- Email: [email protected]
Reperfusion Injury
M1892
of a P (CL/LA) 50:50 reinforced with a PGA fiber mesh and was designed to degrade in six to eight weeks in the body. There was no prior cell seeding onto the graft. Animals were re-laparotomized three months after implantation and gross, histological and blood chemical studies performed. [Results] All recipient pigs survived until they were sacrificed for collection of graft sites three months after implantation. All of them gained weight. On gross examination, the artificial duct was found to have been absorbed and the graft site was indistinguishable from the native extrahepatic bile duct. Stricture was not found on cholangiography, adhesion to surrounding tissue was mild and the graft site could be freed manually. Histology revealed a neo-bile duct growing in the graft site with the epithelium of highly uneven thickness and increased accessory glands compared with the native duct. Blood chemistry data at three months post implantation did not show change from baseline values. [Conclusions] This study demonstrated that biliary stenosis could be treated by resecting the narrowed portion and substituting it with this artificial bile duct. Thus, the artificial duct can be used in place of T-tubes or stents in transplantation surgery and gastrointestinal surgery, or can be used as a prosthesis after surgery for localized lower bile duct cancer.
Endoscopic Retrograde Cholangiopancreatography in the Pre-Operative Evaluation of Living Related Liver Transplant Donors Christopher Kleeman, Burckhardt Ringe, David Anjelly, Asyia S. Ahmad, James C. Reynolds, Ricardo Morgenstern Background: Endoscopic retrograde cholangiopancreatography (ERCP) is routinely used to assess donors for living related liver transplantation (LRLT). This procedure helps to map the biliary anatomy and reveal any anomalies that may alter the surgical approach or exclude patients from donating. It has been shown to be more sensitive than MRCP although it carries substantially more risk. Aim: Determine whether pre-operative ERCP in LRLT donors uncovers clinically significant biliary anomalies with acceptable risk to patients. Methods: We performed a retrospective chart review of all LRLT donors evaluated at our institution since the program's inception 5 years ago. ERCP images were deemed adequate if secondorder branches of the biliary tree were visualized. The biliary anatomy was classified based on the Couinaud system. Complications of ERCP and liver transplant were determined based on the medical record. Results: Thirty patients were evaluated for LRLT between 9/1/02 and 9/1/07. Five were excluded because they did not undergo ERCP: 3 were deemed to be unsuitable donors and 2 underwent emergent transplants. Of the 25 patients who underwent pre-operative ERCP, 4 had failed cannulations. Of the 21 donors who had adequate ERCPs, 8 patients (38%) had abnormal biliary anatomy, including 3 (14%) with triple confluence of the ducts, 2 (10%) with anomalous drainage of the right posterior segmental duct, 3 (14%) with drainage of the right hepatic duct into the cystic duct, and 1 (5%) with an accessory duct. In addition, 2 had aberrant insertion of the cystic duct and 1 had small ducts. MRCP detected 2 out of 8 of these anomalies. There were 3 complications related to ERCP, including self-limited pain (2) and pancreatitis (1) (peak Amylase of 1,153ng/mL). The surgical approach was altered in 1 patient with extension of the cutting plane. Of the 30 recipients, 23 (64%) received segments 5-8, 6 (27%) received segments 2-3, and 1 (5%) received segments 2-4. There were 9 complications in the recipients, including 4 biliary complications. Two complications occurred in patients with normal anatomy, 1 bile stricture occurred in a patient with triple confluence, and 1 bile leak occurred in a patient with an accessory duct but did not involve that segment. Overall, the rate of biliary complications was lower in donors with aberrant anatomy compared to those with normal anatomy (11% vs. 31%). Conclusion: Biliary anomalies were found in 38% of patients undergoing preoperative ERCP for LRLT, only 25% of which were detected by MRCP. However, these anomalies had little impact on the surgical approach and did not correlate with posttransplant biliary complications.
