Intestinal Inflammatory Bowel Disease Research Articles

Phospholipid-drug conjugates as a novel oral drug targeting approach for the treatment of inflammatory bowel disease

The enzyme phospholipase A2 (PLA2) is overexpressed in the inflamed intestine in inflammatory bowel disease (IBD) patients, and in this work we aimed to exploit PLA2 as a prodrug-activating enzyme for a novel PL-drug conjugate, thereby liberating the free drug specifically in the targeted diseased tissue(s). The proposed prodrug contains a drug moiety covalently bound through a linker to the sn-2 position of a phospholipid (PL). The NSAID diclofenac was used as model molecule, and four different linker lengths (2, 4, 6 and 8 –CH2 units) were studied. The four PL-diclofenac conjugates were synthesized and characterized by LC/MS and NMR. PLA2-mediated activation of the prodrugs was analyzed in-vitro, and the remaining intact complex and free drug liberation were assessed after incubation with PLA2. The rate and degree of PLA2-mediated activation were highly dependent on the linker length; 2- and 4-carbon linker conjugates were activated to lower extent than the 6-carbon conjugate, and longer linker again decreased the affinity towards PLA2. The 6-carbon linker conjugate was found to be the optimal and released ~95% of the free drug after incubation with PLA2, whereas only ~20% were delivered by the 2-carbon linker prodrug. The 6-carbon linker conjugate was shown to be stable in intestinal perfusate, fresh plasma, and pH4.0 and 6.8 buffers, but not at pH1.0. In conclusion, the results of this work confirm the feasibility of our general aim to exploit PLA2 as a prodrug-activating enzyme of PL-drug conjugates. This may provide a novel oral drug targeting approach in IBD therapy.

Intestinal Epithelial Sirtuin 1 Regulates Intestinal Inflammation During Aging in Mice by Altering the Intestinal Microbiota

Intestinal epithelial homeostasis is maintained by complex interactions among epithelial cells, commensal gut microorganisms, and immune cells. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD), but the mechanisms of this process are not clear. We investigated how Sirtuin 1 (SIRT1), a conserved mammalian NAD+-dependent protein deacetylase, senses environmental stress to alter intestinal integrity. We performed studies of mice with disruption of Sirt1 specifically in the intestinal epithelium (SIRT1 iKO, villin-Cre+, Sirt1flox/flox mice) and control mice (villin-Cre-, Sirt1flox/flox) on a C57BL/6 background. Acute colitis was induced in some mice by addition of 2.5% dextran sodium sulfate to drinking water for 5-9 consecutive days. Some mice were given antibiotics via their drinking water for 4 weeks to deplete their microbiota. Some mice were fed with a cholestyramine-containing diet for 7 days to sequester their bile acids. Feces were collected and proportions of microbiota were analyzed by 16S rRNA amplicon sequencing and quantitative PCR. Intestines were collected from mice and gene expression profiles were compared by microarray and quantitative PCR analyses. We compared levels of specific mRNAs between colon tissues from age-matched patients with ulcerative colitis (n=10) vs without IBD (n=8, controls). Mice with intestinal deletion of SIRT1 (SIRT1 iKO) had abnormal activation of Paneth cells starting at the age of 5-8 months, with increased activation of NF-κB, stress pathways, and spontaneous inflammation at 22-24 months of age, compared with control mice. SIRT1 iKO mice also had altered fecal microbiota starting at 4-6 months of age compared with control mice, in part because of altered bile acid metabolism. Moreover, SIRT1 iKO mice with defective gut microbiota developed more severe colitis than control mice. Intestinal tissues from patients with ulcerative colitis expressed significantly lower levels of SIRT1 mRNA than controls. Intestinal tissues from SIRT1 iKO mice given antibiotics, however, did not have signs of inflammation at 22-24 months of age, and did not develop more severe colitis than control mice at 4-6 months. In analyses of intestinal tissues, colitis induction, and gut microbiota in mice with intestinal epithelial disruption of SIRT1, we found this protein to prevent intestinal inflammation by regulating the gut microbiota. SIRT1 might therefore be an important mediator of host-microbiome interactions. Agents designed to activate SIRT1 might be developed as treatments for IBDs.

