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Immunology/Infection/Inflammation free papers 091–104

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091 HELICOBACTER PYLORI UPREGULATES MMP-7 IN EPITHELIAL CELLS IN A CAG-DEPENDENT MANNER

J.R. Bebb1, D.P. Letley1, R.J. Thomas1, H.M. Collins2, S.A. Watson2, J.C. Atherton1.Division of Gastroenterology and Institute of Infections and Immunity, Academic Unit of Cancer Studies, 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. Several studies have shown increased expression of MMP-7 in gastric cancer tissue compared to non-cancer tissue. More virulent strains of H.pylori possess the cagPAI (encoding a Type IV secretion system) and an active form of VacA, a toxin that induces vacuolation in vitro. This study examines the effect of H.pylori on MMP-7 expression in HT29 cells.

Methods:H.pylori strains 60190 (cagPAI+, vacA s1/m1), Tx30a (cagPAI-, vacA s2/m2) and the VacA and CagE isogenic mutants of 60190 were co-cultured with HT29 cells for 24 hours. Cell pellets were used for RNA extraction and reverse transcription, and cDNA levels assessed for MMP-7 levels (and GAPDH) by Real Time PCR. HT29 supernatants were assessed for MMP-7 expression by western blot and casein zymography and for other metalloproteinase activity by gelatin zymography. Experiments were performed at least three times.

Results:H.pylori pathogenic strain 60190 increased MMP-7 RNA levels (13-fold vs untreated, p=0.06, unpaired t-test) more than non-pathogenic strain Tx30a. Use of isogenic mutants showed this effect to be VacA-independent but CagE-dependent. Western blot gave a 29kDa band for 60190 and its VacA mutant. Casein zymography revealed a 29kDa band of activity for all samples but reduced intensity for strain Tx30a and the 60190 cagE mutant. This band corresponds to the predicted size for pro-MMP-7. Gelatin zymography showed no differences between treatments.

Conclusion: In HT29 cells H.pylori co-culture leads to upregulation of MMP-7 at both RNA and protein level. This upregulation is partly dependent on an intact Cag Type IV secretion apparatus but is not VacA-dependent. This is a further example of subversion of host systems through Cag-dependent signalling and we speculate that it may be important in the pathogenesis of H.pylori.

092 SERUM IgG4 ANTIBODIES TO COMMON FOOD ANTIGENS ARE ELEVATED IN IRRITABLE BOWEL SYNDROME (IBS)

S. Zar, M.J. Benson, D. Kumar.St George's Hospital Medical School, London, UK

Introduction: Food hypersensitivity is a common perception amongst IBS patients. Data from the dietary elimination and food challenge studies support the role of diet in the pathogenesis of IBS, but there are no well-established tests to identify food hypersensitivity.

Aim: To compare the serum IgG4 antibody titres to common food antigens in IBS patients and healthy controls.

Method: 52 IBS patients [33 diarrhoea-predominant (D-IBS); 13 constipation-predominant (C-IBS); 6 mixed symptoms] and 18 healthy controls were studied. All patients underwent thorough clinical examination, routine blood tests and either a colonoscopy or sigmoidoscopy + Ba enema. Serum samples were tested for IgG4 antibodies to 10 common food articles including milk, eggs, wheat, rice, potatoes, chicken, beef, pork, fish and peanuts. A quantitative assay, carried out in a central laboratory, measured antibody titres in the range of 1.5–30,000MgA/L. Mann Whitney-U test was used to assess difference in the antibody titres to individual food antigens between IBS patients and healthy controls.

Results: IBS patients had significantly higher IgG4 antibody titres to milk (p=0.037), peanuts (p=0.04), beef (p=0.013), pork (p=0.001) and chicken (p=0.009) compared to healthy controls. The D-IBS group had significantly higher titres against peanuts (p=0.014), beef (p=0.017), pork (p=0.002), chicken (p=0.017) and wheat (p=0.038) compared to controls. The difference in milk antibodies was of borderline significance (p=0.058). In the C-IBS group, IgG4 titres were significantly higher for pork (p=0.018) and chicken (p=0.038) compared to controls. D-IBS group had significantly greater IgG4 titres to wheat (p=0.023) compared to C-IBS group. The antibody titres to potatoes, rice, and eggs were not significantly different between the three groups.

