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The Notch-1 Signaling Pathway is Required for Functional Colonic Barrier in wt Mice
Su1824
with compound 48/80 (5ug/ml) +/- tryptase inhibitor and incubated up to 24 hours. The epithelial integrity was evaluated by TER and Transmission Electron Microscopy (TEM), and the expression of junctional proteins was evaluated by Western blot and confocal microscopy. Results: A decrease in JAM-A expression (30%) was observed after 4 hours of incubation with 15 mU tryptase and was concomitant with a significant drop in TER (30 %). Redistribution of JAM-A protein from TJ regions was shown by confocal microscopy. The drop in TER was reversible within 24 hours compared with control samples, however the expression of JAM-A was consistently reduced (40 %). Degranulation of co-cultured HMC-1 cells (40 x 10 5 cells/ml) also induced a decrease in JAM-A expression (30 %) after 6 hours of incubation as demonstrated by Western blot and confocal microscopy. The effect was maintained up to 24 hours and was concomitant with a 40 % drop in TER. In contrast, there was no apparent change in the expression of E-cadherin in response to tryptase either in Caco-2 monolayer experiments or in the co-culture model. Conclusion: A reduction in JAM-A protein expression together with a decrease in epithelial integrity were consistent observations in Caco-2 monolayers either treated with tryptase or exposed to degranulated mast cells. These consistent alterations in JAM-A expression up to 24 hours may shed new light on the regulation of epithelial barrier integrity in IBS.
AGA Abstracts
Crypt Structures Are Protected in BCL-2 Modifying Factor (BMF-/-) Mice During Acute Colitis Katharina Leucht, Michaela Krebs, Christian Ploner, Andreas Villunger, Silvia Kellermeier, Michael Fried, Gerhard Rogler, Martin Hausmann BACKGROUND: Bcl-2 modifying factor (Bmf) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma (Bcl)-2. Bcl-2 is neutralized by Bmf and thereby facilitates cytochrome C release from mitochondria. Recently we reported that translocation of BMF in human intestinal epithelial cells is induced by the loss of cell attachment. This suggests that Bmf may be critical for epithelial cell homeostasis. We investigated the role of Bmf in mice under inflammatory conditions. METHODS: Acute colitis was induced in male C57BL/6-Bmftm1.1Anvi (Bmf-/-) mice weighing 25-32 g with 3.5 % dextran sulfate sodium (DSS). Mice were killed on day nine. A histological score, crypt lengths, colon lengths and percental body weight loss were determined. Immunohistochemistry was performed in human colonic sections with rabbit anti-human Bmf (#ab59906, abcam, 1:100). Intestinal epithelial cells (IEC) were isolated ex vivo. Single cells were separated from whole crypts and the relative amount of Bmf cDNA was calculated by means of real time PCR. RESULTS: Colonic crypt lengths of Bmf-/- mice were significantly increased as compared to wildtype (WT) (156.9 ± 32.0 μm, n = 99 and 139.8 ± 22.4 μm, n = 96, p < 0.05). Upon DSS, weight loss of Bmf-/- mice was significantly decreased from day five to eight as compared to WT mice. Following induction of acute colitis colon length of Bmf-/mice was less reduced. Immunohistochemistry of human IEC showed strong expression of Bmf in the extrusion zone where cells are shed. IEC were isolated ex vivo and whole crypts were separated from single cells. In patients with IBD Bmf mRNA from single cells was significantly increased (4.8 ± 1.5-fold) as compared to crypts. CONCLUSION: Detachment of IEC, the trigger of apoptosis in epithelial cells, is associated with Bmf upregulation. This is likely to play an important role in the regulation of intestinal epithelial cell survival. DSS induces less colitis in Bmf-/- mice. Increased colon and crypt length may indicate an extended lifespan of IEC in Bmf-/- mice compared to WT. This could be protective during onset of inflammation.
