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Introduction: H.pylori is the causative agent of most cases of chronic superficial gastritis in humans and a risk factor for the development of peptic ulcer disease and gastric cancer. Our understanding of the early host response toH.pylori infection is limited. We and others have previously shown increased expression of the anti-microbial peptide hBD2 by gastric epithelial cell lines in response toH.pylori infection. In the present study we investigated the mRNA and protein expression of hBD1 and hBD2 inH.pylori-positive and -negative gastric biopsy samples.
Methods: Gastric biopsies were obtained from patients with H.pylori-positive (n=11) and H.pylori-negative (n=5) gastritis and control subjects (n=8). hBD1 and hBD2 mRNA was quantified by quantitative and semi-quantitative RT-PCR. Immunohistochemistry was performed on archival gastric paraffin-blocked samples from patients with H.pylori- and non-H.pylori-related gastritis.
Results: A marked increase in hBD2 mRNA expression was observed in both groups of gastritis compared to control tissue (H.pylori-positive, p=0.006;H.pylori-negative, p=0.02).The constitutive hBD1 mRNA expression observed in control tissue was also upregulated (H. pylori-positive p=0.035;negative group p=0.01). A parallel increase was also observed in hBD1and hBD2 peptides with the positive staining confined to the surface epithelium.
Conclusion: This is the firstin vivo study showing increased expression of hBD1 and hBD2 in H.pylori-positive and-negative gastritis. These results suggest an important role for these peptides in the innate host defence during inflammatory episodes and infection.
272. CD44 REGULATION AND FUNCTION IN ULCERATIVE COLITITS
We have shown that CD44v6 and CD44v3, variant isoforms of the cell surface glycoprotein CD44, are expressed on the baso-lateral surface of colonic epithelial cells in Ulcerative Colitis (UC) but not in other forms of colonic inflammation including colonic Crohn's Disease (CCD). The regulation of enhanced CD44 expression and the function of CD44 in UC have not been explained. CD44 is known to act as an adhesion molecule, to play a role in lymphocyte activation and to aid lymphocyte homing by binding and sequestering cytokines. Using FACS we have investigated the role of cytokines in inducing the expression of CD44v6 and v3 on the colonic cancer epithelial cell line HT-29. Using a novel lymphocyte adhesion assay we have gone on to investigate the role of CD44v6 and v3 in activated peripheral blood (aPBL) and gut lamina propria lymphocytes (LPL).
Results: IL-4 and IL-13 induced CD44v6 and v3 on HT-29 but IFN-γ, TNF-α, IL-1, 2,6,7,8,and 10 did not. Time course experiments showed that expression was maximal at 12 hours. Co-incubation of HT-29 and IL-4 with hydrocortisone, TNF-α, IFN-γ, IL-2 and 10 resulted in down regulation of CD44v6 and v3. Using a novel adhesion assay we studied the adherence of aPBL and LPL to HT-29 monolayers pre-treated with IL-4 to induce CD44v6 and v3 expression. Monolayers of HT-29 cells were cultured in 96 well plates in the presence of medium alone or medium supplemented with IL-4 to induce CD44v6 expression in test wells. Fluorescently labelled PBL or LPL were added to the test and control wells, allowed to adhere and then washed off. The effect of CD44 expression on PBL and LPL adhesion to HT-29 cells was determined by comparing the residual fluorescence in the presence or absence of CD44 induction. CD44 variant expression resulted in 2–3 fold increase in lymphocyte adhesion. This effect on adhesion could be blocked by pre-treating the HT-29 IL-4 wells with monoclonal antibodies known to block CD44 mediated adhesion.
Summary: Expression of CD44v6 and v3 on colonic epithelial cells is induced by IL-4. This effect can be competed out by TNF-α, IFN-γ, IL-2 and 10 and by hydrocortisone. CD44v6 and v3 mediate adhesion of aPBL and LPL to colonic epithelial cells. This effect can be blocked by antibodies to CD44 that may have a therapeutic role in UC.
273. FISH OIL SUBSTITUTED ELEMENTAL DIET IN ULCERATIVE COLITIS
Introduction: Elemental diet is an effective anti-inflammatory agent in Crohn's disease but not in ulcerative colitis (UC). We have recently shown that elemental diet increases the IL1-receptor antagonist/IL1β ratio in Crohn's disease, but not in ulcerative colitis in vitro. The lipid component of elemental diet is derived from coconut oil, canola oil and safflower oil. We have now investigated the effect of substituting fish oil (EF) for coconut oil, canola oil and safflower oil in elemental diet (EAA) in ulcerative colitisin vitro.
