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T2 Relaxometry Distinguishes Acute Murine DSS Colitis From Chronic Relapsing Colitis. A Novel Tool to Assess Transmural Fibrotic Lesions?
AGA Abstracts
(1.21±0.82% vs. 0.55±0.27%, p=0.03) respectively. Zonulin concentrations correlated urinary L/M ratio in IBD (r=0.86, p<0.01). BDNF correlated with the L/M ratio in healthy controls (r=0.76, p=0.04). IBD patients showed higher cortisol (427.22±113.32 vs. 297.38±114.15 nmol/L, p = 0.03), TNFα (3.12±3.28 vs. 0.99±0.26 pg/ml, p=0.07), and IL-6 (7.63±7.32 vs. 1.26±0.41 pg/ml, p=0.02) than controls. Conclusion: The increased segmental permeability seen in IBD patients was associated with an uncoupling of the relationship between permeability and BDNF evident in healthy controls and a compensatory correlation with increased serum zonulin levels. As neuronal and enteric glial cells regulating neuroimmune signaling, barrier permeability and neuroplasticity are the main source of BDNF, it is likely that dysregulation of BDNF release from glial cells is directly implicated in the inflammatory changes of IP. The correlation between zonulin and IP changes are of relevance due to zonulin's housekeeping functions, which include regulation of tight junction proteins, bacterial colonisation and host-microbiome-immune interactions, all of which are known to be involved in the pathogenesis of IBD. Further studies are required to extend these preliminary data. Grant support by Defence Science & Technology Agency (DSTA), Singapore (1). Reinshagen, M., et al (2002) Curr. Opin. Investig. Drugs 3, 565-568
Mo1736 Renal CA2+ Wasting in Murine Models of Crohn's Disease is Mediated by Concerted Downregulation of Klotho and TRPV5 in Distal Convoluted Tubules Pawel R. Kiela, Robert D. Thurston, Claire B. Larmonier, Rajalakshmy Ramalingam, Daniel Laubitz, Arash Sabetisoofyani, Fayez K. Ghishan Background: Metabolic bone disease and osteopenia/osteoporosis with increased risk of fractures are a common complication in IBD patients. Dysregulated Ca2+ homeostasis is believed to be a contributing factor in the pathogenesis of IBD-associated bone loss. We recently demonstrated experimental colitis results in decreased renal expression of Klotho, a protein with significant role in Ca2+ and vitamin D homeostasis and pathogenesis of osteoporosis. Klotho contributes to renal Ca2+ reabsorption by stabilizing the key Ca2+ channel, TRPV5 on the apical membrane of distal tubule epithelial cells. We therefore investigated whether experimental colitis leads to changes in renal Ca2+ handling and expression of TRPV5 and other key Ca2+ transporters in the kidney. Methods: We utilized three models of murine colitis (all C57BL/6): TNBS colitis, adoptive T-cell transfer model (CD4+CD45RBHi T-cells into RAG1-/- mice), and a model representing a combination of epithelial injury and dysregulated T-cell response (adoptive CD4+ IL-10-/- T-cell transfer into RAG1-/- recipients administered piroxicam in drinking water). Results: All models of colitis were verified by body weight loss, colonic cytokine production (qRT-PCR and explant culture) and histology. Blood analysis of colitic mice revealed normal concentrations of Ca2+, inorganic phosphate (Pi), and PTH. Total serum deoxypyridinoline (tDPD) crosslinks were significantly increased in all colitic mice signifying an increase in bone resorption. Blood and urinalysis demonstrated significantly increased fractional excretion of Ca2+, but not of Pi. Given the increased calcium wasting, we analyzed expression of the renal calcium reabsorption mediators Klotho, TRPV5, TRPV6, PMCA1, and NCX1. Only Klotho and NCX1 showed significantly decreased mRNA expression in all models. However, Western blot analysis indicated that the expression of the distal convoluted tubule-specific and Klothodependent Ca2+ channel, TRPV5, was dramatically decreased along with Klotho protein in colitic mice as compared to their control counterparts. Conclusion: While all murine models of colitis manifest normal blood Ca2+ and Pi, renal Ca2+ reabsorption is inhibited leading to hypercalciuria and a compensatory increase in bone resorption. Further studies will be required to test whether the decreased expression of TRPV5 protein without altered steadystate transcript levels are a direct result of inflammatory cytokine-mediated inhibition of Klotho expression. In this scenario, TNF and IFNγ would act to decrease the expression and enzymatic activity of Klotho, increased sialylation of TRPV5, impaired interaction of TRPV5 and galectin-1, and ultimately lead to the endocytosis and degradation of TRPV5 protein.
Mo1734 IL-6 and Mip-2 Are Important for Neutrophil and Macrophage Recruitment During the Recovery From Colitis in Bacteria-Depleted Bacteria Di Meng, N. Nanda Nanthakumar Conventionally raised mice successfully recover from the mucosal injury caused by DSSinduced colitis by recruiting neutrophil and macrophages into the site of injury. In contrast, we found that bacteria-depleted mice (BD mice), generated by treatment with broad-spectrum oral antibiotics for two weeks, failed to recover from subsequent DSS-induced injury and suffered significant mortality because they fail to recruit neutrophil and macrophages. These observations suggest that interactions between components of the normal gut microflora and the host are required for recovery via neutrophil and macrophages recruitment and recover from DSS-mediated intestinal injury. Monoclonization of gram-negative Bacteroides fragilis and E. coli F-18, but not gram-positive Lactobacillus bacteria in BD mice prevented DSS-induced mortality in BD mice with robust infiltration of neutrophil and macrophage. Since LPS is one component that is common to both these bacteria, we then determined if LPS alone would be sufficient to rescue BD mice from DSS-induced colitis associated mortality. We found that oral treatment with LPS alone was sufficient to reduce DSS-induced mucosal injury and mortality in the BD mice. The data suggest that commensal Gramnegative bacteria are able to activate TLR4-dependent signals that enhance the recovery from DSS-induced colitis in BD mice. In support of this idea, we found that B.fragilis, E.coli (F18) and LPS were unable to rescue the mortality caused by DSS-induced colitis in bacteriadepleted TLR4-/- mice. We investigated the mechanistic basis for these observations and found that DSS treatment resulted in lower intestinal expression of MIP2 and IL-6, and correspondingly lower levels of neutrophil and macrophage recruitment to the damaged mucosa, in bacteria-depleted mice than in control animals. Exogenous MIP2 was able to rescue bacteria-depleted mice from DSS-induced mortality by robust recruitment of neutrophil and macrophage at the site of injury in the bacteria-depleted wild type and IL-6-/- mice. However, LPS was only able to rescue conventional BD wild type mice, but not IL-6-/- bacteriadepleted mice, from injury-induced mortality. These data strongly suggests that IL-6 mediated MIP2-dependent LPS-mediated innate immune response in bacteria-depleted mice preventing mortality. Induction of IL-6 is an important mechanism by which neutrophils and macrophages are recruited to prevent intestinal injury associate-mortality in bacteria-depleted mice. This study sheds light on novel role of the symbiotic microbiota in regulating intestinal innate inflammatory responses and protecting against tissue injury and associated mortality.
