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S1790 Impaired Mucosal Barrier Function in the Small Intestine of the Cystic Fibrosis Mouse
fully polarised spheroids. These spheroids were then exposed to ethanol (21.7 or 43.4 mM), acetaldehyde (100 or 200 μM), culture medium (negative control) or 2 mM EGTA (positive control) for 3 hours. Morphological differentiation was evaluated by transmission electron microscopy. Barrier function was assessed by the flux of fluorescein isothiocyanate-conjugated dextran 4KD (FD4) from the basal to the luminal compartment using live cell imaging. Results: 3D cultured T84 cells form spheroids with the lumen inside, and exhibit a high level of differentiation demonstrated by formation of microvilli and tight junctions facing the lumen. Hence, this model is suitable to study effects of substances at the basal side. This reflects alcohol exposure in the colon after oral intake in humans, as the transport is characterized by a basal to luminal gradient. After 3 hours, the mean fluorescence intensity of FD4 was measured and expressed as the ratio of the luminal over the basal compartment, given as mean values of 7 spheroids from 4 different fields of repeated experiments. A dosedependent increase of the FD4 ratio was found at 21.7 and 43.4 mM ethanol versus control [0.180±0.017 and 0.436±0.008 respectively, vs 0.003±0.000, p<0.001]. Also 100 and 200 μM acetaldehyde increased the ratio dose dependently versus control [0.203±0.038 and 0.391±0.046, respectively vs 0.003±0.000, p<0.001]. Conclusion: T84 cells cultured in 3D represent morphological features of the In Vivo intestine and are a useful model to study barrier function In Vitro. Ethanol and acetaldehyde at physiological concentrations increase intraluminal FD4 concentrations in a dose-dependent manner, pointing to disruption of tight junctions.
Impaired Mucosal Barrier Function in the Small Intestine of the Cystic Fibrosis Mouse Robert C. De Lisle BACKGROUND: The mucosal barrier of the gut is important to protect the body from the large numbers of microbes that inhabit the intestines and the proinflammatory molecules they release. A major factor of barrier function is transepithelial permeability, controlled in part by the tight junction. Another component of the mucosal barrier is that intestinal alkaline phosphatase (IAP) detoxifies its natural substrate endotoxin (lipopolysaccharide, LPS). Loss of gut barrier function can allow LPS access to the circulation which may contribute to inflammation at distant sites in the body. In the genetic disease cystic fibrosis (CF) there is an excessive immune response to infection of the airways, and the airway damage that ensues is the eventual cause of mortality in CF. To begin assessing intestinal mucosal barrier function in CF and its potential relationship to airway inflammation, we are using the Cftr knockout mouse model. METHODS: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. All mice were maintained on a liquid diet (Peptamen) which prevents lethal intestinal obstruction in CF mice. Intestinal permeability was assessed by the passage of gavaged fluorescent-dextran into the blood, and serum protein in the gut lumen. IAP was investigated by histochemistry, qRT-PCR measurement of expression of the mouse IAP gene (Akp3), and enzyme activity. Lung inflammation was assessed histologically, by immunohistochemistry for neutrophils, and by myeloperoxidase (MPO) activity. RESULTS: CF mice had increased intestinal permeability as shown by a 20-fold greater level of plasma rhodamine-dextran at 90min post-gavage. There was also more serum albumin in the CF intestinal lumen. In the CF intestine, histochemical detection of IAP was noticeably less than in WT. There was a 60% reduction in Akp3 mRNA and a 50% reduction in IAP enzyme activity in the CF intestine. The CF mouse lung exhibited mild thickening of the epithelium, increased neutrophils, and increased MPO activity. CONCLUSIONS: The CF mouse intestine has impaired mucosal barrier function as shown by increased permeability to macromolecules and reduced IAP activity. There is also a mild inflammatory state in the uninfected CF mouse lung. To test the hypothesis that impaired gut mucosal barrier function affects the CF lung, future work will determine the extent to which experimental manipulations that improve intestinal barrier function also reduce inflammation of the lungs. This study was supported by NIH grant R21 AI083479 and a pilot project as part of NIH grant P20 RR024214.