M1895
Background/Aims: Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially associated with the preservation of hepatic ATP contents during ischemia. However, despite of the extensive studies performed, the precise molecular mechanisms of these events remain poorly elucidated. The aim of current study was to investigate the proteins involved in I/R injury and IPC in mouse models. Methods: A series of male C57BL/6 mice were randomly divided into three groups and subjected to sham operation, I/R (75 minutes of hepatic ischemia and 2 hours of reperfusion) or IPC (10 minutes of hepatic ischemia followed by 10 minutes of reperfusion) before prolonged I/R, respectively. The liver proteomes were analyzed using two-dimensional electrophoresis (2-DE) combined with MALDI TOF/TOF mass analysis. Results: Twenty-four proteins showing more than 2fold difference were identified in the livers upon I/R injury. Among these proteins, nine proteins were further regulated by IPC when compared with non-pretreated I/R groups. This included metabolic enzymes (BHMT, TTR, ALDH6A1 and ATP5B), molecular chaperones (PDIA1 and GRP78) and extracellular proteins (MUP6 and ALB). One of these proteins, ATP synthase beta subunit (ATP5B) catalyzes the rate-limiting step of ATP formation. The expression level of ATP5B, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Correspondingly, the same tendency of the hepatic ATP level was observed by high-performance liquid chromatography. Therefore, the increasing hepatic ATP5B expression probably attributes to the ATP-preserving effect of IPC. Conclusion: This study provided new clues for understanding the mechanisms of IPC upon I/R injury. The protective role of ATP5B might give evidences for developing new therapeutic approaches against hepatic I/R injury.
M1893 Are Post-Orthotopic Liver Transplant Patients At Higher Risk for Post-ERCPPancreatitis? Murad A. Abu Rajab, S.Ian Gan Objectives: Orthotopic liver transplant patients often undergo endoscopic retrograde cholangio-pancreatography (ERCP) for evaluation of the biliary tree and it is unknown if OLT patients are at higher risk for the development of post-ERCP pancreatitis (PEP). The purpose of this study was to determine if OLT represented an independent risk factor for PEP. Methods: A registry of all liver transplant candidates and recipients at a single tertiary care transplant center (Tufts-New England Medical Center) was reviewed retrospectively.Any patient having an ERCP between 01/2000 and 01/2007 was enrolled in the study and their records were reviewed in detail. Diagnosis of PEP was based on clinical symptoms (abdominal pain)±abdominal CT scan findings. All patients who underwent Roux-en Y anastomosis at time of transplant were excluded from the study. The cohort receiving ERCP prior to transplant was compared to the cohort receiving ERCP post-transplant. Results: 267 patients underwent liver transplantation at Tufts-New England Medical Center over the 7 year period. 9 patients were excluded due to Roux-en Y anastomosis. Seventy-nine percent were male and the mean age was 50.5years at time of transplant. Sixty-two ERCPs were performed on the cohort prior to liver transplant and 146 ERCP were performed after OLT. The indications for post-transplant ERCP included elevated liver function tests (46%), stent exchange (22%), stricture formation (19%) and biliary leak (9%). None of the patients had a history of pancreatitis prior to inclusion. There were no events of PEP in the pre-transplant group (0%). In the post-OLT group, 5 patients (3.4%) developed PEP. The rate of PEP in the post-transplant group was not significantly greater than the pre-transplant group (p = 0.32). The only significant risk factor for PEP was sphincterotomy (p = 0.0018). Sex, age, biliary findings, etiology of liver failure and pancreatic duct cannulation all did not correlate with development of PEP. Conclusion: Post-orthotopic liver transplant patients are not at a higher risk of PEP. Sphincterotomy is the only identifiable risk factor for PEP in this cohort of patients. Subsequent studies are needed to elaborate more risk factors.
