Abigail Henderson

Helicobacter bilis triggers persistent immune reactivity to antigens derived from the commensal bacteria in gnotobiotic C3H/HeN mice

Background: Infection with Helicobacter species has been associated with the development of mucosal inflammation and inflammatory bowel disease (IBD) in several mouse models. However, consensus regarding the role of Helicobacter as a model organism to study microbial-induced IBD is confounded by the presence of a complex colonic microbiota. Aim: To investigate the kinetics and inflammatory effects of immune system activation to commensal bacteria following H bilis colonisation in gnotobiotic mice. Methods: C3H/HeN mice harbouring an altered Schaedler flora (ASF) were selectively colonised with H bilis and host responses were investigated over a 10-week period. Control mice were colonised only with the defined flora (DF). Tissues were analysed for gross/histopathological lesions, and bacterial antigen-specific antibody and T-cell responses. Results: Gnotobiotic mice colonised with H bilis developed mild macroscopic and microscopic lesions of typhlocolitis beginning 3 weeks postinfection. ASF-specific IgG responses were demonstrable within 3 weeks, persisted throughout the 10-week study, and presented as a mixed IgG1:IgG2a profile. Lymphocytes recovered from the mesenteric lymph node of H bilis-colonised mice produced increased levels of interferon γ, tumour necrosis factor α (TNFα), interleukin 6 (IL6) and IL12 in response to stimulation with commensal- or H bilis-specific bacterial lysates. In contrast, DF mice not colonised with H bilis did not develop immune responses to their resident flora and remained disease free. Conclusions: Colonisation of gnotobiotic C3H/HeN mice with H bilis perturbs the host’s response to its resident flora and induces progressive immune reactivity to commensal bacteria that contributes to the development of immune-mediated intestinal inflammation.

Increased CYP4B1 mRNA Is Associated with the Inhibition of Dextran Sulfate Sodium-Induced Colitis by Caffeic Acid in Mice

Susceptibility to inflammatory bowel diseases depends upon interactions between the genetics of the individual and induction of chronic mucosal inflammation. We hypothesized that administration of dietary phenolics, caffeic acid and rutin, would suppress upregulation of inflammatory markers and intestinal damage in a mouse model of colitis. Colitis was induced in C3H/ HeOuJ mice (8 weeks old, 6 male/6 female per treatment) with 1.25% dextran sulfate sodium (DSS) for 6 d in their drinking water. Rutin (1.0 mmol (524 mg)/kg in diet), caffeic acid (1.0 mmol (179 mg)/kg in diet), and hypoxoside extract (15 mg/d, an anticolitic phenolic control) were fed to the mice for 7 d before and during DSS treatment, as well as without DSS treatment. Body weight loss was prevented by rutin and caffeic acid during DSS treatment. Colon lengths in mice fed caffeic acid and hypoxoside during DSS treatment were similar to DSS-negative control. Food intake was improved and myeloperoxidase (MPO) was decreased with each phenolic treatment in DSS-treated mice compared with DSS treatment alone. Colonic mRNA expression of IL-17 and iNOS were inhibited when IL-4 was increased by each phenolic treatment combined with DSS, whereas CYP4B1 mRNA was increased only by caffeic acid in DSS-treated mice, compared with DSS treatment alone. Colonic and cecal histopathology scores of DSS-treated mice were significantly more severe (P < 0.01) than in mice fed caffeic acid before and during DSS treatment, based on mucosal height, necrosis, edema, erosion, and inflammatory cell infiltration. Although both rutin and caffeic acid suppressed the expression of selected inflammatory markers, only caffeic acid protected against DSS-induced colitis, in association with normalization of CYP4B1 expression. The inhibition of DSS-induced colitic pathology by caffeic acid was mediated by mechanisms in addition to anti-inflammatory effects that deserve further study.

Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.

