Article first published online: 15 OCT 2013
Copyright © 2013 American Association for the Study of Liver Diseases
Special Issue: The 64th Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting 2013
Volume 58, Issue S1, pages 368A–377A, October 2013
How to Cite
(2013), Cellular immunobiology. Hepatology, 58: 368A–377A. doi: 10.1002/hep.26838
- Issue published online: 1 OCT 2013
- Article first published online: 15 OCT 2013
Ultra Low Dose Delta 9-tetrahydrocannabinol Protects Mouse Liver from Ischemia Reperfusion Injury
Eylon Lahat2, Edith Hochhauser4, Maya Sultan2, Yosef Sarne3, Mordechai Gutman1, Michal Safran2, Ziv Ben Ari12
1Liver Disease Center, Sheba Medical Center, Ramat Gan, Israel; 2Liver Research Laboratory, Sheba Medical Center, Ramat Gan, Israel; 3Sackler School of Medicine, Tel Aviv University Tel Aviv, Israel; 4Liver and Cardiac Research Laboratory, Felsenstein Medical Research Center, Petah Tiqwa, Israel
Ischemia/reperfusion (I/R) injury is the main cause of both primary graft dysfunction and primary non-function of liver allografts. Delta-9-tetrahydrocannabinol (THC), a cannabinoid, is the active components of marijuana. Cannabinoids has been reported to attenuate myocardial, cerebral and hepatic I/R injury. To date, there are few reports concerning the use of a high dose THC (1-50mg/kg) administered before the induction of ischemic injury in vivo. In this study we examined the role of ultralow dose THC (0. 002mg/kg), injected 2h before I/R induction, in the protection of livers from I/R injury. C57BI Mice were studied in in vivo model of hepatic segmental (70%) ischemia for 60min followed by reperfusion for 3 or 6 hours. Results: THC administration significantly reduced serum liver enzymes level induced by I/R both after 3 and 6 hours of reperfusion compared with untreated I/R mice. Furthermore, THC administration inhibited the cleavage of the hepatic pro-apoptotic caspase-3 protein observed in the untreated mice. In addition, after 6 hours of reperfusion high levels of ERK phosphorylation and the up-regulation of the ERK targeted genes was detected in the livers of untreated mice compared with THC treated mice. Moreover, RNA samples from livers of untreated mice showed elevated levels of the pro-inflammatory NFkB target genes (IL-6, TNFα, MCP-1, IL-1β, IL-1 β, RelB and CIAP2) compared with THC treated mice. Histological findings disclosed significantly less hepatic injury in the THC treated I/R mice and fewer apoptotic hepatocytes cells were identified by morphological criteria compared with untreated mice. Conclusion: very low dose THC can reduce the apoptotic and inflammatory injury induced by hepatic I/R injury. THC may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation, liver resection and trauma.
Ziv Ben Ari - Advisory Committees or Review Panels: MSD, Jenssen, Boehringer Ingelheim, BMS
The following people have nothing to disclose: Eylon Lahat, Edith Hochhauser, Maya Sultan, Yosef Sarne, Mordechai Gutman, Michal Safran
Required levels of liver repopulation with wildtype hepatocytes for amelioration of hyperoxaluria in a mouse model of primary hyperoxaluria-1
Jianqiang Ding1, Xia Wang1, Chandan Guha2, Eduardo Salido3, Jayanta Roy-Chowdhury1, Namita Roy-Chowdhury1
1Departments of Medicine and Genetics, and Marion Bessin Liver Research Center, Albert Einstein College of Med, Bronx, NY; 2Departments of Radiation Oncology and Pathology, Albert Einstein College of Medicine, New York, NY; 3Unidad de Investigacion, Hospital Universitario de Canarias, La Laguna, Spain
BACKGROUND: Primary hyperoxaluria 1(PHI) is characterized by oxalate overproduction by hepatocytes due to mutations of Agxt-1 causing deficiency of alanine: glyoxylate aminotransferase (AGT) activity in hepatocyte peroxisomes. Increased oxalate excretion in urine causes urolithiasis, nephrocalcinosis, renal failure and plasma oxalate accumulation leading to multiorgan disease requiring liver and kidney transplantation. We are developing a hepatocyte transplantation-based therapy for PH1. Oxalate overproduction cannot be reversed simply by adding wildtype hepatocytes, but requires a significant level of replacement of the AGT-deficient host hepatocytes by AGT-competent donor hepatocytes. Here, using an Agxt1'/' mouse model of PHI, we have determined the proportion of AGT-competent hepatocytes that need to be present in the liver for ameliorating hyperoxaluria. METHODS: Agxt1 ٪ mice were subjected to preparative hepatic irradiation (50Gy) to reduce the proliferative capacity of the host hepatocytes. This was followed by transplantation of hepatocytes (106) obtained from congeneic LacZ-transgenic (Rosa26) donor mice, which have normal AGT activity. An adenovector expressing hepatocyte growth factor (10
The following people have nothing to disclose: Jianqiang Ding, Xia Wang, Chandan Guha, Eduardo Salido, Jayanta Roy-Chowdhury, Namita Roy-Chowdhury
Bone Marrow Mesenchymal Stem Cells Support the Regeneration of Transplantable Liver Graft Using Decellularized Whole Organ Scaffold
Yoshie Kadota1, Hiroshi Yagi1, Alejandro Soto-Gutierrez2, Kenta Inomata1, Taizo Hibi1, Yuta Abe1, Minoru Kitago1, Masahiro Shinoda1, Hideaki Obara1, Osamu Itano1, Yuko Kitagawa1; 1 Surgery, Keio University Tokyo, Japan; 2Surgery/ University of Pittsburgh, Pittsburgh, PA
Aim: To generate transplantable liver graft with better cell viability, we evaluated if co-perfusion/culture of hepatocytes and bone marrow mesenchymal stem cells (BM-MSCs) promotes the liver regeneration on decellularized liver scaffold. Methods: First, decellularization protocol was modified to optimize the concentration of enzyme and non-ionic detergent to completely remove the cellular components without destroying the liver's natural extracellular environment and vascular networks. Second, the acellular translucent liver scaffold was resected into hepatic lobes to have the best volume for the examined numbers and proportions of isolated rat hepatocytes and BM-MSCs, which were perfused sequentially via portal vein of the scaffold at the certain velocity to achieve maximum hepatocyte viability. The two different cell types were tracked to detect their locations in the scaffold at different time points by CellTracker Kit and immunofluorescent staining. They were evaluated by histological, biochemical and genetic analyses about the influence of co-perfusion/culture of BM-MSCs with hepatocytes. Results: No leakage was found only from the liver matrix which was decellularized with the optimal concentration of 0. 05% trypsin and 0. 1% Triton X-100, and the total of 50 X 106 hepatocytes and 10 X 106 BM-MSCs (20% of hepatocytes) were finely introduced into the median lobe scaffold at the speed of about 0. 5 ml/min, which reduced the shear stress and improved the viability of engrafted cells. The liver scaffold with BM-MSCs showed that clusters of well-integrated hepatocytes aligned as the original hepatic cords from portal vein to central vein and had good viability. In the portal area, a part of the cells expressed vascular specific growth factors as well as hepatic sinusoid markers, especially along with the decellularized vascular walls, where the CD90 positive and cell tracked-BMMSCs were repopulated. The graft in which BM-MSCs were co-perfused/cultured showed the less apoptosis of hepatocytes and well-maintained ALB/UREA syntheses as well as higher hepatic gene expressions. Conclusion: The modified protocol of decellularization and recellularization could provide well-preserved matrix structure and higher cell viability. In addition, BM-MSCs showed the potential to support the liver regeneration with progenitor characteristics and secretion of growth factors while interacted with hepatocytes and the liver-specific three dimensional matrix structures. The effective approach for the generation of transplantable liver graft with the optimized combination of decellularized scaffold and hepatocytes with BMMSCs was shown.
