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Tu1848 Digestion of Epithelial Tight Junction Proteins by the Commensal Clostridium Perfringens
AGA Abstracts
spheroid that exposes the basolateral membrane of IEC with preserving the cell formation and structure of crypt. Moreover, we have originally generated pIEC from colon. We therefore aim to assess the reaction to flagellin of pIEC via TLR5 on basolateral membrane in small intestine and colon by the alternation of Notch signaling. Methods: pIECs were generated from mouse small intestine and colon by Sato method and original method, respectively. The localization of TLR5 in pIEC was analyzed by immunofluorescence. Flagellin was added to the medium for either 3 hours or 7 days to stimulate TLR5 on basal membrane at the outside of spheroid. Comprehensive genes induced by flagellin were detected by microarray analysis. Notch signaling was suppressed by gamma secretase inhibitor (GSI). Results: pIECs of small intestine and colon were cultured as spheroid shape with keeping cell formation. TLR5 in pIECs was specifically localized on the basal membrane at the outside of the spheroid. Flagellin did not change the cell formation and the phenotypic gene expression of pIECs. However, microarray analysis showed that various genes including cytokines, chemokines and reactive oxygen species were induced by flagellin. Candidate genes were confirmed by quantitative RT-PCR. pIEC generated from MyD88 deficient mice did not react to Flagellin, suggesting that the response to Flagellin is mediated by TLR5 signaling via MyD88. Treatment with GSI inhibited Notch signaling of pIEC, resulting in not only the increase of secretory lineages but also the decrease of absorptive cells and stem cells. Interestingly, Flagellin inducible genes were up-regulated by Notch signal inhibition of pIEC generated from small intestine but not from colon. Conclusion: We have for the first time investigated the natural reaction of IECs by flagellin without the effect of the stromal tissue. IECs play various roles in the invasion of bacteria as a unit at the front line of mucosal defense. Moreover, Notch signaling inhibition in pIECs causes the overreaction to flagellin, suggesting that Notch signaling acceleration of IECs in Crohn's disease patients might weaken flagellin response.
process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the epithelial cells at the concentrations used in this study. To determine whether the proteases from the C. perfringens culture supernatant are directly responsible for the proteolytic cleavage of Ecadherin in C2BBe1 cells, we measured the levels of pro- and active MMP-7, a matrix metalloproteinase known to mediate E-cadherin ectodomain shedding in selected epithelial cells. MMP-7 was not induced or activated in C. perfringens-stimulated C2BBe1 cells. These findings were confirmed by C. perfringens supernatant-induced degradation of the human recombinant E-cadherin. Conclusion: C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins which is likely to enable access to the extracellular matrix components by the paracellular pathway. 1. Pruteanu M, Hyland NP, Clarke DJ, Kiely B, Shanahan F. Degradation of the extracellular matrix components by bacterialderived metalloproteases: implications for inflammatory bowel diseases. Inflamm Bowel Dis. 2011;17:1189-1200. Tu1849 Juxtapositioning of Neutrophils and Macrophages With Colonic Stem Cells, and Mobilisation of Neutrophils to the Surface Epithelium, Following Luminal Microbial Input Dagmara Dyjach, Petr Walczysko, Nikki Horn, Mark Williams, Anastasia Sobolewski BACKGROUND: Recent observations have implicated abnormal host-microbial interactions in the pathogenesis of idiopathic inflammatory bowel diseases (Sartor, 2008).During homeostasis the epithelium forms a barrier between the gut luminal contents (e.g. bacteria) and tissue resident immune cells below. Epithelial stem cell-driven tissue renewal and regeneration is central to maintenance of gut barrier function during homeostasis and infection/injury. Seminal work has shown that recruitment of immune cells to specific epithelial compartments following breach of the epithelial barrier during mucosal injury or infection (Pull et al., 2005, Chieppa et al., 2006). However, during homeostasis when the epithelial barrier is intact, the spatial and temporal characteristics of immune cell recruitment to specific sites along the crypt-axis are not known. HYPOTHESIS: luminal microbialepithelial cell interactions promote recruitment of tissue-resident immune cells to the crypt stem cell niche and the surface epithelium. METHODS: colonic explants from C57BL/6 or LGR5-EGFP mice were placed on Transwell fliters and cultured at air-apical interface. E. coli, MDP