Post-transplant Liver Biopsies Research Articles

Histomorphological spectrum in Post-transplant liver biopsy: A single centre observational study

Histomorphological spectrum in Post-transplant liver biopsy: A single centre observational study

Liver Transplantation in Progressive Familial Intrahepatic Cholestasis with Normal Gamma-Glutamyl Transferase: Evaluation of Post-transplant Steatosis and Steatohepatitis

Background: Progressive familial intrahepatic cholestasis is a disease presenting with severe cholestasis and progressing to the end-stage liver disease later. Liver transplantation is a treatment modality available for progressive familial intrahepatic cholestasis, especially in patients with end-stage liver disease or those who are unsuitable for or have failed biliary diversion. Objectives: To evaluate clinical and pathological characteristics of progressive familial intrahepatic cholestasis patients who had undergone liver transplantation and to determine post-transplant steatosis and steatohepatitis. Methods: We evaluated 111 progressive familial intrahepatic cholestasis patients with normal gamma-glutamyl transferase that performed liver transplantation in Shiraz Transplant Center in Iran between March 2000 and March 2017. Results: The most common clinical manifestations were jaundice and pruritus. Growth retardation and diarrhea were detected in 76.6% and 42.5% of the patients. After transplantation, growth retardation was seen in 31.5% of the patients, and diarrhea in 36.9% of them. Besides, 29.1% of the patients died post-transplant. Post-transplant liver biopsies were taken from 50 patients, and 15 (30%) patients had steatosis or steatohepatitis, five of whom (10%) had macrovesicular steatosis alone, and 10 (20%) had steatohepatitis. Only one patient showed moderate bridging fibrosis (stage III), and none of them showed severe fibrosis. Conclusions: Liver transplantation is the final treatment option for these patients, and it can relieve most clinical manifestations. However, post-transplant mortality rate was relatively high in our center. Diarrhea, growth retardation, and steatosis are unique post-transplant complications in these patients. The rate of post-transplant steatosis and steatohepatitis in patients with liver biopsy in our study was 30%, with a significant difference from previous studies.

Acute Neurological Symptoms After a Liver Transplant

Acute Neurological Symptoms After a Liver Transplant

Sinusoidal dilatation and congestion in post-transplant liver biopsies from patients presenting with transaminitis.

Sinusoidal dilatation and congestion (SDC) in liver biopsy may be obstructive (due to venous outflow impairment) or non-obstructive in nature. The significance of this finding in the post-liver transplant setting remains unexplored. We herein retrospectively analysed all post-transplant liver biopsies showing SDC and examined histopathological features in detail. Association with transaminitis and concurrent graft rejection was assessed. A total of 30 post-transplant liver biopsies from 27 patients showed SDC with atrophy of hepatocyte cords (SDC; incidence 7.4%). All patients had transaminitis. Most patients (n=22; 81.5%) were asymptomatic with deranged liver function tests (LFTs) detected during routine follow-up, raising clinical suspicion of graft rejection. SDC was non-obstructive in 19 (70.4%) and obstructive (due to sinusoidal obstruction syndrome (SOS)) in 8 (29.6%) cases. The incidence of SOS was 2%. SDC was mild, moderate and severe in 18 (66.7%), 7 (25.9%) and 2 (7.4%) cases, respectively. Perivenular and centrilobular sinusoidal fibrosis was seen in the obstructive SDC group (n=3, 11.1%). Concurrent graft rejection was present in 7 (25.9%) cases, of which acute cellular rejection comprised 5 (18.5%), and late acute rejection accounted for 2 cases (7.4%). Serum tacrolimus levels ranged from normal (n=14) to below and above normal (n=5 each). Modulation of immunosuppressive therapy led to normalisation of LFTs in one patient. Obstructive and non-obstructive SDC in post-liver transplant patients presenting with transaminitis mimics graft rejection clinically and may represent a form of drug-induced liver injury. Liver biopsy plays a crucial role in the diagnosis.