The following people have nothing to disclose: Yoshie Kadota, Hiroshi Yagi, Alejandro Soto-Gutierrez, Kenta Inomata, Taizo Hibi, Yuta Abe, Minoru Kitago, Masahiro Shinoda, Hideaki Obara, Osamu Itano, Yuko Kitagawa
Hepatocyte transplantation combined with partial liver resection preconditioning in patients with Crigler-Najjar type I
Carl Jorns1, Antal Nemeth3, Greg Nowak1, Helen Zemack1, LisaMari MÖrk1, Helen Johansson1, Roberto Gramignoli2, BjÖrn Fischler3, Stephen Strom2, Ewa C S. Ellis1, Bo-GÖran Ericzon1
1Transplantation Surgery, Karolinska Institute, Stockholm, Sweden; 2Pathology Karolinska Institute, Stockholm, Sweden; 3Pediatrics, Karolinska Institute, Stockholm, Sweden
CN type I is an autosomal recessive condition due to the deficiency of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). Patients are throughout their lifespan at risk of fatal brain injury due to unconjugated hyperbilirubinemia. Treatment consists of lifelong daily phototherapy of up to 14h per day. Most patients undergo orthotopic liver transplantation as phototherapy becomes less effective after puberty and constitutes a significant impairment in quality of life. We evaluated a 13year-old boy and an 11-year-old girl with CN syndrome type I at our center. Phototherapy of 8 −14h was required to maintain serum bilirubin at 390 to 450 μmol per liter. Patients were accepted to the waiting list for hepatocyte transplantation after ethical committee approval and informed consent. Hepatocytes were isolated under good manufacturing practices from liver tissue obtained from deceased organ donors not accepted for whole organ transplantation or from split or size reduced liver transplantations. Immediately before hepatocyte infusion liver resection of segments 2 and 3 was performed to induce liver regeneration and proliferation of transplanted hepatocytes. Fresh ABO compatible hepatocytes were infused by a portal catheter. Immunosuppression consisted of basiliximab, tacrolimus and steroids. The girl received 5. 3 x 109 viable hepatocytes at one transplantation event and the boy received two infusions from two different donors three months apart with 2.2 and 9 x 109 viable hepatocytes. Serum bilirubin levels increased initially in both patients after the first procedure up to 530 μmol per liter. Thereafter serum bilirubin decreased continuously to 50% of pre-transplant levels for more than 6 months. The boy experienced a sudden increase of serum bilirubin to pre-transplant levels 6 months after the first infusion associated with a scabies infection. Despite intensified phototherapy serum bilirubin did not improve. Due to the risk of encephalopathy we decided together with the family to list him for orthotopic liver transplantation. The girl still remains on significantly decreased serum bilirubin levels and is on the waiting list for further hepatocyte infusions. This case report confirms that hepatocyte transplantation can be a useful treatment for patients with Crigler-Najjar syndrome type I. Pre-conditioning patients with partial hepatectomy prior to cell transplantation is safe. Additional patients will need to be evaluated before conclusions can be drawn on the efficacy of this procedure as compared to traditional hepatocyte transplantation.
Stephen Strom - Stock Shareholder: Stemnion, Yecuris
The following people have nothing to disclose: Carl Jorns, Antal Nemeth, Greg Nowak, Helen Zemack, Lisa-Mari Mörk, Helen Johansson, Roberto Gramignoli, Björn Fischler, Ewa C S. Ellis, Bo-Göran Ericzon
Immuno-modulating effects of Silibinin in liver transplant patients with HCV recurrence
Antonino Castellaneta1, Antonio Massaro1, Maria Rendina1, Nicola Maurizio Castellaneta1, Marianna Zappimbulso1, Francesca Derrico1, Nadia Brambilla2, Giampaolo Giacovelli2, Lucio Rovati2, Massimo D'Amato2, Alfredo Di Leo1
1University of Bari, Bari, Italy; 2Rottapharm, Milan, Italy
Background and Aim: Silibinin (Sil) has been proven to have anti-viral activity in humans and it has been successfully used in our center in a randomized, double-blind, placebo-controlled study for HCV recurrence treatment in liver transplant patients. Sil has also immune-modulating properties by modulating dendritic cells (DC) function. DC are antigen-presentig cells playing, along with regulatory T cells (Treg), a pivotal role in controlling allo-immune response, as well as HCV infection. Immune regulatory molecules expressed by DCs, including PDL1(B7 homologue-1=programmed death ligand-1), ICOS-L, CD39 and the non-classical HLA class I molecule HLA-G and the immunoglobulin-like transcript(ILT)4, have been shown to regulate T cell responses, including the induction of Treg. The PD-1/PD-L1 pathway on Treg is described as one of the mechanisms responsible for balancing HCV T cell responses. So far, the enumeration of DCs and Tregs and the expression of immune regulatory molecules on DC and PD-1 on Treg have not been examined in HCV recurrence and Sil treatment after liver transplant. Aim of the study is to analyze circulating DC subsets and Treg and the expression of costimulatory/coregulatory molecules in liver transplant patients receiving Sil. Material and methods: 15 liver transplant patients with HCV recurrence received iv infusion of Sil (20 mg/kg/day) for 14 consecutive days. We examined by flow cytometry, before and at the end of treatment, the expression of CD86, PD-L1, ICOSL, CD39, HLA-DR, HLA-G, IL-T4 in circulating monocytoid(m) and plasmacytoid(p) DC and of PD-1 on Treg. Results: after Sil treatment we observed a higher pDC/mDC ratio (0. 5±0. 2 vs 0. 7±0. 3, p<0. 5) and pDC exhibited a lower HLA-DR (MFI: 1673±525 vs 1523±531, p<0. 3) and a higher IL-T4 (MFI: 2303±632 vs 2743±718, p<0. 4), CD39 (MFI: 69. 4±7. 6 vs 74. 0±10. 6, p<0. 5; %: 16. 2±8. 7 vs 22. 1 ±9. 4, p<0. 5) and HLAG (MFI: 26. 2±8. 1 vs 36. 1 ±8. 6, p<0. 4) expression as compared with the baseline. No correlation was found between these markers and HCV viral load. In addition, after Sil treatment mDC show a higher ICOSL (MFI: 29. 5±12. 6 vs 36. 2±7. 2, p<0. 4) expression that was inversely correlated to viral load. No changes were detected in Treg frequency and PD-1 expression. Conclusions: this is the first study in liver transplant patients with HCV recurrence showing the impact of Sil on DC and Treg. Findings show changes, not correlated with viral load, in circulating pDC that have previously been associated with tolerogenic conditions, providing new insight into how Sil might regulate allo-immunity. Additional in vitro functional studies are warranted to further explore the tolerogenic potential of Sil.
