Liam O'mahony

Functional modulation of human intestinal epithelial cell responses by Bifidobacterium infantis and Lactobacillus salivarius

Intestinal epithelial cells (IECs) and dendritic cells (DCs) play a pivotal role in antigen sampling and the maintenance of gut homeostasis. However, the interaction of commensal bacteria with the intestinal surface remains incompletely understood. Here we investigated immune cell responses to commensal and pathogenic bacteria. HT‐29 human IECs were incubated with Bifidobacterium infantis 35624, Lactobacillus salivarius UCC118 or Salmonella typhimurium UK1 for varying times, or were pretreated with a probiotic for 2 hr prior to stimulation with S. typhimurium or flagellin. Gene arrays were used to examine inflammatory gene expression. Nuclear factor (NF)‐κB activation, interleukin (IL)‐8 secretion, pathogen adherence to IECs, and mucin‐3 (MUC3) and E‐cadherin gene expression were assayed by TransAM assay, enzyme‐linked immunosorbent assay (ELISA), fluorescence, and real‐time reverse transcriptase–polymerase chain reaction (RT‐PCR), respectively. IL‐10 and tumour necrosis factor (TNF)‐α secretion by bacteria‐treated peripheral blood‐derived DCs were measured using ELISA. S. typhimurium increased expression of 36 of the 847 immune‐related genes assayed, including NF‐κB and IL‐8. The commensal bacteria did not alter expression levels of any of the 847 genes. However, B. infantis and L. salivarius attenuated both IL‐8 secretion at baseline and S. typhimurium‐induced pro‐inflammatory responses. B. infantis also limited flagellin‐induced IL‐8 protein secretion. The commensal bacteria did not increase MUC3or E‐cadherin expression, or interfere with pathogen binding to HT‐29 cells, but they did stimulate IL‐10 and TNF‐α secretion by DCs. The data demonstrate that, although the intestinal epithelium is immunologically quiescent when it encounters B. infantis or L. salivarius, these commensal bacteria exert immunomodulatory effects on intestinal immune cells that mediate host responses to flagellin and enteric pathogens.

Irritable Bowel Syndrome–Type Symptoms in Patients With Inflammatory Bowel Disease: A Real Association or Reflection of Occult Inflammation?

OBJECTIVES:Do gastrointestinal symptoms in patients with inflammatory bowel disease (IBD) in apparent remission reflect the coexistence of irritable bowel syndrome (IBS) or subclinical inflammation? The aims of this study were as follows: (i) to prospectively determine the prevalence of IBS symptoms in IBD patients in remission; and (ii) to determine whether IBS symptoms correlate with levels of fecal calprotectin.METHODS:Remission was defined by physician assessment: Crohns disease (CD) activity index ≤150 and ulcerative colitis disease activity index ≤3, and serum C-reactive protein <10, while off corticosteroids or biologics. Quality of life (QOL) (by inflammatory bowel disease questionnaire), the hospital anxiety and depression scale (HAD), and fecal calprotectin were measured.RESULTS:Rome II criteria for IBS were fulfilled in 37/62 (59.7%) of CD patients and by 17/44 (38.6%) of those with ulcerative colitis (UC). However, fecal calprotectin was significantly elevated above the upper limit of normal in both IBD patient groups, indicating the presence of occult inflammation. Furthermore, calprotectin levels were significantly higher in CD and UC patients with criteria for IBS than in those without IBS-type symptoms. QOL scores were lower and HAD scores higher among UC patients with IBS symptoms in comparison to those who did not have IBS symptoms.CONCLUSIONS:IBS-like symptoms are common in patients with IBD who are thought to be in clinical remission, but abnormal calprotectin levels suggest that the mechanism in most cases is likely to be occult inflammation rather than coexistent IBS.

Sulfate-Reducing Bacteria Colonize Pouches Formed for Ulcerative Colitis but Not for Familial Adenomatous Polyposis

AbstractPURPOSE: Ileal pouch-anal anastomosis remains the “gold standard” in surgical treatment of ulcerative colitis and familial adenomatous polyposis. Pouchitis occurs mainly in patients with a background of ulcerative colitis, although the reasons for this are unknown. The aim of this study was to characterize differences in pouch bacterial populations between ulcerative colitis and familial adenomatous pouches. METHODS: After ethical approval was obtained, fresh stool samples were collected from patients with ulcerative colitis pouches (n = 10), familial adenomatous polyposis (n = 7) pouches, and ulcerative colitis ileostomies (n = 8). Quantitative measurements of aerobic and anaerobic bacteria were performed. RESULTS: Sulfate-reducing bacteria were isolated from 80 percent (n = 8) of ulcerative colitis pouches. Sulfate-reducing bacteria were absent from familial adenomatous polyposis pouches and also from ulcerative colitis ileostomy effluent. Pouch Lactobacilli, Bifidobacterium, Bacteroides sp, and Clostridium perfringens counts were increased relative to ileostomy counts in patients with ulcerative colitis. Total pouch enterococci and coliform counts were also increased relative to ileostomy levels. There were no significant quantitative or qualitative differences between pouch types when these bacteria were evaluated. CONCLUSIONS: Sulfate-reducing bacteria are exclusive to patients with a background of ulcerative colitis. Not all ulcerative colitis pouches harbor sulfate-reducing bacteria because two ulcerative colitis pouches in this study were free of the latter. They are not present in familial adenomatous polyposis pouches or in ileostomy effluent collected from patients with ulcerative colitis. Total bacterial counts increase in ulcerative colitis pouches after stoma closure. Levels of Lactobacilli, Bifidobacterium, Bacteroides sp, Clostridium perfringens, enterococci, and coliforms were similar in both pouch groups. Because sulfate-reducing bacteria are specific to ulcerative colitis pouches, they may play a role in the pathogenesis of pouchitis.

