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M1842 CD44-Hyaluronan Interaction Plays a Critical Role in Biliary Proliferation During the Development of Hepatic Cholestasis
location, and positioning. Morphology: In the non-obese population, BMI correlated well to AWT averaged across all abdominal locations (r=.53*), and at the epigastrum (r=.50), but less so in the obese population (r=.32, and r=.22). Age: Age also interacted with the strength of correlation, with BMI correlating more highly with AWT in those under age 55 overall and at the epigastrum (r=.73* and r=.63* respectively), compared to those aged over 55 (r=.56* and r=.37). Location: Umbilicus had the thinnest AWT, the lowest variability of all measured abdominal locations, and no significant correlation to BMI (r= -.37). Positioning: BMI is highly correlated with AWT when the patient is positioned laterally (r=.81*), compared to supine positions (r=.58*). Procedure: No interaction was found between the type of surgery and AWT. CONCLUSIONS: In previous research we have shown that BMI correlates to AWT and that there are regional differences in the abdominal wall. This study, based on a larger sample, shows that there is a better correlation between BMI and AWT in the young and non-obese populations, and for patients positioned laterally. Data suggest that, because of decreased AWT and AWT-variability, coupled with AWT's low correlation to BMI, placing a port at the umbilicus may be a useful strategy to minimize at-the-trocar wall thickness and AWT variability between patients. The data presented here may be useful in providing further refinement for the understanding of differences in AWT.
M1844 The Effects of Dai-Kenchu-to (TU-100) On Propulsive Motility in the Colon. Michael Wood, Neil H. Hyman, Gary M. Mawe Purpose: Opioid analgesics have long been the primary treatment of postoperative pain. The use of these agents, however, is often associated with adverse effects on gastrointestinal motility including postoperative ileus. Recent efforts have focused on selective antagonists of the opioid-mu receptor. The purpose of this study is to examine the use of the herbal medicine Dai-Kenchu-to (DKT) as a potential treatment for opiate-induced slowing of intestinal transit in an isolated guinea-pig colon model of motility. We then investigated whether DKT could act synergistically with the non-selective opiate receptor antagonist, naloxone to promote propulsive motility. Methods: Isolated segments of distal guinea-pig colon were mounted in a perfusion chamber and imaged with a digital video camera interfaced with a computer. Fecal pellets were placed into the lumen at the oral end of the colonic segment and the rates of motility over a 3-4 cm segment of colon were determined. In addition, intracellular recording were obtained from intact circular muscle. Inhibitory and excitatory junction potentials, evoked by stimulation above or below the recording site, respectively were analyzed. Results: The addition of DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5), a selective mu-receptor agonist, caused a concentration-dependent decrease in colon motility. Naloxone did not affect basal activity, but partially restored motility in the DAMGO treated preparations. DKT (1x10-4-3x10-4 g/ml) also reversed the inhibitory effect of DAMGO treated colon in a concentration dependent manner. At higher concentrations (1x10-3-3x10-3 g/ml), however, this effect was lost. Motility slowed even further when naloxone and DKT were combined, with noticeable disruptions in spatiotemporal patterns. Interestingly, when added alone, DKT caused a reversal of the peristaltic reflex resulting in propulsion of the pellet in an anal to oral direction. In electrophysiological studies, DKT inhibited both excitatory and inhibitory junction potentials. Conclusions: DKT appears to be as effective as naloxone in restoring motility in DAMGO treated colon. These two agents, however, do not appear to have an addictive effect. When used on untreated colon segments, DKT appears to cause disruptions in the intrinsic reflex circuit of the gut by interfering with neuromuscular communication.