Pomegranate polyphenolics reduce inflammation and ulceration in intestinal colitis—involvement of the miR-145/p70S6K1/HIF1α axis in vivo and in vitro

This study investigated the potential role of the p70S6K1/HIF1α axis in the anti-inflammatory activities of pomegranate (Punica granatum L.) polyphenolics in dextran sodium sulfate (DSS)-induced colitis in Sprague–Dawley rats and in lipopolysaccharide (LPS)-treated CCD-18Co colon-myofibroblastic cells. Rats were administered either control (CT) or pomegranate beverage (PG), containing ellagic acid and ellagitannins, then exposed to three cycles of 3% DSS followed by a 2-week recovery period. PG protected against DSS-induced colon inflammation and ulceration (50% and 66.7%, P=.05 and .045, respectively), and decreased the Ki-67 proliferative index in the central and basal regions compared to the control. PG also significantly reduced the expression of proinflammatory cytokines (TNF-α and IL-1β), COX-2, and iNOS at mRNA and protein levels. In addition, the expression of p70S6K1 and HIF1α was reduced, while the tumor suppressor miR-145 was induced by PG. The intestinal microbiota of rats treated with PG showed a significant increase in Ruminococcaceae that include several butyrate producing bacteria (P=.03). In vitro, PG reduced the expression of p70S6K1 and HIF1α and induced miR-145 in a dose-dependent manner. The involvement of miR-145/p70S6K1 was confirmed by treating LPS-treated CCD-18Co cells with miR-145 antagomiR, where the pomegranate polyphenolics reversed the effects of the antagomiR for p70S6K1 mRNA and protein levels. These results suggest that pomegranate polyphenols attenuated DSS-induced colitis by modulating the miR-145/p70S6K/HIF1α axis, indicating potential use in therapeutic treatment of ulcerative colitis.

Cathelicidin-WA Improves Intestinal Epithelial Barrier Function and Enhances Host Defense against Enterohemorrhagic Escherichia coli O157:H7 Infection.

Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines, leading to many intestinal diseases. Cathelicidin peptides suppress intestinal inflammation and improve intestinal epithelial barrier function independently of their antimicrobial activity. In this study, we investigated the effects of Cathelicidin-WA (CWA) on intestinal epithelial barrier function, as well as the underlying mechanism, by using enterohemorrhagic Escherichia coli (EHEC)-infected mice and intestinal epithelial cells. The results showed that CWA attenuated EHEC-induced clinical symptoms and intestinal colitis, as did enrofloxacin (Enro). CWA decreased IL-6 production in the serum, jejunum, and colon of EHEC-infected mice. Additionally, CWA alleviated the EHEC-induced disruption of mucin-2 and goblet cells in the intestine. Interestingly, CWA increased the mucus layer thickness, which was associated with increasing expression of trefoil factor 3, in the jejunum of EHEC-infected mice. CWA increased the expression of tight junction proteins in the jejunum of EHEC-infected mice. Using intestinal epithelial cells and a Rac1 inhibitor in vitro, we demonstrated that the CWA-mediated increases in the tight junction proteins might depend on the Rac1 pathway. Furthermore, CWA improved the microbiota and short-chain fatty acid concentrations in the cecum of EHEC-infected mice. Although Enro and CWA had similar effects on intestinal inflammation, CWA was superior to Enro with regard to improving intestinal epithelial barrier and microbiota in the intestine. In conclusion, CWA attenuated EHEC-induced inflammation, intestinal epithelial barrier damage, and microbiota disruption in the intestine of mice, suggesting that CWA may be an effective therapy for many intestinal diseases.

P064 EBI2 and oxysterols in the development of intestinal lymphoid structures and colitis

P064 EBI2 and oxysterols in the development of intestinal lymphoid structures and colitis

Apple peel polyphenols: a key player in the prevention and treatment of experimental inflammatory bowel disease.

