José Mir

HCV-related fibrosis progression following liver transplantation: increase in recent years

BACKGROUND/AIMS The natural history and predictors of HCV-related disease severity post-transplantation are uncertain. The aims of this study were to define the natural history of post-transplantation HCV infection by assessing the rate of fibrosis progression, to determine if the post-transplantation natural history differs from that observed pre-transplantation, and to identify predictors of post-transplantation disease progression. METHODS Post-transplantation biopsies (mean: 3+/-1.6/patient) from 284 patients were scored according to histologic stage, using the method of Desmet et al. Change in fibrosis score (fibrosis progression/year) post-transplantation was used as the primary outcome. Predictors analyzed included viral factors (genotype and viral load at transplantation), patient demographics, year of transplantation, country of transplantation, pre-transplantation fibrosis progression, immunosuppression and laboratory data. RESULTS There was a linear association between change in fibrosis score and time from transplantation, with a median rate of fibrosis progression per year of 0.3 (0.004-2.19/year). Using parametric time-to-event analysis, the expected median duration to cirrhosis was 10 years. The rate of post-transplantation fibrosis progression was significantly higher than pre-transplantation (0.2/year (0.09-0.8) p<0.0001), and higher in Spanish than US centers (0.48 (0.01-2.19) vs 0.28 (0.004-2.08); p=0.09) despite similar progression rates prior to transplantation. Variables independently associated with post-transplantation progression included year of transplantation (p=0.0001), race (p=0.02), number of methyl-prednisolone boluses (p=0.03), and HCV RNA levels at transplantation (p=0.01). CONCLUSIONS HCV-related disease progression is accelerated in immunocompromised compared to immunocompetent patients, with a progressive increase in patients who have recently undergone liver transplantation. Changes in patient management post-transplantation over time and between transplant centers may account for the increase in fibrosis progression observed in recent years.

De Novo Internal Neoplasms after Liver Transplantation: Increased Risk and Aggressive Behavior in Recent Years?

The goal of the study was to determine the incidence and variables associated with post‐liver transplantation (LT) de novo internal neoplasms development, excluding skin tumors and hepatocellular carcinoma. Medical records were reviewed for recipient/donor demographics, viral serology, cause of liver disease, interval from LT to tumor diagnosis, predisposing factors, immunosuppression and survival. Forty‐one neoplasms (31 solid and 10 hematologic) developed in 772 recipients (5.3%) transplanted between 1991 and 2001. Time to tumor diagnosis was longer in patients transplanted before 1995 than in those transplanted afterwards (58 vs. 22 months; p < 0.05). Hematologic neoplasms (HN) appeared earlier than solid (2 vs. 21 months; p < 0.001), were more prevalent in those transplanted after 1995 than before (32% vs. 12.5%), and had lower survival than solid (2 vs. 21 months, p < 0.001). While HCV was the most frequent indication in HN (70%), alcohol was that of solid tumors (71%). Overall, risk factors for de novo neoplasms included alcohol and immunosuppression (p < 0.01). In patients undergoing LT in recent years, there is a higher incidence of HN with de novo internal neoplasms developing at earlier time‐points than in those transplanted years ago. Risk factors for tumor development include alcohol, HCV and possibly strong immunosuppression.

Potential Impact of Steatosis on Cytochrome P450 Enzymes of Human Hepatocytes Isolated from Fatty Liver Grafts

Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of “cellular steatosis” by incubation of cultured hepatocytes with increasing concentrations (0.25–3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45–65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.

Expanded criteria for liver transplantation in patients with cirrhosis and hepatocellular carcinoma

Orthotopic liver transplantation (OLT) selection for patients with hepatocellular carcinoma (HCC) is a matter of debate. The Milan criteria (MC) have been largely adopted by the international community. The main aim of this study was to evaluate the survival rates and recurrence probabilities of a new proposal for criteria (up to 3 tumors, each no larger than 5 cm, and a cumulative tumor burden ≤ 10 cm). Patients with cirrhosis and HCC included on the waiting list (WL) from 1991 to 2006 were retrospectively analyzed. Outcomes in patients who had tumors within and beyond the MC were compared. The survival analysis was done (1) with the intention‐to‐treat principle and (2) among transplanted patients. A total of 281 patients were included in WL. Twenty‐four cases did not undergo OLT (a dropout rate of 8.5%); all but 1 case had tumors within the MC. Of the 257 transplanted patients, 26 had tumors beyond the MC in the pre‐OLT evaluation. Based on the intention‐to‐treat analysis, the 5‐year survival was 56% versus 66% in patients who had tumors within and beyond the MC, respectively (P = 0.487). Among transplanted patients, the 5‐year survival was 62% versus 69%, respectively (P = 0.734). Through multivariate analysis, microvascular invasion was an independent prognostic factor of poor survival (P = 0.004). The recurrence probabilities at 1 and 5 years were 7% versus 12% and 14% versus 28% in patients with tumors within and beyond the MC, respectively (P = 0.063). The multivariate analysis demonstrated that both poorly differentiated tumors (P < 0.001) and microvascular invasion (P < 0.001) increased the risk of recurrence. The expansion to up to 3 nodules, each up to 5 cm, and a cumulative tumor burden ≤ 10 cm did not result in a reduction of survival in comparison with patients who had tumors within the MC. Liver Transpl 14:1449–1460, 2008.

