times on the module used for training and assessment were recruited in the study. Candidates were given training in a structured way by means of feedback until they were themselves satisfied (minimum 19 min 11 seconds, maximum 33 min 38 seconds, mean 27 min 46 seconds). Training included negotiation of alpha, reverse alpha and N loops. Module 3 or 4 was used at random for teaching purposes. Assessment was made on the same module pre- and post-training.

Results: The data show that in the presence of feedback there is physically and statistically significant improvement in the time taken to complete an episode (p < 0.004), mucosa visualised (p < 0.000), and the efficiency ratio (p < 0.001). There is improvement in the learning curve derived from the differences in efficiency ratios pre- and post-training for each of the episodes completed. The maximum learning was 0.24 and the minimum was −0.4.

Conclusions: This study has demonstrated the influence of structured feedback on performance on the colonoscopy simulator. A statistical description of the learning curve for simulated colonoscopy has been generated. In the presence of teaching in the form of structured feedback, improvements have been demonstrated for all outcome measures. Most importantly the gain in efficiency ratio, employed as a proxy of learning, has been shown to improve significantly with feedback. The colonoscopy teaching laboratory offers a purpose built environment for learning, while maintaining the degree of realism necessary to provide relevant experience. Simulators are economically viable when compared to expert time in theatre. Trainees can be introduced to the concepts such as complication management in a rational manner and be allowed more freedom of scope manipulation prior to expert intervention.

263 SEDATION TRAINING FOR ENDOSCOPISTS: A POSTAL SURVEY OF CURRENT PRACTICE IN NORTHERN ENGLAND

L. Hodgson, E. Stoker, N.P. Thompson.

The Newcastle-upon-Tyne Hospitals NHS Trust

Aims: To assess existing level of sedation training in gastroenterology across the Northern Region in line with current guidelines. The Royal College of Surgeons Report in 1993 suggested there was a lack of formal sedation training courses encompassing maintenance of skills by continuing professional education.

Method: An anonymous postal questionnaire based on current guidelines was sent to all endoscopists in the Northern Region.

Results: 167 questionnaires were distributed. We received 112 (67%) replies of which 100 (89%) were complete and 12 (11%) were partially complete. Respondents comprised consultants 67%, specialist registrars 16%, nurse endoscopists 13%, and other 4%. The average number of years administering sedation was 13 years. Drugs regularly used were midazolam 96%, diazemuls 22%, pethidine 81%, fentanyl 7%, propofol 0%, and other 4%. Informal training was undertaken by 80% of respondents. The training of these respondents was performed by senior colleagues 83%, anaesthetists 4%, self-trained 4%, and other 9%. Formal training was undertaken by 19% of respondents an average of 7 years ago. Training in sedation reversal identified 67% of respondents had undergone informal training of which senior colleagues trained 64%. Basic life support training had been attended by 69% of which 31% of these attended within the previous 12 months. Advanced life support training had been attended by 41% of which 59% of these had attended within the previous 3 years. 89% of respondents ensure oxygen is administered to sedated patients. 74% monitor paediatric and elderly patients differently. 63% of respondents would like to undertake a recognised qualification. 47% of respondents would be prepared to attend yearly simulated sedation incident workshops.

Conclusion: This study identifies the continued lack of formal sedation training that was highlighted by The Royal College of Surgeons Report in 1993. There is a need to implement training to comply with current recommendations and best practice.

264 IS OESOPHAGEAL DILATATION A DYING SKILL? THE IMPACT OF PROTON PUMP INHIBITORS

P.A. Berry, N.I. McNeil.

Ealing Hospital, Uxbridge Road, Southall, Middlesex UB1 3HW, UK

The number of endoscopic oesophageal dilatations performed for benign stricture was observed to have decreased in our unit over the past decade, making it hard for trainees to gain experience. This trend was investigated, and compared to the rise in the use of proton pump inhibitors (PPIs). Comparison was also made with the number of duodenal ulcers diagnosed each year. The number of dilatations performed and new duodenal ulcers identified per year was obtained from records kept in the endoscopy unit for the years 1994 to 2001. As dilatations had been performed on several patients a number of times in quick succession, the number of different patients treated per year was used. Detailed information concerning the quarterly expenditure on all PPIs by the local health authority (Ealing, Hounslow and Hammersmith) was also obtained. Annual expenditure on PPIs rose steadily during the decade, from 1 630 000 in 1994 to 3 200 000 in 2001. The total number of endoscopic procedures remained broadly constant. The number of patients requiring dilatation fell. Only 4 patients presented in 2001, compared to 20 in 1994. The incidence of duodenal ulcer also fell, the trend being most marked in the latter part of the study period (96 in 1997, 59 in 2000). There was strong negative correlation between the amount spent by the health authority on PPIs and the number of dilatations performed (r = −0.68, p < 0.05). Between expenditure on PPIs and number of DUs identified there was less strong negative correlation (r = −0.50, p < 0.05). The reduction in the number of patients requiring dilatation over the past 10 years appears dramatic and sustained. This may be due to the rise in the prescription of powerful acid suppressants. Dilatation of benign oesophageal stricture may become increasingly rare, and trainee endoscopists may lack adequate practise in the technique.

265 DIAGNOSIS OF HELICOBACTER PYLORI IN A DISTRICT GENERAL HOSPITAL (DGH): ACCURACY, COST AND PATIENT PREFERENCE FOR SIX WIDELY AVAILABLE TESTS

C. Gordon, P. Thatcher, P. Youd, A. Stratford, S.R. Gould.

Department of Gastroenterology, Epsom General Hospital, Surrey, UK

Introduction: The detection of H pylori forms part of the care pathway for dyspepsia. However, there are many tests to choose from, and no clear guidance as to which one to use.

Aims: To determine the positive and negative predictive value (PPV and NPV), sensitivity (Sens) and specificity (Spec) and cost of the urea breath test (UBT), in-house prepared rapid urease test (RUT), histology (histo), culture (cult), serology (sero), and faecal antigen (HpSA) in H pylori diagnosis, and to examine the patient’s preference.

Methods: 109 patients were recruited from an open access endoscopy (OGD) service. Samples were taken at OGD for RUT, histology, culture, and serology. UBT and faecal antigen test were carried out within seven days. The gold standard for a positive test for H pylori was taken as a positive UBT with one other positive test. Cost minimisation analysis was performed for all tests. Patient preference was determined via a questionnaire to 100 patients in the general medicine OP department.

Results: The prevalence of H pylori in our population was 16%.

In response to the questionnaire, 20% opted for OGD, 49% for a blood test, 15% for the UBT, and 16% for the stool test.

Discussion: Our area has a low H pylori prevalence, which can decrease the accuracy of all the tests. However, from our data all the tests show similar accuracy apart from the serology, which performs poorly. The patients overall preferred a non-invasive test. The faecal antigen shows a clear cost advantage, the RUT, histology, and culture all require an OGD and this increases the cost markedly. The cost of the UBT is increased by the necessity for a dedicated nurse to do the tests.

Conclusion: On the basis of accuracy, cost, and patient preference, the faecal antigen test is the test of choice for the diagnosis of H pylori.

Abstract 265

266 ENTERAL STENTS FOR THE PALLIATION OF MALIGNANT GASTRIC AND DUODENAL OBSTRUCTION

J.O. Lindsay, P. Vlavianos, H.J.N. Andreyev, D. Westaby.

Department of Gastroenterology, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK

Introduction: Gastric outlet and duodenal obstruction are common complications of advanced upper gastrointestinal malignancy. Relief of obstruction to allow continued enteral feeding and early discharge is the primary therapeutic goal. Surgical bypass has a high success rate, but is not appropriate or feasible in all patients. The endoscopic placement of expandable metal stents has been proposed as an alternative technique to achieve palliation in patients not suitable for surgery.

Aim: To review our experience with gastroduodenal metal stent insertion for the palliation of malignant gastric and duodenal obstruction in patients unsuitable for surgical bypass.

Methods: A retrospective review of the notes of all patients who had a gastroduodenal stent inserted in our unit between March 1999 and October 2002 was performed. In addition, the referring consultant and GP were contacted to obtain follow up information.

Results: 29 patients (17 male, 12 female) with a mean (range) age of 66 (43–93) underwent insertion of an enteral stent for malignant gastric or duodenal obstruction. The primary tumour was in the stomach in 14 (48%), the pancreas in 10 (34%) patients, and metastatic in 5 (17%) patients. A stent was successfully placed in all patients; the sites of placement included the stomach ( n= 5), across the pylorus (n = 6), through a previous surgical anastomosis (n = 3), or the duodenum (n = 15). Two stents blocked after insertion, one required dilatation, and the other patient had a second stent inserted. No patients required surgery. 26 patients have subsequently died, the median (range) survival being 2.8 months (2 weeks to 10 months), 22 patients survived at least one month. 25 (86%) patients were discharged from hospital: 19 home, 6 to a hospice. During follow up 8 (27%) patients returned to a solid diet, 17 (59%) required a soft diet, and 3 (10%) patients tolerated liquids only.

Conclusion: The use of enteral stents achieves good palliation in the majority of patients with malignant gastric or duodenal obstruction, allowing discharge from hospital and re-introduction of an enteral diet.

267 AUDIT OF ERCP INCLUDING SNARE AMPULLECTOMY AS AN ACCESS PROCEDURE

A.C. Daley, P.J. Finch.

Department of Gastroenterology, St Peter’s Hospital, Chertsey KT16 0PZ, UK

Introduction: Difficult cannulation of the bile duct in jaundiced patients can be aided by precut or needle knife sphincterotomy. We have recently used snare ampullectomy instead, because of dissatisfaction with these techniques, and present an audit of our results.

Aim: To audit outcome measures in ERCP patients and assess the safety and efficacy of snare ampullectomy [SA].

Methods: Records of all patients undergoing ERCP in this hospital over 6 years (including 3 years of SA) were reviewed to assess indications, procedures, and outcome including complications (abdominal pain, pancreatitis, bleeding, perforation, delayed discharge, readmission within 7 days) and 30 day mortality.

Results: 724 patients (61% female, median age 67) underwent ERCP by a single endoscopist. Indications included biliary colic (49%), jaundice/abnormal LFTs (38%), and prior pancreatitis with gall stones (11%). Overall success in cannulating the intended duct was 88%. When precut was employed, the success rate was 58%, but with SA, this rose to 88%. The rate of any complication for all ERCPs was 6%, and of pancreatitis was 2%. There were no perforations and 7 bleeds, none requiring transfusion. 30 day mortality was 5% with a median age of 79 years. 43 patients underwent SA including 3 for ampullary neoplasms. In a case with a polya gastrectomy, a stent was inserted to guide the snare, but was cut through by the heating. A stepwise logistic regression model was used to assess the contribution of ES (n = 256), precut (n = 19), and SA to all complications, and revealed significant effects for precut (p < 0.01 odds ratio [OR] 6.2, 95% CI 2.2 to 17.2), SA (p = 0.02 OR 2.9, 95% CI 1.2 to 6.8) and ES (p < 0.01, OR 2.9, 95% CI 1.5 to 5.7). Significant effects in pancreatitis were found for precut (p < 0.01, OR 14.9, 95% CI 4.0 to 56.1), and SA (p = 0.03, OR 4.4, 95% CI 1.2 to 16.3) but not for ES. No significant effect in 30 day mortality was found for precut (p = 0.98), SA (p = 0.24) or ES (p = 0.79).

Conclusion: We feel SA is a safer procedure than precut when access to the bile duct is critical due to jaundice or stones, and appears more effective. It carries a risk of causing pancreatitis, but less so than precut.

268 ENDOSCOPICALLY PLACED METAL STENTS FOR THE PALLIATION OF MALIGNANT GASTRODUODENAL OBSTRUCTION: THE TREATMENT OF CHOICE

A. Holt, C. Hulley, M. Ahmed.

Good Hope Hospital, Sutton Coldfield, Birmingham, UK

Introduction: Self-expanding metal stents (SEMS) have been used to re-canalise a variety of obstructed tubular organs. Their use in the stomach and duodenum has recently been described. We report our single-centre experience with the technique in palliating malignant gastroduodenal obstruction.

Patients and Methods: Stenting was attempted in 16 patients (11 male, 5 female; mean age 79 years, range 64–91years). Eleven patients had inoperable gastric cancer, four patients presented with duodenal obstruction due to pancreatic cancer and one patient had tumour overgrowth of a gastro-jejunostomy. We employed SEMS (Schneider Enteral Wallstent™ in 15 cases and a Flamingo stent in one case, Boston Scientific) over a 0.035” Amplatz wire through a large channel gastroscope or therapeutic duodenoscope. The uncovered Enteral Wallstents were 6 or 9 cm in length and 20 or 22 mm in diameter (after deployment). Positioning of the stent was monitored by fluoroscopy.

Results: Gastroduodenal stenting was technically successful in 14/16 patients and 3/16 patients also had metal biliary stenting. The two failures were due to kinking of the stent assembly in one case and complete duodenal obstruction preventing passage of the guide wire in the other. There were no immediate or late stent related complications. The 14 successfully stented patients all experienced relief from vomiting. 11 patients tolerated semi-solid diet, 1 tolerated liquid diet only, and the remaining 2 patients were too unwell to eat. After stenting, 10 patients were successfully discharged to home (8) or hospice (2). The median time to discharge was 7 days (range 4–26 days). The mean survival post-procedure was 49 days (median 34 days, range 6–210 days).

Conclusion: Gastroduodenal stenting allows effective, minimally invasive palliation of malignant gastroduodenal obstruction. With careful patient selection and appropriate timing, stents relieve the distressing symptom of vomiting and allow safe discharge. The majority of patients tolerate a near normal diet.

269 PROSPECTIVE AUDIT OF ERCP OUTCOMES

J.E. Mawdsley, P. Manoye, A.P. Catterall, P. McIntyre, S.M. Greenfield.

QEII Hospital, Howlands, Welwyn Garden City, Herts, UK

Background: East and North Herts trust serves 500 000 patients; 350 ERCP procedures are performed/year. We present the largest UK prospective audit of 215 ERCPs.

Methods: Data were collected using a standard proforma completed at the time of endoscopy (99% completion). Patients were phoned at 3 and 30 days, or discussed with their medical team if an inpatient, to assess for a procedure related complications.

Results: Cannulation of the desired duct achieved at first attempt in 79% and 82% when the distal duodenum was reached. This improved to 85% with a repeat procedure. Biliary drainage was achieved in 64% of jaundiced patients (44/69). CBD stones were successfully removed in 71% at first procedures and 92% after a second procedure. Bile cytology was positive in (6/11) and bile brushings was positive in 32% (6/19).

Findings: CBD normal (61), CBD stones (58), CBD dilation only (29), CBD stricture (29), duodenal obstruction (7), other (13).

Procedures: Diagnostic (90), standard/needle knife sphincterotomies (50/17), stent insertion (27), CBD stone removal (53).

Complication Rates: Mild pancreatitis occurred in 3 cases (1.5%), Mild haemorrhage at the ampulla was reported in 7 (3.3%) but did not require admission or transfusion. Overnight observation was required in 4 patients (1.9%). Procedure related mortality occurred in 2 cases (0.9%); one patient with pancreatic cancer and liver metastases died from haemorrhage and the second from perforation of the duodenum due to peptic ulceration. Seven deaths (3.3%) occurred within the 30 day follow up period attributable to other causes.

Conclusions: Complication rates were low and similar to other studies. Biliary cytology is worthwhile and more sensitive than brushings. Cannulation rates were similar to recommended JAG guidelines but drainage rates were lower and JAG recommendations may need to be reviewed. Pure diagnostic procedures were common and MRCP was used as a pre-procedure imaging modality in only 10%. This audit might be suitable as a model for the BSG to follow in its proposed prospective National Audit of ERCP.

270 CAN A COLONOSCOPY COMPUTER SIMULATOR DIFFERENTIATE BETWEEN A NOVICE AND EXPERT?

S. Thomas-Gibson, M.E. Vance, B.P. Saunders.

Wolfson Endoscopy Unit, St. Mark’s Hospital, London, UK

Backgound: Computer simulators are being developed as a training tool in colonoscopy. We aimed to establish whether a computer simulator could differentiate between novices and experienced colonoscopists.

Methods: Eight novice and 4 experienced endoscopists took part in the study. Novices (5 nurses, 3 junior doctors) had no previous practical endoscopy experience. Experienced endoscopists were senior doctors (2 consultants, 1 SpR) or nurse consultant (n = 1) (no previous significant simulator experience). All novices were given group teaching in principles of colonoscopy, functions of a colonoscope, and an introduction to the simulator (Immersion Medical). Both groups were given a standard practise case (SPC) with tutoring, then performed the standard test case (STC) without help. Insertion was terminated at 15 minutes if the caecum had not been intubated.

Results: 7 novices (88%) and all experienced endoscopists achieved caecal intubation. Terminal ileal intubation (TII) was achieved in 3 novices (38%). At 5 min the novices were in sigmoid (n = 4) descending (n = 1) splenic flexure (n = 2), and transverse colon (n = 1); all the experienced had intubated the TI and were extubating. There was no difference in percentage time in “no discomfort” or in “moderate/severe discomfort” between the two groups. See Table.

Abstract 270

Conclusions: This simulator can differentiate between novices and experienced endoscopists, novices take longer to achieve caecal intubation and are less likely to achieve TII. However, not all parameters automatically measured by this colonoscopy simulator differentiate between them. Basic principles of colonoscopic insertion can be taught to novices using this simulator.

271 THE DEVELOPMENT OF A MULTIPLE CHOICE QUESTION PAPER FOR TRAINING AND ASSESSMENT IN COLONOSCOPY

S. Thomas-Gibson, M.D. Rutter, N. Suzuki, M.E. Vance, C.B. Williams, B.P. Saunders.

Wolfson Unit for Endoscopy, St Mark’s Hospital, London, UK

Background: Core knowledge is fundamental to good colonoscopy practise.

Methods: As part of a training programme developed at our institution a bank of multiple choice questions (MCQs) was designed. A “curriculum” of topics relevant to colonoscopy was drawn up including gross and endoscopic anatomy; embryology of the colon; patient preparation; instrument functions; technique; endoscopic clinical conditions; complications; and patient follow up. Several hundred questions were designed in MCQ, true/false format. Senior endoscopists performed initial selection and verification of questions. Ambiguous questions were excluded or replaced. Two 30 question papers were formulated, papers A and B, and were combined as a 60 question paper, AB. Each question had 5 stems (a total of 300 questions). Question papers were negatively marked (one mark for a correct answer, minus one mark for an incorrect answer, no marks for a question left blank). 3 groups sat the MCQ: 9 endoscopy nurses (novices), 8 colonoscopy trainees (previous experience of 50–500 colonoscopies) and 3 experienced endoscopists (1 nurse, 2 physicians; 500–5000 previous colonoscopies). The trainees sat paper A or B before an intensive week of colonoscopy training and paper AB at the end of the week.

Results: In the trainee group there was no significant difference in the mean scores for papers A and B pre-training 60.5% versus 49% (p = 0.189).

Conclusions: This MCQ paper can differentiate between endoscopists of different standards. A week of intensive training brings moderately experienced trainees up to the standard of knowledge of experienced endoscopists. This MCQ can be used as a training and assessment tool.

Abstract 271

272 POST-PYLORIC ACCESS ON THE ICU: ENDOSCOPIC PLACEMENT OF THE BENGMARK TUBE

G. Constable, J. Pappachan, M.A. Stroud.

Institute of Human Nutrition, Southampton General Hospital, UK

Introduction: Gastroparesis, high gastric aspirate volumes and retrograde peristalsis are common on the intensive care unit (ICU) and often thwart attempts at enteral feeding. This may result in a higher incidence of nosocomial pneumonia, increased mortality, longer ICU stay and prolonged periods of parenteral nutrition. Post-pyloric feeding improves nutrient delivery and may reduce time to achieve feeding goal rates, but tubes are frequently difficult to place, requiring multiple attempts, endoscopy, and fluoroscopy. Furthermore, transfer of patients to the x ray department is often not possible and fluoroscopy on the ICU is not practical.

Bengmark Tube: The bengmark tube (BT), a self-locating naso-jejunal tube designed for placement at the bedside, is reported to achieve over 90% spontaneous trans-pyloric passage and stay in position longer than alternative tubes. The principle is based upon a preformed large diameter coil that facilitates movement beyond the pylorus and limits regurgitation back through the pylorus, but spontaneous passage requires adequate gastric emptying. In an attempt to overcome the practical difficulties of obtaining post-pyloric access in the ICU we have used a simple technique of endoscopic placement of the BT that takes advantage of the preformed to prevent displacement of the tube as the endoscope is withdrawn, thereby obviating the need for screening, and also to reduce the risk of reflux into the stomach following successful placement.

Method: A suture is applied to the distal end of the BT, which is then flushed with water prior to complete insertion of the wire. The BT is then transferred from nose to mouth by means of laryngoscope and McGills forceps. The BT is grasped at the mouth with forceps deployed in the therapeutic channel of an endoscope and then carried into position under direct vision. Within D2 the wire is partially removed and the coil observed to form. The endoscope is then withdrawn fully prior removal of the wire to prevent tube displacement. Final tube position is confirmed by abdominal radiograph.

Results: This technique has been used successfully in 8/8 patients without procedure related complication. One tube was displaced within 24 hours while turning the patient and required repositioning. One tube became blocked 10 days after placement but was no longer required. The remainder facilitated uncomplicated enteral feeding for a mean of 12 days.

273 CLINICAL OUTCOME FOLLOWING INSERTION OF STENTS FOR THE PALLIATION OF PATIENTS WITH LARGE BOWEL OBSTRUCTION. A DGH EXPERIENCE

I. McNamara, M. Tremelling, I. Dunkley, P. Roberts, R. Dickenson.

Department of Gastroenterology and General Medicine, Hinchingbrooke Hospital, Hinchingbrooke Park, Huntingdon, Cambridgeshire, PE29 6NS, UK

Background: The deployment of self expanding enteral stents via endoscopic and fluoroscopic guidance has emerged as effective treatment of large bowel obstruction in both the acute and elective setting.

Methods: 20 consecutive patients (10 male), with a median age of 72 years were studied between 1997 and 2002 on an intention to treat basis. All patients had symptomatic large bowel strictures.

Results: 26 procedures were performed on 20 patients. A total of 15 stents were inserted with successful placement on the first attempt in 13 procedures. One patient was stented on the second occasion and one patient was stented on the fourth occasion. In 5 patients (6 procedures) a wire could not be passed due to complete occlusion of the lumen or due to the position of the carcinoma which prevented viewing “en face”. The remainder of the failures were due to poor preparation of the colon, or the stenosis being too lax to be amenable to stenting. The cause of the stenosis was malignant in 19 cases with the sites being rectosigmoid (45%), sigmoid colon (40%), splenic flexure (10%), transverse colon (5%). Symptoms improved in all patients successfully stented but in one patient there was distal migration of the stent 3 days later. In the others good palliation was obtained with a median symptom free survival of 7 months. 14 patients died in follow up due to causes unrelated to stent insertion.

Conclusion: Colonic stents have an important place in the management of large bowel obstruction and palliation of colonic carcinoma and should be available in any district general hospital. Despite small numbers we have shown that with experienced endoscopists this is a feasible proposition.

274 PRE-CLINIC SELF-ADMINISTERED HEALTH ASSESSMENT EVALUATION FORMS COULD SAVE TIME AND RESOURCES

P.G. Hardo.

