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Immunology/Infection/Inflammation posters 242–257

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242 IDENTIFICATION OF POSSIBLE GUT HOMING DENDRITIC CELLS IN PERIPHERAL BLOOD BY EXPRESSION OF β7 INTEGRIN

R.J. Rigby1, A.L. Hart1,2, E.D.A. Westcott2, M.A. Kamm2, A. Windsor2, S.C. Knight1, A.J. Stagg1.1Antigen Presentation Research Group, Imperial College at Northwick Park;2St Mark's Hospital, London, UK

Dendritic cells (DC) are amongst the earliest cells to recognise enteric antigens and shape T cell responses. At least in part, DC in the tissues are derived from immature circulating DC populations. Gut DC may be functionally different from DC at other sites and contribute to the special features of intestinal immune responses. Given that there are markers on peripheral blood DC precursors that identify skin homing cells (CD1c and CLA), this study aimed to identify peripheral blood DC destined to home to the gut. We examined DC expression of β7, an integrin associated with mucosal homing of lymphocyte populations.

Methods: DC were identified by multi-colour flow cytometry as an HLA-DR+lineage- (CD3-, CD14-, CD16-, CD19-, CD34-, CD56-) population in peripheral blood and in mononuclear cells extracted from the lamina propria of the colon or small intestine. Co-expression of β7 with CD11c, CD1c and CLA was assessed.

Results: Gut DC from both the small and large intestine expressed β7 integrin. In blood, most DC expressed β7 but they were heterogeneous for the level of expression and for co-expression of the molecules associated with skin homing. Both myeloid and plasmacytoid DC were studied. All CD11c+ `myeloid' DC were β7int, with some also expressing skin homing molecules. CD11c- `plasmacytoid' DC, which are thought to migrate directly into lymphoid tissue, comprised two subpopulations, β7lo and β7hi. Neither of these populations expressed the skin homing markers CLA or CD1c.

Conclusions: It appears that DC with markers associated with homing to the gut mucosa can be identified in the peripheral blood. A population of DC, β7hi CD11c-, that may migrate directly into lymph nodes draining the gut, has been identified. Circulating precursors of gut DC that are functionally committed to specialised roles in the intestine may provide an early target for manipulating mucosal immune responses.

243 NEUTROPHIL RESPIRATORY BURST AND TISSUE PENETRATION ARE NORMAL IN CD DURING G-CSF ADMINISTRATION

M. Harbord1, R. Day2, A. Hankin1, S. Bloom1, A. Forbes2, A.W. Segal1.1Department of Medicine, University College, London;2St Mark's Hospital & Academic Institute, Imperial College, London, UK

Background: Neutrophil migration into the tissues is defective in Crohn's disease (CD). Granulocyte Colony Stimulating Factor (G-CSF) has been used to treat CD. The effects of G-CSF in CD on venous neutrophil respiratory burst (B) and neutrophil tissue penetration (P) are unknown.

Aim: To measure B&P in CD and matched control subjects.

Method: 16 outpatients were enrolled (CD: n=8, 45!8 years, 3 male; control: n=8, 42!7 years, 7 male). Subcutaneous G-CSF (Lenograstim) 5ug/Kg was administered at 24 & 48 hours. Venous neutrophils were purified at 24 & 72 hours by Hypaque-Ficoll gradient centrifugation after erythrocyte osmotic lysis. Oxygen consumption of 1x107 neutrophils was measured in an oxygen electrode after stimulation with autologously opsonised human fecal flora (1x109) or with PMA (1ug). Duplicate skin blisters were induced at 0 & 48 hours by applying 0.1% cantharidin in 25μl acetone to 0.8cm2 paper discs on the forearm, which were covered with parafilm and an adhesive dressing. At 24 and 72 hours the blister fluid was removed. The cellular composition was counted microscopically. Flow cytometry using anti-CD16 and anti-CD14 antibody labelling together with light scatter properties were used to quantitate neutrophils and monocytes/macrophages respectively.

Results: (mean±standard error): Increase in venous monocyte concentration was reduced in CD subjects at 72 hours (1.2±0.11 x 109/l c.f. 1.6±0.15 (p=0.03)). B & P were normal in CD. However in all subjects, B was significantly reduced after G-CSF (flora challenge from 87.1±8.5 to 44.2±6.9 (n.mol.O2/107neutrophils/min) (p=0.008); PMA challenge from 64.8±8.0 to 26.8±3.4 (p=0.002)). P was markedly lower than initial venous neutrophil concentration (CD (21±10%) and controls (14±5%)). P increased with G-CSF (CD from 0.72±0.68 to 3.42±2.51 (106CD16+/ml) (p=0.02); controls from 0.52±0.43 to 3.70±2.68 (p=0.02)) but proportionally less than the increase in venous neutrophil number (CD (12±5%) and controls (15±6%)).