Diets rich in fruits and vegetables may reduce oxidative stress (OxS) and inflammation via several mechanisms. These beneficial effects may be due to their high polyphenol content. The aims of the present study are to evaluate the preventive and therapeutic aspects of polyphenols in dried apple peel powder (DAPP) on intestinal inflammation while elucidating the underlying mechanisms and clinical benefits. Induction of intestinal inflammation in mice was performed by oral administration of the inflammatory agent dextran sulfate sodium (DSS) at 2.5% for 10days. Physiological and supraphysiological doses of DAPP (200 and 400mg/kg/day respectively) were administered by gavage for 10days pre- and post-DSS treatment. DSS-mediated inflammation caused weight loss, shortening of the colon, dystrophic detachment of the epithelium, and infiltration of mono- and poly-morphonuclear cells in the colon. DSS induced an increase in lipid peroxidation, a down-regulation of antioxidant enzymes, an augmented expression of myeloperoxidase (MPO) and cyclooxygenase-2 (COX-2), an elevated production of prostaglandin E2 (PGE2) and a shift in mucosa-associated microbial composition. However, DAPP normalized most of these abnormalities in preventive or therapeutic situations in addition to lowering inflammatory cytokines while stimulating antioxidant transcription factors and modulating other potential healing pathways. The supraphysiological dose of DAPP in therapeutic situations also improved mitochondrial dysfunction. Relative abundance of Peptostreptococcaceae and Enterobacteriaceae bacteria was slightly decreased in DAPP-treated mice. In conclusion, DAPP exhibits powerful antioxidant and anti-inflammatory action in the intestine and is associated with the regulation of cellular signalling pathways and changes in microbiota composition. Evaluation of preventive and therapeutic effects of DAPP may be clinically feasible in individuals with intestinal inflammatory bowel diseases.

ISCHEMICCOLITIS (case report)

Ischemic bowel disease results from an acute or chronic decline of the blood supply to the bowel and may have various clinical presentations, such as intestinal angina, ischemic colitis (IC) or intestinal infarction. Elderly patients with systemic atherosclerosis who are symptomatic for the disease, congestive heart failure and recent aortic or cardiopulmonary bypass surgery are particularly at risk. The clinical evolution and outcome of this disease are difficult to predict because of its polymorphic aspects and the general lack of statistical data. In this paper, we present two cases of patients who were monitored in our clinic. For these patients, we encountered with pivotal changes in the clinical pattern. These evolutions is particularly rare in common clinical practice, and cases are exemplary because it raises discussions about the nature of the condition and therapeutic decisions that should be made at every stage of the disease.

Context-dependent regulation of Th17-associated genes and IFNγ expression by the transcription factor NFAT5.

Stress-activated transcription factors influence T-cell function in different physiopathologic contexts. NFAT5, a relative of nuclear factor κB and the calcineurin-activated NFATc transcription factors, protects mammalian cells from hyperosmotic stress caused by the elevation of extracellular sodium levels. In T cells exposed to hypernatremia, NFAT5 not only induces osmoprotective gene products but also cytokines and immune receptors, which raises the question of whether this factor could regulate other T-cell functions in osmostress-independent contexts. Here we have used mice with a conditional deletion of Nfat5 in mature T lymphocytes to explore osmostress-dependent and -independent functions of this factor. In vitro experiments with CD4 T cells stimulated in hyperosmotic medium showed that NFAT5 enhanced the expression of IL-2 and the Th17-associated gene products RORγt and IL-23R. By contrast, NFAT5-deficient CD4 T cells activated in vivo by anti-CD3 antibody exhibited a different activation profile and were skewed towards enhanced interferon γ (IFNγ) and IL-17 expression and attenuated Treg responses. Using a model of experimental colitis, we observed that mice lacking NFAT5 in T cells exhibited exacerbated intestinal colitis and enhanced expression of IFNγ in draining lymph nodes and colon. These results show that NFAT5 can modulate different T-cell responses depending on stress conditions and stimulatory context.

