Pedro López-Cillero

Impact of donor graft steatosis on overall outcome and viral recurrence after liver transplantation for hepatitis C virus cirrhosis

The aim of this study was to determine the influence of donor graft steatosis on overall outcome, viral recurrence, and fibrosis progression in orthotopic liver transplantation (OLT) for hepatitis C virus (HCV) cirrhosis. One hundred twenty patients who underwent OLT for HCV cirrhosis between 1995 and 2005 were included in the study. Donor steatosis was categorized as absent (0%‐10%; n = 40), mild (10%‐30%; n = 32), moderate (30%‐60%; n = 29), or severe (>60%; n = 19). A Cox multivariate analysis for marginal donor variables and a Model for End‐Stage Liver Disease index were performed. Fibrosis evolution was analyzed in liver biopsies (fibrosis < 2 or ≥2) 3, 6, and 12 months post‐OLT and in the late post‐OLT period. Fifty‐six grafts were lost (46%). The survival of the grafts was inversely proportional to donor liver steatosis: 82%, 72%, and 72% at 1, 2, and 3 years post‐OLT in the absence of steatosis; 73%, 63%, and 58% with mild steatosis; 74%, 62%, and 43% with moderate steatosis; and 62%, 49%, and 42% with severe steatosis (P = 0.012). HCV recurrence was earlier and more frequent in recipients with steatosis > 30% (46% versus 32% at 3 months, P = 0.017; 58% versus 43% at 6 months, P = 0.020; 70% versus 56% at 12 months, P = 0.058; and 95% versus 69% at 3 years post‐OLT, P = 0.0001). Graft survival was lower in alcoholic liver disease recipients versus HCV recipients when steatosis was >30% at 3, 6, and 12 months post‐OLT (P = 0.042) but not when steatosis was <30% (P = 0.53). A higher fibrosis score was obtained 3 months post‐OLT (P = 0.033), 6 months post‐OLT (P = 0.306), 12 months post‐OLT (P = 0.035), and in the late post‐OLT period (P = 0.009). In conclusion, donor graft steatosis influences the outcome of OLT for HCV cirrhosis. HCV recurrence is more frequent and earlier in recipients of moderately and severely steatotic livers. Fibrosis evolution is higher when graft steatosis is >30%. OLT with >30% steatotic donor livers should be precluded in HCV recipients. Liver Transpl 15:37–48, 2009.

A Prospective Study of the Efficacy of Clinical Application of a New Carrier-Bound Fibrin Sealant After Liver Resection

OBJECTIVE To examine the effectiveness of fibrin sealants as supportive treatment to improve hemostasis and decrease the incidence of bile leakage and intra-abdominal collections. DESIGN Prospective, controlled, quasiexperimental study. SETTING Tertiary referral center, University Hospital Reina Sofía. PATIENTS A total of 115 patients (58 in the control group and 57 in the collagen sponge group) scheduled for conventional hepatectomies. INTERVENTIONS Patients were distributed into groups for major and minor hepatectomies with or without application of a carrier-bound collagen sponge on the raw surface of the liver. MAIN OUTCOME MEASURES The main outcome measures were postoperative mortality, incidence and severity of postoperative surgical complications, and length of hospital stay. The secondary outcome measures were postoperative drainage output volume, transfusion requirements, and changes in biochemical parameters (hemoglobin, bilirubin, alanine aminotransferase, and platelet levels). RESULTS The fibrin sealant after major liver resection was effective for decreasing drainage volume (mean [SD] volume, 1124.7 [842.8] mL in the control group and 691.2 [499.5] mL in the collagen sponge group; P = .007) with a higher volume of output by drain each postoperative day in the control patients (P = .003); postoperative blood transfusion requirements (18.9% vs 7.0%, respectively; P = .04); moderate to severe postoperative complications (21% vs 8%, respectively; P = .03); and mean (SD) hospital stay (12.6 [6.7] vs 9.6 [5.1] days, respectively; P = .03). CONCLUSION The use of a new carrier-bound collagen sponge after major liver resection may be recommended because of its clinical and cost-savings effectiveness.

Prediction of graft dysfunction based on extended criteria donors in the model for end-stage liver disease score era.