Mo1737 T2 Relaxometry Distinguishes Acute Murine DSS Colitis From Chronic Relapsing Colitis. A Novel Tool to Assess Transmural Fibrotic Lesions? Christine M. Breynaert, Tom Dresselaers, Clémentine Perrier, Severine Vermeire, Paul J. Rutgeerts, Jan L. Ceuppens, Uwe Himmelreich, Gert A. Van Assche
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BACKGROUND AND AIM: In human Crohn's disease (CD), the value of MRI and CT enterography as a non invasive assessment tool of transmural inflammation and extraluminal complications is increasingly recognized. However, data on MR imaging as a tool to study murine colitis are very limited. The aim of this study was to study whether micro(μ)MR imaging using In Vivo T2 relaxometry is able to distinguish inflammatory and fibrotic lesions in DSS induced acute and chronic colitis. MATERIAL AND METHODS: DSS colitis was induced in 6 week-old C57BL6/J mice. Three groups were compared: control mice (n=6) had normal drinking water, acute colitis mice (n=8) 2% DSS in the drinking water 7 days prior to scanning and chronic colitis mice (n=12) 2 cycles of 7 days of DSS followed by 2 weeks of normal drinking water. Two mice per condition were scanned. The other mice were sacrificed for scoring and FACS analysis of blood and mesenteric lymph nodes (MLN). T2 weighted images and T2 maps of the distal colon were recorded on a 9.4T μMRI system (Bruker). Regions of interest delineating the colon wall were identified on T2 weighted images. Histograms were created from T2 maps in 4 slices per animal. After scanning the distal colon was harvested for histology. Collagen deposition was quantified with MartiusScarlet-Blue staining and measured using ImageJ in 4 cross-sections per mouse. Statistical analysis was performed using student t-test or ANOVA. This study was approved by the Institutional Animal Care Commission and Ethics Committee. RESULTS: The disease activity score normalized in the chronic colitis group compared to the acute colitis group. However, the macroscopic score of the colon (p= 0,035) and the colon weight (p=0,004) were significantly higher in the chronic model compared to the acute model. CD4+foxp3+ cells in peripheral blood decreased in the acute model but normalized in the chronic phase (p= 0.009). IL17+ cells in MLN were significantly higher in the chronic model compared to the acute model (p=0.026), IFNγ+ cells in MLN had the same trend. No difference was seen for IL13+ cells. The histograms of the colon T2 values showed a clear shift towards higher values for the acute colitis mice but lower values for the chronic colitis versus the control condition (figure 1). Histology showed an increased collagen deposition and more αSMA+ cells in the submucosa of the chronic versus the acute colitis mice, indicating more fibrosis and mesenchymal hyperplasia in chronic colitis compared to acute colitis and normal mice. CONCLUSIONS: The immune profile of a chronic DSS model with induction of relapse and remission is clearly different from the acute DSS model. Moreover, these μMRI data
Significance of IgA Anti-OmpC (Outer Membrane Protein C) Antibodies in Inflammatory Bowel Disease. A Single Centre Prospective Study Stanislav Rejchrt, Marcela Drahosova, Darina Kohoutova, Jiri Cyrany, Tomáš Douda, Ilja Tacheci, Rudolf Repak, Jolana Bartova, Marcela Kopacova, Jan Bures Background. Some patients with inflammatory bowel disease (IBD) have been found to have antibodies directed against antigens derived from a variety of bacteria, including Escherichia coli, Pseudomonas and several mycobacteria. The OmpC (outer membrane protein C) is an outer membrane porin, Escherichia coli protein, that is immunoreactive to pANCA antibodies (in subset of ASCA negative patients). Presence of OmpC antibodies might correlate with more complicated disease phenotype. The aim of this prospective study was to investigate antibodies targeting OmpC in Crohn's disease (CD), ulcerative colitis (UC) and healthy controls. Material and methods. The study included a total of 64 CD (27 men, 37 women, aged 20-72, mean 42±15 years) & 20 UC patients (6 men, 14 women, aged 25-74, mean 49±16 years) and 45 controls, healthy blood donors (24 men, 21 women, aged 19-59, mean 37±10 years). Serum IgA anti-OmpC antibodies were investigated by means of ELISA (using QUANTA Lite OMP Plus, Inova Diagnostics, San Diego, USA). Values of 0-20 U/mL were considered negative, values > 25 U/mL were counted as positive. Results. Anti-OmpC antibodies were positive in 43/64 (67%) CD and 14/20 (70%) UC patients, and negative or grey-zone in 37/45 (82%) healthy controls. Values in CD and UC were significantly higher compared to controls (p<0.001) but not significantly different reciprocally (p=0.301). Anti-OmpC antibodies did not correlate with an internal perforating phenotype of CD. Conclusions. Anti-OmpC antibodies have been identified as a potential serologic marker of IBD (but not suitable for differentiating CD and UC). These promising results are stimulating for further research of the possible role of intestinal bacteria in the etiopathogenesis of IBD. Acknowledgements. The study was supported by research project MZO 00179906 from the Ministry of Health.