Conclusion: Efficacy of G-CSF in CD will be affected by the reduction in B. The increase in P during G-CSF does not parallel the increase in venous neutrophil concentration.

244 STREPTOCOCCUS FAECIUM, A POSSIBLE PROBIOTIC BACTERIUM, BUT NOT LACTOBACILLUS ACIDOPHILUS OR ESCHERICHIA COLI (NISSLE), DECREASES PROINFLAMMATORY CYTOKINE PRODUCTION (IFN-γ) BY AN IL-10 DEPENDENT MECHANISM

A.L. Hart1,2, A.J. Stagg1, R. Rigby1, A. Jones1, L. Lammers3, F. Rizzello3, P. Gionchetti3, M. Campieri3, S.C. Knight1, M.A. Kamm2.1APRG, Imperial College;2St Mark's Hospital, London, UK;3University of Bologna, Bologna, Italy

Introduction: Probiotics are effective in the treatment of some inflammatory bowel diseases, but their mechanism of action is unclear.

Methods: A whole blood assay was developed to assess the effect of probiotic bacteria on gamma-interferon (IFNγ) production by polyclonally activated T-cells. Cell wall and soluble fractions of Lactobacillus acidophilus, Streptococcus faecium (S. faecium) and Escherichia coli (Nissle strain) at the equivalent of 108 colony forming units per millilitre were cultured with blood overnight. A neutralising anti-interleukin 10 (IL-10) antibody (20μg/ml) was added to some cultures. Subsequently, production of IFNγ by CD8+ and CD8- T cell populations was determined by intracellular labelling and flow cytometry after 4-hour activation with phorbol-myristate-acetate and ionomycin in the presence of monensin. The production of IL-10 in whole blood cultures was determined by ELISA.

Results: Cell wall, but not soluble components, of S. faecium decreased the proportion and number of IFNγ producing CD8+ and CD8- T-cells. IFNγ production was not altered by the other probiotics. Cell activation assessed by CD69 expression was not affected. The inhibition of IFNγ production by S. faecium was partly dependent on IL-10. However, all three bacteria stimulated IL-10 production in whole blood suggesting that IL-10 is required but not sufficient for the inhibitory effect.

Conclusions: The data indicate that a cell wall component of S. faecium, a gut commensal and putative probiotic, down-regulates T-cell IFNγ production by a mechanism that involves IL-10. This may be a direct effect of the bacteria on the T-cell or may act via additional cell populations.

245 ASCA AND ANCA IN THE DIAGNOSIS OF INFLAMMATORY BOWEL DISEASE AND OTHER DIARRHOEAL ILLNESSES

M. Buckland, M. Mylonaki, D.S. Rampton, H.J. Longhurst.

Depts of Clinical Immunology & Gastroenterology, Barts and the London NHS Trust, London EC1A 7BE, UK

Background: Several studies have examined the utility of ASCA and ANCA either alone or in combination for aiding the diagnosis of inflammatory bowel disease (IBD). ASCA and ANCA in combination are said to be more specific than either alone. ANCA-ASCA+ is the result expected in Crohn's and ANCA+ASCA- in UC. We have tested the utility of ASCA or ANCA alone or in combination in distinguishing between IBD and other diarrhoeal illnesses.

Methods: 150 sera from patients attending a gastroenterology clinic with diarrhoeal disease were screened for ASCA by ELISA using the GENESIS IgA and IgG ASCA kits, and for ANCA by indirect immunofluorescence on ethanol fixed neutrophils. Patients were classified as Crohns, UC or other diarrhoeal illness, independently of the ANCA/ASCA results, according to standard criteria.

Results: IgA ASCA alone has a sensitivity of 0.44 and specificity of 0.94 for Crohn's disease (0.44/0/94). Increases in sensitivity are accompanied by reduction in specificity when IgG ASCA (0.58/0.85) or IgG ASCA/ANCA (0.52/0.90) in combination are added. ANCA alone has a sensitivity of 0.54 and specificity of 0.85 for the diagnosis of UC. The addition of ASCA does not affect sensitivity or specificity (0.54/0.86).