Background. To explain the influence of recipient status combined with the accumulation of extended criteria donor (ECD) variables on the appearance of severe ischemia-reperfusion injury and graft survival in a model for end-stage liver disease (MELD)-based system, we analyzed our most recent consecutive liver transplantations (LTs), dividing them into two periods: 400 LTs (1992–2002; pre-MELD era) and 275 LTs (2002–2007; post-MELD era). Methods. Primary dysfunction (PD) was defined as primary graft failure that required emergency retransplantation or as initial poor function. Donor variables were included in a regression model to assess the probability of PD. Results. Donor age, macrovesicular steatosis more than 30%, and cold ischemia time were associated with allograft dysfunction. Mean probability of PD was 14.8%, 19.2%, 27.5%, and 37.4% for ECD 0, 1, 2, and more than or equal to 3, respectively (P=0.003). Distribution of no-mild, moderate, and severe ischemia-reperfusion injuries among MELD categories was 72.53%, 24.17%, and 3.30% (MELD group=12–19); 56.52%, 36.96%, and 6.5% (MELD group=20–28); and 23.91%, 54.35%, and 21.74% (MELD group ≥29), respectively (P=0.043). The development of PD according to ECD variables was 18.8%, 18.1%, 28.0%, and 35.3% for ECD 0, 1, 2, and more than or equal to 3, respectively (P=0.047). These variables were independent predictors of PD (Cox proportional regression model): ECD 2 (relative risk [RR]=1.59; 95% confidence interval [CI]=1.25–1.62), ECD 3 (RR=2.74; 95% CI=2.38–3.13), MELD 21 to 30 (RR=1.89; 95% CI=1.32–2.06), and MELD more than or equal to 30 (RR=3.38; 95% CI=2.43–3.86). Graft survival decreased, whereas MELD and the number of ECD variables increased. Conclusion. The combination of three or more ECD variables and an MELD more than or equal to 29 is the worst scenario for graft success after LT.

N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by D-galactosamine in primary culture of human hepatocytes

D-Galactosamine (D-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q(10) (Q(10)) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in D-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q(10) (30 microM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1mg/mL) were co-administered with D-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q(10) ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q(10) ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q(10) ratios, and a recovery of mitochondrial complexes I+III and II+III activities and cellular ATP content. The co-administration of Q(10) or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by D-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q(10) and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by D-GalN in cultured hepatocytes.

Alteration of S‐nitrosothiol homeostasis and targets for protein S‐nitrosation in human hepatocytes

The liver is one organ clearly influenced by nitric oxide (NO), and acute and chronic exposure to this substance has been associated with distinct patterns of liver disease. Disruption or deregulation of S‐nitrosothiol (SNO) signalling leads to impairment of cellular function and disease, and this study was aimed to identify potential targets for protein S‐nitrosation during alteration of SNO homeostasis in human hepatocytes. Cells were treated with S‐nitroso‐L‐cysteine (CSNO), an effective physiological nitrosothiol for delivering NO bioactivity to cells. Treatment with CSNO augmented the levels of S‐nitrosoproteins detected both by chemiluminescence and the biotin switch method. CSNO treatment also increased S‐nitrosoglutathione reductase (GSNOR) activity that returned SNO content to basal levels. This increased enzymatic activity was related to augmented levels of ADH‐5 mRNA, the gene encoding for GSNOR in humans. In addition, the treatment with the SNO also increased cell death. Twenty S‐nitrosoproteins were identified in CSNO‐treated hepatocytes, including mitochondrial aldehyde dehydrogenase, protein disulphide isomerase, Hsp60, GRP75 and Raf kinase inhibitor protein. The identification in the S‐nitrosatable proteome of proteins involved in metabolism, maintenance of cellular homeostasis and signalling points to the relevance of protein S‐nitrosation to the physiology and pathophysiology of human hepatocytes.

Cardiotrophin-1 reduces ischemia/reperfusion injury during liver transplant

BACKGROUND Orthotopic liver transplantation (OLT) is currently the elective treatment for advanced liver cirrhosis and acute liver failure. Ischemia/reperfusion damage may jeopardize graft function during the postoperative period. Cardiotrophin-1 (CT-1) has demonstrated cytoprotective properties in different experimental models of liver injury. There is no evidence to demonstrate its potential use in the prevention of the ischemia/reperfusion injury that occurs during OLT. The present study is the first report to show that the administration of CT-1 to donors would benefit the outcome of OLT. MATERIALS AND METHODS We tested the cytoprotective effect of CT-1 administered to the donor prior to OLT in an experimental pig model. Hemodynamic changes, hepatic histology, cell death parameters, activation of cell signaling pathways, oxidative and nitrosative stress, and animal survival were analyzed. RESULTS Our data showed that CT-1 administration to donors increased animal survival, improved cardiac and respiratory functions, and reduced hepatocellular injury as well as oxidative and nitrosative stress. These beneficial effects, related to the activation of AKT, ERK, and STAT3, reduced caspase-3 activity and diminished IL-1β and TNF-α expression together with IL-6 upregulation in liver tissue. CONCLUSIONS The administration of CT-1 to donors reduced ischemia/reperfusion injury and improved survival in an experimental pig model of OLT.