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in subjects with IBD may provoke histamine intolerance with symptoms such as diarrhea, stomach and intestinal pain after food intake.
suggest that In Vivo MRI T2 relaxometry could distinguish between inflammatory and fibrotic lesions in a murine model of IBD, and should be explored in patients with CD.
Selective Glucocorticoid Receptor Agonists (SEGRAs) in Intestinal Disease Promising Effects for Inflammation and Intestinal Tissue Repair In Vitro Kerstin C. Reuter, Stefan Marcel Loitsch, Dieter Steinhilber, Jürgen Stein Despite their excellent anti-inflammatory and immunsuppressive action, treatment of inflammatory bowel disease (IBD) with Glucocorticoids (GC) still carries significant risks in terms of frequently occurring severe side effects, such as inhibition of intestinal tissue repair. Therefore, several selective Glucocorticoid Receptor (GR) Agonists (SEGRAs) have been introduced, offering anti-inflammatory action with fewer side effects. We investigated the effect of the non-steroidal SEGRAs, Compound A (CpdA) and ZK216348 in In Vitro models of IBD and intestinal wound healing. Caco-2, IEC-6 and HEK293T cells were cultured in the presence of various concentrations of Dexamethasone (Dex), CpdA, ZK216348 or specific inhibitors and, where required, were stimulated with TNF-α/ IL-1β [0.5 nM] from 15 min - 24 h. GR translocation was shown by indirect immunfluorescence. NF-κB/ p65 activity was detected by EMSA. Western Blot analysis was used for detection of p65, IκB-α, GR, Annexin-1 expression and evaluation of TGF-β and EGF-pathways. IL-8 and TGF-β1 protein level in cell culture supernatants were evaluated by ELISA technique, mRNA levels of GR and MKP-1 by qPCR. Reporter gene assays were used for trans-activation potency and investigation of TGF-β signalling pathway. Cell migration was determined using an In Vitro wound healing model, cell proliferation by BrdU incorporation assay. Apoptosis of intestinal epithelial cells was assessed using Annexin V/ 7 AAD - FACS analysis. Both Dex and SEGRAs induced the nuclear translocation of GR in Caco-2 cells and revealed no apoptotic potential in used concentrations. However in contrast to SEGRAs, Dex significantly decreased GR expression, mRNA-levels and -stability over time. In terms of transrepression, CpdA and ZK216348 effectively inhibited NF-κB activation and reduced IL-8 secretion of Caco-2 cells, but showed no transactivation potency in pGRE-Luc reporter gene assay. Furthermore, Dex, but not SEGRAs, caused a dose-dependent inhibition of IEC-6 intestinal epithelial cell migration with no effect on cell proliferation in relevant concentrations. The addition of TGFβ1 or EGF partially reversed this effect. TGF-β1 and MAPK signalling pathways important for intestinal wound healing were not affected by SEGRAs but Dex, shown by decreased TGFβ1 peptide levels, Smad-expression and decreased pSBE4-Luc reporter gene activity. Additionally, Dex inhibited EGFR phoshorylation and ERK1/2 phosphorylation by transactivation of MKP-1 transcription and Annexin-1 expression, respectively. Our results illustrate an anti-inflammatory action of CpdA and ZK216348 comparable to Dex with fewer side-effects in respect to intestinal wound healing. We suggest that SEGRAs might offer a new therapeutic option for inflammatory disorders as IBD.
Figure 1 : T2 histograms from the colon for the control, acute and chronic condition Mo1738 Inhibition of NF-kB by a PXR-Dependent Pathway Mediates Counterregulatory Activities of Rifaximin on Innate Immunity in Intestinal Epithelial Cells Andrea Mencarelli, Eleonora Distrutti, Barbara Renga, Giuseppe Palladino, Miriam Barbanti, Stefano Fiorucci Background: Intestinal epithelial cells (IECs) are increasingly recognized as an essential player in the regulation of intestinal immune homeostasis. During inflammatory bowel diseases (IBDs) several chemokines have a dysregulated pattern of expression on IEC contributing to the sustained mucosal inflammation in IBD. Avoiding inappropriate activation of TLR4 via NF-kB inhibition, during IBD, might have a translational readout because epithelial cells are in constant contact with a dense and complex milieu of commensal microorganisms. In addition to the antimicrobial activity, rifaximin a poorly absorbed antibiotic, acts as a gut-specific human PXR ligand Aims. To investigate whether rifaximin regulates epithelial intestinal immune homeostasis through a PXR-dependent mechanism. Methods. CRL-1831 cells, a normal colonic human epithelial cell line (ATCC), were exposed to LPS (1 μg/ml for 20 h) alone and in combination with rifaximin (50 μM). Cytokines and chemokines generation was measured by RT-PCR and ELISA. NF-kB binding activity by EMSA. Additional experiments were carried out in CRL-1831 cells in after PXR silencing (siPXR). Biopsy specimens obtained from CD patients, were cultured with LPS (100 mg/ml) alone or in combination with rifaximin (100 μM) for 18h. Results. CRL-1831 cells express PXR. In comparison to wild type cells, PXR silencing decreased the TGF-β and IP-10 production (P< 0.05) while generation of TNF-α, IL-8, RANTES, PGE2 and MIP-3α and IL-6 mRNA levels were increased (P< 0.05). LPS stimulation further enhanced the production of TNFα, IL-8, Rantes, PGE2, and MIP-3α mRNA levels in siPXR cells (P<0.05). LPS stimulation increased TGF-β production in cell line and siPXR cells. Rifaximin causes a robust attenuation of inflammatory response caused by LPS (P< 0.05), while increased TGF-β (P< 0.05) and IL-6 mRNA (P < 0.05). si PXR abrogated completely the ability of rifaximin to change the IEC's immune profile. Rifaximin cotreatment LPS induced NF-κB DNA binding activityin a PXR-dependent manner. Exposure of colon biopsies to rifaximin reduces the generation of IL-8, Rantes and TNFα caused by LPS and iboosted TGF-β production. Conclusions. Rifaximin regulates IEC immune functions by a PXR mediated mechanism. Attenuation of endotoxin-induced immune activation of epithelial cells might contribute to the immunomodulatory activities of rifaximin in IBDs.
