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old TPH2 KO mice (n = 8) and their WT littermates (n = 8). The severity of TNBS-inducedcolitis (mortality; clinical scores, weight loss) was significantly greater in TPH2 KO than inWT animals. The enhanced severity was also evident in enteric histological scores, leukocyteinfiltration, and cytokine expression. These data are compatible with the ideas that bypromoting inflammation, mucosal 5-HT enhances the defense of the bowel against infection,while neuronal 5-HT makes such an effect less damaging by protecting the ENS frominflammation. Mucosal and neuronal 5-HT may thus exert antagonistic effects on inflamma-tion that are functionally synergistic. Supported by a clinical and translational pilot grantfrom Columbia University (KGM) and NS12969 (MDG).
RAGE-Dependent S100B Protein Modulation of Peripheral and MucosalImmune Cells' Functions in Patients With Ulcerative ColitisCarla Cirillo, Giovanni Sarnelli, Fabio Turco, Alessandra D'Alessandro, Annamaria Mango,Rosario Cuomo
Background and Aim: Glial cells in the gut participate to the local inflammatory responsestriggered by a variety of insults. Whether glial cells are directly affecting the responses ofimmune cells during intestinal inflammatory diseases is still unknown. We aimed to investig-ate the ability of glial-derived S100B protein in mediating immune cells' functions in patientswith ulcerative colitis. Material and Methods: Mucosal immune cells (MIC) were isolatedfrom rectal mucosal biopsies of 10 patients with ulcerative colitis (mean age 47; 6 male and4 female) and then characterized by Flow cytometry technique. In the same subjects,peripheral blood mononuclear cells (PBMC) were isolated. Both MIC and PBMC werestimulated with exogenous S100B protein (0.05-5μM) to evaluate cells' proliferation (bymethylthiazolydiphenyl-tetrazolium bromide conversion assay, after 72h) and responses [bymeasuring interferon gamma levels (IFN-gamma), inducibile nitric oxide synthase (iNOS)and tumor necrosis factor-alpha (TNF-alpha) proteins expression, each after 24h]. Moreover,in the same experimental conditions, we evaluated the expression of the receptor for advancedglycation endproducts (RAGE) both in PBMC and MIC, in order to evaluate if its expressioncanmodulate S100B-mediated effects on immune cells. Results: In PBMC, S100B protein 5μMdid not significantly affect cells' proliferation, but it induced a significant and concentration-dependent increase of IFN-gamma level (89±5 vs 75±6 ng/mL, p<0.05), and of both iNOSand TNF-α protein expression (+32±4% and +41±9% vs. unstimulated, p<0.05 andp<0.01respectively). This finding was associated with the significant increase of RAGEprotein expression after S100B addition (+52±10% vs unstimulated, p<0.01). In MIC, similarconcentrations of S100B protein did not affect cells' proliferation, but, conversely to PBMC,induced a significant and concentration-dependent decrease of IFN-gamma level (42±7 vs61±4 ng/mL, p<0.01), and of both iNOS and TNF-α protein expression (-34±13 and -70±2%vs unstimulated, p<0.05 and p<0.01, respectively). This finding was associated with asignificant decrease of RAGE protein expression after S100B challenge (-52±14% vs unstimu-lated, p<0.05). Conclusions: Although further studies are needed to explain the differentactivity pattern of peripheral and intraepithelial immune cells, we show that enteroglial cellsparticipate to the immune responses in the inflamed gut. We also suggest that S100B-RAGEinteraction represents a crucial mechanism involved in the modulation, either positive ornegative, of immune cells' functions in ulcerative colitis.
Antibodies to an 18-Residue Peptide of Cdt Are Found in the Serum of Rats ina Model of Post-Infectious IBSWalter Morales, Venkata B. Pokkunuri, Jaekyu Sung, Gene Kim, Emily Rooks, ZacharyMarsh, Stacy Weitsman, Patricia Guerry, Christopher Chang, Mark Pimentel
In a recently developed rat model of post-infectious IBS, inoculation with Campylobacterjejuni resulted in chronic altered stool habits and decreased Interstitial Cells of Cajal of thedeep muscular plexus (DMP-ICC). In contrast, rats infected with C. jejuni mutants that failto express cytolethal distending toxin (CDT) had significantly reduced long term effects.Immunohistochemistry with anti-CDT antibody produced high-affinity staining of gut-neuralelements in cross sections of rat ileum, both in previously infected rats, as well as in ratsnever exposed to C. jejuni. This result suggests that anti-CDT-B antibodies may be cross-reacting to a host protein on neuronal elements. In this study, we investigate the developmentof serum antibodies to CDT-B in rats infected with C. jejuni. Methods: Adult male SpragueDawley rats were orally gavaged with 108 CFU of C. jejuni. Three months after clearanceof infection, rats were euthanized, and cardiac blood samples obtained. Another group of20 adult, male Campylobacter-naïve rats served as controls. Serum was separated fromwhole blood by centrifugation. Immunoblots were performed by spotting 10 ul of an 18-residue CDT-B peptide, along with 5 ul of C. jejuni whole cell lysate onto 3 individualImmobilon Psq (Millipore) transfer membranes. Membranes were blocked with 5% non-fatdried milk in TBS-T for 1 hour at room temperature. Each membrane was then incubatedwith a different serum or primary antibody in 0.1% TBS-T as follows: 1:1000 serum fromSprague Dawley rats inoculated with C. jejuni, 1:1000 serum from C. jejuni naive controlSprague Dawley rats, and 1:3000 of a purified antibody against the 18-residue CDT-B peptide(Anaspec). Antibody adherence was detected using a 1:15000 dilution of a Rat IgG-HRP(Millipore), with antibodies incubated in 1:1500 dilution of a rabbit IgG-HRP (GEHealthcare)followed by incubation with Amersham ECL Western blotting detection reagents. Blots werevisualized via ECL autoradiography hyperfilm (Amersham) and developed using a KonicaMinolta SRX-101A. Results: The positive control demonstrated staining of both the CDT-B18 residue amino acid sequence (A) and C. jejuni lysate (B) by anti-CDT-B. Rats previouslyexposed to C. jejuni produced antibodies to both the 18 residue sequence of CDT-B (C) aswell as the C. jejuni lysate (D). In contast, control rats demonstrated no reactivity to theCDT amino acid sequence (E) but mild reactivity to C. jejuni lysate (F). Conclusions: In arat model of post-infectious IBS, rats appear to produce host antibodies to CDT-B (the maintoxin subunit) upon infection with Campylobacter. This antibody may be important in thepathogenesis of post-infectious IBS given previous data suggesting that antibodies to CDTbind enteric neurons.