Su1827 The Notch-1 Signaling Pathway is Required for Functional Colonic Barrier in wt Mice Avantika Chitre, Cecilia Berin, Lloyd Mayer, Stephanie Dahan INTRODUCTION: Gut lympho-epithelial interactions occur in the epithelial layer and in the sub-epithelial space. We have recently shown that the presence of LPLs promotes mucosal barrier function in the RAG1-/- transfer model of colitis putatively due to the Notch-1 signaling pathway. This finding was corroborated In Vitro as the epithelial barrier function correlated with the activation of the Notch-1 signaling pathway In Vitro. Moreover, the activation of Notch-1 extends throughout the epithelium in CD mucosa compared to normal or UC mucosa. AIM: To determine whether the Notch signaling pathway is mandatory for epithelial barrier function In Vivo. METHODS: A specific Notch-1 or a scrambled siRNA sequence was freshly complexed with Lipofectamine 2000. Anesthetized WT mice received 2 intra-rectal injections of complexed siRNA (5 nmol in 20 μl of solution/mouse) 4 days apart. The injected mice were sacrificed one day after the last injection. Colonic tissues were either mounted into Ussing chambers to assess permeability or snap-frozen and subjected to Real-Time PCR for Hes-1, CDX-2, Villin, Occludin, Claudin-5, beta-catenin, and Ecadherin. RESULTS: While no differences in resistance in the distal colon were seen between scrambled and Notch-1 siRNA treated WT mice, there was a significant decrease in flux in the WT mice that received the Notch-1 siRNA (10925 ± 3389 ng of Dextran-FITC/ml/min/ cm2 in the Notch-1 siRNA treated WT mice vs. 2912 ± 1054 ng of Dextran-FITC/ml/min/ cm2 in the scrambled siRNA treated WT mice, p=0.01, n=9). The quantitative analysis of genes related to the Notch-1 signaling pathway showed that Hes-1 and CDX-2 mRNA expression was significantly decreased in the Notch-1 siRNA treated WT mice (p=0.01 and p=0.009, respectively). These findings correlated with a decrease in villin, occludin, and claudin-5 mRNA gene expression (p=0.0002, p=0.0002 and p=0.002, respectively). Moreover, a significant decrease in beta-catenin and E-cadherin was also observed (p<0.0001 and p=0.0004, respectively). CONCLUSION: Local knock down of the Notch-1 signaling pathway led to dysregulated flux across the distal colon but a normal resistance. The genes related to the Notch-1 signaling pathway and the tight junctions were also impaired in the Notch-1 siRNA treated mice. More interestingly, the beta-catenin/E-cadherin mRNA levels were affected by the Notch-1 knock down. These findings might explain why the flux and not the resistance were affected by In Vivo Notch-1 silencing. Thus, the Notch-1 signaling pathway is critical for the epithelium in order to achieve differentiation and functional barrier integrity.
Su1825 Increased Intestinal Permeability and Disturbance of Tight-Junctions in an Acute Graft Versus Host Disease Model in Mice Rainer Noth, Julia Lange-Grumfeld, Jochen Hampe, Stefan Schreiber, Alexander Arlt Background Hematopoietic stem cell transplantation is increasingly performed for acute and chronic leukemia and other hematologic malignancies. One of the severe side effects of the procedure are graft versus host reactions leading among other complications to gastrointestinal symptoms including diarrhea, nausea, vomiting, abdominal cramping, anorexia and gastrointestinal bleeding. Up to now little is known about the pathophysiological mechanisms evoking these GI-symptoms, especially the manifestation in the small intestine. Murine acute graft versus host disease (GvHD) has long been investigated as an animal model for the immune pathomechanisms of human GVHD after allogeneic bone marrow transplantation and due to its characteristic changes in the intestinal histology consisting of lymphocytic infiltrates, crypt hyperplasia, and villous atrophy, it serves as a model for immunologically mediated atrophic-hyperregenerative diseases of the intestine. Aim To evaluate effects of a murine GvHD on the intestinal barrier functions in the small intestine. Results The analysis of the gastrointestinal permeability by measuring blood levels of mannitol (a marker of absorption of small molecules representative for the transcellular permeability) and lactulose (a marker of abnormal absorption of large molecules representative for the paracellular permeability) after infusion through gastrointestinal tubes we were able to show that GvHD animals had significant higher blood levels of mannitol and lactulose if compared to control animals. The quotient of these two sugars indicated that the GvHD predominantly leads to an increased paracellular intestinal permeability. By using electron microscopy we were able to show that the intercellular space of the tight junctions of GvHD animals increased indicating a disturbance of the tight junctions. By PCR we could show that the extracellular tight junction protein occludin in contrast to the intracellular tight junction protein ZO-1 was significantly lower expressed. Immunhistochemistry revealed that this lower expression resulted in a disturbed assembly of the tight junctions in the GvHD animals. Conclusions Our analysis of the intestinal barrier functions of the small intestine in a murine model of GvHD revealed a disorganization of the tight junctions due to lower expression of occludin and consecutive increase in the intestinal permeability. This mechanism may contribute to the frequent gastrointestinal symptoms and malabsortion in kachexia in GvHD.