Identification of candidate biomarkers for hepatocellular carcinoma in plasma of HCV‐infected cirrhotic patients by 2‐D DIGE

The current methods available for screening and detecting hepatocellular carcinoma (HCC) have insufficient sensitivity and specificity, and only a low percentage of diagnosis of small tumours is based on these assays. Because HCC is usually asymptomatic at potentially curative stages, identification of biomarkers for the early detection of HCC is essential to improve patient survival.

Nitric oxide mimics transcriptional and post-translational regulation during α-Tocopherol cytoprotection against glycochenodeoxycholate-induced cell death in hepatocytes

BACKGROUND & AIMS Reactive oxygen species (ROS) and nitric oxide (NO) exert a relevant role during bile acid-induced hepatotoxicity. Whether α-Tocopherol regulates oxidative and nitrosative stress, bile acid transporter expression and their NO-dependent post-translational modifications, and cell death were assessed in vitro and in vivo. METHODS α-Tocopherol and/or NO donors (DETA-NONOate or CSNO, and V-PYRRO/NO) were administered to glycochenodeoxycholic acid (GCDCA)-treated cultured human hepatocytes or to bile duct obstructed rats. Cell injury, superoxide anion (O⁻₂) production, as well as inducible nitric oxide synthase (NOS-2), cytochrome P4507A1 (CYP7A1), heme oxygenase-1, (HO-1) and bile acid transporter expression were determined. Cysteine S-nitrosylation and tyrosine nitration of Na(+)-taurocholate co-transporting polypeptide (NTCP), as well as taurocholic acid (TC) uptake were also evaluated. RESULTS GCDCA-induced cell death was associated with increased (O⁻₂) production, NTCP and HO-1 expression, and with a reduction of CYP7A1 and NOS-2 expression. α-Tocopherol reduced cell death, (O⁻₂) production, CYP7A1, NTCP, and HO-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and tyrosine nitration, and reduced TC uptake in hepatocytes. α-Tocopherol and V-PYRRO/NO reduced liver injury and NTCP expression in obstructed rats. CONCLUSIONS The regulation of CYP7A1, NTCP, and HO-1 expression may be relevant for the cytoprotective properties of α-Tocopherol and NO against mitochondrial dysfunction, oxidative stress and cell death in GCDCA-treated hepatocytes. The regulation of NO-dependent post-translational modifications of NTCP by α-Tocopherol and NO donors reduces the uptake of toxic bile acids by hepatocytes.

Cytoprotective properties of rifampicin are related to the regulation of detoxification system and bile acid transporter expression during hepatocellular injury induced by hydrophobic bile acids

Background/PurposeRifampicin has been used for the treatment of patients with jaundice and pruritus. This study evaluated the effect of rifampicin on the expression of different detoxification systems and bile acid transporters during in-vivo and in-vitro experimental models of cholestasis.MethodsRifampicin was administered to glycochenodeoxycholic acid (GCDCA)-treated human hepatocytes and bile duct-obstructed rats. Different parameters related to cell death, and the expression of phase I and II drug metabolizing enzymes (DME) and bile acid transporters were determined.ResultsThe induction of hepatocellular injury induced by cholestasis was associated with a reduction in cytochrome P4503A4 (CYP3A4), CYP7A1, and UDP-glucuronosyltransferase 2B4 (UGT2B4) expression, as well as an increase in import (Na+-taurocholate co-transporting polypeptide, NTCP) system expression. The beneficial properties of rifampicin were associated with an increase in DME and export bile acid systems (multidrug resistance-associated protein 4, MRP4, and bile acid export pump to bile duct, BSEP) expression, as well as a reduction in NTCP expression.ConclusionsThe beneficial effect of rifampicin in cholestasis is associated with an increase in DME expression involved in toxic, bile acid and cholesterol metabolism, as well as a reduction in the bile acid importing system in hepatocytes.

S-nitrosation of proteins during d-galactosamine-induced cell death in human hepatocytes

Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocytes.