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Comparative Expression of Vitamin D Receptor and Cathelicidin in Human Intestinal Mucosa in Persons With and Without Inflammatory Bowel Disease
Tu1829
Comparative Expression of Vitamin D Receptor and Cathelicidin in Human Intestinal Mucosa in Persons With and Without Inflammatory Bowel Disease Robert Kizer, Devendra K. Agrawal
Relation Between Endoscopic and Histological Activity of the Intestinal Inflammatory Bowel Disease (IBD) and Angiogenic and Lymphangiogenic Factors Alicia Algaba, Pablo M. Linares, Maria Encarnacion Fernandez Contreras, Ámparo Ordoñez, Javier Trápaga Porrero, Iván Guerra, Maria Chaparro, José Luis Rodríguez Agullo, Javier P. Gisbert, Fernando Bermejo
Background: Vitamin D receptor (VDR) expression has been demonstrated to be lower in explanted colons of patients with Ulcerative Colitis (UC) compared to patients without Inflammatory Bowel Disease (IBD). Similar studies are not available with Crohn's disease (CD). Vitamin D has been shown to induce expression of the antimicrobial protein cathelicidin in multiple cell lines, whereas expression of cathelicidin has been shown to be blunted in patients with CD. Objectives: To quantify the expression of the VDR, CYP24A and CYP27B, and cathelicidin in colonic and ileal mucosa of patients with IBD when compared to healthy controls, and to correlate this expression with endoscopically visualized mucosal inflammation in patients with IBD. Methods: Patient undergoing colonoscopy for colorectal cancer screening were invited to participate in this study, as were patients undergoing colonoscopy for either diagnosis or follow-up of IBD. Endoscopic findings were noted. Biopsies were obtained from the rectum and terminal ileum in all patients. Biopsies were obtained from areas of active inflammation in patients with IBD. Pathology reports were reviewed when biopsies were obtained for diagnostic or surveillance purposes. Real Time PCR was performed on samples for VDR, CYP24A, CYP27B and cathelicidin. Immunofluorescent staining was also performed for VDR expression. Results: Forty-two patients were recruited: 27 without IBD, 10 with UC, and 5 with CD. Five patients had endoscopic evidence of active disease with erythema, nodularity, or ulceration. PCR revealed measurable expression of VDR, CYP24A, and cathelicidin in controls and subjects. Minimal expression of CYP27B was present in either controls or subjects. Intestinal mucosa from patients with IBD had decreased mRNA transcripts of VDR compared to those without IBD (p <0.05). There was a trend of increased mRNA transcripts of cathelicidin in the intestinal mucosa from subjects with IBD compared to controls. Results were not statistically significant. Immunostaining revealed prominent VDR expression in normal intestinal epithelium. Intestinal epithelial VDR expression by immunostaining is decreased in endoscopically apparent areas of active IBD when compared with controls. Epithelial VDR expression by immunostaining in IBD samples without active disease was similar to controls. Conclusions: VDR is expressed in healthy intestinal epithelial cells. Intestinal epithelial VDR expression is decreased in patients with active UC and CD when compared to healthy controls, and when compared to IBD patients with inactive disease. Since vitamin D administration enhances VDR expression, a clinical study with vitamin D administration in patients with UC and CD is warranted to confirm the role of VDR in the pathophysiology of IBD.