Deregulation of the Purine Pathway in Pre-Transplant Liver Biopsies Is Associated with Graft Function and Survival after Transplantation

The current shortage of livers for transplantation has increased the use of marginal organs sourced from donation after circulatory death (DCD). However, these organs have a higher incidence of graft failure, and pre-transplant biomarkers which predict graft function and survival remain limited. Here, we aimed to find biomarkers of liver function before transplantation to allow better clinical evaluation. Matched pre- and post-transplant liver biopsies from DCD (n = 24) and donation after brain death (DBD, n = 70) were collected. Liver biopsies were analysed using mass spectroscopy molecular phenotyping. Discrimination analysis was used to parse metabolites differentiated between the two groups. Five metabolites in the purine pathway were investigated. Of these, the ratios of the levels of four metabolites to those of urate differed between DBD and DCD biopsies at the pre-transplantation stage (q < 0.05). The ratios of Adenosine monophosphate (AMP) and adenine levels to those of urate also differed in biopsies from recipients experiencing early graft function (EGF) (q < 0.05) compared to those of recipients experiencing early allograft dysfunction (EAD). Using random forest, a panel consisting of alanine aminotransferase (ALT) and the ratios of AMP, adenine, and hypoxanthine levels to urate levels predicted EGF with area under the curve (AUC) of 0.84 (95% CI (0.71, 0.97)). Survival analysis revealed that the metabolite classifier could stratify six-year survival outcomes (p = 0.0073). At the pre-transplantation stage, a panel composed of purine metabolites and ALT could improve the prediction of EGF and survival.

Use of Nakanuma staging and cytokeratin7 staining for diagnosing recurrent primary biliary cholangitis after living-donor liver transplantation.

Diagnosis of primary biliary cholangitis (PBC), which recurs in approximately 30% of liver transplant recipients, is histology-based, but no staging system has been established for recurrent PBC (rPBC). We used the Nakanuma staging system and cytokeratin7 (CK7) staining to examine post-transplant liver biopsy specimens retrospectively and to evaluate histological features of rPBC. From 107 patients who underwent living donor liver transplantation for PBC, 60 recipients with 214 liver biopsies after 1-year post transplant were enrolled. Fibrosis, bile duct loss (BL), cholangitis activity, hepatitis activity, and CK7-positive hepatocytes were scored. Nakanuma staging was based on fibrosis and BL scores. We examined the correlation of scores and clinicolaboratory data among rPBC patients. We also evaluated whether chronological change of stage was correlated with liver-related failure. Of 214 biopsies, 52 were protocol biopsy; 162 were episodic. Higher BL, cholangitis activity, and hepatitis activity scores were associated with rPBC diagnosis. At median follow up of 10.0years (range 1.4-18.7years), 29 (48%) patients were diagnosed with rPBC at 4.6years (range 1.3-14.5years). Liver-related failure occurred in five rPBC cases; three from rPBC, and two from chronic rejection. At rPBC diagnosis, higher BL and CK7 scores were more frequent in patients who developed liver-related failure than in other patients (P = 0.04, P < 0.01, respectively). In failure patients, the Nakanuma stage increased over time, and reached up to stage4, whereas the Scheuer stage did not reach above stage3. Nakanuma staging is associated with rPBC and disease progression. Scores for BL and CK7 might be early markers for progressive rPBC.

Serum miR-33a is associated with steatosis and inflammation in patients with non-alcoholic fatty liver disease after liver transplantation.