or LPS were added luminally. Following 1 hour of culture, tissue was fixed, cryosectioned and stained with immunofluorescent antibodies. Confocal microscopy was used for visualization of the distribution and recruitment of 7/4-positive neutrophils and F4/80-positive macrophages within the colonic mucosa. RESULTS:E-Cadherin expression and fluorescent dextran studies showed that the epithelial barrier of the mucosal explants was intact following 1 hour culture and that the viability (caspase activity) of the tissue was comparable to control (n=6). Neutrophil recruitment to the mucosal surface and the stem cell niche occurred within 1h of luminal MDP or LPS treatment (n=7, p<0.01), constituting a 1.5-fold increase in the mucosal neutrophil number (n=7, p<0.001). F4/80+ macrophage cell number (shown to be cd11c negative in initial studies) in the mucosa was not affected (n=7). Significantly however, both neutrophils and F4/80+ cells were located closer to the crypt-base epithelium in MDP (n=5, p<0.001 and n=5, p<0.05 respectively) and LPS-treated tissue (n=5, p<0.05 and p<0.001 respectively). Further analysis using Lgr5-EGFP mice demonstrated that neutrophils and macrophages moved closer to GFP positive stem cells following luminal addition of either LPS (n=4, p<0.001, n=2 p<0.05) or E.coli (n=3, p<0.05). CONCLUSION: These findings demonstrate that following luminal interactions between microbes and the intact surface epithelium, neutrophils and macrophages are positioned juxtaposed to intestinal stem cells, while neutrophils are also mobilised to underneath the surface epithelium. Ongoing work is investigating the functional consequences to epithelial tissue renewal and barrier function.
Tu1847 Dysregulated Responses to Microbial DNA are Associated With Altered Microflora in Patients With Crohn's Disease Naomi Hotte, Saad Y. Salim, Robert Tso, Levinus A. Dieleman, Richard N. Fedorak, Karen Madsen Dysfunctional interactions between the gut microbiome and host epithelium are believed to be important in inflammatory bowel disease (IBD). A critical role for the intestinal epithelium lies in its ability to discriminate between pathogens and commensals and respond appropriately. In this study, we examined responses of mucosal biopsies from patients with Crohn's disease (CD) and ulcerative colitis (UC) to isolated bacterial DNA and correlated the host microenvironment with tissue responses. Methods: Biopsies were obtained from non-inflamed areas of the transverse colon in healthy controls (HC: n=20), and CD (n=23) and UC (n=21) patients. Biopsies were either snap-frozen or cultured for 2 hrs with DNA from Lactobacillus plantarum (LP), a probiotic strain, or Salmonella dublin (SD), a pathogen. Gene expression was analyzed under basal conditions and in response to DNA using Human Immune TaqMan Low Density Arrays. Mucosal-associated microflora was analyzed using terminal restriction fragment length polymorphism. The relationship between tissue response to bacterial DNA and specific microbes was investigated using Spearman rank correlation analysis. Gene networks were analyzed using the Ingenuity Pathways Analysis application. Frequency of single nucleotide polymorphisms (SNPs) in NOD2 and TLR9 was assessed. Results: There were no significant differences between the groups in age, gender, disease duration, or types of drug therapy. LP DNA induced an anti-inflammatory response in both HC and UC patients, with an up-regulation of IL-10 and involvement of STAT3 and STAT6 in HC and a down-regulation of IL12B with involvement of NF-kB and STAT1 in UC patients. In contrast, in CD patients, LP DNA induced an upregulation of IL17A and other pro-inflammatory mediators. SD DNA induced numerous chemokines in all groups, as well as inducing IL-17 specifically in CD patients. CD patients had altered microflora with reduced Clostridia and increased Erysipelotrichi and Mollicutes. Principle component analysis showed CD patients to cluster independently from both UC and HC based upon their microflora profile. In CD patients positive correlations between gene expression of several pro-inflammatory mediators and the presence of Flavobacteria and Sphingobacteria were seen. There were no differences in SNP frequencies or expression levels of TLR9 or NOD2 between the groups. Conclusions: Crohn's disease patients exhibit significant alterations of their gut microflora which correlates with dysregulated responses to bacterial DNA. A larger number of correlations exists in CD patients between gene expression and the presence of specific microbial species. These findings suggest that the response to bacterial DNA may depend not only on the specific type of bacterial DNA, but also on the luminal environment of the host.
