Human Liver Transplantation Research Articles

A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components

Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration.

High serum Aspartate transaminase levels on day 3 postliver transplantation correlates with graft and patient survival and would be a valid surrogate for outcome in liver transplantation clinical trials.

Aspartate transaminase, a liver specific enzyme released into serum following acute liver injury, is used in experimental organ preservation studies as a measure of liver IR injury. Whether post-operative serum transaminases are a good indicator of IR injury and subsequent graft and patient survival in human liver transplantation remains controversial. A single centre prospectively collected liver transplant database was analysed for the period 1988-2012. All patients were followed up for 5 years or until graft failure. Transaminase levels on the 1st, 3rd and 7th post-operative days were correlated with the patient demographics, operative outcomes, post-operative complications and both graft and patient survival via a binary logistic regression analysis. Graft and patient survival at 3 months was 80.3% and 87.5%. AST levels on the 3rd (P = 0.005) and 7th (P = 0.001) post-operative days correlated with early graft loss. Patients were grouped by their AST level (day 3): <107iU, 107-1213iU, 1213-2744iU and >2744iU. The incidence of graft loss at 3 months was 10%, 12%. 27% and 59% and 1-year patient mortality was 12%, 14%, 27% and 62%. Day 3 AST levels correlate with patient and graft outcome post-liver transplantation and would be a suitable surrogate endpoint for clinical trials in liver transplantation.

Rapamycin Attenuates Mouse Liver Ischemia and Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress

The roles of endoplasmic reticulum (ER) stress in liver ischemia and reperfusion injury (IRI) have been well recognized. However, the impact of rapamycin (Rapa), a broadly used immunosuppressive agent in human liver transplantation, on ER stress during IRI remains unclear. This study was designed to investigate the roles of Rapa in the regulation of ER stress in vivo and in vitro. In a mouse liver partial warm ischemia and reperfusion mode, we demonstrated that Rapa markedly protected livers from IRI, as evidenced by serum alanine aminotransferase (sALT) levels and liver histology. Then we also confirmed the protection of Rapa from thapsigargin (Tg)-induced cell death in primary hepatocytes. Both in vivo and in vitro experiments showed that the ER stress markers were markedly up-regulated by IRI and Tg treatment, whereas they were down-regulated by Rapa pretreatment, as monitored by Western blot at the protein levels and by quantitative reverse transcription polymerase chain reaction (RT-PCR) at the messenger RNA (mRNA) levels. In addition, it was also revealed that Rapa was able to remarkably inhibit the mammalian target of rapamycin (mTOR) pathway and enhance autophagy both in IR-stressed livers and Tg-treated primary hepatocytes. Thus, these results suggest that Rapa protects livers from IRI through inhibiting the ER stress pathway.

Bone marrow-derived progenitor cells in de novo liver regeneration in liver transplant.

The study was designed (1) to examine the hypothesis that circulating progenitor cells play a role in the process of de novo regeneration in human liver transplants and that these cells arise from a cell population originating in, or associated with, the bone marrow and (2) to investigate whether the transplanted liver volume has an effect on the circulating recipient-derived progenitor cells that generate hepatocytes during this process. Clinical data and liver tissue characteristics were analyzed in male individuals who underwent sex-mismatched adult-to-adult living donor liver transplantation using dual left lobe grafts. Dual left lobe grafts were examined at the time of transplantation and 19 to 27 days after transplantation. All recipients showed recovery of normal liver function and a significant increase in the volume of the engrafted left lobes after transplantation. Double staining for a Y-chromosome probe and the CD31 antigen showed the presence of hybrid vessels composed of recipient-derived cells and donor cells within the transplanted liver tissues. Furthermore, CD34-expressing cells were observed commingling with Y-chromosome+ cells. The ratio of recipient-derived vessels and the number of Y+ CD34+ cells tended to be higher when smaller graft volumes underwent transplantation. These findings suggest that the recruitment of circulating bone marrow-derived progenitor cells could contribute to vessel formation and de novo regeneration in human liver transplants. Moreover, graft volume may be an important determinant for the active mobilization of circulating recipient-derived progenitor cells and their contribution to liver regeneration.