Background & aimsMiR-33a has emerged as a critical regulator of lipid homeostasis in the liver. Genetic deficiency of miR-33a aggravates liver steatosis in a preclinical model of non-alcoholic fatty liver disease (NAFLD), and relative expression of miR-33a is increased in the livers of patients with non-alcoholic steatohepatitis (NASH). It was unknown whether miR-33a is detectable in the serum of patients with NAFLD. We sought to determine whether circulating miR-33a is associated with histological hepatic steatosis, inflammation, ballooning or fibrosis, and whether it could be used as a serum marker in patients with NAFLD/NASH.MethodsWe analysed circulating miR-33a using quantitative PCR in 116 liver transplant recipients who underwent post-transplant protocol liver biopsy. Regression analysis was used to determine association of serum miR-33a with hepatic steatosis, inflammation, ballooning and fibrosis in liver biopsy.ResultsLiver graft steatosis and inflammation, but not ballooning or fibrosis, were significantly associated with serum miR-33a, dyslipidemia and insulin resistance markers on univariate analysis. Multivariate analysis showed that steatosis was independently associated with serum miR-33a, ALT, glycaemia and waist circumference, whereas inflammation was independently associated with miR-33a, HbA1 and serum triglyceride levels. Receiver operating characteristic analysis showed that exclusion of serum miR-33a from multivariate analysis resulted in non-significant reduction of prediction model accuracy of liver steatosis or inflammation.ConclusionsOur data indicate that circulating miR-33a is an independent predictor of liver steatosis and inflammation in patients after liver transplantation. Although statistically significant, its contribution to the accuracy of prediction model employing readily available clinical and biochemical variables was limited in our cohort.

Sinusoidal and pericellular fibrosis in adult post-transplant liver biopsies: association with hepatic stellate cell activation and patient outcome

Post-transplant sinusoidal fibrosis (SF) and pericellular fibrosis (PCF) have not been extensively investigated in adults. Fifty-two post-transplant liver biopsies from 28 consented patients (12 men, mean age 49, range 33–67 years) were studied. Tissue morphology, including an arbitrary summative fibrosis score was assessed in detail. Collagen proportionate area (CPA) and alpha-smooth muscle actin (α-SMA) immunostain were evaluated by digital image analysis (DIA). Anti-keratin 7, anti-C4d and anti-sonic hedgehog (Shh) immunostains were scored semi-quantitatively. SF was observed in 36/52 (69.2%) biopsies and most of these (20/36, 55.6%) had centrilobular fibrosis (CLF). PCF was seen in 7/52 (13.5%) biopsies exclusively in cases with CLF. CPA was significantly correlated with time since liver transplantation (p = 0.043), summative fibrosis score and its main components but not with α-SMA. α-SMA-positive area significantly correlated with the Banff rejection score (p = 0.022) and centrilobular inflammatory changes were more severe in cases with CLF (p = 0.003). Hepatocyte ballooning of cholestatic type was associated with PCF (p = 0.016) and Shh expression (p < 0.001). Sinusoidal fibrosis is a frequent occurrence in post-transplant adult livers, with predilection toward centrilobular areas. Graft age and oxidative stress may contribute to SF development, while hepatocyte ballooning may be implicated in PCF development. Hepatic stellate cell (HSC) activation is likely affected by centrilobular inflammation.

Allograft Liver Biopsy

Although not ideal, liver biopsy is the best available method for evaluation of the liver and is considered the gold standard. The most common use of liver biopsies in the posttransplantation setting is for diagnosis of allograft dysfunction presenting with abnormal liver chemistry tests. The causes of allograft dysfunction differ according to time. Early on, preservation and reperfusion injury, infection, donor-related disease, and acute rejection are more common. Later, disease recurrence, de novo disease, and chronic rejection are seen more frequently. A complete history and physical examination are followed by ultrasonography with Doppler. If biliary or vascular causes are suspected, further imaging is performed and stents or surgery planned. If these tests are not diagnostic, a liver biopsy is performed. In addition to diagnosis of allograft dysfunction, protocol liver biopsy can be helpful particularly to diagnose disease recurrence, particularly the immune-mediated diseases, as well as to evaluate the patient for eligibility for immunosuppression minimization and possible withdrawal. Given the risks and cost associated with liver biopsy, several methods are used for evaluation of fibrosis and rejection in the liver allograft. Although very promising, these methods have not been widely validated and are not ready for clinical use. This review contains 9 figures, 2 tables, and 54 references. Key Words: biopsy to diagnose allograft liver dysfunction, disease recurrence after liver transplant, immunosuppression withdrawal after liver transplant, liver biopsy to guide immunosuppression minimization, noninvasive methods to evaluate liver allograft, posttransplant diagnostic liver biopsy, preservation and reperfusion injury, protocol liver biopsy after transplant