M1896 Deletion of CD39 On Natural Killer Cells Attenuates Warm Hepatic Ischemia/ Reperfusion Injury Guido Beldi, Yara Banz, Alexander Kroemer, Annette Pexa, Yan Wu, Keichi Enjyoji, Daniel Candinas, Xian C. Li, Simon C. Robson Background: CD39 (nucleoside triphosphate diphosphohydrolase (NTPDase)-1) is an ectonucleotidase, expressed by the vasculature and defined immune cell subsets involved in liver ischemia reperfusion injury (IRI) e.g. natural killer NK and NKT cells. CD39, in tandem with CD73/ecto-5'-nucleotidase, modulates purinergic signaling via nucleotide P2-receptors and also generates extracellular adenosine. Pharmacological agonists for the adenosine-2A receptors upon NKT cells are thought to block interferon-gamma (IFN gamma) release and provide protective effects post-warm partial liver IRI. Aim: To examine consequences of CD39 expression on NK or NKT cells and study effects on hepatic IRI, changes in IFN gamma production and cytotoxicity. Methods and Results: CD39 is the dominant ectonucleotidase in both NK and NKT cells. NK cells lack expression of CD73, unlike NKT cells that express both ecto-enzymes. Hepatic NK but not NKT cell populations are preferentially increased in mice null for CD39 when compared to wild type controls; CD11bhiCD27low subsets of NK cells (viz. less aggressive, long-lived) accounted for these increases. Effects of CD39 on liver injury were evaluated in a model of partial warm hepatic ischemia. CD39 and IFN gamma null mice are protected equivalently against early biochemical manifestations of liver injury seen with warm partial hepatic IRI in wild type mice. Adoptive transfers were also performed into Rag2/common gamma null mice (deficient in T cells, B cells and NK cells) using NK and NKT cells of wild type and mutant mice with targeted deletion of either CD39 or IFN gamma. Our data show that relative protective effects of CD39 deletion are seen with transfer of NK but not NKT cells. The improvements in IRI outcomes with adoptive transfer of IFN gamma null NK cells are comparable to CD39 null NK cells; both are associated with decreased neutrophil infiltration and tissue injury. Deletion of CD39 results in aberrant NK cell responses In Vitro with decreased secretion of IFN gamma in response to exogenous IL-12 and IL-18. Furthermore, secretion of IFN gamma from CD39 null NK cells and cytotoxic activity can be significantly decreased by the non- hydrolysable ATPyS, are unaffected by adenosine and restored by bypass of P2Y-receptor responses In Vitro. Conclusion: NK and NKT cells both express CD39 but differentially express CD73. CD39 modulates NK numbers and their function In Vitro. Importantly, CD39 deletion on NK cells is protective in hepatic IRI. Disordered purinergic signaling with P2Y-receptor desensitization results in decreased IFN gamma secretion by CD39 null NK cells with less injury post hepatic IRI.
M1894 Development of An Artificial Bile Duct Made of Bioabsorbable Polymer to Be Used for Treatment of Biliary Stenosis Masayasu Aikawa, Mitsuo Miyazawa, Katsuya Okada, Yasuko Toshimitsu, Yoshihide Otani, Isamu Koyama It has been reported that choledochoenterostomy to treat biliary stenosis and other disease is associated, in the long term, with postoperative bile duct cancer at an incidence of about 5 to 10%. Along with the recent widespread use of laparoscopic cholecystectomy and livingdonor liver transplantation, complications involving the biliary system are increasing. Stent or T-tube insertion is a common treatment for bile duct stenosis by which the papilla of Vater can be preserved. However, there are some disadvantages with both stents and Ttubes, and new means of treatment have been called for. We investigated whether an artificial bile duct made of bioabsorbable polymer could substitute for a narrowed bile duct. [Methods] Hybrid pigs were laparotomized under general anesthesia and the extrahepatic bile duct was identified. Then a portion of the duodenal side of the bile duct was resected, 3 cm in major axis, and substituted by a bioabsorbable polymer tube of the same size. It was made
A-805
AASLD Abstracts
AASLD Abstracts
Proteomic Analysis of Hepatic Ischemia/Reperfusion Injury and Ischemic Preconditioning in Mice Revealed the Protective Role of Atp5b Xuequn Zhang, Chengfu Xu, Chaohui Yu, Guohua Lu, Shaohua Chen, Liming Xu, Wei Ding, Qiaojuan Shi, Youming Li
of a P (CL/LA) 50:50 reinforced with a PGA fiber mesh and was designed to degrade in six to eight weeks in the body. There was no prior cell seeding onto the graft. Animals were re-laparotomized three months after implantation and gross, histological and blood chemical studies performed. [Results] All recipient pigs survived until they were sacrificed for collection of graft sites three months after implantation. All of them gained weight. On gross examination, the artificial duct was found to have been absorbed and the graft site was indistinguishable from the native extrahepatic bile duct. Stricture was not found on cholangiography, adhesion to surrounding tissue was mild and the graft site could be freed manually. Histology revealed a neo-bile duct growing in the graft site with the epithelium of highly uneven thickness and increased accessory glands compared with the native duct. Blood chemistry data at three months post implantation did not show change from baseline values. [Conclusions] This study demonstrated that biliary stenosis could be treated by resecting the narrowed portion and substituting it with this artificial bile duct. Thus, the artificial duct can be used in place of T-tubes or stents in transplantation surgery and gastrointestinal surgery, or can be used as a prosthesis after surgery for localized lower bile duct cancer.