Antonino Castellaneta - Grant/Research Support: Rottapharm
Nadia Brambilla - Employment: Rottapharm
Giampaolo Giacovelli - Employment: Rottapharm
Lucio Rovati - Employment: Rottapharm S. p. A.
Massimo D'Amato - Employment: Rottapharm
The following people have nothing to disclose: Antonio Massaro, Maria Rendina, Nicola Maurizio Castellaneta, Marianna Zappimbulso, Francesca Derrico, Alfredo Di Leo
Quantification of liver of liver perfusion is a sensitive approach to non-invasive evaluation of donor liver viability
Maria-Louisa Izamis1 , 2, Christina Keravnou1, Damianos Christofides1, Michalakis A. Averkiou1 , 3
1University of Cyprus, Nicosia, Cyprus; 2Massachusetts General Hospital, Boston, MA; 3University of Washington, Seattle, WA
In the United States, less than a third of the 30, 000 patients with liver failure will receive a transplant this year. Machine perfusion is an investigational tool that can expand the donor pool by improving and quantifying liver viability, essential for the safe recovery of discarded livers. We have demonstrated that perfusate biochemical markers and liver biopsies provide highly sensitive indicators of viability; however, they only reflect an average overview of organ performance or focal information, respectively. To test whether greater measurement specificity can be achieved across the entire organ, we introduced dynamic contrast-enhanced ultrasound (DCEUS) for real-time, non-invasive, non-ionizing visualization of liver anatomy and perfusion. Here we use DCEUS to describe trends in perfusion as a function of ischemic severity, perfusion time, and treatment choice. A bolus of contrast passing through either the portal vein or hepatic artery was quantified with parameters such as wash-in time, time to peak intensity, and mean transit time. We observed that as exposure to warm ischemia in nonheparinized porcine livers (a model of uncontrolled cardiac death) increased from 30-60-90 minutes, the measured parameters differed significantly between groups and tended towards normal (0 minutes warm ischemia) at a dose-dependent rate. The microcirculation improved over time with visible vasodilation and lysis of blood clots with streptokinase and sonothrombolysis. Bile production correlated with the extent of arterial perfusion while resistance to flow was indicative of the extent of portal perfusion. In conclusion, DCEUS is a simple technique for visualizing the liver's anatomy and quantifying perfusion quality, in addition to providing novel diagnostic and prognostic metrics of viability. DCEUS placed online biochemical and hemodynamic measurements, otherwise non-specific markers of liver function, in context such that appropriate treatment could be provided in real-time. DCEUS also served as a treatment modality itself, overall enhancing the role of machine perfusion as a platform for organ-tailored optimization.
Maria-Louisa Izamis - Patent Held/Filed: Cell, Tissue and Organ Resource Core
The following people have nothing to disclose: Christina Keravnou, Damianos Christofides, Michalakis A. Averkiou
Hepatocyte producing plasminogen activator-1 (PAI-1) regulates adhesion formation after hepatectomy in a murine model and in patients undergoing hepatectomy
Koichiro Ohashi1, Tomohiro Yoshimoto2, Hisashi Kosaka1, Tadamichi Hirano1, Yuji Iimuro1, Shuhei Nishiguchi4, Kenji Nakanishi3, Jiro Fujimoto1
1Surgery, Hyogo College of Medicine, Nishinomiya, Japan; 2Laboratory of Allergic Diseases, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; 3Immunology and Medical Zoology Hyogo College of Medicine, Nishinomiya, Japan; 4Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan
Background/Aim: Although abdominal adhesion after hepatectomy is a serious problem, it has not been extensively studied. The aim of this study was to elucidate the molecular mechanisms underlying adhesion formation after hepatectomy in a murine model and in patients undergoing hepatectomy. Methods: We performed partial hepatectomy of left lobe of the using bipolar forceps to develop an experimental mouse model of abdominal adhesions. Moreover, we used cecal cauterization abdominal adhesion model to examine the role of liver for intestinal adhesion. Adhesion was estimated by a standard scoring system; score 0, no adhesion to score 5, very thick vascularized adhesion. Antibodies to CD4 and interferon- ۷(IFN-۷), IFN-۷ KO, natural killer T (NKT) cell-deficient, and PAI-1 KO mice were used. Recombinant hepatocyte growth factor (HGF) was tested for its potential preventive effect on adhesions. Liver specimens were obtained during surgery from patients undergoing hepatectomy. IFN-۷, tissue-PA (tPA) and PAI-1 were measured and fluorescence immuno-staining was performed. Results: Adhesion formation depended on IFN-۷, and NKT KO mice only developed a few adhesions. Adhesion was completely inhibited in PAI-1 KO mice. PAI-1 was increased in the liver after hepatectomy, followed by diminution of tPA in wild-type mice. Interestingly, PAI-1 was overexpressed not only in the remnant left lobe of the liver but also in the right lobe which has not been injured. Moreover, PAI-1 was also increased in the liver after cecal cauterization. HGF strongly inhibited abdominal adhesion after hepatectomy by reducing IFN-y and PAI-1 and increasing tPA. In liver specimens obtained from patients, NKT cells had accumulated in the liver after hepatectomy, and mRNA of PAI-1 was significantly increased in the liver specimens. Histological analysis revealed that PAI-1 was markedly stained in hepatocyte in the liver specimens. Conclusion: IFN-y plays important role for abdominal adhesion formation after hepatectomy, acting via the reciprocal balance of PAI-1 and tPA. PAI-1 was produced by hepatocyte, and PAI-1 was increased in any portion of liver after hepatectomy. PAI-1 upregulation in the liver was also noted in intestinal adhesion model. This molecular mechanism also regulates adhesion formation in patients following hepatectomy. These results indicate that the IFN-۷ and PAI-1 are possible therapeutic targets and HGF could prevent postoperative adhesion formation after hepatectomy.
The following people have nothing to disclose: Koichiro Ohashi, Tomohiro Yoshimoto, Hisashi Kosaka, Tadamichi Hirano, Yuji Iimuro, Shuhei Nishiguchi, Kenji Nakanishi, Jiro Fujimoto
Transplantation of cell sheets made from hepatic differentiated human mesenchymal stem cells by a small compound inhibitor of Wnt/beta-catenin signal ameliorates acute liver failure in mice
Noriko Itaba, Yoshiaki Matsumi, Kaori Okinaka, Yohei Kono, Goshi Shiota
Department of Genetic Medicine and Regenerative Therapeutics, Tottori University, Yonago, Japan
Aims: Human mesenchymal stem cells (hMSCs) have regenerative potential by producing trophic factors as well as hepatic differentiation capacity, and cell-based therapies utilizing hMSCs are expected to be an alternative treatment for liver transplantation. For future clinical applications, we focused on Wnt/beta-catenin signal inhibitors since suppression of Wnt/beta-catenin signaling by siRNA enhances hepatic differentiation of hMSCs. In this study, we screened 10 small compounds inhibiting Wnt/beta-catenin signal as candidate compounds driving hMSCs to transdifferentiate into functional hepatocytes, and examined whether cell sheets made from hMSCs by Wnt/beta -catenin signal inhibitors can ameliorate acute liver failure in mice. Methods: First, the effects of Wnt/beta-catenin signal-inhibiting small compounds on TCF4/beta-catenin transcriptional activities were screened by reporter assay in UE7T-13 hMSC cells. Differentiation capacities were assessed by RT-PCR analysis and functional assays. Cell sheets were fabricated by differentiation procedure on temperature-responsive polymer-grafted culture dishes and then transplanted into NOD/SCID mice. One, two, and three layered cell sheets were transplanted onto two sites of liver surface in group 1, 2 and 3, respectively and sham operated mice in group 4 were compared as controls. All mice were administrated carbon tetrachloride on day 1. Liver function tests were performed on day 2, 4 and 8, and mice were followed up to day 8. Results: Hexachlorophene potently inhibited TCF4/betacatenin transcriptional activity and enhanced hepatocyte-specific gene expressions, such as albumin, C3, C4, and APOE. Glycogen storage and urea synthesis were also induced by hexachlorophene. Transplantation of hexachlorophene-induced hepatic cell sheets resulted in significant reduction of serum aminotransferases in group 3, 2, 1 in this order, compared to group 4 on day 4 (P<0. 01, each). Total bilirubin on day 2 was also decreased in group 3, 2 and 1 in this order (P<0. 01, each). Furthermore, survival rate was remarkably improved in group 2 and 3 (P<0. 05). Mitotic and Ki 67-labelled hepatocytes were significantly increased in cell sheets-transplanted mice. RT-PCR analysis showed several human-specific humoral factors such as SCF, HGF, APOE, and C3 were expressed in the graft tissues. Conclusions: Hexachlorophene is a potent inducer of hepatic differentiation of hMSCs. Transplantation of cell sheets manipulated by hexachlorophene promotes liver regeneration by producing trophic factors including liver-specific serum proteins.
The following people have nothing to disclose: Noriko Itaba, Yoshiaki Matsumi, Kaori Okinaka, Yohei Kono, Goshi Shiota
Partial hepatectomy accelerates the progression of nonalcoholic fatty liver disease in mice
Golnar Karimian, Marc Kirschbaum, Susanne Veldhuis, Robert J. Porte, Ton Lisman
Hepatobiliary Surgery and Liver Transplantation, University of Groningen, University Medical Center Groningen, Groningen, Netherlands
Background and Aim: Hepatic steatosis is the main feature of non-alcoholic fatty liver disease (NAFLD). Severe steatosis and progression to non-alcoholic steatohepatitis (NASH) results in hepatocyte damage and liver dysfunction. Factors involved in progression of simple steatosis to NAFLD and NASH are incompletely understood, but likely involve a ‘multiple hit' mechanism. As the number of individuals with mild to moderate liver steatosis is increasing, the number of patients with steatosis that require a partial hepatectomy for malignant disease is increasing. We hypothesized that partial hepatectomy would affect the progression of steatotic liver disease and have investigated the effect of partial hepatectomy on liver regeneration and the progression of the NAFLD status in mice with mild steatosis. Methods: C57BL/6JolaHsd mice were fed a choline deficient L-amino acid defined diet (CD-AA) for a maximum of 3 weeks. Mice fed a choline sufficient L-amino acid defined diet (CS-AA) were used as controls. Two weeks after the start of the diet, mice underwent partial hepatectomy or a sham operation.
Mice were sacrificed at several time points after the operation and blood and liver samples were taken for analysis. Results: The CD-AA diet induced mild hepatic steatosis by 3 weeks as demonstrated by histological examination and an elevated NAFLD activity score (1. 8 ± 0. 7) in the sham group. Mice in the CD-AA sham group had significantly higher basal levels of aminotransferases in plasma compared to the CS-AA group by 3 weeks (P <0. 05). After partial hepatectomy, aminotransferase levels in plasma increased significantly (p <0. 05) in both CDAA and CS-AA groups over a 2-hour period but returned to basal levels over time. Liver mass restoration over time was not different between the CD-AA and CS-AA groups. Interestingly, in the CD-AA group NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3. 7 ± 1. 3 vs. 1. 8 ± 0. 7; P<0. 05). In addition, malondialdehyde (MDA) levels in liver tissue of the CD-AA but not of the CS-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham mice (P <0. 05). Conclusion: Mild liver steatosis does not impair liver regeneration. However, partial hepatectomy does substantially accelerate the progression of NAFLD, which may have clinical consequences for humans with steatosis that require partial hepatectomy.