S1793 Validation of Colonic Mucosal Permeability Measurements by Urinary Saccharide Excretion After Oral Ingestion in Healthy Controls by Comparison With Intracolonic Infusion Archana S. Rao, Michael Camilleri, Irene A. Busciglio, Deborah J. Eckert, Duane D. Burton, Banny S. Wong, Ravinder J. Singh, Jesse Lamsam, Alan R. Zinsmeister Background: We have recently shown that urinary excretion of the saccharides lactulose (L) and mannitol (M) occurs more than two hours after ingestion. This reflects colonic mucosal permeability since simultaneous radioscintigraphy documented the presence of >50% of the substrates in the human colon at 2h. Aim: To compare the L:M ratio after oral (liquid and delayed release capsule formulations) versus intracolonic administration of these sugars in healthy controls. Methods: Ten healthy controls ingested 1g of lactulose and 0.2g of mannitol delivered in radiolabeled solution or methacrylate-coated capsules. After colon cleansing with a PEG electrolyte-based oral colonic lavage solution on the day prior to study, they also underwent colonoscopy under conscious sedation with i.v. midazolam for intracolonic administration of sugars. Radiolabeled solution containing the sugars was infused into the transverse colon; scintigraphic imaging documented that the infusate had reached the ascending colon. All subjects underwent all three studies (liquid, capsule, and intracolonic administration of sugars) on separate occasions, and the order of testing was randomized. Urine collections were obtained from 0-8h after intracolonic infusion and 2-8h after oral ingestion to preferentially evaluate colonic permeability; saccharide excretion was measured by HPLC tandem mass spectrometry. Scintiscans were obtained to document arrival of liquid sugars into the colon. Primary endpoint for analysis was the L:M ratio. Paired t-test was used to compare the L:M ratio for the different modes of administration. Results: After liquid administration, isotope reached the colon after 54.0±9.8min (range 30-120min). There was a significant difference in the L:M ratio after colonic infusion compared to oral solution. No significant difference in the L:M ratio was observed between administration of saccharides via direct colonic infusion and oral ingestion with delayed release capsule. Conclusion: Urinary lactulose and mannitol measurements are different when sugars are directly infused into the colon versus ingested orally in liquid formulation. However, lactulose and mannitol excretion to assess colonic permeability are not significantly different when administered via delayed release, methacrylate-coated capsules or direct intracolonic infusion. Cumulative Excretion of Sugars (mg) and L:M Ratio (Mean±SEM)
S1791 A Novel Animal Model for HIV Enteropathy Brendan Halloran, Marie Claire Arrieta, Ferdinand Maingat, Jon Meddings, Christopher Power Background: The pathogenesis underlying HIV enteropathy remains uncertain although both direct HIV infection and systemic inflammation involving the gut have been implicated. HIV infection of the gut is posited to be associated with microbial translocation leading to immunosuppression. Like HIV, feline immunodeficiency virus (FIV) is a lentivirus which causes immunosuppression through infection and depletion of CD4+ T cells. Herein we investigated the extent to which chronic FIV infection affected gut function. Hypothesis: Lentivirus infection results in aberrant gut function including enteropathy in conjunction with immunosuppression. Methods: Specific pathogen-free cats were infected with a recombinant FIV infectious molecular clone (FIV-Ch) at 10,000 TCID50/ml at post-natal day 1 and sacrificed at week 12. Blood CD4+/CD8+ T cell counts, plasma viral load, weight gain and plasma bacterial (E. coli) RNA levels were analyzed. The presence of enteropathy was evaluated by non-invasive, site specific measurements of gastrointestinal permeability as well as endpoint determinations of histology and tissue markers of inflammation. Results: FIVinfected animals exhibited delayed weight gain with occasional diarrhea over the 12 week period compared to mock-infected animals (p<0.01). CD4+ T cell counts were suppressed in FIV-infected animals at weeks 8 and 12 post-infection (p<0.05) while CD8+ T cell counts were increased at week 8 (p<0.01). Small intestine epithelial dysfunction was evident at 12 weeks post-infection in terms of the lactose/mannitol ratio in the FIV-infected animals (0.203 +/- 0.065) compared to mock-infected (0.06+/- 0.06) animals (p<0.01). Plasma bacterial RNA levels were detected in all animals but did not differ between experimental groups. Median plasma viral load in the FIV-infected group was 10,000 copies/ml but was not detected in mock-infected animals. Conclusions: FIV infection resulted in immunosuppression and a high viral burden in conjunction with delayed weight gain. These findings were associated with selectively increased small bowel permeability without evidence of gastric and colonic dysfunction or microbial translocation. Whether this enteropathy is a consequence of direct viral effect of the gut or indirect effect of systemic inflammation or opportunistic infection remains unclear but ongoing studies of gut immune activation and morphological changes will elucidate these issues. This model may lend itself to further studies into the pathogenesis of HIV enteropathy, permitting the development of new diagnostic and therapeutic strategies. These studies were supported by the Canadian Institutes of Health Research.
