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A Lactobacillus Rhamnosus GG (LGG)-Derived Protein Promotes intestinal Development via Activation of EGF Receptor in Intestinal Epithelial Cells
AGA Abstracts
increased ileal and colonic luminal 5-HT, as well as blood serotonin, and increased SERT mRNA. SERT protein was also regulated in B. dentium colonized mouse colon, compared with germ-free controls. Conclusions: These data demonstrate that the commensal microbe B. dentium is capable of regulating key components of the intestinal serotonergic system. As downregulation of SERT has been implicated in the pathophysiology of several functional gut disorders, our data support the consideration of next generation probiotics as potential therapies for serotonin-associated disorders.
441 COMBINED ESOPHACAP CYTOLOGY WITH MUC2 IMMUNOHISTOCHEMISTRY FOR SCREENING BARRETT'S ESOPHAGUS, DYSPLASIA AND CARCINOMA Zhongren Zhou, Donna Russell, Irina Kalatskaya, Norman E. Marcon, Paul Krzyzanowsk, Maria Cirocco, Virginia Litle, Tony E. Godfrey, Lincoln D. Stein The incidence of esophageal adenocarcinoma (EAC) has increased 700% in the US over the last 30 years. Detection of BE and its progression to EAC currently requires endoscopy with biopsy. Only 5-10% EAC patients were under surveillance. A simple and inexpensive test to detect BE or EAC is needed. We recruited adult (18+) patients in both Canada and United States for EsophaCap study. The EsophaCap is a gelatin capsule containing a compressed sponge attached to a tether. When swallowed, the capsule dissolved in the stomach over a period of 5 minutes and the sponge expands. The tether was then pulled to retrieve the sponge and collect esophageal mucosal cells. Samples were sent to the cytology laboratory where Thin Pap and cell block were prepared. MUC2 immunohistochemistry (IHC) was confirmed by tissue microarray (TMA) and used to detect goblet cells in EsophaCap samples. HPV was tested by PCR. Patients underwent routine endoscopic examination with biopsy in the same day. Total 170 cases were received for cytology study. 29 EsophaCap cases with biopsy were submitted for setting up the cytology diagnostic criteria (Table 1). Based on these criteria, 141 cases were evaluated by pathologists blinded to patient information and surgical biopsy diagnosis. (Table 2). Matched surgical biopsy diagnosis was received for analysis. The EsophaCap thin smear and cell block contained predominantly normal squamous cells and some glandular cells.The number and the percentage of glandular cell groups in each cell block were counted. In TMA, MUC2 showed 100% sensitivity and 99.5% specificity for detecting BE and 66.9% sensitivity and 99.5% specificity for detecting BE and beyond including LGD, HGD and EAC. 50% EAC cases were negative for MUC2. HPV was only identified in one patient. Combined EsophaCap cytology and MUC2 IHC for screening high risk population for BE and beyond: For analysis, we evaluated a combination of cytology and MUC2 IHC to screen high risk population for BE and beyond including LGD and HGD and EAC. Using surgical diagnosis we set up columnar cell metaplasia (CM), squamous epithelium (SE), squamous cell carcinoma (SCC), squamous dysplasia (SD) as negative and BE, LGD, HGD and EAC as positive. For EsophaCap samples a cytology diagnosis of no columnar cell (NCC), columnar cell with no goblet cell (CCNGC), SCC, atypical squamous cells (ASC) and a negative MUC2 IHC were required to be considered negative. Intestinal metaplasia with no high grade dysplasia (IMNHGD), atypical glandular cells (AGC), suspicious for EAC (SFEAC), EAC or a positive MUC2 IHC were considered to be positive. The sensitivity and specificity for detecting BE or beyond were 66% and 77%. Combined MUC2 immunostain and cytology diagnosis has high sensitivity and specificity for screening BE and beyond. Table 1 Diagnostic categories of esophageal glandular cells in EsophaCap cytology sample
439 TRIM21 NEGATIVELY REGULATES INTESTINAL MUCOSAL INFLAMMATION THROUGH INHIBITION OF TH1/TH17 CELL IMMUNE RESPONSES IN INFLAMMATORY BOWEL DISEASE Guangxi Zhou, Lin Yu, Wenjing Yang, Tianming Yu, Liang Chen, Zhanju Liu Objective Tripartite motif-containing (TRIM) 21 is reported to be involved in the pathogenesis of systemic lupus erythematosus and Sjögren's syndrome. However, the exact roles of TRIM21 in the pathogenesis of inflammatory bowel disease (IBD) are still unknown. Methods TRIM21 expression in inflamed mucosa from patients with IBD was examined by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Peripheral blood CD4+ T cells were transfected with lentivirus-expressing TRIM21 (LV-TRIM21) or lentivirus-expressing antiTRIM21 (LV-anti-TRIM21), respectively, and cytokine expression was determined by qRTPCR and ELISA. Experimental colitis murine models, including trinitrobenzene