Effects of 25-Hydroxycholesterol Sulfation on Liver Regeneration in Normal and Partial Hepatectomy (PHX) Mouse Models

Mo1065

Introduction In phenylketonuria (PKU) patients, the intellectual quotient outcome is directly related to the levels of phenylalanine during infancy. Best prognosis is associated with maintenance of average phenylalanine levels below 400 μmol/L (6.6 mg/dl) in children less than 10 years of age. There is currently no specific treatment beside protein restriction and phenylalanine free products. The metabolism of phenylalanine is exclusively liver based. Hence, liver cell transplantation is an attractive and logical option to help controlling phenylalanine levels in the most severely affected patients. Best results of liver cell transplantation are achieved with good quality cells, freshly isolated, from young donors and short ischemic time. This is made extremely infrequent due to lack of organs offered for liver cell transplantation. Material and Methods The candidate patient was a 6 years old male with severe PKU. He was poorly equilibrated despite a close medical, dietary and psychosocial follow up. Genotype P281L / IVS10-11G>A confirmed the diagnosis of severe PKU with no residual activity of PAH, and no improvement was obtained under Tetrahydrobiopterin treatment. The child had frequent elevated levels of phenylalanine above the safe limit for the last three years. Tolerance to phenylalanine was very low, and normal levels could only be reached when the child was hospitalized. In this context, we performed liver cell transplantation, using 1.7 billion fresh cells from a 14 months old girl with type 1b glycogen storage. The child received a second infusion of 0.8 billion fresh cells seven and a half months later from a healthy donor. Results Following this, mean phenylalanine level 3 months before transplantation, 11.1 ± 3.8 mg/dl (n=11), decreased up to 3.5 ± 1.8 mg/dl (n=11) after the 2 cell infusions, returning within normal limits. In parallel, the half life of phenylalanine, as evaluated by a loading test, decreased from 41.6 hours before cell therapy, to 19.1 hours after second cell transplantation. Conclusion This is the first demonstration in man that liver cell therapy can significantly improve phenylketonuria in severe patients.

The diagram shows that 27.1% of non donors expressed that they would become organs donors should they were assigned few extra MELD points in case they needed liver transplant (Positive conversion) vs. 3.8% of donors expressed that they would become organs non donors should extra few MELD points assigned to donors (Negative conversion). Mo1064 Frequency and Risk Factors of Venous Thromboembolism in Liver Transplant Recipients - A 15-Year Single Center Experience Augustine Salami, Waqas Qureshi, Marwan Abouljoud Purpose Venous thromboembolism is a potentially fatal complication of major abdominal surgeries. Orthotopic liver transplant is a major abdominal surgery carried out as a treatment for end stage liver disease. Due to the depletion of clotting factors in end stage liver disease, the patients are in a relative coagulopathic state. It is known that there is a slower recovery of depleted coagulation and anticoagulation proteins after the transplantation in these patients. There is also a controversy in the use of VTE prophylaxis in these patients. It is not well studied that whether this population is at increased or decreased risk of a VTE event after a liver transplant even though the fibrin formation is found to be normal in these patients. This study was carried out to find out the frequency of VTE in this patient population and to compare it with the already published literature regarding other major abdominal surgeries. Methods A retrospective review of 917 patients who received 1001 orthotopic liver transpants between june 1995 to march 2010 at a single tertiary center was carried out. All liver transplant patients greater than 18 years of age with symptomatic VTE occuring up to one year after liver transplantation were included. Both upper and lower extremity DVT were identified. The diagnosis of VTEwas made using Doppler ultrasound and computed tomography. PE was diagnosed by high-probability ventilation/perfusion scan and intravenous contrast computed tomography of the chest. Data regarding known risk factors of VTE such as thrombophilia, recent hospitalization, etiology of cirrhosis and other co-morbid conditions was collected. Results Out of 917 patients, a total of 45 events occured in 42 patients(4.58%). 12 (1.58%)patients had pulmonary embolism and 33 (3.3%) patients had DVT events. On Cox regression analysis the absence of an alcohol diagnosis( Hazard Ratio(HR) 0.328, 95% CI 0.13-0.83), the presence of diabetes(HR-3.361, 95% CI 1.766.42) , a history of VTE (HR-8.062 , 95%CI 3.37-19.27) , and the presence of the ESRD(HR 3.676 ,95% CI 1.34-10.01)were significant predictors of the DVT/PE outcome. No particular diagnosis, history of malignancy, presence of thrombophilia was associated with increased risk of VTE. The analysis was controlled for the known risk factors as given above. Conclusion The 4.58 % yearly incidence of VTE is comparable to the incidence in major abdominal surgery (5-10% per year). This data also shows that there is increased risk of VTE in these patients if they have a comorbid conditions of diabetes mellitus, previous VTE and ESRD. This data also implies that a similar aggressive strategy for prophylaxis of VTE should be used in orthotopic hepatic transplant patients as is done for other major intraabdominal surgeries. Hazard ratios for risk factors of VTE