Conclusion: Serum IgG4 antibodies to common food antigens like beef, pork, chicken, peanuts, wheat and milk are elevated in IBS patients. In keeping with the observation in other atopic conditions, this finding suggests the possibility of a similar pathophysiological role for IgG4 antibodies in IBS patients. The observation that the difference in antibody titres was predominantly observed in the D-IBS group further strengthens this theory.

093 IS THERE A ROLE FOR MMP19 IN GUT INFLAMMATION?

J.S.R. Jennings, P. Komolmit, P.J. Hamlin, N. Scott, A.F. Markham, P.A. Robinson, P.D. Howdle.Academic Unit of Medicine, University of Leeds, Clinical Sciences Building, St James's Hospital, Leeds LS9 7TF, UK

Introduction: The matrix metalloproteinases (MMPs) comprise a family of 26 enzymes, which remodel components of the extracellular matrix (ECM). There is evidence that MMPs 1,2,3 & 9 are over expressed in the gut in inflammatory bowel disease (IBD). The gene encoding MMP19 lies on chromosome 12q14 close to the IBD 2 susceptibility locus. We therefore investigated the expression of MMP19 protein in normal and diseased gut by immunohistochemistry.

Methods: Biopsy specimens were obtained at endoscopy from patients with Ulcerative Colitis (n=6), Crohns Disease (n=11), Colonic Carcinoma (n=2), Pseudomembranous Colitis (n=2), Coeliac Disease (n=3) and normal controls (n=15). Immunohistochemistry was performed on paraffin embedded sections using a polyclonal rabbit anti-human MMP19 hinge region antibody (Sigma) in accordance with standard techniques.

Results: In normal tissue, staining for MMP19 was observed in the cytoplasm of epithelial cells in surface and crypt mucosa. Within the crypt, we also identified cytoplasmic staining of cells of neuroendocrine origin. Some pigment was seen in the stroma and lamina propria, including the cytoplasm of mononuclear cells. This pattern was consistent in the rectum, colon, ileum, duodenum and stomach. In the colon, the cytoplasm of pericryptal fibroblasts was prominently stained. In the duodenum, the cytoplasm of Brunners gland cells was stained. The specimens from IBD and coeliac patients demonstrated increased staining of surface and crypt mucosal epithelial cells and stromal tissue. This was not observed in colonic carcinoma or pseudomembranous colitis. No differences were seen between crohns disease and ulcerative colitis.

Discussion: This is the first demonstration of MMP19 protein in the gut. We have identified expression in epithelial cells of the mucosa, stromal tissue including mononuclear cells and pericryptal fibroblasts. Increased expression of MMP19 was identified in IBD and coeliac disease.

094 ATTENUATION OF THE HEPATIC INFLAMMATORY RESPONSE TO PORTAL ENDOTOXAEMIA IN OBSTRUCTIVE JAUNDICE USING A NOVEL ANTI-ENDOTOXIN PEPTIDE.

M.A. Taylor1, J.M. Black1, A. Wallace2, M. Hoper1, W.D.B. Clements1, N.V. McFerran2, T. Diamond1, M.C. Regan1.1Department of Surgery and 2Centre for Peptide and Protein Engineering, The Queen's University of Belfast, Northern Ireland

Background: In obstructive jaundice (OJ), hepatic proinflammatory cytokines such as TNFα and IL6 are released in response to portal endotoxaemia. Exaggeration of this response may occur following a “second hit” such as surgical intervention, leading to organ dysfunction. The aim of this study was to develop novel anti-endotoxin peptides and assess their efficacy in reducing the hepatic proinflammatory response to a second hit of portal endotoxaemia in OJ.