Tu1850 Expression of Toll-Like Receptor (TLR)-2 and -4 in the Intestinal Crypt Epithelial Cells in Inflammatory Bowel Disease (IBD) Matthew Brown, Yashwant R. Mahida TLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but may exacerbate established colitis. Epithelial stem cells and their progeny are located in crypts of the small and large intestine (SI and LI respectively). We have investigated TLR2 and TLR4 expression in ulcerative colitis (UC) and Crohn's disease crypt epithelial cells. Methods. SI and LI mucosal samples were obtained from operation resection specimens and were histologically normal (>5 cm from cancer; normal controls) or inflamed (Crohn's disease or UC). Additionally, histologically normal (from proximal LI) and inflamed (mild - severe, distal LI) mucosal samples were obtained from 5 colectomy specimens with left-sided UC. Intestinal epithelial crypt cells (IEC) were isolated and disaggregated using EDTA and pancreatin. Expression of mRNA transcripts was studied by conventional and real-time RT-PCR. For the latter, relative quantification of TLR2 and TLR4 transcripts was deduced by comparing cycle threshold (Ct) value of each sample to mean Ct value of the relevant control group, normalized to housekeeping gene hypoxanthine phosphatidyl ribosyltransferase (HPRT). Cell surface TLR protein was studied by flow cytometry and expressed as median fluorescence intensity (MFI) after subtraction of MFI value from isotype controls. TLR expression was also studied by immunofluorescent (IF) staining of tissue sections. Data are expressed as median (inter-quartile range; IQR). Results. Following demonstration of TLR2 and TLR4 mRNA by conventional RT-PCR, relative quantification of the transcripts was undertaken by real-time RT-PCR (data in Table). In paired analyses, TLR2 or TLR4 mRNA expression was not significantly different in IEC from normal UC mucosa compared to IEC from inflamed UC mucosa. Compared to IEC from normal control LI, IEC from Crohn's LI showed significantly greater expression of cell surface TLR2 protein [MFI 10.1 (2.4-29.0) versus 33.1 (17.7-107.5) p=0.05] and TLR4 protein [MFI 12.1 (4.9-25.3) versus 40.9 (23.2-103.5), p=0.04]. IF staining confirmed TLR2 and TLR4 protein expression on the luminal surface of crypt epithelial cells. Conclusions. 1.TLR2 mRNA expression was significantly increased in IEC from inflamed LI in both UC
Tu1848 Digestion of Epithelial Tight Junction Proteins by the Commensal Clostridium Perfringens Mihaela Pruteanu, Fergus Shanahan Background & Aim: The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease (IBD) but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens, have the proteolytic capacity for host matrix degradation.1 This included reduced transepithelial resistance after exposure of rat distal colon to culture supernatants of C. perfringens in Ussing chambers. Here, we examined the C. perfringensderived proteolytic activity against epithelial tight junction proteins. Methods: The colonic epithelial cell line C2BBe1 (brush border expressing) was deployed. Confluent or polarized C2BBe1 cells were treated for various times with different concentrations of C. perfringens filtered sterile culture supernatant and the levels of tight junction (occludin and JAM-1) and adherens junction (E-cadherin) proteins were determined by western blotting. A resazurinbased cytotoxicity assay was used to exclude a cytotoxic effect from the culture supernatant on the intestinal epithelial cells. Results: The protein levels of occludin, JAM-1 and fulllength E-cadherin (using antibodies against either the extracellular or cytoplasmic domain of E-cadherin) decreased in C2BBe1 cells treated with C. perfringens culture supernatant. Ecadherin ectodomain shedding in C. perfringens-stimulated C2BBe1 cells was detected with antibodies against the extracellular domain of E-cadherin and we demonstrate that this
AGA Abstracts
S-860