* 0-8h urine collection; # liquid vs infusion, p=0.008; ** capsule vs infusion, p=ns S1794 Distinct Phenotypes of the Placodes- And Neural Crest-Derived Nociceptors Innervating the Mouse Esophagus Lenka Surdenikova, Christina Nassenstein, Fei Ru, Marian Kollarik The mouse is an indispensable model for the study of afferent signaling from the esophagus but the phenotypes of the esophageal afferent nerves in the mouse are incompletely understood. We addressed the hypothesis that the vagal afferent TRPV1+ nociceptive nerves in the mouse esophagus originate from both embryonic neural crest and placodes, and have distinct phenotypes. Retrograde tracing studies in the Wnt1Cre/R26R mice that allows for identification of the neural crest-derived cells revealed that 17±4% of jugular/nodose ganglia (JNG) neurons projecting into the mouse esophagus were derived from neural crest (total 250 neurons from 9 JNGs analyzed). Our previous studies in the guinea pig vagal system showed that the expression of the purinergic receptor subunit P2X2 discriminates between the TRPV1+ nociceptive neurons derived from placodes (P2X2+) and neural crest (P2X2-). Functional studies in the Wnt1Cre/R26R JNG neurons were consistent with this observation. Single cell RT-PCR performed on the afferent neurons retrogradely labeled from the mouse esophagus (n=172) showed that putative neural crest-derived (TRPV1+/P2X2-) and putative placodes-derived (TRPV1+/P2X2+) TRPV1-positive JNG nociceptors comprise 16% and 13% of vagal afferent neurons innervating the esophagus, respectively. The putative neural crestderived JNG nociceptors (TRPV1+/P2X2-) expressed preprotachykinin-A gene products (PPTA), GFL co-receptor GFRα3, and trophic factor receptor TrkA , but rarely TrkB (Table
S1792 Effects of Ethanol and Acetaldehyde on Epithelial Integrity in a Three Dimensional (3D) Epithelial Cell Culture Model Elhaseen Elamin, Daisy Jonkers, Freddy Troost, Kati Juuti-Uusitalo, Sven C. van IJzendoorn, Hans Duimel, Jos L. Broers, Fons K. Verheyen, Jan Dekker, Ad Masclee Introduction: A decreased intestinal barrier function and translocation of endotoxins are involved in the pathogenesis of alcoholic liver diseases. Ethanol and its metabolite acetaldehyde are found to disrupt tight junction structures in an In Vitro two dimensional monolayer of Caco2 cells. However, this model has limitations due to diminished differentiation, cell signaling, and non-physiological morphology. Aim: To investigate in a (3D) model of human colon carcinoma cells (T84), the effects of ethanol and acetaldehyde on epithelial integrity, in concentrations of ethanol and acetaldehyde found in the systemic and colonic circulation after ingestion of 10-30 g of alcohol. Methods: T84 cells were grown for 5 days to induce
S-275
AGA Abstracts
AGA Abstracts
S1790
Impaired Mucosal Barrier Function in the Small Intestine of the Cystic Fibrosis Mouse Robert C. De Lisle BACKGROUND: The mucosal barrier of the gut is important to protect the body from the large numbers of microbes that inhabit the intestines and the proinflammatory molecules they release. A major factor of barrier function is transepithelial permeability, controlled in part by the tight junction. Another component of the mucosal barrier is that intestinal alkaline phosphatase (IAP) detoxifies its natural substrate endotoxin (lipopolysaccharide, LPS). Loss of gut barrier function can allow LPS access to the circulation which may contribute to inflammation at distant sites in the body. In the genetic disease cystic fibrosis (CF) there is an excessive immune response to infection of the airways, and the airway damage that ensues is the eventual cause of mortality in CF. To begin assessing intestinal mucosal barrier function in CF and its potential relationship to airway inflammation, we are using the Cftr knockout mouse model. METHODS: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. All mice were maintained on a liquid diet (Peptamen) which prevents lethal intestinal obstruction in CF mice. Intestinal permeability was assessed by the passage of gavaged fluorescent-dextran into the blood, and serum protein in the gut lumen. IAP was investigated by histochemistry, qRT-PCR measurement of expression of the mouse IAP gene (Akp3), and enzyme activity. Lung inflammation was assessed histologically, by immunohistochemistry for neutrophils, and by myeloperoxidase (MPO) activity. RESULTS: CF mice had increased intestinal permeability as shown by a 20-fold greater level of plasma rhodamine-dextran at 90min post-gavage. There was also more serum albumin in the CF intestinal lumen. In the CF intestine, histochemical detection of IAP was noticeably less than in WT. There was a 60% reduction in Akp3 mRNA and a 50% reduction in IAP enzyme activity in the CF intestine. The CF mouse lung exhibited mild thickening of the epithelium, increased neutrophils, and increased MPO activity. CONCLUSIONS: The CF mouse intestine has impaired mucosal barrier function as shown by increased permeability to macromolecules and reduced IAP activity. There is also a mild inflammatory state in the uninfected CF mouse lung. To test the hypothesis that impaired gut mucosal barrier function affects the CF lung, future work will determine the extent to which experimental manipulations that improve intestinal barrier function also reduce inflammation of the lungs. This study was supported by NIH grant R21 AI083479 and a pilot project as part of NIH grant P20 RR024214.