Methods: Endotoxin specific peptides were generated using biopanning of a pVIII random linear phage library with Lipopolysaccharide from Salmonella minnesota Re995. A test peptide, P6, was developed which was shown to inhibit LPS-induced TNFa secretion by human monocytic cells. Bile duct ligation was performed on 9 Male Wistar rats who were randomised to receive either (a) endotoxin (LPS) alone (n=4) or (b) LPS + P6 peptide (n=5), during in-situ hepatic perfusion performed 1 week post surgery. Aliquots of effluent perfusate were collected for cytokine analysis at 80, 100 and 120 minutes.

Results: See table.

Abstract 094

Conclusion: This novel anti-endotoxin peptide offers an exciting new therapeutic strategy for preventing an exaggerated endotoxin-induced inflammatory response at the time of surgery in patients with OJ.

095 THE CD14+/CD16+ BLOOD MONOCYTE SUB-SET AND NOT GENETIC PRE-DISPOSITION INFLUENCES THE INCREASED SOLUBLE CD14 RECEPTOR EXPRESSION ASSOCIATED WITH SEVERE ACUTE PANCREATITIS

S.H. Rahman, G. Salter, M. Larvin, M.J. McMahon.Academic Unit of Surgery, The General Infirmary, Leeds LS1 3EA, UK

Background: The soluble form of CD14 (sCD14) is derived from a 55kDa membrane bound glycoprotein on monocytes, and enhances endothelial cytokine responses to lipopolysaccharides (LPS). It is a mediator of the systemic inflammatory response syndrome (SIRS). We investigated the role of sCD14 expression in the SIRS associated with acute pancreatitis (AP), to determine if altered expression was due to a C260T polymorphism in the CD14 promoter gene, or attributable to an altered monocyte sub-population.

Methods: Peripheral blood samples in patients with AP were assayed for sCD14 (24 and 72-hr from onset) and correlated with clinical severity (Atlanta Criteria), and SIRS (Acute Physiology Score, APS). Peripheral blood mononuclear cells (PBMC) were isolated to identify immunophenotypes using immunofluorescence flow cytometry. Leukocyte DNA was genotyped for the CD14 polymorphism using PCR.

Results: Severe AP (n=20) was associated with an early sustained increase in plasma sCD14 [median 67μgl-1 (R:25–216)] compared to mild attacks (n=70) [median:50μgl-1 (R:24–103), p<0.001), and healthy controls (n=40) [median:51μgl-1 (R:23–78), p<0.001]. Soluble CD14 strongly correlated with APS at 24hr (r2=0.38, p<0.001) and 72hr (r2=0.56, p<0.001). The proportion of monocytes in PBMC isolates was increased in severe attacks (p<0.05), but the early increase in CD14 only correlated with the relative expansion in the population of CD14+/CD16+ monocytes (r2=0.57, p<0.001). No differences in CD14 genotype were detected between 245 patients with AP (68 severe) and 143 controls even after stratification for disease severity.

Conclusions: Severe AP is associated with increased sCD14 expression that may be secondary to alterations in monocyte sub-sets, possibly in response to LPS, but appears not to be genetically pre-determined.

096 CHOLERA TOXIN (CT), ESCHERICHIA COLI HEAT LABILE TOXIN (LT) AND HEAT STABLE TOXIN (STA) HAVE A DIRECT EFFECT ON THE NEURONAL-LIKE PC12 CELL LINE

A.C. Casburn-Jones1, S.C. Barnett2, U.F. Fitzgerald2, M.J.G. Farthing1.1Dept Medicine, 2Dept Neuroscience, University of Glasgow, UK

Evidence exists that CT, LT and STa mediate intestinal secretion through an enteric neural reflex arc. Enterotoxins may activate a secretory response by binding directly to the enteric nervous system. To investigate the direct affect of these toxins on neurons we used PC12 cells, a neurogenic cell line derived from a rat phaeochromocytoma. PC12 cells have been used as a model system for neuronal differentiation and neurite outgrowth. After stimulation with nerve growth factor (NGF) PC12 cells change from a chromaffin-like to a neuronal-like phenotype.