Conclusions: (1) The high specificity of IgA ASCA for Crohn's suggests that the presence of this antibody should trigger appropriate further investigation. However, its low sensitivity means that a negative result does not exclude Crohn's. For this reason, the small loss of specificity resulting from adding IgG ASCA or ANCA will result in a loss of utility, despite the increase in sensitivity. (2) The low sensitivity and specificity of these assays for UC suggests that their utility in this setting is low. (3) While IgA ASCA screening may have value in diagnosing Crohn's, IgG ASCA and ANCA are not useful in routine diagnosis of patients with diarrhoeal disease.

246 INCREASED EXPRESSION OF α-DEFENSINS WITH INCREASING SEVERITY OF TROPICAL ENTEROPATHY

W. Dhaliwal1, M. Bajaj-Elliott1, R. Poulsom2, D. Ghosh3, C. Bevins3, P. Kelly1,4.1Dept Adult & Paediatric Gastroenterology, St Bart's & Royal London School of Medicine, London, UK;2Imperial Cancer Research Fund, Lincoln's Inn, London, UK;3Dept Immunology, Cleveland Clinic Foundation, Cleveland, Ohio, USA;4University of Zambia School of Medicine, Lusaka, Zambia

Background: Tropical enteropathy is a term used to describe a different villous and crypt architecture to that seen in temperate climates. Its significance is uncertain. Paneth cells in the human small intestine express a powerful array of antimicrobial molecules, notably the human intestinal α-defensins HD5 and HD6. The objective of the current study was to determine if expression of HD5 and HD6 is related to enteropathy as this might indicate that tropical enteropathy is an adaptive response to a hostile environment.

Methods: Biopsies (n=84 in the first year, n=44 second year and n= 36 third year) were obtained from a longitudinal study of enteropathy and infection in an impoverished township in Misisi, Lusaka. Defensin mRNA was quantified by competitive RT-PCR (expressed as log transcripts /μg total RNA). mRNA and peptide expression was localised by in situ hybridisation and immunostaining. Immunoblotting was used to analyse expression of HD5 isoforms.

Results: Peptide and mRNA expression were only seen in the Paneth cell compartment. Mucosal architecture and defensin mRNA expression showed considerable variation in the 84 biopsies analysed at baseline. In cross-sectional analysis, HD5 mRNA and villous height (VH) were inversely correlated (Spearman's ρ = -0.57, p = 0.003). Over one year, change in HD5 and change in VH showed a similar correlation (ρ = -0.50, p = 0.002). VH varied with time of year and this seasonality was seen in HD5 mRNA expression levels, in a reciprocal relationship to VH. A similar pattern was seen for HD6. Isoform expression of these defensins did not vary with the severity of enteropathy.

Conclusions: Increased severity of tropical enteropathy correlated with increased quantity of α-defensin mRNA in jejunal biopsies, consistent with the hypothesis that tropical enteropathy is an adaptive response to microbial challenge.

247 THE CLINICAL SPECTRUM OF GASTROINTESTINAL INVOLVEMENT IN PATIENTS WITH PRIMARY HUMORAL IMMUNODEFICIENCY; A CLINICAL SURVEY OF PATIENTS FROM IRANIAN PRIMARY IMMUNODEFICIENCY REGISTRY

Aghamohammadi Asghar, Moein Mostafa, Farhoudi Abolhasan, Pourpak Zahra, Rezaei Nima, Abolmaali Kamran, Movahedi Masoud, Gharagouzlou Mohammad, Mahmoudi Maryam, Hojjati Ashrafi Taha.Department of Immunology, Allergy and Asthma, Children Medical Center Hospital, Tehran University of Medical Sciences

Background: Primary Humoral ImmunoDeficiencies (PHID) are currently increasingly recognized, thanks to novel advances in the immunology and its laboratory techniques. Gastrointestinal involvement, together with respiratory infections, account for most of the complications and the main cause of hospitalizations in such patients.

Objectives: To determine the clinical spectrum of gastrointestinal involvement in patients with PHID.

Method: We have reviewed the data from the clinical files of patients with PHID, diagnosed according to WHO criteria, who were enrolled in Iranian Primary Immunodeficiency Registry.