Gastroenterology Unit, Benenden Hospital, Kent, UK

Background: The use of self-administered health assessment questionnaires maybe a useful instrument in an outpatient setup, especially when the general practitioner’s (GP) referral letters contain insufficient information. They can also improve doctor–patient communication. We therefore routinely use an in-house designed health assessment form (HAF) in the outpatient’s clinic.

Aims: To evaluate patient satisfaction with the HAF.

Methods: The two page HAF contains questions on common GI symptoms/signs together with information on previous tests, operations, family and social history, drug history and the impact of current illness on quality of life. Enough space is left for comments. All patients completed the HAF before they were assessed. When a GP letter was lacking information, a HAF was sent to the patient to be returned by post for prioritisation. A total 173 consecutive patients (111F/62M) were anonymously asked to evaluate the value of HAD before leaving the hospital. They completed a simple questionnaire about its relevance, usefulness, clarity, and ease of completion. A satisfaction scale from: 1 = not at all to 5 = yes, definitely, was used and a space for comment was allocated.

Results: 36% were male and 64% female. The very small proportion (2–3%) who felt that the HAF was not very relevant or useful (score 1) were mostly patients with minor or no GI problems. The majority of patients liked the HAF and wrote constructive comments.

Conclusion: Most patients found the HAF valuable in preparing them for the consultation and a useful way to think about their problems before seeing the doctor. Based on our 6 years experience with the HAF (which we have modified as a result of this audit) we found it particularly helpful for: prioritising the urgency for consultation; speeding up the consultation process; and avoiding duplication of investigation. We achieve 99% patients’ compliance in completing the HAF. We recommend the use of a “self-administered health assessment questionnaire” as an integral part of the outpatient GI assessment.

Abstract 274

275 AUDIT OF THERAPEUTIC PANCREATIC ENDOSCOPY

A.I. Thuraisingam, H.L. Smart.

Royal Liverpool Hospital, Prescot Street, Liverpool L7 8XP, UK

Background and Aims: Therapeutic pancreatic endoscopy is a specialised technique used to treat a range of pancreatic disease. There are few prospective, randomised studies of pancreatic endotherapy and most data are derived from uncontrolled series. We assessed current practice relating to pancreatic endotherapy at ERCP in a regional teaching hospital and examined our procedural outcomes.

Methods: A retrospective case note audit was undertaken of all patients, over a three and a half year period, in whom therapeutic endoscopy was attempted or performed. Data were collected regarding patient demographics; endoscopists; indications; use of pre-procedural imaging; procedure performed; success rate; 30 day complication rate; and 1 year outcome.

Results: Sixty-three out of 1906 ERCPs (3.3%), performed in this period, involved pancreatic endotherapy. These were undertaken in 32 patients by 5 principal endoscopists. There were 21 men and 11 women with a mean age of 49 years (range 11–74 years). Incomplete data were available for one patient. Imaging in the 3 months prior to endotherapy included: 43 CT scans; 27 ultrasounds; 25 ERCPs; 5 endoluminal ultrasounds; and 2 MRIs. The procedural indications were: 25 for pseudocyst drainage (9 with another indication); 26 for stent change; 8 for pancreatic duct stricture; 7 for pancreaticopleural fistula; 3 for pancreatic ascites; and 3 for pancreatic duct trauma/disruption. Overall 49 of 63 procedures (78%) were completed successfully, with 2 patients having repeated failed procedures. 19 of 32 patients required more than one procedure. Complications included one episode each of pancreatitis, infection, peritonitis, and bleeding. There was no 30 day mortality. 18 of 21 patients followed up for over 1 year were alive.

Discussion: Pancreatic endotherapy remains an uncommonly performed specialised procedure. The predominant indications in this series relate to complications of pancreatic ductal disruption in chronic pancreatitis. Patients often undergo multiple imaging investigations and may require repeat procedures for treatment. Endoscopic management can be effective and our data compares well with the available evidence.

276 COLONOSCOPY TRAINING: 90% CAECAL INTUBATIO—HOW EASY? HOW LONG?

A. Kumar, W. Ashraf.

King George Hospital, London, UK

Background: JAG and ASGE recommended guidelines advocate over 90% caecal intubation rate for successful colonoscopy training. Most GI fellows in structured training programmes in the USA achieve this in 2–3 years. There is a lack of structured colonoscopy training programmes in the UK, hence a poor overall caecal intubation rate in the UK (57–77% at best)1.

Aim: To determine the time and effort needed to achieve required competence in colonoscopy with structured training.

Method: A trust registrar in surgery who had no previous endoscopy experience underwent formal training offered by an experienced and well trained colonoscopist. Training was offered for one NHS session per week. The training programme included formal training in Upper GI endoscopy and flexible sigmoidoscopy (8 months) followed by colonoscopy training. Each procedure was recorded on computer Endoscopy Reporting System (version S11.05/13 1204/1999).

Results: There was one significant postpolypectomy bleed (10th day). There were no perforations.

Conclusion: 90% caecal intubation rate at colonoscopy is easily achievable with a structured training programme utilising only one NHS session per week. A very low risk of complications with fully supervised training. Training non-gastroenterologists is an attractive option to deal with long waiting lists and impending CRC screening programme.

  1. IBNC Audit. Gut 2002;50(suppl 1):A036.

Abstract 276

277 VARIATIONS IN REPORTING COLONOSCOPY FINDINGS BY DIFFERENT ENDOSCOPISTS AT A SINGLE CENTRE

H.L. Spencer, S.A. Riley.

Northern General Hospital, Sheffield, UK

Introduction: Colonoscopy is a fundamental investigation for the gastroenterologist. Recently training guidelines have been issued to promote technical and diagnostic competence. Few studies have addressed the consistency or importance of interpretational skills.

Methods: A prospective study was carried out of all colonoscopies from Oct 2001 to Sept 2002, performed on a joint medical and surgical list. Using the “Infoflex” database, demographic details, completeness of examination, and final endoscopic diagnosis were recorded and analysed by endoscopist (4 surgical consultants, 4 medical consultants, 6 specialist registrars, and 2 nurse endoscopists). 5 further endoscopists who had done less than 20 procedures, were excluded from the analysis.

Results: 1776 colonoscopies were performed. Of these, 38 were done by the excluded endoscopists. The frequency of reporting common diagnoses was very variable and the differences between endoscopists were highly significant when analysed using χ2 (see table).

Conclusions: Within our centre there are major variations in reporting colonoscopy findings. While it is reassuring that there was no major difference in the frequency of tumours, the variation in polyp reporting is particularly worrying as it may have serious implications for patients’ health. Further studies are required to assess the determinants of endoscopic reporting and diagnosis.

278 FOLLOW UP AUDIT OF COLONOSCOPY COMPLETION RATE: IT TAKES TIME TO IMPROVE

P.J. Mullen.

The Princess Elizabeth Hospital, Guernsey

Background: An initial audit of 357 colonoscopies1 demonstrated what was thought, at the time, to be a well below par overall completion rate of 73.9%, adjusted to 80.5% if operator independent failures (eg poor bowel preparation, stricture, obstructing tumour) were excluded.

Action undertaken: Efforts were made to improve by: more frequent alteration of patient position; better loop avoidance; the use of “jiggling”; earlier and repeated de-looping manoeuvres; and increased use of manual abdominal pressure.

Methods: The records of all procedures performed by the same operator over the 4 years following the previous audit were reviewed retrospectively. The prime data collected were patient demographics, completion rate type of colonoscope used, and total procedural time (“door-to-door”).

Results: There was no significant difference in patient demographics of the 653 procedures. The completion rate was significantly improved to 85.9% overall and 90.2% adjusted (p < 0.005). This improvement was still seen when comparing only procedures done with a CF230L colonoscope: 85.9% cf. 74.5% overall (p < 0.001) and 89.7% cf. 81.3% adjusted (p < 0.005). There were significantly more procedures taking 40 min or more (21.6% cf. 14.2%, p < 0.05) in the second audit period.

Conclusion: While certain aspects of technique are clearly important, it may be that workload constraints are primarily responsible for low completion rates. More acceptable standards appear feasible if time is made available for the difficult cases.

  1. Mullen PJ, Hanaghan J. Failure at colonoscopy. Gut 1999;44(suppl 1):T59.

279 COLONOSCOPY COMPLETION RATES: A COMPARISON BETWEEN STANDARD AND VARIABLE STIFFNESS COLONOSCOPES

P.J. Mullen.

The Princess Elizabeth Hospital, Guernsey

Introduction: Faster caecal intubation times and reduced patient discomfort have been reported with the variable stiffness colonoscope when compared to a C200HL colonoscope1. In this study of 100 cases, there was no demonstrable difference in completion rates.

Aim: To compare the CF240AL variable stiffness and standard CF230L colonoscopes with respect to completion rates and total procedural time.

Method: The records of all colonoscopies undertaken by a single operator since the purchase of a CF240AL colonoscope in April 2000 up until the end of September 2002 were reviewed retrospectively. Prime data collected were: patient demographics, completion rates (overall, and adjusted for operator independent failures such as poor bowel preparation, stricture, obstructing tumour), and total procedural time (door-to-door”).

Results: There were 191 procedures with the CF230L and 214 with the CF240AL. There was no difference in either the patient demographics, the overall (86.4% cf. 86.9%) and adjusted (91.7% cf. 91.6%) completion rates, or the procedural times. Over the past 12 months, however, the completion rates were better with the CF240AL than the CF230L: 92.7% (97.8%) cf. 90.2% (93.2%) (difference NS).

Conclusion: This study reveals no difference in completion rates or procedural times between the two types of colonoscope. This unexpected result probably reflects a learning curve with the CF240AL and a type 2 statistical error.

  1. Brooker JC, Saunders BP, Shah SG, Williams CB. A new variable stiffness colonoscope makes colonoscopy easier: a randomised controlled trial. Gut 2000;46:801–5.

280 A METHOD OF ACCURATELY SITING OESOPHAGEAL PROSTHESES WITHOUT RADIOLOGY

P. Kooner, R. Aljabari, K. Besherdas, N. van Someren.

Department of Gastroenterology, Chase Farm Hospital, Enfield EN2 8JL, UK

Background: Expanding metal oesophageal stents are an effective method of palliating inoperable oesophageal carcinoma. Unlike colonic, duodenal, or biliary stents, oesophageal stents are deployed from an introducer whose diameter is too large to pass the biopsy channel of any endoscope. Such prostheses are usually placed using fluoroscopic guidance, and inaccurate stent placement is a frequent occurrence because of difficulties arising from correct identification of the tumour margins and stent radiopaque markings. We describe a method of deploying such stents under direct endoscopic vision with a high degree of accuracy.

Methods: A preliminary survey of the proximal tumour margin and remaining oesophageal lumen is made using an endoscope. A stiff guidewire (eg 0.35” ‘Tiger’ wire) is passed through the oesophageal lumen until it lies within the stomach. Often the endoscope will be too large to cross the stricture and enter the stomach, and free passage of the wire is the key to ensuring that the wire lies in the correct position. No dilatation of the stricture is necessary. The endoscope markings are used to gauge the length of the stricture, and a stent of appropriate length is selected. The endoscope is withdrawn, leaving the guidewire in situ, and then reintroduced alongside the guidewire. The stent with its introducer is passed over the guidewire, and into the stricture under direct endoscopic view. The stent is then deployed while viewing through the endoscope, and correct placement ensured.

Results: We placed 24 oesophageal stents (Boston Scientific) between 2000 and 2002 using radiological guidance, and 25 using the direct view method. All stents were placed satisfactorily.

Conclusions: The direct view method of oesophageal stent placement has results equivalent to fluoroscopically guided placement, and avoids the use of x rays. We also feel that the procedure is easier and takes less time.

281 RESEARCH TRENDS IN BRITISH GASTROENTEROLOGY: PUBLICATION RATES IN NEWLY APPOINTED NHS CONSULTANTS OVER A 9 YEAR PERIOD

A.D. Hopper, R.J. Atkinson, L. Prtak, D.S. Sanders.

Department of Gastroenterology, Royal Hallamshire Hospital, Sheffield, UK

Background: The amount of published research that has been performed by a specialist registrar (SpR) at the time of appointment to a consultant post has no set expectations. It has been suggested that research is diminishing in all specialities in the United Kingdom.

Aims: To observe any publication trends in newly appointed NHS consultants over a 9 year period. In addition, we assessed whether there were differences between district general hospital (DGH) appointments versus teaching centre hospitals (TCH).

Methods: All consultant appointments and location of SpR training were noted from February 1993 to April 2001 (obtained from trainees in gastroenterology). A PubMed and Embase search was performed on each individual to note the number and type of publications up to 19 months post-appointment (previously described as the median time from submission to publication). The consultant name was then matched with his/her entry in the BSG handbook and any higher degree noted (PhD, MD or, MA). If no degree was documented the individual’s department was contacted to ascertain whether this information had not been supplied to the BSG. It was noted whether the appointment was at a DGH or TCH.

Results: During the study period there were 362 appointments: 210 were NHS consultants appointments (52 excluded: consultant transfers n = 41 and academic appointments n = 11). There was a significant year by year reduction in the number of publications. 39% of consultants were appointed in the region where they trained. A consultant was just as likely to have a higher degree if appointed to a DGH (68%, 80/118) or TCH (72%, 66/92). Consultants appointed to TCHs had a significantly greater number of publications (mean 15.6) compared with DGH consultants (mean 10.9, χ2, p = 0.01).

Conclusion: There is a year by year, significant decreasing trend in the publication rate of SpRs’ at the time of their consultant appointment. This could reflect diminished research funding opportunities, changing ratios of trainees to posts, or the impact of the Calman SpR system.

Abstract 277

Abstract 281

282 CLEANING AND DISINFECTION OF GASTROINTESTINAL ENDOSCOPES: CURRENT PRACTICE IN THE UK

L.J. Gilby, M. Mulcahy, M.C. Allison.

Gastroenterology Unit, Royal Gwent Hospital and St Joseph’s Hospital, Newport, South Wales, UK

Introduction: Until recently most endoscopy units in Europe and the USA have employed glutaraldehyde based disinfectants. Occupational exposure to glutaraldehyde is associated with hypersensitivity reactions prompting one manufacturer (Johnson & Johnson) to withdraw one of its widely used products (Cidex™). The Medical Devices Agency has recently revised its recommendations for endoscope decontamination. There have also been concerns about possible transmission of prion proteins by endoscopes, prompting the recommendation that enzymatic detergents are used during mechanical precleaning.

Methods: We undertook a telephone survey of 107 hospitals in the UK to gather information on decontamination practice and the range of disinfectants and detergents in current use. A range of size of hospital was chosen, including seven private hospitals. Of the NHS hospitals there were 60 from England, and 20 each from Scotland and Wales.

Results: All units surveyed perform manual cleaning before automatic endoscope disinfection. Manual cleaning is done using an enzymatic detergent by 63 (59%) of units and using ordinary detergent or soap solutions such as washing-up liquid by 28 (26%). The type of detergent used was unknown for 16 units. Aldehyde based disinfectants are used by 69 (64%); within this group 48 continue to use glutaraldehyde based solutions, 2 use dialdehydes and 19 use ortho-phthalaldehyde (an aldehyde with greater mycobactericidal activity and lower sensitisation risk than glutaraldehyde). Of the remainder 20 units use peracetic acid disinfectants, 11 use superoxidised saline, and 8 use chlorine dioxide. We identified 20 different brands of automatic endoscope reprocessor.

Conclusions: Mechanical cleaning followed by automatic endoscope reprocessing is now standard practice. Recommendations for the use of enzymatic detergents are only partly being adhered to. While most centres use aldehyde based disinfectants, a wide variety of other agents are in common usage. There is a need for more research, both on the optimal use of these agents and in development of further disinfectants with high microbicidal activity and low risk to patients and staff.

283 SAFE EARLY DISCHARGE OF PATIENTS WITH LOW RISK UPPER GASTROINTESTINAL HAEMORRHAGE—A CHANGE IN PRACTICE

R.S. Hodgson, M.J. Carter, A.A. Barrett, A. Ainsworth, A.P. Catterall, P.B. McIntyre, S.M. Greenfield.

Queen Elizabeth II Hospital, Herts, UK

Approximately 650 bed days are used by patients admitted with upper gastrointestinal haemorrhage (UGIH) per annum in this hospital serving a population of 250 thousand. 145 of these are for patients with low risk UGIH (pre-endoscopy Rockalls scores 0 and 1). There are few data on clinical outcomes and no published prospective studies in this group. A previous hospital audit confirmed safe outcome of low risk UGIH. We investigated the outcome of outpatient management of patients with low risk UGIH.

Methods: Hospital policy was changed from 1 June 2002 to avoid admission of patients presenting with low risk UGIH. An UGIH clerking proforma was developed to aid and direct assessment of patients and for audit purposes. All patients with UGIH have a pre- and post-endoscopy Rockall score calculated. Low risk patients (score of 0 or 1) are assessed by a senior member of the on-call medical team before discharge. Emergency outpatient UGI endoscopy is arranged on the next working morning. All patients are phoned 30 days later.

Results: In 18 weeks 53 patients presented with UGIH. 26 had pre-endoscopy scores of 0 or 1 (49%, 14M:12F), age range 18–78 years (mean 50). 13/26 were discharged (50%), only one did not present for UGI endoscopy as planned. None required admission following endoscopy. 7/13 (58%) had a normal endoscopy, 2 required OPA, 3 further investigations. 1 patient (diagnosis DU) had possible ongoing melaena at 30 days in spite of OPA review (had not taken prescribed medication). Of the 13 low risk patients admitted, 11 had UGI endoscopy, 3/11 (27%) were normal. All admissions were for other medical issues. The median length of hospital stay was 5 days.

Conclusions: Discharge from A&E of low risk patients presenting with UGIH with plans for early emergency outpatient UGI endoscopy appears safe and cost effective. An estimated 72 bed-days may be saved per annum in this hospital. In a UK population of 58.8 million this represents potentially a saving of 17 thousand bed-days per annum.

284 ADULTS WITH FOREGUT DUPLICATION CYSTS DIAGNOSED BY ENDOSOCPIC ULTRASOUND (EUS) CAN BE SAFELY MANAGED WITH AN EXPECTANT POLICY

L. Langmead, S. James, I.D. Norton, D.B. Jones.

Department of Gastroenterology, Concord Hospital, University of Sydney, Australia

Background: Enteric duplication cysts (EDC) are rare congenital anomalies that lie within or adjacent to the wall of the gastrointestinal tract. The majority of EDC have characteristic sonographic patterns on EUS. Because the management for adults diagnosed with foregut EDC at EUS is controversial, we aimed to assess their long term outcome.

Methods: We reviewed the indications and EUS findings for patients diagnosed with EDC over 5 years. Outcome was established from medical records and/or a questionnaire sent to referring physicians.

Results: Of 768 EUS examinations, 27 patients (10 males) were diagnosed with probable EDC. Median age was 51 years (range, 21–77). Presenting symptoms included dysphagia (n = 7), dyspepsia (6), reflux symptoms (5), and chest pain (2). Abnormal upper GI endoscopy (n = 21), with possible extrinsic compression (10) or submucosal tumour (8), was the commonest indication for EUS. Other prior investigations were CT (n = 7), barium meal (2), MRI (1) CXR (1), abdominal US (1). At EUS, most EDC were in the oesophagus (proximal (n = 4), mid (7), distal (11)). Other sites were proximal stomach (2) and duodenum (3). Maximal diameter ranged from 10–60 mm. Septation was seen in 2 cysts, sediment in 3, and probable haemorrhage in 4. Follow up data were available in 21 of 27, with diagnosis of EDC subsequently revised in 5/21. 3 patients had surgery after further imaging. In a patient with early oesophageal cancer, coincident EDC was not confirmed. A leiomyoma and a bronchial inclusion cyst were found in the others. CT after EUS suggested an alternative diagnosis in 2 patients (1 leiomyoma and 1 partial situs invertus with anomalies of azygous vasculature). In 16 patients (including 4 in whom follow up imaging supported the initial diagnosis of EDC) an expectant policy was employed. All 16 patients remain well 6 months to 4 years after EUS without development of new symptoms or complications related to the EDC.

Conclusion: Adult patients with asymptomatic, uncomplicated EDC of the foregut at EUS can be managed expectantly.

285 INPATIENT COLONOSCOPY AND FLEXIBLE SIGMOIDOSCOPY—AN INFERIOR SERVICE?

C. Metcalf, R. Graham, B. Macfarlane, J. Meyrick-Thomas, S. Dracup, J. McNeice, A. Leahy.

Watford General Hospital, West Hertfordshire Hospitals NHS Trust, UK

Introduction: A recent retrospective audit of 3455 colonoscopies within our unit found that non-elective inpatient procedures were less likely to be completed to the caecum (77%) than in outpatients (89%). Despite this finding, abnormal examinations were more common in inpatients. Endoscopists and nursing staff have long suspected that inpatients might be less adequately prepared and informed than outpatients. We therefore conducted a prospective audit to investigate such disparities.

Aim: To compare the quality of inpatient to outpatient lower gastrointestinal endoscopy.

Method: A prospective audit was performed using standardised questionnaires completed by patients, endoscopy nursing, and medical staff. Bowel preparations used were Fleet Phospha-soda (colonoscopy: COL) and phosphate enema (flexible sigmoidoscopy: FS). All outpatients received procedural information leaflets, while all inpatients had access to ward based information sheets. Statistical analyses employed were the students t-test and chi-square test.

Results: Procedures audited were 183 COL and 111 FS (34 inpatient, 260 outpatient). Inpatients were less likely to understand the indication and reason for the procedure or to recall receiving an information sheet (p < 0.05). For both COL and FS the bowel was less well prepared in inpatients (COL p < 0.03, FS p < 0.001). Inpatients had a higher mean American Society for Anesthesiology score (p < 0.001). There was no difference between the two groups with regards to side effects of the bowel preparation, sedation used, and duration or discomfort of the procedure as assessed by patient, endoscopy nurse, or endoscopist.

Conclusions: Inpatients are frailer and less well prepared for lower GI endoscopy in terms of both the quality of the bowel preparation and information they receive. This has implications regarding the consenting process and procedural completion rates. Endoscopy units should consider new ways of working in order to improve inpatient preparation.

Pancreas posters 286–288

286 A COMPARISON OF THE PANCREOLAURYL RATIO WITH FAECAL PANCREATIC ELASTASE-1 FOR ASSESSING PANCREATIC EXOCRINE FUNCTION

D. Elphick, D. Bullimore, K. Kapur.

Department of Gastroenterology, Barnsley District General Hospital, Gawber Road, Barnsley, S. Yorkshire, UK

Introduction: An ELISA kit for measurement of Faecal Pancreatic Elastase-1 (FE1) has recently been introduced for the investigation of pancreatic insufficiency. We compared this test with the urine pancreolauryl ratio (PLR). The clinical response to pancreatic enzyme supplements is used as the standard against which they are compared.

Methods: 45 patients under investigation for chronic diarrhoea were included in the study. All had urinary PLR measured as an outpatient. This has been the standard investigation for pancreatic insufficiency in our hospital to date. These patients also had a FE1 level measured. 33 patients (with either a high clinical suspicion of pancreatic insufficiency, a PLR < 20 or a FE1 < 200 μg/l) were given a trial of pancreatic supplements (Creon) at standard dose, and their clinical response assessed. A subjective improvement in diarrhoea and objective evidence of weight gain (at least 5% gain over 6 months) was required to record a positive response.

Results: We found a strong correlation between FE1 level and clinical response to Creon (p = 0.013 by χ2 analysis).

However, a similar analysis yielded no significant correlation between PLR and clinical response to Creon (χ2 p = 0.15).