Editorial introductions

Editorial introductions

Human immunodeficiency virus and liver transplantation: Hepatitis C is the last hurdle.

1Departments of Surgery and MedicineUniversity of California San FranciscoSan FranciscoCA *Address reprint requests to: Norah Terrault, M.D., Departments of Surgery and Medicine, University of California San Francisco, S357, 513 Parnassus Avenue, San Francisco, CA 94143. E‐mail: [email protected]; fax: +1‐415‐476‐0659.

How case reports have contributed to the progress of surgery.

Editorial The now well-known Zollinger-Ellison syndrome was recognized by Professor Zollinger at Ohio State University and his student, Dr. Ellison, based on their insightful observations of two young women who suffered from refractory peptic ulcers requiring repeated surgery [1]. The index case for Kasabach-Merritt syndrome was a 2-month-old male infant who underwent radiation therapy for a huge and growing capillary hemangioma in the left thigh causing extensive purpura [2]. Dr. Kasabach was a radiologist and Dr. Merritt was a colleague pediatrician at Columbia University. The first successful right lobectomy of the liver was performed by Professor Honjo at Kyoto University in 1949 to remove a large liver metastasis from rectal cancer in a 22-year-old man [3]. Professor Honjo reported this epoch-making progress in liver surgery as a case report in an English journal. The first three cases of human liver transplantation were reported by Professor Starzl from the University of Colorado in 1963 [4]. Nevertheless, case reports or case series are ranked as low level 4 evidence [5] in the hierarchy of evidence-based medicine. Most of the top medical or surgical journals now restrict the acceptance of case reports to an extreme degree because such reports, in general, are seldom cited and thus would reduce the journal's impact factor. However, we must recognize how previous reports of a few unusual cases, observed and treated by excellent physicians, have contributed greatly to the progress of medical science. In addition to the outstanding case reports mentioned above, there have been a number of amazing discoveries made by surgeons in their daily practice. Sir James Cantlie, a surgeon at the University of Hong Kong, started his anatomical research on the liver after

The three first liver transplantations in Norway, and the road leading to them.

Although renal transplantation was a therapeutic reality in Norway from 1969, organ transplantation was largely regarded as experimental surgery from its introduction in the early 1950s until the licensing of ciclosporin in 1982. After the first successful renal transplantation in 1954, 13 years elapsed before a liver and a heart were successfully transplanted, both in 1967. Inspired by the pioneers Thomas Starzl in Denver, Colorado, and Roy Yorke Calne in Cambridge, early in 1968 Snorre Aune, Gunnar Schistad and Andreas Skulberg began experimental studies on pigs at Ullevål Hospital to develop a surgical technique for liver transplantation. They collaborated with a team at Rikshospitalet led by Audun Flatmark and performed transplantations there every other week, and every other week at Ullevål. It took over one year of weekly animal experiments before the first transplanted pig survived. The first three transplantations on humans in Norway were performed at Ward 2, Ullevål Hospital in 1969, 1970 and 1972. The first patient died shortly after surgery, the second after 24 days, the third 54 days after transplantation. Snorre Aune, Gunnar Schistad and Andreas Skulberg were awarded the Michael Skielderup gold medal in 1972 for this pioneering work. The article is based on the author's own experience as an assistant surgeon and junior partner in the animal experiments and the first human liver transplantations in Norway, and a survey of relevant publications.

Galectin-1 in Stable Liver Transplant Recipients

IntroductionThe achievement of a state of tolerance and minimization of the immunosuppressive load form part of the “Holy Grail” in solid organ transplantation. Galectin-1 recently has been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of galectin-1 in stable liver transplant recipients. MethodsSerum levels of galectin-1 were determined in 30 stable liver transplant recipients who had been free of rejection episodes for at least 8 years. Fifteen patients with an acute rejection episode and 34 healthy subjects were used as the control group. ResultsThe concentrations of galectin-1 were significantly higher in stable liver transplant recipients compared with healthy subjects and with the acute rejection group. ConclusionsThese preliminary results indicate that galectin-1 is upregulated in stable liver transplant recipients. Thus, our results extend the recent findings that galectin-1 may play an immune-suppressive role in liver transplantation. It remains to be established whether it might help to induce tolerance in liver transplantation.