Results: We analyzed 125 patients (84 males), with the diagnoses of primary antibody deficiency including common-variable immunodeficiency (64 pts), x-linked agammaglobulinemia (29 pts), IgA deficiency (20 pts), IgG-subclass deficiency (8 pts), and hyper-IgM syndrome (4 pts). The mean age of the patients at the time of study was 11 years. In the evolution of their disease, 78 cases (62.4%) have had involvement. Diarrhea, being the most common type of involvement was seen in 70 patients (56%). Seventeen of these patients (24.2%) have progressed to chronic diarrhea. Giardiosis and hepatitis were seen in 12 (9.6%) and 7 (5.6%), respectively. Also, we had 5 cases of chronic active hepatitis and 3 cases of ulcerative colitis. Among nonspecific symptoms, hepatomegally was seen in 32 patients and splenomegally in 28 patients. Celiac disease was seen in 2 cases with the diagnosis of selective IgA deficiency.

Conclusion: Following the respiratory tract, gastrointestinal tract constitute the second site of involvement in patients with primary humoral immunodeficiency. Even some patients may present with recurrent diarrhea as the first manifestation of immunodeficiency disorders.

248 THE RESPONSE TO PROTON PUMP INHIBITOR THERAPY IS GENETICALLY DETERMINED

E.J. Dickson, A.J. Morris1, C. Craig, J. Eskdale, G. Gallagher, R.C. Stuart.University Depts of Surgery and1Medicine, Glasgow Royal Infirmary, UK

Aim: The metabolism of PPIs by the CYP2C19 enzyme is genetically determined with considerable inter-individual variation. The aim of this study was to establish the relative frequencies of fast, medium and slow metabolisers, and to examine PPI dose with respect to metabolic capacity.

Methods: 535 patients with benign acid disorders on acid suppression therapy, and 106 patients with gastro-oesophageal cancer were genotyped by PCR and restriction enzyme digest to determine their capacity to metabolise PPIs. The benign group was further subdivided according to H. pylori status and presence of peptic ulcer.

Results: The frequency of each metabolic group was similar in the benign and cancer patients. Rapid Extensive (fast) metabolisers 73.5% benign, 76.4% cancer: Extensive (medium) metabolisers 23.7% benign, 20.8% cancer; Poor (slow) metabolisers 2.8% benign, 2.8% cancer. Thus 26.5% of patients on acid suppression therapy are medium or slow metabolisers and have a reduced capacity to metabolise PPIs. In the H. pylori negative peptic ulcer group 87.5% of the fast metabolisers were on 20mg omeprazole and 12.5% were on 10mg. This may be a reflection of lack of symptomatic response to a lower dose. In the medium / slow group 53.8% were on 20mg omeprazole, and 46.2% on 10mg (p=0.023, x2 test)

Conclusions: A significant proportion of patients on acid suppression therapy have a reduced ability to metabolise PPIs. These patients may require a lower dose of PPI. The clinical implications are treatment failure for fast metabolisers on low dose PPI, and adverse effects and unnecessary financial cost in the medium / slow metabolisers.

Abbreviations: PPI = proton pump inhibitor, PCR = polymerase chain reaction.

249 HELICOBACTER PYLORI INFECTION IN CHILDHOOD REDUCES THE RISK OF ATOPIC DISORDERS IN ADULT LIFE: THE BRISTOL HELICOBACTER PROJECT

C.A. McCune, A.J. Lane, R.F. Harvey, L.J. Murray, I.M. Harvey, M. Egger, J.L. Donovan, P.N. Nair.Department of Social Medicine, University of Bristol & Frenchay Hospital, Bristol, UK

Background: The prevalence of atopic disorders including asthma has increased dramatically over the last 20 years. Exposure to early childhood infections, including gastrointestinal infections, may influence the developing immune system in such a way as to reduce the risk of later development of asthma, eczema and similar atopic diseases.

Aim: We investigated the hypothesis that Helicobacter pylori (HP) infection is associated with a decreased prevalence of atopy (asthma, allergic rhinitis and eczema).

Methods: 26,203 individuals aged 20–59 years from 7 primary care centres in the SW of England were invited to participate in a randomised controlled trial of HP eradication. 10,537 agreed to participate and underwent a 13C urea breath test. 3,244 individuals (2,165 HP –ves, 1,079 HP +ves) supplied medication details on a validated questionnaire. Inhaled/oral bronchodilators, inhaled corticosteroids or cromoglicate therapy were used as surrogate markers for asthma. Similarly oral antihistamines and topical corticosteroids were used as markers for allergic rhinitis and eczema respectively.