Conclusion: FE1 more accurately predicts response to pancreatic enzyme supplements than PLR. This may be due to the inaccuracy inherent in performing the urine PLR as an outpatient. We have now adopted the FE1 for assessment of pancreatic exocrine function.

Abstract 286

287 COMPARISON OF URINARY PANCREOLAURYL AND FAECAL ELASTASE-1 TESTS IN THE ASSESSMENT OF EXOCRINE PANCREATIC INSUFFICIENCY

V. Sharma1, E. Thorpe2, C. Samuell2, S.P. Pereira1.

1Department of Gastroenterology, 2Department of Chemical Pathology, The Middlesex Hospital, UCL Hospitals NHS Trust, London, UK

The urinary pancreolauryl (UPL) test is used widely for the non-invasive diagnosis of exocrine pancreatic insufficiency, but is time consuming and reported to be unreliable in mild to moderate exocrine insufficiency. A highly sensitive enzyme linked immunosorbent assay for faecal elastase-1 (FE1) using two specific monoclonal antibodies is now commercially available.

Aim: To compare UPL and FE1 test results in a group of patients with well characterised (CT, MRCP ± ERCP) chronic pancreatitis with and without overt exocrine pancreatic insufficiency.

Methods: Paired UPL and FE1 data were obtained in 51 patients (36 M, 15 F; mean age 52 year, range 29–77 year). Standard cutoff values for the UPL test (< 20% fluorescein recovery from the orally administered ester abnormal, 20–30% equivocal) and FE1 test (< 200 μg/g faeces abnormal) were used.

Results: There was concordance between the two tests in 31 (61%) patients (both UPL and FE1 normal in 12, abnormal in 19). Two of the 12 patients with normal UPL/FE1, and all 19 in whom both tests were abnormal, had moderate or severe morphological changes in the pancreas on CT/ERCP (Cambridge classification). Six patients had equivocal UPL results, none of whom had overt exocrine insufficiency and only one of whom had a low FE1. Two patients (4%) had normal UPL but low FE1 (both had moderate/severe morphological changes), while 12 (24%) had low UPL but normal FE1 (2 moderate/severe).

Conclusions: The FE1 test is simpler to perform than the UPL test, and correlates well with abnormalities of the pancreas seen on structural imaging. There was a disparity between FE1 and UPL results in patients with structurally mild disease, but FE1 appears to be a sensitive and specific indirect test for mod/severe pancreatic insufficiency

288 PROPHYLACTIC PANCREATIC DUCT STENTING IN PATIENTS WITH SUSPECTED SPHINCTER OF ODDI DYSFUNCTION BUT NORMAL SPHINCTER OF ODDI MANOMETRY

P. Prasad, S. Varadarajulu, W. Foody, D. Sabol, E. Rawls, M. Payne, R. Hawes, P. Cotton.

Medical University of South Carolina, Charleston, USA

Background: Although post-ERCP pancreatitis is a well recognised complication among patients with sphincter of Oddi dysfunction (SOD), preliminary data suggest that the rate of pancreatitis could be as high as 10% to 15% even among patients suspected to have SOD but have normal sphincter of Oddi manometry (SOM) studies. The aim of this study is to determine if placement of a temporary pancreatic stent in patients with suspected SOD but normal SOM reduces pancreatitis rates.

Methods: Evaluation of all patients who underwent ERCP for suspected SOD but had a normal SOM study (both biliary and pancreatic) over a 4 year period (1998–2002). If SOM was normal (basal sphincter pressure < 40 mm Hg), the decision to place a temporary, 3 Fr, single pig-tail pancreatic stent (Wilson-Cook, Winston-Salem, NC) was made by the individual endoscopist. Patients who underwent any therapeutic intervention, such as stent extraction or orifice dilation, during the same setting were excluded from the study. Post-ERCP pancreatitis was defined as per standard consensus criteria. Abdominal x ray was done routinely after 14 days to verify spontaneous stent passage.

Results: A total of 147 patients with normal biliary and pancreatic manometry studies were identified from the ERCP database. The rate of post-ERCP pancreatitis was 0% (0/26) among patients who underwent prophylactic pancreatic stenting compared to 10.7% (13/121) among those who were not stented. No pancreatic stent induced complications were encountered.

Conclusions: Routine temporary pancreatic duct stenting appears to decrease the rate and severity of pancreatitis among patients suspected to have SOD but have normal SOM studies. The procedure appears safe. A randomised controlled trial is warranted to confirm these findings.

Gastroduodenal posters 289–307

289 VARIATION IN HELICOBACTER PYLORI STRAINS FROM IRAN AND ZAMBIA

M. Raeiszadeh1,2, N. Fernando1, J. Holton1, D. Vaira3, F. Siavoshi4, A. Hosseini2, P. Kelly5.

1Department of Bacteriology, RF&UCLMS, London, UK, 2Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, 3First Medical Clinic, University of Bologna, Italy, 4Department of Microbiology, Faculty of Science, Tehran University, 5Department of Adult and Paediatric Gastroenterology , St Bartholomew’s & Royal London School of Medicine, London, UK

Different genotypes of H pylori have been observed in different locations of the world and identification of these genotypes may be important for understanding the clinical outcome of infection, the efficacy of antibiotic treatment, laboratory diagnosis, and possibly human migration patterns. Data on the vacA diversity from Iran and Africa are sparse. We compared the vacuolating cytotoxin alleles (vacA) and the cytotoxin associated gene A (cagA) in H pylori isolates from Iran and Zambia by means of PCR. A total of 31 H pylori isolates were studied from Iranian dyspeptic and 23 from Zambian dyspeptic patients. There was an equal distribution of s1 and s2 alleles in Iran, where s1c was the predominant s1 subtype (10 of 16 s1 strains) with the m2 region predominating (26 out of 26 strains, with 5 stains unable to be typed in the “m” region). Isolates from Zambia mostly carried the s1 allele (21 out of 23) with the s1b allele being the most frequent (18 out of 21 ) and in the m region the m1 allele predominating (17 out of 21) with 2 stains non-typable for the m region. Using primer pairs for cagA 3’ region, the percentages of cagA positive strains in Iran and Zambia were 41% (13 out of 31) and 86% ( 20 out of 23), respectively. Most of the cagA positive strains (85%) from Iran were of the non-Asian type and only two isolates were of the pure East Asian type. All of the 20 cagA positive strains from Zambia were of the non-Asian type and had the s1 allele. In Iran the cagA gene was found at a higher frequency in the s1 allele type (11 out of 16) as compared to the s2 strains (2 out of 15). Overall there was a strong association between the cagA marker and the s1 allele (p < 0.001).

290 DOES CAGA HELICOBACTER PYLORI INCREASE GASTRIC MUCUS LAYER THICKNESS? A PRELIMINARY STUDY

M.S. Al-Marhoon1, S. Nunn2, M.E. Denyer3, S.M. Kelly4, A. Rielly5, R.W. Soames1.

1School of Biomedical Sciences, University of Leeds, LS2 9JT, 2University of Durham, 3Seacroft Hospital, Leeds, 4York District Hospital, 5St James’s University Hospital, Leeds

Background and Aims: Infection with cytotoxin associated gene A (cagA) H pylori is associated with increased risk of distal gastric cancer. There are contradictory opinions concerning the effect of H pylori on the gastric mucus layer thickness. The present study explores whether cagA positive (cagA+) H pylori increases gastric mucus layer thickness.

Methods: Fifty patients, not on acid suppressive drugs, NSAIDs, or with gastric ulcer or cancer, were recruited from endoscopy clinics. Six biopsies were obtained from each patient. 18 μm thick cryostat sections were cut and stained using the modified Periodic Acid Schiff/Alcian Blue technique.1 Mucus thickness measurements were made using light microscopy and computer software. H pylori diagnosis was based on histology, CLOtest, and culture, whereas cagA strain was determined by PCR.

Results: There were 18 (36%) H pylori positive (HP+) and 32 (64%) H pylori negative (HP-) patients with mean (±SD) mucus thickness of 68.2 ± 36.9 μm and 77.7±54.8 μm, respectively. In the HP+ group 5 were cagA+ and 11 cagA-, with a mean mucus thickness of 93.7 ± 61.0 μm and 53.8 ± 14.8 μm, respectively. There was no significant difference in mucus thickness between cagA+ and HP- controls (p = 0.63 Mann-Whitney U test). Age and sex matching between the HP+ and HP- groups revealed no difference in mucus thickness or between cagA+ and cagA- (p = 0.46), however in patients younger than age 50 a significant difference in mucus thickness between cagA+ (101.3 ± 18.9 μm) and cagA- (54.3 ± 9.5 μm) was observed (p < 0.05). Excluding older age and atrophy from the analysis showed no difference in mucus thickness between cagA+ and cagA- groups.

Conclusions: In H pylori infected patients less than 50 years old those with cagA+ strains have a significantly greater mean gastric mucus layer thickness than those with cagA-, suggesting that cagA+ strains may be more virulent as they are protected by an increased mucus gel layer.

  1. Jordan, Newton, Pearson, et al. Clin Sci 1998;95:97–106.

291 HELICOBACTER PYLORI SEROLOGY IN AN URBAN MULTIETHNIC POPULATION: TEST AND TREAT OR GUESS AND TREAT?

R. Dor, C. Blanshard.

Academic Unit of Gastroenterology, Homerton University Hospital, Homerton Row, London E9 6SR, UK

Background: Our current guidelines advocate a “test and treat” in young patients with dyspepsia without alarm symptoms. Most GPs use H pylori serology as the test of choice, which has not been validated for our population.

Aims: To determine the sensitivity and specificity of the serological assay in a multiethnic population.

Patient and Methods: 240 patients (97 native Caucasians and 143 non-Caucasians), with dyspepsia and no previous history of H pylori treatment, referred to the hospital for an upper gastrointestinal endoscopy, had blood sent for H pylori serology. At endoscopy biopsies were taken for rapid urease testing ( CLO™ ) along with histology. Patients’ ethnic group was also established by a short questionnaire.

Results: 145 patients were found to be positive for H pylori on the basis of serology, however, only 128 proved positive histologically or by urease testing.

Conclusions:H pylori serology is an unreliable test for predicting H pylori infection. This is particularly so in a multiethnic population where the specificity is much lower. Therefore, other non-invasive, more reliable ways of diagnosing H pylori infection should be used preferentially.

Abstract 291

292 EFFECT OF RED WINE, MINOR CONSTITUENTS, AND ALCOHOL ON THE GASTRIC EMPTYING AND THE METABOLIC EFFECTS OF A SOLID DIGESTIBLE MEAL

L. Benini, L. Salandini, G. Rigon, N. Tacchella, F. Brighenti, I. Vantini.

Internal Medicine A, Department of Biomedical and Surgical Sciences, University of Verona, 37134 Verona, Italy

Wine but not alcohol increases gastric acid secretion. Our aim was to clarify the influence of a moderate dose of wine, of its minor constituents, and of alcohol, on gastric emptying; and postprandial serum levels of glucose, insulin, triglycerides, NEFA, and acetate.

Materials and Methods: 12 healthy subjects were studied on four different days after ingestion, with the test meal, of 183 ml water; alcohol, 11.9% v/v; dealcoholated red wine; or red wine, alcoholic concentration 11.9% v/v. Gastric emptying of an 800 Cal solid meal was studied by a previously validated ultrasonographic method; serum parameters were studied at 30’ intervals for 240 min, calculating the area under the curve. The ANOVA for repeated measurements and, if positive, the student’s t test for paired data were used for statistical analysis, considering 0.05 as the limit for significance. Mean ± 1SEM are shown.

Results: gastric emptying was significantly influenced by the beverages (p < 0.05, ANOVA). It was significantly delayed after wine (259 ± 13.1 min) compared with water (199.1 ± 12.1, p < 0.05). Alcohol (221 ± 13.5, p < 0.05) and dealcoholated wine (226.8 ± 13.3, p = 0.055) produced an intermediate delay. The postprandial increase of glucose was significantly higher after ingestion of water than after the other beverages (p < 0.01), independently from the insulin response, not significantly different during the four tests. Triglycerides were slightly higher after ingestion of the alcoholic beverages than after water, but were markedly reduced after dealcolated wine compared with the other beverages (p < 0.05 for all contrasts). Both alcohol containing beverages produced a highly significant postprandial increase of serum acetate.

Conclusions: Red wine produces a marked delay in gastric emptying, only partially explained by its alcoholic content. Moreover, red wine, alcohol, and minor wine constituents reduce the postprandial increase of serum glucose, only minor wine constituents reduce that of triglycerides.

293 INFECTION OF MONGOLIAN GERBILS WITH CHINESE HELICOBACTER PYLORI STRAINS WITH FUNCTIONAL CAG PATHOGENICITY ISLANDS

J. Wang1,2, A.H.T. Jeremy1, M.A. Aboshkiwa1, P.A. Robinson1, J.E. Crabtree1.

1Molecular Medicine Unit, St James’s University Hospital, Leeds, UK, 2Gastroenterology Department, People’s Hospital, Beijing University, People’s Republic of China

Introduction: To date only a few H pylori strains have been demonstrated to colonise Mongolian gerbils. The aims of this study were to establish stable colonisation of strains of H pylori from China in Mongolian gerbils and to assess the function of the cag pathogenicity island of infecting strains.

Methods: Fresh clinical H pylori isolates from Chinese patients were inoculated into gerbils. At 4 to 6 weeks post-inoculation, infection status was evaluated by microbial culture, biopsy urease test and pathology. Sequencing of glmM and random amplified polymorphic DNA (RAPD) fingerprinting of DNA from cultured H pylori were used to evaluate the genetic identity of pre-inoculated and post-inoculated strains. The ability of pre- and post-inoculated strains to stimulate IL-8 transcription in L5F11 gastric epithelial cells was analysed using an IL-8 luciferase reporter assay.

Results: Three of five inoculated clinical isolates colonised the Mongolian gerbils and induced chronic antral gastritis by 4 weeks post-infection. Each of the three pre- and post-inoculation cagA+ strains had identical glmM sequences and RAPD profiles, and stimulated luciferase secretion from L5F11 epithelial cells. The strain, which caused severe pathological changes, was selected for repeat infection to prove reproducible and stable colonisation. The cagA+, vacA s1c/m2a Chinese strain 42GX gave stable colonisation in the Mongolian gerbil and induced severe gastritis.

Conclusions: This study demonstrates that low passage H pylori clinical isolates will successfully colonise Mongolian gerbils and the ability of the strains to stimulate IL-8 transcription in human gastric epithelial cells is maintained following infection in the Mongolian gerbil.

294 ACUTE PRESENTATION OF GASTRIC CANCER: A SIGNIFICANT AND INDEPENDENT PROGNOSTIC MARKER

G. Blackshaw1, P. Edwards1, J. Barry1, C. Gent1, M. Allison2, W. Lewis1.

1Department of Surgery, 2Department of Gastroenterology, Royal Gwent Hospital, Newport, NP20 2UB, UK

Aims: The aim of our study was to determine the outcomes of patients admitted to hospital as emergencies with acute complications of their undiagnosed cancers, and to compare their outcomes with those of patients diagnosed via conventional outpatient referrals.

Methods: Eighty-eight patients (median age 73 years, 55 months) admitted to hospital as emergencies with acute complications of their undiagnosed gastric cancers were studied prospectively and compared with 170 patients (71 years, 114 months) with gastric cancer diagnosed as out patients or open access endoscopy.

Results: In a multivariate analysis, acute presentation (HR 2.70, 95% CI 2.07 to 3.33, p = 0.002) and the stage of gastric cancer (HR 1.773, 95% CI 1.457 to 2.089, p < 0.0001) were found to be significantly and independently associated with poor survival.

Conclusion: Although the one in three patients presenting acutely had not suffered undue diagnostic delay, they were less likely to undergo potentially curative surgery and to survive long term. Acute presentation with gastric cancer remains common, and if the findings of this study are representative, is an independent and significant prognostic marker of an aggressive form of gastric cancer.

Abstract 294

295 COST EFFECTIVENESS OF H PYLORI “TEST AND TREAT” VERSUS PPI FOR UNINVESTIGATED DYSPEPSIA: MODELLING AND META-ANALYSIS IN THE DESIGN OF THE MRC-CUBE TRIAL

B.C. Delaney1, P. Moayyedi2, R.F.A. Logan3, M. Qume1, A. Roalfe1, S. Wilson1, P. Barton4.

1Department of Primary Care and General Practice, Birmingham, UK, 2City Hospital NHS Trust, Birmingham, UK, 3Department of Public Health and Epidemiology, University of Nottingham, UK, 4Health Economics Unit, The University of Birmingham, UK

Background: Only a small number of trials have directly addressed the management of patients with “uninvestigated” dyspepsia. The design of trials to determine cost effectiveness is more complex than efficacy trials, and can be enhanced by pre-trial modelling.

Methods: A simulation model of 72 alternative management strategies for a dyspeptic patient was constructed. The model simulated the flow of individual patients with potential upper GI disorders through potential investigations and therapies, including endoscopy, non-invasive testing for H pylori, acid suppression therapy, and H pylori eradication. Epidemiological data and meta-analyses were used to specify parameters within the model. The model was used to establish the important comparisons for future research and to establish likely effect sizes in terms of cost and efficacy. Sensitivity analysis explored potential effect modifiers and the MRC-CUBE study was designed and powered on the basis of the model.

Results: Recent trials have supported some of the model’s findings, in that only empiric acid suppression with either an antacid or a PPI, or H pylori “test and treat”, followed by acid suppression were cost effective. Test and treat was more effective, but more costly than empiric acid suppression alone. Based on the model, the minimum effect size would be 7%, assuming a maximum willingness to pay for a patient free of dyspepsia for a year of 100, and a control event rate of 70%. 1940 patients would be required for an α of 0.05 and β 0.1.

Conclusions: The MRC-CUBE trial is about to start recruitment in 60 practices, a large, multi-centre trial being justified on the basis of the model. As well as informing the selection of appropriate comparisons, pre-trial modelling enables cost effectiveness trials to be powered for cost as well as effectiveness outcomes.

296 IS THE NICE GUIDANCE ON THE USE OF PROTON PUMP INHIBITORS (PPIs) FOLLOWED IN CLINICAL PRACTICE?

H.R. Ferguson, B.E. Loo, A. Varghese.

Department of Gastroenterology, Causeway Hospital, Coleraine, Northern Ireland

Introduction: Millions of pounds are spent each year in the NHS on prescribing PPIs for dyspepsia. The National Institute for Clinical Excellence (NICE) produced guidance on the use of PPIs in July 2000 to improve clinical practice and cost effectiveness.

Aim: To establish current practice in prescribing PPIs.

Methods: We identified medical admissions over a 2 week period in September 2002. The charts of those patients on PPIs were reviewed and data collected.

Results: 91 patients were admitted of which 40 (44%) were on a PPI. In this group the average age was 71.2 years (range 41–96 years). 65% were male. The average length of therapy was 128 weeks (range 1–624 weeks). In 25 (62%) there was a clear indication for PPI use (recently diagnosed peptic ulcer, previous ulcer on NSAID, dyspepsia) but in 15 (38%) there was no compelling indication (NSAID with no peptic ulcer disease, alcohol abuse, on clopidogrel). 22 patients (55%) were on healing doses of PPI. In 6 (15%) this was for an appropriate reason (active peptic ulcer, uncontrolled reflux symptoms). 16 patients (40%) were prescribed inappropriately high doses (NSAID prophylaxis, controlled dyspepsia with no attempt made to reduce to maintainance doses). 22 patients (55%) had previous upper GI endoscopy, 10 (25%) were unfit for investigation, 1 (2%) had not attended, 7(18%) were suitable but had no previous investigations performed. 12 patients had a diagnosis of gastritis, duodenitis or peptic ulcer disease made at endoscopy. Of these 2 were not tested for Helicobacter pylori infection.

Conclusion: A substantial proportion of patients were prescribed PPI therapy with no clear indication, or in inappropriately high doses. 7 patients were on long term PPIs without a confirmed clinical diagnosis, and 2 with possible H pylori associated pathology were not tested. The NICE guidance on the use of PPIs is not followed in clinical practice.

297 THE VALUE OF DRUGS TO TREAT UPPER GASTROINTESTINAL SYMPTOMS: WHAT ARE PATIENTS WILLING TO PAY?

M. Follows1, A.T.R. Axon1, D.M. Chalmers1, P. Moayyedi2.

1Centre for Digestive Diseases, Leeds General Infirmary, Leeds, UK; 2Gastroenterology Unit, City Hospital, Dudley Road, Birmingham, UK

Introduction: Over £500 000/year is spent on anti-secretory therapy in the UK and until recently they were the most expensive drug class in the NHS budget. The treatment of upper gastrointestinal symptoms does not save life so we need to be sure that this money is being spent appropriately. We have previously shown that quality adjusted life years are not sufficiently sensitive to measure the value of treating dyspepsia symptoms. We have therefore assessed directly how much dyspepsia patients are willing to pay for cure of their symptoms. Methods: Unselected patients with dyspepsia attending for endoscopy were interviewed. Demographic and income data were collected and also a validated dyspepsia questionnaire was administered. Patients were asked to place a monetary value on a hypothetical drug that had a 20, 50, or 80% chance of curing their symptoms. The patient bid higher or lower from a randomly selected starting point between £5 and 50 until they were satisfied that the correct price had been reached.

Results: 193 patients completed the interview (mean age = 50, range 19–76 years; 89 (46%) male). Patients were willing to pay £11.52 (95% CI = 9.60 to 13.45) for a drug with a 20% chance of curing their symptoms, £20.24 (95% CI = 18.15 to 22.34) for a 50% chance of curing their symptoms and £30.13 (95% CI = 27.88 to 32.37) for a drug with an 80% chance of curing their symptoms. These costs did not statistically significantly alter with increasing severity of dyspepsia or endoscopy diagnosis. In a multiple regression model including age, sex, income, educational status, endoscopy diagnosis, dyspepsia score, most troublesome symptom, and starting bid the only significant predictor of the amount patients were willing to pay was the starting bid (0.23; 95% CI = 0.09 to 0.37 per unit increase in starting bid).

Conclusions: These data suggest lower priced drugs such as generic H2 receptor antagonists and prokinetics are value for money even with low probabilities of treatment success. Proton pump inhibitors are too expensive when the probability of cure is low but these are at an acceptable cost when the chance of success is more than 50%.

298 CAN PATIENTS WITH AN UPPER GI BLEED BE DISCHARGED?

A. Sourianarayanane, A.H. Shenoy, D.N. Foster.

Rochdale Infirmary, Whitehall Street, Rochdale OL12

There are various scoring systems for upper GI bleeding to predict mortality, including the Rockall score. The newer Blatchford score predicts the need for intervention. These scores help in management of patients with least intervention requirement who are otherwise investigated including endoscopies after hospitalisation, has not been studied in district general hospital setting where most of the patients are managed.

Aims: To validate the Blatchford scoring system in a different population group and also compare it with the Rockall scoring system, in identifying the population at least risk, of requiring intervention.

Methods: All patients presenting to the hospital with the diagnosis of upper GI bleeding during a 12 month period were included in this study. They were scored using Blatchford and Rockall systems and the results compared with the outcome.

Results: Of 17 249 general medical patients admitted, 225 had upper GI bleeding. As in other studies there was a close correlation between mortality and the initial Rockall score (eg Score 2 in his study mortality was 5.6%; in this study 12.1%. Score 5 in his 39.6%; in this 30% mortality). Surprisingly a relationship also existed between the Rockall score and the need for intervention and length of stay, not found in previous studies. As in the original study the Blatchford score predicted the need for intervention and length of stay. With a Blatchford score of 0, the need for intervention in this study was 3.8% versus 1.8% in the Blatchford study; a score of 6 was associated with intervention in 62.5% versus 50.4% in the Blatchford study.

