Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products.

BACKGROUND AIMS: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). METHODS: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. RESULTS: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. CONCLUSIONS: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Metformin modifies glutamine metabolism in an in vitro and in vivo model of hepatic encephalopathy.

AIM: to analyze the effect of metformin on ammonia production derived from glutamine metabolism in vitro and in vivo. METHODS: twenty male Wistar rats were studied for 28 days after a porto-caval anastomosis (n = 16) or a sham operation (n = 4). Porto-caval shunted animals were randomized into two groups (n = 8) and either received 30 mg/kg/day of metformin for two weeks or were control animals. Plasma ammonia concentration, Gls gene expression and K-type glutaminase activity were measured in the small intestine, muscle and kidney. Furthermore, Caco2 were grown in different culture media containing glucose/glutamine as the main carbon source and exposed to different concentrations of the drug. The expression of genes implicated in glutamine metabolism were analyzed. RESULTS: metformin was associated with a significant inhibition of glutaminase activity levels in the small intestine of porto-caval shunted rats (0.277 ± 0.07 IU/mg vs 0.142 ± 0.04 IU/mg) and a significant decrease in plasma ammonia (204.3 ± 24.4 µg/dl vs 129.6 ± 16.1 µg/dl). Glucose withdrawal induced the expression of the glutamine transporter SLC1A5 (2.54 ± 0.33 fold change; p < 0.05). Metformin use reduced MYC levels in Caco2 and consequently, SLC1A5 and GLS expression, with a greater effect in cells dependent on glutaminolytic metabolism. CONCLUSION: metformin regulates ammonia homeostasis by modulating glutamine metabolism in the enterocyte, exerting an indirect control of both the uptake and degradation of glutamine. This entails a reduction in the production of metabolites and energy through this pathway and indirectly causes a decrease in ammonia production that could be related to a decreased risk of HE development.

Oxidized low-density lipoprotein antibodies/high-density lipoprotein cholesterol ratio is linked to advanced non-alcoholic fatty liver disease lean patients.

BACKGROUND AND AIM: A small but significant proportion of patients with normal body mass index show non-alcoholic fatty liver disease (NAFLD). Oxidized low-density lipoprotein (LDL) is a powerful immunogenic molecule, which causes oxidative stress and produces antibodies (oxLDL-ab). We aimed to analyze the role of oxLDL-ab on histological features in lean-NAFLD patients. METHODS: Seventy-two biopsy-proven NAFLD patients were included. Lean patients showed body index mass of <30 kg/m(2) . Liver biopsies were assessed by one pathologist blinded to clinical data. Histological features were non-alcoholic steatohepatitis (NASH), steatosis, hepatocellular ballooning, and liver fibrosis. Metabolic and hepatic profiles were analyzed, and lipid-lowering medication was recorded. OxLDL-ab levels were measured by ELISA. OxLDL-ab-based lipid indexes analyzed: oxLDL-ab/total cholesterol ratio; oxLDL-ab/LDL-c ratio; oxLDL-ab/high-density lipoprotein cholesterol (HDL-c) ratio; and oxLDL-ab/oxLDL ratio. RESULTS: Lean-NAFLD patients presented 26.5% (9/34) of NASH. OxLDL-ab/HDL-c ratio (r = 0.570; n = 34; P = 0.001) correlated with NAS score and was the only variable associated with NASH in the multivariate analysis [odds ratio, OR, 1.10 (95% confidence interval, CI: 1.01-1.21); P = 0.039]. Severe steatosis was present in 41.2% (14/34) of lean-NAFLD patients. OxLDL-ab/HDL-c ratio was higher in patients with grade-III steatosis (54.9 (37.3-124.6)) than those with grade II (37.1 (20.2-71.1)) and grade I (17.7 (13.1-22.8)) (P = 0.018). Hepatocellular ballooning was present in 20.6% (7/34) of lean-NAFLD patients, and OxLDL-ab/HDL-c ratio (OR 1.03 [95% CI: 1.01-1.05]; P = 0.050) was independently associated with histological features. OxLDL-ab/HDL-c ratio was higher in patients with advanced fibrosis (39.8 (22.9-121.6) vs 17.7 (13.9-30.9); P = 0.025), increasing gradually with the fibrosis stage (P = 0.042) and remained in the final multivariate model [OR 1.05 (95% CI: 1.00-1.11); P = 0.05]. However, in obese-NAFLD patients, oxLDL/HDL-c ratio was not associated with histological features. CONCLUSIONS: Oxidized low-density lipoprotein antibodies/high-density lipoprotein cholesterol ratio could represent an interesting biomarker associated with NASH, hepatocellular ballooning, and liver fibrosis, in lean patients. OxLDL-ab/HDL-c could play an important role for distinguishing patients with and without NAFLD complications.