Mucosal gene expression profiles following the colonization of immunocompetent defined-flora C3H mice with Helicobacter bilis: a prelude to typhlocolitis

An aberrant immune response to the commensal microbiota is widely hypothesized to contribute to the development of inflammatory bowel disease. Helicobacter bilis colonization of defined-flora mice has been shown to trigger host immune responses to the commensal flora. However, the magnitude of the effects on mucosal homeostasis following colonization with H. bilis has not been determined. Using microarray analysis, differential gene expression within the cecal mucosa was assessed at 15, 30, or 45 days following H. bilis colonization using Affymetrix Genechips. H. bilis colonization induced marked upregulation of genes associated with protein metabolism, immune responses, and downregulation of genes associated with fatty acid metabolism and detoxification which peaked at 15 days postinfection. A set of genes associated with glycoprotein synthesis and detoxification including Fut2, B3galt5, Ceacam12, Cyp4b1, and Ugt8a were uniquely identified and found to be similarly expressed following the induction of typhlocolitis by dextran sodium sulfate or Brachyspira hyodysenteriae. This study provides preliminary evidence as to the types of factors or changes in the intestinal mucosa that potentially predispose the host to the development of typhlocolitis.

Mo1708 Serum-Derived Bovine Immunoglobulin/Protein Isolate Attenuated DSSInduced Colitis in a Defined Floral Model

Introduction. A colovesical fistulas present an important clinical problems. They usually occur as a complication of diverticulosis, IBD or malignant process. The pentadecapeptide BPC 157 , in clinical trials for IBD therapy, has already shown the effectiveness in healing colocutaneous fistulas ( J. Pharmacol Sci 2008 ) , gastrocutaneous fistulas ( Dig Dis Sci 2009 ). Therefore we suggest it as a possible therapy of colovesical fistulas. Matherials and methods. Wistar Albino rats were randomly assigned, at 5 cm from anocutanous borderline, with the diameter of 3 mm. The surgical procedure was performed according to rules brought by the Local Ethical Committee. Medication of BPC 157 (10ug/kg, 10ng/kg) was per-orally, in drinkingwater and intraperitoneally, once daily, the first dose immediately after the operation, the last dose at 24 h before sacrifice, at 7, 15, 30 post-operative day, with the biomechanical, functional, macroscopic and microscopic assessment. Results. Biomechanical analysis :we measured pressure of the fistulas rupture for all groups of animals with the system for central venous pressure assesment .In the control group pressures for fistulas rupture were similar for every experimental period : ≤1mL H2O, 1mL±0,3 mL, 2 mL±0,5 ml H2O. In the second group( BPC per-orally in drinking water 10ug/kg and 10ng/kg ) water pressures were 1mL±0,3; 2mL±0,3 and 5mL±0,5 but without fistulas rupture at the and of 30 days experimental period.In the third group( BPC intraperitoneally 10ug/kg and 10ng/kg ) water pressure was increased from 2mL±0,5mL until 5 mL±0,5 at the and of 15 days experimental period. In most animals fistulas were healed at the end of 30 days experimental period. Macroscopic analysis: We measured diameters of fistulas on the colonic and the bladder side.In the control group diameters of fistulas were similar for both sides for all experimental periods : 3mm ±0.5mm on the colon and bladder side,3mm±1mm and 2,5±0,5mm. In the second group fistulas diameter was 3±0,2 mm for both sides ( 7 days ),2mm±0,5 mm for colon and 2,5±0,5 mm for bladder ( 15 days ), 1,5mm±0,5 for colon, 2mm±0,5 mm for bladder side ( 30 days ). In the third group fistulas were reduced : 2,5mm±0,2mm colon, 3mm±0,5 bladder ( 7 days ), 1,5mm±0,5mm colon; 2mm±0,5mm bladder ( 15 days ) and at the end oft he 30 days experimental period in most animals fistulas were healed er reduced diameter at 1mm±0,3mm for both sides.. Conclusion. According to these results the pentadecapeptide BPC 157 could present a new possible pathway in therapy of colovesical fistulas.