Mycobacterium avium subsp. Paratuberculosis (MAP) as a modifying factor in Crohn's disease

Background: Crohns disease (CD) is a multifactorial syndrome with genetic and environmental contributions. Mycobacterium avium subspecies paratuberculosis (MAP) has been frequently isolated from mucosal tissues of patients with CD but the cellular immune response to this bacterium has been poorly described. Our aim was to examine the influence of MAP on T‐cell proliferation and cytokine responses in patients with inflammatory bowel disease (IBD). Methods: Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph node cells (MLNCs) were obtained from IBD patients and non‐IBD controls. PBMC T‐cell proliferation in response to MAP was determined using CFSE labeling and flow cytometry. The specificity of cytokine responses to MAP was controlled by parallel exposure to Listeria monocytogenes (LM) or Salmonella typhimurium (ST). Results: Coincubation of PBMCs with MAP induced significantly more T‐cell proliferation (P < 0.0001) in PBMCs isolated from CD patients compared to PBMCs obtained from ulcerative colitis (UC) patients or healthy volunteers. In addition, PBMCs from CD patients secreted significantly higher (P < 0.05) levels of tumor necrosis factor‐alpha (TNF‐&agr;; 2302 ± 230 pg/mL) and interleukin (IL)‐10 (299 ± 48 pg/mL) in response to MAP compared to UC patients (TNF‐&agr;: 1219 ± 411 pg/mL; IL‐10: 125 ± 19 pg/mL) and controls (TNF‐&agr;: 1447 ± 173 pg/mL; IL‐10: 127 ± 12 pg/mL). No difference in cytokine responses was observed in response to LM or ST. MLNCs from both CD and UC patients secreted significantly more TNF‐&agr; and IL‐8 in response to MAP compared to MLNCs from non‐IBD control patients. Conclusions: Increased proliferation of T cells and an altered cytokine response suggest that prior exposure to MAP and engagement of the immune system is common in patients with CD. This does not imply causation but does support further examination of this bacterium as an environmental modifying factor. (Inflamm Bowel Dis 2009)

Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria

BackgroundHuman intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-κB activation were measured using enzyme-linked immunosorbent assays.ResultsCompared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-κB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-κB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis- induced CCL20 secretion.ConclusionThis study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

Portrait of a canine probiotic Bifidobacterium - from gut to gut.

The gastrointestinal environment is a complex interactive system involving the host, ingested dietary components, and numerous microbial species. We hypothesized that isolation and screening of Lactobacilli and Bifidobacteria adherent to healthy canine gastrointestinal tissue would yield strains with commensal activity in canines. The aims of this study were (1) to isolate a bank of commensal organisms from the canine gastrointestinal tract; (2) to screen these novel microbial isolates for potential probiotic effects; (3) to select one organism from these screens and test its impact on the canine microbiota. Lactic acid bacteria (LAB) were isolated from resected canine gastrointestinal tissue and screened in vitro for putative probiotic activities. Murine studies examined gastrointestinal transit and inhibition of Salmonella typhimurium translocation. One strain was progressed to a canine study where its impact on the gastrointestinal microbiota was determined. Of the 420 isolates from the canine gut, 62 strains were characterised as LAB. Following assessment of the strain bank with regard to pH sensitivity, bile resistance, pathogen inhibition and survival following freeze-drying, four Lactobacillus strains and two Bifidobacteria strains were selected for further examination. Bifidobacterium animalis AHC7 adhered to epithelial cells, transited the murine gastrointestinal tract to high numbers and significantly reduced S. typhimurium translocation. B. animalis AHC7 consumption significantly reduced the carriage of Clostridia, in particular Clostridium difficile, in dogs. This study describes the isolation and screening of canine-derived bacterial strains with commensal traits. The results demonstrate that B. animalis AHC7 has significant potential for improving canine gastrointestinal health.

