Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

<p>Abstract</p> <p>Background</p> <p>Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.</p> <p>Methods</p> <p>Six genes, beta-2-microglobulin (<it>B2M</it>), glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), hydroxymethyl-bilane synthase (<it>HMBS</it>), hypoxanthine phosphoribosyl-transferase 1 (<it>HPRT1</it>), succinate dehydrogenase complex, subunit A (<it>SDHA</it>) and ubiquitin C (<it>UBC</it>), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both <it>geNorm </it>and <it>NormFinder </it>software.</p> <p>Results</p> <p><it>HMBS </it>and <it>GAPDH </it>were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. <it>HMBS, GAPDH </it>and <it>UBC </it>were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of <it>HMBS, B2M</it>, <it>SDHA </it>and <it>GAPDH </it>was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.</p> <p>Conclusion</p> <p>Of six genes studied, <it>HMBS </it>was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.</p

Bioelectrical impedance analysis in clinical practice: implications for hepatitis C therapy BIA and hepatitis C

<p>Abstract</p> <p>Background</p> <p>Body composition analysis using phase angle (PA), determined by bioelectrical impedance analysis (BIA), reflects tissue electrical properties and has prognostic value in liver cirrhosis. Objective of this prospective study was to investigate clinical use and prognostic value of BIA-derived phase angle and alterations in body composition for hepatitis C infection (HCV) following antiviral therapy.</p> <p>Methods</p> <p>37 consecutive patients with HCV infection were enrolled, BIA was performed, and PA was calculated from each pair of measurements. 22 HCV genotype 3 patients treated for 24 weeks and 15 genotype 1 patients treated for 48 weeks, were examined before and after antiviral treatment and compared to 10 untreated HCV patients at 0, 24, and 48 weeks. Basic laboratory data were correlated to body composition alterations.</p> <p>Results</p> <p>Significant reduction in body fat (BF: 24.2 ± 6.7 kg vs. 19.9 ± 6.6 kg, genotype1; 15.4 ± 10.9 kg vs. 13.2 ± 12.1 kg, genotype 3) and body cell mass (BCM: 27.3 ± 6.8 kg vs. 24.3 ± 7.2 kg, genotype1; 27.7 ± 8.8 kg vs. 24.6 ± 7.6 kg, genotype 3) was found following treatment. PA in genotype 3 patients was significantly lowered after antiviral treatment compared to initial measurements (5.9 ± 0.7° vs. 5.4 ± 0.8°). Total body water (TBW) was significantly decreased in treated patients with genotype 1 (41.4 ± 7.9 l vs. 40.8 ± 9.5 l). PA reduction was accompanied by flu-like syndromes, whereas TBW decline was more frequently associated with fatigue and cephalgia.</p> <p>Discussion</p> <p>BIA offers a sophisticated analysis of body composition including BF, BCM, and TBW for HCV patients following antiviral regimens. PA reduction was associated with increased adverse effects of the antiviral therapy allowing a more dynamic therapy application.</p

SPARC expression is associated with hepatic injury in rodents and humans with non-alcoholic fatty liver disease

Mechanisms that control progression from simple steatosis to steato-hepatitis and fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) are unknown. SPARC, a secreted matricellular protein, is over-expressed in the liver under chronic injury. Contribution of SPARC accumulation to disease severity is largely unknown in NAFLD. We assessed the hypothesis that SPARC is increased in livers with more necrosis and inflammation and could be associated with more fibrosis. qrt-PCR, immunohistochemistry, and ELISA were employed to localize and quantify changes in SPARC in 62 morbidly obese patients with NAFLD/NASH and in a mouse model of diet-induced-NASH. Results were correlated with the severity of NAFLD/NASH. In obese patients 2 subgroups were identified with either high SPARC expression (n = 16) or low SPARC expression (n = 46) in the liver, with a cutoff of 1.2 fold expression. High expression of SPARC paralleled hepatocellular damage and increased mRNA expression of pro-fibrogenic factors in the liver. In line with these findings, in the NASH animal model SPARC knockout mice were protected from inflammatory injury, and showed less inflammation and fibrosis. Hepatic SPARC expression is associated with liver injury and fibrogenic processes in NAFLD. SPARC has potential as preventive or therapeutic target in NAFLD patients.Fil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Atorrasagasti, María Catalina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Onorato, Agostina Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Peixoto, Estanislao. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Schlattjan, Martin. Universitat Essen; AlemaniaFil: Sowa, Jan Peter. Universitat Essen; AlemaniaFil: Sydor, Svenja. Universitat Essen; AlemaniaFil: Gerken, Guido. Universitat Essen; AlemaniaFil: Canbay, Ali. Universitat Essen; Alemani

Evaluation of Biomarkers of NAFLD in a Cohort of Morbidly Obese Patients

Hepatocyte apoptosis is a key event in nonalcoholic fatty liver disease (NAFLD), and serum apoptotic markers are emerging as surrogate markers for NAFLD. We studied the role of caspase-cleaved cytokeratin18 in the diagnosis of fibrosis in a cohort of 127 morbidly obese patients and also performed a review of the literature biomarkers of NAFLD and fibrosis. Here, we found that cleaved caspase 18 correlated with liver steatosis and liver injury as assessed by serum transaminase levels. Furthermore, hepatocyte apoptosis as assessed by cleaved CK18 and TUNEL staining correlated with the extent of fibrosis as assessed by Sirius Red staining and serum hyaluronic acid. These results underscore the important role of hepatocyte apoptosis in the pathogenesis of fibrosis in NAFLD, which led to the utilization of surrogate markers for apoptosis in the noninvasive diagnosis of NAFLD. We furthermore reviewed current literature of biomarkers of NAFLD and fibrosis