Current developments in pediatric liver transplantation.

In 1953, the pioneer of human orthotopic liver transplantation (LT), Thomas E Starzl, was the first to attempt an orthotopic liver transplant into a 3 years old patient suffering from biliary atresia. Thus, the first LT in humans was attempted in a disease, which, up until today, remains the main indication for pediatric LT (pLT). During the last sixty years, refinements in diagnostics and surgical technique, the introduction of new immunosuppressive medications and improvements in perioperative pediatric care have established LT as routine procedure for childhood acute and chronic liver failure as well as inherited liver diseases. In contrast to adult recipients, pLT differs greatly in indications for LT, allocation practice, surgical technique, immunosuppression and post-operative life-long aftercare. Many aspects are focus of ongoing preclinical and clinical research. The present review gives an overview of current developments and the clinical outcome of pLT, with a focus on alternatives to full-size deceased-donor organ transplantation.

Accurate assessment of liver steatosis in animal models using a high throughput Raman fiber optic probe.

Due to the shortage of healthy donor organs, steatotic livers are commonly used for transplantation, placing patients at higher risk for graft dysfunction and lower survival rates. Raman Spectroscopy is a technique which has shown the ability to rapidly detect the vibration state of C-H bonds in triglycerides. The aim of this study is to determine whether conventional Raman spectroscopy can reliably detect and quantify fat in an animal model of liver steatosis. Mice and rats fed a methionine and choline-deficient (MCD) and control diets were sacrificed on one, two, three and four weeks' time points. A confocal Raman microscope, a commercial Raman (iRaman) fiber optic probe and a highly sensitive Raman fiber optic probe system, the latter utilizing a 785 nm excitation laser, were used to detect changes in the Raman spectra of steatotic mouse livers. Thin layer chromatography was used to assess the triglyceride content of liver specimens, and sections were scored blindly for fat content using histological examination. Principal component analysis (PCA) of Raman spectra was used to extract the principal components responsible for spectroscopic differences with MCD week (time on MCD diet). Confocal Raman microscopy revealed the presence of saturated fats in mice liver sections. A commercially available handheld Raman spectroscopy probe could not distinguish the presence of fat in the liver whereas our specially designed, high throughput Raman system could clearly distinguish lobe-specific changes in fat content. In the left lobe in particular, the Raman PC scores exhibited a significant correlation (R(2) = 0.96) with the gold standard, blinded scoring by histological examination. The specially designed, high throughput Raman system can be used for clinical purposes. Its application to the field of transplantation would enable surgeons to determine the hepatic fat content of the donor's liver in the field prior to proceeding with organ retrieval. Next steps include validating these results in a prospective analysis of human liver transplantation implant biopsies.

Biomarker differences between cadaveric grafts used in human orthotopic liver transplantation as identified by coulometric electrochemical array detection (CEAD) metabolomics.

Metabolomics in systems biology research unravels intracellular metabolic changes by high throughput methods, but such studies focusing on liver transplantation (LT) are limited. Microdialysate samples of liver grafts from donors after circulatory death (DCD; n=13) and brain death (DBD; n=27) during cold storage and post-reperfusion phase were analyzed through coulometric electrochemical array detection (CEAD) for identification of key metabolomics changes. Metabolite peak differences between the graft types at cold phase, post-reperfusion trends, and in failed allografts, were identified against reference chromatograms. In the cold phase, xanthine, uric acid, and kynurenine were overexpressed in DCD by 3-fold, and 3-nitrotyrosine (3-NT) and 4-hydroxy-3-methoxymandelic acid (HMMA) in DBD by 2-fold (p<0.05). In both grafts, homovanillic acid and methionine increased by 20%-30% with each 100 min increase in cold ischemia time (p<0.05). Uric acid expression was significantly different in DCD post-reperfusion. Failed allografts had overexpression of reduced glutathione and kynurenine (cold phase) and xanthine (post-reperfusion) (p<0.05). This differential expression of metabolites between graft types is a novel finding, meanwhile identification of overexpression of kynurenine in DCD grafts and in failed allografts is unique. Further studies should examine kynurenine as a potential biomarker predicting graft function, its causation, and actions on subsequent clinical outcomes.