Allograft Liver Biopsy

Although not ideal, liver biopsy is the best available method for evaluation of the liver and is considered the gold standard. The most common use of liver biopsies in the posttransplantation setting is for diagnosis of allograft dysfunction presenting with abnormal liver chemistry tests. The causes of allograft dysfunction differ according to time. Early on, preservation and reperfusion injury, infection, donor-related disease, and acute rejection are more common. Later, disease recurrence, de novo disease, and chronic rejection are seen more frequently. A complete history and physical examination are followed by ultrasonography with Doppler. If biliary or vascular causes are suspected, further imaging is performed and stents or surgery planned. If these tests are not diagnostic, a liver biopsy is performed. In addition to diagnosis of allograft dysfunction, protocol liver biopsy can be helpful particularly to diagnose disease recurrence, particularly the immune-mediated diseases, as well as to evaluate the patient for eligibility for immunosuppression minimization and possible withdrawal. Given the risks and cost associated with liver biopsy, several methods are used for evaluation of fibrosis and rejection in the liver allograft. Although very promising, these methods have not been widely validated and are not ready for clinical use. This review contains 9 figures, 2 tables, and 54 references. Key Words: biopsy to diagnose allograft liver dysfunction, disease recurrence after liver transplant, immunosuppression withdrawal after liver transplant, liver biopsy to guide immunosuppression minimization, noninvasive methods to evaluate liver allograft, posttransplant diagnostic liver biopsy, preservation and reperfusion injury, protocol liver biopsy after transplant

Predictors of De Novo Nonalcoholic Fatty Liver Disease After Liver Transplantation and Associated Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) can occur de novo in patients undergoing liver transplantation (LT) for indications other than NAFLD, and it has been increasingly recognized as a complication in the post-LT setting. This study aims to better characterize de novo NAFLD after LT by identifying risk factors for its development, describing incidence and extent of fibrosis, assessing the diagnostic utility of noninvasive serum fibrosis algorithms, and comparing survival to those without NAFLD. This was a retrospective single-center analysis of de novo NAFLD in a post-LT cohort. Those whose primary indication for LT was nonalcoholic steatohepatitis (NASH) were excluded. Risk factors were analyzed by univariate and multivariate analyses. De novo NAFLD and fibrosis were assessed on posttransplant liver biopsies, and noninvasive fibrosis scores were calculated from concomitant blood tests. After applying the exclusion criteria, 430 for-cause post-LT biopsies were evaluated; 33.3% (n = 143) had evidence of de novo steatosis and/or NASH at a median of 3.0 years after transplant. On multivariate analysis, body mass index (BMI; odds ratio [OR], 1.12; P < 0.001), diabetes mellitus (OR, 3.01; P = 0.002), hepatitis C virus (OR, 4.61; P < 0.001), weight gain (OR, 1.03; P = 0.007), and sirolimus use (OR, 3.11; P = 0.02) were predictive of de novo NAFLD after LT. Significant fibrosis (≥F2) was present in almost 40% of the cohort. Noninvasive serum fibrosis scores were not useful diagnostic tests. There was no significant difference in the short-term or longterm survival of patients who developed de novo NAFLD. In conclusion, diabetes, BMI, weight gain after LT, and sirolimus-based immunosuppression, in keeping with insulin resistance, were the only modifiable factors associated with development of de novo NAFLD. A significant proportion of patients with de novo NAFLD had fibrosis and given the limited utility of noninvasive serum fibrosis algorithms, alternative noninvasive tools are required to screen for fibrosis in this population. There was no significant difference in the short-term or longterm survival of patients who developed de novo NAFLD.