Methods: Colonoscopic rectosigmoid biopsies in six patients with UC were incubated in an organ culture model for 24 hours, by diluting elemental diet-fish oil (EF) or EAA (E028, SHS,UK) with modified Waymouth‘s complete medium (MB705/1) in dilutions of 1:20, 1:10, 1:5. Control was media alone without any added EF or EAA. The tissue viability was assessed by adding Bromodeoxyuridine (BrdU) to the culture fluid. The in-vitro uptake was confirmed by further immunohistochemical processing of the tissue and confirmation of BrdU labelled cells. The supernatant was collected after 24h and frozen at -70°C for immunoassay. ELISA was performed for pro-inflammatory (IL-1β) and anti-inflammatory (IL1-ra) cytokines in the stored supernatants.
Results: Incubation of the organ culture specimens for 24 hours with EAA in 1:20,1:10 and 1:5 dilutions did not increase the IL1ra/IL1β ratio. In contrast, the IL1-ra/IL1β ratio increased when organ culture specimens were incubated for 24 hours with EF. The median IL1-ra/IL1β ratio (range) was 8.14 (2.67–17.67) when incubated with media alone. This increased to 46.42 (2.2–127.3) with 1:20 EF (p=0.02), to 18.2 (13.6–215.4) with 1:10 EF (p=0.01) and to 43.1 (18.9–111.7) with 1:5 EF (p=0.04), (Mann-Whitney test).
Conclusion: Fish oil substituted elemental diet increases the anti-inflammatory to pro-inflammatory cytokine balance in vitro, unlike standard elemental diet in ulcerative colitis. Clinical trials may be warranted if palatability problems can be overcome. This study corroborates a recentin vivo study showing reduction of disease activity in distal proctocolitis by n-3 PUFA administration.
274. MORPHOMETRIC ANALYSIS OF MACROPHAGES CONTAINING INORGANIC PARTICLES IN INFLAMMATORY BOWEL DISEASE
Introduction: We have recently shown that microparticles can be transported across the intestinal epithelium. Such uptake of microparticles is more avid in inflamed epithelium compared to normal epithelium. We describe the presence and distribution of such particles within macrophages in IBD affected and normal intestine and nature of such particles.
Methods: Paraffin embedded sections from 12 surgically resected intestine (4 Crohn’s disease, 4 ulcerative colitis and 4 normal from unaffected segment of cancer) were stained with H & E and cells with particles were located. In confocal laser scanning microscope (LSM) non-biological nature of particles was determined. Electron spectroscopic imaging with X-ray microanalysis (EDX) with transmission electron microscopy (TEM) was done to determine the composition of the particles. Macrophages were detected in three step immunoperoxidase method using monoclonal antibody for CD68 antigen. Area of macrophages was measured under color detection system in image analysis (25 fields at x 400).
Results: Most macrophages in IBD, both Crohn's and UC contained inorganic particles which are non-biological. In contrast only a few particles were seen in one normal tissue. The particles were exclusively located in lysosomes within the cytoplasm of the macrophages. EDX analysis with TEM showed these particles to consist of titanium, silicon and aluminum. The area of tissue (μm2) occupied by CD68 stained macrophages was significantly higher in IBD affected tissues compared to normal tissues [mean± SE: Crohn's (858±57), UC (582±29) and normal (308±20), p<0.01]. Proportion of macrophages (mean ±SE) in the tissues was: Crohn's (6.5±0.4), UC (4.4±0.2 and normal (2.3±0.15), p<0.01.
Conclusion: IBD affected tissues contained particles of titanium, silicon and aluminum. This study confirms previous reports on presence of similar particulate material in IBD tissue and clearly shows that particles are exclusively present within the cytoplasm of macrophages. This study for the first time shows that macrophage area was increased in IBD-affected tissues, especially in Crohn's disease. Whether the presence of particles activates the macrophages to liberate pro-inflammatory cytokines and perpetuate inflammation requires further study.