Ulinastatin ameliorates acute kidney injury following liver transplantation in rats and humans.

Acute kidney injury (AKI) is a common complication following orthotopic liver transplantation (OLT) that evidently affects prognosis. However, no effective treatment exists for AKI. The aim of the present study was to elucidate whether ulinastatin application during OLT in humans can reduce kidney damage and improve renal function. In addition, the underlying mechanisms of ulinastatin were investigated on a rat autologous OLT (AOLT) model. In total, 60 patients undergoing an OLT were randomly selected to receive ulinastatin (U group; n=30) or saline (C group; n=30) during the OLT surgery. The patient demographics, AKI incidence rate, recovery indicators and renal injury indexes were measured during the perioperative period. In addition to the clinical trials, 40 rats were subjected to an AOLT and were divided into the control (C-R), sham-operation and ulinastatin treatment groups. Pathological renal damage, biomarkers of inflammation and oxidative stress were measured to investigate the effects and possible mechanisms of ulinastatin on AKI. In the clinical trials, ulinastatin application was shown to attenuate the incidence of AKI following OLT (P<0.05) and reduce the serum levels of cystatin C and urinary β2 microglobulin within 24 h of the OLT (P<0.05). Furthermore, ulinastatin was found to significantly improve the recovery of patients by reducing the time spent in the intensive care unit (P<0.01 vs. C group), the ventilation time and the hemodialysis rates (P<0.05 vs. C group). In the rat AOLT model, ulinastatin application was also shown to relieve renal pathological damage by reducing the serum cystatin C and creatinine levels. Notably, the levels of tumor necrosis factor-α, interleukin-6, hydrogen peroxide and reactive oxygen species were evidently reduced, while the level of superoxide dismutase was increased in the ulinastatin groups (P<0.05, vs. C-R group). In conclusion, ulinastatin application was demonstrated to protect against AKI following OLT by inhibiting inflammation and oxidation.

The Inhibition of Aldose Reductase Attenuates Hepatic Ischemia-Reperfusion Injury Through Reducing Inflammatory Response

We aim to investigate the role of aldose reductase (AR) in hepatic ischemia-reperfusion injury (IRI) of normal and fatty livers and to explore the underlying mechanisms. Hepatic IRI is a typical inflammatory response during liver surgery. It contributes to liver graft failure or nonfunction after transplantation. Increasing evidence implicates that AR plays a key role in a number of inflammatory diseases. However, the role of AR in hepatic IRI is still unknown. Intragraft AR expression profile and the association with liver graft injury were investigated in both human and rat liver transplantation using normal or fatty graft. The direct role of AR in hepatic IRI was studied in the AR knockout mice IRI model with or without fatty liver. They were further validated by the simulated IRI in vitro model using fatty LO2 cells with or without AR inhibitor zopolrestat and primary peritoneal macrophages isolated from AR knockout and wild-type mice. Gene expression of inflammatory cytokines/chemokines, the infiltration of macrophages/neutrophils, and NF-κB pathway activation were compared among different groups. AR was overexpressed in liver graft after human and rat liver transplantation and correlated with consequent liver injuries. The knockout of AR significantly attenuated hepatic sinusoidal damage and apoptosis in both normal and fatty livers after IRI. The expression of proinflammatory cytokines/chemokines and neutrophil chemoattractants, infiltration of macrophage and neutrophil, and activation of inflammation-associated NF-κB and JNK pathway were downregulated in AR knockout mice. Furthermore, the inhibition of AR effectively suppressed macrophage migration and decreased lipopolysaccharide (LPS)-induced production of proinflammatory cytokines/chemokines in isolated macrophages. The deficiency of AR attenuated hepatic IRI in both normal and fatty livers by reducing liver inflammatory responses.

Histological Markers of Ischemia and Reperfusion Injury in Macrosteatotic Grafts in Human Liver Transplantation.

Histological Markers of Ischemia and Reperfusion Injury in Macrosteatotic Grafts in Human Liver Transplantation.

T Cell and Myeloid Chimerism Without GVHD in Human Intestinal and Liver Transplantation: A Prospective Study.