An Overview of Liver Transplant Pathology: Data from a Tertiary Referral Centre in Western India

Introduction and aim. 1. Study of liver explants - Etiologic types of end-stage chronic liver disease (ESCLD) and acute liver failure (ALF) in adults and children. 2. Assessment of donor steatosis and incidental granulomas. 3. Post-transplant liver biopsies.Material and methods. Specimens of 180 explant hepatectomies, 173 donor wedge and 30 core liver biopsies, and 58 post transplant liver biopsies received in our department from April 2013 to March 2017.Results. 1. Most common causes of ESCLD in adults were: alcohol related (30.32%), hepatitis virus related (18.71%) and non-alcoholic steatohepatitis related (18.06%); and in children < 12 years were: biliary atresia (27.27%), autoimmune disease (18.18%) and Wilson’s disease (18.18%). Most common causes of ALF in adults and children were anti-tubercular therapy induced and idiopathic, respectively. 2. Prevalence rate of moderate steatosis (between 30-60%) was 4.28%. Incidental granulomas were seen in 5 cases. 3. Most common diagnoses of post-transplant biopsies in adults included acute cellular rejection (ACR) (36.17%), recurrence of viral disease (8.51%) and moderate non-specific portal triaditis (8.51%). Among children < 12 years, most common diagnoses included unremarkable liver parenchyma, ACR and ischemia/reper fusion injury.Conclusion. 1. Alcohol- and hepatitis- virus related ESCLD, and biliary atresia are leading indications for liver transplantation in adults and children, respectively. 2. Prevalence of 4.28% of moderate steatosis, is much lower than that documented in western literature. Only 5 cases of incidental granulomas is unexpectedly low in a country endemic for tuberculosis. 3. Most common diagnoses of post-transplant liver biopsies in adults has been acute rejection, which is similar to the findings from much larger published series.

FRI-036 - Long-term post-transplant liver biopsies of children: central perivenulitis is associated with circulating donor-specific antibodies and tissue C4D deposition

FRI-036 - Long-term post-transplant liver biopsies of children: central perivenulitis is associated with circulating donor-specific antibodies and tissue C4D deposition

Liver transplant pathology: When the things are grave!

Post transplant liver biopsies (PTLB) form a cornerstone in management of transplant cases. They provide critical information regarding rejection or reoccurrence of diseases. With the development in understanding of transplant pathology new updates have come in reporting guidelines of liver transplant biopsy specimens. The recent updates also include criteria for reporting of antibody mediated rejection in liver. This review aims to cover all those aspects.

Negative Correlation Between Hepatitis C Virus (HCV) and Let-7 MicroRNA Family in Transplanted Livers: The Role of rs868 Single-Nucleotide Polymorphism

BackgroundGenetic alterations of TGF-β pathway members, including its transmembrane receptor, TGFBR1, may influence the course of HCV infection. Rs868 is a single-nucleotide polymorphism of the 3′UTR region of TGFBR1, located in a binding site for the conserved let-7/miR98 microRNA family. Previously, we demonstrated a favorable course of hepatitis C recurrence after liver transplantation in rs868 AG genotype of the transplanted liver when compared to rs868 AA. The aim of the present study was to confirm the biological effect of rs868.Material/MethodsHepG2 cell line was transfected with luciferase vectors cloned with 3′UTR of TGFBR1 gene encompassing different rs868 alleles. Post-transplant liver biopsies from 61 patients with HCV-related end-stage liver disease were evaluated histopathologically and analyzed for the expression of TGFBR1 mRNA, let-7/miR98 microRNAs, HCV RNA load, and rs868 genotype.ResultsLuciferase expression was significantly lower in the A allele-containing vector. TGFBR1 mRNA and HCV RNA load were correlated negatively with let-7/miR98 microRNAs and this correlation was significantly stronger for rs868 AG compared to AA genotype. A strong positive correlation was demonstrated between TGFBR1 and HCV in both genotypes. In AG heterozygotes, let-7/miR98 microRNAs showed a strong negative correlation with periportal or periseptal interface hepatitis (Ishak A score).ConclusionsThere is a negative correlation between let-7/miR98 microRNAs and HCV viral load and TGFBR1 mRNA after liver transplantation. In the rs868 AG heterozygotes, this correlation was stronger and there was a negative correlation between let-7/miR98 and Ishak A score, which is in concordance with the previously demonstrated protective role of this genotype in post-transplant hepatitis C recurrence.