sulphonic acid (TNBS)-induced acute colitis in TRIM21-/- mice and a chronic colitis model in Rag-1-/mice reconstituted with TRIM21-/- CD4+CD45RBhigh T cells, were established to determine the potential role of TRIM21 in intestinal mucosal inflammation. Levels of STAT1 and STAT3 in colonic CD4+ T cells after TNBS exposure were determined by Western blot. Results TRIM21 expression was highly decreased in inflamed mucosa from patients with IBD compared with healthy controls. TRIM21 was mainly expressed in CD4+ T cells. Transfection with LV-TRIM21 into peripheral CD4+ T cells from patients with IBD significantly inhibited T helper (Th) 1 and Th17 cell immune responses compared with CD4+ T cells transfected with controls, whereas transfection with LV-anti-TRIM21 had opposite effects. Moreover, TRIM21-/- mice were found to develop more severe mucosal inflammation after TNBS exposure compared with WT mice, characterized by more loss of body weight, severe diarrhea, higher levels of pathological scores, and more infiltration of CD4+ T cells and macrophages. Levels of IFN-γ and IL-17A were markedly increased in lamina propria CD4+ T cells from TRIM21-/- mice after TNBS exposure. Phosphorylated levels of STAT1 and STAT3 were also up-regulated in colonic CD4+ T cells from TRIM21-/- mice compared with WT mice in TNBS induced colitis. Moreover, Rag-1-/- mice reconstituted with TRIM21-/CD4+CD45RBhigh T cells also demonstrated more severe colitis than WT controls. Conclusion Our data reveal that TRIM21 plays an important protective role in intestinal mucosal inflammation of IBD through inhibiting Th1 and Th17 cell differentiation. Therefore, TRIM21 may serve as a therapeutic target for treatment of human IBD.
440 A LACTOBACILLUS RHAMNOSUS GG (LGG)-DERIVED PROTEIN PROMOTES INTESTINAL DEVELOPMENT VIA ACTIVATION OF EGF RECEPTOR IN INTESTINAL EPITHELIAL CELLS Xi Shen, Liping G. Liu, D. Brent Polk, Richard M. Peek, Sari Acra, Fang He, Fang Yan Background and Aim. Our studies demonstrate that colonization of conventionally raised mice with LGG promotes intestinal development before weaning. We have identified an LGG-derived protein, p40, which protects intestinal epithelial cells from injury in an epidermal growth factor (EGF) receptor dependent manner, thereby ameliorating colitis in mice. Since activation of EGF receptor promotes organ development and growth, this study aimed to determine the roles and mechanisms of p40 functional maturation in the mouse intestine. Methods. We generated pectin/zein beads for specifically delivering p40 to the small intestine and the colon. Delivery of p40 in 2-week old pups was examined by labeling p40 with NHS-rhodamine and detecting fluorescent intensity in tissues. EGF receptor activation was examined by Western blot analysis using an anti-phospho-specific-EGF receptor antibody. Wt, Egfrfl/fl-Vil-Cre with constitutive EGF receptor deletion in the intestinal epithelial cells, and Egfrfl/fl (littermate control) pups were gavaged with p40-containing beads at 0.25, 0.5, and 0.75 µg/day during postnatal days 2-6, 7-13, and 14-21, respectively. Beads without p40 were used as control. The intestinal tissues were prepared for immunostaining with antibodies to Ki67, Muc2, and IgA and for RNA isolation for real-time PCR analysis of gene expression. The fecal IgA level was examined using ELISA. Results. p40 was recovered from the small intestine and the colon at 2 and 6 hours after single dosage of p40 gavage, respectively. Accordingly, EGF receptor was activated at these time points in the small intestinal and colonic epithelial cells. p40-treated wt pups showed increased bodyweight gain and exhibited increased length of villi and depth of crypts from 2 to 3-weeks of age, but no differences were found in older mice, as compared to pups with non-p40 bead treatment. Proliferation assessed by Ki67 positive cells and differentiation by Muc2-positive cells and Muc2 gene expression in the small intestinal and colonic epithelial cells were significantly increased in pups with p40 treatment. p40 increased ZO-1 membrane localization in the ilium and expression of the tight junction protein claudin 3, suggesting maturation of barrier function. Fecal IgA levels and the number of IgA-expressing cells in lamina propria of the small intestine in 3-week old mice with p40 treatment were both significantly higher than those of age-matched mice with non-p40 bead treatment. Furthermore, The effects of p40 on promoting proliferation, differentiation, tight junction formation and IgA production were observed in Egfrfl/fl pups, but not in Egfrfl/fl-Vil-Cre pups. Conclusion. These findings reveal a previously unrecognized mechanism by which the intestinal microbiota-derived factors contribute to intestinal development through activation of EGF receptor in intestinal epithelial cells.