As suggested in the Blatchford study group of patients with Hb > 13.0 gm/dl in men and > 12.0 gm/dl in women, urea < 6.5mmol/l, systolic BP > 110 mm Hg, and pulse < 100, (15.56% of patients) the need for intervention is low at 2.8% and in those < 65 years of age (8.9% of patients) is 0%. Similarly no intervention is needed in patients with value of 0 in both Blatchford and initial Rockall scoring system.

Conclusion: The Rockall scoring system is useful for assessing risk of mortality while the Blatchford scoring system predicts the need for intervention. A combination of them could help in early discharge of some 9% of patients with less intervention requirements.

299 IMPROVEMENT OF DYSPEPTIC SYMPTOMS AND QUALITY OF LIFE IN HEALTHY INDIVIDUALS AFTER ADMINISTRATION OF ARTICHOKE LEAF EXTRACT (ALE)

G. Marakis2, A. Walker2, J.C.L. Booth1, J. Wright 2.

1Department of Gastroenterology, The Royal Berkshire Hospital, Reading, UK; 2Hugh Sinclair Unit of Human Nutrition, University of Reading, Reading UK

Background: Dyspepsia is a common condition but can be a difficult symptom to treat although antacids or pro-kinetics are often used. Recent data from Germany suggest that high doses of artichoke leaf extract (ALE) can reduce symptoms of dyspepsia.

Aim: To investigate the efficacy of low dose intervention with ALE on amelioration of dyspeptic symptoms and improvement of quality of life.

Design of Study: Open, dose ranging postal study in patients with self-reported dyspepsia recruited through the media (articles in local and national newspapers).

Methods: The Nepean Dyspepsia Index and the State-Trait Anxiety were assessed at baseline and after 2 months of treatment with ALE to assess the efficacy of the intervention. Volunteers were randomly allocated to receive either 320 or 640 mg of the extract daily.

Results: Of the 516 participants, 454 completed the study. In both dosage groups, there was a significant reduction of all dyspeptic symptoms, with an average reduction of 40% in global dyspepsia score. There were no differences in the outcome measures between the two groups. Health related quality of life was significantly improved in both groups.

Conclusion: This open study demonstrated that ALE may ameliorate upper gastrointestinal symptoms and improve quality of life in healthy subjects suffering from dyspepsia. Further double blind, placebo controlled studies are needed to confirm these results. Nevertheless, these results hold promise of a safe new approach to dyspepsia in primary health care.

300 DIAGNOSTIC DELAY IN GASTRIC CANCER IS RELATED TO BOTH INDICES OF DEPRIVATION AND BLIND ACID SUPPRESSION

G. Blackshaw1, P. Edwards1, J. Barry1, C. Gent1, M. Allison2, W. Lewis1.

1Department of Surgery, 2Department of Gastroeneterology, Royal Gwent Hospital, Newport, UK

Aims: To examine the time taken to diagnose gastric cancer, identify the source of delay, and assess its clinical importance.

Methods: Two hundred and fifty eight consecutive patients (median age 73 years, 55 months) with cancer of the stomach were studied prospectively. The main measures of outcome were the interval from the onset of symptoms to histological diagnosis, final pathological stage of the tumour, and whether potentially curative resection was possible. Indices of multiple deprivation related to electoral wards were obtained from the office of national statistics.

Results: The median delay from first symptoms to diagnosis was 15 weeks (range 1–175). One hundred of the patients (39%) had incurable stage IV tumours at presentation, although no relation was found between diagnostic delay and tumour stage. Sixty seven patients (26%) had received blind acid suppression therapy from their general practitioners, which resulted in further and significant delays in diagnosis (median 20 compared with 12 weeks, p = 0.025). Diagnostic delay correlated significantly with indices of multiple deprivation for electoral divisions, as described by the Office for National Statistics (delay in consulting a doctor p = 0.044, total delay in diagnosis p = 0.025). Delays in diagnosis in patients from electoral wards with deprivation scores of 10 or less were significantly shorter than the delays in patients with scores of greater than 30 (7 compared with 20 weeks, p = 0.024).

Conclusion: Long delays remain common in the diagnosis of cancer of the stomach. Measures in the community directed specifically at areas of high social deprivation, to educate the public and doctors alike, are required if patients with gastric cancer are to be diagnosed at an early and potentially curable stage.

301 ANTIBIOTIC RESISTANCE TO HELICOBACTER PYLORI IN NORTH WALES

J.P.L. Ong, W. Ahmed, M. Walker, D. Owens.

Department of Gastroenterology, Ysbyty Gwynedd Hospital, Bangor, North Wales, UK

Background: Resistance to antibiotics can be a major problem in the treatment of bacteria infections. As the use of antibiotics increases, bacterial resistance to these agents is rising and in many cases is responsible for the failure of treatment regimes. Although the treatment of H Pylori infection requires the use of more than one antibiotic to obtain adequate eradication rates, the efficacy of currently used antibiotics combination has been shown to be reduced by resistance to one of the antibiotics used.

Aim: To evaluate the antibiotic resistance to H Pylori in the past 4 years in North Wales.

Method: Central database of all patients who are H Pylori positive over the past 4 years in two district general hospitals in North Wales. Minimum inhibitory concentration (MIC) of amoxycillin, metronidazole, tetracycline, and erytromycin were determined by agar dilution method.

Results: A total of 888 H Pylori organism isolated. Of those, 147 had no sensitivities due to failure of the organism to grow on subculture. Primary resistance rates were: metronidazole 12.6%, erythromycin 6.7%, amoxycillin 0.6%, and tetracycline 0.7%. There was an overlap with organisms resistant to both metonidazole+erythromycin (6.8% of total of these groups); metronidazole+tetracycline (2.3% of total of these groups) and erythromycin+amoxicillin (1.9% of total of these groups).

Conclusion: A proportion of patients from North Wales were infected with resistant strain of H Pylori. The higher resistance rate to metronidazole and this used in combination with erythromycin have both proved not to be effective for eradication therapy of H Pylori. Amoxicillin and tetracycline are useful component of treatment regimens in this area. No significant change in resistance rates during the period of investigation were observed. Antibiotic resistance monitoring is very important to ensure effective eradication of H Pylori infection.

302 PATIENT AND GP USE OF NSAIDs IN MUSCULOSKELETAL PAIN: MISPERCEPTIONS OF RISK

A.L. Blower1, A.D. Woolf2, N. Amin3, A.J. Carr4, (introduced by M Guslandi5) on behalf of the Arthritis Action Group.

1Royal Albert Edward Infirmary, Wigan, UK; 2Royal Cornwall Hospital, UK; 3White Lodge Medical Practice, UK; 4University of Nottingham, UK; 5Istituto Scientifico S. Raffaele, Italy

Background: In the UK, up to 2000 NSAID users a year die from gastrointestinal (GI) side effects. Little is known about the perceptions of risk and benefit associated with NSAID management of musculoskeletal pain (MP) in primary care. The Arthritis Action Group (AAG) survey was undertaken to increase understanding of the management of MP in 8 European countries. Data from UK GPs and patients are presented here.

Methods: A telephone survey was conducted with 1483 doctors and 5803 chronic pain sufferers in 8 European countries. In the UK, 200 GPs and 798 MP sufferers were surveyed. All doctors and patients were randomly selected. The survey was based on a structured questionnaire that asked about health status (SF-12), usual management of MP, risks and benefits of treatment and beliefs about treatment.

Results: Arthritis is the most common cause of MP. In first line management of MP, 61% of GPs use analgesia alone and 35% use NSAIDs alone. Cox-2 NSAIDs are used by 2% of GPs. 26% of patients taking prescription NSAIDs supplement with OTC medication. Most GPs (85%) are concerned about NSAID related side effects and routinely screen patients for some known risk factors but only tell patients about the most common side effects (64%). Most patients who are taking NSAIDs (71%) are unconcerned about side effects and only 51% are aware of any side effects of NSAIDs. However, among those who change medications, 42% do so because of GI side effects. Patients’ perceptions of their risk of serious GI side effects is poor: 44% of patients who are at increased/critical risk of side effects perceive their risk as moderate or high. GPs and patients believe that the side effects of NSAIDs can be worse than the condition and few believe that they are safe from side effects if they take NSAIDs as prescribed. Patients are concerned about tolerance and addiction to NSAIDs.

Conclusions: Many patients remain unaware of their personal risk, despite general concerns about NSAIDs. They require more and better information about the specific personal risks and benefits of treatment.

303 INTENSIVE STUDY OF SERIAL ENDOSCOPIC AND HISTOLOGICAL CHANGES OCCURRING OVER 48 HOURS IN HUMANS GIVEN NAPROXEN

M.W. James, J.R. Bebb, C.T. Atherton, N. Bailey-Flitter, A. Zaitoun, C.J. Hawkey.

Division of Gastroenterology, University Hospital, Nottingham, UK

Introduction: Animal studies support a central role for leukocytes in acute NSAID associated injury, which can be abrogated by pre-treatment with anti-leukocyte antibodies. However, with chronic human use inflammatory cells are seldom seen without H pylori infection. Because appreciation of a role for leukocytes in early human pathogenesis would be important and to test the relevance of animal models of NSAID damage to humans we performed an intensive endoscopic and histological evaluation of the acute effects of naproxen in humans.

Methods: Eight healthy H pylori –ve human volunteers were randomised to receive naproxen 500 mg twice daily or no treatment for 48 h. Endoscopy was performed before and 3, 12, and 48 hours after dosing. Serial antral biopsies were analysed by blinded histology.

Results: Increased neutrophil margination and vascular plugging were not seen over a time course in which they are prominent in animal models. Any changes were mild and showed no evidence of progressive evolution, with no differences between naproxen and placebo. The table shows the number of subjects with individual changes at any time point.

Conclusion:In H pylori –ve human volunteers, acute naproxen therapy does not replicate the changes seen in animal models, calling their relevance into question. How H pylori +ve individuals respond remains to be determined.

Abstract 303

304 FUNDIC EXPRESSION OF GHRELIN IN RELATION TO H pylori STATUS

A. Bassi1, H. Kamarova2, L. Pazmany2, K. Bodger1.

1Aintree Centre for Gastroenterology, 2Dept of Medicine, University Hospital Aintree, Liverpool, UK

Background: Ghrelin, a hormone secreted mainly from the gastric fundus, is an inducer of growth hormone release and acts as an orexigenic gut-brain signal to stimulate appetite. We speculate that H pylori infection may downregulate gastric ghrelin expression, thereby providing a potential mechanism for extraintestinal effects of the infection (eg growth delay in childhood).

Methods: Subjects attending for gastroscopy were recruited. Two fundic biopsies were taken for RNA extraction and two antral biopsies for H pylori status. RNA was reverse transcribed and cDNA subject to quantitative real-time PCR with results normalised for the housekeeping gene hHPRT. Ghrelin primers were: Fwd: 5’-ACAACTCCT TGCAGCTCC-3’; Rev:5’-ATCTTCATGAAGGTAGRCAGTC-3’.

Results: Nine subjects (4 HpPos) have been studied so far (Age: 32–73 years; BMI 23–32 kg/m2). A trend towards lower median expression (ghrelin/HPRT ratio) in H pylori positive versus negative subjects is apparent (p = NS).

Conclusions: Based on these preliminary findings, H pylori infection may be associated with downregulation of gastric fundic ghrelin expression. If this observation is confirmed (and is associated with reduced gastric ghrelin secretion) this phenomenon may contribute to extraintestinal manifestations of the infection via reduced GH release and/or via disturbances in the regulation of appetite and energy homeostasis.

305 PREVALENCE AND APPLICABILITY OF PROTON PUMP INHIBITOR PRESCRIPTION IN SECONDARY CARE

R. Hebbar, Z. Ahmed, M.S. Zulfiker, S. Scott-Thomas, N.D. Hawkes.

Department of Medicine, Prince Charles Hospital, Merthyr Tydfil, UK

Introduction: NICE has issued guidance on the prescription of proton pump inhibitors (PPIs), which play an important role in managing the dyspeptic patient but constitute a major expenditure for the NHS.

Aims: To establish the prevalence of PPI prescription in patients admitted to medical and surgical wards, indication for PPI use, applicability of prescription, and potential cost savings.

Methods: A 1 month, prospective, proforma based study. A team of pharmacists reviewed daily all patients admitted to the designated study wards and noted details of PPI prescription. A trial investigator further reviewed the indication and applicability of prescription according to agreed criteria based on NICE and BSG guidance documents. Cost estimates were based on information in the British National Formulary (No. 44). Statistical analysis was performed using SPSS version 11.

Results: Of the 960 admissions, 98 (10.2 %) were taking PPI medication, 36 males, mean age 68.4 years (range 18–94). 60/98 (62%) were long term prescriptions and 42/98 (42.9%) did not follow NICE guidance. Endoscopic investigation increased the proportion of appropriate prescriptions compared with clinical based decisions (χ = 14.5, p < 0.001). Details are shown for each indication (see table). Estimated annual savings for this cohort over a 12 month period were 13 420. Assuming constant admission and PPI prevalence rates, this represents a potential annual saving of 161 042 for the community drug budget.

Conclusions: We found a prevalence of 10.2% of patients on PPI medication. NICE guidance was not followed in 43% of this inpatient cohort taking PPIs. Specialist review of inpatients can play a significant role in reducing overall cost of long term PPI therapy in the community.

Abstract 305

306 MASTIC GUM HAS NO EFFECT ON HELICOBACTER PYLORI LOAD IN VIVO

J.R. Bebb1, N.F. Bailey-Flitter1, D.A.A. Ala’Aldeen2, J.C. Atherton1.

1Division of Gastroenterology, 2Department of Microbiology, University Hospital, Nottingham, UK

Background: Mastic gum is a resinous exudate obtained from the stem and leaves of Pistacia lentiscus. It is widely used in Middle Eastern and Mediterranean countries as a chewing gum and food additive. We have previously shown1 that it is bactericidal against H pylori in vitro and this has been independently confirmed. Since then mastic has been widely marketed both in the USA and Europe as a cure for H pylori infection and peptic ulcers.

Methods and Patients: Nine H pylori positive patients were recruited from routine day case endoscopy lists. No patients had current or previous gastroduodenal ulceration or had taken antibiotics, bismuth compounds, or proton pump inhibitors for 6 weeks prior to the trial. A 13C urea breath test was performed prior to, on day 14 of and 5 weeks after treatment with mastic capsules 1 g four times a day.

Results: Eight patients completed the trial protocol (one withdrew after 5 days due to nausea). All nine patients remained H pylori positive by UBT immediately after mastic treatment with unchanged UBT values (pre-treatment mean ± SEM 19.4 ± 3.2 per ml, post-treatment 18.5 ± 3.4 per ml). Eight patients attended for UBT 5 weeks after treatment finished, again with unchanged UBT values (18.2 ± 3.6 per ml). Other than the patient who withdrew, two patients reported mild adverse symptoms; one complained of fatigue and the second of constipation and bloating.

Conclusion: This study shows that high dose mastic gum has no significant effect against H pylori in vivo.

  1. Levine JA, Ray A, Jensen MD. Relation between chubby cheeks and visceral fat. New Engl J Med 1998;339:1946-7.

307 THE ROLE OF SURGERY AFTER NEOADJUVANT CHEMOTHERAPY IN LOCALLY ADVANCED OESOPHAGOGASTRIC CANCER

A.G.K. Li, E. Fernandes, J.K.F. Wong, E. Choke, M. Nicolson, K.G.M. Park.

Aberdeen Royal Infirmary, Foresterhill Site, Aberdeen, Scotland, UK

Background: Surgical resection of carcinoma of oesophagus or stomach provides the best means of disease control when a complete resection is possible. Moreover, the majority of patients present at a late stage, and a surgical approach is not often possible. While patients with operable oesophageal and gastric cancers may benefit from neo-adjuvant chemotherapy followed by surgery, it is not clear whether patients who present with inoperable disease that is downstaged with chemotherapy benefit from subsequent surgery.

Aim: To determinate the benefit of surgery after downstaging with chemotherapy in locally advanced OG cancer.

Methods: Between 1994 and 1998, a single institution, non randomised study, evaluated the outcome of patients who underwent surgery after chemotherapy downstaging, compared to the control group without subsequent surgery. 26 patients with inoperable, locally advanced oesophagogastric tumours were treated with a standard regimen of either pirubicin, cisplatin and infusional 5 flurouracile (ECF), or mitomycin, cisplatin and infusional 5 flurouracile (MCF). After completion of chemotherapy (3–8 cycles) 13/26 patients were down staged sufficiently to allow a surgical resection to be undertaken. Patients were followed up until death or for a minimum of 45 months.

Results: The overall mean survival in the patients who underwent a resection was 33.1 months (8.1–81.8 months), whereas in the non-resected patients the mean of survival was 13.2 months (3.5–55 months). In the resected group 9 died of disseminated malignancy and and 4 remain disease free 41.3–66.6 months following surgery. Of the 13 non-resected patients 1 remains alive after 55 months. The remainder have died of progressive local (n = 5) or disseminated disease (n = 6).

Conclusion: In a subgroup of patients with locally advanced oesophagogastric cancers, surgical resection following down staging with combination chemotherapy can provide long term survival. It is likely that this is due to better local disease control.

Neurogastroenterology/motility posters 308–318

308 IDIOPATHIC SLOW TRANSIT CONSTIPATION: A RARITY IN MALES

C.K. Rayner1, M.A. Kamm1, C.H. Knowles2, S.M. Scott2, P.J. Lunniss2.

1 St Mark’s Hospital, Harrow, Middlesex; 2 The Academic Department of Surgery, The Royal London Hospital

Colonic transit is delayed in about half of patients presenting to specialist units with severe constipation. Most patients are female, and a majority (about two thirds) have no identifiable cause. There is almost no information regarding males with slow transit constipation. We reviewed the records of patients with constipation referred to two large centres specialising in functional bowel disorders, over 4 years (Centre 1) and 8 years (Centre 2) respectively. Colonic transit was assessed by validated radiological marker studies. Constipation was considered idiopathic when dilated bowel (megarectum) and neurologic injury (central, spinal, or pelvic) were excluded. See Table 1. Not only were the minority of patients referred for constipation male, but proportionally fewer males than females had slow colonic transit (*p < 0.05). Of males with slow transit, only 8 from Centre 1, and 2 from Centre 2, were idiopathic (10 of 43, or 23%) (see Table 2).

Abstract 308 Table 1

Abstract 308 Table 2

In the small number of males presenting with constipation, colonic transit is delayed in the minority, and most of these have an identifiable cause, in distinction to women. Either men are less exposed to insults that impair colonic function, or have a greater reserve in resisting their effects.

309 EVIDENCE FOR INCREASED POST-PRANDIAL PLASMA 5-HYDROXYTRYPTAMINE (5-HT) CONCENTRATION IN FUNCTIONAL DYSPEPTICS AND RELATIONSHIP TO GENDER AND SYMPTOMS

R. Lea1, L.A. Houghton1, P.J. Whorwell1, P. Whitaker2.

1 Department of Medicine, University Hospital of South Manchester; 2 Chemical Pathology, Leicester Royal Infirmary

Recent studies suggest that patients with irritable bowel syndrome (IBS) have elevated levels of plasma 5-HT following meal ingestion compared with healthy controls. Moreover, patients who exhibit post-prandial symptoms have higher plasma 5-HT levels compared with those who do not, suggesting a possible relationship between post-prandial symptoms and abnormalities in 5-HT release. Given functional dyspepsia (FD) belongs to the same family of disorders as IBS, the aims of this study were to assess platelet depleted plasma (PDP) 5-HT concentrations under fasting and fed (760 kcal meal) conditions in 11 patients with FD (aged 30–61 years; 6 males) and 12 healthy volunteers (aged 20–48 years; 5 males) and assess any relationship to gender and epigastric pain/discomfort (p/d).

Results: Under fasting conditions FD patients had similar levels of PDP 5-HT to controls (FD (n = 11): 4.65 ng/ml (geometric mean) vs controls (n = 12): 4.57ng/ml; ratio FD:controls (95%CI), 1.02 (0.14,1.26)). However, under fed conditions, although the peak PDP 5-HT concentration for the whole group of FD patients was not significantly different from controls (11.19 ng/ml vs 8.43 ng/ml; 1.33(0.69, 2.54); p = 0.37, patients who reported post-prandial p/d had a higher PDP 5-HT peak (n = 8: 14.74 ng/ml) than both patients who did not report p/d (5.36ng/ml; ratio with:without p/d 2.75(1.24, 6.10); p = 0.02) and controls (8.43 ng/ml; ratio FD with p/d:controls, 1.75 (0.79, 3.87); p = 0.14). This was as a consequence of a higher PDP 5-HT peak in the female FD patients with p/d (36.56 ng/ml), as all other females (FD and controls (n = 9): 7.71 ng/ml; ratio FD with p/d:FD and controls, 4.74 (2.5, 9.0); p < 0.001) and all males (FD and controls (n = 11): 8.07 ng/ml; ratio female FD with p/d: all males, 4.53 (2.19,9.35); p < 0.001) had lower PDP 5-HT peaks. Lastly, symptom severity tended to correlate with peak PDP 5-HT concentration (rho = 0.37; p = 0.08).

Conclusions: These data provide evidence for a role for 5-HT in post-prandial symptoms in patients with FD, particularly females.

310 SMOKING IS ASSOCIATED WITH THE DEVELOPMENT OF POST-INFECTIOUS IRRITABLE BOWEL SYDROME

S.D. Parry, J.R. Barton, M.R. Welfare.

University of Newcastle, Faculty of Medicine, North Tyneside Hospital, Rake Lane, North Shields NE29 8NH, UK

Introduction: Irritable bowel syndrome (IBS) can be triggered by psychological stress, alterations in gut motor function and/or visceral perception. Previous studies suggest 4–32% of people develop IBS after bacterial gastroenteritis but the exact mechanisms underlying post-infectious IBS are not clear.

Aim: To look into the role of possible causative factors in the development of post-infectious functional gastrointestinal disorders, namely IBS, functional dyspepsia, or functional diarrhoea.

Methods: A prospective cohort study. There were 128 subjects without a prior FGID under study and with recent stool positive bacterial gastroenteritis who consented to participate. The presence or not of IBS, functional dyspepsia or functional diarrhoea was diagnosed at the start and on follow up at 6 months using self-complete Rome II modular questionnaires. Data on demographics, social class, infecting organism, smoking and alcohol use, anxiety, depression (via Hospital Anxiety and Depression Scale) and life events and impact (via Life Events Survey) were collected at the start of the study.

Results: One hundred and seven Rome II questionnaires were returned at follow up. Twenty-five subjects developed a FGID, 16 who had symptoms consistent with IBS. Of the factors mentioned, only smoking was significantly associated with the development of a post-infectious FGID (odds ratio 4.1; CI 1.4 to 11.6). The odds ratio of a smoker developing post-infectious IBS was 3.6 (CI 1.12 to 11.60). The odds ratios of a smoker developing functional diarrhoea or functional dyspepsia were not statistically significant.

Conclusion: Post-infectious IBS appears to be associated with smoking and we did not find evidence of association with life events, anxiety, or depression. Smoking is known to moderate gut immunity in other disorders such as ulcerative colitis and Crohn’s disease. Our study adds to increasing evidence for an organic basis to post-infectious IBS, perhaps moderated via inflammatory pathways.