Mo1741 Crohn's Disease Paneth Cells Exhibit an Increase of LC3 Storage Elodie Thachil, Jean-Pierre Hugot, Régine Paris, Frédérick Barreau, Maryline Roy, Dominique Berrebi, Jérôme Viala Background/Aims: An induction deficiency of autophagy has been reported for the NOD2 and ATG16L1 risk alleles associated to Crohn's Disease (CD). However, little is known about autophagy in normal and CD intestinal tissues. The aim of this study was to describe the autophagy processes in gut biopsies from CD patients and controls. Methods: 23 pediatric CD patients were selected at diagnostic before any treatment and were compared to 13 healthy controls, 3 ulcerative colitis and 9 celiac disease patients. Patients and controls were genotyped for NOD2, ATG16L1, and IRGM main CD risk alleles. Autophagy was investigated by immunostaining of LC3, Beclin1 and P62 in four locations including duodenum, ileum, left and right colons. Results: No expression of autophagic markers was observed in controls intestinal mucosa while a strong expression of LC3 was detected within Paneth cells of CD patients, especially in biopsies from ileum and duodenum (3.3% vs 60.2% (P< 0.01) of Paneth cells are LC3 positive in duodenum and 4.8% vs 45.8% (P<0.01) in ileum of controls and CD patients respectively). LC3 staining was not correlated to inflammation level and patients genotypes. LC3 expression was weakly detected in duodenal and ileal biopsies of celiac disease and ulcerative colitis patients. However, Beclin1 and P62 immunohistochemical stainings were not observed in CD patients and controls Paneth cells. Conclusion: CD Paneth cells exhibit a strong level of LC3 expression, in inflamed and non-inflamed parts of the gut. However, the absence of other autophagic markers suggests that a LC3 dependent vesicular mechanism, independent of autophagy process, may be in cause.
Mo1739 Decreased Diamine Oxidase Release Into the Intestinal Lymph in Rats With Experimental Colitis Yasuhisa Sakata, Yong Ji, Patrick Tso BACKGROUND: Diamine oxidase (DAO) is one of the histamine-degrading enzymes, which exists in intestinal tract and is thought to eliminate ingested histamine. Impairment of the enzymatic function of DAO causes histamine intolerance. Several studies suggested that DAO and histamine were involved in the pathogenesis of inflammatory bowel disease (IBD). We previously reported that DAO release was regulated by nutrients, especially with fat absorption. Recently, in lymph fistula rats, we showed that the ingestion of dietary fat stimulated intestinal mast cell activation and this resulted in histamine release into lymph. AIM: The aim of this study was to determine if DAO release after meal was impaired in experimental colitis. METHODS: Male Sprague-Dawley rats (n=7) were given 5% dextran sulfate sodium (DSS) in drinking water for 5 days. Following a 2-day recovery period during which DSS was omitted, the main mesenteric lymph duct was cannulated. Rats received a 3-ml bolus dose of saline or Ensure (3.24kcal) through the intraduodenal feeding tube and lymph was collected continuously. The lymphatic level of glucose, triglyceride, cholesterol, nonesterified fatty acid (NEFA), phospholipid and protein were measured. DAO activity was measured by a radiometric assay using 3H- labeled putrescine as substrate. The colon and the small intestine were removed for measurement of myeloperoxidase (MPO) activities and for the evaluation of tissue damage. RESULTS: Infusion of Ensure increased the rate of DAO output into the intestinal lymph, which reached a peak value by 1 hour. The peak rate of lymphatic DAO output was significantly lower in rats with acute DSS-induced colitis (225.01±21.70mU/h) than rats without colitis (366.44±38.50 mU/h, P<0.01). Tissue MPO activity in the colon was significantly increased in rats with colitis as compared to rats without colitis (51.04±10.67 mU/mg tissue vs. 8.53±1.86 mU/mg tissue, P<0.01) but not in the small intestine. In rats with acute colitis, tissue damages of the small intestine were not observed and nutrients output into the intestinal lymph were not disturbed. CONCLUSION: Acute colonic inflammation without small intestinal involvement altered the response of DAO release into the intestinal lymph by nutrients. Reduced DAO activity
Mo1742 Hypoxia, Adenosine, and Adenosine Receptor 2B as Pathophysiological Regulators of Gut Enterochromaffin Cell Serotonin Synthesis and Secretion in Active Crohn's Disease Bernhard Svejda, Maria Brønstad, Erik Solligard, Roswitha Pfragner, Irvin Modlin, Bjorn I. Gustafsson, Mark Kidd Background: Serotonin synthesized and secreted by gut enterochromaffin (EC) cells regulates immune cell proinflammatory production; EC cells therefore play a regulatory role in inflammatory bowel disorders (IBD). These cells are also chemomechanosensors and respond to adenosine released during peristalsis and bowel motion with serotonin secretion via activation of ADORA2 receptors. Nucleotides are also released by hypoxia, a common mucosal phenomenon in IBD. We hypothesized that the elevated EC cell serotonin signaling noted in Crohn's disease was due to exposure to hypoxia. Methods: Human samples including whole mucosa (normal: n=5, Crohn's disease: n=8), isolated normal and Crohn's disease EC cells, and the EC cell tumor cell line KRJ-I were studied. Real-time PCR and western blot (WB) were used to characterize ADORA2A/V, tryptophan hydroxylase 1 (Tph1 - the rate-limiting step in serotonin synthesis) and HIF1a expression. In KRJ-I, the effect of the ADORA agonist (NECA), 2A/B antagonists (SCH442416/MRS1754) and hypoxia (0, 30 mins and 120 mins)
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AGA Abstracts
AGA Abstracts
Mo1740
(1.21±0.82% vs. 0.55±0.27%, p=0.03) respectively. Zonulin concentrations correlated urinary L/M ratio in IBD (r=0.86, p<0.01). BDNF correlated with the L/M ratio in healthy controls (r=0.76, p=0.04). IBD patients showed higher cortisol (427.22±113.32 vs. 297.38±114.15 nmol/L, p = 0.03), TNFα (3.12±3.28 vs. 0.99±0.26 pg/ml, p=0.07), and IL-6 (7.63±7.32 vs. 1.26±0.41 pg/ml, p=0.02) than controls. Conclusion: The increased segmental permeability seen in IBD patients was associated with an uncoupling of the relationship between permeability and BDNF evident in healthy controls and a compensatory correlation with increased serum zonulin levels. As neuronal and enteric glial cells regulating neuroimmune signaling, barrier permeability and neuroplasticity are the main source of BDNF, it is likely that dysregulation of BDNF release from glial cells is directly implicated in the inflammatory changes of IP. The correlation between zonulin and IP changes are of relevance due to zonulin's housekeeping functions, which include regulation of tight junction proteins, bacterial colonisation and host-microbiome-immune interactions, all of which are known to be involved in the pathogenesis of IBD. Further studies are required to extend these preliminary data. Grant support by Defence Science & Technology Agency (DSTA), Singapore (1). Reinshagen, M., et al (2002) Curr. Opin. Investig. Drugs 3, 565-568