Endoscopic Retrograde Cholangiopancreatography in the Pre-Operative Evaluation of Living Related Liver Transplant Donors Christopher Kleeman, Burckhardt Ringe, David Anjelly, Asyia S. Ahmad, James C. Reynolds, Ricardo Morgenstern Background: Endoscopic retrograde cholangiopancreatography (ERCP) is routinely used to assess donors for living related liver transplantation (LRLT). This procedure helps to map the biliary anatomy and reveal any anomalies that may alter the surgical approach or exclude patients from donating. It has been shown to be more sensitive than MRCP although it carries substantially more risk. Aim: Determine whether pre-operative ERCP in LRLT donors uncovers clinically significant biliary anomalies with acceptable risk to patients. Methods: We performed a retrospective chart review of all LRLT donors evaluated at our institution since the program's inception 5 years ago. ERCP images were deemed adequate if secondorder branches of the biliary tree were visualized. The biliary anatomy was classified based on the Couinaud system. Complications of ERCP and liver transplant were determined based on the medical record. Results: Thirty patients were evaluated for LRLT between 9/1/02 and 9/1/07. Five were excluded because they did not undergo ERCP: 3 were deemed to be unsuitable donors and 2 underwent emergent transplants. Of the 25 patients who underwent pre-operative ERCP, 4 had failed cannulations. Of the 21 donors who had adequate ERCPs, 8 patients (38%) had abnormal biliary anatomy, including 3 (14%) with triple confluence of the ducts, 2 (10%) with anomalous drainage of the right posterior segmental duct, 3 (14%) with drainage of the right hepatic duct into the cystic duct, and 1 (5%) with an accessory duct. In addition, 2 had aberrant insertion of the cystic duct and 1 had small ducts. MRCP detected 2 out of 8 of these anomalies. There were 3 complications related to ERCP, including self-limited pain (2) and pancreatitis (1) (peak Amylase of 1,153ng/mL). The surgical approach was altered in 1 patient with extension of the cutting plane. Of the 30 recipients, 23 (64%) received segments 5-8, 6 (27%) received segments 2-3, and 1 (5%) received segments 2-4. There were 9 complications in the recipients, including 4 biliary complications. Two complications occurred in patients with normal anatomy, 1 bile stricture occurred in a patient with triple confluence, and 1 bile leak occurred in a patient with an accessory duct but did not involve that segment. Overall, the rate of biliary complications was lower in donors with aberrant anatomy compared to those with normal anatomy (11% vs. 31%). Conclusion: Biliary anomalies were found in 38% of patients undergoing preoperative ERCP for LRLT, only 25% of which were detected by MRCP. However, these anomalies had little impact on the surgical approach and did not correlate with posttransplant biliary complications.