Su1828 Induction of Gastrokine-2 Expression in the Intestinal Tract of Gastrokine-1 Deficient Mice Shirley Paski, James P. Lodolce, Lauren Kolodziej, Sarah Bartulis, Mark Logsdon, Jonathan Chang, Wesley Grimm, Margaret Walsh-Reitz, F Gary Toback, David L. Boone INTRODUCTION: Gastrokine-1 (GKN1) is an 18-kDa protein expressed predominantly in the stomach where it is secreted into the lumen. A synthetic 21-mer peptide corresponding to a central domain of GKN1 (GKN1-peptide) is mitogenic, motogenic, and cytoprotective in the GI tract of mice exposed to indomethacin. The objectives of this study were to evaluate the: (1) effectiveness of GKN1-peptide at treating lipopolysaccharide (LPS)-induced increases in intestinal permeability in mice and (2) essential function of GKN1 by phenotyping GKN1 knockout mice. METHODS: To evaluate the protective effect of GKN1-peptide, 25mg/kg ip GKN1-peptide was given 5d, 24h, or 6h before treatment with 4mg/kg ip LPS. Intestines were removed 60min after LPS treatment, and barrier function was assessed by flux of 4kDa FITC dextran over 75min. To evaluate the therapeutic effect of GKN1-peptide, mice were injected with LPS followed by GKN1-peptide 15h later. Tight junction proteins occludin and ZO-1 were visualized with immunofluorescence and laser scanning confocal microscopy. To develop GKN1-/- mice, the GKN1 gene was ablated in entirety and replaced by a BAC-based vector expressing the β-galactosidase from the E. coli lacZ gene and neomycin phosphotransferase coding sequences. GKN1+/- embryonic stem cells from C57BL/6 mice were implanted into C57BL/6 embryos to create GKN1+/- mice. The growth and development of the first generation of GKN-/- mice was observed over 24 weeks. Mucosal histology and gene expression of GKN1, ZO-1, and occludin were studied in stomach, small intestine, and colon. Expression of GKN2 and GKN3 mRNA, which share homology to GKN1, were also evaluated. RESULTS: Treatment with LPS markedly increased FITC-dextran flux and caused altered localization of TJ proteins consistent with increased intestinal permeability. GKN1-peptide attenuated LPS-induced barrier disruption when injected 5d, 24h, and 6h before LPS. Giving GKN1-peptide 15h after LPS therapeutically reversed LPS-induced barrier disruption. GKN1-/- mice grow and develop normally compared to GKN1+/+ or GKN1+/littermates, showing no spontaneous gastroenteritis. Gastrointestinal histology and gene expression of ZO-1 and occludin did not differ between GKN-/- and GKN+/- mice. GKN2
Su1826 Mast Cell Tryptase Reduces Junctional Adhesion Molecule-a (JAM-A) Expression in Human Intestinal Epithelial Cells: Implications for the Mechanisms of Barrier Dysfunction in Irritable Bowel Syndrome (IBS) Ewa Wilcz, Siobhan McClean, Maria O'Sullivan Introduction: Alterations in mast cell numbers and in intestinal epithelial permeability have both been implicated in the pathogenesis of IBS. Tryptase, released from mast cells, has been shown to induce changes in paracellular permeability and in tight junction (TJ) proteins. The expression, however, of the junctional proteins JAM-A and E-cadherin in response to mast cell mediators has not been investigated in IBS, and may represent new insights into the regulation of epithelial barrier integrity. Aims: To investigate the expression of JAM-A and E-cadherin proteins 1) in Caco-2 monolayers exposed to the mast cell mediator, tryptase and 2) in a newly developed Caco-2/HMC-1 cells co-culture model. Methods: Caco-2 cells were grown as polarised monolayers up to 23 days on semi-permeable filters. Tryptase (3 mU or 15 mU) was added to the apical side of Caco-2 monolayers and incubated up to 24 hours. Epithelial integrity was monitored by measurement of transepithelial resistance (TER). The expression of JAM-A and E-cadherin, was assessed by Western blot and immunofluorescent confocal microscopy. In parallel studies, Caco-2 cells were co-cultured with human mast cells (HMC-1) up to 23 days. On the day of experiment HMC-1 cells were degranulated