Mo1066 Effects of 25-Hydroxycholesterol Sulfation on Liver Regeneration in Normal and Partial Hepatectomy (PHX) Mouse Models Xin Zhang, Qianming Bai, Douglas M. Heuman, William M. Pandak, Shunlin Ren Background: The acidic pathway of bile acid biosynthesis, initiated by CYP27A1 in the mitochondria, appears to be necessary for normal liver regeneration, but the mechanism is unknown. CYP27A1 catalyzes formation of 25-hydroxycholesterol (25HC), which subsequently may be sulfated in cytosol via the sulfotransferase SULT2B1b. The product of this pathway, 25HC-3-sulfate (25HC3S) has potent regulatory properties on lipid metabolism, inflammation and other basic cellular functions. The expression of SULT2B1b is substantially increased by PHX. Hypothesis: 25HC3S may play a role in regulation of liver regeneration. Methods: C57BL/6 mice underwent partial hepatectomy (PHX). Prior to PHX, mice were either infected with recombinant adenovirus encoding CMV-SULT2B1b (48 hours pre-PHX) or intravenously injected with 25HC3S (two hours pre-PHX). Gal adenovirus and vehicle solution were used as controls. During PHX, left lateral and median lobes were completely excised. At 24 hours post-PHX (72 hours post transfection) mice were sacrificed. Expression of SULT2B1b and of the proliferation marker PCNA in liver tissues were determined by western blot analysis and immunostaining, respectively. Expression of genes involved in cell proliferation was determined by real-time RT-PCR. Results: The human SULT2B1b gene was successfully overexpressed in the liver after transfection: mRNA levels increased by 100 to 200-fold and protein levels by 15 to 20-fold at 72 hours post transfection. In mice transfected with SULT2B1b who underwent PHX at 48 hours post transfection, the percentage of PCNA positive cells measured 24 hours post-PHX was increased by 4-fold compared to gal-adenovirus transfected controls 24 hours post PHX. In addition, SULT2B1b overexpression was associated at 24 hours post-PHX with increased mRNA expression for a variety of genes involved in liver proliferation including FoxM1b (+ 228%), HGF (+ 200%), CDC25b (+ 535%), CyclinA (+ 300%), and C-myc (+170%). Compared to PHX controls, 25HC3S treated mice 24 hours post PHX exhibited a 1.8-fold increase in PCNA positivity. HGF (+123%) and Cyclin A (+240%) also were increased, while other proliferative markers did not differ significantly. Conclusion: These results indicated that sulfated oxysterol, 25HC3S, whether administered exogenously or synthesized endogenously (following SULT2B1b overexpression), can promote liver regeneration in mice. Mo1067 Sphingosine Kinase-2 is Involved in Cold Storage/Reperfusion Injury During Orthotopic Rat Liver Transplantation Yanjun Shi, Hasibur Rehman, Charles D. Smith, Zhi Zhong Background: Ischemia/reperfusion (IR) injury plays a key role in primary graft failure after liver transplantation (LT). Our previous study showed that inhibition of sphingosine kinase2 (SK2) by ABC294640 attenuates hepatic warm IR injury. Aim: Therefore, in this study we further investigated the roles of SK2 in hepatic injury after cold storage/transplantation. Methods: The effect of SK2 inhibition was evaluated in an orthotopic rat LT model. Livers were explanted and stored in the UW cold storage solution with ABC294640 (100 μM) for 18 h. ABC294640 was also injected into the recipients (50 mg/kg, iv) immediately after LT. Rats were observed for 4 weeks post-operatively for survival. Results: Sphingosine-1phosphate increased ~3-fold after LT, indicating activation of SK. ABC294640 blunted increases of sphingosine-1-phosphate by half. Eighteen hours after transplantation, serum alanine aminotransferase increased from the basal level of ~75 U/L to ~6,300 U/L and bilirubin increased from 0.25 mg/dL to ~2.3 mg/dL. Hepatic necrotic cell death increased from 0% to ~25%. TUNEL-positive apoptotic cells increased from 0.2% to 4% after transplantation. These results confirm that cold storage/transplantation causes severe liver injury. Treatment with ABC294640 blunted ALT release by ~60% and hyperbilirubinemia by ~80% and decreased necrosis by ~69% and apoptosis by ~80%. Thirty day-survival was ~10% in

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AASLD Abstracts

AASLD Abstracts

Hepatocyte Transplantation Transforms Severe Phenylketonuria to Mild Hyperphenylalaninemia Françoise N. Smets, Xavier Stephenne, Guillaume Debray, Renaud Menten, Raymond Reding, Mustapha Najimi, Etienne M. Sokal