Aim: To correlate the levels of the main angio and lymphangiogenic factors (ALF) in serum and colonic mucosa biopsies cultures supernatant (CCS) with the endoscopic and histological activity in patients with IBD. Methods: A transversal study in 60 patients with IBD that underwent a colonoscopy because of medical criteria. From each patient, 3 different types of samples were obtained: A blood sample for ALF determination in serum, several samples of colonic tissue for their 24h culture and supernatant determination of the same ALF and other biopsies for histological analyses. In those patients in which activity was observed during the colonoscopy and, in which the disease extension allowed it, samples from affected and non-affected mucosa were also taken. The patients were classified into four groups based on the endoscopic activity (endoscopic Mayo subscore for ulcerative collitis and SESCD for Crohn's disease): non-activity of the disease, mild, moderate and severe activity. Considering histological findings, patients were also classified into four groups: Quiescent IBD, mild, moderate and severe lesion. VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, PlGF, Ang1, Ang2 and Tie2 concentrations both in serum and CCS were determined by ELISA assay. Results: 49% of the patients did not have endoscopic activity, 23% moderate, 20% mild and 8% severe activity. There were significant differences between the concentrations of VEGFA (p<0.001), VEGFC (p<0.05), VEGFD (p<0.001), PlGF (p<0.001), VEGFR1 (p<0.01), Ang-1 (p<0.001), Ang-2 (p<0.001) and Tie-2 (p<0.001) in CCS based on endoscopic activity. These concentrations increase parallel to the endoscopic activity. According to histology, 45% of the patients had quiescent IBD, 25% had a moderate lesion, 22% mild and 8% severe lesions. Levels of all the studied proteins (except VEGFR3) were also significantly higher in those patients with severe histological lesions: VEGFA (p<0.001), VEGFC (p<0.01), VEGFD (p<0.001), PlGF (p<0.001), VEGFR1 (p<0.01), VEGFR2 (p<0.05), Ang1(p<0.001), Ang-2 (p<0.001) and de Tie-2 (p<0.001). The concordance between endoscopic and histological activity was of 88.14%. In 67% of the patients with activity, biopsies of affected and non-affected mucosa were taken for comparison. Significant differences in Ang1 (p<0.05), Ang2 (p<0.001) and VEGFR3 (p<0.05) levels were observed. These levels were higher in the affected than in the non-affected mucosa. Significant differences were demonstrated in the VEGFA (p<0.05) and Ang-1 (p<0.001) serum levels depending on the endoscopic activity observed. Conclusions: The levels of angio and lymphangiogenic factors and their receptors in tissue culture supernatant and VEGFA and Ang-1 in serum correlate with the endoscopic and histologic activity of IBD. Therefore serum determination of VEGFA and Ang-1 could be useful to avoid unnecessary colonoscopies. Tu1830 Murine Guanylate Cyclase C Signaling Regulates Colonic Injury and Inflammation Kris A. Steinbrecher, Eleana Harmel-Laws, Monica P. Garin-Laflam, Elizabeth A. Mann, Mitchell B. Cohen Background: The transmembrane receptor Guanylate Cyclase C (GC-C) is activated by its secreted ligand guanylin (Gn) to produce cGMP and stimulate intestinal secretion. This intestinal epithelial cell (IEC) signaling pathway regulates proliferative/apoptotic homeostasis and maintains IEC monolayer barrier function. We therefore hypothesized that ligandinduced activation of GC-C provides resistance to colonic inflammation. Methods: We utilized the dextran sodium sulfate (DSS) colonic injury model (5 days of 3% DSS) to challenge control, GC-C-/-, and Gn-/- mice (all 10th generation C57BL/6). We measured clinical parameters and histopathology. Proliferation and apoptosis were assessed using immunohistochemistry (IHC). Western blotting and IHC measured resistin-like molecule β (RELMβ) production, and cytokine analysis was performed by ELISA. In some studies, recombinant RELMβ peptide was administered once daily by enema. In a second model, GC-C-/- mice were bred with interleukin (IL)-10 deficient animals. Histological disease scores were assessed in IL-10-/- and GC-C-/-IL-10-/- mice at 6 and 8 weeks of age. Results: Unexpectedly, GC-C-/- mice were resistant to DSS-induced colonic injury. Relative to wildtype (WT) controls, GC-C-/- animals had less rectal bleeding, diarrhea, and colon atrophy. GC-C-/- mice had minimal histological damage, enhanced IEC proliferation, reduced apoptosis, and little induction of TNFα or IFNγ. Gn-/- mice responded similarly. The secreted goblet cell protein RELMβ facilitates DSS-induced injury through activation of infiltrating immune cells which produce TNFα and IFNγ. In contrast to WT mice, RELMβ was not induced by DSS treatment in GC-C-/- animals, despite similar numbers of alcian blue-stained goblet cells and similar expression of the goblet cell marker Muc2 in both genotypes. Supplementation of active RELMβ peptide by enema reversed the resistance of GC-C-/- mice to DSS. Conversely, our analysis of GC-C-/-IL-10-/- mice indicated that GC-C was necessary to suppress spontaneous colitis. Unlike IL-10-/- controls, pathology in GC-C-/-IL-10-/animals was apparent early and, by eight weeks of age, GC-C-/-IL-10-/- mice had severely altered mucosal architecture, crypt abscesses, and hyperplastic epithelia in the submucosa of the colon. TNFα and IFNγ mRNA were elevated in GC-C-/-IL-10-/- mucosa versus that of IL-10-/- mice. Conclusions: GC-C signaling markedly and differentially regulates the response to DSS and IL-10-/- induced colitis. While the lower colonic RELMβ production in GC-C-/- mice may be protective in response to DSS treatment, we speculate that defects in small intestinal barrier function facilitate early disease onset in spontaneous colitis models. This differential response must be considered when investigating the therapeutic targeting of GC-C in the treatment of gastrointestinal inflammatory disease.
Immunostaining for Vitamin D Receptor (VDR) in rectal mucosa from a healthy control.
Immunostaining for VDR in ascending colon mucosa from patient with active IBD in the ascending colon.