Hypovitaminosis D and its relation to demographic and laboratory data among hepatitis C patients.

BACKGROUND: The relationship between 25-hydroxyvitamin D [25(OH)D] serum levels and response to antiviral therapy and laboratory data in HCV infection remains unclear. The aim of this study was to determine pre-treatment 25(OH)D serum level among HCV infected individuals and to evaluate the association between vitamin D status, virological response, and laboratory data. MATERIAL AND METHODS: Baseline serum 25(OH)D levels were measured in 237 chronic HCV infected patients (139 female, age 53.7 ± 11.2 years) using chemiluminescence immunoassay. Correlations between serum 25(OH)D levels, virological and laboratory data regarding HCV infection as well as sustained virological response (SVR) to antiviral therapy were evaluated. RESULTS: Mean serum values of 25(OH)D was 26.2 ± 12 ng/mL and prevalence of vitamin D deficiency (< 30 ng/mL) was 66.2%. Advanced age (> 55 years), high mean values of LDL, total cholesterol, HDL and low mean values of alkaline phosphatase and hemoglobin were statistically associated to vitamin D deficiency. Antiviral treatment was underwent by 133 HCV patients and 44.3% of them achieved SVR. Most of individuals that presented SVR also presented 25(OH)D level higher than 30ng/mL (55.9%). SVR was associated to low mean values of LDL, total cholesterol and platelets; high mean values of ALT, AST and low fibrosis grade. CONCLUSIONS: In conclusion, low vitamin D levels were observed among HCV infected patients and was associated to laboratory findings, however baseline 25(OH)D level is not independently associated with SVR.

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

BACKGROUND: 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. AIM: The aim of this study was to identify candidate markers for HCC development in the plasma from hepatitis C virus (HCV)-infected cirrhotic patients. METHODS: We compared protein expression profiles of plasma samples from HCV-infected cirrhotic patients with and without HCC, using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF mass spectrometry. The 2-D DIGE results were analysed statistically using Decyder™ software, and verified by western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: In the plasma of HCV-infected HCC patients, we observed decreased expression of complement component 9, ficolin-3 (FCN3), serum amyloid P component (SAP), fibrinogen-gamma and immunoglobulin gamma-1 chain, and increased expression of vitronectin (VTN) and galectin-3 binding protein (G3BP) by DIGE analysis. ELISA confirmed DIGE results for VTN and G3BP but not for SAP or FCN3 in a larger patient population. CONCLUSIONS: The proteins VTN and SAP are candidate biomarkers for HCC development in HCV-infected cirrhotic patients.

Irisin, a link among fatty liver disease, physical inactivity and insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in industrialized countries. The increasing prevalence of NAFLD mirrors the outbreak of obesity in western countries, highlighting the connection between these two conditions. Nevertheless, there is currently no specific pharmacotherapy for its treatment. Accepted management begins with weight loss and exercise. Moreover, exercise can provide metabolic benefits independently of weight loss. It is known how long-term aerobic training produces improvements in hepatic triglycerides, visceral adipose tissue and free fatty acids, even if there is no weight reduction. A recent study from Boström et al. unravels a potential molecular mechanism that may explain how exercise, independently of weight loss, can potentially improve metabolic parameters through a new messenger system (irisin) linking muscle and fat tissue. Irisin has been proposed to act as a hormone on subcutaneous white fat cells increasing energy expenditure by means of a program of brown-fat-like development. Moreover, it was also shown that irisin plasma concentration was higher in people who exercise, suggesting a molecular mechanism by which exercise may improve metabolism. The present systematic review is based on the possibility that irisin might represent a hypothetical connection between NAFLD pathogenesis and disease progression.

Role of diabetes mellitus on hepatic encephalopathy.

Hepatic encephalopathy is the main cognitive dysfunction in cirrhotic patients associated with impaired prognosis. Hyperammonemia plus inflammatory response do play a crucial role on hepatic encephalopathy. However, in some patients HE appeared without hyperammonemia and patients with increased levels of ammonia could not show cognitive dysfunction. This has led to investigate other factors that could act in a synergistic way. Diabetes mellitus and insulin resistance are characterized by releasing and enhancing these pro-inflammatory cytokines and, additionally, has been related to hepatic encephalopathy. Indeed, patients with diabetes showed raised risk of over hepatic encephalopathy in comparison with non-cirrhotics. Type 2 diabetes mellitus could impair hepatic encephalopathy by different mechanisms that include: a) increasing glutaminase activity; b) impairing gut motility and promoting constipation, intestinal bacterial overgrowth and bacterial translocation. Despite of insufficient clarity about the practicability of anti-diabetic therapy and the most efficacious therapy, we would have to pay a special attention to the management of type 2 diabetes mellitus and insulin resistance in cirrhotic patients.