M1842 CD44-Hyaluronan Interaction Plays a Critical Role in Biliary Proliferation During the Development of Hepatic Cholestasis Gordon D. Wu, Yao He, Haimei Wang, Hong Wang, John M. Vierling, Andrew S. Klein
SSAT Abstracts
Background: High levels of CD44 and its ligand hyaluronic acid (HA) are present in cholestatic livers. The role of CD44-hyaluronic acid (HA) interaction in biliary pathology, however, is poorly undrstood. Methods: A rat model of cholestatic liver induced by bile duct ligation (BDL) was employed for the studies. Histological distribution of CD44-expressing cells and disposition of extracellular hyaluronic acid (HA) were examined in cryostat sections of the livers with immunofluorescence or histochemistry. Biliary epithelial cells (BEC), hepatic stellate cells (HSC), CD31+ hepatic endothelial cells (HEC) and ED2+ Kupffer cells (KC) were isolated and examined for expression of CD44 standard (s) and variant (v) isoforms with quantitative real time PCR. The regulatory role of CD44-HA interaction in biliary proliferation was investigated in biliary epithelial cell (BEC) cultures. Results: BDL livers developed intensive intrahepatic bile duct proliferation. Epithelia lining the proliferative bile ducts were strongly positive for CD44. CD44 expression by the proliferative BEC was associated with markedly increased interstitial HA accumulation. Quantitative PCR revealed that CD44 expressed by the cholestatic livers increased 26-fold over the control (p<0.01). BEC isolated from the cholestatic livers expressed high levels of CD44 mRNA, which was 3-fold, 17-fold, and 19-fold as that expressed by sinusoidal endothelia, Kupffer cells and hepatic stellar cells (P<0.01, respectively). BEC significantly increased expression of CD44 (p<0.01) and cell proliferation marker ki-67 (p<0.05) in responses to hyaluronan stimulation in cultures. Cellular proliferation assay demonstrated that cholangiocyte propagation accelerated upon HA stimulation, and was antagonized by anti-CD44 treatment (p<0.05). Conclusion: The study provides compelling evidence to suggest that proliferative BEC lining the intrahepatic bile ducts are the major source of hepatic CD44. CD44-HA interaction, by enhancing biliary proliferation, plays a pathogenic role in the development of cholestatic liver diseases.
M1845 Effect of NOD2/CARD15 Mutations On Ileal Gene Expression Profiles in Crohn's Disease Casey K. McCullough, Christina M. Hamm, Elizabeth Gorbe, Jonathan T. Unkart, Ellen Li, Qing qing Gong, Candace R. Miller, Thaddeus S. Stappenbeck, Christian Stone, David W. Dietz, Steven R. Hunt BACKGROUND: Three mutations (Leu1007InsC, R702W, G908R) in the nucleotide-binding oligomerization domain (NOD)2/caspase recruitment domain (CARD)15 have been associated with an increased risk of Crohn's disease (CD) and ileal disease location. Analysis of the effect of NOD2/CARD15 mutations on ileal gene expression could provide further insight on the pathogenesis of ileal CD. METHODS: We performed microarray analysis on normal ileal mucosa from three NOD2- (none of the three mutations) normal control subjects, and on inflamed and uninflamed ileal mucosa from three NOD2- CD patients, and three NOD2+ (at least one of three mutations) CD patients. All patients were nonsmokers. In the six CD patients, both inflamed and macroscopically uninflamed tissue biopsies were obtained from fresh pathologic specimens at the time of their initial ileocolic resection. The probes prepared from the ileal RNA and the common reference ileal RNA (from a fourth NOD2- control subject) were hybridized with the Agilent Whole Human Genome array using the two-color protocol. The data was analyzed using Statistical Analysis of Microarrays (SAM) software. RESULTS: SAM analysis detected no significant difference between NOD2+ and NOD2uninflamed mucosa. We identified a number of genes that were upregulated in the NOD2+ inflamed ileal mucosa relative to NOD2- inflamed ileal mucosa. One of the identified target genes, CYBB, encodes cytochrome b-245, beta polypeptide, which is a component of the microbicidal oxidase system in phagocytes (q=0.04). In addition, we observed altered expression of genes (q<0.05) in the uninflamed mucosa in CD patients compared to control subjects that was independent of NOD2 status. CONCLUSIONS: We have identified target genes that are upregulated in inflamed ileal mucosal biopsies from NOD2+ CD patients relative to inflamed mucosa from NOD2- CD patients. One of these genes, CYBB, plays an integral role in mucosal defense. These experiments have also detected altered expression of genes in macroscopically normal ileal mucosa from CD patients compared to control subjects, prior to upregulation of pro-inflammatory genes associated with CD activity.