Mo1736 Renal CA2+ Wasting in Murine Models of Crohn's Disease is Mediated by Concerted Downregulation of Klotho and TRPV5 in Distal Convoluted Tubules Pawel R. Kiela, Robert D. Thurston, Claire B. Larmonier, Rajalakshmy Ramalingam, Daniel Laubitz, Arash Sabetisoofyani, Fayez K. Ghishan Background: Metabolic bone disease and osteopenia/osteoporosis with increased risk of fractures are a common complication in IBD patients. Dysregulated Ca2+ homeostasis is believed to be a contributing factor in the pathogenesis of IBD-associated bone loss. We recently demonstrated experimental colitis results in decreased renal expression of Klotho, a protein with significant role in Ca2+ and vitamin D homeostasis and pathogenesis of osteoporosis. Klotho contributes to renal Ca2+ reabsorption by stabilizing the key Ca2+ channel, TRPV5 on the apical membrane of distal tubule epithelial cells. We therefore investigated whether experimental colitis leads to changes in renal Ca2+ handling and expression of TRPV5 and other key Ca2+ transporters in the kidney. Methods: We utilized three models of murine colitis (all C57BL/6): TNBS colitis, adoptive T-cell transfer model (CD4+CD45RBHi T-cells into RAG1-/- mice), and a model representing a combination of epithelial injury and dysregulated T-cell response (adoptive CD4+ IL-10-/- T-cell transfer into RAG1-/- recipients administered piroxicam in drinking water). Results: All models of colitis were verified by body weight loss, colonic cytokine production (qRT-PCR and explant culture) and histology. Blood analysis of colitic mice revealed normal concentrations of Ca2+, inorganic phosphate (Pi), and PTH. Total serum deoxypyridinoline (tDPD) crosslinks were significantly increased in all colitic mice signifying an increase in bone resorption. Blood and urinalysis demonstrated significantly increased fractional excretion of Ca2+, but not of Pi. Given the increased calcium wasting, we analyzed expression of the renal calcium reabsorption mediators Klotho, TRPV5, TRPV6, PMCA1, and NCX1. Only Klotho and NCX1 showed significantly decreased mRNA expression in all models. However, Western blot analysis indicated that the expression of the distal convoluted tubule-specific and Klothodependent Ca2+ channel, TRPV5, was dramatically decreased along with Klotho protein in colitic mice as compared to their control counterparts. Conclusion: While all murine models of colitis manifest normal blood Ca2+ and Pi, renal Ca2+ reabsorption is inhibited leading to hypercalciuria and a compensatory increase in bone resorption. Further studies will be required to test whether the decreased expression of TRPV5 protein without altered steadystate transcript levels are a direct result of inflammatory cytokine-mediated inhibition of Klotho expression. In this scenario, TNF and IFNγ would act to decrease the expression and enzymatic activity of Klotho, increased sialylation of TRPV5, impaired interaction of TRPV5 and galectin-1, and ultimately lead to the endocytosis and degradation of TRPV5 protein.
Mo1734 IL-6 and Mip-2 Are Important for Neutrophil and Macrophage Recruitment During the Recovery From Colitis in Bacteria-Depleted Bacteria Di Meng, N. Nanda Nanthakumar Conventionally raised mice successfully recover from the mucosal injury caused by DSSinduced colitis by recruiting neutrophil and macrophages into the site of injury. In contrast, we found that bacteria-depleted mice (BD mice), generated by treatment with broad-spectrum oral antibiotics for two weeks, failed to recover from subsequent DSS-induced injury and suffered significant mortality because they fail to recruit neutrophil and macrophages. These observations suggest that interactions between components of the normal gut microflora and the host are required for recovery via neutrophil and macrophages recruitment and recover from DSS-mediated intestinal injury. Monoclonization of gram-negative Bacteroides fragilis and E. coli F-18, but not gram-positive Lactobacillus bacteria in BD mice prevented DSS-induced mortality in BD mice with robust infiltration of neutrophil and macrophage. Since LPS is one component that is common to both these bacteria, we then determined if LPS alone would be sufficient to rescue BD mice from DSS-induced colitis associated mortality. We found that oral treatment with LPS alone was sufficient to reduce DSS-induced mucosal injury and mortality in the BD mice. The data suggest that commensal Gramnegative bacteria are able to activate TLR4-dependent signals that enhance the recovery from DSS-induced colitis in BD mice. In support of this idea, we found that B.fragilis, E.coli (F18) and LPS were unable to rescue the mortality caused by DSS-induced colitis in bacteriadepleted TLR4-/- mice. We investigated the mechanistic basis for these observations and found that DSS treatment resulted in lower intestinal expression of MIP2 and IL-6, and correspondingly lower levels of neutrophil and macrophage recruitment to the damaged mucosa, in bacteria-depleted mice than in control animals. Exogenous MIP2 was able to rescue bacteria-depleted mice from DSS-induced mortality by robust recruitment of neutrophil and macrophage at the site of injury in the bacteria-depleted wild type and IL-6-/- mice. However, LPS was only able to rescue conventional BD wild type mice, but not IL-6-/- bacteriadepleted mice, from injury-induced mortality. These data strongly suggests that IL-6 mediated MIP2-dependent LPS-mediated innate immune response in bacteria-depleted mice preventing mortality. Induction of IL-6 is an important mechanism by which neutrophils and macrophages are recruited to prevent intestinal injury associate-mortality in bacteria-depleted mice. This study sheds light on novel role of the symbiotic microbiota in regulating intestinal innate inflammatory responses and protecting against tissue injury and associated mortality.