Impact of age on liver regeneration response to injury after partial hepatectomy in a rat model.

BACKGROUND: Liver resection is a feasible treatment for multiple liver diseases. There is no evidence about the impact of age on liver regeneration. OBJECTIVE: To assess the effect of age on liver regeneration in an experimental in vivo animal model of 70%-partial hepatectomy. METHODS: Forty young (Y) and old (O) Wistar male rats (n = 80) were distributed into four groups [controls (C), sham operated (SO), hepatectomy 6 h (H6), and 48 h (H48)]. Different morphometric and biochemical factors, oxidative and nitrosative stress, lipid peroxidation, cytokines kinetics, and histopathologic tissular parameters were determined. RESULTS: Early postoperative mortality was higher in aged rats (P = 0.049). Morphometric determinations, liver regeneration index, and total volume weight were favorable to young rats. Serum transaminase levels were higher in aged rats. Parameters of necrosis (measured by histopathologic injury [HI: 0-I-II-III]), regeneration (measured by bromodeoxyuridine-BrdU incorporation) and apoptosis (determined by the TDT-mediated dUTP nick end labeling-TUNEL) were well-synchronized in young rats. Parameters of oxidative stress such as reduced (GSH), oxidized (GSSG) glutathione and lipid peroxidation (measured by hepatic malondialdehyde -MDA-) were lower in young animals throughout the studied period. Nitrosative stress measured by nitric oxide (NO) end-products was higher in late stages in resected old rats. Pro-inflammatory cytokines (TNF- α) reached higher and earlier levels in aged rats while pro-regenerative cytokines (IL-6) were significantly higher in early stages for young rats and in late stages for aged rats. The levels of TGF-ß were higher in young rats. CONCLUSION: Liver regeneration is delayed and reduced in aged animals submitted to liver resection.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca2+ on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca2+ pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca2+ entrance was induced by A23187 in HepG2. Cell death, Ca2+ mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca2+ concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and l-NAME enhanced, GCDCA-induced cell death. The reduction of Ca2+ entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca2+ entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca2+ concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca2+-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

The reduction of cell death and proliferation by p27(Kip1) minimizes DNA damage in an experimental model of genotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer worldwide. The expression of p27 has been related to reduced severity of tumor grade and recurrence of HCC. The study assessed the role of p27 on the cell proliferation and death, and DNA mutagenesis in experimental genotoxicity induced by aflatoxin B1 (AFB(1)) in cultured hepatocytes obtained from control and p27(Kip1) deficient mice. The overexpression of p27 was assessed with wild type p27(Kip1) expression vector in HepG2 cells. The expression of p27, p21 and p53 was assessed in well and poorly-differentiated liver tumors. DNA damage and cell death induced by AFB(1) were related to a reduction of p27 and p21 expression in cultured hepatocytes. AFB(1)-induced nuclear phosphorylated (Ser 10) p27 degradation was related to a rise of nuclear KIST, Rsk-1 and Rsk-2 expression and cytoplasm phosphorylated (Thr 198) p27 expression. The overexpression of p27 reduced cell proliferation, cell death and DNA damage in AFB(1)-treated hepatocytes. The enhanced survival of patients with well differentiated compared to poorly-differentiated tumors was related to high expression of p27, p21 and p53 in liver sections. The study showed that the p27 reduced cell proliferation and death, as well as the accumulation of DNA damage in hepatocarcinogenesis.

Mitochondrial-driven ubiquinone enhances extracellular calcium-dependent nitric oxide production and reduces glycochenodeoxycholic acid-induced cell death in hepatocytes.

Ca(2+) mobilization, nitric oxide (NO), and oxidative stress have been involved in cell death induced by hydrophobic bile acid in hepatocytes. The aim of the study was the elucidation of the effect of the antioxidant mitochondrial-driven ubiquinone (Mito Q) on the intracellular Ca(2+) concentration, NO production, and cell death in glycochenodeoxycholic acid (GCDCA)-treated HepG2 cells. The role of the regulation of the intracellular Ca(2+) concentration by Ca(2+) chelators (EGTA or BAPTA-AM), agonist of Ca(2+) entrance (A23187) or NO (L-NAME or NO donor), was assessed during Mito Q cytoprotection in GCDCA-treated HepG2 cells. Cell death, NO synthase (NOS)-1, -2, and -3 expression, Ca(2+) mobilization, and NO production were evaluated. GCDCA reduced the intracellular Ca(2+) concentration and NOS-3 expression and enhanced cell death in HepG2. NO donor prevented and L-NAME enhanced GCDCA-induced cell death. The reduction of Ca(2+) entry by EGTA, but not its release from intracellular stores by BAPTA-AM, reduced the expression of NOS-3 and enhanced cell death in control and GCDCA-treated cells. Mito Q prevented the reduction of intracellular Ca(2+) concentration, NOS-3 expression, NO production, and cell death in GCDCA-treated HepG2 cells. The conclusion is that the recovery of Ca(2+)-dependent NOS-3 expression by Mito Q may be considered an additional cytoprotective property of an antioxidant.