M1843 Decreased PLD2 Expression Correlates with NM404 Retention in Human Colorectal Cancer Xenografts Joseph Nwankwo, Jaime H. McCord, Mary Wentworth, Jamey Weichert, Sharon M. Weber Introduction: Radioiodinated NM404 (12-(4-iodophenyl)-octadecylphosphocholine), a second-generation phospholipid ether analog, has displayed remarkable tumor selectivity in rodent tumor models. This novel agent is retained within tumor cells for a prolonged time, thus allowing for clinical imaging applications that may enhance preoperative staging. Phospholipid ether analogs appear to be a substrate for phospholipase D (PLD) but not phospholipase A or C based on preliminary data. We hypothesized that the mechanism of prolonged retention of NM404 is likely due to a decrease in its breakdown secondary to either decreased PLD1 or PLD2 expression. Methods: Three human colorectal cancer cell lines, DLD-1, HT-29 and LS-180, were simultaneously implanted subcutaneously (1 x 106 cells) into SCID mice. When tumor size reached 0.5 cm, animals were injected i.v. with 124I-NM404 (80-100 µCi in 0.1 ml). MicroPET and microCT images were obtained at 24 and 72 hrs. post injection, while quantification of NM404 retention was determined by ROI analysis of the PET scans. Cell lines were analyzed for PLD1 and PLD2 mRNA using quantitative PCR. qPCR for the PLD gene transcripts involved 50 cycles of amplification by real-time PCR to give PCR products of 560 bp for PLD1 and 557 bp for PLD2. Results: On microPET imaging, tumor conspicuity was excellent in all 3 tumor types at both 24 and 72 hours post injection. For NM404 retention, tumor-to-muscle ratios of 11.1, 6.0, and 4.0 were obtained for LS180, DLD-1, and HT-29, respectively. qPCR results from three separate experiments for PLD1 and PLD2 in the colorectal cancer cell lines were determined and expressed as a ratio to the level of gene expression in the normal CCD-18co colorectal cells after normalizing to the S26 house-keeping gene. There was no difference in PLD1 expression in the colorectal cell lines compared to CCD-18co. However, expression of PLD2 was markedly decreased in all three cell lines, with PLD2 values of 0.05 ± 0.02 (p = 0.006): 0.47 ± 0.19 (p = 0.083); and 0.14 ± 0.06 (p = 0.021) for LS-180, DLD-1, and HT-29, respectively. Thus a decreased PLD2 expression level correlated with increased retention levels of NM404. Conclusion: Human colorectal tumors showed excellent tumor conspicuity when utilizing 124I-NM404 microPET. This appears to be associated with a decrease in expression of PLD2. Further evaluation of the mechanism of selective retention of NM404 is needed in order to define a molecular profile that may classify tumors as more sensitive to NM404, enabling the selection of a subset of patients that would benefit from preoperative staging with NM404.