Mo1737 T2 Relaxometry Distinguishes Acute Murine DSS Colitis From Chronic Relapsing Colitis. A Novel Tool to Assess Transmural Fibrotic Lesions? Christine M. Breynaert, Tom Dresselaers, Clémentine Perrier, Severine Vermeire, Paul J. Rutgeerts, Jan L. Ceuppens, Uwe Himmelreich, Gert A. Van Assche
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BACKGROUND AND AIM: In human Crohn's disease (CD), the value of MRI and CT enterography as a non invasive assessment tool of transmural inflammation and extraluminal complications is increasingly recognized. However, data on MR imaging as a tool to study murine colitis are very limited. The aim of this study was to study whether micro(μ)MR imaging using In Vivo T2 relaxometry is able to distinguish inflammatory and fibrotic lesions in DSS induced acute and chronic colitis. MATERIAL AND METHODS: DSS colitis was induced in 6 week-old C57BL6/J mice. Three groups were compared: control mice (n=6) had normal drinking water, acute colitis mice (n=8) 2% DSS in the drinking water 7 days prior to scanning and chronic colitis mice (n=12) 2 cycles of 7 days of DSS followed by 2 weeks of normal drinking water. Two mice per condition were scanned. The other mice were sacrificed for scoring and FACS analysis of blood and mesenteric lymph nodes (MLN). T2 weighted images and T2 maps of the distal colon were recorded on a 9.4T μMRI system (Bruker). Regions of interest delineating the colon wall were identified on T2 weighted images. Histograms were created from T2 maps in 4 slices per animal. After scanning the distal colon was harvested for histology. Collagen deposition was quantified with MartiusScarlet-Blue staining and measured using ImageJ in 4 cross-sections per mouse. Statistical analysis was performed using student t-test or ANOVA. This study was approved by the Institutional Animal Care Commission and Ethics Committee. RESULTS: The disease activity score normalized in the chronic colitis group compared to the acute colitis group. However, the macroscopic score of the colon (p= 0,035) and the colon weight (p=0,004) were significantly higher in the chronic model compared to the acute model. CD4+foxp3+ cells in peripheral blood decreased in the acute model but normalized in the chronic phase (p= 0.009). IL17+ cells in MLN were significantly higher in the chronic model compared to the acute model (p=0.026), IFNγ+ cells in MLN had the same trend. No difference was seen for IL13+ cells. The histograms of the colon T2 values showed a clear shift towards higher values for the acute colitis mice but lower values for the chronic colitis versus the control condition (figure 1). Histology showed an increased collagen deposition and more αSMA+ cells in the submucosa of the chronic versus the acute colitis mice, indicating more fibrosis and mesenchymal hyperplasia in chronic colitis compared to acute colitis and normal mice. CONCLUSIONS: The immune profile of a chronic DSS model with induction of relapse and remission is clearly different from the acute DSS model. Moreover, these μMRI data
Significance of IgA Anti-OmpC (Outer Membrane Protein C) Antibodies in Inflammatory Bowel Disease. A Single Centre Prospective Study Stanislav Rejchrt, Marcela Drahosova, Darina Kohoutova, Jiri Cyrany, Tomáš Douda, Ilja Tacheci, Rudolf Repak, Jolana Bartova, Marcela Kopacova, Jan Bures Background. Some patients with inflammatory bowel disease (IBD) have been found to have antibodies directed against antigens derived from a variety of bacteria, including Escherichia coli, Pseudomonas and several mycobacteria. The OmpC (outer membrane protein C) is an outer membrane porin, Escherichia coli protein, that is immunoreactive to pANCA antibodies (in subset of ASCA negative patients). Presence of OmpC antibodies might correlate with more complicated disease phenotype. The aim of this prospective study was to investigate antibodies targeting OmpC in Crohn's disease (CD), ulcerative colitis (UC) and healthy controls. Material and methods. The study included a total of 64 CD (27 men, 37 women, aged 20-72, mean 42±15 years) & 20 UC patients (6 men, 14 women, aged 25-74, mean 49±16 years) and 45 controls, healthy blood donors (24 men, 21 women, aged 19-59, mean 37±10 years). Serum IgA anti-OmpC antibodies were investigated by means of ELISA (using QUANTA Lite OMP Plus, Inova Diagnostics, San Diego, USA). Values of 0-20 U/mL were considered negative, values > 25 U/mL were counted as positive. Results. Anti-OmpC antibodies were positive in 43/64 (67%) CD and 14/20 (70%) UC patients, and negative or grey-zone in 37/45 (82%) healthy controls. Values in CD and UC were significantly higher compared to controls (p<0.001) but not significantly different reciprocally (p=0.301). Anti-OmpC antibodies did not correlate with an internal perforating phenotype of CD. Conclusions. Anti-OmpC antibodies have been identified as a potential serologic marker of IBD (but not suitable for differentiating CD and UC). These promising results are stimulating for further research of the possible role of intestinal bacteria in the etiopathogenesis of IBD. Acknowledgements. The study was supported by research project MZO 00179906 from the Ministry of Health.