311 MEDICINES TAKEN BY PATIENTS WITH IRRITABLE BOWEL SYNDROME (IBS)

M. Kinnear1, D. Steinke1, G. Smith2, I.D. Penman2.

1 Lothian Pharmacy Practice Unit and University of Strathclyde, UK; 2 Gastrointestinal Unit, Western General Hospital, Edinburgh, UK

Introduction: Many studies have evaluated the prescribed medicines taken by patients with IBS. However, there is a paucity of information about over-the-counter and complementary therapies taken. This study describes medicines taken by patients with IBS from a self-administered diary and evaluates differences in medication taken by those referred to secondary care and those not referred.

Method: A self-selected group of patients with IBS (n = 595) submitted a structured 10 question daily diary for 6 months. The cohort was stratified by those who reported a referral to secondary care (n = 381) and those who did not report referral (n = 203) at baseline evaluation. Eleven patients could not be stratified and were excluded from analysis. Medicines taken at any time during the study period were evaluated and were defined as prescribed or non-prescribed (including herbal).

Results: At any time over the study period 425 of 595 people took a “medicine”. In total there were 1589 “medicines” taken, 1335 (84%) had a BNF identifiable ingredient. Of the 381 secondary care patients, 280 (73%) patients were taking “medicine”. The majority of patients (58%) were taking an antispasmodic (161/280). Other medicines include laxatives (35%), antidiarrhoeals (29%), and analgesics (18%). There were 145 (71%) patients taking “medicines” of the 203 patients treated in primary care. The most frequently taken medicines were antispasmodics (75% 110/145), laxatives (36%), antacids (18%), and analgesics (14%). Herbal and non-prescribed therapies were used similarly proportion (31%) in each group (88/280 referred, 45/145 not referred) with Acidophilus, aloe vera and digestive salts being the most frequent.

Conclusions: “Medicine” usage is similar whether or not IBS sufferers are managed in primary or secondary care. A larger proportion of patients treated in primary care took antispasmodics, but use of other medicines was similar. A significant number of IBS patients take non-prescribed or herbal products, the proportion using these was the same in both groups. Study was funded in part by GlaxoWellcome.

312 ROLE OF HEPATOBILIARY SCINTIGRAPHY IN THE MANAGEMENT OF SPHINCTER OF ODDI DYSFUNCTION

E.W. Seward, M.J. Guinane, M.K. Khela, E. Yazaki, D.F. Evans, C.A. Ainley.

Wingate Institute, 26 Ashfield Street, London E1 2AJ, UK

Sphincter of Oddi dysfunction (SOD) is a cause of post-cholecystectomy pain. Current gold-standard diagnosis is by measurement of basal pressure by sphincter of Oddi manometry (SOM), and if elevated an endoscopic sphincterotomy (ES) is performed. Both these procedures carry significant complication rates. Hepatobiliary scintigraphy has been promoted as a means of assisting management.

Aim: to evaluate hepatobiliary scintigraphy (HBS) as a means of screening for SOD and predicting treatment response.

Methods: 21 post-cholecystectomy patients were screened by HBS prior to standard evaluation with SOM during ERCP. HBS was performed using a standard protocol1. A semi-quantitative scoring system classified scans as positive (SOD) or negative (no SOD). Two observers reviewed all 21 scintigrams. SOM was regarded as diagnostic of SOD if sphincter base pressure was > 40 mm Hg. If elevated, an ES was performed and a successful result was defined as an improvement in symptoms.

Results: 17 of our patients were female, average age 42.4 + 4.0 years. Agreement was 100% between observers.

Conclusions: HBS can be interpreted reliably between observers. HBS is a poor screening tool in isolation, but may prove a useful adjunct to manometry in predicting ES outcome.

  1. Sostre J Nuc Med 1992:04

Abstract 312 Table 1

HBS results vs SOM results

Abstract 312 Table 2

HBS result vs. sphincterotomy outcome

313 ALTERED BOWEL HABIT DUE TO TIME ZONE SHIFTS: A PILOT STUDY

S.K. Arthur1, D.F. Evans1, P. Farrand2, E. Yazaki1.

1 Department of Adult and Paediatric Gastroenterology, Barts and the London School of Medicine and Dentistry; 2 Institute of Health Studies, University of Plymouth

Introduction: Circadian rhythms for protein transcription have recently been identified in the gut.1 Environmental synchronizers may reset biological clocks, leading to alteration in gut functions, but alteration in bowel transit due to time zone shifts experienced in long haul flight is yet to be reported.

Methods: Six men (Age 33.2 +/- 10.6 years) and 3 women (36.7 +/- 7.2) (mean +/- 1 SD) flew from London to California, USA, and back. As a pre-condition to joining the study, they were on no drugs for digestive diseases. We elicited responses pre-, in-, and post- flight and during foreign stay about their bowel habit, stool form scale and abdominal symptoms using validated questionnaires. Paired t-tests were used to examine significant changes in sleep and stool form scale from the pre-flight level. Stool form scale may be used as a guide to intestinal transit time.2

Results: Sleep duration and times pre-flight, during foreign stay and post flight were not significantly different (p > 0.05). Additionally, there was no reason to believe that the stool changes were attributable to a drastic change in diet. Abdominal symptoms were unremarkable and did not change during foreign stay or on return home from abroad. All our subjects were normal and had an uneventful stay abroad. Average stool consistency changed significantly (p < 0.01) from a lumpy to smooth consistency to a smooth to soft consistency during foreign stay and persisted 4–5 days into post-flight. The respective stool form scales were pre-flight 2.8 +/- 1.0, foreign stay 3.5 +/-1.1, post-flight 3.7 +/-1.2.

Summary and Conclusion: During a time zone shift of +/- 8h, while the sleep clock was synchronized with the external clock, gut transit, as measured by the stool form scale, failed to synchronize with the external clock, leading to altered bowel habit. Biologic clocks that regulate gut functions may not adapt as readily to time shifts, as does the sleep clock.

  1. Scheving LA. Biological clocks and the digestive system. Gastroenterol 2000;119:536–49.

  2. Lewis SJ Heaton KW. Stool form scale as a useful guide to intestinal transit time. Scand J Gastroenterol 1997;32: 920–24.

314 ASSESSMENT OF REFLUX IN NEUROLOGICALLY-IMPAIRED CHILDREN USING THE MULTIPLE INTRALUMINAL IMPEDANCE PROCEDURE

M. Thomson, D. Rawat, G. Ball, T. Wenzl, R. Del Buono.

Centre for Paediatric Gastroenterology, Royal Free Hospital, Pond Street, London, NW3 2QG, UK

Background: In addition to acid gastro-oesophageal reflux (GOR), non-acid GOR (pH > 4) may be clinically relevant in neurologically impaired children. However, standard pH metry can only detect the former. We have therefore used the pH independant technique of intraluminal electrical impedance to quantify all reflux events.

Aim: The aim of this study was to quantify acid and non-acid reflux in a group of these patients using a new catheter related technique.

Methods: Ten children (9 cerebral palsy, 1 Trisomy 21) fed intra-gastrically underwent 12 h studies of intra-oesophageal 6 channel impedance and dual channel pH monitoring. All patients were off medication influencing gastric pH. Recordings were analysed for the frequency of acid and non-acid GOR and the height reached by the refluxate.

Results: 369 reflux events were detected with the combined technique. 191 (51.8%) were non-acid events (mean pH 5.6) and of these 138 (72.2%) reached the uppermost (1) impedance channel. Of the 178 acid reflux events (mean pH 3.1), 81.5% reached the uppermost channel.

Conclusions: Over half of reflux events in neurologically impaired children are non-acidic and therefore are missed using standard pH metry. Most of these refluxes reached the upper oesophagus. Simultaneous intraoesophageal impedance and pH measurements proves to add valuable information that may improve therapeutic management in this patient group.

315 SIGNIFICANCE OF ANXIETY IN IBS OUTPATIENTS: RELATION TO BOWEL SYMPTOMS AND FINAL DIAGNOSIS

J.R. Boulton-Jones, R.C. Spiller.

Queens Medical Centre, Derby Road, Nottingham NG7 2UH, UK

Introduction: Although it is well recognised that the probability of consultation with irritable bowel syndrome (IBS) is increased by anxiety, it is unclear what influence anxiety has on severity of symptoms.

Aims: To prospectively evaluate the relationship between anxiety, symptoms, and final diagnosis in patients presenting with symptoms of IBS.

Methods: Hospital anxiety and depression (HAD) scores were measured prospectively in 178 new out-patients referred with symptoms consistent with IBS. Investigations to exclude other diagnoses were instituted as clinically indicated. The final diagnosis was obtained from review of electronic records 0.3–3 years later. Patients were categorised by mode of onset (post-infectious (PI-IBS)), or predominant bowel habit (diarrhoea (d-IBS), constipation (c-IBS), alternating (alt-IBS)). The anxiety and depression scores were compared for each group. A healthy control group (n = 40) was included.

Results: 28 patients had alternative diagnoses made including: bile salt malabsorption (5), lactose intolerance (4), microscopic colitis (4), ulcerative colitis (1), pancreatic disease (3), diverticular disease (2), and coeliac disease (1) leaving 150 patients with IBS (38 c-IBS, 49 d-IBS, 20 alt-IBS, 37 PI-IBS 6 IBS unspecified). Abnormal anxiety levels were present in 68.6% (severe in 12%, moderate in 22.4% and mild in 34.4%). Raised depression scores were detected in 26.3% (severe in 2.5%, moderate in 10.7% and mild in 13.1%). The mean (± SEM) A and D scores were 9.3 (±0.35) and 4.5 (±0.32) respectively for all IBS patients, which was significantly greater than values in control patients (4.0 (±0.4) and 1.6 (±0.33) (p < 0.001)) but not from those in whom other diagnoses were made (9.9 (±0.78) and 6.36 (±0.68)). There were no significant differences in anxiety or depression scores between the subtypes of IBS. Anxiety scores did not correlate with number of stools passed or days per week of bloating or pain.

Conclusions: Anxiety is common in patients presenting with IBS-type symptoms but this does not differentiate them from those with underlying organic disease. There is no significant relationship between severity of reported symptoms and anxiety.

316 INVESTIGATION OF THE REPRODUCIBILITY OF CEREBRAL ACTIVITY TO OESOPHAGEAL STIMULATION

S.J. Coen1, L.J. Gregory1,2, D. Hall2, L. Yaguez1, E. Amaro1, S. Smale3, S.C.R. Williams1, D.G. Thompson2, Q. Aziz2.1Institute of Psychiatry, London, UK; 2GI Sciences, University of Manchester, UK; 3King’s College Hospital, London, UK

Introduction: The cerebral activation during functional magnetic resonance imaging (fMRI) sessions can be influenced by many variables, including scanner noise, subject movement, and cognition, such as habituation and learning. Although the cerebral processing of visceral sensation has been studied, information regarding the trial re-trail reliability is not available.

Aims: The purpose of this study was to assess the reproducibility of the functional neural correlates of oesophageal sensation, using fMRI.

Methods: 7 healthy volunteers participated in the study. The protocol consisted of two conditions; non-painful (50% of the difference between sensory and pain thresholds) and painful (pain threshold) stimulation intensities using phasic balloon distension in the distal oesophagus. The order of presentation of these intensities was counterbalanced. A modified block design was employed for each intensity where “active” and “rest” phases were repeated five times. This experimental procedure was repeated on two additional occasions (three scans in total) to investigate the consistency of cerebral activation over time.

Results: In response to painful stimulation, highly reproducible activation was seen in the anterior cingulate gyrus (ACG) (BA24, 32), bilateral insula, supplementary motor cortex (SMA), thalamus, primary and secondary sensory cortices (SI&SII) and dorsolateral prefrontal cortex. Further analysis revealed a significant (p < 0.05) decrease in cerebral activity from the first to the final scan in the anterior cingulate (ACG), SMA and SI. Activation in response to non-painful stimulation was seen in similar regions to those seen during painful stimulation, with the exception of the thalamus, but was found to be more variable in the ACC, SI, SII, and SMA in comparison to painful stimulation.

Conclusions: Painful stimulation of the oesophagus produces robust activity in many brain regions previously associated with visceral pain. Non-painful stimulation results in a similar pattern of cerebral activation, but is variable between scan time, possibly suggesting that individuals are more likely to habituate to a non-painful stimulus, rather than a more salient painful stimulus.

317 EVIDENCE OF OESOPHAGEAL STIMULUS INTENSITY DEPENDANT RESPONSE IN THE HUMAN ANTERIOR CINGULATE AND PRIMARY SOMATOSENSORY CORTEX

S.J. Coen1, L, J. Gregory1,2, D. Hall2, L. Yaguez1, E. Amaro1, S. Smale3, S.C.R. Williams1, D.G. Thompson2, Q. Aziz2.1Institute of Psychiatry, London, UK; 2GI Sciences, University of Manchester, UK; 3King’s College Hospital, London, UK

Introduction: Functional Magnetic Resonance Imaging (fMRI) can be used to objectively quantify perception of visceral sensation. However, the neural correlates of varying, quantifiable, intensities of visceral stimulation have remain unclear, and the regions involved in encoding of stimulation intensity are not fully understood.

Aims: To determine the neural correlates of varying intensities of oesophageal stimulation using fMRI.

Methods: 7 healthy volunteers participated in the study. The protocol consisted of four conditions. During each condition one of four balloon distension intensities, obtained by dividing the difference between sensory (0%) and pain thresholds (100%) into 4 levels at 25% increments ie 25%, 50%, 75%, and 100% were used to stimulate the distal oesophagus. A modified block design was employed where each “active” and “rest” phase was repeated five times. This design was employed four times, once for each intensity. Behavioural data measuring the subjective perception of the stimulus was acquired using visual analogue scales (VAS).

Results: VAS scores increased progressively with increasing stimulation intensities (p < 0.001). In response to 100% and 75% stimulation intensity, activation was seen in the anterior cingulate gyrus (ACG) (BA 24, 32), bilateral insula, supplementary motor cortex (SMA), thalamus, primary and secondary sensory cortices (SI and SII) and dorsolateral prefrontal cortex (DLPFC). 50% and 25% intensity (non-painful) stimuli activated the same regions to a lesser extent, with the exception of the thalamus, and SII, and additional activation in the inferior frontal gyrus. Further analysis revealed that there was a significant trend (p < 0.05) of an increase in cerebral activity with an increase in stimulation intensity, in the ACG (BA24), and SI (bilateral).

Conclusions: Visceral stimulation results in a complex pattern of cerebral activation that is similar across varying levels of stimulation intensity. The ACG and SI, both show evidence of stimulus dependent response, which may be a result of encoding of stimulus intensity, unpleasantness of the stimulus, or levels of attention.

318 INCREASED EXPRESSION OF GALANIN IN MUCOSAL NERVES OF PATIENTS WITH PAINFUL DIVERTICULAR DISEASE

J. Simpson1, F. Sundler2, D. Jenkins3, R.C. Spiller4.1Department of Surgery, University Hospital, Nottingham, UK; 2Department of Physiological Sciences, Lund University, Sweden; 3Department of Histopathology, University Hospital, Nottingham, UK; 4Division of Gastroenterology, University Hospital, Nottingham, UK

Background: Galanin is a neuropeptide distributed widely throughout the central and peripheral nervous system. In particular, its presence has been demonstrated in dorsal root ganglia, spinal dorsal horn neurones and enteric nerves. It is known to have a modulatory function on nociception and peripheral nerve injury has been shown to upregulate its synthesis. We have previously shown that resection sections for complicated diverticular disease shown evidence of neural damage and regeneration within the enteric nervous system (ENS).

Aim: To quantify the level of galanin expressed by mucosal nerves in patients with symptomatic and asymptomatic diverticular disease.

Method: Ten symptomatic and ten asymptomatic patients underwent flexible sigmoidoscopy and multiple peridiverticula biopsies were obtained. Standard fluorescent immuno-histochemical methods were used. Mucosal nerves were identified using PGP9.5 and peripherin. Using digitised image analysis, the level of galanin immunoreactivity within mucosal nerves was expressed as percentage area of lamina propia.

Results: Median age was 68.5 years (range 49–70) with no difference between groups. Symptomatic patients experienced recurrent abdominal pain on a median of 6 (range 3–12) days/month. Duration of the pain was 4 (0.75–12) hours. No pain was experienced by the asymptomatic group. The percentage area of galanin immunoreactivity within the lamina propia was 0.155% in symptomatic patients compared with 0.004% in controls (p < 0.0001 Mann-Whitney).

Conclusion: There is an increased expression of galanin within colonic mucosal nerves in patients with painful diverticular disease. Although the direct effect of this change in the development of abdominal pain is as yet unclear, the increased expression implies previous or ongoing injury to the ENS in symptomatic patients, possibly due to inflammation.

Radiology posters 319–322

319 METHODOLOGICAL STUDY TO ASSESS REPEAT PLANAR WHITE CELL SCANNING IN MONITORING THE EFFICACY OF TREATMENT IN INFLAMMATORY BOWEL DISEASE

A. Poullis1, A.G. Irwin1, M. Dearing1, J. Gane1, A.J. Britten1, S. Heenan1, P. Soni2 W. Vennart2, J.D. Maxwell1.1St George’s Hospital, London, SW17, UK; 2Pfizer, UK

Introduction: Radio-labelled white cell scans (WCS) provide non-invasive and accurate quantification of inflammatory bowel disease (IBD) activity in both large and small bowel and may be useful in the objective evaluation of treatments for IBD. Clinical activity scores are partly subjective and do not accurately reflect underlying inflammation.

Aims: A methodological study to assess the efficacy of repeated planar WCS in monitoring the response to treatment of active IBD and comparing to clinical activity scores.

Patients: 9 subjects with active ulcerative colitis (UC) (5 mild, 3 moderate, 1 severe) and 9 subjects with active Crohn’s disease (CD) (5 mild, 3 moderate, 1 severe) were recruited.

Methods: 99 mTc-HMPAO WCS were carried out immediately before and 2 weeks after treatment. Prior to each scan disease activity scores for UC (Powell-Tuck index (PTI) and Mayo clinic score (MCS)) or CD (Crohn’s disease activity index (CDAI), Harvey Bradshaw Index (HBI) and van Hees activity index (VHAI)) were calculated. Scan scores for total bowel activity were calculated by using a validated visual grading system at 1 hour. Two independent observers scored each scan and the final score was agreed by consensus.

Results: In IBD subjects following anti-inflammatory treatment 11/18 (61%) of WCS improved, 4/18 (22%) remained unchanged and 3/18 (17%) deteriorated. In UC subjects there was agreement for direction of change in scan score and PTI in 4/9 (44%) and MCS in 3/9 (33%). In the CD subjects there was better agreement between WCS score and clinical scores (CDAI in 7/9 (78%); HBI in 5/9 (56%); VHAI in 5/9 (56%)).

Conclusion: Clinical scores in IBD agree poorly with objective measures of bowel inflammation, and while valuable in assessing the patients general well being have limited application in assessing the anti-inflammatory effect of therapeutic agents. Non-invasive white cell scanning is a more reliable method to monitor the anti-inflammatory efficacy of treatments for active IBD.

320 PERCEIVED PREOPERATIVE STAGE OF OESOPHAGEAL CANCER. PROSPECTIVE EVALUATION OF EUS AND SPECIAL INTEREST CT

G. Blackshaw1, S. Weaver1, P. Edwards1, W. Lewis1, M. Allison1, A. Roberts2, G. Thomas1.1Departments of Surgery and Radiology, Royal Gwent Hospital, Newport, Cardiff; 2The Department of Radiology, University Hospital of Wales, Cardiff

Aims: Endoluminal ultrasound (EUS) has been championed as the answer to the limitations of computed tomography (CT) in assessing tumour infiltration (T stage) and lymph node involvement (N stage) in patients with oesophageal cancer. However, the precise strength of agreement between EUS, special interest CT, and the true histopathological stage has not been reported.

Methods: Sixty consecutive patients (median age 61 year, 35 m, 41 adeno, 19 squamous cell) with oesophageal cancer were studied prospectively. Each patient underwent preoperative CT (Siemens Somatom +4) performed by the specialist multidisciplinary team consultant radiologist, and EUS (Olympus UM-20, and MH-908-I). The strength of the agreement between the perceived radiological stage and the true histopathological stage of the resected specimen was determined by the weighted Kappa statistic (Kw).

Results: When CT and EUS concurred, the accuracy of the radiological stage was enhanced twofold (T Kw 0.51, N Kw 0.80, p < 0.0001).

Conclusion: These data reinforce the fact that the modalities of CT and EUS are complimentary, and highlights the value of multidisciplinary teams in refining preoperative diagnoses and optimising the treatment of patients with oesophageal cancer.

Abstract 320

321 RENDEZVOUS PROCEDURE OR PTC AND METAL STENT FOR BILIARY OBSTRUCTION—IMPLICATIONS FOR PROCEDURAL COSTS AND DURATION OF INPATIENT STAY

G.R. MacFaul1, R. Matthialagan1, M. Gibson2, P. Torrie2, A.S. Mee2.1Department of Gastroenterology, 2Department of Radiology, Royal Berkshire Hospital, Reading, UK

Background and Aims: After failed endoscopic treatment for malignant biliary obstruction, non-surgical approaches include a rendezvous (combined) procedure (CP) incorporating percutaneous cholangiography (PTC) then ERCP, or initial PTC and insertion of a metal stent. We hypothesised that PTC and stenting would lead to reduced procedural costs, morbidity, and duration of inpatient stay.

Methods: Between 1994 and 1998 15 patients had a CP, and between 1998 and 2000 15 patients went onto PTC and stenting, following failed ERCP(s). Notes of both groups were analysed for diagnosis, duration of stay including delay from definitive procedure to discharge/death, and individual procedural costs.

Results: Full data were available on 14 patients in each group. The average length of stay from first ERCP to discharge or death was 11.2 days (5–26) in the CP group, with a delay from CP to discharge of 4.7 days (1–19). Similar durations in the PTC group were 10.4 days (2–34) and 5.6 days (2–12), respectively. Average procedural costs were 1752 with CP (5 patients required two ERCPs prior to the CP), and 1548 for PTC and stenting. Average age was 71.8 years (41–89) and 78.5 years (55–94), respectively. Obstruction was due to proven pancreatic cancer in 86% of the CP group and 50% of the PTC group. Cholangiocarcinoma was found in 4 patients (26%) who had a metal stent. 2 patients from the CP group died (at 2 and 8 days) and 1 from the PTC group (day 5 post-PTC).

Conclusion: PTC and metal stenting lead to decreased procedural costs and length of inpatient stay compared to the rendezvous procedure. It was also safely undertaken in a more elderly cohort of patients and we therefore suggest it should be the procedure of choice in the relief of malignant biliary obstruction after failed ERCP.

322 SEGMENTAL ASSESSMENT OF COLONIC TRANSIT STUDIES: AN INDICATOR OF EVACUATORY DISORDERS?

M.T. Eltringham, E. McCauley, I.M. Bain, J.Y. Yiannakou.University Hospital of North Durham, Durham, UK

Background: Colonic transit study is a widely used investigation in patients with chronic constipation. It has been suggested that it is possible to determine from the distribution of markers in various regions of the colon whether the patient has an evacuatory disorder or colonic inertia. The aim of the study is to assess whether this is the case.

Methods: 40 patients who attended a specialist constipation clinic were investigated using colonic transit studies and radionuclide proctography. The patients all had prolonged transit and were divided into 2 groups, normal or prolonged evacuation rate on the basis of the proctogram. The transit studies from the 2 groups were assessed by two different methods: number of markers in the left and right colon; and number of markers in 3 regions (right colon, proximal left colon and rectosigmoid).