SSAT Abstracts
M1847 The Effect of Toll Like Receptor (TLR) 9 Agonist, CpG Oligodeoxynucleotides (ODN), On Abdominal and Gastrotomy Wound Healing in a Murine Model Ik-Yong Kim, Xiaohong Yan, Raymond Baxter, Thomas R. Gardner, Chandana Shantha Kumara HM, Aqeel Ahmed, Carlos Cordon-Cardo, Richard L. Whelan Introduction: The synthetic TLR9 ligand, CpG ODN (CpG) has been used clinically as an anti-cancer immunotherapy. It has largely been used in the adjuvant or stage 4 setting. It has been shown experimentally that CpG, when given alone prior to surgery (preop), limits postoperative tumor growth. Perioperative (Periop) CpG might also limit post-surgical immunosuppression. Prior to human periop studies CpG's impact on wound healing must be determined. This study's purpose was to assess abdominal wall wound and anastomotic healing after periop CpG. Methods: Thirty Balb/C mice were randomized into 2 groups and given CpG 1826 (20 ug/dose) or PBS via i.p. both PreOP (7, 5, 3, and 1 day before surgery) and on PostOP day (POD) 1, 2, and 3. All mice underwent a laparotomy and gastrotomy that were carefully suture closed. The mice were sacrificed on POD7 and 21 and their abdominal pelts and stomachs excised. Tensometry was used to determine the peak force and total energy required to disrupt the abdominal wall wounds while the bursting pressure of the gastric wounds was measured via saline infusion. The Sircol assay was used to determine soluble collagen content and a blinded pathologist used a wound healing scoring system histologically to assess the pelts. Results: There were no significant differences in the
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M1844 The Effects of Dai-Kenchu-to (TU-100) On Propulsive Motility in the Colon. Michael Wood, Neil H. Hyman, Gary M. Mawe Purpose: Opioid analgesics have long been the primary treatment of postoperative pain. The use of these agents, however, is often associated with adverse effects on gastrointestinal motility including postoperative ileus. Recent efforts have focused on selective antagonists of the opioid-mu receptor. The purpose of this study is to examine the use of the herbal medicine Dai-Kenchu-to (DKT) as a potential treatment for opiate-induced slowing of intestinal transit in an isolated guinea-pig colon model of motility. We then investigated whether DKT could act synergistically with the non-selective opiate receptor antagonist, naloxone to promote propulsive motility. Methods: Isolated segments of distal guinea-pig colon were mounted in a perfusion chamber and imaged with a digital video camera interfaced with a computer. Fecal pellets were placed into the lumen at the oral end of the colonic segment and the rates of motility over a 3-4 cm segment of colon were determined. In addition, intracellular recording were obtained from intact circular muscle. Inhibitory and excitatory junction potentials, evoked by stimulation above or below the recording site, respectively were analyzed. Results: The addition of DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5), a selective mu-receptor agonist, caused a concentration-dependent decrease in colon motility. Naloxone did not affect basal activity, but partially restored motility in the DAMGO treated preparations. DKT (1x10-4-3x10-4 g/ml) also reversed the inhibitory effect of DAMGO treated colon in a concentration dependent manner. At higher concentrations (1x10-3-3x10-3 g/ml), however, this effect was lost. Motility slowed even further when naloxone and DKT were combined, with noticeable disruptions in spatiotemporal patterns. Interestingly, when added alone, DKT caused a reversal of the peristaltic reflex resulting in propulsion of the pellet in an anal to oral direction. In electrophysiological studies, DKT inhibited both excitatory and inhibitory junction potentials. Conclusions: DKT appears to be as effective as naloxone in restoring motility in DAMGO treated colon. These two agents, however, do not appear to have an addictive effect. When used on untreated colon segments, DKT appears to cause disruptions in the intrinsic reflex circuit of the gut by interfering with neuromuscular communication.