AGA Abstracts
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in subjects with IBD may provoke histamine intolerance with symptoms such as diarrhea, stomach and intestinal pain after food intake.
suggest that In Vivo MRI T2 relaxometry could distinguish between inflammatory and fibrotic lesions in a murine model of IBD, and should be explored in patients with CD.
Selective Glucocorticoid Receptor Agonists (SEGRAs) in Intestinal Disease Promising Effects for Inflammation and Intestinal Tissue Repair In Vitro Kerstin C. Reuter, Stefan Marcel Loitsch, Dieter Steinhilber, Jürgen Stein Despite their excellent anti-inflammatory and immunsuppressive action, treatment of inflammatory bowel disease (IBD) with Glucocorticoids (GC) still carries significant risks in terms of frequently occurring severe side effects, such as inhibition of intestinal tissue repair. Therefore, several selective Glucocorticoid Receptor (GR) Agonists (SEGRAs) have been introduced, offering anti-inflammatory action with fewer side effects. We investigated the effect of the non-steroidal SEGRAs, Compound A (CpdA) and ZK216348 in In Vitro models of IBD and intestinal wound healing. Caco-2, IEC-6 and HEK293T cells were cultured in the presence of various concentrations of Dexamethasone (Dex), CpdA, ZK216348 or specific inhibitors and, where required, were stimulated with TNF-α/ IL-1β [0.5 nM] from 15 min - 24 h. GR translocation was shown by indirect immunfluorescence. NF-κB/ p65 activity was detected by EMSA. Western Blot analysis was used for detection of p65, IκB-α, GR, Annexin-1 expression and evaluation of TGF-β and EGF-pathways. IL-8 and TGF-β1 protein level in cell culture supernatants were evaluated by ELISA technique, mRNA levels of GR and MKP-1 by qPCR. Reporter gene assays were used for trans-activation potency and investigation of TGF-β signalling pathway. Cell migration was determined using an In Vitro wound healing model, cell proliferation by BrdU incorporation assay. Apoptosis of intestinal epithelial cells was assessed using Annexin V/ 7 AAD - FACS analysis. Both Dex and SEGRAs induced the nuclear translocation of GR in Caco-2 cells and revealed no apoptotic potential in used concentrations. However in contrast to SEGRAs, Dex significantly decreased GR expression, mRNA-levels and -stability over time. In terms of transrepression, CpdA and ZK216348 effectively inhibited NF-κB activation and reduced IL-8 secretion of Caco-2 cells, but showed no transactivation potency in pGRE-Luc reporter gene assay. Furthermore, Dex, but not SEGRAs, caused a dose-dependent inhibition of IEC-6 intestinal epithelial cell migration with no effect on cell proliferation in relevant concentrations. The addition of TGFβ1 or EGF partially reversed this effect. TGF-β1 and MAPK signalling pathways important for intestinal wound healing were not affected by SEGRAs but Dex, shown by decreased TGFβ1 peptide levels, Smad-expression and decreased pSBE4-Luc reporter gene activity. Additionally, Dex inhibited EGFR phoshorylation and ERK1/2 phosphorylation by transactivation of MKP-1 transcription and Annexin-1 expression, respectively. Our results illustrate an anti-inflammatory action of CpdA and ZK216348 comparable to Dex with fewer side-effects in respect to intestinal wound healing. We suggest that SEGRAs might offer a new therapeutic option for inflammatory disorders as IBD.
Figure 1 : T2 histograms from the colon for the control, acute and chronic condition Mo1738 Inhibition of NF-kB by a PXR-Dependent Pathway Mediates Counterregulatory Activities of Rifaximin on Innate Immunity in Intestinal Epithelial Cells Andrea Mencarelli, Eleonora Distrutti, Barbara Renga, Giuseppe Palladino, Miriam Barbanti, Stefano Fiorucci Background: Intestinal epithelial cells (IECs) are increasingly recognized as an essential player in the regulation of intestinal immune homeostasis. During inflammatory bowel diseases (IBDs) several chemokines have a dysregulated pattern of expression on IEC contributing to the sustained mucosal inflammation in IBD. Avoiding inappropriate activation of TLR4 via NF-kB inhibition, during IBD, might have a translational readout because epithelial cells are in constant contact with a dense and complex milieu of commensal microorganisms. In addition to the antimicrobial activity, rifaximin a poorly absorbed antibiotic, acts as a gut-specific human PXR ligand Aims. To investigate whether rifaximin regulates epithelial intestinal immune homeostasis through a PXR-dependent mechanism. Methods. CRL-1831 cells, a normal colonic human epithelial cell line (ATCC), were exposed to LPS (1 μg/ml for 20 h) alone and in combination with rifaximin (50 μM). Cytokines and chemokines generation was measured by RT-PCR and ELISA. NF-kB binding activity by EMSA. Additional experiments were carried out in CRL-1831 cells in after PXR silencing (siPXR). Biopsy specimens obtained from CD patients, were cultured with LPS (100 mg/ml) alone or in combination with rifaximin (100 μM) for 18h. Results. CRL-1831 cells express PXR. In comparison to wild type cells, PXR silencing decreased the TGF-β and IP-10 production (P< 0.05) while generation of TNF-α, IL-8, RANTES, PGE2 and MIP-3α and IL-6 mRNA levels were increased (P< 0.05). LPS stimulation further enhanced the production of TNFα, IL-8, Rantes, PGE2, and MIP-3α mRNA levels in siPXR cells (P<0.05). LPS stimulation increased TGF-β production in cell line and siPXR cells. Rifaximin causes a robust attenuation of inflammatory response caused by LPS (P< 0.05), while increased TGF-β (P< 0.05) and IL-6 mRNA (P < 0.05). si PXR abrogated completely the ability of rifaximin to change the IEC's immune profile. Rifaximin cotreatment LPS induced NF-κB DNA binding activityin a PXR-dependent manner. Exposure of colon biopsies to rifaximin reduces the generation of IL-8, Rantes and TNFα caused by LPS and iboosted TGF-β production. Conclusions. Rifaximin regulates IEC immune functions by a PXR mediated mechanism. Attenuation of endotoxin-induced immune activation of epithelial cells might contribute to the immunomodulatory activities of rifaximin in IBDs.