M1895
Background/Aims: Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially associated with the preservation of hepatic ATP contents during ischemia. However, despite of the extensive studies performed, the precise molecular mechanisms of these events remain poorly elucidated. The aim of current study was to investigate the proteins involved in I/R injury and IPC in mouse models. Methods: A series of male C57BL/6 mice were randomly divided into three groups and subjected to sham operation, I/R (75 minutes of hepatic ischemia and 2 hours of reperfusion) or IPC (10 minutes of hepatic ischemia followed by 10 minutes of reperfusion) before prolonged I/R, respectively. The liver proteomes were analyzed using two-dimensional electrophoresis (2-DE) combined with MALDI TOF/TOF mass analysis. Results: Twenty-four proteins showing more than 2fold difference were identified in the livers upon I/R injury. Among these proteins, nine proteins were further regulated by IPC when compared with non-pretreated I/R groups. This included metabolic enzymes (BHMT, TTR, ALDH6A1 and ATP5B), molecular chaperones (PDIA1 and GRP78) and extracellular proteins (MUP6 and ALB). One of these proteins, ATP synthase beta subunit (ATP5B) catalyzes the rate-limiting step of ATP formation. The expression level of ATP5B, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Correspondingly, the same tendency of the hepatic ATP level was observed by high-performance liquid chromatography. Therefore, the increasing hepatic ATP5B expression probably attributes to the ATP-preserving effect of IPC. Conclusion: This study provided new clues for understanding the mechanisms of IPC upon I/R injury. The protective role of ATP5B might give evidences for developing new therapeutic approaches against hepatic I/R injury.
M1893 Are Post-Orthotopic Liver Transplant Patients At Higher Risk for Post-ERCPPancreatitis? Murad A. Abu Rajab, S.Ian Gan Objectives: Orthotopic liver transplant patients often undergo endoscopic retrograde cholangio-pancreatography (ERCP) for evaluation of the biliary tree and it is unknown if OLT patients are at higher risk for the development of post-ERCP pancreatitis (PEP). The purpose of this study was to determine if OLT represented an independent risk factor for PEP. Methods: A registry of all liver transplant candidates and recipients at a single tertiary care transplant center (Tufts-New England Medical Center) was reviewed retrospectively.Any patient having an ERCP between 01/2000 and 01/2007 was enrolled in the study and their records were reviewed in detail. Diagnosis of PEP was based on clinical symptoms (abdominal pain)±abdominal CT scan findings. All patients who underwent Roux-en Y anastomosis at time of transplant were excluded from the study. The cohort receiving ERCP prior to transplant was compared to the cohort receiving ERCP post-transplant. Results: 267 patients underwent liver transplantation at Tufts-New England Medical Center over the 7 year period. 9 patients were excluded due to Roux-en Y anastomosis. Seventy-nine percent were male and the mean age was 50.5years at time of transplant. Sixty-two ERCPs were performed on the cohort prior to liver transplant and 146 ERCP were performed after OLT. The indications for post-transplant ERCP included elevated liver function tests (46%), stent exchange (22%), stricture formation (19%) and biliary leak (9%). None of the patients had a history of pancreatitis prior to inclusion. There were no events of PEP in the pre-transplant group (0%). In the post-OLT group, 5 patients (3.4%) developed PEP. The rate of PEP in the post-transplant group was not significantly greater than the pre-transplant group (p = 0.32). The only significant risk factor for PEP was sphincterotomy (p = 0.0018). Sex, age, biliary findings, etiology of liver failure and pancreatic duct cannulation all did not correlate with development of PEP. Conclusion: Post-orthotopic liver transplant patients are not at a higher risk of PEP. Sphincterotomy is the only identifiable risk factor for PEP in this cohort of patients. Subsequent studies are needed to elaborate more risk factors.