Method: PC12 cells were grown on poly-L-lysine coated coverslips in 24–well plates in defined serum-free medium (termed SATO) for 24 h. CT, LT and STa, (0.01–2.0μg/ml), were added to each well, in the presence or absence of 50ng/ml NGF. Morphological effects were assessed 48–72 hours, in 10 fields per coverslip and recorded with a Zeiss Axiovert 100 camera. Only cells excluding trypan blue were included in the analysis.

Results: NGF induces neurite outgrowth: 60.7% of PC12 cells in the presence of NGF have neurite(s) =1cell diameter compared to 1.84% of control, (p<0.005). CT and LT alone induce morphological alterations similar to NGF-differentiated PC12 cells: CT and LT induce neurite outgrowth and branched spikes of tips of neurites. 1.0μg/ml CT and 1.0 μg/ml LT result in 16.4% and 25.8 % of cells bearing neurite(s) =1cell diameter, respectively, (p<0.005). STa alone has no significant effect on neurite outgrowth. Enterotoxins and NGF have an additive effect: 1.0μg/ml CT and 1.0 μg/ml LT increase neurite outgrowth with NGF by 55% and 15.4% respectively a (p<0.05). This effect was not seen with STa, however STa did increase cell diameter in the presence of NGF (p<0.05).

Conclusion: These observations support the hypothesis that enterotoxins have a direct effect on neuronal cells. CT and LT induce neurite outgrowth in PC12 cells and enhance the neuronal differentiation effects of NGF. STa does not induce neurite outgrowth alone but does appear to change the morphology of PC12 cells in the presence of NGF.

097 INTESTINAL DENDRITIC CELLS INCREASE T CELL EXPRESSION OF α4β7 INTEGRIN

A.J. Stagg1, M.A. Kamm2, S.C. Knight1.1Antigen Presentation Research Group, Imperial College at Northwick Park; 2St Mark's Hospital, London, UK

Integrin α4β7 binds to MadCAM-1 and contributes to homing of lymphocytes to mucosal tissues. Monoclonal antibodies to α4β7 ameliorate gut inflammation, indicating the functional importance of this homing. Circulating naïve T cells express low levels of α4β7 whereas memory T cells comprise α4β7+, primed in mucosal tissues, and α4β7- subsets. Differentiation of α4β7lo naïve cells into α4β7+ or α4β7- may be influenced by antigen presenting cells, such as the dendritic cell. Induction of α4β7 following activation of mouse cells with the APC-dependent stimulus soluble anti-CD3 was examined.

Methods: Flow cytometry using the dye CFSE, which segregates equally between daughter cells at each cell division, was used to distinguish responding cells. Cells expressing α4β7 were identified by double staining with the antibody DATK32 and absolute cell numbers determined using FlowCount fluorospheres.

Results: Almost all mouse T cells freshly isolated from mesenteric (MLN) and peripheral (PLN-axillary, brachial and inguinal) nodes stained weakly for α4β7 but a subpopulation became α4β7hi upon activation with anti-CD3 in a cell cycle- and accessory cell-dependent manner. Precursor frequency analysis revealed that a small proportion (1.6±0.5%) of the starting cells gave rise to α4β7hi cells after culture. Both the proportion and number of dividing cells expressing α4β7 was consistently greater for MLN than PLN (five experiments). Typically 2–3 fold fewer PLN than MLN were α4β7hi. Peyer's patch cultures displayed intermediate values. In crossover experiments using highly purified T cells, MLN DC induced significantly (p<0.01) more α4β7hi cells than PLN DC irrespective of the source of responding T cells.