275. GUT BACTERIA AND ULCERATIVE COLITIS—THE ENEMY WITHIN
Introduction: Although ulcerative colitis (UC) appears to be the results of unrestrained inflammatory response, the antigen (s) which initiates and perpetuate this chronic, relapsing disorder remain uncertain. It has been suggested that ubiquitous luminal bacterial products are somehow involved in the pathogensis of this disease. Furthermore, compelling evidence has been provided that not the mere presence of bacteria per se that induces colitis, but certain bacteria are needed, such asBacteroides spp. We tested this hypothesis by using human model of patients with ileal pouch-anal anastomosis (IPAA, these patients virtually have no colon) and with active UC with recently developed physiologically representative method. We have also assessed the biological potentials of the bacterial products by using peripheral blood mononuclear cells (PBMC).
Methods: 19 patients with IPAA [10 with normally functioning pouch (NFP), median age 39 (28–73) and 9 patients with pouchitis, median age 36 (28–74) and 11 patients with active UC, median age 29 (19–66) as well as 12 age-matched healthy controls were investigated. All patients and controls underwent whole gut lavage (WGL) using polyethylene glycol-based solution drunk at a standard rate of 1litre/h. The first clear effluent was collected and processed. IgA antibody against B. fragilis and a ‘cocktail’antigen [(Endotoxin core antigens were prepared from a range of Gram-negative bacteria (E. coli,Klebseilla pneumonae,Pseudomonas aeruginosa, andSalmonella typhi)] were measured by ELISA. TNF-α and IL-8 production were assessed by bioassay and radio-immunoassay respectively.
Results: The median value of IgA EndoCAb (antibody against endotoxin core antigens) in UC patients were significantly higher than that of controls and NFP (p<0.004 and p<0.003 respectively). Likewise, IgA EndoCAb concentration was significantly higher in pouchitis than that of NFP (p<0.008). The IgA antibody against B. fragilis in UC and pouchitis were significantly higher than that of NFP (p<0.04 and p<0.02). Both groups of bacterial antigens induces considerable amount of TNF-α and IL-8 in PBMC.
Conclusion: The study confirms that there is an unrestrained immune response against luminal bacterial antigens in the gut mucosa in patients with UC. The luminal antigens can induce inflammatory cytokines. Breakdown of tolerance towards anaerobic Gm-ve colonic bacteria may be a pathogenic mechanism of UC.
276. REGULATION OF CHEMOTAXIS BY ALPHA CHEMOKINES AND THEIR RECEPTORS IS ABNORMAL IN ULCERATIVE COLITIS
Introduction: Alpha chemokines are molecules produced by activated gastrointestinal mucosa, vascular endothelium and circulating leukocytes which regulate chemotaxis of leukocytes into areas of inflammation. This study examines the contribution of alpha chemokines and their receptors to chemotaxis in patients with ulcerative colitis (UC).
Methods: The cellular expression of alpha chemokine receptors CXCR1 and CXCR2 on circulating leukocytes was measured by flow cytometry using conjugated monoclonal antibodies. An in-vitro chemotaxis assay was used to quantify the effects of alpha chemokines produced by UC patients and healthy volunteers, together with the effects of CXCR1 and CXCR2 blockade by purified monoclonal antibodies.
Results: Polymorph expression of CXCR1 and CXCR2 in UC patients (n=32) was significantly lower than in controls (n=15) but similar in lymphocytes and monocytes. UC patients with inactive disease had no significant difference in receptor expression compared to those with active disease (p>0.1). Interleukin-8 was found to be the most potent chemokine for all groups, but was significantly less active in UC than in controls (p<0.02). Chemotaxis mediated by interleukin-8, GRO-alpha and ENA-78 was significantly reduced by CXCR1 and CXCR2 blockade (67/42/25% and 35/48/38% respectively).
Conclusions: Patients with UC demonstrate reduced expression of alpha chemokine receptors on polymorphs and this does not appear to be a consequence of disease activity. Furthermore, the response to IL-8 is attenuated in UC patients. Therapeutic inhibition of CXC receptors in ulcerative colitis warrants further investigation.
277. ALPHA-DEFENSIN EXPRESSION IN HUMAN JEJUNUM VARIES IN HIV INFECTION AND TROPICAL ENTEROPATHY
Introduction: Paneth cells secrete α-defensins, endogenous antimicrobial peptides, but the extent to which expression varies in human populations or disease states is unknown. We set out to determine whether expression of human defensins (HD) 5 and 6 varies with HIV or parasitic infection or correlates with mucosal structure or function in jejunal biopsies from 84 adults participating in an ongoing study of tropical enteropathy in a poor urban community in Lusaka, Zambia. Approval for the studies was obtained from two ethics committees.