M1842 CD44-Hyaluronan Interaction Plays a Critical Role in Biliary Proliferation During the Development of Hepatic Cholestasis Gordon D. Wu, Yao He, Haimei Wang, Hong Wang, John M. Vierling, Andrew S. Klein
SSAT Abstracts
Background: High levels of CD44 and its ligand hyaluronic acid (HA) are present in cholestatic livers. The role of CD44-hyaluronic acid (HA) interaction in biliary pathology, however, is poorly undrstood. Methods: A rat model of cholestatic liver induced by bile duct ligation (BDL) was employed for the studies. Histological distribution of CD44-expressing cells and disposition of extracellular hyaluronic acid (HA) were examined in cryostat sections of the livers with immunofluorescence or histochemistry. Biliary epithelial cells (BEC), hepatic stellate cells (HSC), CD31+ hepatic endothelial cells (HEC) and ED2+ Kupffer cells (KC) were isolated and examined for expression of CD44 standard (s) and variant (v) isoforms with quantitative real time PCR. The regulatory role of CD44-HA interaction in biliary proliferation was investigated in biliary epithelial cell (BEC) cultures. Results: BDL livers developed intensive intrahepatic bile duct proliferation. Epithelia lining the proliferative bile ducts were strongly positive for CD44. CD44 expression by the proliferative BEC was associated with markedly increased interstitial HA accumulation. Quantitative PCR revealed that CD44 expressed by the cholestatic livers increased 26-fold over the control (p<0.01). BEC isolated from the cholestatic livers expressed high levels of CD44 mRNA, which was 3-fold, 17-fold, and 19-fold as that expressed by sinusoidal endothelia, Kupffer cells and hepatic stellar cells (P<0.01, respectively). BEC significantly increased expression of CD44 (p<0.01) and cell proliferation marker ki-67 (p<0.05) in responses to hyaluronan stimulation in cultures. Cellular proliferation assay demonstrated that cholangiocyte propagation accelerated upon HA stimulation, and was antagonized by anti-CD44 treatment (p<0.05). Conclusion: The study provides compelling evidence to suggest that proliferative BEC lining the intrahepatic bile ducts are the major source of hepatic CD44. CD44-HA interaction, by enhancing biliary proliferation, plays a pathogenic role in the development of cholestatic liver diseases.
M1845 Effect of NOD2/CARD15 Mutations On Ileal Gene Expression Profiles in Crohn's Disease Casey K. McCullough, Christina M. Hamm, Elizabeth Gorbe, Jonathan T. Unkart, Ellen Li, Qing qing Gong, Candace R. Miller, Thaddeus S. Stappenbeck, Christian Stone, David W. Dietz, Steven R. Hunt BACKGROUND: Three mutations (Leu1007InsC, R702W, G908R) in the nucleotide-binding oligomerization domain (NOD)2/caspase recruitment domain (CARD)15 have been associated with an increased risk of Crohn's disease (CD) and ileal disease location. Analysis of the effect of NOD2/CARD15 mutations on ileal gene expression could provide further insight on the pathogenesis of ileal CD. METHODS: We performed microarray analysis on normal ileal mucosa from three NOD2- (none of the three mutations) normal control subjects, and on inflamed and uninflamed ileal mucosa from three NOD2- CD patients, and three NOD2+ (at least one of three mutations) CD patients. All patients were nonsmokers. In the six CD patients, both inflamed and macroscopically uninflamed tissue biopsies were obtained from fresh pathologic specimens at the time of their initial ileocolic resection. The probes prepared from the ileal RNA and the common reference ileal RNA (from a fourth NOD2- control subject) were hybridized with the Agilent Whole Human Genome array using the two-color protocol. The data was analyzed using Statistical Analysis of Microarrays (SAM) software. RESULTS: SAM analysis detected no significant difference between NOD2+ and NOD2uninflamed mucosa. We identified a number of genes that were upregulated in the NOD2+ inflamed ileal mucosa relative to NOD2- inflamed ileal mucosa. One of the identified target genes, CYBB, encodes cytochrome b-245, beta polypeptide, which is a component of the microbicidal oxidase system in phagocytes (q=0.04). In addition, we observed altered expression of genes (q<0.05) in the uninflamed mucosa in CD patients compared to control subjects that was independent of NOD2 status. CONCLUSIONS: We have identified target genes that are upregulated in inflamed ileal mucosal biopsies from NOD2+ CD patients relative to inflamed mucosa from NOD2- CD patients. One of these genes, CYBB, plays an integral role in mucosal defense. These experiments have also detected altered expression of genes in macroscopically normal ileal mucosa from CD patients compared to control subjects, prior to upregulation of pro-inflammatory genes associated with CD activity.