The following people have nothing to disclose: Golnar Karimian, Marc Kirschbaum, Susanne Veldhuis, Robert J. Porte, Ton Lisman
Hypothermic perfusion improves recovery of isolated hepatocytes from ischemia-damaged rat and steatotic human livers
Mark G. Clemens2, Cathy Culberson2, Joshua D. Wheaton1, Steven Hoynowski3, John W Ludlow3, Charles Lee2
1University of North Carolina at Charlotte, Charlotte, NC; 2HepatoSys Inc, Charlotte, NC; 3ZenBio, Research Triangle Park, NC
Primary human hepatocytes remain the gold standard for testing liver metabolism and toxicity. However, with increased use of marginal livers for therapeutic transplant, availability of livers that yield high quality hepatocytes is becoming limited. We have developed a hypothermic machine perfusion (HMP) process that restores function to ischemia-damaged livers leading to improved survival of transplants in a rat model. We therefore tested if this system could improve isolation of hepatocytes from marginal donors. In rat studies, livers (n=6/group) were subjected to 120 min warm ischemia (WI) followed by either 24 hr simple cold storage (SCS) or 24 hr SCS + 5 hr perfusion with a recovery solution (HMP). Hepatocytes were then isolated by collagenase digestion. HMP improved yield by 50% and improved viability from 66. 6% to 86. 5% (p<0. 05). Isolated cells were plated on collagen coated plates. Plateability of the cells was improved by HMP from 38. 5% to 72. 2% vs. SCS. Function of the cells was tested by ethoxycoumarin O-deethylase (ECOD) activity and urea production. HMP cells showed improved both phase I and phase II ECOD activity (90 vs 51 pmole/106 cells/min) for phase II, HMP vs SCS, p<0. 05). Urea production by HMP cells was also more than double that of SCS (p<0. 05). These results suggested that HMP provides improved yield, viability and function of hepatocytes isolated from ischemia damaged rat livers. We then tested the procedure in a series of three human livers. Livers not accepted for transplant were obtained from an OPO with cold storage times of 16-24 hrs. They were divided into two segments with one digested immediately and the other placed on HMP for 3 hrs and then digested. On a grading scale in which a score of <6 is acceptable for cell isolation, the liver scores were 5, 10 and 15. With a score of 5, HMP and SCS showed similar yield and viability but HMP cells had a 40% greater attachment after cryopreservation. The second liver (score of 10) was steatotic and 20 min WI. HMP improved yield (6 x 108 vs 1. 4 x 108 cells) and viability (73 vs 57%). HMP also improved ECOD activity after cryopreservation (233 vs 77. 5 pmol/106 cells/min). The third liver had a WI of 60 min and >50% steatosis. SCS yielded no viable cells while HMP yielded 4. 3x108 cells from 500 g liver. Although plating efficiency was low after cryopreservation (10%) additional storage of cells in HMP solution increased it to 24%. The results demonstrate that HMP after the SCS process can improve cell isolation from both rat and human DCD livers. Also, additional hypothermic storage in the HMP solution improved viability and plating of human hepatocytes.
Mark G. Clemens - Management Position: HepatoSys Inc; Stock Shareholder: HepatoSys Inc
John W. Ludlow - Consulting: Zen Bio Inc.
Charles Lee - Management Position: HepatoSys Inc.
The following people have nothing to disclose: Cathy Culberson, Joshua D. Wheaton, Steven Hoynowski
Micro cell-compatible hydrogels, allow for long term hepatocytes engraftment after intravascular transplantation
Julie Carmel1, Omri Nayshool2, Tarek Saadi1, Arie Arish3, Zakhar Bramnik3, Uri Kaplan4, Iris Mironi-Harpaz5, Dror Seliktar5, Yaacov Baruch 1 , 2
1Liver Unit, Rambam - Health Care Campus, Haifa, Israel; 2Bruce Rappaport Faculty of Medicine, Technion- Israel Institute of Technology, Haifa, Israel; 3Department of Surgery B, Rambam - Health Care Campus, Haifa, Israel; 4Department of Surgery, Haemek Medical Center, Afula, Israel; 5Department of Biomedical Engineering, Technion- Israel Institute of Technology, Haifa, Israel
Introduction: Hydrogel cell construct made of fibrinogen (Fib) crosslinked with poly ethylene glycol (PEG) and diacrylates side chains, form a hydrogel when exposed to UV light. After intravascular injection, the hydrogel cell construct may retain transplanted cells in the portal radicles space, protecting them from shear stress and immediate immunological pressure and thus may improve engraftment. Aims: Long term (up to 3 weeks) in vivo engraftment assessment of intraportal transplantation of micro hydrogel constructs with adult parenchymal cells. Methods: Evaluation of engraftment efficiency in rat models, SD or F344 DPPIV(-) rats after partial 34% hepatectomy (PHP) or CCL4 acute intoxication. 6X1-06 cells transplanted as free cells or as cell hydrogel constructs (200-700 μm) intraportaly. The engraftment efficiency was evaluated using real time qPCR for Y chromosome, histochemistry and histology. We also studied the durability of the microcapsules after transplantation into the spleen and its effect on cell departure to the liver, in the DPPIV(-) model. Results: Both in the liver and in the spleen, cell constructs were present up to 3 days. Survival of transplanted encapsulated cells was much better over 21 days compared to isolated cell transplantation (2. 8±0. 4% vs. 54. 6±5% P<0. 01). Groups of transplanted cells were seen in the CCL4 F344 DPPIV(-) rats model, immediately after injection within the microcapsules, and later as groups close to the portal veins. The number of cells leaving the spleen to the liver was lower when cells were transplanted within microcapsules. Conclusions: Long term survival and engraftment of intravascular transplanted adult hepatocytes is much better in within hydrogel cell micro construct. The presence of cells grouped at the portal radicles support our concept that cells engraft through the portal radical and not the sinusoids, and the polymers enhance this effect.
Yaacov Baruch - Consulting: Coeruleus Ltd, MSD
The following people have nothing to disclose: Julie Carmel, Omri Nayshool, Tarek Saadi, Arie Arish, Zakhar Bramnik, Uri Kaplan, Iris Mironi-Harpaz, Dror Seliktar
Ex vivo cultured human CD34+ cells promotes hepatic regeneration in chronically injured rat liver
Toru Nakamura1,2,Takuji Torimura2, Hiroshi Masuda1, 2, Hideki Iwamoto1,2, Hironori Koga1, 2, Mitsuhiko Abe1, 2, Yu Ikezono1, 2, Osamu Hashimoto1, 2, Takato Ueno3, Michio Sata1
1Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan; 2Liver Cancer Division, Research Center for Innovative Cancer Therapy, Kurume University, Kurume, Japan; 3Asakura Medical Association Hospital, Asakura, Japan
Background: In 61th AAASL meeting, we demonstrated that the transplantation of human steady state peripheral CD34+ cells into an immunodeficient rat liver fibrosis model reduced liver fibrosis by suppressing activated hepatic stellate cells and increasing MMP activity, and led to hepatic regeneration. Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with decompensated liver cirrhosis. The aim of this study was to investigate the efficacy of cell transplantation therapy with ex vivo expanded human CD34+ cells for carbon tetrachloride (CCl4)-induced liver fibrosis model. Methods: Human granulocyte-colony stimulation factor-mobilized peripheral CD34+ cells of patients with liver cirrhosis were isolated by magnetic cell sorting system. Recipient nude rats were injected i. p. with CCl4 twice weekly for 3 weeks before initial treatment. Then, saline, 5x104, 2x105, or 1x106 freshly isolated (no expansion) and expanded CD34+ cells/kg body weight were transplanted via spleen, respectively. The administration of CCl4 was continued for three more weeks until the rats were sacrificed. Examination items were as follows. 1)FACS, real-time PCR and gene array analysis of freshly isolated and expanded CD34+ cells, 2) morphometry of fibrotic areas in Azan-Mallory stained liver, 3) immunohistochemistry using anti-α-smooth muscle actin (SMA), CD31, keratin19, albumin and PCNA antibodies, and 4) the expression of metalloproteinase and tissue inhibitor of metalloproteinase-1 by gelatin zymography and real-time PCR. Results: For 7 days in culture, CD34+ cells were effectively expanded to 8-fold. Increased expression of VE-cadherin, KDR and Tie2 was determined by FACS analysis. The expression of VEGF, transforming growth factor-α, fibroblast growth factor-2, endothelial nitric oxide synthase and angiopoietin-2 in expanded CD34+ cells was increased compared with that in freshly isolated CD34+ cells. Gene array analysis showed that the most up-regulated gene in expanded CD34+ cells compared with freshly isolated CD34+ cells was integrin-3β. Expanded CD34+ cell transplantation reduced liver fibrosis with the decrease of αSMA positive cells. The transplanted cells differentiated into CD31+ and smooth myosin heavy chain-1+ cells. The transplantation of expanded CD34+ cells significantly up-regulated the number of PCNA positive hepatocyte compared with the transplantation of freshly isolated CD34+ in 3 different groups of cell number, respectively. Conclusion: These observations suggest that ex vivo expanded CD34+ cell transplantation may become a promising therapeutic strategy for patients with decompensated liver cirrhosis.
Michio Sata - Speaking and Teaching: MSD K. K., Chugai Pharmaceutical Co.,
The following people have nothing to disclose: Toru Nakamura, Takuji Torimura, Hiroshi Masuda, Hideki Iwamoto, Hironori Koga, Mitsuhiko Abe, Yu Ikezono, Osamu Hashimoto, Takato Ueno
Longitudinal non-invasive in vivo imaging of transplanted hepatocytes using the sodium iodide symporter gene
Raymond D. Hickey Shennen A. Mao, Jaime Glorioso, Bruce Amiot, Stephen J. Russell, Scott L. Nyberg
Mayo Clinic, Rochester, MN
Hepatocyte transplantation is a potential treatment for a myriad of liver disorders that are currently only curable by liver transplantation. A major limiting factor of hepatocyte transplantation is an inability to non-invasively and longitudinally monitor engraftment and expansion of transplanted cells. We hypothesized that the sodium iodide symporter (NIS) gene could be used to visualize transplanted hepatocytes and set out to test this reporter system in a rodent model of liver repopulation. FAH+ C57BI/6J mouse hepatocytes were transduced ex vivo using a lentiviral vector containing the mouse Slc5a5 (NIS) gene under the control of a liver specific promoter. Transduction efficiencies of 70-80% were achieved and NIS-labeled cells could robustly concentrate radiolabeled iodine in vitro. Next, NIS-labeled hepatocytes were transplanted into congenic fumarylacetoacetate hydrolase knockout (Fah-KO) mice. NTBC was removed from the diet to stimulate a selective repopulating advantage for FAH+ donor cells. NIS-labeled hepatocytes were readily imaged in vivo non-invasively by single-photon emission computed tomography (SPECT) imaging. We observed a temporal increase in radiolabeled tracer in the liver correlating with an increase in hepatocyte repopulation after intra-splenic injection of cells. Additionally, NIS-imaging was able to specifically identify the extrahepatic biodistribution of transplanted hepatocytes in Fah-KO mice after intra-peritoneal injection. This work is the first to demonstrate the efficacy of NIS-labeling in the field of hepatology. We anticipate that NIS-labeling of cells has broad application as a tool for monitoring engraftment and expansion of transplanted cells in various cell-based therapies for liver disorders, not only in small animals, but in larger preclinical models also.
Stephen J. Russell - Board Membership: Imanis Life Sciences LLC; Management Position: Imanis Life Sciences LLC; Stock Shareholder: Imanis Life Sciences LLC
The following people have nothing to disclose: Raymond D. Hickey, Shennen A. Mao, Jaime Glorioso, Bruce Amiot, Scott L. Nyberg
Ischemic postconditioning (IPostC): a new strategy to protect the liver against ischemia-reperfusion injury
Julia Schewe, Lisa Selzner, Elisabeth-Ingrid Liss, Christian J. Steib, Alexander L. Gerbes
Department of Medicine II, University of Munich, Campus Grosshadern, Munich, Germany
Introduction: The protective effect of ischemic postconditioning (IPostC) after transplantation has been shown in heart diseases; up to now only little data exist for the liver. The aim of the current study is to investigate the effect of IPostC in healthy and fatty livers following 24hours of cold ischemia. Methods: Male SD rats received a high-fat-diet (70% energy from fat) for four weeks to induce a fatty liver compared to controls fed with conventional breeding diet (10% energy from fat). The livers were examined histologically using HE staining. Isolated liver perfusion was performed: stabilization period of 30min. followed by 24h of cold ischemia at 4°C and reperfusion for 120min. at 37°C. In healthy and fatty livers the following three groups (each n=8) were investigated. Group 1: 120min. reperfusion; group 2: IPostC 8x20sec. at start of reperfusion; group 3: IPostC 4x60sec. at start of reperfusion. To display the cell damage lactate dehydrogenase (LDH) in the perfusate and bile flow were measured (mean ± SEM; *p<0. 05). Statistical analysis of the data was performed using Students t-test. Results: Fatty livers showed histologically mild inflammation (grade 2), individual periportal necrosis and a moderate to severe fat storage. Cell damage was reduced by IPostC (LDH-efflux [all results mU/min x g liver] healthy liver group 1: 8223 ± 807 vs. group 2: 4420 ± 661* vs. group 3: 5290 ± 509*; fatty liver group 1: 9771 ± 545 vs. group 2: 7516 ± 926* vs. group 3: 7466 ± 588*) and bile flow increased (bile flow [all results ml/min x g liver] healthy liver group 1: 3, 97 ± 0, 93 vs. group 2: 5, 39 ± 0, 58 vs. group 3: 6, 51 ± 0, 83*; fatty liver group 1: 2, 14 ± 0.53 vs. group 2: 4, 21 ± 0, 86* vs. group 3: 4, 39 ± 0, 76*). Conclusion: IPostC has a protective effect on healthy and fatty livers and could significantly improve graft function after liver transplantation. It was also shown, that a few long cycles (4x60sec.) have similar protective effects as many short cycles (8x20sec.), which appears more feasible in practice and should be tested in the clinical situation.