P048 NGS performance on a transplanted liver biopsy sample: A case study

A male pediatric patient was diagnosed with biliary atresia and was transplanted with liver from a deceased donor. The surgery went well, but years later the patient developed antibodies to Cw12, DR14, DQ7 and DQA1∗04:01 and various DP alleles. The need for high resolution typing to determine the identity of de novo DSA and the lack of any remaining pre-transplant sample on a deceased donor led to the use of NGS to determine chimeric typing. Genomic DNA was extracted from a post-transplant liver biopsy from this patient by using Gentra Puregene Tissue kit. We used the Immucor NGS FLEX HLA typing system for amplification and sequencing of HLA genes HLA -A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 followed by analysis with MIA FORA software. The NGS methodology is a clonal sequencing approach which solves the phase ambiguity problems inherent with Sanger-based sequencing when alleles are amplified and sequenced together. The genomic DNA extracted from the transplanted liver biopsy is chimeric, with residual recipient DNA at very low but detectable levels. The presence of chimeric reads is the result of co-amplification of both donor and recipient HLA genes. The majority of sequencing reads with zero mismatches belonged to the donor and were concordant with the deceased donor HLA type provided by UNOS. Unclaimed sequencing reads appeared as grey background and derived from the recipient HLA type (previously obtained with pre-transplant samples). In this case and a few other chimeric cases examined in our laboratory, the MIA FORA NGS software detected the HLA type of donor as the called alleles and showed some unclaimed reads that did not align to the donor type but rather to the HLA type of recipient. We note that if the donor and recipient HLA genes are perfectly matched in one or both alleles then they are indistinguishable. The clonal sequencing nature of the NGS reveals PCR primer specificity by identifying the sequences of all co-amplified genomic regions within the intended target region in the same biopsy sample. This case demonstrates the clinical utility of high resolution HLA typing by NGS technology in the clinical setting to de convolute a solid organ biopsy admixture i.e. the ability to do chimeric typing.

Transmission of Hepatitis A Virus through Combined Liver-Small Intestine-Pancreas Transplantation.

Although transmission of hepatitis A virus (HAV) through blood transfusion has been documented, transmission through organ transplantation has not been reported. In August 2015, state health officials in Texas, USA, were notified of 2 home health nurses with HAV infection whose only common exposure was a child who had undergone multi-visceral organ transplantation 9 months earlier. Specimens from the nurses, organ donor, and all organ recipients were tested and medical records reviewed to determine a possible infection source. Identical HAV RNA sequences were detected from the serum of both nurses and the organ donor, as well as from the multi-visceral organ recipient's serum and feces; this recipient's posttransplant liver and intestine biopsy specimens also had detectable virus. The other organ recipients tested negative for HAV RNA. Vaccination of the donor might have prevented infection in the recipient and subsequent transmission to the healthcare workers.

Retrospective analysis of post-transplant liver biopsies – From diagnosis to therapy – Can we guide further? Experience from a tertiary care center in India