M1843 Decreased PLD2 Expression Correlates with NM404 Retention in Human Colorectal Cancer Xenografts Joseph Nwankwo, Jaime H. McCord, Mary Wentworth, Jamey Weichert, Sharon M. Weber Introduction: Radioiodinated NM404 (12-(4-iodophenyl)-octadecylphosphocholine), a second-generation phospholipid ether analog, has displayed remarkable tumor selectivity in rodent tumor models. This novel agent is retained within tumor cells for a prolonged time, thus allowing for clinical imaging applications that may enhance preoperative staging. Phospholipid ether analogs appear to be a substrate for phospholipase D (PLD) but not phospholipase A or C based on preliminary data. We hypothesized that the mechanism of prolonged retention of NM404 is likely due to a decrease in its breakdown secondary to either decreased PLD1 or PLD2 expression. Methods: Three human colorectal cancer cell lines, DLD-1, HT-29 and LS-180, were simultaneously implanted subcutaneously (1 x 106 cells) into SCID mice. When tumor size reached 0.5 cm, animals were injected i.v. with 124I-NM404 (80-100 µCi in 0.1 ml). MicroPET and microCT images were obtained at 24 and 72 hrs. post injection, while quantification of NM404 retention was determined by ROI analysis of the PET scans. Cell lines were analyzed for PLD1 and PLD2 mRNA using quantitative PCR. qPCR for the PLD gene transcripts involved 50 cycles of amplification by real-time PCR to give PCR products of 560 bp for PLD1 and 557 bp for PLD2. Results: On microPET imaging, tumor conspicuity was excellent in all 3 tumor types at both 24 and 72 hours post injection. For NM404 retention, tumor-to-muscle ratios of 11.1, 6.0, and 4.0 were obtained for LS180, DLD-1, and HT-29, respectively. qPCR results from three separate experiments for PLD1 and PLD2 in the colorectal cancer cell lines were determined and expressed as a ratio to the level of gene expression in the normal CCD-18co colorectal cells after normalizing to the S26 house-keeping gene. There was no difference in PLD1 expression in the colorectal cell lines compared to CCD-18co. However, expression of PLD2 was markedly decreased in all three cell lines, with PLD2 values of 0.05 ± 0.02 (p = 0.006): 0.47 ± 0.19 (p = 0.083); and 0.14 ± 0.06 (p = 0.021) for LS-180, DLD-1, and HT-29, respectively. Thus a decreased PLD2 expression level correlated with increased retention levels of NM404. Conclusion: Human colorectal tumors showed excellent tumor conspicuity when utilizing 124I-NM404 microPET. This appears to be associated with a decrease in expression of PLD2. Further evaluation of the mechanism of selective retention of NM404 is needed in order to define a molecular profile that may classify tumors as more sensitive to NM404, enabling the selection of a subset of patients that would benefit from preoperative staging with NM404.
SSAT Abstracts
M1847 The Effect of Toll Like Receptor (TLR) 9 Agonist, CpG Oligodeoxynucleotides (ODN), On Abdominal and Gastrotomy Wound Healing in a Murine Model Ik-Yong Kim, Xiaohong Yan, Raymond Baxter, Thomas R. Gardner, Chandana Shantha Kumara HM, Aqeel Ahmed, Carlos Cordon-Cardo, Richard L. Whelan Introduction: The synthetic TLR9 ligand, CpG ODN (CpG) has been used clinically as an anti-cancer immunotherapy. It has largely been used in the adjuvant or stage 4 setting. It has been shown experimentally that CpG, when given alone prior to surgery (preop), limits postoperative tumor growth. Perioperative (Periop) CpG might also limit post-surgical immunosuppression. Prior to human periop studies CpG's impact on wound healing must be determined. This study's purpose was to assess abdominal wall wound and anastomotic healing after periop CpG. Methods: Thirty Balb/C mice were randomized into 2 groups and given CpG 1826 (20 ug/dose) or PBS via i.p. both PreOP (7, 5, 3, and 1 day before surgery) and on PostOP day (POD) 1, 2, and 3. All mice underwent a laparotomy and gastrotomy that were carefully suture closed. The mice were sacrificed on POD7 and 21 and their abdominal pelts and stomachs excised. Tensometry was used to determine the peak force and total energy required to disrupt the abdominal wall wounds while the bursting pressure of the gastric wounds was measured via saline infusion. The Sircol assay was used to determine soluble collagen content and a blinded pathologist used a wound healing scoring system histologically to assess the pelts. Results: There were no significant differences in the
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