Methods: HD5 and HD6 mRNA expression was evaluated by quantitative RT-PCR, and peptide expression by immunohistochemistry using anti-HD5 kindly provided by Drs. Ganz and Porter (UCI, Irvine, CA). Mucosal morphometry was performed on well-orientated formalin-fixed sections.
Results: mRNA expression (log transcripts per μg total RNA) varied from undetectable to 7. HD5 (but not HD6) mRNA was lower in HIV seropositive adults (median 5.0, interquartile range 0–6.1) than in HIV seronegative adults (5.7, 4.9–6.9; p=0.01). HIV seropositive adults were also more likely to have undetectable HD5 mRNA than seronegative adults (OR 4.0, 95%CI 1.4–1.2; p=0.008). HD5 mRNA (but not HD6) was inversely correlated with both villous height and epithelial surface area by rank correlation (ρ=-0.41, p=0.003) but only in HIV seronegative adults. Correlation between HD5 and HD6 expression increased with increasing grade of enteropathy (ρ=0.04 in minimal but ρ=0.85 in severe enteropathy). Expression did not differ in the presence or absence of parasitic infection. By immunohistochemistry peptide levels varied less between samples and no correlation was found with mRNA expression.
Discussion: HD5 expression is altered in enteropathy and HIV infection, but the regulatory mechanism(s) remain to be identified.
278. INTERLEUKIN-10 AND TGF-β1 REGULATE INTERFERON-γ-MEDIATED INHIBITION OF CRYPTOSPORIDIUM PARVUMDEVELOPMENT IN ENTEROCYTES
Introduction: Activation of macrophages by interferon (IFN)-γ to kill intracellular pathogens is down-regulated by interleukin (IL)-10, transforming growth factor (TGF)-β1 and IL-4. IFN-γ also activates enterocytes to inhibit development of intracellular pathogens, but the mechanisms regulating this are poorly understood. This investigation examined the modulatory effect of IL-4, IL-10 and TGF-β1 on IFN-γ-mediated inhibition of development of Cryptosporidium parvum.
Methods: The human enterocyte cell line, HT 29, was grown to confluence in 24-well plates at 37oC. Human recombinant IFN-γ with or without either IL-4, IL-10 or TGF-β1 was added to cell cultures starting 24h before infection (-24h) with C. parvum; alternatively, IFN-γ was added at -48h and the modulatory cytokine introduced at -24h, or vice versa. Tweny-four hours after infection with oocysts the monolayers were fixed with methanol and stained with Giemsa so that numbers of intracellular parasites could be determined microscopically.
Results: Treatment of enterocytes with IFN-γ starting 24h or 48h before infection inhibited parasite reproduction by 60–70%. Addition of TGF-β1 or IL-10 to cell cultures at the same time as, but not 24h after, IFN-γ reduced the inhibitory activity of IFN-γ in a dose-dependent manner. In contrast, IL-4 had no significant effect on IFN-γ activity.
Discussion: These results suggest that IL-10 and TGF-β1, but not IL-4, may be important in down-regulation of Th1-mediated activation of gut epithelium infected by intracellular pathogens. Once activated by IFN-γ, however, enterocytes may be refractory to modulation by IL-10 or TGF-β1.
279. MAP KINASE INHIBITION REVERSES CRYPTOSPORIDIUM PARVUM INDUCED IL-8 SECRETION BY ENTEROCYTES
Introduction: C.parvum is a major cause of diarrhoea world-wide. The parasite has previously been shown to induce the expression of the C-X-C chemokine IL-8 by infected enterocytes. Mitogen-activated protein (MAP) kinase activation is important in signalling the regulation of inflammatory gene expression in response to a variety of external stimuli including pathogens. We determined the role of MAP kinases in mediating infection and in modulating the induction of IL-8 by enterocytes infected withC.parvum using selective inhibitors in our established in vitro model.
Methods: Cells of the intestinal cell line HCT 8 were grown to confluence in 24 well plates. Cells were treated with either non-selective MAP kinase inhibitor apigenin (Apig, 25μM), selective p38 kinase inhibitor SB203580 (20μM), selective MEK inhibitor PD98059 (50μM), the inactive related compound SB202474 (negative control, 20μM), or medium alone with or without DMSO. Treated cells were infected with C.parvum(0.25–2x106 oocysts or purified sporozoites/well) and incubated 24–48h. Infection was quantified by immunofluorescence assay, using an anti-C.parvum polyclonal antibody 24h after parasite inoculation. ELISA was used to quantify IL-8 protein in supernatants collected 48h after parasite inoculation.