The following people have nothing to disclose: Julia Schewe, Lisa Selzner, Elisabeth-Ingrid Liss, Christian J. Steib, Alexander L. Gerbes
Hepatic stellate cells improve survival and engraftment of hepatocytes after transplantation
Ange-Clarisse Dusabineza1, Noemi Van Hul1, Vanessa Legry1, Dung N. Khuu2, Etienne M. Sokal2, Leo A. van Grunsven3, Mustapha Najimi2, Isabelle A. Leclercq1
1laboratory of hepatogastroenterology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium; 2Laboratory of Pediatric Hepatology & Cell Therapy, Université catholique de Louvain, Brussels, Belgium; 3Liver cell biology Lab, Department of Cell Biology, Vrije Universiteit Brussel, Brussels, Belgium
Background: Human primary hepatocytes are used for liver cell therapy. However, only a small fraction of infused cells do engraft, limiting the benefit of cell transplantation. Aim: we tested whether co-transplantation of hepatocytes with hepatic stellate cells (HSC) could improve hepatocyte attachment in vitro or engraftment in vivo. Method: Human primary hepatocytes and HSC were isolated from healthy liver donors and from explanted livers with single metabolic defects. Hepatocytes were co-cultured with or without HSC (quiescent, after culture activation or immortalized LX2 cells), directly or in a transwell system (20: 1α hepatocytes: HSC ratio). Cell attachment was evaluated 24h after seeding. SCID mice were transplanted with hepatocytes alone or with HSC or LX2 (20: 1 hepatocytes: HSC ratio), and sacrificed 6h or 4w later. By immunostaining, we assessed human hepatocyte engraftment (anti-human albumin, alb) differentiation (anti-ornithine transcarbamylase, OTC), polarity (anti-CD1 0), and proliferation (BrdU incorporation). Anti-aSMA and Sirius red staining were used to highlight HSC and extracellular matrix (ECM) deposition. Results: Co-culture with HSC improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSC. Four weeks after transplantation to SCID mice, human alb+ hepatocytes were found scattered, occupying 0. 66% of the tissue section. By contrast, when human hepatocytes were transplanted in a mixture with HSC or LX2 cells, they formed clusters and were more numerous (1. 17±0. 59% or 3. 89±2. 56 (p=0. 05), respectively). Analysis of human alb mRNA expression in transplanted livers confirmed those results. The presence of HSC ameliorated the number of hepatocytes entrapped in the host liver at the early time point post-transplantation but not their in situ proliferation, as the cumulative incorporation of BrdU during 4 weeks in engrafted hepatocytes was similar whether transplanted alone or together with HSC. Engrafted hepatocytes co-expressed human alb/OTC and formed CD10+ hybrid canaliculi with adjacent endogenous mouse hepatocytes. Importantly, 4w post-transplantation, we found no accumulation of αSMA+ cells, ECM deposition or mRNA expression of human MMP9, αSMA or collagen α1 genes. Conclusion: In vitro, HSC improve the attachment and survival of hepatocytes. This effect is mediated by HSC-derived soluble factors as well as by direct contact between hepatocytes and HSC or the matrix they produce. In vivo, co-transplantation of HSC significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing without generating fibrosis.
Etienne M. Sokal - Board Membership: Promethera Biosciences; Management Position: Promethera Biosciences; Patent Held/Filed: Promethera Biosciences
Mustapha Najimi - Consulting: Promethera Biosciences; Stock Shareholder: Promethera Biosciences
The following people have nothing to disclose: Ange-Clarisse Dusabineza, Noemi Van Hul, Vanessa Legry, Dung N. Khuu, Leo A. van Grunsven, Isabelle A. Leclercq
Decreased TRAIL expression is a dominant feature of human HCC
Katja Straub, Khaleda Khairzada, Guido Gerken, Kerstin Herzer
Gastroenterology and Hepatology, Universityhospital Essen, Essen, Germany
Background: The proapoptotic molecule TRAIL has gained attention for its ability to induce apoptosis in liver cancer cells without damaging normal liver cells. It may play an important role in preventing development and outgrowth of liver tumors. HCC is recognized as one of the most common and most malignant cancers worldwide. However, the molecular mechanisms causing the sequence of events of its development are still poorly understood. To clarify the clinical implication of TRAIL for HCC development, the expression of TRAIL was analysed in a large series of human HCCs. Methods: 70 patients undergoing partial liver resection or LTx because of HCC were included. HCC probes and surrounding non-tumorous liver tissue was macrodissected and analysed for expression of TRAIL by qrtPCR. The same was done in several hepatoma cell lines. TRAIL expression was correlated with ethiology of liver disease, tumor spread and AFP levels before surgery. Liver tissue with fibrosis or cirrhosis not developing HCC as well as benign liver tumors were used as control. Results: Expression of the TRAIL mRNA was reduced or abolished in 60% of all tumors compared to surrounding non-tumorous liver tissue. Seperated by ethiology of liver disease, decreased levels of TRAIL correlate with Hepatitis C virus infection in 2/3 of cases. As well, female patients with NASH display decreased TRAIL expression in 80% of all NASH-based tumors. Intrestingly, cirrhotic livers with background of autoimmune disorders of the liver (AIH, PBC, PSC) do rarely show HCC development in general and corresponding liver tissue rather shows normal or increased TRAIL levels on the cell surface. Low TRAIL-expression levels do not significantly correlate with higher AFP levels. Conclusions: Our results suggest, that TRAIL protein loss goes in line with HCC development. Predominately Hepatitis C virus-induced mechanisms result in liver tumor development, as well as alimentary liver disorders. Loss of TRAIL expression seems to influence aggressiveness of tumor growth. Furthermore, TRAIL expression is not compromised in those liver diseases rarely developing HCCs such as autoimmune liver diseases. Thus, a decrease in TRAIL expression may significantly contribute in HCC development and growth.