Abstract 3297: Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition

Abstract Background. Intestinal mucus layer disruption and gut microflora modification in conjunction with tight junction (TJ) changes can increase colonic permeability that allows bacterial dissemination and intestinal and systemic disease. Employing Citrobacter rodentium (CR) as a model system to mimic the pathogenic mechanisms of Enteropathogenic and Enterohaemorrhagic E. coli, we showed previously that a functional cross-talk between the Notch and Wnt/β-catenin pathways regulates hyperplasia and/or colitis following CR infection. Aim: To test the hypothesis that CR infection combined with chronic Notch inhibition will alter the composition of the colonic mucus to promote dysbiosis, tight junction disruption, loss of multipotent intestinal stem cells (ISCs) and colitis. Material/methods: Immune-competent NIH:Swiss outbred mice were infected with CR (108 CFUs) and treated with Notch blocker Dibenzazepine [DBZ, i.p. @ 10μmol/Kg body weight]. Mucus composition, fecal 16S rDNA analysis and components of TJ integrity were measured and correlated with changes in components of the Notch pathway. 6% Pectin diet served as a SCFA-delivery system to the colon. Results: In the colons of CR-infected/DBZ-treated mice, mucus analysis revealed dramatic alterations in the composition of trace O-glycans and complex type and hybrid N-glycans, compared to CR-infected alone that preceded alterations in 16S rDNA microbial community structure. Indeed, mucin degrading and colitogenic bacterium, Akkermansia muciniphila exhibited dramatic increases in the feces of CR-infected and DBZ-treated mice. At the same time, colons from CR-infected/DBZ-treated mice had decreased expression of antimicrobial peptides Angiogenin-4, Retnlβ, Intelectin-1 and Intelectin-2, respectively. Expression levels of both TJ and adherens junction proteins (e.g., Claudin-5, ZO-1, ZO-2, E-Cadherin, β-catenin) also decreased significantly in the colonic crypts of CR-infected/DBZ-treated mice that paralleled loss of Notch signaling. Both C3H/HeN mice that exhibit exaggerated response to CR infection and T- and B-cell deficient Rag-1 mice following CR-infection and DBZ-treatment, exhibited dramatic increases in paracellular permeability, goblet cell metaplasia and exacerbation of colitis. Chronic blockade of the Notch pathway depleted colonic epithelium of ISC markers Dclk1 and Lgr5 concomitant with exacerbation of colitis. 6% Pectin diet ameliorated colitis by restoring barrier integrity via recruitment of Bacteroides vulgatus, Streptococcus gordonii and Lactococcus lactis, bacteria with potential anti-inflammatory functions. Conclusions: 1. These studies help us delineate the mechanistic basis of colitis development in the aftermath of chronic Notch inhibition. 2. Our findings also caution against the use of Notch inhibitors for patients suffering from colitis-associated colon cancer. Citation Format: Ishfaq Ahmed, Badal Roy, Rita-marie McFadden, Shrikant Anant, Seth Septer, Shahid Umar. Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3297.

Preserving viability of Lactobacillus rhamnosus GG in vitro and in vivo by a new encapsulation system

Probiotics have shown beneficial effects on health and prevention of diseases in humans. However, a concern for application of probiotics is the loss of viability during storage and gastrointestinal transit. The aim of this study was to develop an encapsulation system to preserve viability of probiotics when they are administrated orally and apply Lactobacillus rhamnosus GG (LGG) as a probiotic model to evaluate the effectiveness of this approach using in vitro and in vivo experiments. LGG was encapsulated in hydrogel beads prepared using pectin, a food grade polysaccharide, glucose, and calcium chloride, and lyophilized by freeze-drying. Encapsulated LGG was cultured in vitro under the condition that mimicked the physiological environment of the human gastrointestinal tract. Compared to non-encapsulated LGG, encapsulation increased tolerance of LGG in the acid condition, protected LGG from protease digestion, and improved shelf time when stored at the ambient condition, in regard of survivability and production of p40, a known LGG-derived protein involved in LGG's beneficial effects on intestinal homeostasis. To evaluate the effects of encapsulation on p40 production in vivo and prevention of intestinal inflammation by LGG, mice were gavaged with LGG containing beads and treated with dextran sulphate sodium (DSS) to induce intestinal injury and colitis. Compared to non-encapsulated LGG, encapsulated LGG enhanced more p40 production in mice, and exerted higher levels of effects on prevention of DSS-induced colonic injury and colitis and suppression of pro-inflammatory cytokine production. These data indicated that the encapsulation system developed in this study preserves viability of LGG in vitro and in vivo, leading to longer shelf time and enhancing the functions of LGG in the gastrointestinal tract. Thus, this encapsulation approach may have the potential application for improving efficacy of probiotics.

Intestinal micro-ecology and ulcerative colitis

Intestinal micro-ecology and ulcerative colitis

Quantitative Analysis of Intestinal Flora of Uygur and Han Ethnic Chinese Patients with Ulcerative Colitis.

Aim. To study the correlation between intestinal flora and ulcerative colitis by analyzing the abundance of Bacteroides, Fusobacterium, Clostridium, Bifidobacterium spp., and Faecalibacterium prausnitzii in the intestinal of ulcerative colitis (UC) patients and healthy controls with Uygur and Han ethnic. Methods. Bacterial genomic DNA was extracted from fecal samples and analyzed with real-time fluorescence quantitative polymerase chain reaction (PCR) to identify the abundance of Bacteroides, Fusobacterium, Clostridium, Bifidobacterium spp., and Faecalibacterium prausnitzii. Results. The samples from UC patients, Uygur and Han ethnic combined, had higher abundance of Bacteroides (P = 0.026) but lower Clostridium (P = 0.004), Bifidobacterium spp. (P = 0.009), and Faecalibacterium prausnitzii (P = 0.008) than those from healthy controls. Among UC patients, Bacteroides population was raised in acute UC patients (P ≤ 0.05), while the abundance of Clostridium, Bifidobacterium spp., Fusobacterium, and Faecalibacterium prausnitzii decreased (P ≤ 0.05) compared with the remission. In both UC patients group and control group, no difference was observed in the abundance of these 5 bacteria between the Han and the Uygur group. Conclusions. Variations in the abundance of these five bacterial strains in intestines may be associated with the occurrence of UC in Uygur and Han populations; however, these variations were not associated with ethnic difference.

DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation.

Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.

Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease.

To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease. Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn's disease (n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps (n = 6) and colorectal cancer (n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine (PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A (HEXA) and sialidase-3 (NEU3) were measured in intestinal mucosa using western blot and compared among subject groups. Relative GM3 ganglioside content was 2-fold higher (P < 0.05) in intestine from patients with inflammatory bowel disease (IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue (P < 0.05). Control intestine exhibited 3-fold higher (P < 0.01) relative GD1a ganglioside content than IBD intestine. GD3 and GD1a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine (P < 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold (P < 0.05) and NEU3 was increased 8.3-fold (P < 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1a and PC. This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.

Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity.

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKβ, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKβ(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.

Syndecan-1 Acts in Synergy with Tight Junction Through Stat3 Signaling to Maintain Intestinal Mucosal Barrier and Prevent Bacterial Translocation.

Intestinal epithelial tight junction (TJ) is the principal determinant of mucosal permeability, defects of which have been correlated to inflammatory bowel disease. In this study, we investigated whether syndecan-1 (Sdc1), the predominant cell surface heparan sulfate proteoglycan, affects TJ proteins to protect intestinal barrier function. The role of Sdc1 in barrier function was examined in cultured colonic epithelial cells and dextran sodium sulfate-induced colitis mouse model. Barrier function was determined by transepithelial electrical resistance, bacterial translocation, and FITC-dextran flux. Canonical TJ proteins ZO-1 and occludin were measured by Western blot and immunofluoresence. Role of the Stat3 pathway was detected by Western blot and chromatin immunoprecipitation. Overexpressed Sdc1 in Caco-2 cells attenuated transepithelial electrical resistance reduction, prevented bacterial translocation, and repressed FITC-dextran flux, whereas Sdc1 knockdown in HT29 cells resulted in a greater loss of barrier function. Supplementation of exogenous Sdc1 in colitis mice ameliorated the activity of colitis and barrier defect. Mechanistically, Sdc1 significantly modulated expressions of ZO-1 and occludin by activating Stat3, which directly bound to the promoter regions of ZO-1 and occludin. Furthermore, ZO-1 and occludin were found to bind to each other, and their repression could induce Sdc1 upregulation. Sdc1 plays an important role in protecting the intestinal barrier function and preventing bacterial translocation, in synergy with TJ through Stat3 signaling in an Sdc1/Stat3/ZO-1 and occludin feedback loop. Sdc1 participates in the mechanism that is related to intestinal barrier function and colitis and represents a therapeutic target for novel anti-inflammatory bowel disease strategies.