spheroid that exposes the basolateral membrane of IEC with preserving the cell formation and structure of crypt. Moreover, we have originally generated pIEC from colon. We therefore aim to assess the reaction to flagellin of pIEC via TLR5 on basolateral membrane in small intestine and colon by the alternation of Notch signaling. Methods: pIECs were generated from mouse small intestine and colon by Sato method and original method, respectively. The localization of TLR5 in pIEC was analyzed by immunofluorescence. Flagellin was added to the medium for either 3 hours or 7 days to stimulate TLR5 on basal membrane at the outside of spheroid. Comprehensive genes induced by flagellin were detected by microarray analysis. Notch signaling was suppressed by gamma secretase inhibitor (GSI). Results: pIECs of small intestine and colon were cultured as spheroid shape with keeping cell formation. TLR5 in pIECs was specifically localized on the basal membrane at the outside of the spheroid. Flagellin did not change the cell formation and the phenotypic gene expression of pIECs. However, microarray analysis showed that various genes including cytokines, chemokines and reactive oxygen species were induced by flagellin. Candidate genes were confirmed by quantitative RT-PCR. pIEC generated from MyD88 deficient mice did not react to Flagellin, suggesting that the response to Flagellin is mediated by TLR5 signaling via MyD88. Treatment with GSI inhibited Notch signaling of pIEC, resulting in not only the increase of secretory lineages but also the decrease of absorptive cells and stem cells. Interestingly, Flagellin inducible genes were up-regulated by Notch signal inhibition of pIEC generated from small intestine but not from colon. Conclusion: We have for the first time investigated the natural reaction of IECs by flagellin without the effect of the stromal tissue. IECs play various roles in the invasion of bacteria as a unit at the front line of mucosal defense. Moreover, Notch signaling inhibition in pIECs causes the overreaction to flagellin, suggesting that Notch signaling acceleration of IECs in Crohn's disease patients might weaken flagellin response.
process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the epithelial cells at the concentrations used in this study. To determine whether the proteases from the C. perfringens culture supernatant are directly responsible for the proteolytic cleavage of Ecadherin in C2BBe1 cells, we measured the levels of pro- and active MMP-7, a matrix metalloproteinase known to mediate E-cadherin ectodomain shedding in selected epithelial cells. MMP-7 was not induced or activated in C. perfringens-stimulated C2BBe1 cells. These findings were confirmed by C. perfringens supernatant-induced degradation of the human recombinant E-cadherin. Conclusion: C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins which is likely to enable access to the extracellular matrix components by the paracellular pathway. 1. Pruteanu M, Hyland NP, Clarke DJ, Kiely B, Shanahan F. Degradation of the extracellular matrix components by bacterialderived metalloproteases: implications for inflammatory bowel diseases. Inflamm Bowel Dis. 2011;17:1189-1200. Tu1849 Juxtapositioning of Neutrophils and Macrophages With Colonic Stem Cells, and Mobilisation of Neutrophils to the Surface Epithelium, Following Luminal Microbial Input Dagmara Dyjach, Petr Walczysko, Nikki Horn, Mark Williams, Anastasia Sobolewski BACKGROUND: Recent observations have implicated abnormal host-microbial interactions in the pathogenesis of idiopathic inflammatory bowel diseases (Sartor, 2008).During homeostasis the epithelium forms a barrier between the gut luminal contents (e.g. bacteria) and tissue resident immune cells below. Epithelial stem cell-driven tissue renewal and regeneration is central to maintenance of gut barrier function during homeostasis and infection/injury. Seminal work has shown that recruitment of immune cells to specific epithelial compartments following breach of the epithelial barrier during mucosal injury or infection (Pull et al., 2005, Chieppa et al., 2006). However, during homeostasis when the epithelial barrier is intact, the spatial and temporal characteristics of immune cell recruitment to specific sites along the crypt-axis are not known. HYPOTHESIS: luminal microbialepithelial cell interactions promote recruitment of tissue-resident immune cells to the crypt stem cell niche and the surface epithelium. METHODS: colonic explants from C57BL/6 or LGR5-EGFP mice were placed on Transwell fliters and cultured at air-apical interface. E. coli, MDP or LPS were added luminally. Following 1 hour of culture, tissue was fixed, cryosectioned and stained with immunofluorescent antibodies. Confocal microscopy was used for visualization of the distribution and recruitment of 7/4-positive neutrophils and F4/80-positive macrophages within the colonic mucosa. RESULTS:E-Cadherin expression and fluorescent dextran studies showed that the epithelial barrier of the mucosal explants was intact following 1 hour culture and that the viability (caspase activity) of the tissue was comparable to control (n=6). Neutrophil recruitment to the mucosal surface and the stem cell niche occurred within 1h of luminal MDP or LPS treatment (n=7, p<0.01), constituting a 1.5-fold increase in the mucosal neutrophil number (n=7, p<0.001). F4/80+ macrophage cell number (shown to be cd11c negative in initial studies) in the mucosa was not affected (n=7). Significantly however, both neutrophils and F4/80+ cells were located closer to the crypt-base epithelium in MDP (n=5, p<0.001 and n=5, p<0.05 respectively) and LPS-treated tissue (n=5, p<0.05 and p<0.001 respectively). Further analysis using Lgr5-EGFP mice demonstrated that neutrophils and macrophages moved closer to GFP positive stem cells following luminal addition of either LPS (n=4, p<0.001, n=2 p<0.05) or E.coli (n=3, p<0.05). CONCLUSION: These findings demonstrate that following luminal interactions between microbes and the intact surface epithelium, neutrophils and macrophages are positioned juxtaposed to intestinal stem cells, while neutrophils are also mobilised to underneath the surface epithelium. Ongoing work is investigating the functional consequences to epithelial tissue renewal and barrier function.
Tu1847 Dysregulated Responses to Microbial DNA are Associated With Altered Microflora in Patients With Crohn's Disease Naomi Hotte, Saad Y. Salim, Robert Tso, Levinus A. Dieleman, Richard N. Fedorak, Karen Madsen Dysfunctional interactions between the gut microbiome and host epithelium are believed to be important in inflammatory bowel disease (IBD). A critical role for the intestinal epithelium lies in its ability to discriminate between pathogens and commensals and respond appropriately. In this study, we examined responses of mucosal biopsies from patients with Crohn's disease (CD) and ulcerative colitis (UC) to isolated bacterial DNA and correlated the host microenvironment with tissue responses. Methods: Biopsies were obtained from non-inflamed areas of the transverse colon in healthy controls (HC: n=20), and CD (n=23) and UC (n=21) patients. Biopsies were either snap-frozen or cultured for 2 hrs with DNA from Lactobacillus plantarum (LP), a probiotic strain, or Salmonella dublin (SD), a pathogen. Gene expression was analyzed under basal conditions and in response to DNA using Human Immune TaqMan Low Density Arrays. Mucosal-associated microflora was analyzed using terminal restriction fragment length polymorphism. The relationship between tissue response to bacterial DNA and specific microbes was investigated using Spearman rank correlation analysis. Gene networks were analyzed using the Ingenuity Pathways Analysis application. Frequency of single nucleotide polymorphisms (SNPs) in NOD2 and TLR9 was assessed. Results: There were no significant differences between the groups in age, gender, disease duration, or types of drug therapy. LP DNA induced an anti-inflammatory response in both HC and UC patients, with an up-regulation of IL-10 and involvement of STAT3 and STAT6 in HC and a down-regulation of IL12B with involvement of NF-kB and STAT1 in UC patients. In contrast, in CD patients, LP DNA induced an upregulation of IL17A and other pro-inflammatory mediators. SD DNA induced numerous chemokines in all groups, as well as inducing IL-17 specifically in CD patients. CD patients had altered microflora with reduced Clostridia and increased Erysipelotrichi and Mollicutes. Principle component analysis showed CD patients to cluster independently from both UC and HC based upon their microflora profile. In CD patients positive correlations between gene expression of several pro-inflammatory mediators and the presence of Flavobacteria and Sphingobacteria were seen. There were no differences in SNP frequencies or expression levels of TLR9 or NOD2 between the groups. Conclusions: Crohn's disease patients exhibit significant alterations of their gut microflora which correlates with dysregulated responses to bacterial DNA. A larger number of correlations exists in CD patients between gene expression and the presence of specific microbial species. These findings suggest that the response to bacterial DNA may depend not only on the specific type of bacterial DNA, but also on the luminal environment of the host.