AGA Abstracts
S-104
increased ileal and colonic luminal 5-HT, as well as blood serotonin, and increased SERT mRNA. SERT protein was also regulated in B. dentium colonized mouse colon, compared with germ-free controls. Conclusions: These data demonstrate that the commensal microbe B. dentium is capable of regulating key components of the intestinal serotonergic system. As downregulation of SERT has been implicated in the pathophysiology of several functional gut disorders, our data support the consideration of next generation probiotics as potential therapies for serotonin-associated disorders.
441 COMBINED ESOPHACAP CYTOLOGY WITH MUC2 IMMUNOHISTOCHEMISTRY FOR SCREENING BARRETT'S ESOPHAGUS, DYSPLASIA AND CARCINOMA Zhongren Zhou, Donna Russell, Irina Kalatskaya, Norman E. Marcon, Paul Krzyzanowsk, Maria Cirocco, Virginia Litle, Tony E. Godfrey, Lincoln D. Stein The incidence of esophageal adenocarcinoma (EAC) has increased 700% in the US over the last 30 years. Detection of BE and its progression to EAC currently requires endoscopy with biopsy. Only 5-10% EAC patients were under surveillance. A simple and inexpensive test to detect BE or EAC is needed. We recruited adult (18+) patients in both Canada and United States for EsophaCap study. The EsophaCap is a gelatin capsule containing a compressed sponge attached to a tether. When swallowed, the capsule dissolved in the stomach over a period of 5 minutes and the sponge expands. The tether was then pulled to retrieve the sponge and collect esophageal mucosal cells. Samples were sent to the cytology laboratory where Thin Pap and cell block were prepared. MUC2 immunohistochemistry (IHC) was confirmed by tissue microarray (TMA) and used to detect goblet cells in EsophaCap samples. HPV was tested by PCR. Patients underwent routine endoscopic examination with biopsy in the same day. Total 170 cases were received for cytology study. 29 EsophaCap cases with biopsy were submitted for setting up the cytology diagnostic criteria (Table 1). Based on these criteria, 141 cases were evaluated by pathologists blinded to patient information and surgical biopsy diagnosis. (Table 2). Matched surgical biopsy diagnosis was received for analysis. The EsophaCap thin smear and cell block contained predominantly normal squamous cells and some glandular cells.The number and the percentage of glandular cell groups in each cell block were counted. In TMA, MUC2 showed 100% sensitivity and 99.5% specificity for detecting BE and 66.9% sensitivity and 99.5% specificity for detecting BE and beyond including LGD, HGD and EAC. 50% EAC cases were negative for MUC2. HPV was only identified in one patient. Combined EsophaCap cytology and MUC2 IHC for screening high risk population for BE and beyond: For analysis, we evaluated a combination of cytology and MUC2 IHC to screen high risk population for BE and beyond including LGD and HGD and EAC. Using surgical diagnosis we set up columnar cell metaplasia (CM), squamous epithelium (SE), squamous cell carcinoma (SCC), squamous dysplasia (SD) as negative and BE, LGD, HGD and EAC as positive. For EsophaCap samples a cytology diagnosis of no columnar cell (NCC), columnar cell with no goblet cell (CCNGC), SCC, atypical squamous cells (ASC) and a negative MUC2 IHC were required to be considered negative. Intestinal metaplasia with no high grade dysplasia (IMNHGD), atypical glandular cells (AGC), suspicious for EAC (SFEAC), EAC or a positive MUC2 IHC were considered to be positive. The sensitivity and specificity for detecting BE or beyond were 66% and 77%. Combined MUC2 immunostain and cytology diagnosis has high sensitivity and specificity for screening BE and beyond. Table 1 Diagnostic categories of esophageal glandular cells in EsophaCap cytology sample
439 TRIM21 NEGATIVELY REGULATES INTESTINAL MUCOSAL INFLAMMATION THROUGH INHIBITION OF TH1/TH17 CELL IMMUNE RESPONSES IN INFLAMMATORY BOWEL DISEASE Guangxi Zhou, Lin Yu, Wenjing Yang, Tianming Yu, Liang Chen, Zhanju Liu Objective Tripartite motif-containing (TRIM) 21 is reported to be involved in the pathogenesis of systemic lupus erythematosus and Sjögren's syndrome. However, the exact roles of TRIM21 in the pathogenesis of inflammatory bowel disease (IBD) are still unknown. Methods TRIM21 expression in inflamed mucosa from patients with IBD was examined by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Peripheral blood CD4+ T cells were transfected with lentivirus-expressing TRIM21 (LV-TRIM21) or lentivirus-expressing antiTRIM21 (LV-anti-TRIM21), respectively, and cytokine expression was determined by qRTPCR and ELISA. Experimental colitis murine models, including trinitrobenzene sulphonic acid (TNBS)-induced acute colitis in TRIM21-/- mice and a chronic colitis model in Rag-1-/mice