Blood chimerism, thought to be due to the transfer of “passenger leukocytes” from donor to host, has been reported sporadically among visceral transplant recipients, usually in association with GVHD. We hypothesized that the large-volume transfer of lymphocytes inherent to multivisceral transplant (MVTx) would correlate with a higher degree of mixed chimerism compared to isolated liver (iLTx) or intestinal transplant (iITx). We used multicolor flow cytometry to perform lineage chimerism analyses on peripheral blood leukocytes from iLTx (n=10), iITx (n=4) and MVTx (n=3) recipients sampled up to one year post-transplant, regardless of clinical status. The Area Under the Curve (AUC) for chimerism among various hematopoietic lineages was calculated by integrating the percent donor chimerism over time for each lineage. With the exception of one MVTx and one iLTx, all subjects lost mixed chimerism within one year. T cell chimerism was more common among ITx (iITx+MVTx), detected in 6/7 compared to 3/10 iLTx recipients (Fischer's exact test, p=0.05), with only a single episode of self-limited GVHD. T cell chimerism was significantly higher among ITx (mean AUC: 2097 vs 73 %xdays; p=0.005) with a median peak of 14.4 versus 1.7% among iLTx (p=0.02). Myeloid lineage chimerism was present exclusively among iLTx and MVTx (6/13 recipients, median peak: 5.25%), suggesting that its presence requires a hepatic allograft. MVTx were found to have the highest overall level of donor chimerism compared to iLTx and iITx for T (MVTx vs iLTx: p=0.01, MVTx vs iITx: p=0.1), B (MVTx vs iLTx: p=0.01, MVTx vs iITx: p=0.06) and myeloid (MVTx vs iLTx: p=0.08, MVTx vs iITx: p=0.03) lineages. ITx experiencing biopsy-proven moderate-to-severe rejection (n=2) demonstrated lower levels of chimerism than those with no or mild rejection (n=5) (mean AUC: 123 vs 2887 %xdays). This study contests the notion that T cell chimerism after ITx occurs in association with GVHD and suggests that T cell progenitors may originate in ITx whereas myeloid progenitors may be enriched in hepatic allografts. The possible immunoregulatory effects of increased mixed chimerism after MVTx, which may account for the lower rate of severe intestinal rejection in this population, are currently under investigation.

HCV-Induced Immune Responses Influence the Development of Operational Tolerance After Liver Transplantation in Humans

Pathogen-induced immune responses prevent the establishment of transplantation tolerance in experimental animal models. Whether this occurs in humans as well remains unclear. The development of operational tolerance in liver transplant recipients with chronic hepatitis C virus (HCV) infection allows us to address this question. We conducted a clinical trial of immunosuppression withdrawal in HCV-infected adult liver recipients to elucidate (i) the mechanisms through which allograft tolerance can be established in the presence of an ongoing inflammatory response and (ii) whether anti-HCV heterologous immune responses influence this phenomenon. Of 34 enrolled liver recipients, drug withdrawal was successful in 17 patients (50%). Tolerance was associated with intrahepatic overexpression of type I interferon and immunoregulatory genes and with an expansion of exhausted PD1/CTLA4/2B4-positive HCV-specific circulating CD8(+) T cells. These findings were already present before immunosuppression was discontinued and were specific for HCV infection. In contrast, the magnitude of HCV-induced proinflammatory gene expression and the breadth of anti-HCV effector T cell responses did not influence drug withdrawal outcome. Our data suggest that in humans, persistent viral infections exert immunoregulatory effects that could contribute to the restraining of alloimmune responses, and do not necessarily preclude the development of allograft tolerance.

Method for finding metabolic properties based on the general growth law. Liver examples. A general framework for biological modeling.