Development and Application of a Novel Real‐Time PCR Assay for Citrobacter rodentium Quantification

Citrobacter rodentium is a murine pathogen causing transmissible colonic hyperplasia and used to model intestinal colitis including foodborne Escherichia coli O157:H7 pathogenesis. Several probiotics have been found to alleviate infection and Bifidobacterium bifidum MIMBb75 may be effective because it strongly adheres to colonocytes and modifies the gut microbiota. C. rodentium is typically quantified by classical culturing; the aim of this study was to establish a novel molecular assay based on quantitative real-time PCR. Primers were designed to target the C. rodentium-specific espB gene. Specificity was validated in silico and experimentally with a range of intestinal bacteria. A standard curve was constructed using serially diluted C. rodentium DNA depicting a linearity range (R2 > 0.99) of 102 to 107 cells per PCR reaction (efficiency 96%) and sensitivity of 104 cells/g feces. To assess effects of B. bifidum MIMBb75 on C. rodentium fecal load, C57BL/6J mice were gavaged with 109 cells of B. bifidum MIMBb75 in PBS or PBS alone daily from 7 days before to 10 days post infection (PI) with 109 cells of C. rodentium. Fecal counts of C. rodentium at day 10 PI were quantified with the novel qPCR assay and did not differ significantly between the treatment and control group (6.0 ± 0.4 and 6.4 ± 0.1 log cells/g feces, respectively). This was confirmed by classical culturing. This is the first real-time PCR assay specific for C. rodentium and it can be used to determine its load and distribution along the intestinal tract in response to preventive and therapeutic interventions. Other probiotics were unable to reduce fecal load yet mitigated inflammation thus, further research is warranted. *co-first authors Funding JP Bickell Foundation, NSERC, NSERC USRA

Berberine blocks the relapse of Clostridium difficile infection in C57BL/6 mice after standard vancomycin treatment.

Vancomycin is a preferred antibiotic for treating Clostridium difficile infection (CDI) and has been associated with a rate of recurrence of CDI of as high as 20% in treated patients. Recent studies have suggested that berberine, an alternative medical therapy for gastroenteritis and diarrhea, exhibits several beneficial effects, including induction of anti-inflammatory responses and restoration of the intestinal barrier function. This study investigated the therapeutic effects of berberine on preventing CDI relapse and restoring the gut microbiota in a mouse model. Berberine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis. The disease activity index (DAI), mean relative weight, histopathology scores, and levels of toxins A and B in fecal samples were measured. An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum. Berberine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of vancomycin treatment. Therapy consisting of vancomycin and berberine combined prevented weight loss, improved the DAI and the histopathology scores, and effectively decreased the mortality rate. Berberine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI. Our data indicate that a combination of berberine and vancomycin is more effective than vancomycin alone for treating CDI. One of the possible mechanisms by which berberine prevents a CDI relapse is through modulation of the gut microbiota. Although this conclusion was generated in the case of the mouse model, use of the combination of vancomycin and berberine and represent a novel therapeutic approach targeting CDI.

Activation of Epidermal Growth Factor Receptor Mediates Mucin Production Stimulated by p40, a Lactobacillus rhamnosus GG-derived Protein

The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfr(wa5) mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.

Inflammatory bowel disease is a disorder of ganglioside metabolism (959.4)

AIM To demonstrate that the metabolism of ganglioside is altered in inflammatory bowel disease (IBD). METHODS Sections of intestinal mucosa from terminal ileum and/or colon were obtained from subjects with ulcerative colitis or inflammatory Crohn’s disease undergoing surgical resection for complications related to IBD. Control groups consisted of tissue samples of apparently normal intestinal mucosa from subjects with colorectal cancer and apparently normal intestinal mucosa from subjects with benign adenoma polyposis. Gangliosides of the intestinal mucosa were characterized using LC‐QQQ‐MS and compared among subject groups. Ganglioside catabolism enzymes HEXA and NEU3 were measured in the intestinal mucosa by western blot. RESULTS GM3 was significantly higher (P&lt;0.01) by 2.1 fold in IBD intestine compared to apparently normal intestinal mucosa. IBD intestine exhibited 2.7 fold lower (P&lt;0.001) GD1a content than control intestine. The mean relative expression of HEXA in IBD intestine was increased 1.7‐fold (P&lt;0.08) and the mean relative expression of NEU3 in IBD intestine was increased 8.3‐fold (P&lt; 0.001) compared to normal intestine. CONCLUSION Metabolism of gangliosides is altered in the intestinal mucosa of subjects with IBD. Investigation into utility of dietary ganglioside as a potential treatment of IBD is warranted.Grant Funding Source: Supported by Broad Foundation and CIHR