Results: Those individuals found to be HP positive were less likely to be taking a medication for asthma, eczema or allergic rhinitis (crude OR 0.70 (0.54-0.91), p<0.007 and adjusted OR 0.75 (0.57,0.99), p<0.05).

Conclusions: Childhood infection with Helicobacter pylori is associated with a reduced risk of atopic disorders in adult life.

250 PROBIOTIC BACTERIA STIMULATE NATURAL KILLER (NK) CELL ACTIVATION AND CYTOKINE PRODUCTION, AN EARLY STEP IN INNATE IMMUNITY

A.L. Hart1,2, R. Rigby1, A.J. Stagg1, K. Lammers3, F. Rizzello3, P. Gionchetti3, M. Campieri3, S.C. Knight1, M.A. Kamm2.1APRG, Imperial College;2St Mark's Hospital, London, UK;3University of Bologna, Bologna, Italy

Introduction: Probiotics are effective in the treatment of inflammatory bowel diseases, but their mechanism of action is unclear. Given that NK cells have a central regulatory role in host defence against bacteria, in particular in models of colitis, we studied the effect of probiotic bacteria on NK cell activation and cytokine production.

Methods: Whole blood and mononuclear cells from colonic biopsies were cultured overnight with Lactobacillus acidophilus, Streptococcus faecium and Escherichia coli (Nissle strain). Expression of the activation antigen CD69 and intracellular cytokines (IFNγ, IL-10 and IL-4) were assessed by flow cytometry.

Results: At high concentrations (108 colony forming units per millilitre), cell wall fractions of all three probiotic bacteria induced expression of CD69 on greater than 97% of blood NK (CD3-CD8+) cells. The dose required for 50% maximal CD69 expression differed between the bacteria: Escherichia coli>Lactobacillus acidophilus>Streptococcus faecium CD69 expression was induced to a lesser extent on CD8+ (35%) and CD4+ (20%) T cells. The soluble fraction of Escherichia coli (Nissle strain) but not Streptococcus faecium also activated NK cells. IFNγ, IL-10 and IL-4 production by NK cells was detected on exposure to the different bacteria, demonstrating the functional significance of this NK activation. In colonic tissue, there was a high baseline expression of CD69 by NK cells, indicating that these cells are activated in vivo, possibly as a result of local exposure to commensal bacterial antigens. This activation was further increased by all of the probiotic bacteria.

Conclusions: Probiotic bacteria modulate innate immunity by activating and stimulating cytokine production by NK cells. The effect varied with different bacterial dose, fraction and probiotic species. Modulation of innate immunity, including NK cells, may contribute to the therapeutic action of probiotic bacteria in intestinal inflammation.

251 INTERFERON-γBLOCKS THE INTERLEUKIN-1 AND BACTERIALLY MEDIATED INDUCTION OF HUMAN β-DEFENSIN 2 EXPRESSION IN GASTRIC AND INTESTINAL EPITHELIAL CELLS

M. Bajaj-Elliott1, P. Fedeli1, D. O'Neil2 .1Department of Adult & Paediatric Gastroenterology, St Bartholomew's and the Royal London School of Medicine & Dentistry, London, UK;2Division of Gut Microbiology and Immunology, The Rowett Research Institute, Aberdeen, UK

Introduction: β-defensins are endogenous antibiotics secreted by the epithelia of mucosal surfaces, where their expression is augmented by infection and inflammation. We have previously shown marked induction of human-β defensin 2 (hBD-2) expression in gastric and intestinal epithelial cell lines by various pathogenic stimuli. In the present study we have explored the role of IFN-γ; one of the major cytokines expressed during chronic Th1-mediated GI inflammation (e.g. Crohn's disease and gastritis) in the regulation of hBD-2 gene expression.

Methods: hBD-2 mRNA expression was quantified by RT-PCR. Immunohistochemistry was performed on archival paraffin-blocked samples from patients with varying grades of gastritis (grade I-III) and from IBD (inflammatory bowel disease) tissue.

Results: In marked contrast to the known stimulatory effect of IL-1 on hBD2 expression, IFN-γ did not induce hBD2 in a panel of gastric and intestinal cell lines. Interestingly, pre-exposure of cells to IFN-γ completely abolished the effects of IL-1 and pathogenic-stimuli on hBD2 gene expression. This inhibitory effect of IFN-γ was however, time-dependent. We also observed an inverse relationship between defensin peptide expression and the degree of inflammation in biopsy samples.