Conclusions: In addition to their other immunoregulatory roles, dendritic cells can shape immune responses by influencing the homing of the lymphocytes they activate. Modulating lymphocyte migration via the activating DC may be useful in therapy of intestinal inflammation and development of mucosal vaccines.

098 STEROID ENHANCING EFFECT OF THE INTERLEUKIN-2 ANTAGONIST BASILIXIMAB ON LYMPHOCYTE STEROID SENSITIVITY

S.D. Hearing, C.J. Crowley, T.J. Creed, M.N. Norman, C.M. Dayan.University Division Medicine, Bristol Royal Infirmary, Bristol, UK

Up to 30% of patients with severe ulcerative colitis (UC) fail to respond to steroid therapy. We have previously shown that lymphocytes from these patients (and approximately 20% of normal subjects) have reduced sensitivity to steroids when measured in vitro1. Interleukin-2 (IL-2) is released by activated T lymphocytes and acts as a natural steroid antagonist, promoting proliferation and reducing apoptosis. We therefore hypothesised that basiliximab (BAS), a clinically well-tolerated chimeric monoclonal IL-2 receptor blocking antibody might enhance lymphocyte steroid sensitivity in resistant subjects.

Methods: Peripheral blood lymphocytes were isolated from 32 subjects (25 healthy volunteers and 7 patients with quiescent UC), on 41 occasions, using buoyant density centrifugation. Lymphocyte steroid sensitivity was assessed by measuring the antiproliferative effect of dexamethasone (DEX) on phytohaemagglutinin stimulated lymphocytes. Maximum steroid induced suppression was expressed as a % of control lymphocyte proliferation (Imax). Imax was measured in the absence and presence of BAS (1mg/L).

Results: The addition of BAS significantly enhanced the anti-proliferative effect of DEX (Wilcoxon signed-rank test, p < 0.0001) (see table). All 8 steroid-resistant subjects (Imax <60%) were modulated (in vitro) to steroid-sensitive by the addition of BAS.

Abstract 098

Conclusion: BAS has a marked in vitro steroid enhancing effect. As we have previously demonstrated an association between lymphocyte steroid resistance and poor response to steroids in severe UC1, this raises the possibility that BAS may be an effective adjuvant therapy in steroid-resistant subjects. A clinical trial of basiliximab in steroid-resistant UC is planned.

1Hearing et al (1999) Gut 45:382–8.

099 EXPRESSION OF TUMOUR NECROSIS FACTOR α (TNFα) AND LYSOZYME IN NECROTIZING ENTEROCOLITIS (NEC)

S.G. Lala, D. Turton, S. Keshav.Department of Medicine, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK

Background: NEC is an acute inflammatory disease of premature neonates. Risk factors for NEC include prematurity, bacterial colonisation, formula feeding and hypoxia, but its aetiology remains unknown. TNFα is a potent proinflammatory cytokine associated with the development of NEC in animal models. In neonates with NEC, plasma TNFα levels are not consistently elevated and do not correlate with disease severity. However plasma TNFα levels do not accurately reflect cellular activity. We therefore analysed TNFα gene expression in situ in intestinal tissue from neonates with severe NEC (Bell stage IIIB), and compared this with the expression of lysozyme, an antibacterial enzyme produced by Paneth cells in the small intestine.

Methods: We performed in situ hybridisation, using digoxigenin labelled riboprobes, on paraffin embedded intestinal tissue from eight neonates with NEC and four control subjects. Sense and anti-sense probes were used, and macrophages were identified immunohistochemically using an antibody to CD 68.

Results: The intestinal architecture was disrupted in all cases of NEC and necrosis, ulceration and haemorrhage were the dominant histopathological features. Increased TNFα expression was present in all cases of NEC (8/8) whilst normal intestinal tissue and control NEC sections showed no TNFα expression. TNFα expression was most intense in infiltrating macrophages in the lamina propria and around areas of pneumatosis intestinalis. TNFα expression was also noted in Paneth cells, epithelial cells and circulating macrophages and correlated with the histological severity of NEC. Lysozyme gene expression was noted in sections containing Paneth cells.