Results: C.parvuminduced an inoculum-dependent increase in IL-8 in serum-starved HCT 8 cells, with a peak at 48h and 1x106 sporozoites (2629.5 pg/mL, ± 90.1) compared with untreated control.s (1334.1 pg/mL, ± 21.6). MAP kinase inhibitors did not modify infection of HCT 8 cells. However MAP kinase inhibition was found to partially reverseC.parvum induced IL-8 induction (Apig 83.2 % ± 0.4, p<0.01; SB203580 50.0 % ± 4.2, p<0.05; PD98059 35.7 % ± 4.9, p<0.05).
Discussion: Our findings indicate that MAP kinase inhibition may partially reverse the induction of IL-8 byC.parvum in vitro. In contrast MAP kinase inhibition had no action on parasite infection of enterocytes. We hypothesise that MAP kinases function as upstream mediators of NF-κB activation and resultant IL-8 gene induction followingC.parvum enterocyte infection.
Acknowledgements: RCGP is a Wellcome Trust Training Fellow.
280. PRO-INFLAMMATORY CYTOKINES INHIBIT CRYPTOSPORIDIUM PARVUMINFECTION OF ENTEROCYTES
Introduction: We have previously shown IFN-γ may directly inhibit C.parvuminfection of human intestinal cell lines. In this study we have examined the action of other pro-inflammatory cytokines, IL-1β, TNF-α, IFN-α, IL-15, and the anti-inflammatory cytokine TGF-β1 on parasite infection of enterocytes in our established in vitro model. These cytokines may all be produced by enterocytes and are therefore potential mediators of the innate mucosal host immune response to infection.
Methods: HT29 intestinal cells grown on coverslips were infected with C.parvumoocysts (2x105 oocysts/well) as previously described. Cell were pre-treated with IFN-α_(0.01–1 ng/mL); IL-15 (1–100ng/mL); IL-1β (0.1 ng/mL); TNF-α (1ng/mL); or TGF-β1 (5 ng/mL) for 24h prior to parasite inoculation. Infection was quantified by immunofluorescence assay, using an anti-C.parvum polyclonal antibody and Giemsa staining. Inhibition or enhancement of infection in the treated wells was calculated as percentage of infection in untreated controls.
Results: All of the pro-inflammatory cytokines assessed had an inhibitory effect on parasite infection. IFNα maximal inhibition was 35.3% ± 10.7 (ANOVAp<0.001). IL-15 maximal inhibition was 40.1% ± 8.1 (ANOVA p<0.001). IL-18 maximal inhibition was 31.1% ± 7.1 (ANOVAp<0.001). IL-1β inhibition was 33.0 % ± 8.1 (p<0.001). TNF-α inhibition was 61.0 % ± 7.8 (p<0.01). In contrast, TGF-β1 enhanced infection by 36.6 % ± 7.8 (p<0.05).
Discussion: We have demonstrated that a range of pro-inflammatory cytokines may inhibit in vitro infection of intestinal epithelial cells by the parasiteC.parvum. Production of these cytokines by the enterocyte offers a potential innate mechanism of of mucosal immunity to infection.
Acknowledgements: RCGP is a Wellcome Trust Research Fellow.
281. THE ROLE OF IL-12 AND IFN-γ IN IMMUNITY TO CITROBACTER RODENTIUM INFECTION
Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC)Escherichia coli are important human pathogens. The formation of “attaching and effacing” (A/E) lesions on gut enterocytes is central to the pathogenesis of EHEC and EPEC, a feature shared with the non-invasive enteric mouse pathogenCitrobacter rodentium. AlthoughC. rodentium is a non-invasive pathogen, previous studies (Higgins et al, Infect Immun 67:3031) have shown that infection is associated with a mucosal Th1 immune response. We have therefore examined the role IL-12 and IFN-γ in host defense againstC. rodentium infection. Following infection, IL-12p40 and IFN-γ deficient mice had higher numbers of C. rodentium in mucosal and systemic tissues than wild type mice, and 25% of the IL-12 deficient mice died. The striking features of the pathology in IL-12 deficient mice were firstly, patchy lesions present in the mucosa, with some areas showing a huge infiltrate of CD4+ cells and mucosal hyperplasia, and other areas with few CD4+ cells and no colonic hyperplasia; secondly bacteria were seen at the base of the glands in the IL-12 deficient mice compared to controls, which correlated with the absence of epithelial mouse β-defensin 3 expression. IFN-γ and TNF-α mRNA transcripts were also seen in the colon of IL-12 deficient mice although phosphorylated Stat4 and mature IL-18 were not increased. These studies are the first to show an important role for IL-12 and IFN-γ in limiting non-invasive bacterial infection of the colonic epithelium, and also demonstrate that Th1 activation in the gut wall can occur in the absence of IL-12 and IL-18.