reconstituted with TRIM21-/- CD4+CD45RBhigh T cells, were established to determine the potential role of TRIM21 in intestinal mucosal inflammation. Levels of STAT1 and STAT3 in colonic CD4+ T cells after TNBS exposure were determined by Western blot. Results TRIM21 expression was highly decreased in inflamed mucosa from patients with IBD compared with healthy controls. TRIM21 was mainly expressed in CD4+ T cells. Transfection with LV-TRIM21 into peripheral CD4+ T cells from patients with IBD significantly inhibited T helper (Th) 1 and Th17 cell immune responses compared with CD4+ T cells transfected with controls, whereas transfection with LV-anti-TRIM21 had opposite effects. Moreover, TRIM21-/- mice were found to develop more severe mucosal inflammation after TNBS exposure compared with WT mice, characterized by more loss of body weight, severe diarrhea, higher levels of pathological scores, and more infiltration of CD4+ T cells and macrophages. Levels of IFN-γ and IL-17A were markedly increased in lamina propria CD4+ T cells from TRIM21-/- mice after TNBS exposure. Phosphorylated levels of STAT1 and STAT3 were also up-regulated in colonic CD4+ T cells from TRIM21-/- mice compared with WT mice in TNBS induced colitis. Moreover, Rag-1-/- mice reconstituted with TRIM21-/CD4+CD45RBhigh T cells also demonstrated more severe colitis than WT controls. Conclusion Our data reveal that TRIM21 plays an important protective role in intestinal mucosal inflammation of IBD through inhibiting Th1 and Th17 cell differentiation. Therefore, TRIM21 may serve as a therapeutic target for treatment of human IBD.
440 A LACTOBACILLUS RHAMNOSUS GG (LGG)-DERIVED PROTEIN PROMOTES INTESTINAL DEVELOPMENT VIA ACTIVATION OF EGF RECEPTOR IN INTESTINAL EPITHELIAL CELLS Xi Shen, Liping G. Liu, D. Brent Polk, Richard M. Peek, Sari Acra, Fang He, Fang Yan Background and Aim. Our studies demonstrate that colonization of conventionally raised mice with LGG promotes intestinal development before weaning. We have identified an LGG-derived protein, p40, which protects intestinal epithelial cells from injury in an epidermal growth factor (EGF) receptor dependent manner, thereby ameliorating colitis in mice. Since activation of EGF receptor promotes organ development and growth, this study aimed to determine the roles and mechanisms of p40 functional maturation in the mouse intestine. Methods. We generated pectin/zein beads for specifically delivering p40 to the small intestine and the colon. Delivery of p40 in 2-week old pups was examined by labeling p40 with NHS-rhodamine and detecting fluorescent intensity in tissues. EGF receptor activation was examined by Western blot analysis using an anti-phospho-specific-EGF receptor antibody. Wt, Egfrfl/fl-Vil-Cre with constitutive EGF receptor deletion in the intestinal epithelial cells, and Egfrfl/fl (littermate control) pups were gavaged with p40-containing beads at 0.25, 0.5, and 0.75 µg/day during postnatal days 2-6, 7-13, and 14-21, respectively. Beads without p40 were used as control. The intestinal tissues were prepared for immunostaining with antibodies to Ki67, Muc2, and IgA and for RNA isolation for real-time PCR analysis of gene expression. The fecal IgA level was examined using ELISA. Results. p40 was recovered from the small intestine and the colon at 2 and 6 hours after single dosage of p40 gavage, respectively. Accordingly, EGF receptor was activated at these time points in the small intestinal and colonic epithelial cells. p40-treated wt pups showed increased bodyweight gain and exhibited increased length of villi and depth of crypts from 2 to 3-weeks of age, but no differences were found in older mice, as compared to pups with non-p40 bead treatment. Proliferation assessed by Ki67 positive cells and differentiation by Muc2-positive cells and Muc2 gene expression in the small intestinal and colonic epithelial cells were significantly increased in pups with p40 treatment. p40 increased ZO-1 membrane localization in the ilium and expression of the tight junction protein claudin 3, suggesting maturation of barrier function. Fecal IgA levels and the number of IgA-expressing cells in lamina propria of the small intestine in 3-week old mice with p40 treatment were both significantly higher than those of age-matched mice with non-p40 bead treatment. Furthermore, The effects of p40 on promoting proliferation, differentiation, tight junction formation and IgA production were observed in Egfrfl/fl pups, but not in Egfrfl/fl-Vil-Cre pups. Conclusion. These findings reveal a previously unrecognized mechanism by which the intestinal microbiota-derived factors contribute to intestinal development through activation of EGF receptor in intestinal epithelial cells.
AGA Abstracts
S-104