Results: 17 patients had prolonged evacuation rates and 23 were normal. The mean values for numbers of transit study markers on the whole left colon (TSMLtotal), proximal left colon (TSNPL), and rectosigmoid (TSMRS) are shown in Table 1. The number of markers in the rectosigmoid is also expressed as a percentage of the total number of markers retained throughout the colon (RS%TC) and percentage of total left colonic markers (RS%L).

Abstract 322

Discussion: There is no significant difference between the abnormal and normal evacuation rate groups in terms of total numbers of left sided or rectosigmoid markers. This indicates that the segmental method of assessing radiological marker transit studies is no more accurate in indicating an evacuatory disorder than the left or right method.

Neoplasia posters 323–339

323 “THE 2 WEEK UPPER GI CANCER RULE.” HOW SUCCESSFUL IS THIS? A RETROSPECTIVE AUDIT IN A DISTRICT GENERAL HOSPITAL

R.J. Warner, D. Das.Department of Gastroenterology, Stepping Hill Hospital, Stockport, UK

Introduction: The NHS Executive introduced a 2 week standard for cancer referrals in an attempt to speed up diagnosis and improve patient care. This has now been implemented, but are we improving our care because of it, or is it just an additional headache for endoscopy units?

Aims: To assess how many referrals: 1) were endoscoped (OGD) within 2 weeks, 2) identified cancer, 3) for upper Gastrointestinal (GI) cancer came via our cancer office—the Bobby Moore Oncology Unit.

Methods: We performed a retrospective audit of all patients referred via the Bobby Moore unit in a 12 month period. Faxed referrals were made on a standard proforma that was distributed to all local GPs. Data were collected looking at referral information and OGD results.

Results: 146 referrals were received (age range 23–92), of which 144 (98.6%) were offered OGD within 2 weeks. 10 patients did not attend 1st appointment. 10/146 (6.8%) had cancer, 6 oesophageal, 2 gastric, 1 pharyngeal and 1 direct spread from transverse colon. Over the same period 61 oesophageal and 43 gastric cancers were identified via alternative routes, ie only 7.7% of upper GI cancers came on the cancer proforma. Recent onset dyspepsia in patients > 45 years as the only symptom (28/146) yielded 0 cancers, whereas 11% of patients with dysphagia had cancer.

Conclusions: We only detected 7.7% of upper GI cancers via our cancer proforma. Patients who did not attend in our study did not have cancer on subsequent OGD. Dyspepsia alone did not yield any cancers. Although offering this rapid access service we may be delaying OGDs for patients with cancer referred from other sources.

324 TAPE RECORDED ENHANCED OESOPHAGOGASTRIC CANCER CONSULTATIONS FACILITATE PATIENTS’ MEMORY AND PERCEPTION OF TREATMENT OPTIONS

G. Blackshaw, P. Edwards, C. Gent, M. Allison, W. Lewis.Royal Gwent Hospital, Newport, NP20 2UB, UK

Background: Consultations to convey a diagnosis of oesophagogastric cancer may be a difficult and traumatic time for patients. Indeed, as many as 50% of patients are displeased with the information received. One of the key recommendations of the Bristol report was the provision of audio tape recording facilities.

Aims: The aim of this study was to assess the value of tape recording the first consultation, to determine whether re-listening to the information given, might aid patients’ memory of key facts, and reduce anxiety prior to therapy.

Methods: Two groups of patients were studied: a historical control group of 15 patients (median age 66 years, 8 months, 5 oesophageal, 10 gastric cancers) who did not receive tape recordings of their consultations was compared with 10 consecutive patients (median age 68 years, 8 months, 6 oesophageal, 4 gastric cancers) who had their consultations tape recorded. All patients completed a hospital anxiety and depression (HAD) questionnaire 1–2 weeks after the consultation.

Results: Patients who had received tape recordings of their consultations were less likely to forget key facts regarding their diagnosis and treatment options (0 patients) compared with patients who had not received a tape (5 patients, χ2 = 4.167, df 1, p = 0.04). HAD questionnaire scores were also lower in patients who had received a tape compared with patients who had not (median (range) HAD A 5.5 (0–160) v 6 (2–14), HAD D 3.5 (0–13) v 4.5 (1–11), respectively, p = n.s.).

Conclusion: All patients who received a tape found that listening to the consultation again with relatives was helpful. Tape recorded enhanced interviews improved patients factual memory and in general reduced levels of anxiety prior to therapy.

325 AUDIT OF THE 2 WEEK RULE (TWR) FOR SUSPECTED UPPER GASTROINTESTINAL (UGI) CANCER AND THE PATHWAYS TO DIAGNOSIS

J Barbour, G. Leontiadis, A. Saeed, S. Kadis (introduced by N. P. Thompson).Department of Gastroenterology, Queen Elizabeth Hospital, Gateshead, UK

Aims: To audit TWR for UGI cancers in a north east district general hospital (DGH) and to determine the proportion of UGI cancers diagnosed outside the TWR referral system.

Methods: Prospective audit of all TWR over a 20 week period (Oct 2001 to March 2002) in our DGH (catchment population approximately 250 000). All upper GI cancers diagnosed over the same time period were also audited.

Results: 172 patients (mean age 62 years, range 17–89 years, 84 male, 88 female) were referred for suspected UGI cancer under the TWR referral system during the audit period. The mean delay from faxed referral to endoscopy was 11 days (range 1–30 days), with 86% of referrals assessed within 2 weeks. A total of 35 UGI cancers were diagnosed in our DGH during this period. Of those, 17 (49%) were diagnosed within the TWR referral system (mean delay 11 days, range 4–18) and 11 (31%) following emergency admission (mean delay from admission to endoscopy 5 days, range 1–12). Of the remaining 7 patients with UGI cancers, 2 were picked up by appropriate surveillance, 1 did not follow the TWR referral pathway due to an administrative error (delay 86 days) and 4 patients were referred by the GP outside TWR referral pathway despite having suspicious symptoms and were seen in the outpatient clinic first (mean delay from clinic to endoscopy 45 days, range 24–83, principally due to investigations requested prior to endoscopy).

Conclusion: 86% of referrals under the TWR system for UGI cancers in our hospital were assessed within 2 weeks. All cancer patients other than those diagnosed through surveillance had suspicious symptoms. Delay can be reduced by GPs and hospital consultants using the TWR referral system for all patients with suspicious symptoms.

326 IMPACT OF 2 WEEK WAIT REFERRAL ON THE MANAGEMENT OF UPPER GASTROINTESTINAL MALIGNANCY

M.S. Aung, J. Vuojic, S. Anwar, M.H. Shiwani (introduced by M. Hayat).Barnsley District General Hospital, Gawber Road, Barnsley, UK

Objective: To audit 2 week wait referral (TWR) for suspected upper gastrointestinal malignancy (UGIM) and also to evaluate whether this system identifies patients with suspicion of cancer at early stage of the disease.

Method: In a district general hospital all patients referred through TWR desk from September 2000 to December 2001 were studied. All newly diagnosed patients with UGIM referred through the conventional routes in the same period were included. Case notes of all the patients with proven UGIM were studied and data were collected.

Result: Of the 307 TWR only 29 (9.4%) patients were diagnosed with UGIM and the majority, 91.6 % patients, have alternative diagnosis. Total number of patients diagnosed with UGIM through TWR and conventional routes were 105. Of these 105 UGIM, the majority 76 (72%) patients were referred through conventional routes including A&E: 43(57 %), clinic: 20 (26%) and direct access endoscopy: 13 (17 %). Median appointment time, referral time to diagnosis, and waiting time to treatment were 12, 21, and 35 days, respectively for TWR group and 25, 34, and 55 days, respectively for conventional group. Staging of the disease and treatment offered was noted using various modalities (see Table).

Conclusion: TWR standards are achievable. This system generates more work but does not provide any benefit to diagnose disease at early stage, hence does not have any impact on long term survival.

Abstract 326

327 RAPID ACCESS UPPER GI CANCER SERVICE (RAUGICS): WORKLOAD, NON-ATTENDANCE, AND PREDICTIVE VALUE OF REFERRAL CRITERIA

N. Kapoor, A. Bassi, R. Sturgess, K. Bodger.Aintree Centre for Gastroenterology, University Hospital Aintree, Liverpool, UK

Background: The NHS 2 week rule requires rapid evaluation of patients with suspected upper GI cancer based on the presence of pre-determined alarm features. We report a single centre experience of implementing a RAUGICS in a university hospital serving a population of 330 000.

Methods: Details of all referrals to the RAUGICS were recorded prospectively on a dedicated database, including demographics, referral indications (from standard referral proforma), OGD results, and outcome. DNA rates (failure to attend) were also monitored. Accuracy of GP stated referral criteria were verified by self-administered symptom questionnaire in a sample of patients.

Results: Predictive value of referral criteria for cancer (18 month data; 1852 patients; mean age 59 years; cancer yield: 3.8%): Logistic regression analysis revealed that dysphagia (OR 3.1), weight loss (OR 2.6) and age > 55 years (OR 9.5) were significant predictors of cancer, whereas so-called “high-risk dyspepsia” had negative predictive value within this cohort (OR 0.1). Workload data (12 month period): 1207 patients were referred. All initial non-attenders were sent a 2nd appointment, hence 1462 slots (121% of referral numbers) were allocated. 1137 patients (94.2%) attended for OGD (74% being undertaken by nurse endoscopists). There were 53 cases of cancer (yield: 4.6%). Of 1030 patients who were allocated a follow up clinic appointment after OGD, 240 (23%) failed to attend. The presence of significant pathology at OGD did not influence subsequent clinic DNA rate (serious disease: 20% vs normal OGD: 24%). GP stated referral criteria: symptom questionnaires confirmed the presence of designated alarm features in 97% of patients (n = 65).

Conclusions: Despite effectively targeting rapid access diagnosis to a high risk population for cancer (4.6% prevalence), the high workload of our RAUGICS is compounded by a DNA rate of 21%, with similar levels of non-attendance at the follow up clinic. Application of narrower referral criteria for “2 week rule” investigation (eg exclusion of “high risk dyspepsia” alone), more selective targeting of patients for follow-up clinic, and strategies to reduce DNA rate may improve cost effectiveness while maintaining yield of cancer. As expected, most of the activity of RAUGICS involves dealing with benign conditions as the referral criteria have poor sensitivity and specificity for cancer.

328 ANGIOGENESIS IN RECTAL CANCER: CAN PREOPERATIVE ENDOSCOPIC BIOPSIES PREDICT TUMOUR BEHAVIOUR?

S. Chaudhri, K. Jain, H.M. Arthur, M.K. Bennett, A.F. Horgan.Departments of Colorectal Surgery and Pathology, Freeman Hospital and Institute of Human Genetics, Newcastle University, Newcastle-upon-Tyne, UK

Introduction: Preoperative radiotherapy is now the accepted treatment for a subgroup of patients with rectal cancer who are at high risk for local recurrence and poor survival. At present cross-sectional imaging is the only preoperative means of identifying this high risk subgroup. Rectal tumours with high microvessel density (MVD) in the resected specimen have been shown to be associated with poor survival. The aim of this study was to compare angiogenesis using MVD in preoperative endoscopic rectal cancer biopsy specimens (PERCBS) with the corresponding resected cancer specimen.

Methods: Immunohistochemistry using CD31 monoclonal antibody was undertaken on paraffin sections from preoperative biopsies and microvessel density was compared with corresponding resected tumours from 25 patients with rectal cancer. Statistical analysis was done using linear regression and paired t test.

Results: In patients who had not undergone radiotherapy prior to surgery there was a highly significant correlation (p < 0.0001; r2 = 0.812) between MVD in biopsies (range 35–231/μm2; median 118) and resected tumour specimens (range 27–177/μm2; median 112). MVD was significantly lower in post radiotherapy resected specimens compared with PERCBS (p < 0.0255).

Conclusion: Our results suggest that MVD in PERCBS is a reliable predictor of tumour MVD and could also provide useful prognostic tool with implication for planning of adjuvant of radiotherapy. Also in this study preoperative radiotherapy appears to downstage angiogenic activity as assessed by MVD, the significance of which will need further investigation.

329 GI SYMPTOMS DEVELOPING AFTER PELVIC RADIOTHERAPY REQUIRE GASTROENTEROLOGICAL REVIEW

H.J.N. Andreyev, Z. Amin, P. Blake, D. Dearnaley, D. Tait, P. Vlavianos.Imperial College, Chelsea & Westminster & Royal Marsden Hospitals, London, UK

Background: After pelvic radiotherapy, at least 30% patients develop permanent change in bowel habit affecting quality of life. Most are never referred to a gastroenterologist.

Aim and Methods: To analyse the cause for GI symptoms following pelvic radiotherapy we prospectively recorded all final diagnoses from a consecutive series of patients with symptoms starting at any time after their treatment referred over 24 months to a gastroenterology clinic.

Results: 167 patients have completed investigations. 51 women, median age 62 (22–81 years), 116 men, median age 70 (31–85 years). Radiotherapy ended 2.5 years (0–13) earlier in women and 2 years (0–21) in men. Primary tumours were urological (n = 117), gynaecological (n = 43), and gastrointestinal (n = 9). Reasons for referral included: rectal bleeding (n = 109), loose stool or incontinence (n = 69), tenesmus or discomfort (n = 33), change in habit (n = 18), vomiting (n = 13), or other (n = 4). After investigation, diagnoses could be grouped into 3 categories, definitely related, possibly related or clearly unrelated to previous radiotherapy. Diagnoses directly related included proctitis (n = 92), sigmoid ulceration (n = 2), and obstruction (n = 4). Findings possibly related included hyperplastic polyposis (n = 13), bacterial overgrowth (n = 7), squamous polyps (n = 3), fatty acid malabsorption (n = 2), pancreatic insufficiency (n = 2), bile salt malabsorption, collagenous colitis, and relapsed ulcerative colitis (n = 1 each). Diagnoses unrelated to radiotherapy included a new primary tumour (n = 6), relapse of the primary cancer (n = 7), adenomatous polyps (n = 19), diverticular disease (n = 37), hypothyroidism (n = 2), infection (n = 2), irritable bowel syndrome (n = 2), alcohol (n = 2), rectal mucosal prolapse, and bleeding piles (n = 1 each). In 2 patients, no diagnosis was made. Gastroenterological referral changed the diagnosis in 64% and allowed institution of curative therapy in 27%.

Conclusion: Untreatable radiation toxicity must not be assumed to be the cause for GI symptoms after pelvic radiotherapy. Evaluation of new GI symptoms arising after pelvic radiotherapy by an experienced gastroenterologist is mandatory as it allows diagnostic investigations to be performed and appropriate treatment to be instituted.

330 IS SURVIVING SIGNIFICANT IN THE P53 APOPTOTIC PATHWAY AFTER SHORT COURSE RADIOTHERAPY?

H. Ishikawa1,2, I.C. Talbot2.1First Department of Surgery, Nara Medical University, Kashihara, Japan; 2Academic Department of Pathology & ICRF, St. Mark’s Hospital, Harrow, UK

Aim: We have shown the significance of the p53 apoptotic pathway in short course radiotherapy. Recently, survivin, a novel anti-apoptotic gene, has been identified but its role in vivo is still unclear. We have examined the role of survivin with reference to apoptotic cell death and the expression of apoptosis regulating proteins in short course radiotherapy.

Methods: 26 patients underwent operation from June 1982 to October 1984 after short course radiotherapy (15Gy). Patients were aged between 27 and 77 years (median 57), 11 were male. Tumour stages were (Dukes) A (2), B (11), C (13). Sections of paired biopsies and post-irradiated surgical specimens of each tumour were immunohistochemically stained for p53, Bcl-2, BCL-XL, Bax, P21/WAF1, and survivin. The apoptotic index (AI) was the percentage of positive tumour cells using the TUNEL method.

Results: After radiotherapy, the average AI increased significantly (2.2 vs 7.5; p < 0.01). Survivin immunostaining was positive in 15/26 (57.7%) of biopsies and in 8/26 (30.8%) of surgical specimens. Bax immunostaining was positive in 3/26 (11.5%) of biopsies and in 15/26 (57.7%) of surgical specimens. There were no correlation of expression between survivin and other proteins in both biopsies and surgical specimens. In surgical specimens, the average AI of survivin-negative subgroup (n = 8) was higher than that of negative one (n = 18) (7.87 vs 6.7; p > 0.05). In biopsies the average AI was almost equal irrespective of survivin expression. However, in surgical specimens, the combination of p53-low/survivin-negative expression formed a group with the highest AI of all other subgroups.

Conclusion: Apoptotic cell death and down-regulation of survivin protein were induced by radiation in vivo. These results suggest that survivin has a role, possibly regulatory, in the p53-apoptotic pathway after short course preoperative radiotherapy.

331 LATE TOXICITY FOLLOWING SHORT COURSE PREOPERATIVE RADIOTHERAPY FOR OPERABLE RECTAL CANCER

M. King1, S. Tolan1, S. Giridharan1, C. McConkey2, A. Hartley1, J.I. Geh1 (introduced by M. Cox).1Cancer Centre, Queen Elizabeth Hospital, Birmingham B15 2TH, UK; 2Cancer Research UK Clinical Trials Unit, University of Birmingham, UK

Introduction: Short course preoperative radiotherapy (SCPRT) reduces local recurrence following total mesorectal excision (TME). In the Dutch TME trial the absolute reduction was 5.8% at 2 years (2.4% vs. 8.2%). This benefit should be weighed against acute and late side effects following combined treatment. We have previously reported acute toxicity in a series of 176 consecutive patients. The purpose of this study was to report late side effects in the same cohort of patients.

Method: The hospital notes of 176 patients who underwent TME preceded by SCPRT were examined. Side effects occurring more than 3 months after the start of SCPRT were graded using the EORTC/RTOG late radiation toxicity system. A multivariate analysis was performed to identify associated factors.

Results: Of 176 patients, 15 died within 3 months of SCPRT and 3 patients were lost to follow up. At a median follow up of 40 months severe (grade 3–4) toxicity was seen in 20 (13%) of 158 assessable patients (gastrointestinal 13 (8%); urological 3 (2%); thromboembolic 3 (2%); musculoskeletal 1 (1%)). On multivariate analysis, abdomino-perineal (AP) resection (p < 0.001) and Dukes A tumours (p = 0.05) were associated with less severe toxicity.

Conclusions: In this retrospective series the rate of late grade 3–4 toxicity following SCPRT and TME was 13%. Although AP resection is associated with a lower incidence of late physical side effects this may be counterbalanced by the impact of a stoma on quality of life. These factors should be considered when deciding on the most appropriate management for operable rectal cancers.

332 TNF α MODULATION OF E-CADHERIN IN AN EX VIVO MODEL OF BARRETT’S METAPLASIA

E. Harper1,2, R. Harrison1, B.T. Cooper2, J.A.Z. Jankowski1, R.T. Spychal2.1The Epithelial Laboratory, University of Birmingham, UK; 2City Hospital, Birmingham, UK and Digestive Diseases Centre, Leicester, UK

Introduction: The majority of oesophageal adenocarcinoma arises from Barrett’s metaplasia (BM) following a metaplasia-dysplasia-carcinoma sequence. E-cadherin expression is down regulated along this progression, and TNFα expression is increased. We have demonstrated in an intestinal cell culture model that TNFα stimulation results in down regulation of E-cadherin. We aim to investigate the modulation of E-cadherin by TNFα in an ex-vivo tissue culture model.

Methods: Specimens of Barrett’s metaplasia were obtained endoscopically. Each biopsy was divided, half for histology and half for tissue culture. Matched normal squamous and duodenal epithelium was also taken from each patient. Biopsies were divided and randomly assigned to culture with or without TNFα at 50 ng/ml by standard tissue explant methods. Specimens were then harvested at time points and processed for Western blotting. LDH levels in the culture media were used as a measure of viability, along with morphological assessment and immunohistochemistry (IHC) with Ki-67 antibody.

Results: Nine sets of biopsies were cultured. LDH assay, morphology and Ki-67 IHC confirmed viability at 18 hours. By 24 hours media LDH levels had increased and there was evidence of necrosis, with reduced Ki-67 expression indicating a reduction in cell division. E-cadherin protein expression as assessed by Western blotting did not show any evidence of down regulation in response to TNFα when compared to uncultured specimens or those cultured in TNFα free media. This was also true for normal squamous and duodenal mucosa.

Conclusion: We demonstrate the short life span of explant tissue samples ex vivo. In addition the effects of TNFα on E-cadherin expression are in contrast to our previous work. This may reflect the fact that BM has a TNFα rich microenvironment and that its receptors are already maximally stimulated. It is also possible that ex vivo TNFα does not modulate E-cadherin expression within the period of experimentation.

333 EXPRESSION OF C-KIT (CD117) IN NEUROENDOCRINE TUMOURS—A TARGET FOR THERAPY?

K. Khan, V. Kostoula, K. Savage, M. Stubbs, M. McStay, A.P. Dhillon, M.E. Caplin.

Royal Free and University College Medical School, London, UK

Introduction: Glivec (imatinib mesylate) is currently being used in clinical trials for the treatment of gastrointestinal stromal tumours and is known to specifically inhibit the tyrosine coupled receptor encoded by the c-kit proto-oncogene. Glivec is currently being assessed as a therapeutic possibility in other c-kit positive tumours.

Aim: To assess the expression of c-kit (CD117) in neuroendocrine tumours.

Methods: Immunohistochemistry was performed on paraffin embedded sections from 62 consecutive NET patients: 36 carcinoid, 16 pancreatic NET, 5 paraganglioma and 5 medullary carcinoma of thyroid. C-kit (CD117) polyclonal antibody (Dakocytomation) raised against synthesised c-kit peptide was used followed by a horseradish peroxidase detection step for immunohistochemistry. Appropiate negative controls were carried out simultaneously.

Results: 36% of carcinoids and 18% of pancreatic NET demonstrated expression of c-kit (CD117). No staining was observed on paragangliomas and medullary carcinoma of thyroid.

Conclusion: Immunohistochemical studies have demonstrated the presence of c-kit (CD117) in carcinoid and pancreatic neuroendocrine tumours, with the most expression observed in carcinoid. With limited treatment availability, Glivec may have therapeutic efficacy in the treatment of selected tumour patients.

334 GERMLINE MUTATIONS IN THE BASE EXCISION REPAIR GENE MYH—A NEW POLYPOSIS DISORDER

R. Davies1, N. Al-Tassan1, S. Jones1, N.H. Chmiel2, J. Maynard1, N. Fleming1, A.L. Livingston2, G.T. Williams1, A.K. Hodges1, S. Dolwani1, S.S. David2, J.P. Cheadle1, J.R. Sampson1.

1University Hospital of Wales, Cardiff, UK; 2Department of Chemistry, University of Utah, Salt Lake City, USA

Background: Multiple colorectal adenoma families have been identified that do not conform to classical FAP or HNPCC. We studied the somatic mutation profile of adenomas from affected siblings in one such family to establish the nature of their inherited defect.

Method: Family ‘N’ consisted of seven siblings, 1 with colon cancer at the age of 46, which was associated with adenomas. Screening his siblings identified two with approximately 50 adenomatous colorectal polyps at the ages of 59 and 55, respectively. 11 tumours from these 3 affected siblings were screened for somatic APC gene mutations.