Bevacizumab Allows Preservation of Liver Function and its Regenerative Capacity after Major Hepatectomy.

BACKGROUND: Parallel to the safety of liver resections, new chemotherapy drugs have emerged for the control of liver metastases. However, there is unclear evidence about the combination of intensive BVZ-therapy and extended resections. The main aim was to analyse the impact of Bevacizumab (BVZ) in terms of liver safety and tolerability in two experimental models: a basal-toxicity situation and after major hepatectomy. METHODS: Eighty male-Wistar rats were grouped as toxicity analysis (sham-operated rats-OS-) and regeneration after- surgery analysis (hepatectomy rats-H-). Eight further subgroups were created according to sacrifice (6- hours-6h- or 24-hours-24h-) and dose (µg) of BVZ (none, 100, 200, 400). Several measurements were performed, including biochemical serum samples, histopathological analysis, cytokines (IL-6, TNF-α, TGF-ß), oxidative-stress (GSH/GSSG, ATP), lipid-peroxidation (TBARS) and epidermal and vascular endothelium growth-factors (EGF and VEGF). RESULTS: In the toxicity analysis, safe results with BVZ were observed, with no significant differences among the groups. A trend towards a lower oxidative status was observed in the OS 6 h-100, -200 and -400 versus the OS 6 h-none group. Similar results were observed in the hepatectomy model, with stable oxidative-stress-index and IL-6, TNF- α, and TGF- ß levels. Despite higher lipid peroxidation status, overall regeneration was preserved. As expected, VEGF was almost undetectable in BVZ-treated groups after resection, but not in the non-resection group. CONCLUSION: It was concluded that liver status was not impaired by BVZ even at the high-dose. Similarly, liver regeneration after extended hepatectomy in BVZ-treated animals was well-preserved. Extended liver resections may be encouraged in BVZ-treated patients due to its excellent tolerability and good liver regeneration status.

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.

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats.

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.

The differential effect of PGE(1) on d-galactosamine-induced nitrosative stress and cell death in primary culture of human hepatocytes.

The pre-administration of PGE(1) reduced inducible nitric oxide synthase (NOS-2) expression and cell death induced by d-galactosamine (d-GalN) in cultured rat hepatocytes. The present study evaluated the role of nitric oxide (NO) during PGE(1) treatment in fully established d-GalN-induced cytotoxicity in cultured human hepatocytes. Human hepatocytes were isolated from liver resections by classic collagenase perfusion. PGE(1) (1 microM) was administered at 2 h before d-GalN (40 mM), or 2 or 10 h after d-GalN in cultured hepatocytes. The production of NO was inhibited by N-omega-nitroso-l-arginine methyl ester (l-NAME) (0.5 mM). Various parameters related to oxidative and nitrosative stress, mitochondrial dysfunction, NF-kappaB activation, NOS-2 expression and cell death were evaluated in hepatocytes. NO mediated mitochondrial disturbances, nitrosative stress and cell death in d-GalN-treated hepatocytes. The administration of PGE(1) 10 h after d-GalN enhanced NF-kappaB activation, NOS-2 expression and nitrosative stress. Although PGE(1) administered at 2 h before or 2h after d-GalN reduced apoptosis and necrosis, its administration 10 h after d-GalN had no beneficial effect on cell death. In conclusion, the administration of PGE(1) during advanced d-GalN cytotoxicity induced nitrosative stress and lost its cytoprotective properties in cultured human hepatocytes.

Effect of Quercetin on Hepatitis C Virus Life Cycle: From Viral to Host Targets.