AGA Abstracts
S-504
with compound 48/80 (5ug/ml) +/- tryptase inhibitor and incubated up to 24 hours. The epithelial integrity was evaluated by TER and Transmission Electron Microscopy (TEM), and the expression of junctional proteins was evaluated by Western blot and confocal microscopy. Results: A decrease in JAM-A expression (30%) was observed after 4 hours of incubation with 15 mU tryptase and was concomitant with a significant drop in TER (30 %). Redistribution of JAM-A protein from TJ regions was shown by confocal microscopy. The drop in TER was reversible within 24 hours compared with control samples, however the expression of JAM-A was consistently reduced (40 %). Degranulation of co-cultured HMC-1 cells (40 x 10 5 cells/ml) also induced a decrease in JAM-A expression (30 %) after 6 hours of incubation as demonstrated by Western blot and confocal microscopy. The effect was maintained up to 24 hours and was concomitant with a 40 % drop in TER. In contrast, there was no apparent change in the expression of E-cadherin in response to tryptase either in Caco-2 monolayer experiments or in the co-culture model. Conclusion: A reduction in JAM-A protein expression together with a decrease in epithelial integrity were consistent observations in Caco-2 monolayers either treated with tryptase or exposed to degranulated mast cells. These consistent alterations in JAM-A expression up to 24 hours may shed new light on the regulation of epithelial barrier integrity in IBS.
AGA Abstracts
Crypt Structures Are Protected in BCL-2 Modifying Factor (BMF-/-) Mice During Acute Colitis Katharina Leucht, Michaela Krebs, Christian Ploner, Andreas Villunger, Silvia Kellermeier, Michael Fried, Gerhard Rogler, Martin Hausmann BACKGROUND: Bcl-2 modifying factor (Bmf) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma (Bcl)-2. Bcl-2 is neutralized by Bmf and thereby facilitates cytochrome C release from mitochondria. Recently we reported that translocation of BMF in human intestinal epithelial cells is induced by the loss of cell attachment. This suggests that Bmf may be critical for epithelial cell homeostasis. We investigated the role of Bmf in mice under inflammatory conditions. METHODS: Acute colitis was induced in male C57BL/6-Bmftm1.1Anvi (Bmf-/-) mice weighing 25-32 g with 3.5 % dextran sulfate sodium (DSS). Mice were killed on day nine. A histological score, crypt lengths, colon lengths and percental body weight loss were determined. Immunohistochemistry was performed in human colonic sections with rabbit anti-human Bmf (#ab59906, abcam, 1:100). Intestinal epithelial cells (IEC) were isolated ex vivo. Single cells were separated from whole crypts and the relative amount of Bmf cDNA was calculated by means of real time PCR. RESULTS: Colonic crypt lengths of Bmf-/- mice were significantly increased as compared to wildtype (WT) (156.9 ± 32.0 μm, n = 99 and 139.8 ± 22.4 μm, n = 96, p < 0.05). Upon DSS, weight loss of Bmf-/- mice was significantly decreased from day five to eight as compared to WT mice. Following induction of acute colitis colon length of Bmf-/mice was less reduced. Immunohistochemistry of human IEC showed strong expression of Bmf in the extrusion zone where cells are shed. IEC were isolated ex vivo and whole crypts were separated from single cells. In patients with IBD Bmf mRNA from single cells was significantly increased (4.8 ± 1.5-fold) as compared to crypts. CONCLUSION: Detachment of IEC, the trigger of apoptosis in epithelial cells, is associated with Bmf upregulation. This is likely to play an important role in the regulation of intestinal epithelial cell survival. DSS induces less colitis in Bmf-/- mice. Increased colon and crypt length may indicate an extended lifespan of IEC in Bmf-/- mice compared to WT. This could be protective during onset of inflammation.