282. URINALYSIS FOR INTERLEUKIN-8 IN THE NON-INVASIVE DIAGNOSIS OF ACUTE AND CHRONIC INTRA- AND EXTRA-ABDOMINAL INFLAMMATORY CONDITIONS
Background: In preliminary studies, we have shown that interleukin-8 (IL-8) levels rise in patients with active inflammatory bowel disease. To explore its diagnostic potentials, we measured urinary IL-8 levels in a range of acute and chronic inflammatory conditions , in both adults and children.
Methods: IL-8 was measured by ELISA in random urine samples (1 ml each), carrying code numbers, and taken from 208 patients: 177 adults and 31 children. Active inflammation was diagnosed clinically, with the aid of acute phase responses and, where appropriate, using radiology, endoscopy, and histology.
Results: In adults, the median urinary IL-8 level was 75 pg/ml in those with active chronic inflammatory bowel disease (n=25) compared with 20 pg/ml in patients with inactive disease (n=28), and 9 pg/ml in controls (n=14; P=0.001). Patients with active chronic rheumatoid arthritis (n=21) had a median IL-8 level of 93 pg/ml, compared with 11 pg/ml in those with inactive arthritis (n=22; P=0.001). The median IL-8 value in subjects with non-specific abdominal pain or constipation (n=20) was 8 pg/ml compared with 107 pg/ml in patients with acute cholecystitis (n=15; P<0.0001), 127 pg/ml in those with appendicitis (n=8; P=0.0001), 584 pg/ml in patients with urinary tract infection (n=12; P<0.0001), and 191 pg/ml in those with diverticulitis or gastroenteritis (n=12; P=0.0001). Children with non-specific conditions (n=10) had a median IL-8 level of 10 pg/ml compared with 199 pg/ml in those with non-viral inflammation/ infection (n=12; P=0.0001), and 7 pg/ml in patients with viral upper respiratory tract infection (n=9; P=0.7). In the study group as a whole, urinary IL-8 values correlated positively with peripheral blood white cell count, erythrocyte sedimentation rate, and C-reactive protein.
Conclusions: Urinary IL-8 measurement helps in the non-invasive assessment of active inflammation. It is simple and has the potential to be used by both clinicians and patients.
Acknowledgements: The work is protected by an international patent application.
283. INCREASED SUSCEPTIBILITY OF TNFRP55 DEFICIENT MICE TO CITROBACTER RODENTIUM(MOUSE EPEC) INFECTION
Infection of mice with the intestinal bacterial pathogenCitrobacter rodentium (murine EPEC) results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of IBD. In these latter models, and in patients with Crohn's disease, neutralisation of TNF-α is of therapeutic benefit. Since there is no information on the role of TNF-α in either immunity to non-invasive bacterial pathogens such as Citrobacter, nor in the role of TNF-α in infectious colitis, we have investigated Citrobacter infection in TNFRp55-/- mice. In wild-type C57BL/6 mice Citrobacter infection does not alter weight gain, despite the presence of a high bacterial burden in the colon. In contrast, in TNFRp55-/- mice, there are higher bacterial burdens, but weight loss only occurs late in infection. Bacteria are however cleared showing that TNF-α is not needed for protective anti-bacterial immunity. The most striking feature of infection in TNFRp55-/- mice however is the markedly enhanced pathology, with increased mucosal weight and thickness, increased T cell infiltrate and a markedly greater mucosal Th1 response. The enhanced bacterial burden is probably due to the increased mucosal surface area in the TNFRp55-/- mice. To help explain the enhanced Th1 associated immunopathology we examined IL-12 expression. IL-12 p40 transcripts were markedly elevated in Citrobacter infected TNFRp55-/- mice and this was associated with enhanced STAT4 phosphorylation. TNF-α may therefore play a protective role in the intestine in response to bacterial infection by down-regulating tissue-damaging Th1 responses.
284. IN VITRO MODELLING OF INTESTINAL BACTERIAL COMMUNITIES COLONISING MUCIN
The large intestine is covered with a protective mucus coating that harbours bacterial populations believed to be distinct from those in the gut lumen. While little is known of their composition or metabolic activities, they are likely to play an important role in mucus breakdown.
Aims: To investigate the formation of mucin biofilm communities, and to study physiological and enzymological factors that enable mucin degrading species to compete for substrates in biofilms.
Methods: Colonisation of mucin gels by faecal bacteria was studied using a two-stage chemostat, simulating conditions of nutrient stress characteristic of the proximal (vessel 1) and distal (vessel 2) colons. Establishment of bacterial communities in the gels was investigated using selective culture methods, scanning electron microscopy and confocal laser scanning microscopy, in association with fluorescently-labelled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin degrading enzymes and measurements of residual mucin sugars (Dionex).
Results: Mucin gels were rapidly colonised by heterogeneous bacterial populations, especially members of theB. fragilis group, enterobacteria and clostridia. Planktonic anaerobe: facultative anaerobe ratios were 5000:1, whereas in the mucin gels, this dropped to 3:1 (vessel 1) and 6:1 (vessel 2), which is similar to the rectal mucosa. N-Acetyl neuraminidase, β-galactosidase and N-acetyl β-glucosaminidase rapidly appeared in the gels. Apart from N-acetylgalactosamine in vessel 1, all mucin oligosaccharide sugars were digested by colonic bacteria. Destruction of mucin was most extensive by organisms growing under strongly carbon-limited conditions in vessel 2. Galactose and N-acetyl glucosamine were utilized most effectively. Acetate and butyrate production by planktonic bacteria in vessels 1 and 2 was more rapid than in microbial biofilm communities, which mainly produced acetate.
Conclusions: Intestinal bacterial populations colonising mucin surfaces in the chemostats were shown to be phylogenetically and metabolically distinct from their planktonic counterparts. In vitro modelling of the microbiota in this way has wider applicability, for example, in studying the effects of antibiotics and drugs on gut biofilms.
285. MIGRATION OF PRECURSORS OF INTRAEPITHELIAL LYMPHOCYTES (IELS) FROM HUMAN COLONIC LAMINA PROPRIA (LP)
Background: Together with epithelial cells, IELs represent the first cells of intestinal defence. IELs express the integrins αEβ7 and a restricted repertoire of αβ T-cell receptors (TCR), as shown by restricted variable (V) region usage of the TCR β-chain (Vβ). In addition to V region, TCR αβ genes also consist of diversity (D), joining (J), and constant (C) regions. The epithelial basement membrane contains many discrete pores through which IEL precursors migrate in vivofrom the lamina propria (LP) into the epithelium. Using anex-vivo model, we have investigated the migration of IEL precursors from the colonic lamina propria.
Methods: Human colonic mucosal samples (n=11) were denuded of the epithelium and placed in medium. T cells migrating out of the lamina propria (via basement membrane pores) were collected after 1 h and 16 h culture and studied by FACS and quantitative PCR (for ratios of αE:TCR Cα mRNA). TCR Vβ region usage was investigated by spectratyping using 24 family-specific PCRs. The number of peaks (reflecting the sizes of VDJ regions in the PCR products) for each member of TCR Vβ family was determined. Data are expressed as mean (±SEM).
Results: A greater proportion of CD8+ T cells expressed αE integrin than CD4+ cells [0–1 h culture: 38.1(±5.4)% vs 13.8(±5.4)%, p=0.005 and 1–16 h culture: 38.0(±5.1)% vs 11.8(±2.7)%, p<0.001]. The ratio of αE:TCR Cα mRNA transcripts in cells migrating out of the lamina propria over 0–1 h culture of denuded mucosal samples was significantly greater than in cells collected after the 1–16 h culture period [8.7(±2.2) vs 1.8(±0.5); p=0.024]. Amplified TCR Vβ transcripts of cells migrating over 0–1 h culture period showed fewer peaks than those migrating over 1–16 h period [3.1(± 0.3) vs 6.4(±0.3); p<0.001].