Results: 18 somatic inactivating APC gene mutations were identified in these 11 tumours. Remarkably, 15 of these were G:C→T:A transversions. This type of mutation was significantly over-represented when compared to somatic mutations so far described in sporadic and FAP associated adenomas / adenocarcinomas. Similar mutations had previously been reported in DNA repair deficient yeast and bacteria. In E coli a series of enzymes function synergistically to protect cells from the deleterious effects of guanine oxidation. Analysis of the human homologue of mutY (MYH) showed that the affected siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These occur at sub-polymorphic frequency in the general population. Assays of glycosylase activity of the mutant proteins showed their activity was reduced significantly. This provided a link between inherited variants of MYH and the pattern of somatic APC mutation found in family N and implicated defective base excision repair in adenoma formation. Since the original report of family N, we have identified over 20 further families with multiple colorectal adenomas and biallelic germline MYH mutations, confirming the role of MYH in some families with colorectal polyposis.

Conclusion: Germline mutations of the base excision repair gene MYH are a cause of multiple colorectal adenomas and carcinoma.

335 VEGF-C EXPRESSION AND TUMOUR SPROUTING ARE USEFUL INDICATORS FOR NODAL INVOLVEMENT IN SUBMUCOSAL INVASIVE COLORECTAL CARCINOMAS

H. Ishikawa1, H. Fujii1, F. Koyama1, T. Mukougawa1, H. Matsumoto1, T. Kobayashi1, H. Kuniyasu2, Y. Nakajima1.

1First Department of Surgery, Nara Medical University, Kashihara, Japan; 2Oncological Pathology, Nara Medical University, Kashihara, Japan

Aim: It is difficult to estimate the adequacy of endoscopic resection for submucosal invasive colorectal carcinoma (SICC) because about 10% of patients with SICCs have nodal involvement. VEGF-C is suggested to have an important role for tumour lymphangiogenesis and tumour sprouting has regained attention as an indicator of lymphatic invasion (LI). To investigate risk factors of nodal involvement in SICC, we have examined the relationship between nodal involvement and tumour status of VEGF expression and tumour sprouting along with clinicopathological factors.

Methods: Sixty-eight consecutive patients with SICCs underwent either surgical or endoscopic resection between 1990 and 2001. Patients were aged between 38 and 80 years (median 65), 44 were male. 36 tumours were polypoid. Tumours were classified by the absolute amount of submucosal invasion both in their depth and width (depth; sm1 ≤ 0.5 mm, 0.5 < sm2 < 1.0 mm, 1.0 mm ≤ sm3, width; sma ≤ 1.0 mm, 1.0 < smb < 3.0 mm, 3.0 mm ≤ smc) and were immunohistochemically stained for VEGF-C and Cytokeratin 8/18.

Results: Of 68 tumours 7 had nodal involvement. Among clinocopathologocal factors, the ratio of nodal involvement of polypoid tumours (1/35) was lower than that of non-polypoid (6/35; p = 0.044). Tumours were divided into sm1(14), sm2(29), sm3(25) in the depth, sma(18), smb(27), smc(23) in the width. There was no nodal involvement in sm1 and sm2a(6). As tumour invasion increased in depth and width, positive cases of LI, venous invasion (VI) and nodal involvement increased. VEGF-C expression showed positive correlation with LI, VI and nodal involvement and tumour sprouting showed positive correlation with LI and nodal involvement. Both VEGF-C expression (6/7, 85.7%) and tumour sprouting (5/7, 71.4%) showed high specificity for nodal involvement.

Conclusion: VEGF-C expression and tumour sprouting are useful in predicting nodal involvement in SICC.

336 EXPRESSION AND UPTAKE OF PARATHYROID HORMONE RELATED PEPTIDE IN HEPATOCELLULAR CARCINOMA

M. McStay, K. Savage, M. Stubbs, K. Khan, A. Dhillon, M. Caplin.

Royal Free and University College Medical School, London, UK

Background: PTH-related protein (PTHrP) was identified in 1982 as the major factor responsible for humoral hypercalcaemia of malignancy (HHM). Since then, it has been found to be expressed in malignancies such as breast and prostate cancer, which are not commonly associated with HHM, and has been found to regulate their growth. Preliminary studies have suggested a role for PTHrP in hepatocellular carcinoma.

Aim: To assess the expression of PTHrP and its corresponding PTH/PTHrP type 1 receptor (PTH1R) in human hepatocellular carcinoma resection specimens and cell lines.

Methods: Immunocytochemical localisation of PTHrP and PTH1R was performed by the APAAP method on paraffin sections from 16 consecutive, pre-operatively eucalcaemic, patients with well-defined hepatocellular carcinoma, and on the hepatocellular carcinoma cell lines, PLC/PRF/5, HepG2 and MCA RH 7777. Murine monoclonal antibodies to PTHrP(1–10) and PTH1R were used, and specificity was demonstrated by pre-absorbance of antibodies with epitope. Western immunoblotting, using the same anti-PTHrP antibody, was used to assess the expression of PTHrP by the cell lines. PTHrP(1–10) peptide was labelled with Alexa Fluor 488 and incubated with the cell lines for 1 hour at 37°C. Cells were counterstained with fluorescent nuclear stain and examined under fluorescence microscope with appropriate filters.

Results: All of the tumour specimens and cell lines showed positive cytoplasmic staining for PTHrP and PTH1R. Variable staining for both peptide and receptor was seen in the background liver of the tumour specimens, but in 11 cases the staining was stronger in the tumour. Cellular PTHrP was detected by Western blotting in all of the cell line extracts. Uptake of labelled PTHrP was seen in some cells in each of the cell lines examined. The pattern of uptake was predominantly cytoplasmic, but nuclear localisation was also seen in some cells.

Conclusion: PTHrP and PTH1R are expressed by hepatocellular carcinoma tumour cells. This implies a possible autocrine/paracrine role for PTHrP that may regulate tumour growth and differentiation.

337 CARRIERS OF LKB1 MUTATIONS IN PEUTZ-JEGHERS SYNDROME ARE AT A HIGH RISK OF CANCER

W. Lim1, N. Hearle1, S.V. Hodgson2, R.K.S. Phillips3, R.S. Houlston1.

1Section of Cancer Genetics, Institute of Cancer Research, Sutton SM2 5NG, UK; 2Department of Clinical Genetics, Guy’s Hospital, London SE1 9RT, UK; 3Polyposis Registry, St. Mark’s Hospital, Watford Road, Harrow HA1 3UJ, UK

Introduction: Germline mutations in the LKB1 tumour suppressor gene cause Peutz-Jeghers syndrome (PJS) a rare dominant disorder. In addition to hamartomatous gastrointestinal polyps and pigmented perioral lesions, PJS is associated with an increased risk of tumours at multiple sites. Follow up information on carriers is limited. Genetic heterogeneity makes counselling and management in PJS difficult. Here we report the analysis of the LKB1 locus in 33 PJS families, and estimation of cancer risks in carriers and non-carriers.

Methods: Thirty-three index patients with PJS were ascertained from within United Kingdom. Clinical information including details of any cancer in first- and second-degree relatives was collected on all patients. There was no selection of cases for a family history of cancer. Conformation sensitive gel electrophoresis was used for the initial screen of LKB1 mutations, followed by direct sequence analysis for mutational characterisation. Estimates of cancer risks were obtained from survival analyses and standardised mortality ratios using life table methods.

Results: Germline mutations of LKB1 were identified in 52% of cases. This observation reinforces the hypothesis of a second PJS locus. In carriers of LKB1 mutations the risk of cancer was markedly elevated. The risk of developing any cancer in carriers by age 65 was 47% (95% CI: 27 to 73%) with elevated risks of both gastrointestinal and breast cancer.

Conclusion: PJS with germline mutations in LKB1 are at a very high relative and absolute risk of multiple gastrointestinal and non-gastrointestinal cancers. To obtain precise estimates of cancer risk associated with PJS requires further studies of genotype-phenotype especially with respect to LKB1 negative cases as this group is likely to be heterogeneous.

338 EFFECT OF FIBRE DIET ON NEOPLASIA; CELL PROLIFERATION AND CRYPT FISSION IN MIN MICE

O. Bashir1, A.J. FitzGerald2, R.A, Goodlad2.

1Gastroenterology Department, Imperial College Faculty of Medicine, Hammersmith Hospital, London, UK; 2Histopathology Unit, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

Background: The precise nature and magnitude of the relationship between specific fibre intake and risk of colorectal cancer have not been clearly established. Some studies have shown increased risk rather than protection in man and in animals. One possible explanation is that dietary fibre can modify the process of crypt fission, by which crypts can duplicate themselves, and increase cell proliferation which, can be a promoter of carcinogenesis.

Methods: We have used the Min mouse (C57BK/6J-ApcMin) to investigate the actions of a chow diet, a fibre-free semisythetic diet and semi-synthetic diets supplemented with apple fibre or wheat bran fibre on polyp progression, cell proliferation and crypt fission. Forty Min mice, 4 weeks old, were divided into 4 groups and fed the four diets for 8 weeks. The number and size of polyps in the small and large intestines were scored as were the number of native mitoses and the percentage of branching crypts.

Results: The guts of Min mice fed the fibre-free semi-synthetic (SS) diets were lighter than those of the chow fed. There were fewer polyps in the semisythetic diet compared to the chow fed (p < 0.050). There were more polyps in small intestine of the Min mice fed apple fibre (p = 0.07) and bran (p = 0.014). In the colon there were 50% more polyps in the apple fibre group (NS) and 188% more in the bran fed. There was a significant increase in cell proliferation in the colon of the apple fed group with little change in colonic crypt fission. Crypt fission in the small intestine was significantly decreased by the apple and bran fibres (p < 0.05).

Conclusion: Both apple fibre and bran were associated with increased polyp number in the small intestine and colon but the actions of altered crypt fission are still not clear.

339 ANALYSIS OF BILE IN PATIENTS WITH AND WITHOUT PANCREATICOBILIARY MALIGNANCY BY IN VITRO 31-PHOSPHORUS NMR SPECTROSCOPY

S.A. Khan1, I.J. Cox2, D. Bansi3, A. Thillainayagam3, H.C. Thomas1, S.D. Taylor-Robinson1,2.

1Liver Unit, St Mary’s Campus; 2Department of Imaging Sciences, Hammersmith Campus; 3Gastroenterology Unit, Hammersmith and Charing Cross Campuses, Faculty of Medicine, Imperial College, London, UK

Background: Mortality rates for cholangiocarcinoma have been widely reported to be increasing. In vitro phosphorus (31P) NMR spectroscopy can be used to analyse body fluids for metabolite abnormalities and the presence of xenobiotics. Several NMR spectroscopy studies on organs and tissue extracts have highlighted differences in the ratio of phospholipid metabolites (phosphodiesters, PDE and phosphomonoesters, PME) between individuals with and without cancer. Decreases in resonances contributing to the PDE region are typically seen in malignancy, often with increases in resonances from the PME region, reflecting increasingly rapid cell turnover of tumour cells. Such a study of has not previously been performed on bile.

Methods:31P NMR spectra were obtained from bile samples from 24 patients, collected at ERCP. 13 patients had an underlying malignancy: pancreatic carcinoma, cholangiocarcinoma, or metastatic liver disease. 11 cases had non-malignant pathology.

Results: A combination of a reduced glycerophosphorylcholine (GPC) signal from the PDE region with the presence of a resonance from the PME region, was seen in bile in 38% of cancer patients, but in only 25% of non-cancer patients. A low or absent GPC signal level was present in 77% of samples from cancer and only 50% of non-cancer patients.

Conclusion: Although the difference in PME/PDE resonances between cancer and non-cancer patients did not reach statistical significance, the patterns found are consistent with previous NMR studies of changes in PME/PDE resonances and their constituent components in organs and tissue extracts. This is the first study to show that 31P NMR spectroscopy of bile can potentially be used to assess differences in phospholipid content between cancer and non-cancer patients. This has implications for the development of novel diagnostic and prognostic strategies.

Colorectal posters 340–357

340 RENAL TUBULAR PROTEINURIA IN PATIENTS WITH IRRITABLE BOWEL SYNDROME

A.J. O’Brien1, E.J. Lamb2, A.F. Muller1.

1Department of Gastroenterology; 2Department of Clinical Biochemistry, Kent and Canterbury Hospital, Canterbury, Kent, UK

Introduction: The irritable bowel syndrome (IBS) is a heterogeneous disorder involving genetics, infection, mucosal inflammation, and visceral hypersensitivity.1 We have previously demonstrated tubular proteinuria in patients with inflammatory bowel disease (IBD).2 This study examined whether tubular proteinuria may be a feature of IBS.

Methods: 53 control subjects (age range 20–65 years) and 21 patients with IBS (M:F 9:12 age range 16–64 years (NS)) were recruited. Subjects with known renal disease, hypertension, diabetes, or microbiological evidence of urinary infection were excluded. The IBS group patients all fulfilled the Rome II criteria for diagnosis. None gave a history of preceding gastroenteritis. Many patients underwent radiological or endoscopic evaluation. All subjects provided the second voided urine of the morning. Urinary concentrations of the protein α1-microglobulin (α1-M) were measured using rate nephelometric immunoassay and corrected for urinary concentration by measurement of creatinine (upper reference limit 1.5 mg/mmol). Blood samples were analysed for biochemical and haematological indices including C-reactive protein. Statistical analysis was by unpaired T-test.

Results: None of the IBS patients over a 3 year follow up period were reclassified with IBD. All had normal haematochemical parameters. Mean (+/- SD) urinary α1-M concentrations were significantly higher in IBS than controls (IBS (1.17 +/- 0.65 mg/mmol; controls 0.75 +/- 0.36 mg/mmol, p <0.01) and exceeded 1.5 mg/mmol in 7 patients.

Conclusions: Urinary α1-M concentration is elevated in IBS, suggesting the presence of renal tubular injury.

  1. Talley NJ, Spiller RC. Lancet 2002;360:555–64.

  2. Fraser JS et al. Aliment Pharmacol Ther 2001;15:1131–37.

341 CLOSTRIDIUM DIFFICILE ASSOCIATED DIARRHOEA (CDAD), ONSET IN THE COMMUNITY AND HOSPITAL

S.S. Johal, J. Hammond, Y.R. Mahida.

Division of Gastroenterology, University Hospital, Queens Medical Centre, Nottingham, UK

CDAD is usually considered to be a problem that develops in patients following hospitalisation for another condition that requires administration of antibiotics. Little is known about patients that require admission to hospital following the onset of CDAD in the community. We have compared patients that developed CDAD following admission to hospital with those in whom the diarrhoea started in the community.

Method: Patients admitted to hospital or developing diarrhoea as inpatients were studied. CDAD was diagnosed in the presence of diarrhoea (> 3 liquid motions in 24 h) and either positive stool C difficile cytotoxin test or pseudomembranes were seen at sigmoidoscopy and the diarrhoea responded to vancomycin or metronidazole.

Results: Over a period of 30 months, 136 patients with CDAD (50 male and 86 female) of median age 82 year (range 24 – 97 year) were studied, of which 131 (96%) had taken broad spectrum antibiotic(s) prior to the onset of diarrhoea. In 38 (28%) diarrhoea developed in the community and in 98 (72%) diarrhoea developed while inpatient. Of the patients admitted with diarrhoea, 33 (86.8%) had been hospitalised over the preceding 12 months compared with 56 (57.1%) that developed diarrhoea as inpatient (p < 0.001). Prior to treatment, patients who developed diarrhoea in the community had a higher daily stool frequency (7.2 (±0.8) vs 4.6 (±0.2), p < 0.001) and shorter period of hospitalisation (23 (±3) vs 49 (±4) days, p < 0.001), but there was no difference in age, the duration of diarrhoea, CRP, or WBC. Of the patients who developed diarrhoea in the community, 21 (55.3%) were admitted from their own home with 23 (60.5%) returning there on discharge. Respective figures for patients that developed CDAD in hospital: 68 (69.4%) and 30 (30.6%, p < 0.001).

Conclusions: Of hospitalised patients with CDAD, a significant proportion developed the disease in the community. A majority of these had been hospitalised over the preceding 12 months, raising the possibility of acquisition of toxigenic C difficile during a previous hospital admission, with the development of CDAD following the administration of antibiotics in the community.

342 RAPID DIAGNOSIS OF GI INFECTION FROM FLATUS

C.S. Probert1, P. Jones2, N. Ratcliffe2.

1University of Bristol, UK; 2UWE, Bristol, UK

Clostridium difficile affects ca.15 000 people pa in England and Wales and results in 2100 bed days lost per DGH pa costs. Part of the problem is the delay in treatment—8.2 days in a recent series. Small round structured virus (SRSV/Norwalk) outbreaks result in ward closures. Early diagnosis will facilitate treatment or isolation.

We have undertaking a study of the volatile compounds in stool of patients with diarrhoea (38) and healthy controls (6). Using a novel solid phase microextraction technique linked to gas chromatography and mass spectroscopy, we have characterised the key volatile compounds in normal stool and diarrhoea due to C difficile, SRSV, rotavirus, and Campylobacter jejuni. Diagnostically useful volatiles were: indole, 3-methylindole (3MI), 2-furancarbox-aldehyde (2FC), 5-methyl-2-furancarboxaldehyde (5M2FC), ethyl dodecanoate (EDD), ammonia (Amm), the terpenes, and hydrocarbons (HC). The strength of association is shown using χ2 test, sensitivity and specificity, positive predictive value (PPV) and negative predictive values (NPV). Grouping some compounds increased their diagnostic value.

These observations suggest that volatiles from stool samples could be used to rapidly diagnosis the cause of infectious diarrhoea. A device based on a portable MS machine could be used or an e-nose built. Our observation may lead to near patient testing on wards or in the field.

Abstract 342

343 FAECAL CALPROTECTIN LEVELS ARE ALTERED IN NEONATED WITH ACUTE NECROTISING ENTEROCOLITIS

D. Carroll1, R.D. Spicer1, P. Cairns2, A. Corfield3.

1Department of Paediatric Surgery, Bristol Children’s Hospital, UK; 2Institute of Child Health, St Michael’s Hospital, UK; 3Division of Medicine, University of Bristol, UK

Introduction: Faecal calprotectin levels are known to be markedly elevated in children and adults with inflammatory bowel disease. Faecal calprotectin levels are an indirect marker of gastrointestinal inflammation. Necrotising entercolitis (NEC) is a severe condition of neonatal like characterised by gastrointestinal inflammation and ischaemia. Optimal management for this condition relies upon prompt recognition and early institution of conservative treatment. This study aims to evaluate the potential usefulness of faecal calprotectin levels to diagnose NEC.

Methods: Stool samples were collected from neonates with written parental consent on D1, D7 and D14 of life. Any children with suspected NEC had additional stool samples collected. Feed type, gestational age, and postnatal age were recorded. Stool samples were stored frozen at −20°C and analysed using the PhiCal ELISA based kit. Faecal calprotectin levels for each patient were calculated against a standard curve run with each assay. Statistical analysis of data was performed using SPSS for Windows version 10.

Results: The presence of NEC is associated with a statistically significant increase in faecal calprotecitn levels compared to matched controls (p < 0.001). Faecal calprotectin levels in infants are considerably higher than the reported adult reference range. This is most marked in stool samples collected on the first day of life. Faecal calprotectin levels fall as postnatal age increases.

Conclusions: Faecal calprotectin levels are a useful tool for diagnosing NEC in infants. Faecal calprotectin levels are significantly higher in neonatal life than in healthy adult controls.

344 L-ERYTHRO METHOXAMINE IS MORE POTENT AT INDUCING PORCINE INTERNAL ANAL SPHINCTER (IAS) CONTRACTION IN VITRO COMPARED WITH PHENYLEPHRINE: IMPLICATIONS FOR TOPICAL THERAPIES FOR FAECAL INCONTINENCE

O.M. Jones, J. Thompson, A.F. Brading, N.J.M. Mortensen.

Departments of Colorectal Surgery and Pharmacology, University of Oxford, UK

Introduction: Topical phenylephrine (an alpha-1 adrenoceptor agonist) increases resting anal canal pressure in normal and incontinent subjects, but requires high concentrations of gel, around 10–40%. This can cause local side effects, including perianal burning. This study examined if methoxamine might be a more potent alternative to phenylephrine.

Methods: IAS tissue was cut into strips, suspended in a superfusion organ bath, and allowed to equilibrate. Strips were subjected to drugs under testing for 20 seconds, sufficient to obtain stable tone. Phenylephrine, methoxamine racemate, and its four stereoisomers were all evaluated, and concentration-response curves constructed.

Results: In vitro, methoxamine racemate (1:1:1:1 ratio of its four isomers) and phenylephrine both caused contraction of IAS strips, blocked by phentolamine, an alpha-adrenoceptor antagonist. EC50 values for the two drugs were comparable (74.7±16.5 mM vs 58.3±13.4 mM, respectively; p > 0.05). However, when methoxamine racemate was separated into its four constituent isomers by chromatography, L-erythro methoxamine was significantly more potent than the other three isomers (EC50 17.6±3.7 mM; p < 0.01). Furthermore, this was also more potent than methoxamine racemate and phenylephrine (p < 0.01). This stereoisomer has been synthesised by a novel chemical synthetic pathway, which retains the potency of the chromatographically separated fraction (EC50 10.5±2.0 mM).

Conclusion: L-erythro methoxamine is a possible treatment for incontinence and is four times more potent than phenylephrine. This compound has potential to produce more substantial rises in anal resting pressure with fewer local side effects. Further trials are underway examining the efficacy of L-erythro methoxamine in vivo.

345 ACUTE APPENDICITIS—A DISEASE IN DECLINE: A STUDY OF HOSPITAL ADMISSIONS IN ENGLAND

A. Dhar1, J. Hoare2, A. Majeed3, R.C.N. Williamson4, J.D. Maxwell1, J.Y. Kang1.

1St George’s Hospital, London, UK; 2Office for National Statistics, UK; 3University College, London, UK; 4Hammersmith Hospital, London, UK

Background: The incidence of acute appendicitis declined in Western countries from the 1930s to the 1980s. However, it has been argued that this could in part be due to some patients being reclassified as having non-specific abdominal pain (NSAP) or mesenteric lymphadenitis (ML).

Aim: To determine time trends in hospital admissions for acute appendicitis in England from 1989/90 to 1999/2000.

Methods: Hospital episode statistics for admissions were obtained from the Department of Health.

Results: Between 1989/90 and 1999/2000, age-standardised hospital admission rated for acute appendicitis decreased by 12% for males and 19% for females. The admission rates for ML decreased by 40% and 41%, respectively. Coding for NSAP was changed in 1995/1996. From 1989/90 to 1994/1995, NSAP admissions increased by 4.7% for males and 4.9% for females. Over the same period admissions for acute appendicitis decreased by 8.3% for males and 11.4% for females. The increase in NSAP admissions occurred among subjects aged > 34 years, with a decline among those aged 5–19 years. There was a decline in appendicitis among all age groups, especially marked for those aged < 25 and > 84 years.

Conclusions: Admission rates for acute appendicitis have continued to fall in the 1990s. This fall cannot be accounted for by an increased tendency to diagnose ML or NSAP.