Mo1741 Crohn's Disease Paneth Cells Exhibit an Increase of LC3 Storage Elodie Thachil, Jean-Pierre Hugot, Régine Paris, Frédérick Barreau, Maryline Roy, Dominique Berrebi, Jérôme Viala Background/Aims: An induction deficiency of autophagy has been reported for the NOD2 and ATG16L1 risk alleles associated to Crohn's Disease (CD). However, little is known about autophagy in normal and CD intestinal tissues. The aim of this study was to describe the autophagy processes in gut biopsies from CD patients and controls. Methods: 23 pediatric CD patients were selected at diagnostic before any treatment and were compared to 13 healthy controls, 3 ulcerative colitis and 9 celiac disease patients. Patients and controls were genotyped for NOD2, ATG16L1, and IRGM main CD risk alleles. Autophagy was investigated by immunostaining of LC3, Beclin1 and P62 in four locations including duodenum, ileum, left and right colons. Results: No expression of autophagic markers was observed in controls intestinal mucosa while a strong expression of LC3 was detected within Paneth cells of CD patients, especially in biopsies from ileum and duodenum (3.3% vs 60.2% (P< 0.01) of Paneth cells are LC3 positive in duodenum and 4.8% vs 45.8% (P<0.01) in ileum of controls and CD patients respectively). LC3 staining was not correlated to inflammation level and patients genotypes. LC3 expression was weakly detected in duodenal and ileal biopsies of celiac disease and ulcerative colitis patients. However, Beclin1 and P62 immunohistochemical stainings were not observed in CD patients and controls Paneth cells. Conclusion: CD Paneth cells exhibit a strong level of LC3 expression, in inflamed and non-inflamed parts of the gut. However, the absence of other autophagic markers suggests that a LC3 dependent vesicular mechanism, independent of autophagy process, may be in cause.
Mo1739 Decreased Diamine Oxidase Release Into the Intestinal Lymph in Rats With Experimental Colitis Yasuhisa Sakata, Yong Ji, Patrick Tso BACKGROUND: Diamine oxidase (DAO) is one of the histamine-degrading enzymes, which exists in intestinal tract and is thought to eliminate ingested histamine. Impairment of the enzymatic function of DAO causes histamine intolerance. Several studies suggested that DAO and histamine were involved in the pathogenesis of inflammatory bowel disease (IBD). We previously reported that DAO release was regulated by nutrients, especially with fat absorption. Recently, in lymph fistula rats, we showed that the ingestion of dietary fat stimulated intestinal mast cell activation and this resulted in histamine release into lymph. AIM: The aim of this study was to determine if DAO release after meal was impaired in experimental colitis. METHODS: Male Sprague-Dawley rats (n=7) were given 5% dextran sulfate sodium (DSS) in drinking water for 5 days. Following a 2-day recovery period during which DSS was omitted, the main mesenteric lymph duct was cannulated. Rats received a 3-ml bolus dose of saline or Ensure (3.24kcal) through the intraduodenal feeding tube and lymph was collected continuously. The lymphatic level of glucose, triglyceride, cholesterol, nonesterified fatty acid (NEFA), phospholipid and protein were measured. DAO activity was measured by a radiometric assay using 3H- labeled putrescine as substrate. The colon and the small intestine were removed for measurement of myeloperoxidase (MPO) activities and for the evaluation of tissue damage. RESULTS: Infusion of Ensure increased the rate of DAO output into the intestinal lymph, which reached a peak value by 1 hour. The peak rate of lymphatic DAO output was significantly lower in rats with acute DSS-induced colitis (225.01±21.70mU/h) than rats without colitis (366.44±38.50 mU/h, P<0.01). Tissue MPO activity in the colon was significantly increased in rats with colitis as compared to rats without colitis (51.04±10.67 mU/mg tissue vs. 8.53±1.86 mU/mg tissue, P<0.01) but not in the small intestine. In rats with acute colitis, tissue damages of the small intestine were not observed and nutrients output into the intestinal lymph were not disturbed. CONCLUSION: Acute colonic inflammation without small intestinal involvement altered the response of DAO release into the intestinal lymph by nutrients. Reduced DAO activity
Mo1742 Hypoxia, Adenosine, and Adenosine Receptor 2B as Pathophysiological Regulators of Gut Enterochromaffin Cell Serotonin Synthesis and Secretion in Active Crohn's Disease Bernhard Svejda, Maria Brønstad, Erik Solligard, Roswitha Pfragner, Irvin Modlin, Bjorn I. Gustafsson, Mark Kidd Background: Serotonin synthesized and secreted by gut enterochromaffin (EC) cells regulates immune cell proinflammatory production; EC cells therefore play a regulatory role in inflammatory bowel disorders (IBD). These cells are also chemomechanosensors and respond to adenosine released during peristalsis and bowel motion with serotonin secretion via activation of ADORA2 receptors. Nucleotides are also released by hypoxia, a common mucosal phenomenon in IBD. We hypothesized that the elevated EC cell serotonin signaling noted in Crohn's disease was due to exposure to hypoxia. Methods: Human samples including whole mucosa (normal: n=5, Crohn's disease: n=8), isolated normal and Crohn's disease EC cells, and the EC cell tumor cell line KRJ-I were studied. Real-time PCR and western blot (WB) were used to characterize ADORA2A/V, tryptophan hydroxylase 1 (Tph1 - the rate-limiting step in serotonin synthesis) and HIF1a expression. In KRJ-I, the effect of the ADORA agonist (NECA), 2A/B antagonists (SCH442416/MRS1754) and hypoxia (0, 30 mins and 120 mins)
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AGA Abstracts
AGA Abstracts
Mo1740