Conclusions: Our present study suggests that during Th1-driven chronic GI inflammation, IFN-γ may act as a biological `off-switch' for hBD2 expression. Downregulation of host innate defence during infection and inflammation may represent one strategy employed by potential pathogens in evading the host immune response.

252 DYSPEPSIA IS MORE COMMON IN ELDERLY WOMEN

K. Sundaram1,2, E. Rink2, M.A. Mendall1.1Mayday University Hospital;2St Georges Hospital Medical School, UK

Background: Dyspepsia is said to be a common symptom in the general population. This has not previously been formally studied in the older population, despite them having a higher prevalence of Helicobacter pylori (HP) infection.

Aims: To conduct a questionnaire survey of the general population over 60 years in the London Borough of Croydon in order to document the prevalence of, and risk factors for dyspeptic symptoms.

Method: A total of 1860 individuals over the age of 60 years were chosen at random from the lists of general practitioners across the London Borough of Croydon. They were sent a single sheet questionnaire. This ascertained basic demographic details and asked whether subjects suffered from waterbrash, vomiting, bloating, nausea, upper abdominal pain or heartburn on more than one occasion or for more than one day in the previous month. Participants were also asked how often they took any medication for these symptoms and requested to send a sample of saliva collected in a cotton salivette (Sarstedt) by return of post. The saliva samples were analysed for antibodies to HP in order to determine seropositivity as previously described (Gut 2000; 46 (suppl II), A67: W141). Analyses were made using Chi squared / Fisher's Exact test, or Mann Whitney U test.

Results: In total 1116 subjects (60%) returned both the questionnaire and a usable sample. Of these 616 (55%) were women. 284 (25%) were determined as seropositive to HP infection. 516 (46%) had had symptoms of dyspepsia more than once in the previous month, and 370 (33%) specifically reported reflux related symptoms (waterbrash / heartburn). HP infection was detected in 25% of both these groups. The prevalence of dyspeptic symptoms was not related to HP seropositivity, age, smoking, social class, BMI nor NSAID use. It was more common in women (51 vs 39%, p< 0.0001). In particular women had more symptoms of bloating (25 vs 13%, p< 0.0001) and nausea (11 vs 5%, p< 0.015). These differences remained significant even after subjects on occasional or regular medication for dyspepsia were excluded from the analysis.

Conclusion: The prevalence of self reported dyspepsia in older individuals is high, and is more common in women. It is not related to NSAID use, smoking or HP infection, and deserves further investigation.

253 CHOLERA TOXIN (CT), ESCHERICHIA COLI HEAT LABILE (LT) AND HEAT STABLE TOXIN (STA) HAVE AN INDIRECT EFFECT ON DISTAL INTESTINAL FLUID TRANSPORT IN THE RAT SMALL INTESTINE

M.R. Banks1, A.C. Casburn-Jones2, M.J.G. Farthing2.1St Bartholomew's and the Royal London Hospital;2Glasgow University, UK

Background: CT, LT and STa induce intestinal secretion directly via cyclic AMP and cyclic GMP dependent pathways respectively. Increasing evidence exists that these enterotoxins may mediate intestinal secretion through a local intramural neural reflex arc. To investigate this neural enterotoxin- induced intestinal secretion, we measured the effects of CT, LT and STa on intestinal fluid and electrolyte transport in distal non-contiguous and transected intestinal segments separately.

Methods: A model of distant ab-oral secretion was created in an-aesthetised 200g male Whistar rats. CT (50 μg/ml), LT(50 μg/ml), STa (2μg/ml) and saline (control) were placed independently in a proximal intestinal loop (an isolated 15cm jejunal loop) separated from a distal intestinal loop (a 15cm ileal loop) by tissue glue (Inderal); the luminally placed glue maintained neurological and vascular but not luminal continuity. The distal loop was perfused with a plasma electrolyte solution containing 14C-polyethylene glycol as a non-absorbable marker to measure changes in fluid and electrolyte transport. The experiment was repeated with the distal loop transected, for each enterotoxin.

Results: In controls, absorption in the distal loop ranged between 75 and 112 μl/min/g. Following application of CT, LT and STa to the proximal loop, distal loop absorption was reduced by 28%, 55% and 20% respectively (p<0.05). Following transection of the distal loop, the application of CT, LT and STa had no significant effect compared to control, on distal intestinal fluid or electrolyte transport.