We propose a method for finding metabolic parameters of cells, organs and whole organisms, which is based on the earlier discovered general growth law. Based on the obtained results and analysis of available biological models, we propose a general framework for modeling biological phenomena and discuss how it can be used in Virtual Liver Network project. The foundational idea of the study is that growth of cells, organs, systems and whole organisms, besides biomolecular machinery, is influenced by biophysical mechanisms acting at different scale levels. In particular, the general growth law uniquely defines distribution of nutritional resources between maintenance needs and biomass synthesis at each phase of growth and at each scale level. We exemplify the approach considering metabolic properties of growing human and dog livers and liver transplants. A procedure for verification of obtained results has been introduced too. We found that two examined dogs have high metabolic rates consuming about 0.62 and 1 gram of nutrients per cubic centimeter of liver per day, and verified this using the proposed verification procedure. We also evaluated consumption rate of nutrients in human livers, determining it to be about 0.088 gram of nutrients per cubic centimeter of liver per day for males, and about 0.098 for females. This noticeable difference can be explained by evolutionary development, which required females to have greater liver processing capacity to support pregnancy. We also found how much nutrients go to biomass synthesis and maintenance at each phase of liver and liver transplant growth. Obtained results demonstrate that the proposed approach can be used for finding metabolic characteristics of cells, organs, and whole organisms, which can further serve as important inputs and constraints for many applications in biology (such as protein expression), biotechnology (synthesis of substances), and medicine.

Humanized Mice for Studying Human Immune Responses and Generating Human Monoclonal Antibodies.

The new-generation humanized (Hu) mouse models permit multilineage human hematopoiesis and generate T cells, B cells, macrophages, and dendritic cells required for a coordinated human immune response. Therefore, any desired antigen or human-specific pathogens that can infect humanized mice can be used to generate human antibody responses. Two leading humanized mouse models are currently being used. The Hu-HSC model uses the transplantation of human hematopoietic stem cells (HSCs), whereas the BLT mouse model is created by transplantation of human fetal liver, thymus, and HSC. A number of human pathogens such as HIV-1, dengue, Epstein-Barr virus, and hepatitis C virus have been studied in these systems. Responder antigen-specific B cells from these animals can be collected and used to generate human monoclonals by B-cell immortalization or by single-cell PCR methods to "rescue" antibody-producing genes for ectopic expression. Both models generate cellular and humoral immune responses. However, the antibodies generated are primarily of the IgM type because of the inefficient immunoglobulin class switch resulting in the suboptimal production of antigen-specific affinity-matured IgG. The current Hu mouse models thus far have permitted the analysis of human "antibodyome," and recent reports demonstrated their utility in generating human monoclonal antibodies. Ongoing efforts at further refinements are expected to make these systems more efficient in the near future.

A Randomized Clinical Trial Testing the Anti-Inflammatory Effects of Preemptive Inhaled Nitric Oxide in Human Liver Transplantation

Decreases in endothelial nitric oxide synthase derived nitric oxide (NO) production during liver transplantation promotes injury. We hypothesized that preemptive inhaled NO (iNO) would improve allograft function (primary) and reduce complications post-transplantation (secondary). Patients at two university centers (Center A and B) were randomized to receive placebo (n = 20/center) or iNO (80 ppm, n = 20/center) during the operative phase of liver transplantation. Data were analyzed at set intervals for up to 9-months post-transplantation and compared between groups. Patient characteristics and outcomes were examined with the Mann-Whitney U test, Student t-test, logistic regression, repeated measures ANOVA, and Cox proportional hazards models. Combined and site stratified analyses were performed. MELD scores were significantly higher at Center B (22.5 vs. 19.5, p<0.0001), surgical times were greater at Center B (7.7 vs. 4.5 hrs, p<0.001) and warm ischemia times were greater at Center B (95.4 vs. 69.7 min, p<0.0001). No adverse metabolic or hematologic effects from iNO occurred. iNO enhanced allograft function indexed by liver function tests (Center B, p<0.05; and p<0.03 for ALT with center data combined) and reduced complications at 9-months (Center A and B, p = 0.0062, OR = 0.15, 95% CI (0.04, 0.59)). ICU (p = 0.47) and hospital length of stay (p = 0.49) were not decreased. iNO increased concentrations of nitrate (p<0.001), nitrite (p<0.001) and nitrosylhemoglobin (p<0.001), with nitrite being postulated as a protective mechanism. Mean costs of iNO were $1,020 per transplant. iNO was safe and improved allograft function at one center and trended toward improving allograft function at the other. ClinicalTrials.gov with registry number 00582010 and the following URL:http://clinicaltrials.gov/show/NCT00582010.