Conclusions: Increased TNFα expression characterises acute NEC and this expression is sustained in established disease. These findings support data implicating TNFα in the pathogenesis of NEC in animal models. Treatment of NEC with anti-TNFα monoclonal antibodies may therefore be justified in the setting of a controlled clinical trial.

100 A PRO-INFLAMMATORY CYTOKINE COCKTAIL INCREASES CCK RELEASE FROM STC-1 CELLS VIA AN EFFECT ON INTRACELLULAR CALCIUM

F.C. Leslie, J.T. McLaughlin, S. Kazmi, A. Varro2, G. Warhurst1, G.J. Dockray2, D.G. Thompson.GI Science and 1Gut Barrier Group, Hope Hospital, Manchester; 2Physiological Laboratory, University of Liverpool, UK

Background: How mucosal inflammation produces anorexia is poorly understood, modulation of enteroendocrine cell function is a plausible mechanism. Cholecystokinin (CCK) produces symptoms of nausea and satiety when infused parenterally and is involved in IL-1 induced anorexia.

Methods: The CCK secreting cell line, STC-1 was incubated with the pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α alone or in combination. CCK secretion was measured by radioimmunoassay. As CCK secretion is mediated by increases in intracellular calcium ([Ca 2+]i), changes in [Ca 2+]i in response to 70 mM K+ (a receptor-independent stimulus of secretion) were assessed using a dual wavelength ratio imaging technique (Fura-2). To look at effects on gene expression we studied the effects of combined cytokines on activity of CCK promoter-reporter constructs using a luciferase assay system and luminometry.

Results: Pre-incubation with the single cytokines IFN-γ, IL-1β and TNF-α for 2 hours produced small increases in basal CCK secretion. IFN-γ (6.25U/ml) produced a 38.8±4.6% increase in CCK secretion compared to control (p=0.02), IL-1β (0.125ng/ml) produced a 16.9±1.5% increase (p=0.005) and TNF-α (5ng/ml) produced a 13.7±1.83% increase (p=ns). However when a cocktail of IFN-γ, IL-1β and TNF-α was pre-incubated together this produced an increase of 109.4±24.0% over basal secretion after 2 hours incubation (p=0.001). This increase was maintained after 4 hours with a 70.7±24.7% increase (p=0.02) but diminished at 8 hours to a 43.6±8.0% increase (p=0.055). The cytokine cocktail had no effects on basal [Ca 2+]i, but pre-incubation for 2 hours produced a 23.9±2.9% (p=0.03) increase in the [Ca 2+]i response to K+ compared to control. However the cytokine cocktail did not stimulate CCK reporter-promoter activity during the same period of pre-incubation.

Conclusions: Short-term incubation with a cocktail of the pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α has a synergistic effect increasing basal CCK secretion in the STC-1 cell line. This occurs via a mechanism involving changes in intracellular calcium. This may have implications for symptom genesis, particularly anorexia in proximal GI inflammation.

Dr Leslie is sponsored by the Wellcome Trust & Dr McLaughlin by the DDF.

101 FUNCTIONAL IL-18 RECEPTOR EXPRESSION BY HUMAN INTESTINAL EPITHELIAL CELLS REQUIRED FOR INHIBITION OF CRYPTOSPORIDIUM PARVUM DEVELOPMENT BY IL-18

R.C.G. Pollok, V. McDonald, M.J.G. Farthing, M. Bajaj-Elliott.St Bartholomew's & The Royal London School of Medicine and Dentistry; University of Glasgow

Introduction: IL-18 is a cytokine with both Th1 polarising and proinflammatory actions. IL-18 binds the heterodimeric IL-18 receptor (R) to mediate its action. We have previously observed infection of intestinal epithelial cells (IECs) by the intracellular parasite C.parvum may be inhibited by proinflammatory cytokines (IFN-γ, TNF-α and IL-1β). We hypothesised IECs expressed functional IL-18R and parasite development could thereby be directly inhibited by IL-18.