Conclusions: These observations support a non-direct mechanism of CT, LT and STa- induced intestinal secretion. This mechanism is likely to be through intrinsic or extrinsic neurones. Transection of the intestinal wall abolished the distal effect on intestinal transport by CT, LT and STa supporting the hypothesis that intrinsic intramural neurones play a key role in enterotoxin-induced intestinal secretion. Further work is required to define the circuitry of these intrinsic neural pathways.

254 PATHOGENIC BACTERIA STIMULATE COLONIC DENDRITIC CELLS TO PRODUCE PRO-INFLAMMATORY IL-12 WHILE THE RESPONSE TO PROBIOTIC BACTERIA IS TO PRODUCE ANTI-INFLAMMATORY IL-10

R. Rigby1, M.A. Kamm2, S.C. Knight1, A.L. Hart2, A.J. Stagg1.1APRG, Imperial College;2St Mark's Hospital, London, UK

Intestinal dendritic cells (DC) sample luminal contents and play a central role in the regulated response to the commensal gut flora. This is mediated in part by cytokine production following exposure to bacterial products, and results in a balance between pro- and anti-inflammatory responses. Cytokine production by murine colonic DC was assessed after stimulation by a component of pathogenic bacteria and a putative probiotic bacterium.

Methods: DC were identified as CD11c+MHC class II+ cells in mononuclear cell preparations obtained by collagenase digestion of colonic tissue. Production of IL-12, IL-10 and IL-4 in response to LPS (1μg/ml), cell walls from Bifidobacteria longum (at the equivalent of 107 per ml) or medium alone was determined by intracellular staining. DC were also isolated by immunomagnetic separation on the basis of CD11c expression.

Results: Approximately 3% of colonic cells were DC. They were CD40+CD80+CD86+ and stimulated a primary mixed leucocyte reaction following overnight culture. A small proportion (<5%) of unstimulated DC produced IL-12. LPS, but not B. longum, increased the proportion of IL-12 producing DC by 4–10 fold. In contrast, IL-10 was not detected in unstimulated cultures or in response to LPS, but was induced in over 50% of DC in the presence of B. longum. Addition of LPS to B. longum stimulated cultures, downregulated production of IL-10 by approximately 50% indicating cross-regulation of the responses induced by these two stimuli. IL-4 production by DC was not detected under any conditions.

Conclusions: Colonic dendritic cells, which are early and central regulators of mucosal immunity, respond to bacterial stimulation with the production of both pro-and anti-inflammatory cytokines. However different bacteria or bacterial components stimulate opposing responses, and therefore have the potential to determine the subsequent immune response. This provides further supportive evidence for the use of probiotic bacteria in altering gut immune regulation.

255 CHANGES IN EPITHELIAL BARRIER AND CYTOKINE RELEASE IN RESPONSE TO C. DIFFICILE TOXIN A

S.S. Johal, K. Solomon, J. Webb, S. Dodson, Y.R. Mahida.Division of Gastroenterology, University Hospital, Nottingham, UK

Following infection with toxin-secreting C. difficile, presentation can range from severe colitis to no colonic inflammation. We investigated changes in epithelial barrier and release of transforming growth factor-β (TGF-β) and interleukin-8 (IL-8) in response to C. difficile toxin A.

Methods: Monolayers of T84 cells were pre-exposed to purified C. difficile toxin A for 3 h and cytokine release was assessed after subsequent culture for 24 h. TGF-β and IL-8 levels were determined using specific bioassay and ELISA respectively. Barrier function was assessed by measurement of transepithelial electrical resistance.

Results: See table for TGF-β and IL-8 conc. in culture supernatants, mean (±SEM). TGF-β1 was the predominant isoform released. Within 24 h of pre-exposure to ≥10 ng/ml toxin A, there was complete and irreversible loss of electrical resistance across T84 monolayers. At conc. <10ng/ml of toxin A, loss of monolayer resistance was followed by recovery that was dependent upon the conc. of toxin originally applied. This recovery of epithelial barrier was significantly enhanced in the presence of 1 ng/ml recombinant TGF-β1 (maximal % increase in resistance, 0.01 ng/ml toxin A: 179%; 0.1 ng/ml toxin: 156%; 1ng/ml toxin: 232%. All p<0.04).