Su1827 The Notch-1 Signaling Pathway is Required for Functional Colonic Barrier in wt Mice Avantika Chitre, Cecilia Berin, Lloyd Mayer, Stephanie Dahan INTRODUCTION: Gut lympho-epithelial interactions occur in the epithelial layer and in the sub-epithelial space. We have recently shown that the presence of LPLs promotes mucosal barrier function in the RAG1-/- transfer model of colitis putatively due to the Notch-1 signaling pathway. This finding was corroborated In Vitro as the epithelial barrier function correlated with the activation of the Notch-1 signaling pathway In Vitro. Moreover, the activation of Notch-1 extends throughout the epithelium in CD mucosa compared to normal or UC mucosa. AIM: To determine whether the Notch signaling pathway is mandatory for epithelial barrier function In Vivo. METHODS: A specific Notch-1 or a scrambled siRNA sequence was freshly complexed with Lipofectamine 2000. Anesthetized WT mice received 2 intra-rectal injections of complexed siRNA (5 nmol in 20 μl of solution/mouse) 4 days apart. The injected mice were sacrificed one day after the last injection. Colonic tissues were either mounted into Ussing chambers to assess permeability or snap-frozen and subjected to Real-Time PCR for Hes-1, CDX-2, Villin, Occludin, Claudin-5, beta-catenin, and Ecadherin. RESULTS: While no differences in resistance in the distal colon were seen between scrambled and Notch-1 siRNA treated WT mice, there was a significant decrease in flux in the WT mice that received the Notch-1 siRNA (10925 ± 3389 ng of Dextran-FITC/ml/min/ cm2 in the Notch-1 siRNA treated WT mice vs. 2912 ± 1054 ng of Dextran-FITC/ml/min/ cm2 in the scrambled siRNA treated WT mice, p=0.01, n=9). The quantitative analysis of genes related to the Notch-1 signaling pathway showed that Hes-1 and CDX-2 mRNA expression was significantly decreased in the Notch-1 siRNA treated WT mice (p=0.01 and p=0.009, respectively). These findings correlated with a decrease in villin, occludin, and claudin-5 mRNA gene expression (p=0.0002, p=0.0002 and p=0.002, respectively). Moreover, a significant decrease in beta-catenin and E-cadherin was also observed (p<0.0001 and p=0.0004, respectively). CONCLUSION: Local knock down of the Notch-1 signaling pathway led to dysregulated flux across the distal colon but a normal resistance. The genes related to the Notch-1 signaling pathway and the tight junctions were also impaired in the Notch-1 siRNA treated mice. More interestingly, the beta-catenin/E-cadherin mRNA levels were affected by the Notch-1 knock down. These findings might explain why the flux and not the resistance were affected by In Vivo Notch-1 silencing. Thus, the Notch-1 signaling pathway is critical for the epithelium in order to achieve differentiation and functional barrier integrity.
Su1825 Increased Intestinal Permeability and Disturbance of Tight-Junctions in an Acute Graft Versus Host Disease Model in Mice Rainer Noth, Julia Lange-Grumfeld, Jochen Hampe, Stefan Schreiber, Alexander Arlt Background Hematopoietic stem cell transplantation is increasingly performed for acute and chronic leukemia and other hematologic malignancies. One of the severe side effects of the procedure are graft versus host reactions leading among other complications to gastrointestinal symptoms including diarrhea, nausea, vomiting, abdominal cramping, anorexia and gastrointestinal bleeding. Up to now little is known about the pathophysiological mechanisms evoking these GI-symptoms, especially the manifestation in the small intestine. Murine acute graft versus host disease (GvHD) has long been investigated as an animal model for the immune pathomechanisms of human GVHD after allogeneic bone marrow transplantation and due to its characteristic changes in the intestinal histology consisting of lymphocytic infiltrates, crypt hyperplasia, and villous atrophy, it serves as a model for immunologically mediated atrophic-hyperregenerative diseases of the intestine. Aim To evaluate effects of a murine GvHD on the intestinal barrier functions in the small intestine. Results The analysis of the gastrointestinal permeability by measuring blood levels of mannitol (a marker of absorption of small molecules representative for the transcellular permeability) and lactulose (a marker of abnormal absorption of large molecules representative for the paracellular permeability) after infusion through gastrointestinal tubes we were able to show that GvHD animals had significant higher blood levels of mannitol and lactulose if compared to control animals. The quotient of these two sugars indicated that the GvHD predominantly leads to an increased paracellular intestinal permeability. By using electron microscopy we were able to show that the intercellular space of the tight junctions of GvHD animals increased indicating a disturbance of the tight junctions. By PCR we could show that the extracellular tight junction protein occludin in contrast to the intracellular tight junction protein ZO-1 was significantly lower expressed. Immunhistochemistry revealed that this lower expression resulted in a disturbed assembly of the tight junctions in the GvHD animals. Conclusions Our analysis of the intestinal barrier functions of the small intestine in a murine model of GvHD revealed a disorganization of the tight junctions due to lower expression of occludin and consecutive increase in the intestinal permeability. This mechanism may contribute to the frequent gastrointestinal symptoms and malabsortion in kachexia in GvHD.