Methods: mRNA transcripts of the receptor sub-units IL-1Rrp and AcPL and the adapter molecule MyD88 were measured by semi-quantitative PCR in the transformed IEC lines Caco-2, HCT-8, and HT-29 and freshly isolated human IECs. The specificity of the PCR reaction was confirmed by restriction digest or sequence analysis. The action of a panel of proinflammatory cytokines on receptor sub-unit expression by IECs was similarly determined. Finally, the action of exogenous IL-18 (2–200 ng/mL) on C.parvum development in IECs was studied using a previously described in vitro model of infection (Gastro 119: 1234).

Results: Transcripts of IL-1Rrp, AcPL and MyD88 were detectable in all transformed IEC lines tested. Expression of the receptor sub-units by isolated human IECs was variable and dependent on the origin of the cells. Restriction digests confirmed the specificity of the PCR products for the receptor sub-units. Functionality of the IL-18R expressed by HT-29 and HCT-8 cells was also demonstrated since exogenous IL-18 significantly inhibited parasite development (p<0.0001)

Conclusion: We describe for the first time functional expression of the IL-18R by both cultured and isolated human IECs. We speculate IL-18 has a previously unknown proinflammatory action on enterocytes and may be an important innate mucosal defence mechanism in the control of intracellular enteric pathogens. Further work is required to confirm this action in vivo.

RCGP funded by a Wellcome Trust Research Training Fellowship.

102 MORBIDITY AND MORTALITY REDUCTION USING NITAZOXANIDE IN ZAMBIAN CHILDREN WITH CRYPTOSPORIDIOSIS: A RANDOMISED CONTROLLED TRIAL

B. Amadi1, M. Mwiya1, J. Musuku1, A. Watuka1, S. Sianongo2, A. Ayoub4, P. Kelly3,5.1Dept Paediatrics, 2Dept Pathology and 3Dept Medicine, University of Zambia School of Medicine, Lusaka, Zambia; 4Dept Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK; 5Dept Adult & Paediatric Gastroenterology, St Bart's & Royal London School of Medicine & Dentistry, London, UK

Background: Cryptosporidiosis in children in the developing world causes persistent diarrhoea and malnutrition and is associated with increased mortality. Worldwide it affects children and the immunocompromised, and waterborne outbreaks can be very large, but there is no effective treatment. We conducted a randomised double-blind placebo-controlled trial to evaluate the safety and efficacy of nitazoxanide, a new broad-spectrum antiparasitic drug, in young children with diarrhoea caused by Cryptosporidium parvum.

Methods: HIV seronegative children age 12 to 35 months with cryptosporidial diarrhoea were admitted to the University Teaching Hospital, Lusaka, Zambia, and randomised to receive nitazoxanide (100 mg twice daily as an oral suspension for 3 days) or placebo.

Results: Fifty children were recruited for the study, and 47 with cryptosporidiosis confirmed at randomisation were included. 39 (83%) of these 47 children were malnourished. Seven days after initiation of therapy, diarrhoea had resolved in 14 (56%) of 25 children receiving nitazoxanide compared to 5 (23%) of 22 receiving placebo (p=0.037). Thirteen (52%) of 25 patients receiving nitazoxanide had 2 negative stool examinations for C. parvum between 7 and 10 days following initiation of treatment compared to 3 (14%) of 22 receiving placebo (p=0.007). Four children (18%) out of 22 in the placebo group died during the 10-day course of the study compared to none of 25 in the nitazoxanide group (p=0.041). Children receiving nitazoxanide did not experience significant adverse events.

Conclusions: A 3 day course of nitazoxanide significantly improved the resolution of diarrhoea, parasitological response and mortality, even in malnourished children.