Lessons learned from anatomic variants of the hepatic artery in 1,081 transplanted livers

The aim of this study is to contribute our experience to the knowledge of the anatomic variations of the hepatic arterial supply. The surgical anatomy of the extrahepatic arterial vascularization was investigated prospectively in 1,081 donor cadaveric livers, transplanted at La Fe University Hospital from January 1991 to August 2004. The vascular anatomy of the hepatic grafts was classified according to Michels description (Am J Surg 1966;112:337‐347) plus 2 variations. Anatomical variants of the classical pattern were detected in 30% of the livers (n = 320). The most common variant was a replaced left artery arising from the left gastric artery (9.7%) followed by a replaced right hepatic artery arising from the superior mesenteric artery (7.8%). In conclusion, the information about the different hepatic arterial patterns can help in reducing the risks of iatrogenic complications, which in turn may result in better outcomes not only following surgical interventions but also in the context of radiological treatments. Liver Transpl 13:1401–1404, 2007.

Effect of Calcineurin Inhibitors in the Outcome of Liver Transplantation in Hepatitis C Virus-Positive Recipients

Background. There is a paucity of good studies evaluating the impact of calcineurin inhibitors on posttransplantation outcome in hepatitis C virus (HCV)-infected liver transplant (LT) recipients. Methods. We sought to determine whether there are differences on posttransplantation survival and histologic recurrence in HCV-LT recipients based on initial immunosuppression (IS) by conducting a prospective study comparing tacrolimus (Tac) versus cyclosporine-based IS in patients undergoing LT between 2001 and 2007. Protocol liver biopsies were performed. Results. Baseline characteristics (demographics, liver function at LT, genotype distribution, donor, surgery, and IS except for the type of calcineurin inhibitor) did not differ between groups. Severe disease (defined as bridging fibrosis, cirrhosis, cholestatic hepatitis, or allograft loss or death because of recurrent disease in the first year) was present in 67 of 253 (26.5%) and was equally distributed in the CsA and Tac groups (27% vs. 26%; P=0.68). Two thirds of protocol biopsies performed at 1 year showed some fibrosis without differences between CsA and Tac groups (75% vs. 70%). Advanced fibrosis (bridging fibrosis and cirrhosis) was diagnosed in 30% CsA and 24.5% Tac patients (P=NS). No differences in survival at 1 and 7 years were observed (83% and 67% vs. 78% and 64%, respectively, P=0.4). In summary, in patients undergoing LT for HCV-related liver disease, posttransplantation outcome is not related to the calcineurin inhibitor used.

Cirrhosis of mixed etiology (hepatitis C virus and alcohol): Posttransplantation outcome—Comparison with hepatitis C virus–related cirrhosis and alcoholic‐related cirrhosis

Hepatitis C virus (HCV)‐related liver disease is enhanced by alcohol consumption. Of HCV‐related liver transplantation (LT) recipients, 25% have a history of alcohol intake. The purpose of this research was to determine whether LT outcome differs between patients with cirrhosis of mixed etiology compared to HCV or alcohol alone. Of 494 LT (1997‐2001), recipient/donor features, post‐LT histological, metabolic complications [hypertension, diabetes–diabetes mellitus (DM)], and de novo tumors were compared in 3 groups [HCV‐related cirrhosis = 170 (HCV group), alcohol‐related cirrhosis (alcohol group) = 107, and cirrhosis of mixed etiology (mixed group) = 60]. Protocol biopsies were done in HCV patients. Severe recurrent HCV disease was defined as: 1‐year fibrosis >1, cholestatic hepatitis, recurrent cirrhosis, or HCV‐related liver retransplantation (reLT) within 5 years. Patients in the mixed group were younger (mean age: HCV group = 59 years; mixed group = 49 years; alcohol group = 53 years; P < 0.05) and mainly men (% men: HCV group = 51%; mixed group = 97%; alcohol group = 87%). Hepatocellular carcinoma (HCC) was more frequent in HCV patients (HCV group = 44%; mixed group = 35%; alcohol group = 18%; P = 0.05). Five‐year survival was lowest in the HCV group (HCV group = 49% versus mixed group = 73% versus alcohol group = 76%; and P < 0.01 for the HCV group versus the alcohol group or the HCV group versus the mixed group; P = 0.74 for the alcohol group versus the mixed group). Metabolic complications and de novo tumors were more frequent in the alcohol groups. Severe HCV disease was similar in the HCV+ groups (HCV group = 45%; mixed group = 45%; P = 0.66). Patients with in the mixed group were more frequently treated with antivirals (32% versus HCV group = 18%; P = 0.03). In HCV patients, factors independently associated with lower survival were older donor age, LT indication (HCV alone), and increased body mass index (BMI). Antiviral therapy was a protective factor. Post‐LT survival was lower in the isolated HCV group compared to the alcohol or mixed groups despite a similar recurrence of HCV disease. A greater use of antiviral therapy in the mixed group may explain these differences. The incidence of metabolic complications and de novo tumors was greater in the alcohol groups. Liver Transpl 15:79–87, 2009.

Functional Assessment of the Quality of Human Hepatocyte Preparations for Cell Transplantation

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM ClNH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.

Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using β-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

Influence of Preservation Solution on the Isolation and Culture of Human Hepatocytes from Liver Grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 ± 41% vs. 79 ± 14%, p < 0.05), yield of cells (4.0 ± 5.2 × 106 vs. 8.2 ± 5.6 × 106 cells/g, p < 0.05), and protein content at 24 h of culture (0.6 ± 0.6 vs. 1.2 ± 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.