Su1828 Induction of Gastrokine-2 Expression in the Intestinal Tract of Gastrokine-1 Deficient Mice Shirley Paski, James P. Lodolce, Lauren Kolodziej, Sarah Bartulis, Mark Logsdon, Jonathan Chang, Wesley Grimm, Margaret Walsh-Reitz, F Gary Toback, David L. Boone INTRODUCTION: Gastrokine-1 (GKN1) is an 18-kDa protein expressed predominantly in the stomach where it is secreted into the lumen. A synthetic 21-mer peptide corresponding to a central domain of GKN1 (GKN1-peptide) is mitogenic, motogenic, and cytoprotective in the GI tract of mice exposed to indomethacin. The objectives of this study were to evaluate the: (1) effectiveness of GKN1-peptide at treating lipopolysaccharide (LPS)-induced increases in intestinal permeability in mice and (2) essential function of GKN1 by phenotyping GKN1 knockout mice. METHODS: To evaluate the protective effect of GKN1-peptide, 25mg/kg ip GKN1-peptide was given 5d, 24h, or 6h before treatment with 4mg/kg ip LPS. Intestines were removed 60min after LPS treatment, and barrier function was assessed by flux of 4kDa FITC dextran over 75min. To evaluate the therapeutic effect of GKN1-peptide, mice were injected with LPS followed by GKN1-peptide 15h later. Tight junction proteins occludin and ZO-1 were visualized with immunofluorescence and laser scanning confocal microscopy. To develop GKN1-/- mice, the GKN1 gene was ablated in entirety and replaced by a BAC-based vector expressing the β-galactosidase from the E. coli lacZ gene and neomycin phosphotransferase coding sequences. GKN1+/- embryonic stem cells from C57BL/6 mice were implanted into C57BL/6 embryos to create GKN1+/- mice. The growth and development of the first generation of GKN-/- mice was observed over 24 weeks. Mucosal histology and gene expression of GKN1, ZO-1, and occludin were studied in stomach, small intestine, and colon. Expression of GKN2 and GKN3 mRNA, which share homology to GKN1, were also evaluated. RESULTS: Treatment with LPS markedly increased FITC-dextran flux and caused altered localization of TJ proteins consistent with increased intestinal permeability. GKN1-peptide attenuated LPS-induced barrier disruption when injected 5d, 24h, and 6h before LPS. Giving GKN1-peptide 15h after LPS therapeutically reversed LPS-induced barrier disruption. GKN1-/- mice grow and develop normally compared to GKN1+/+ or GKN1+/littermates, showing no spontaneous gastroenteritis. Gastrointestinal histology and gene expression of ZO-1 and occludin did not differ between GKN-/- and GKN+/- mice. GKN2
Su1826 Mast Cell Tryptase Reduces Junctional Adhesion Molecule-a (JAM-A) Expression in Human Intestinal Epithelial Cells: Implications for the Mechanisms of Barrier Dysfunction in Irritable Bowel Syndrome (IBS) Ewa Wilcz, Siobhan McClean, Maria O'Sullivan Introduction: Alterations in mast cell numbers and in intestinal epithelial permeability have both been implicated in the pathogenesis of IBS. Tryptase, released from mast cells, has been shown to induce changes in paracellular permeability and in tight junction (TJ) proteins. The expression, however, of the junctional proteins JAM-A and E-cadherin in response to mast cell mediators has not been investigated in IBS, and may represent new insights into the regulation of epithelial barrier integrity. Aims: To investigate the expression of JAM-A and E-cadherin proteins 1) in Caco-2 monolayers exposed to the mast cell mediator, tryptase and 2) in a newly developed Caco-2/HMC-1 cells co-culture model. Methods: Caco-2 cells were grown as polarised monolayers up to 23 days on semi-permeable filters. Tryptase (3 mU or 15 mU) was added to the apical side of Caco-2 monolayers and incubated up to 24 hours. Epithelial integrity was monitored by measurement of transepithelial resistance (TER). The expression of JAM-A and E-cadherin, was assessed by Western blot and immunofluorescent confocal microscopy. In parallel studies, Caco-2 cells were co-cultured with human mast cells (HMC-1) up to 23 days. On the day of experiment HMC-1 cells were degranulated
AGA Abstracts
S-504