S-371 AGA Abstracts
Increases in Ileal Mast Cells in Patients With Diarrhoea Predominant IrritableBowel Syndrome May Be Due to a Relative Reduction in Mucosa-AssociatedLactobacilliGareth Parkes, Neil B. Rayment, Isabel Woodman, Barry N. Hudspith, Miranda Lomer,Jonathan Brostoff, Kevin Whelan, Jeremy D. Sanderson
INTRODUCTION: There is substantial evidence for gastrointestinal (GI) immune upregul-ation and altered GI microbiota in patients with irritable bowel syndrome (IBS). The aimwas to compare the immune cell populations in patients with IBS and controls and tocorrelate differences with the adjacent microbiota in small and large intestine. METHODS:Patients with IBS (Rome III), and healthy controls were recruited. Patients underwentileocolonoscopy at which paired ileal and rectal biopsies were snap frozen. Rectal microbiol-ogical analysis was performed using FISH as described previously (1). Ileal microbiologicalanalysis was performed on an adjacent biopsy using qPCR as described previously (2).Immunohistochemistry on sections cut from the same biopsy was used to enumerate mastcells, intra-epithelial and lamina-propria lymphocytes and macrophages. Symptom data wasrecorded using a validated questionnaire. RESULTS: 37 patients with IBS (27 diarrhoeapredominant (IBS-D) and 10 constipation predominant (IBS-C)) and 23 healthy controlswere recruited. There were significantly more mucosal mast cells in the rectum and theileum in the IBS-D group than in IBS-C patients and controls median see figure. There wasa inverse correlation between the number of ileal mast cells and the number of mucosa-associated lactobacilli (p=0.005) although this was not seen in rectal samples. In additionthe severity of diarrhoea positively correlated with the number of macrophages (p=0.001),mast cells (p=0.02) and IELs (p=0.01). CONCLUSIONS This is the first data to correlatealterations in the mucosa-associated microbiota with the adjacent immune cells in IBS.Increases in ileal mast cells in patients with IBS-D maybe due to reductions in adjacentlactobacilli. Increasing severity of diarrhoea positively correlated with increases in rectalmucosal immune cell populations.
Migration of Eosinophils and CCR2-/Cd68-Double Positive Cells Into theDuodenal Mucosa of Patients With Post-Infectious Functional DyspepsiaSeiji Futagami, Mayumi Shimpuku, Tetsuro Kawagoe, Masafumi Kusunoki, Katya Gudis,Kazumasa Miyake, Katsuhiko Iwakiri, Sheila E. Crowe, Choitsu Sakamoto
Background/Aims: Recent studies have shown that post-infectious functional dyspepsia (FD)symptoms may persist after elimination of gastrointestinal infection as well as post-infectiousIBS accompanying colonic inflammation. However, it is unclear whether intestinal chronicinflammation could contribute to clinical symptoms of certain FD patients such as post-infectious FD. To determine the relationship between local inflammation of the duodenumand clinical symptoms, we evaluated the infiltration of several phenotypes of duodenalinflammatory cells as well as gastric motility using 13C urea breath test in post-infectiousFD patients. Methods: We enrolled 136 consecutive patients diagnosed with FD accordingto Rome III criteria, and 20 healthy controls. Gastric motility was evaluated by gastricemptying time (T-max) using the 13C-acetate breath test. Upper abdominal symptomsincluding epigastric pain, epigastric burning, postprandial fullness, abdominal distention,and early satiety were assessed by questionnaire scores. We obtained biopsy specimens fromthe stomach and duodenum during upper gastrointestinal endoscopy. Histological gastritisand duodenitis was assessed as mild, moderate, or severe according to previous report.Characteristics of inflammatory cells and neuroendocrine cells were determined immunohis-tochemically with antibodies to CD3, CD68, CCR2, Vdelta1 TCR, and serotonin. Results:Endoscopic duodenitis was observed in only 5.7% of post-infectious FD patients. However,the rate of histological duodenitis in FD patients was significantly higher, with rates of 17%for mild, 26% for moderate and 57% for severe grades of duodenitis. There was no significantdifference in duodenal inflammation and Tmax value in the post-infectious FD patients withor without H. pylori infection. There was a significant correlation between epigastric burningand the degree of duodenitis in post-infectious FD patients. The degree of duodenitis ofpost-infectious FD patients was significantly greater than that of healthy volunteers. Inaddition, CD68-positive cell number in post-infectious FD patients was significantly increasedcompared to the numbers in subjects with EPS, or PDS and in healthy volunteers. CCR2/CD68-double positive cell number in post-infectious FD patients was significantly (p=0.009)increased compared to those in healthy volunteers. Conclusions: Migration of inflammatorycells, in particular, duodenal CCR2-positive macrophages may play an important role in thepathophysiology of post-infectious FD patients.