Abstract 255

Conclusions: In intestinal epithelial cells, (i) exposure to low concentrations of toxin A induces the release of TGF-β, which facilitates recovery of barrier function (ii) exposure to high concentrations of toxin A induces the expression of the neutrophil chemoattractant IL-8 and rapid, irreversible loss of barrier function. Thus the level of exposure of intestinal epithelial cells to C. difficile toxins may determine the development and severity of colitis.

256 NITRIC OXIDE (NO) RELEASED BY GLYCO-SNAP-1 SIGNIFICANTLY INHIBITS THE LPS STIMULATED PRODUCTION OF IL-1β BUT NOT TNF but not tnf α BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCs)

J. Burdsall, J. Smith, C.J. Hawkey.University of Nottingham, Division of Gastroenterology, UK

Introduction: NO donating anti-inflammatory drugs such as NO-NSAIDS and Nitroprednisolone are currently being evaluated for enhanced anti-inflammatory properties believed to be related to NO release. PBMC's are recruited to sites of inflammation in conditions such as ulcerative colitis or Crohn's disease. We therefore investigated the effect of NO release on these cells by measuring the production of pro-inflammatory cytokines IL-1β and TNFα after stimulation with LPS.

Methods: Human PBMC's were purified using Histopaque. The isolated cells were re-suspended in RPMI to a final concentration of 1x106 per ml. The cells were then pre-incubated with the NO donor (G-SNAP-1) over a concentration range of 0 to 1mM for 30mins prior to the addition of LPS (1μg/ml). After overnight incubation (37°C, 5% CO2), the cells were centrifuged and the supernatant IL-1β and TNFα concentrations measured by ELISA (RnD DuoSet).

Results: There was a dramatic and highly significant inhibition of IL-1β but not TNFα release in a concentration related response ((P<0.001 One Way ANOVA analysis, IL-1β,IC50 = 61.7μM, SEM 0.04) (see fig).

Conclusion: NO released from G-SNAP-1 is a potent inhibitor of pro- inflammatory mediator IL-1β production by human PBMC's but not TNFα.

257 DIAGNOSTIC YIELD OF STOOL ANALYSIS AND ENDOSCOPY IN HIV POPULATION PRESENTING WITH DIARRHOEA

D. Datta, B. Gazzard.St Stephen's Centre, Chelsea and Westminster Hospital, London, UK

Background: HIV patients commonly attend with history of diarrhoea. The use of highly active antiretroviral therapy (HAART) has led to a change in the epidemiology of diarrhoea in HIV patients. The patients with negative stool studies are frequently referred for endoscopic evaluation. We aimed to determine the diagnostic yield of stool analysis and endoscopy in HIV patients presenting with diarrhoea.

Methods: We retrospectively reviewed 525 HIV positive patients who presented with diarrhoea from January 1, 2000 to august 31,2001 at Chelsea & Westminster hospital. Patients were divided in 3 groups - Group 1) CD4 count ≤ 200 , 2) CD4 >200 - <350 3) CD4≥ 350. Patients who had 2 or more stool examinations were included. We also reviewed the endoscopy and biopsy findings of the patients who had negative stool studies.

Results: Of 86 patients with CD4 count ≤ 200 ( Group 1) the stool examination was diagnostic in 30 patients ( 34.8%) - the commonest diagnosis being cryptosporidiosis ( 10 patients- 11.6%), other causes were E histolytica , giardiasis, campylobacter, salmonella, C diff toxin, microsporidia, isospora, rotavirus. Of 30 patients, 12 patients (40%) were on HAART, however only 2 patients (20%) presenting with cryptosporidiosis were on HAART. In group 2 ( CD4 >200 - <350 ) and group 3 (CD4≥ 350), diagnostic yield of stool examination was in 37 patients(24.34%) and 74 patients (25.78%) respectively. The cause most frequently found in group 2 and group 3 was giardiasis ( 14 patients - 9.2%) and campylobacter ( 20 patients -6.96% ) respectively. 64 patients ( 16.6% ) with negative stool studies were referred for endoscopy. Upper GI endoscopy and lower GI endoscopy with biopsies was diagnostic in 12 (30%) and 19 ( 50% ) patients respectively.

Conclusion: In our population stool analysis in HIV patients with higher CD4 count has a low diagnostic yield. With the advent of HAART the infectious causes of diarrhoea are probably less important. Our study also showed that upper and lower GI endoscopies with biopsies are useful diagnostic investigations in patients with chronic diarrhoea.

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