Background and Aim: Post-transplant liver biopsies form a critical part of management of complications arising post-transplant. The objective of this study was to analyze the Indian experience in pathologic diagnosis of liver biopsies after orthotopic liver transplantation (OLT) with special emphasis on cases presenting with intrahepatic cholestasis (IHC). Type, incidence, and timing of major complications were analyzed. All cases with IHC were retrospectively analyzed with clinical inputs to look for cryptic clues in subclassifying such cases. Materials and Methods: Forty-five post-transplant liver biopsies from 39 OLT patients were retrospectively analyzed from May 2015 to May 2016. All biopsies were stained with hematoxylin and eosin and Masson's Trichrome, and other stains were performed as required. Results: The number of liver biopsies performed for each patient ranged from 1 to 3. The timing of these biopsies varied from 5 days to >4 years post-transplant. Of the 39 patients who underwent post-transplant liver biopsies, most common etiology of a liver transplant was hepatitis C virus (HCV)-related chronic liver disease in 66.6% cases. The common complications post-transplant were acute cellular rejection (ACR) (33.3%), biliary stricture (13.3%), HCV recurrence (11.1%), plasma cell hepatitis (4.4%), chronic hepatitis (4.4%), IHC (22.2%), and others. On analysis of post transplant biopsy cases with IHC, we found that patients with high baseline HCV RNA levels had recurrences presenting only with prominent IHC without fibrosis and ballooning of hepatocytes. These changes might represent early stages of fibrosing cholestatic hepatitis (FCH). Conclusions: This study evaluated the types, incidence, and timing of major complications occurring after OLT. ACR remains major complication following transplant. The presence of IHC on biopsy, especially in HCV-positive patients, should prompt anti-HCV therapy even if other features of FCH were not found.

Oval Cells Contribute to Fibrogenesis of Marginal Liver Grafts under Stepwise Regulation of Aldose Reductase and Notch Signaling.

Background and Aims: Expanded donor criteria poses increased risk for late phase complications such as fibrosis that lead to graft dysfunction in liver transplantation. There remains a need to elucidate the precise mechanisms of post-transplant liver damage in order to improve the long-term outcomes of marginal liver grafts. In this study, we aimed to examine the role of oval cells in fibrogenic development of marginal liver grafts and explore the underlying mechanisms.Methods: Using an orthotopic rat liver transplantation model and human post-transplant liver biopsy tissues, the dynamics of oval cells in marginal liver grafts was evaluated by the platform integrating immuno-labeling techniques and ultrastructure examination. Underlying mechanisms were further explored in oval cells and an Aldose reductase (AR) knockout mouse model simulating marginal graft injury.Results: We demonstrated that activation of aldose reductase initiated oval cell proliferation in small-for-size fatty grafts during ductular reaction at the early phase after transplantation. These proliferative oval cells subsequently showed prevailing biliary differentiation and exhibited features of mesenchymal transition including dynamically co-expressing epithelial and mesenchymal markers, developing microstructures for extra-cellular matrix degradation (podosomes) or cell migration (filopodia and blebs), and acquiring the capacity in collagen production. Mechanistic studies further indicated that transition of oval cell-derived biliary cells toward mesenchymal phenotype ensued fibrogenesis in marginal grafts under the regulation of notch signaling pathway.Conclusions: Oval cell activation and their subsequent lineage commitment contribute to post-transplant fibrogenesis of small-for-size fatty liver grafts. Interventions targeting oval cell dynamics may serve as potential strategies to refine current clinical management.

Host conditioning and rejection monitoring in hepatocyte transplantation in humans

Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Here we aimed to use preparative liver-directed radiation therapy, and continuous monitoring for possible rejection in an attempt to overcome these limitations. Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression. Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67+ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343±48μM (normal 30-119μM) to 854±25μM (treatment goal ≤360μM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900μM during supervised follow-up, but graft loss occurred after follow-up became inconsistent. Radiation preconditioning and serial rejection risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure. Hepatocyte transplantation can potentially be used to treat genetic liver disorders but its application in clinical practice has been impeded by inefficient hepatocyte engraftment and the inability to monitor rejection of transplanted liver cells. In this study, we first show in non-human primates that pretreatment of the host liver with radiation improves the engraftment of transplanted liver cells. We then used this knowledge in a series of clinical hepatocyte transplants in patients with genetic liver disorders to show that radiation pretreatment and rejection risk monitoring are safe and, if optimized, could improve engraftment and long-term survival of transplanted hepatocytes in patients.