Conclusions: Following loss of the surface epithelium, majority of the αE-expressing T cells migrating out of the human colonic lamina propria are CD8+. Cells migrating out of the lamina propria during the 1st h of culture express higher levels of αE transcripts and show a restricted repertoire of TCR Vβ. These cells are likely to be IEL precursors.
286. A ROLE FOR PLATELET ACTIVATING FACTOR AND TUMOR NECROSIS FACTOR IN THE REGULATION OF MMP'S IN THE GUT; A POTENTIAL EXPERIMENTAL MODEL FOR NECROTISING ENTEROCOLITIS
Introduction: Necrotising enterocolitis is a major cause of morbidity and mortality among pre-term infants, occurring in up to 10% at all neonatal intensive care unit admissions. The symptomatic spectrum of NEC can range from mild intestinal ischemia to severe bowel infarction and necrosis in addition to a range of systemic symptoms. Many studies have implicated MMPs in tissue injury in the gut, in particular stromelysin-1 (MMP-3) which is produced in excess by cytokine and mediator activated stromal cells. Several pro-inflammatory mediators and cytokines [Tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and platelet activating factor (PAF) and IL-1β and IL-8], have been detected in blood and in surgical specimens from infants with NEC.
Aim: To set up a human model for NEC and to ascertain the effects for PAF and TNFα on the expression levels of key MMPs.
Materials and methods: Human foetal gut explants and a foetal gut derived myofibroblast cell line were cultured and stimulated with PAF-16 and TNFα and the culture supernatents analysed by western blotting.
Results: Under basal conditions the expression of MMPs 1, 3 and 9 are low. Upon the addition of PAF the levels of MMPs 1, 3 and 9 increase moderately in a dose dependent fashion with the highest level of expression being achieved under physiological conditions (18ng/ml). When a physiological dose of TNFα alone is added, the levels of MMP 1, 3 and 9 increase beyond the levels observed with PAF alone. However, PAF plus TNFα, synergise to dramatically increase the levels of MMP expression beyond the levels observed when either factor is used alone. In contrast, MMP-2 and TIMPs remain constitutively expressed. This profile has been observed in both foetal gut explants and myofibroblast cells.
Conclusions: PAF and TNFα act synergistically as potent mediators of MMP expression. This suggests a potential role for MMPs in disease-associated tissue destruction in an experimental model of NEC.
287. EXPRESSION AND FUNCTIONS OF PURINERGIC RECEPTOR P2X7 IN COLONIC MACROPHAGES AND T CELLS FROM NORMAL AND INFLAMMATORY BOWEL DISEASE MUCOSA
Introduction: P2X7receptor is an ATP-gated ion channel which can form large membrane pores and has been implicated in cytokine release and apoptosis in inflammatory cells. We therefore investigated its expression, activation and inhibition in normal and diseased colonic macrophages and T cells.
Methods: Expression of P2X7receptor mRNA was studied by RT-PCR. Concentration-dependent changes in pore formation (ethidium bromide influx), apoptosis (annexin V staining) and IL-1β release (sandwich ELISA) were used as functional assays. Ultra-structural changes were studied by transmission electron microscopy.
Results: P2X7 receptor mRNA was present in all colonic macrophages and T cells studied. Pore formation and apoptosis were induced by purinergic ligands, with a potency order typical for P2X7 receptor agonists (BzATP>ATP>2MeSATP). The concentration of ATP to induce 50% apoptosis was 84 μM for macrophages and 20 μM for T cells respectively (n=4). ATP-induced apoptosis could be specifically inhibited both by apyrase hydrolysis or by the P2X7 receptor antagonist, oxidised ATP, KN-62 and by P2 pan receptor antagonist PPADS, but not the P2Y receptor antagonist. Functional evidence of apoptosis and pore formation was associated with loss of cell membrane continuity. In LPS-primed macrophages, ATP caused capase-1 activation and rapid concentration-dependent release of mature IL-1β, which was inhibited by o-ATP (IC50=30 μM). The proportion of unstimulated macrophages and T cells recovered from ulcerative colitis (72%, n=5) and Crohn's disease (70%, n=4) tissues that were apoptotic was significantly higher than for normal (32%, n=8), and not further increased by BzATP. o-ATP reduced the spontaneous IL-1β release in IBD samples.
Conclusions: Expression and functional data support the presence of P2X7 receptor on colonic mucosal macrophages and T lymphocytes. This may play a role in the immunopathology of inflammatory bowel diseases.
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