S1793 Validation of Colonic Mucosal Permeability Measurements by Urinary Saccharide Excretion After Oral Ingestion in Healthy Controls by Comparison With Intracolonic Infusion Archana S. Rao, Michael Camilleri, Irene A. Busciglio, Deborah J. Eckert, Duane D. Burton, Banny S. Wong, Ravinder J. Singh, Jesse Lamsam, Alan R. Zinsmeister Background: We have recently shown that urinary excretion of the saccharides lactulose (L) and mannitol (M) occurs more than two hours after ingestion. This reflects colonic mucosal permeability since simultaneous radioscintigraphy documented the presence of >50% of the substrates in the human colon at 2h. Aim: To compare the L:M ratio after oral (liquid and delayed release capsule formulations) versus intracolonic administration of these sugars in healthy controls. Methods: Ten healthy controls ingested 1g of lactulose and 0.2g of mannitol delivered in radiolabeled solution or methacrylate-coated capsules. After colon cleansing with a PEG electrolyte-based oral colonic lavage solution on the day prior to study, they also underwent colonoscopy under conscious sedation with i.v. midazolam for intracolonic administration of sugars. Radiolabeled solution containing the sugars was infused into the transverse colon; scintigraphic imaging documented that the infusate had reached the ascending colon. All subjects underwent all three studies (liquid, capsule, and intracolonic administration of sugars) on separate occasions, and the order of testing was randomized. Urine collections were obtained from 0-8h after intracolonic infusion and 2-8h after oral ingestion to preferentially evaluate colonic permeability; saccharide excretion was measured by HPLC tandem mass spectrometry. Scintiscans were obtained to document arrival of liquid sugars into the colon. Primary endpoint for analysis was the L:M ratio. Paired t-test was used to compare the L:M ratio for the different modes of administration. Results: After liquid administration, isotope reached the colon after 54.0±9.8min (range 30-120min). There was a significant difference in the L:M ratio after colonic infusion compared to oral solution. No significant difference in the L:M ratio was observed between administration of saccharides via direct colonic infusion and oral ingestion with delayed release capsule. Conclusion: Urinary lactulose and mannitol measurements are different when sugars are directly infused into the colon versus ingested orally in liquid formulation. However, lactulose and mannitol excretion to assess colonic permeability are not significantly different when administered via delayed release, methacrylate-coated capsules or direct intracolonic infusion. Cumulative Excretion of Sugars (mg) and L:M Ratio (Mean±SEM)
S1791 A Novel Animal Model for HIV Enteropathy Brendan Halloran, Marie Claire Arrieta, Ferdinand Maingat, Jon Meddings, Christopher Power Background: The pathogenesis underlying HIV enteropathy remains uncertain although both direct HIV infection and systemic inflammation involving the gut have been implicated. HIV infection of the gut is posited to be associated with microbial translocation leading to immunosuppression. Like HIV, feline immunodeficiency virus (FIV) is a lentivirus which causes immunosuppression through infection and depletion of CD4+ T cells. Herein we investigated the extent to which chronic FIV infection affected gut function. Hypothesis: Lentivirus infection results in aberrant gut function including enteropathy in conjunction with immunosuppression. Methods: Specific pathogen-free cats were infected with a recombinant FIV infectious molecular clone (FIV-Ch) at 10,000 TCID50/ml at post-natal day 1 and sacrificed at week 12. Blood CD4+/CD8+ T cell counts, plasma viral load, weight gain and plasma bacterial (E. coli) RNA levels were analyzed. The presence of enteropathy was evaluated by non-invasive, site specific measurements of gastrointestinal permeability as well as endpoint determinations of histology and tissue markers of inflammation. Results: FIVinfected animals exhibited delayed weight gain with occasional diarrhea over the 12 week period compared to mock-infected animals (p<0.01). CD4+ T cell