S-843
AGA Abstracts
AGA Abstracts
Tu1828
Comparative Expression of Vitamin D Receptor and Cathelicidin in Human Intestinal Mucosa in Persons With and Without Inflammatory Bowel Disease Robert Kizer, Devendra K. Agrawal
Relation Between Endoscopic and Histological Activity of the Intestinal Inflammatory Bowel Disease (IBD) and Angiogenic and Lymphangiogenic Factors Alicia Algaba, Pablo M. Linares, Maria Encarnacion Fernandez Contreras, Ámparo Ordoñez, Javier Trápaga Porrero, Iván Guerra, Maria Chaparro, José Luis Rodríguez Agullo, Javier P. Gisbert, Fernando Bermejo
Background: Vitamin D receptor (VDR) expression has been demonstrated to be lower in explanted colons of patients with Ulcerative Colitis (UC) compared to patients without Inflammatory Bowel Disease (IBD). Similar studies are not available with Crohn's disease (CD). Vitamin D has been shown to induce expression of the antimicrobial protein cathelicidin in multiple cell lines, whereas expression of cathelicidin has been shown to be blunted in patients with CD. Objectives: To quantify the expression of the VDR, CYP24A and CYP27B, and cathelicidin in colonic and ileal mucosa of patients with IBD when compared to healthy controls, and to correlate this expression with endoscopically visualized mucosal inflammation in patients with IBD. Methods: Patient undergoing colonoscopy for colorectal cancer screening were invited to participate in this study, as were patients undergoing colonoscopy for either diagnosis or follow-up of IBD. Endoscopic findings were noted. Biopsies were obtained from the rectum and terminal ileum in all patients. Biopsies were obtained from areas of active inflammation in patients with IBD. Pathology reports were reviewed when biopsies were obtained for diagnostic or surveillance purposes. Real Time PCR was performed on samples for VDR, CYP24A, CYP27B and cathelicidin. Immunofluorescent staining was also performed for VDR expression. Results: Forty-two patients were recruited: 27 without IBD, 10 with UC, and 5 with CD. Five patients had endoscopic evidence of active disease with erythema, nodularity, or ulceration. PCR revealed measurable expression of VDR, CYP24A, and cathelicidin in controls and subjects. Minimal expression of CYP27B was present in either controls or subjects. Intestinal mucosa from patients with IBD had decreased mRNA transcripts of VDR compared to those without IBD (p <0.05). There was a trend of increased mRNA transcripts of cathelicidin in the intestinal mucosa from subjects with IBD compared to controls. Results were not statistically significant. Immunostaining revealed prominent VDR expression in normal intestinal epithelium. Intestinal epithelial VDR expression by immunostaining is decreased in endoscopically apparent areas of active IBD when compared with controls. Epithelial VDR expression by immunostaining in IBD samples without active disease was similar to controls. Conclusions: VDR is expressed in healthy intestinal epithelial cells. Intestinal epithelial VDR expression is decreased in patients with active UC and CD when compared to healthy controls, and when compared to IBD patients with inactive disease. Since vitamin D administration enhances VDR expression, a clinical study with vitamin D administration in patients with UC and CD is warranted to confirm the role of VDR in the pathophysiology of IBD.
Aim: To correlate the levels of the main angio and lymphangiogenic factors (ALF) in serum and colonic mucosa biopsies cultures supernatant (CCS) with the endoscopic and histological activity in patients with IBD. Methods: A transversal study in 60 patients with IBD that underwent a colonoscopy because of medical criteria. From each patient, 3 different types of samples were obtained: A blood sample for ALF determination in serum, several samples of colonic tissue for their 24h culture and supernatant determination of the same ALF and other biopsies for histological analyses. In those patients in which activity was observed during the colonoscopy and, in which the disease extension allowed it, samples from affected and non-affected mucosa were also taken. The patients were classified into four groups based on the endoscopic activity (endoscopic Mayo subscore for ulcerative collitis and SESCD for Crohn's disease): non-activity of the disease, mild, moderate and severe activity. Considering histological findings, patients were also classified into four groups: Quiescent IBD, mild, moderate and severe lesion. VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, PlGF, Ang1, Ang2 and Tie2 concentrations both in serum and CCS were determined by ELISA assay. Results: 49% of the patients did not have endoscopic activity, 23% moderate, 20% mild and 8% severe activity. There were significant differences