Quercetin is a natural flavonoid, which has been shown to have anti hepatitis C virus (HCV) properties. However, the exact mechanisms whereby quercetin impacts the HCV life cycle are not fully understood. We assessed the effect of quercetin on different steps of the HCV life cycle in Huh-7.5 cells and primary human hepatocytes (PHH) infected with HCVcc. In both cell types, quercetin significantly decreased i) the viral genome replication; ii) the production of infectious HCV particles and iii) the specific infectivity of the newly produced viral particles (by 85% and 92%, Huh7.5 and PHH respectively). In addition, when applied directly on HCV particles, quercetin reduced their infectivity by 65%, suggesting that it affects the virion integrity. Interestingly, the HCV-induced up-regulation of diacylglycerol acyltransferase (DGAT) and the typical localization of the HCV core protein to the surface of lipid droplets, known to be mediated by DGAT, were both prevented by quercetin. In conclusion, quercetin appears to have direct and host-mediated antiviral effects against HCV.

Imaging biomarkers for steatohepatitis and fibrosis detection in non-alcoholic fatty liver disease.

There is a need, in NAFLD management, to develop non-invasive methods to detect steatohepatitis (NASH) and to predict advanced fibrosis stages. We evaluated a tool based on optical analysis of liver magnetic resonance images (MRI) as biomarkers for NASH and fibrosis detection by investigating patients with biopsy-proven NAFLD who underwent magnetic resonance (MR) protocols using 1.5T General Electric (GE) or Philips devices. Two imaging biomarkers (NASHMRI and FibroMRI) were developed, standardised and validated using area under the receiver operating characteristic curve (AUROC) analysis. The results indicated NASHMRI diagnostic accuracy for steatohepatitis detection was 0.83 (95% CI: 0.73-0.93) and FibroMRI diagnostic accuracy for significant fibrosis determination was 0.85 (95% CI: 0.77-0.94). These findings were independent of the MR system used. We conclude that optical analysis of MRI has high potential to define non-invasive imaging biomarkers for the detection of steatohepatitis (NASHMRI) and the prediction of significant fibrosis (FibroMRI) in NAFLD patients.

Plasma protein biomarkers of hepatocellular carcinoma in HCV-infected alcoholic patients with cirrhosis.

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers in the world, with limited options for treatment unless timely diagnosed. Chronic hepatitis C virus (HCV) infection and persistent heavy alcohol consumption are independent risk factors for HCC development, which may induce a specific protein expression pattern different from those caused separately. The aim of the study was to identify protein biomarkers for the detection of HCC in HCV-infected alcoholic patients with cirrhosis in order to improve survival. We compared protein expression profiles of plasma samples from 52 HCV-infected alcoholic patients with and without HCC, using 2-D DIGE coupled with MALDI-TOF/TOF mass spectrometry. The 2-D DIGE results were analyzed statistically using Decyder software, and verified by western-blot and ELISA. In plasma samples from HCV-infected alcoholic patients, we found significantly differential expression profiles of carboxypeptidase-N, ceruloplasmin (CP), complement component 4a (C4a), fibrinogen-alpha (FGA), immunoglobulin mu chain C region, serum albumin, and serum paraoxonase/arylesterase 1 (PON1). Deregulation of plasma/serum levels of the identified proteins was associated to HCV, ethanol consumption, and/or HCC progression. In the validation through ELISA, C4a serum concentration was increased in HCC patients (2.4±1 ng/mg vs 1.8±0.6 ng/mg; p = 0.029), being the only independent predictor of HCC in the multivariate analysis (OR = 2.15; p = 0.015), with an AUROC = 0.70. The combination of C4a, FGA, CP and PON1 improved slightly the predictive ability of C4a alone (AUROC 0.81). In conclusion, we identified proteins related to acute-phase response, oxidative stress, or immune response, whose differential expression in plasma may be attributed to the presence of HCC. Among them, C4a, and its combination with CP, FGA and PON1, could be considered as potentially reliable biomarkers for the detection of HCC in HCV-infected alcoholic patients.

Evaluation of potential antigenotoxic, cytotoxic and proapoptotic effects of the olive oil by-product "alperujo", hydroxytyrosol, tyrosol and verbascoside.

Olive oil is an integral ingredient of the "Mediterranean diet". The olive oil industry generates large quantities of a by-product called "alperujo" (AL) during the two-phase centrifugation system developed in the early nineties. AL could be a potent exploitable source of natural phenolic antioxidants. Our results showed that AL and its distinctive phenols hydroxytyrosol, tyrosol and verbascoside were not genotoxic in the Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster and exerted antigenotoxic activity against DNA oxidative damage generated by hydrogen peroxide (H2O2). Alperujo and hydroxytyrosol also exhibited notable antiproliferative and caspase 3-dependent proapoptotic effects toward the human tumoral cell line HL60. AL can provide a cheap and efficient source of chemopreventive phenolic compounds with strong antioxidant properties, becoming a promising and potent therapeutic drug in the future.