Abstract 345

346 COST COMPARISON OF TREATING EARLY AND LATE COLORECTAL NEOPLASIA

S. Ahmad, E. Phillips, K. Vellacott (introduced by M. Denyer).

Department of Colorectal Surgery, Royal Gwent Hospital, Newport, UK

The objective of this paper is to compare the cost of treating early colorectal neoplasia, to that of late neoplasia with an attempt to show that treating early colorectal neoplasia is more economical in comparison to late colorectal neoplasia. While we are debating the most effective method of screening in order to improve survival, very few people have explored the economical aspects of treating colorectal cancer (CRC) at different stages of its progression. In current literature there is no evidence to suggest that treating early CRC is more cost economical. The majority of the medical literature concerning the treatment of CRC concentrates on the best available options in terms of patient outcome rather than the cost incurred. While this should inevitably be the case, it is important that in an increasingly cost conscious health service, the price of the treatment must also be a consideration. A retrospective analysis of 471 patients over a 3 year period was performed by calculating the cost of inaugural treatment and the subsequent management during a 5 year follow up phase or until the death of the patient with CRC or polyps. Among these, 304 had adenomatous polyps or early carcinoma (adenomas, Duke’s A and B) and 167 had late stage cancer (Duke’s C, D and T4). The median cost of treatment and follow up was significantly lower for early than late stage colorectal neoplasia (10 607 and 13 891, respectively, Mann-Whitney U test, p = 0.0002). The main contributors to the high cost of the late stage cancer were oncological treatment, more admissions with complications or local recurrences, non-clinical cost (eg stoma care, palliative care), and in certain cases, oncological surgery. In view of these findings, early detection is certainly the desired goal. The growing popularity of screening is justified considering it would be both instrumental in decreasing the mortality as well as the cost of treatment of CRC.

347 THE USE OF METALLIC STENTS IN LARGE BOWEL OBSTRUCTION

W.-K. Syn, M.M. Ahmed.

Good Hope Hospital, Sutton Coldfield, Birmingham, B75 7RR, UK

Introduction: There has been an expansion in the use of metallic stents in the management of large bowel obstruction.

Aims: To evaluate results of colonic stent placement in our centre.

Methods: We reviewed all patients who underwent colonic stent placement at our hospital between March 2000 and August 2002. Stenting was performed under fluoroscopic and endoscopic guidance. Enteral Wallstents (6 cm or 9 cm long × 20–22 mm diameter, uncovered, Boston Scientific) were used in the majority. All patients had intravenous midazolam (mean dose 4.7 mg).

Results: There were 13 patients (7 female, mean age 74.4 years, range: 42–92 year) undergoing 14 procedures. All patients presented with large bowel obstruction. The sites of obstruction were rectum (n = 1), rectosigmoid (n = 9), descending colon (n = 2), and ascending colon (n = 1). 9/13 patients had adenocarcinoma, the others had colonic obstruction due to ovarian cancer (2), Non-Hodgkins Lymphoma (1), and diverticular stricture (1). The mean length of the obstructing lesion was 6.2 cm (range 3–14.5 cm). Technical success was achieved in 12 stenting attempts. One patient had 2 stents placed simultaneously for a long rectosigmoid tumour, another had 2 successive stents (3 months apart) for recurrent tumour. Of these 12 technical successes, 10 had sustained clinical and radiological improvement. One patient had early bleeding (requiring transfusion) and subsequent stent migration. 6/13 patients also underwent adjuvant chemoradiotherapy. In the 10 patients with technical and clinical success, stenting was the definitive palliative procedure. The length of hospital stay was 1–5 days. Mean survival was 128 days in those who received adjuvant chemoradiotherapy and 40 days in those who did not. The remaining 4 with technical or clinical failure underwent emergency surgical decompression.

Conclusion: Metallic stents are effective, safe and minimise hospital stay. Stenting obviates the need for emergency surgery in a group of generally elderly patients with significant co-morbidity.

348 MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1/CCL2) AND THE MACROPHAGE INFILTRATE ASSOCIATED WITH HUMAN COLORECTAL CANCER

C. Bailey, R. Negus, D. Peck, A. Darzi.

Academic Surgical Unit, Imperial College of Science, Technology and Medicine, St Mary’s Campus, Praed Street, Paddington, London W2 1NY

The chemokines are a group of more than 40 chemotactic cytokines that are responsible for leukocyte trafficking under normal and pathological conditions. They are small molecules (∼70 kDa) that show structural similarities both at the primary and tertiary levels. In particular they are characterised by conserved amino terminal cysteine residues that may be single (C), adjacent (CC) or separated by another amino acid (CXC). Chemokine signalling is mediated via serpentine receptors. Many human tumours contain a leukocyte infiltrate, including colorectal cancer, of which macrophages may form a significant part. In the 1980s and 1990s a variety of tumours were found to express chemokines that may account for the type and distribution of these infiltrates. In particular another epithelial cancer, ovarian, was found to express and produce monocyte chemoattractant protein-1 (MCP-1, CCL2), a potent monocyte/macrophage chemoattractant. MCP-1/CCL2 was expressed predominantly by tumour cells but also by some infiltrating macrophages. We have examined MCP-1/CCL2 production in human colorectal cell lines by specific ELISA and have found constitutive or inducible expression (in response to TNF-α) in 2/3. MCP-1/CCL2 expression has also been examined in RNA extracted from whole tumours and was present in 7/7. In the cell line that did not express MCP-1/CCL2, HT-29, monocyte chemoattractant activity was still present when supernatants were tested using the monocytic cell line THP-1 in Boyden chamber migration assays. We have demonstrated that MCP-1/CCL2 can be produced by colorectal cell lines and is expressed by tumours. MCP-1/CCL2 may therefore be involved in determining both the type and distribution of the leukocyte infiltrate in these malignancies.

349 DIVERTICULAR DISEASE: RACIAL DIFFERENCES IN PREVALENCE

A. Dhar1, R.J. Leicester2, M.J. Benson1, D. Kumar2, D. Melville2, P. Neild1, C.J. Tibbs1, J.D. Maxwell1, J.Y. Kang1.

1Department of Gastroenterology; 2Department of Colorectal Surgery, St Georges Hospital, London SW17 0QT, UK

Introduction: Diverticular disease (DD) is said to be uncommon in Africans and in Asians.

Aim: To determine if patients of Indian subcontinent racial origin (ISC Asians) have a lower prevalence of DD compared to other patients (non-Asians).

Method: All colonoscopies performed at St George’s Hospital from October 1999 to October 2002 were studied. ISC Asians were identified by name.

Results: 3013 patients were included. There were 245 ISC Asians and 2768 non-Asians. ISC Asians were younger and more likely to be male (mean age 55.1 years, M:F = 1:0.7) compared to non-Asians (mean age 60.1 years, M:F = 1:1.2). ISC Asians and non-Asians with DD had similar sex ratios (1:1.2 and 1:1.1, respectively). Indications for colonoscopy were similar in the two groups.

Conclusions: There is a lower prevalence of DD among ISC Asians presenting for colonoscopy, compared to non-Asians. This cannot be explained by sex or age differences. If confirmed, our findings may provide opportunities for research into the pathogenesis of DD.

Abstract 349

350 COLONIC MICROFLORA METABOLITES ALTER THE PROTECTIVE CAPACITY OF THE COLONIC MUCUS LAYER IN VIVO

I.A. Brownlee1, M.E. Havler2, P.W. Dettmar2, A. Allen1, J.P. Pearson1.

1Cell & Molecular Biosciences, Newcastle University, NE2 4HH, UK; 2Reckitt Benckiser Healthcare (UK) Ltd, Hull, HU8 7DS, UK

This study aimed to determine the effects of reactive oxygen species (ROS) and butyrate on the protective capacity of the colonic mucus layer in vivo. An intravital microscopy technique was used to measure the maximal thickness and replenishment rates (rate of mucus secretion after partial removal of the layer by suction) of the colonic mucus layer of anaesthetised male Wistar rats (n = 5 for each treatment) over 5 h under 7 mM butyrate (normal concentration in the rat colon), as well as ROS solutions containing 0.5 mM Fe++/EDTA mixed with 5–50 mM H2O2. These effects were compared to saline controls (n = 5). Maximal mucus thickness was not affected by 7 mM butyrate or 10 mM H2O2, but 5 mM H2O2 significantly increased maximal mucus thickness, whereas ROS solutions containing more than 10 mM peroxide (ie 17.5, 25, and 50 mM) significantly reduced the thickness of the mucus barrier (50% in the case of 50 mM H2O2). 7 mM butyrate significantly increased mucus layer replenishment rate over the first hour of measurement (×3 faster than the saline control. Measurement of the effect of ROS on colonic mucus layer replenishment was hampered by release of plasma exudate and red blood cells from the colonic mucosa. This suggests that low millimolar concentrations of butyrate and ROS may increase the protective capacity of the colonic mucus layer, possibly as a cytoprotective response. Higher levels of ROS appear to be damaging to both the mucus layer and mucosa, especially once the mucus layer is depleted. This study shows that bacterial metabolites may alter the protective potential of the colonic mucus barrier.

351 THE RISK OF DYSPLASIA IN POST-SURGICAL COLORECTAL MUCOSA IN ULCERATIVE COLITIS

M.D. Rutter, B.P. Saunders.

Wolfson Unit for Endoscopy, St Mark’s Hospital, Harrow, UK

Background and Aim: Although panproctocolectomy (PPC) is the operation of choice in longstanding extensive ulcerative colitis (UC), other procedures are occasionally chosen, leaving the patient at risk of dysplasia/cancer in the colorectal remnant. We aimed to assess this risk.

Methods: Operative details of all patients in a major UC surveillance programme were reviewed. All patients undergoing surgery other than PPC were studied. Follow up data were obtained from our prospective surveillance database, case notes, colonoscopy and histology reports, GPs, other hospitals, and the Office of National Statistics. Details included indication for surgery, surgical procedure, and post-surgical outcome.

Results: Twenty-one patients were studied (11 male, 10 female). Mean post-surgical follow-up was 6.3 years. Median age at operation was 50 (range 28–80). Median duration of colitis was 21 years (range 9–52). Indication for surgery was dysplasia (3), cancer (7), and symptomatic colitis/bleeding (11; surgical specimen showing cancer in 1, dysplasia in 2, and no dysplasia in 8). Nine patients had colectomy with ileostomy, 8 patients colectomy with ileorectal anastomosis, and 4 had segmental resection of neoplasia. Eleven patients remained well with no dysplasia. Four underwent surgical resection of the rectal remnant due to symptoms, no dysplasia being found. Six patients died: one from metastatic progression of initial Dukes’ C cancer, and 5 from unrelated causes, one of whom had had post-operative rectal high grade dysplasia but had refused further surgery. This solitary patient with post-operative dysplasia had originally undergone ileorectal anastomosis for high grade dysplasia in the sigmoid, but had also had rectal high grade dysplasia 2 years previously.

Conclusion: Patients with ulcerative colitis who retain part of the colorectum post-operatively remain at risk of cancer; however this study suggests the risk is low, even in patients with previous colorectal neoplasia.

352 HISTOLOGICAL PREDICTORS OF SURVIVAL IN RECTAL CANCER IN THE SHORT TERM

M.T.P.R. Perera1, J. Hewawisenthi2, A. Pathmeshwaran3, K.I. Deen1.

1University Department of Surgery, Colombo North Teaching Hospital, Ragama; 2Department of Pathology; 3Department of Community Medicine, University of Kelaniya, Sri Lanka

Introduction: Rectal cancer is associated with poor overall survival at 05 years. We aimed to explore the histological features associated with poor survival in the short term, in patients having an operation for rectal cancer between March 1995 and September 2002.

Patients and Methods: 84 patients (40 male, 44 female, median age 55 years, range 22–85) with rectal cancer situated (median, range) 6 cms, (1–15) from the anal verge underwent anterior resection (n = 60), abdomino-perineal resection/total reconstruction (n = 7), Hartmann operation (n = 3), restorative proctocolectomy (n = 7), and subtotal colectomy (n = 7). All specimens were evaluated by a single pathologist after haematoxylin and eosin staining of paraffin sections. The following data were obtained: TNM stage, serosal involvement by tumour, involvement of resection margins—radial, mesorectal excision, and distal resection, angioinvasion, lymphatic, and perineural involvement. The association between histology and survival was evaluated by Cox’s regression and Kaplan Meier analysis.

Results: The median follow up was 18 months (range 5–86 months). Overall operative mortality was 4 (4.7%). Cancer related mortality has been 26 (31%) of 84. Independent predictors of poor survival were; T stage (p = 0.028 Cox regression) and presence of metastasis (p = 0.02). Nodal involvement alone was not an independent predictor of survival (N0, p = 0.48; N3, p = 0.42). Similarly, invasion of blood vessels, lymphatics, and nerves around tumour were not independent predictors of short term survival (p = 0.64, p = 0.64, p = 0.28, respectively).

Conclusion: Patients with rectal cancer were likely to have reduced short term survival in rectal tumours, which invaded beyond the submucosa, and in those with tumour involving peritoneum. The combined presence of metastasis with tumour beyond submucosa further reduced survival. Lymph node involvement by tumour reduced survival only if combined with any of the above predictive parameters; they were not independent predictors of survival.

353 HISTOLOGICAL AGREEMENT BETWEEN PATHOLOGISTS FOR DIAGNOSING COLORECTAL NEOPLASMS: THE EFFICACY OF THE VIENNA CLASSIFICATION

N. Suzuki1,2, A. Price1, I. Talbot1, K. Wakasa2, S. Ishiguro3, B. Saunders1.

1St Mark’s Hospital, London, UK; 2Osaka City University Hospital, Japan; 3Osaka Medical Centre for Cancer, Osaka, Japan

Background: The use of different nomenclature and differences in histological interpretation between Western and Japanese pathologists have caused considerable problems in the diagnosis, treatment and outcome for gastrointestinal epithelial neoplasms. In order to resolve this discrepancy, the Vienna classification (VC) has been developed.

Aim: To clarify the discrepancy between UK and Japanese pathologists and to assess the efficacy of the VC for the diagnosis of colorectal neoplasms.

Methods: 350 colorectal neoplasms (340 polypectomy and 10 biopsy specimens) were examined by two British (UK1, UK2) and two Japanese (J1, J2) pathologists using both conventional (mildly, moderately, or severely dysplastic adenomas and cancer) and the VC.

Result: Under conventional classification, there was moderate to good agreement (κ = 0.63, 0.56) between the pathologists within each country compared to only fair to moderate agreement (κ = 0.29–0.52) between UK and Japanese pathologists. By adopting the VC a better mutual agreement was established between the UK and Japanese pathologists (κ = 0.50–0.64). Japanese pathologists tended to assign a higher grade of dysplasia in both classifications (see Table).

Conclusions: The discrepancy caused by different nomenclatures with their possible knock-on differences for clinical management was improved by using the VC. The reason for the higher grading made by Japanese pathologists needs analysis but might be related to more reliance on cytological parameters.

Abstract 353

354 AWARENESS AND KNOWLEDGE OF COLORECTAL CANCER: IMPLICATIONS FOR FUTURE COLORECTAL CANCER SCREENING PROGRAMMES

A.M. Lennon, M. McMenamin, H. Barry, D. Keegan, H. Purcell, D.P. O’Donoghue, H. Mulcahy.

Centre for Colorectal Diseases, St Vincent’s Hospital, Dublin, Royal Brompton Hospital, London

Introduction: Cancer screening is rarely successful in populations with little knowledge of the disease being screened for. CRC screening will likely be implemented in many countries over the next 10 years, requiring accompanying educational programmes. Data are unavailable from any country on awareness and knowledge of CRC within the general population.

Aim: To determine awareness and knowledge of CRC in the Irish population.

Methods: A nationwide survey of 2355 adults (1250 females), comprising almost 0.1% of the entire adult population. Survey conducted in all 26 Irish regions in 2001/2002. Patients completed a questionnaire with questions on different aspects of CRC. Questions were asked about heart disease and breast cancer to provide control data.

Results: Awareness of heart disease and breast cancer was high (73% and 81%, respectively had heard of these diseases in the recent past). In contrast, only 40% had heard of CRC (14% magazines/newspapers, 12% TV/radio, 22% family/friends, 6% medical profession, 7% other sources). In relation to knowledge, 79% knew that a positive family history was associated with increased risk. However, irritable bowel syndrome (60%) and stress (62%) were thought to be greater risk factors for CRC than polyps (59%). 42% could name at least 1 colorectal cancer symptom, 53% thought that most patients were under 60 years of age at diagnosis, 61% were aware of surgery as a treatment, 45% estimated 5 year survival as above 40%, and 46% were aware that screening tests were available. Factors associated with CRC knowledge included having read about the disease in the recent past (p = 0.01), older age (p < 0.001), and higher educational status (p < 0.001).

Conclusions: This nationwide survey shows that awareness and knowledge of CRC is limited, especially in certain groups. This may have important implications for the success of future screening programmes and will allow us to target certain population groups for intensive education about CRC and the risks and benefits of screening.

355 IMPROVEMENT IN COLORECTAL CANCER SURVIVAL OVER THE PAST 20 YEARS: THERE ARE MORE QUESTIONS THAN ANSWERS

G. Bennett, J. Hyland, K. Sheahan, J.J. Murphy, A. White, D.P. O’Donoghue, H.E. Mulcahy.

Centre for Colorectal Diseases, St Vincent’s University Hospital, Elm Park, Dublin 4

Introduction: There has been a 1000% increase in the output of colorectal cancer research over the past 30 years and many diagnostic, surgical, and oncological advances. These might be expected to have had a favourable impact on survival but it is not known if this is the case.

Aim: To determine colorectal cancer survival trends over a 20 year period in a single institution.

Patients: 1853 consecutive patients admitted to St Vincent’s University Hospital, Dublin between 1983 and 2002 were prospectively entered onto a database. Demographic, clinical, and pathological data were collected and patients were followed until death. For analysis, the study group was divided into tertiles based upon year of diagnosis.

Results: Age rose over the study period (p < 0.001) while the percentage of patients undergoing tumour resection decreased from 96% to 88% (p < 0.001). There were no changes in sex (p = 0.35), tumour site (p = 0.52), or overall tumour stage (0.58). However, on subsection analysis, the proportion of stage II tumours decreased and stage III tumours increased in frequency over time (p = 0.02). Cancer related survival improved over the period from 45% to 56% (p = 0.005). On substage analysis, only those with stage III cancers had a significant improvement in survival (p = 0.05).

Conclusions: There has been a modest improvement in colorectal cancer survival over the past 20 years that may be related to a number of discrete factors. The improvement appears greatest in stage III tumours but this is as likely to be related to stage migration over the 20 years as it is to any therapeutic advances.

356 PELVIC TRAUMA AND RECTOCELES: DO THEY CAUSE SEVERE CONSTIPATION?

M.T. Eltringham, E. McCauley, A. Mackie, I.M. Bain, J.Y. Yiannakou.

University Hospital of North Durham, Durham, UK

Introduction: There are a host of causes attributed to chronic constipation while some patients remain idiopathic. Labour, pelvic surgery, and consequent rectoceles have been seen as causes but this remains controversial and few studies have shown a definite link.

Methods: 53 consecutive female patients with severe constipation (SC) were assessed using a proforma including a detailed history of the effect, on their symptoms of pelvic trauma (PT) including childbirth, and pelvic surgery. Patients with PT were asked whether it had; triggered symptoms of SC, worsened them if present previously, or had no effect. All of the patients had radionuclide proctograms and data were obtained on the presence and size of rectoceles.

Results: 40/53 (75%) of patients with SC had experienced PT. Of these 12/40 (30%) felt their constipation was triggered by it, 10/40 (25%) had symptoms preceding PT but felt it worsened their symptoms, and 18/40 (45%) felt there was no relationship between PT and SC. Patients with a history of PT had a greater likelihood of having a rectocele (85%) compared with the group with no PT (52%). Rectocele size was significantly greater in the PT group, 3.1 cm versus 2.2 cm (p = 0.02). Rectocele size was no greater for patients in “triggered” (3.13 cm) or “worsened” (2.97 cm) groups than in those with “no effect” (2.95 cm) (p = 0.96).

Conclusions: Pelvic trauma is a significant factor in the aetiology of SC. However, the mechanism of this is unclear. Rectoceles are a common finding in women with SC, and are likely to be greater in size if the patient has experienced pelvic trauma. However, it seems unlikely that the presence or size of rectocoele is a major factor in the pathogenesis of SC.

357 ABDOMINAL SUTURE RECTOPEXY WITHOUT LARGE BOWEL RESECTION FOR RECTAL PROLAPSE DOES NOT RESULT IN CONSTIPATION; DATA FROM PROSPECTIVE BOWEL FUNCTION EVALUATION, ANORECTAL PHYSIOLOGY, AND TRANSIT STUDIES

V.Y. Sabaratnam, G. Rathnayake, K.I. Deen.

University Department of Surgery, North Colombo General Hospital, Sri-Lanka

Introduction: Traditionally, suture rectopexy has been combined with sigmoid resection for rectal prolapse to prevent postoperative constipation. Furthermore, preservation of lateral ligaments will not result in constipation. Suture rectopexy alone without resection is being popularised. No studies have shown the influence of suture rectopexy without resection on colonic transit, and postoperative constipation.

Patients and Methods: Forty-six patients (median (range) age 32 (19–82) years) with rectal prolapse underwent suture rectopexy alone without division of lateral ligaments from March 1999. Prospectively, bowel function and anorectal physiology (ARP) were evaluated before and after surgery in a subset of 15 patients. Follow up (median, range) has been 12 (1–42) months.

Results: Follow up was complete in 36 patients. Recurrent prolapse was seen in 5 (full thickness 3 (8.3%); mucosal prolapse 2 (5.5%)). Physiological data in a subset of 15 patients revealed no significant difference in anorectal physiology before and 3 months after the operation (see Table). Similarly there was no significant difference in the rate of evacuation of transit markers on day 3 and 5 (see Table).

Abstract 357

Conclusion: Abdominal suture rectopexy without resection for rectal prolapse improves constipation symptoms, and does not result in significant change in colonic transit. We recommend this procedure either by open operation or by laparoscopy.

Nutrition posters 358–363

358 A SIMPLE AND SAFE METHOD OF TRANSCUTANEOUS GASTROSTOMY REPLACEMENT

M.B. Frenz1, G. Siuda1, A.S. McIntyre2, S.P.L. Travis1.

1Gastroenterology Unit, John Radcliffe Hospital Oxford, OX3 9DU, UK; 2Wycombe Hospital, HP11 2TT, UK

Introduction: The replacement of gastrostomy (PEG) feeding tubes often requires endoscopic procedures as the fistula closes soon after the accidental or elective removal of the PEG. Transcutaneous replacement of PEG tubes often leads to reduction in PEG size because of contraction of the fistula tract.

Aim: To assess the use and safety of a guide wire and vascular dilators for transcutaneous PEG replacement.

Methods: Patients referred for either elective PEG replacement or re-insertion of accidentally removed PEGs received the new balloon PEG (MIC-Key Peg, Vygon UK, in 11 cases and Corflow, Merck UK in one case) using the transcutaneous approach. The gastrostomy fistula was dilated with vascular dilators after a standard ERCP guide wire was passed through the fistula. The replacement PEG was inserted over the guide wire and the position checked by aspiration of gastric contents.

Results: A total of 12 PEGs were replaced in 8 patients. In 9 episodes the PEG had been accidentally removed and patients presented after a delay of 16–24 hours. In 3 cases the PEG had to be replaced because of malfunction and the old PEG was removed using the cut and push technique. Sedation was only used in one patient. The gastrostomy fistula was dilated to 2 Fr larger than the replacement PEG. In 9 cases the size of the replacement PEG was identical to that of the old PEG (14 Fr in 8 patients and 10 Fr in one patient). In two cases the replacement PEG was smaller than the original PEG (20 Fr to 14 Fr and 18 Fr to 16 Fr). In one case the replacement PEG was larger than the original PEG (9 Fr to 12 Fr). All patients tolerated the procedure well and had no complications except minor bleeding during dilatation, which was self-limiting. In 8 of the 12 patients the procedure was carried out as an outpatient procedure.

Comments: This technique of PEG replacement appears safe and well tolerated by patients. It reduces the need for repeat endoscopic procedures and sedation in most cases. Vascular dilators are easily available and less expensive than other dilators. It is a simple outpatient proc