between the concentrations of VEGFA (p<0.001), VEGFC (p<0.05), VEGFD (p<0.001), PlGF (p<0.001), VEGFR1 (p<0.01), Ang-1 (p<0.001), Ang-2 (p<0.001) and Tie-2 (p<0.001) in CCS based on endoscopic activity. These concentrations increase parallel to the endoscopic activity. According to histology, 45% of the patients had quiescent IBD, 25% had a moderate lesion, 22% mild and 8% severe lesions. Levels of all the studied proteins (except VEGFR3) were also significantly higher in those patients with severe histological lesions: VEGFA (p<0.001), VEGFC (p<0.01), VEGFD (p<0.001), PlGF (p<0.001), VEGFR1 (p<0.01), VEGFR2 (p<0.05), Ang1(p<0.001), Ang-2 (p<0.001) and de Tie-2 (p<0.001). The concordance between endoscopic and histological activity was of 88.14%. In 67% of the patients with activity, biopsies of affected and non-affected mucosa were taken for comparison. Significant differences in Ang1 (p<0.05), Ang2 (p<0.001) and VEGFR3 (p<0.05) levels were observed. These levels were higher in the affected than in the non-affected mucosa. Significant differences were demonstrated in the VEGFA (p<0.05) and Ang-1 (p<0.001) serum levels depending on the endoscopic activity observed. Conclusions: The levels of angio and lymphangiogenic factors and their receptors in tissue culture supernatant and VEGFA and Ang-1 in serum correlate with the endoscopic and histologic activity of IBD. Therefore serum determination of VEGFA and Ang-1 could be useful to avoid unnecessary colonoscopies. Tu1830 Murine Guanylate Cyclase C Signaling Regulates Colonic Injury and Inflammation Kris A. Steinbrecher, Eleana Harmel-Laws, Monica P. Garin-Laflam, Elizabeth A. Mann, Mitchell B. Cohen Background: The transmembrane receptor Guanylate Cyclase C (GC-C) is activated by its secreted ligand guanylin (Gn) to produce cGMP and stimulate intestinal secretion. This intestinal epithelial cell (IEC) signaling pathway regulates proliferative/apoptotic homeostasis and maintains IEC monolayer barrier function. We therefore hypothesized that ligandinduced activation of GC-C provides resistance to colonic inflammation. Methods: We utilized the dextran sodium sulfate (DSS) colonic injury model (5 days of 3% DSS) to challenge control, GC-C-/-, and Gn-/- mice (all 10th generation C57BL/6). We measured clinical parameters and histopathology. Proliferation and apoptosis were assessed using immunohistochemistry (IHC). Western blotting and IHC measured resistin-like molecule β (RELMβ) production, and cytokine analysis was performed by ELISA. In some studies, recombinant RELMβ peptide was administered once daily by enema. In a second model, GC-C-/- mice were bred with interleukin (IL)-10 deficient animals. Histological disease scores were assessed in IL-10-/- and GC-C-/-IL-10-/- mice at 6 and 8 weeks of age. Results: Unexpectedly, GC-C-/- mice were resistant to DSS-induced colonic injury. Relative to wildtype (WT) controls, GC-C-/- animals had less rectal bleeding, diarrhea, and colon atrophy. GC-C-/- mice had minimal histological damage, enhanced IEC proliferation, reduced apoptosis, and little induction of TNFα or IFNγ. Gn-/- mice responded similarly. The secreted goblet cell protein RELMβ facilitates DSS-induced injury through activation of infiltrating immune cells which produce TNFα and IFNγ. In contrast to WT mice, RELMβ was not induced by DSS treatment in GC-C-/- animals, despite similar numbers of alcian blue-stained goblet cells and similar expression of the goblet cell marker Muc2 in both genotypes. Supplementation of active RELMβ peptide by enema reversed the resistance of GC-C-/- mice to DSS. Conversely, our analysis of GC-C-/-IL-10-/- mice indicated that GC-C was necessary to suppress spontaneous colitis. Unlike IL-10-/- controls, pathology in GC-C-/-IL-10-/animals was apparent early and, by eight weeks of age, GC-C-/-IL-10-/- mice had severely altered mucosal architecture, crypt abscesses, and hyperplastic epithelia in the submucosa of the colon. TNFα and IFNγ mRNA were elevated in GC-C-/-IL-10-/- mucosa versus that of IL-10-/- mice. Conclusions: GC-C signaling markedly and differentially regulates the response to DSS and IL-10-/- induced colitis. While the lower colonic RELMβ production in GC-C-/- mice may be protective in response to DSS treatment, we speculate that defects in small intestinal barrier function facilitate early disease onset in spontaneous colitis models. This differential response must be considered when investigating the therapeutic targeting of GC-C in the treatment of gastrointestinal inflammatory disease.
Immunostaining for Vitamin D Receptor (VDR) in rectal mucosa from a healthy control.
Immunostaining for VDR in ascending colon mucosa from patient with active IBD in the ascending colon.
S-843
AGA Abstracts
AGA Abstracts
Tu1828