Heidi Yeh

Geographic inequity in access to livers for transplantation.

Background. Liver transplantation offers life-saving therapy for patients with decompensated liver disease or T2 hepatocellular carcinomas. In the United States, deceased donor livers are primarily allocated by Model for End-Stage Liver Disease (MELD) score within each of the countrys more than 50 donation service areas (DSAs). Variation in DSA size, population, and organ availability have engendered concern that unequal access to deceased donor livers across DSAs contributes to geographic variability in outcome. Methods. To determine the extent to which DSA variability in organ availability correlated with combined waitlist and posttransplant mortality, we analyzed retrospectively national waitlist and posttransplant data for a 7-year period after implementation of the current MELD-based allocation system. Results. Marked variation among DSAs was evident in death rate (3.3-fold), transplant rate (20-fold), and mean transplant MELD (>10 points). Death rate correlated with organ availability was assessed by transplant rate and transplant MELD. DSAs with low organ availability included the countrys largest cities, had more new listings per capita, larger waitlists, more transplant centers per DSA, and a higher proportion of black and Asian patients. DSAs of organ shortage were also characterized by more frequent dual listing at another transplant center, more living donor liver transplants, and increased average length of the transplant admission. Conclusions. Geographic differences in deceased donor organ availability contribute to variation in overall death rate of liver transplant patients, shape the clinical practice of transplant, and influence the resources consumed per transplant. Geographic variation in organ access results primarily from rates of listing rather than donation. Our findings highlight the need to restructure organ distribution areas to achieve equal access to deceased donor livers for transplantation in the United States.

Subnormothermic Machine Perfusion for Ex Vivo Preservation and Recovery of the Human Liver for Transplantation

To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h of oxygenated subnormothermic machine perfusion (21°C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11–1.94] to 6.74 [4.15–8.16] mL O2/min kg liver), lactate levels (4.04 [3.70–5.99] to 2.29 [1.20–3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6–87.5] pmol/mg preperfusion to 167.5 [151.5–237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production, was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters postischemia.

TGF-β-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti‐T cell immunoglobulin domain and mucin domain‐1 and anti‐CD45RB antibody treatment results in tolerance to full MHC‐mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen‐specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4+CD25− T cells than with naive B cells. We also show that Breg cells express the TGF‐β associated latency‐associated peptide and that Breg‐cell mediated graft prolongation post‐adoptive transfer is abrogated by neutralization of TGF‐β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C‐C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody‐induced transplantation tolerance, Breg cells promote graft survival by promoting Treg‐cell development, possibly via TGF‐β production.

Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.

BACKGROUND & AIMS The peribiliary glands of large bile ducts have been identified as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. We aimed to determine whether injury to the peribiliary glands of donor livers is a risk factor for development of non-anastomotic biliary strictures (NAS) after liver transplantation. METHODS In 128 liver transplant procedures, biopsies were taken from the donor bile duct and injury was assessed using an established histological grading system. Histological severity of injury was subsequently compared in liver grafts that later developed biliary structures vs. uncomplicated liver grafts. RESULTS Luminal biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in only 16.4%. Periluminal peribiliary glands were more severely injured than deep peribiliary glands located near the fibromuscular layer (>50% loss in 56.9% vs. 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to grafts without NAS (>50% loss in 50.0% vs. 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to grafts without NAS (>50% vascular changes in 57.1% vs. 20.3%; p=0.006). CONCLUSION Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insufficient regeneration due to loss of peribiliary glands or impaired blood supply may explain the development of biliary strictures.

Anti‐CD45RB/Anti‐TIM‐1‐Induced Tolerance Requires Regulatory B Cells

The role of B cells in transplant tolerance remains unclear. Although B‐cell depletion often prolongs graft survival, sometimes it results in more rapid rejection, suggesting that B cells may have regulatory activity. We previously demonstrated that tolerance induction by anti‐CD45RB antibody requires recipient B cells. Here, we show that anti‐CD45RB in combination with anti‐TIM‐1 antibody has a synergistic effect, inducing tolerance in all recipients in a mouse islet allograft model. This effect depends on the presence of recipient B cells, requires B‐cell IL‐10 activity, and is antigen‐specific. These data suggest the existence of a regulatory B‐cell population that promotes tolerance via an IL‐10‐dependent pathway.