M1896 Deletion of CD39 On Natural Killer Cells Attenuates Warm Hepatic Ischemia/ Reperfusion Injury Guido Beldi, Yara Banz, Alexander Kroemer, Annette Pexa, Yan Wu, Keichi Enjyoji, Daniel Candinas, Xian C. Li, Simon C. Robson Background: CD39 (nucleoside triphosphate diphosphohydrolase (NTPDase)-1) is an ectonucleotidase, expressed by the vasculature and defined immune cell subsets involved in liver ischemia reperfusion injury (IRI) e.g. natural killer NK and NKT cells. CD39, in tandem with CD73/ecto-5'-nucleotidase, modulates purinergic signaling via nucleotide P2-receptors and also generates extracellular adenosine. Pharmacological agonists for the adenosine-2A receptors upon NKT cells are thought to block interferon-gamma (IFN gamma) release and provide protective effects post-warm partial liver IRI. Aim: To examine consequences of CD39 expression on NK or NKT cells and study effects on hepatic IRI, changes in IFN gamma production and cytotoxicity. Methods and Results: CD39 is the dominant ectonucleotidase in both NK and NKT cells. NK cells lack expression of CD73, unlike NKT cells that express both ecto-enzymes. Hepatic NK but not NKT cell populations are preferentially increased in mice null for CD39 when compared to wild type controls; CD11bhiCD27low subsets of NK cells (viz. less aggressive, long-lived) accounted for these increases. Effects of CD39 on liver injury were evaluated in a model of partial warm hepatic ischemia. CD39 and IFN gamma null mice are protected equivalently against early biochemical manifestations of liver injury seen with warm partial hepatic IRI in wild type mice. Adoptive transfers were also performed into Rag2/common gamma null mice (deficient in T cells, B cells and NK cells) using NK and NKT cells of wild type and mutant mice with targeted deletion of either CD39 or IFN gamma. Our data show that relative protective effects of CD39 deletion are seen with transfer of NK but not NKT cells. The improvements in IRI outcomes with adoptive transfer of IFN gamma null NK cells are comparable to CD39 null NK cells; both are associated with decreased neutrophil infiltration and tissue injury. Deletion of CD39 results in aberrant NK cell responses In Vitro with decreased secretion of IFN gamma in response to exogenous IL-12 and IL-18. Furthermore, secretion of IFN gamma from CD39 null NK cells and cytotoxic activity can be significantly decreased by the non- hydrolysable ATPyS, are unaffected by adenosine and restored by bypass of P2Y-receptor responses In Vitro. Conclusion: NK and NKT cells both express CD39 but differentially express CD73. CD39 modulates NK numbers and their function In Vitro. Importantly, CD39 deletion on NK cells is protective in hepatic IRI. Disordered purinergic signaling with P2Y-receptor desensitization results in decreased IFN gamma secretion by CD39 null NK cells with less injury post hepatic IRI.
M1894 Development of An Artificial Bile Duct Made of Bioabsorbable Polymer to Be Used for Treatment of Biliary Stenosis Masayasu Aikawa, Mitsuo Miyazawa, Katsuya Okada, Yasuko Toshimitsu, Yoshihide Otani, Isamu Koyama It has been reported that choledochoenterostomy to treat biliary stenosis and other disease is associated, in the long term, with postoperative bile duct cancer at an incidence of about 5 to 10%. Along with the recent widespread use of laparoscopic cholecystectomy and livingdonor liver transplantation, complications involving the biliary system are increasing. Stent or T-tube insertion is a common treatment for bile duct stenosis by which the papilla of Vater can be preserved. However, there are some disadvantages with both stents and Ttubes, and new means of treatment have been called for. We investigated whether an artificial bile duct made of bioabsorbable polymer could substitute for a narrowed bile duct. [Methods] Hybrid pigs were laparotomized under general anesthesia and the extrahepatic bile duct was identified. Then a portion of the duodenal side of the bile duct was resected, 3 cm in major axis, and substituted by a bioabsorbable polymer tube of the same size. It was made
A-805
AASLD Abstracts
AASLD Abstracts
Proteomic Analysis of Hepatic Ischemia/Reperfusion Injury and Ischemic Preconditioning in Mice Revealed the Protective Role of Atp5b Xuequn Zhang, Chengfu Xu, Chaohui Yu, Guohua Lu, Shaohua Chen, Liming Xu, Wei Ding, Qiaojuan Shi, Youming Li