103 SPECIFIC CYTOTOXIC T LYMPHOCYTE IMMUNITY AGAINST TELOMERASE IN COLORECTAL CANCER

L. Titu, R.L. Loveday, R.P. Baker, J. Greenman, J.T.T. Monson.Academic Surgical Unit, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, UK

Background: Characterisation of the cytotoxic T lymphocyte (CTL) activity against tumour antigens is crucial for understanding cancer immune surveillance mechanisms. Although telomerase activity is found in 90–100% of colorectal cancers, there is insufficient data on the prevalence of specific anti-telomerase immunity.

Methods: The CTL response against two HLA-A2-restricted epitopes of human telomerase reverse transcriptase (hTERT) was studied ex vivo in 16 HLA-A2+ colorectal cancer patients and 6 healthy subjects. An interferon gamma (IFN-?) ELISPOT assay was used to quantify the amplitude of the specific CTL response against telomerase, after incubation of peripheral blood mononuclear cells (PBMC) with hTERT peptides. PBMC incubated with or without phorbol 12-myristate -13-acetate (PMA) served as positive and negative controls, respectively. The level of the specific CTL response against HLA-A2-restricted peptides of Influenza A and CEA was also determined to allow comparison.

Results: Specific CTL against one of the two hTERT peptides were found in 6 (38%) of the 14 colorectal cancer patients, with 4 (25%) of these recognising both peptides. The frequencies of telomerase-specific CTL were between 15–45/106 PBMC. In addition, 11 (69%) patients presented CTL reactive with the Influenza A peptide and 5 patients (31%) had CTL that responded to stimulation with the CEA peptide. CTL active against telomerase were found both in patients with disseminated disease, as well as in patients with early disease. None of the healthy volunteers studied presented CTL capable of recognising telomerase and/or CEA antigens, but three (50%) recognised Influenza A.

Conclusions: The present study demonstrates that patients with colorectal cancer are capable of mounting a specific cellular immune response against telomerase antigens. Our findings suggest that the lack of an effective immune response against tumour cells that possess telomerase activity is not due to the absence of CTL directed against telomerase epitopes, but probably to a defective presentation mechanism for these antigens.

104 INCREASED MUCOSA-ASSOCIATED & INTRA-EPITHELIAL BACTERIA IN COLON CANCER

H.M. Martin, B.J. Campbell, C.A. Hart, J.M. Rhodes.Department of Medicine, University of Liverpool, Liverpool L69 3BX, UK

Introduction: It has been suggested that mucosa-associated bacteria may be important in the pathogenesis of colon cancer (Swidsinski et al. Gastroenterol.1998;115,281). Such bacteria might be recruited as a result of interaction between bacterial adhesins and the altered colonic mucosal glycoconjugates found in colon cancer and pre-cancer.

Methods: Mucosa-associated and intra-epithelial bacteria were isolated from biopsies taken from patients with colon cancer and from histologically normal controls using the gentamicin protection method. Bacteria were cultured on MacConkey agar, identified using API 20E bacterial identification kits and assayed for agglutination of sialidase-treated red blood cells which express the TF cancer-associated carbohydrate antigen, Galβ1–3GalNAcα-.

Results: A significant increase of mucosa-associated and intra-epithelial bacteria was found in colon cancer (see table). 73% of intra-epithelial bacteria were identified as being Gram-negative (mostly E. coli). Bacteria from 3 of the cancer cases, including 2 from the distant mucosa, but none of the normal controls, tested positive for TF adhesin activity. This is the first report of TF-binding intestinal bacteria in humans. Further studies on an isolate of TF-adhesin-positive non-pathogenic E. coli (HM44), which we have shown to lack conventional pathogenicity islands, from a colon cancer case show that it is able to invade HT29 human colon cancer cells.

Abstract 104

Conclusions: These results support the hypothesis that altered mucosal carbohydrate expression (such as TF antigen) in colon cancer and distant “unaffected” mucosa may lead to recruitment of E. coli. Bacteria which lack conventional markers of pathogenicity but which can invade intestinal epithelial cells could be relevant to carcinogenesis.

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