Tu1850 Expression of Toll-Like Receptor (TLR)-2 and -4 in the Intestinal Crypt Epithelial Cells in Inflammatory Bowel Disease (IBD) Matthew Brown, Yashwant R. Mahida TLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but may exacerbate established colitis. Epithelial stem cells and their progeny are located in crypts of the small and large intestine (SI and LI respectively). We have investigated TLR2 and TLR4 expression in ulcerative colitis (UC) and Crohn's disease crypt epithelial cells. Methods. SI and LI mucosal samples were obtained from operation resection specimens and were histologically normal (>5 cm from cancer; normal controls) or inflamed (Crohn's disease or UC). Additionally, histologically normal (from proximal LI) and inflamed (mild - severe, distal LI) mucosal samples were obtained from 5 colectomy specimens with left-sided UC. Intestinal epithelial crypt cells (IEC) were isolated and disaggregated using EDTA and pancreatin. Expression of mRNA transcripts was studied by conventional and real-time RT-PCR. For the latter, relative quantification of TLR2 and TLR4 transcripts was deduced by comparing cycle threshold (Ct) value of each sample to mean Ct value of the relevant control group, normalized to housekeeping gene hypoxanthine phosphatidyl ribosyltransferase (HPRT). Cell surface TLR protein was studied by flow cytometry and expressed as median fluorescence intensity (MFI) after subtraction of MFI value from isotype controls. TLR expression was also studied by immunofluorescent (IF) staining of tissue sections. Data are expressed as median (inter-quartile range; IQR). Results. Following demonstration of TLR2 and TLR4 mRNA by conventional RT-PCR, relative quantification of the transcripts was undertaken by real-time RT-PCR (data in Table). In paired analyses, TLR2 or TLR4 mRNA expression was not significantly different in IEC from normal UC mucosa compared to IEC from inflamed UC mucosa. Compared to IEC from normal control LI, IEC from Crohn's LI showed significantly greater expression of cell surface TLR2 protein [MFI 10.1 (2.4-29.0) versus 33.1 (17.7-107.5) p=0.05] and TLR4 protein [MFI 12.1 (4.9-25.3) versus 40.9 (23.2-103.5), p=0.04]. IF staining confirmed TLR2 and TLR4 protein expression on the luminal surface of crypt epithelial cells. Conclusions. 1.TLR2 mRNA expression was significantly increased in IEC from inflamed LI in both UC
Tu1848 Digestion of Epithelial Tight Junction Proteins by the Commensal Clostridium Perfringens Mihaela Pruteanu, Fergus Shanahan Background & Aim: The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease (IBD) but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens, have the proteolytic capacity for host matrix degradation.1 This included reduced transepithelial resistance after exposure of rat distal colon to culture supernatants of C. perfringens in Ussing chambers. Here, we examined the C. perfringensderived proteolytic activity against epithelial tight junction proteins. Methods: The colonic epithelial cell line C2BBe1 (brush border expressing) was deployed. Confluent or polarized C2BBe1 cells were treated for various times with different concentrations of C. perfringens filtered sterile culture supernatant and the levels of tight junction (occludin and JAM-1) and adherens junction (E-cadherin) proteins were determined by western blotting. A resazurinbased cytotoxicity assay was used to exclude a cytotoxic effect from the culture supernatant on the intestinal epithelial cells. Results: The protein levels of occludin, JAM-1 and fulllength E-cadherin (using antibodies against either the extracellular or cytoplasmic domain of E-cadherin) decreased in C2BBe1 cells treated with C. perfringens culture supernatant. Ecadherin ectodomain shedding in C. perfringens-stimulated C2BBe1 cells was detected with antibodies against the extracellular domain of E-cadherin and we demonstrate that this
AGA Abstracts
S-860