counts were suppressed in FIV-infected animals at weeks 8 and 12 post-infection (p<0.05) while CD8+ T cell counts were increased at week 8 (p<0.01). Small intestine epithelial dysfunction was evident at 12 weeks post-infection in terms of the lactose/mannitol ratio in the FIV-infected animals (0.203 +/- 0.065) compared to mock-infected (0.06+/- 0.06) animals (p<0.01). Plasma bacterial RNA levels were detected in all animals but did not differ between experimental groups. Median plasma viral load in the FIV-infected group was 10,000 copies/ml but was not detected in mock-infected animals. Conclusions: FIV infection resulted in immunosuppression and a high viral burden in conjunction with delayed weight gain. These findings were associated with selectively increased small bowel permeability without evidence of gastric and colonic dysfunction or microbial translocation. Whether this enteropathy is a consequence of direct viral effect of the gut or indirect effect of systemic inflammation or opportunistic infection remains unclear but ongoing studies of gut immune activation and morphological changes will elucidate these issues. This model may lend itself to further studies into the pathogenesis of HIV enteropathy, permitting the development of new diagnostic and therapeutic strategies. These studies were supported by the Canadian Institutes of Health Research.
* 0-8h urine collection; # liquid vs infusion, p=0.008; ** capsule vs infusion, p=ns S1794 Distinct Phenotypes of the Placodes- And Neural Crest-Derived Nociceptors Innervating the Mouse Esophagus Lenka Surdenikova, Christina Nassenstein, Fei Ru, Marian Kollarik The mouse is an indispensable model for the study of afferent signaling from the esophagus but the phenotypes of the esophageal afferent nerves in the mouse are incompletely understood. We addressed the hypothesis that the vagal afferent TRPV1+ nociceptive nerves in the mouse esophagus originate from both embryonic neural crest and placodes, and have distinct phenotypes. Retrograde tracing studies in the Wnt1Cre/R26R mice that allows for identification of the neural crest-derived cells revealed that 17±4% of jugular/nodose ganglia (JNG) neurons projecting into the mouse esophagus were derived from neural crest (total 250 neurons from 9 JNGs analyzed). Our previous studies in the guinea pig vagal system showed that the expression of the purinergic receptor subunit P2X2 discriminates between the TRPV1+ nociceptive neurons derived from placodes (P2X2+) and neural crest (P2X2-). Functional studies in the Wnt1Cre/R26R JNG neurons were consistent with this observation. Single cell RT-PCR performed on the afferent neurons retrogradely labeled from the mouse esophagus (n=172) showed that putative neural crest-derived (TRPV1+/P2X2-) and putative placodes-derived (TRPV1+/P2X2+) TRPV1-positive JNG nociceptors comprise 16% and 13% of vagal afferent neurons innervating the esophagus, respectively. The putative neural crestderived JNG nociceptors (TRPV1+/P2X2-) expressed preprotachykinin-A gene products (PPTA), GFL co-receptor GFRα3, and trophic factor receptor TrkA , but rarely TrkB (Table
S1792 Effects of Ethanol and Acetaldehyde on Epithelial Integrity in a Three Dimensional (3D) Epithelial Cell Culture Model Elhaseen Elamin, Daisy Jonkers, Freddy Troost, Kati Juuti-Uusitalo, Sven C. van IJzendoorn, Hans Duimel, Jos L. Broers, Fons K. Verheyen, Jan Dekker, Ad Masclee Introduction: A decreased intestinal barrier function and translocation of endotoxins are involved in the pathogenesis of alcoholic liver diseases. Ethanol and its metabolite acetaldehyde are found to disrupt tight junction structures in an In Vitro two dimensional monolayer of Caco2 cells. However, this model has limitations due to diminished differentiation, cell signaling, and non-physiological morphology. Aim: To investigate in a (3D) model of human colon carcinoma cells (T84), the effects of ethanol and acetaldehyde on epithelial integrity, in concentrations of ethanol and acetaldehyde found in the systemic and colonic circulation after ingestion of 10-30 g of alcohol. Methods: T84 cells were grown for 5 days to induce
S-275
AGA Abstracts
AGA Abstracts
S1790