Injury to Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures

C1650 The Knockout of Rap1 Accelerates Liver Regeneration After Transplantation. C. Li, C. Lo, K. Ng, X. Qi, W. Geng, Y. Ma, X. Liu, H. Liu, K. Man. Surgery, The University of Hong Kong, Hong Kong, China. Introduction and aim: Impaired of liver regeneration severe affects the tissue repair following liver surgery. Cytokine, growth factor and metabolic networks play important roles in theprogression of liver regeneration. Rap1 is a part of the shelterin complex at mammalian telomeres, involved in protecting chromosome ends and promoting gene silencing. Recent research revealed its novel roles in the regulation of metabolism andinfl ammatory response through binding to extra-telomeric sites. We recently also showed that the knockout of Rap1 attenuated liver graft injury after transplantation. Here, we aimed to investigate the role of Rap1 in liver regeneration and to explore underlying mechanism. Methods:Clinically, the intragraft Rap1 expression and its correlation among liver function, infl ammatory cytokines/chemokines, and macrophage infi ltration were analyzed in human liver graft biopsies after transplantation. To investigate the direct role of Rap1 in hepatocyte proliferation and liver regeneration, Rap1 knockoutand wild type mice were subjected to major hepatectomy plus partial hepatic ischemia/ reperfusion injury (IRI). Mouse hepatocyte proliferation, histological damage, liver function and gene expressions were compared between Rap1 knockoutand wild type group. Results: Intrahepatic Rap1 expression was increased in small-for-size graft in comparison to normal graft (2.9 vs 1.9 folds of normal liver, p<0.05), and associated with higher expressions of infl ammatory cytokines/chemokines and more infi ltrations of macrophage and neutrophil at 2 hours after transplantation. Furthermore, overexpression of Rap1 was signifi cantly correlated with impaired liver function after transplantation (ALT: day 1, 3, 4 p<0.05; AST: day 0, 3, 4, 5 p<0.05). In mouse model, the knockout of Rap1 signifi cantly accelerated liver regeneration at day 2 after major hepatectomy and hepatic IRI compared to wild type group (Ki67: 56 vs 11/ HPF, p<0.05; PCNA: 46 vs 29/HPF, p<0.05). The knockout of Rap1 also attenuated histological damage and liver function. Furthermore, reduced expression of PPAR-α and increased expressions of G0S2 and IL1β were detected in Rap1 knockout mice. Conclusion: The knockout of Rap1 accelerates liver regeneration and associated with the regulation of cell cycle control, PPAR-α and cytokine signaling. Abstract# C1651 Injury of Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures. N. Karimian,1 S. op den Dries,1,3 A. Westerkamp,1 A. Gouw,2 J. Markmann,3 T. Lisman,1 H. Yeh,3 K. Uygun,3 P. Martins,3 R. Porte.1 1Department of Surgery, Section of Hepatopancreatobiliary Surgery and Liver Transplantation, University Medical Center Groningen (UMCG), Groningen, Netherlands; 2Department of Pathology, University Medical Center Groningen (UMCG), Groningen, Netherlands; 3Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston. C1651 Injury of Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures. N. Karimian,1 S. op den Dries,1,3 A. Westerkamp,1 A. Gouw,2 J. Markmann,3 T. Lisman,1 H. Yeh,3 K. Uygun,3 P. Martins,3 R. Porte.1 1Department of Surgery, Section of Hepatopancreatobiliary Surgery and Liver Transplantation, University Medical Center Groningen (UMCG), Groningen, Netherlands; 2Department of Pathology, University Medical Center Groningen (UMCG), Groningen, Netherlands; 3Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston. Peribiliary glands of large bile ducts have been identifi ed as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. It is unknown whether injury of the peribiliary glands is a risk factor for the development of non-anastomotic biliary strictures (NAS) after liver transplantation. Moreover, it is unknown whether pretransplant biliary injury is different in livers donated after brain death (DBD) or cardiac death (DCD). In 128 liver transplant procedures, biopsies were taken from the extrahepatic bile duct and injury was assessed using a systematic histological grading system. Histological injury was correlated with the occurrence of posttransplant biliary strictures and a comparison wasmade between DBD (n=97) and DCD livers (n=29). Biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in 16.4%. Periluminal peribiliary glands were more severely injured than the deep peribiliary glands located near the fi bromuscular layer (>50% loss in 56.9% versus 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to uncomplicated grafts (>50% loss in 50.0% versus 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to uncomplicated grafts (>50% vascular changes in 57.1% versus 20.3%; p=0.006). Comparison of DBD and DCD livers revealed signifi cantly more vascular injury in the latter (p=0.005). Conclusion: Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insuffi cient regeneration due to loss of peribiliary glands and blood supply may explain the development of biliary strictures. Abstract# C1652 N-Acetylcysteine Treatment Attenuates ROS Mediated Endoplasmic Reticulum Stress and Apoptosis During Liver Ischemia Reperfusion Injury. H. Lu, F. Zhang, J. Rao, Y. Sun, X. Qian, L. Lu, X. Wang. Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University(NJMU); Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing, China. Background: The protective effects of N-acetylcysteine (NAC) treatment have been demonstrated during ischemia reperfusion injury (IRI) in several organs, but its underlying mechanism has not been suffi ciently elucidated. Our previous data has shown that ER stress is critical for the development of liver IRI. This study investigated effects of NAC on ER stress and tissue injury during liver IRI. Methods: Mice were injected with NAC (300mg/kg, ip) 2 hours before ischemia. The level of reactive oxygen species (ROS) was analyzed by GSH and MDA. Hepatic injury was evaluated based on sALT and histopathology. Apoptosis was analyzed by TUNEL staining and Caspase-3 activities. ER stress molecules (GRP78, ATF4 and CHOP) were determined by real-time PCR and western blotting in vivo and in vitro. Antiapoptotic molecules related to ER stress (Bcl-2 and Bcl-xl) were assessed after reperfusion. To analyze the roles of NAC on ROS-mediated ER stress and apoptosis, LDH was examined in cultured hepatocytes treated by H2O2 or Thapsigargin (TG). Results: NAC treatment signifi cantly attenuated ROS-mediated liver injury after IRI. Importantly, ROS-mediated ER stress was signifi cantly inhibited in NAC treated mice after IRI. As demonstrated by experiments in vitro, NAC treatment signifi cantly reduced the upregulation of ER stress molecules after H2O2 treatment. Furthermore, NAC treatment signifi cantly reduced caspase-3 activity after reperfusion, which was in line with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment signifi cantly inhibited LDH release from hepatocytes treated by H2O2 or TG. Conclusions: This work provides a new evidence for the protective effects of NAC treatment on hepatocytes from IRI. Through the inhibition of ROS-mediated ER stress, NAC may be critical for inhibiting the ER stress related apoptosis pathway. Abstract# C1653 Inhibition of 12/15-Lipoxygenase Prevents Cell Death After Hepatic Ischemia and Reperfusion. M. Drefs, M. Thomas, A. Khandoga, F. Haak, J. Andrassy, M. Guba, J. Werner, M. Rentsch. Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital of the University of Munich, Munich, Germany. Introduction Graft failure secondary to ischemia-reperfusion-injury (IRI) still represents a major complication in liver transplantion. 12/15-Lipoxygenase (LOX), a protein of the glutathione-peroxidase-4 (GPx-4) signaling cascade, has been proven to substantially mediate cerebral postischemic apoptosis. Aim of this study was to investigate the impact of 12/15-LOX inhibition on hepatic IRI and thus gain insights into GPx-4 dependent signaling in the liver. Methods Livers of C57BL/6 mice were exposed to 60 minutes of warm ischemia by clamping the common pedicle of the median and left lateral liver lobe and subsequent reperfusion for 90 minutes. Baicalein, an inhibitor of 12/15-LOX, was administered intraperitoneally 30 minutes before operation (group 1). Controls were treated with vehicle dimethylsulfoxide(DMSO) (group 2) or untreated (group 3).Tissue samples were analyzed by TUNEL assay and Western Blot for pro-apoptotic proteins p44/42 MAP kinase (ERK1/2), Jun-amino-terminal kinase (JNK), Poly-ADP-ribose polymerase (PARP) and Caspase-3. Results Analysis of hepatic cell death by fl uorescence TUNEL labeling showed a signifi cant reduction of apoptotic cells in liver samples pretreated with baicalein (group 1: -64.8%; p<0,001) and with DMSO (group 2: -23.2%) compared to untreated samples (group 1). Western Blot analysis revealed a considerable downregulation of ERK1/2 (-36,7%) and PARP (-73,8%), as well as a slight reduction of JNK and Caspase-3 after Baicalein administration. The pretreatment with DMSO also showed a slight down-regulation regarding the investigated pro-apoptotic protein cascade, however in a discrete fashion. Conclusion Inhibition of 12/15-LOX leads to a signifi cant decr