Mechanisms of adherence of a probiotic Lactobacillus strain during and after in vivo assessment in ulcerative colitis patients

In a pilot-scale, open-label study to determine the ability of well-characterized probiotic Lactobacillus salivarius UCC118 cells to adhere to human epithelial cells in situ, the bacterial strain was administered to ulcerative colitis patients at approximately 109 CFU/day for 12 days. Microbiological analysis of biopsy specimens demonstrated that the ingested bacteria effectively adhered to both inflamed and non-inflamed mucosa of the large bowel in significant numbers. In previous reports, we have described the ability of the lactobacilli to adhere to enterocytic epithelial cells in vitro. In this study, we found that the bacteria adhered at higher levels to differentiated rather than undifferentiated epithelial monolayers; and that stationary phase lactobacilli were found to adhere to eukaryotic HT-29 and Caco-2 epithelial cells at greater levels than log phase bacterial cells. Pretreatment of the Lactobacillus cells with proteolytic enzymes abolished attachment, indicating the potential involvement of surface/exposed protein(s) as bacterial adhesin(s). SDS-PAGE (denaturing) techniques determined that the proteolytic treatment resulted in degradation of a cell wall-associated protein of approximately 84 kDa. The proteinaceous factor was purified by both anion-exchange chromatography and by gel extraction after SDS-PAGE electrophoresis, and under in vitro assay conditions proved capable of adherence and significant inhibition of bacterial attachment to enterocytic epithelial cells.

Technical Advance: Function and efficacy of an α4-integrin antagonist using bioluminescence imaging to detect leukocyte trafficking in murine experimental colitis

Leukocyte trafficking is a therapeutic target in IBD. The integrins α4β7 and α4β1 regulate leukocyte migration into tissues and lymphoid organs. Current strategies rely on biologics, such as mAb, to inhibit leukocyte recruitment. Here we show the in vivo therapeutic effects of a small molecule α4‐integrin antagonist (GSK223618A) in a leukocyte‐trafficking model and a murine model of colitis. Leukocytes isolated from MLNs of transgenic β‐actin‐luc+ mice were injected i.v. into recipients with DSS‐induced colitis. Recipient mice were orally gavaged with vehicle or an α4‐integrin antagonist 1 h pre‐adoptive transfer, followed by bioluminescence whole body and ex vivo organ imaging 4 h post‐transfer. To confirm its therapeutic effect, the α4‐integrin antagonist was given orally twice daily for 6 days to mice with DSS‐induced colitis, starting on Day 3. Clinical, macroscopic, and histological signs of inflammation were assessed and gene‐expression profiles analyzed. Using bioluminescence imaging, we tracked and quantified leukocyte migration to the inflamed gut and demonstrated its inhibition by a small molecule α4‐integrin antagonist. Additionally, the therapeutic effect of the antagonist was confirmed in DSS‐induced colitis in terms of clinical, macroscopic, and histological signs of inflammation. Gene expression analysis suggested enhancement of tissue healing in compound‐treated animals. Inhibition of leukocyte trafficking using small molecule integrin antagonists is a promising alternative to large molecule biologics. Furthermore, in vivo bioluminescence imaging is a valuable strategy for preclinical evaluation of potential therapeutics that target leukocyte trafficking in inflammatory diseases.

Infection burden and immunological responses are equivalent for polymeric and metallic implant materials in vitro and in a murine model of fracture-related infection: INFECTION OF POLYMERIC AND METAL IMPLANTS

The development of an infection is a major complication for some patients with implanted biomaterials. Whether the material or surface composition of the used biomaterial influences infection has not been directly compared for key biomaterials currently in use in human patients. We conducted a thorough in vitro and in vivo investigation using titanium (Ti) and polyether-ether-ketone (PEEK) as both commercially available and as modified equivalents (surface polished Ti, and oxygen plasma treated PEEK). Complement activation and cytokine secretion of cell of the immune system was assessed in vitro for all materials in the absence and presence of bacterial stimulants. In a follow-up in vivo study, we monitored bacterial infection associated with clinically available and standard Ti and PEEK inoculated with Staphylococcus aureus. Complement activation was affected by material choice in the absence of bacterial stimulation, although the material based differences were largely lost upon bacterial stimulation. In the in vivo study, the bacterial burden, histological response and cytokine secretion suggests that there is no significant difference between both PEEK and Ti. In conclusion, the underlying material has a certain impact in the absence of bacterial stimulation, however, in the presence of bacterial stimulation, bacteria seem to dictate the responses in a manner that overshadows the influence of material surface properties.

Recent Developments and Highlights in Mechanisms of Allergic Diseases: Microbiome

All body surfaces are exposed to a wide variety of microbes, which significantly influence immune reactivity within the host. This review provides an update on some of the critical novel findings that have been published on the influence of the microbiome on atopic dermatitis, food allergy and asthma. Microbial dysbiosis has consistently been observed in the skin, gut and lungs of patients with atopic dermatitis, food allergy and asthma, respectively, and the role of specific microbes in allergic disorders is being intensively investigated. However, many of these discoveries have yet to be translated into routine clinical practice.