The following people have nothing to disclose: Katja Straub, Khaleda Khairzada, Guido Gerken, Kerstin Herzer
Drastic partial hepatectomy outcomes in A20 heterozygous mice can be overcome by dietary manipulation: A lesson for the clinic
Cleide G. Da Silva, Peter Studer, Eva Csizmadia, Darlan C. Minussi, Kathleen Daniels, Christiane Ferran
Surgery, Beth Israel Deaconess Medical Center, Boston, MA
Increased free fatty acids (FA) metabolism following partial hepatectomy (PH) is required to meet energy demands of liver regeneration (LR). Impact of dietary lipid intake on LR depends on composition: diets enriched in olive/fish oils increase, while high fat diets decrease hepatocyte proliferation after PH by causing steatosis and inflammation. We previously showed that overexpression of the NFқB inhibitory and hepatoprotective protein A20 improves FA metabolism. This culminates in decreased oxidative stress and increased energy production, thereby improving mouse survival following severe liver ischemia/ reperfusion injury, and lethal radical hepatectomy. In contrast, partial loss of A20 (heterozygous (HT) mice) delays LR and increases lethality (42%) following PH, through impaired lipid metabolism and increased inflammation. In this study, we evaluated the impact of a fish oil (FO) diet on LR. A20 HT and wild type (WT) mice were fed 7% FO or soybean (SO) oil diets for 4 weeks prior to 2/3 PH. We noted significantly less proliferating (Ki67+) hepatocytes and heightened macrosteatosis (Oil Red O staining) correlating with increased lethality (>15% vs. 0%) in SO fed HT, as compared to WT mice, 48h after PH. Remarkably, FO feeding of HT mice abrogated death post PH by improving hepatocyte proliferation and reducing steatosis. This benefit related to FO lowering higher Fatty Acid Synthase mRNA levels noted in SO fed HT mice, as compared to WT. This reduced de novo lipogenesis, as evidenced by lower levels of palmitic acid. In addition, increased fatty acid uptake observed in SO fed HT after surgery was normalized by FO diet, as demonstrated by reduced content of the essential fatty acids linoleic and alpha-linoleic. Finally, FO diet reduced proinflammatory arachidonic acid and increased anti-inflammatory eicosapentaenoic and docosahexaenoic fatty acids in livers of HT mice as compared to SO diet. This decreased liver inflammation after PH, as evaluated by mRNA levels of Serum Amyloid A1. WT mice faired similarly well, regardless of diet. This is the first demonstration that dietary manipulation of lipid composition prior to PH restores hepatocyte proliferative ability, improving outcome in A20 HT mice. The clinical relevance of these findings is emphasized by recently described gene polymorphisms associated with A20's decreased expression or function. We propose that FO rich diets offer a safe and inexpensive means to overcome genetic predisposition in patients with unfavorable A20 polymorphisms, allowing for better outcomes following liver transplantation, mainly living donor liver transplantation.
The following people have nothing to disclose: Cleide G. Da Silva, Peter Studer, Eva Csizmadia, Darlan C. Minussi, Kathleen Daniels, Christiane Ferran
Inhibition of TNF-α in Intact Animals by Etanercept (ETN) Suppressed Cell Transplantation-induced Inflammation and Improved Liver Repopulation by Transplanted Hepatocytes
Preeti Viswanathan1, Sriram Bandi2, Sanjeev Gupta2
1Wediatric Gastroenterology and Hepatology Childrens Hospital at Montefiore, Albert Einstein College of Medicine, New York, NY; 2Medicine and Pathology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NY
To improve outcomes of liver cell therapy, superior engraftment of transplanted cells and liver repopulation is critical. As hepatocyte transplantation in liver rapidly induces microcirculatory disturbances, e. g., release of inflammatory chemokines/cytokines, with clearance of most transplanted cells, we hypothesized that inhibition of master regulators, such as TNF-α, will be effective. Previously, bosentan, blocker of endothelin (ET)−1 receptors A/B, or darusentan, blocker of ETRA, improved liver repopulation after treatment of cells in vitro or of animals in vivo, respectively, without abolishing hepatic inflammation. This made it appropriate to examine combined approaches with assays in DPPIV- rats receiving freshly isolated syngeneic F344 rat hepatocytes via spleen. In ETN pretreated rats, cell transplantation did not alter onset of hepatic ischemia or endothelial injury and activity of neutrophils, Kupffer cells or hepatic stellate cells, but major effects were observed by gene arrays in expression of inflammatory chemokines/cytokines. After cell transplantation, we examined cell engraftment with morphometric analysis of livers stained for DPPIV activity. Groups of control and etanercept-treated rats were established with tissue analysis 1, 2, 4 and 7 d, 1 mo and mo after cells. In ETN-treated rats, transplanted cell numbers increased several-fold, p<0. 001, and subsequently remained constant, indicating cells did not proliferate after ETN alone. Next, to elicit effects of ETN on kinetics of liver repopulation, we used retrorsine/PH-conditioned rats. This showed significant acceleration of liver repopulation after ETN, p<0. 001. We then determined whether ETN could be beneficial by priming of cells in vitro since incubation of primary hepatocytes for h with ETN resulted in their protection in subsequent cell culture from TNF-α cytotoxicity. This cytoprotection by ETN was greater than after ETRA/B blockade by bosentan. However, transplantation into retrorsine/PH-conditioned rats of bosentanprimed cells, but not of ETN-primed cells, produced superior liver repopulation, p<0. 05. When cells primed with bosentan were transplanted into ETN-treated rats, liver repopulation further improved, p<0. 05. Conclusions: Cell transplantationinduced chemokine/cytokine release involving TNF-α and had major effects in transplanted cell clearance. This mechanism was amenable to intervention with ETN for gains in cell engraftment and liver repopulation. Priming of hepatocytes with bosentan to block ETRA/B followed by transplantation of cells in ETN-treated animals yielded superior liver repopulation. This will help in optimization of cell therapy strategies.
The following people have nothing to disclose: Preeti Viswanathan, Sriram Bandi, Sanjeev Gupta
Human SIRPα expression by porcine LSEC overcomes CD47-SIRPa species incompatibilities reducing human platelet phagocytosis in vitro
Leela L. Paris, Luz M. Reyes, Ray K. Chihara, Richard A. Sidner, Ross L. Blankenship, Susan M. Downey A. Joseph Tector
Surgery Indiana University School of Medicine, Indianapolis, IN
Background: Xenotransplantation using genetically engineered porcine livers could eliminate the shortage of donor organs for liver transplantation. The immediate barrier to clinical application of porcine liver xenotransplantation is thrombocytopenia caused by liver sinusoidal cell phagocytosis. Platelet homeostasis and phagocytosis is partially controlled by CD47-Signal Regulatory Protein alpha (SIRPα) negative regulation pathways. The aim of this study was to determine if xenogeneic platelet phagocytosis can be prevented by minimizing interspecies incompatibilities through expression of human SIRPα on porcine liver sinusoidal endothelial cells (LSEC). Methods: Expression of SIRPα was examined on LSEC by PCR and confocal microscopy. CD47 levels on platelets were examined by flow cytometry, as was binding of the extracellular domains of porcine and human CD47 to porcine cells. Platelet phagocytosis was measured following artificial activation by porcine CD47. Phagocytosis of human platelets was examined in porcine LSEC transiently transfected with human SIRPα. Results: SIRPα is expressed on LSEC. Artificial activation of the pathway using the extracellular domain of porcine SIRPα resulted in less human platelet uptake. Flow cytometry showed that binding differences between human and porcine SIRPα and CD47 exist. Expression of human SIRPα in porcine LSEC lead to decreased human platelet phagocytosis. Conclusions: Interspecies incompatibilities in CD47-SIRPa signaling contribute to xenogeneic platelet phagocytosis by porcine LSEC. Expression of human SIRPα by porcine cells reduces xenogeneic platelet phagocytosis. These findings are a significant contribution to the development of a pig with an organ suitable for xenotransplantation.
The following people have nothing to disclose: Leela L. Paris, Luz M. Reyes, Ray K. Chihara, Richard A. Sidner, Ross L. Blankenship, Susan M. Downey, A. Joseph Tector