Donor age and cold ischemia interact to produce inferior 90-day liver allograft survival.

Background. Expanded regional sharing of liver allografts may increase cold ischemia and allograft failure, particularly with livers from older donors. The aim of this study was to examine whether older donor age and cold ischemic time interact to produce inferior allograft survival. Methods. We undertook a retrospective cohort study of adult liver transplants in the United States performed between December 1, 1995 and December 31, 2005, using data from the Organ Procurement and Transplantation Network. The primary outcome was allograft failure within 90 days. Results. Forty-four thousand seven hundred fifty-six liver transplant recipients were analyzed. Older age was defined as 45 years or more, and prolonged cold ischemia was defined as 12 hours or more. Using data from the pre-Model for End Stage Liver Disease (MELD), post-MELD and combined eras, three separate analyses of the interaction between older donor age and prolonged cold ischemia were performed. In multivariable logistic regression, the interaction of age 45 years or more and cold ischemia more than or equal to 12 hr reached statistical significance in the combined (OR 1.24, CI 1.08–1.42, P<0.01) and pre-MELD (OR 1.26, CI 1.08–1.46, P<0.01) datasets, but not in the smaller post-MELD dataset (OR 1.18, CI 0.81–1.72, P=0.38). In the combined dataset, recipients of livers from donors aged 45 years or more and cold ischemia more than or equal to 12 hr showed an adjusted absolute risk of allograft failure at 90 days of 17.3% (odds ratio 1.84), compared with 11.1% for recipients of livers from donors older than 45 years and cold ischemia less than 12 hr. Conclusions. These findings suggest that older donor age and prolonged cold ischemia interact to increase liver allograft failure at 90 days. Proposals to expand regional sharing of older livers should be regarded with caution.

Transplant Center Volume and Outcomes After Liver Retransplantation

Liver retransplantation surgery has a high rate of allograft failure due to patient comorbidities and technical demands of the procedure. Success of liver retransplantation could depend on surgeon experience and processes of care that relate to center volume. We performed a retrospective cohort study of adult liver retransplantation procedures performed from January 1, 1996 through December 31, 2005 using registry data from the Organ Procurement Transplantation Network. The primary outcome was 1‐year allograft failure. Liver transplant centers were categorized as small, intermediate or high volume by dividing overall liver transplants into three tertiles of approximately equal size. Mean annual volume of overall liver transplants was <50 for low‐volume centers, 50–88 for intermediate‐volume centers and >88 for high‐volume centers. The primary analysis consisted of 3977 liver retransplantation patients. The unadjusted risk of 1‐year allograft failure was 37.8%. In multivariable logistic regression, the risk of 1‐year allograft failure was not significantly different between low‐ (reference), intermediate‐ (OR 0.86, CI 0.72–1.03, p = 0.11) and high‐volume centers (OR 0.88, CI 0.74–1.04, p = 0.14). Results were similar when the analysis was limited to retransplantation performed >160 days after initial transplantation. Center volume is an imprecise surrogate measure for 1‐year outcomes after liver retransplantation.

Cellular Immunity Delimits Adenoviral Gene Therapy Strategies for the Treatment of Neoplastic Diseases

AbstractBackground: Adenoviral gene therapy is a promising new approach for the treatment of neoplastic diseases. To design rational clinical trials and distinguish the effects of therapeutic transgene expression from those caused by viral infection alone, the immune response to the vector must be understood. In these experiments, we further define cellular immunity to recombinant adenovirus. Methods: The immune response to hepatic adenoviral gene transfer was studied in infected mice by depleting T cells with an anti-CD3 antibody, measuring splenocyte cytokine production, determining the impact of transgene expression on inflammation, and assessing liver MHC protein expression. Results: The cellular immune response to recombinant adenovirus is (1) averted by T lymphocyte depletion, (2) marked by a TH1 response with increased IL-2 production, (3) directed against both the transgene product and viral proteins, and (4) associated with increased hepatocyte MHC Class I expression. Conclusions: It is necessary to take into consideration the constraints imposed by the immunogenicity of recombinant adenovirus and its transient transgene expression in the clinical application of adenoviral gene transfer for the treatment of cancer.

Islet alone versus islet after kidney transplantation: metabolic outcomes and islet graft survival.

Background. Isolated islet transplantation with infusions from two to three donor pancreata and Edmonton immunosuppression consistently achieves insulin independence in patients with type 1 diabetes. The success of this protocol has been attributed to a novel combination of immunosuppressive agents and avoidance of steroids; however, the outcome of islet transplantation may differ in kidney transplant recipients who are already immunosuppressed. Methods. We compared the metabolic outcomes and graft survival of islet transplantation in our program where nine patients underwent islet transplantation alone treated with Edmonton immunosuppression and eight patients received islet after kidney (IAK) transplants under standard kidney transplant immunosuppression often including steroids. Results. Transplants in the IAK and islet transplantation alone setting demonstrated similar islet potency (islet equivalents/unit insulin reduction) and recipients from both groups routinely gained insulin independence, functional islet mass, and duration of graft survival, however, seemed superior in the IAK group. Conclusions. These results suggest that better islet graft function and survival may be attained using non-Edmonton rather than Edmonton immunosuppression and can include maintenance steroid therapy.