Optimisation of antioxidants extraction from soybeans fermented by Aspergillus oryzae

4 figuras, 7 tablasThe extraction of antioxidant compounds from soybeansfermented with Aspergillusoryzae was optimised using a factorial design. A kinetic study of the total phenolic production and DPPH radical scavenging activity was first performed at the points selected in the factorial design. In both cases, the experimental profiles were fitted to a modified first-order kinetic model. To investigate the combined effects of temperature and solvent concentration on the extraction, the parameters obtained from the fitted kinetic models were used as response variables in a rotatable second-order design with quintuple replications in the centre of the experimental domain. The results obtained indicate that temperature had the most significant effect. The response surfaces show a maximum in the experimental domain studied. The optimum conditions for the extraction of total phenolic content were 65.3 °C and 73.1% ethanol, in which 56.2 mg of GAE/g were predicted. A scavenging activity of 81.6% DPPH radical was predicted at the optimum conditions of 61.6 °C and 60% ethanolDrs. Pablo Fuciños and José Antonio Vázquez has been awarded a postdoctoral grant (Programa de bolsas para estadías fóra de Galicia, 2007 and 2008 respectively) by the Dirección Xeral de Investigación, Desenvolvemento e Innovación, Xunta de Galicia, Spain.Peer reviewe

Liver Injury Indicating Fatty Liver but Not Serologic NASH Marker Improves under Metformin Treatment in Polycystic Ovary Syndrome

Objective. Polycystic ovary syndrome (PCOS) is associated with obesity and insulin resistance (IR), key features of nonalcoholic steatohepatitis (NASH). Cytokeratin 18 fragments (M30) have been established as a serum marker for NASH. The insulin sensitizer metformin improves hepatic IR. This study evaluates the influence of MF on serologic NASH (sNASH) in patients with PCOS. Patients and Methods. In 89 patients, metabolic parameters, liver injury indicating fatty liver (LIFL), and M30 were assessed at baseline and after metformin treatment. Patients with initial IR were subdivided into dissolved (PCOS-exIR) and persistent IR (PCOS-PIR) after treatment and compared to an initially insulin sensitive PCOS group (PCOS-C). Results. Improvement of LIFL prevalence could be seen in PCOS-C and PCOS-exIR compared to PCOS-PIR (−19.4, resp., −12.0% versus 7.2%, Chi2 = 29.5, P<0.001) without change in sNASH prevalence. In PCOS-PIR, ALT levels increased significantly accompanied by a nominal, nonsignificant M30 increase. Conclusions. Metformin improves LIFL in subgroups of patients with PCOS without influencing sNASH. This could either indicate a missing effect of metformin on NAFLD or slowed disease progression. Further studies are needed to elucidate NAFLD in the context of PCOS and potential therapeutic options

Interferon alfa for chronic hepatitis B infection: Increased efficacy of prolonged treatment

Interferon alfa (IFN-a) is the primary treatment for chronic hepatitis B. The standard duration of IFN-a therapy is considered 16 weeks; however, the optimal treatment length is still poorly defined. We evaluated the efficacy and acceptability of prolonged IFN-a treatment in patients with chronic hepatitis B. To investigate whether treatment prolongation could enhance the rate of hepatitis B e antigen (HBeAg) seroconversion, we conducted a prospective, controlled, multicenter trial in wh

Hepatocyte KLF6 expression affects FXR signalling and the clinical course of primary sclerosing cholangitis

Background & Aims: Primary sclerosing cholangitis (PSC) is characterized by chronic cholestasis and inflammation, which promotes cirrhosis and an increased risk of cholangiocellular carcinoma (CCA). The transcription factor Krueppel-like-factor-6 (KLF6) is a mediator of liver regeneration, steatosis, and hepatocellular carcinoma (HCC), but no data are yet available on its potential role in cholestasis. Here, we aimed to identify the impact of hepatic KLF6 expression on cholestatic liver injury and PSC and identify potential effects on farnesoid-X-receptor (FXR) signalling. Methods: Hepatocellular KLF6 expression was quantified by immunohistochemistry (IHC) in liver biopsies of PSC patients and correlated with serum parameters and clinical outcome. Liver injury was analysed in hepatocyte-specific Klf6-knockout mice following bile duct ligation (BDL). Chromatin-immunoprecipitation-assays (ChIP) and KLF6-overexpressing HepG2 cells were used to analyse the interaction of KLF6 and FXR target genes such as NR0B2. Results: Based on IHC, PSC patients could be subdivided into two groups showing either low (80%) hepatocellular KLF6 expression. In patients with high KLF6 expression, we observed a superior survival in Kaplan-Meier analysis. Klf6-knockout mice showed reduced hepatic necrosis following BDL when compared to controls. KLF6 suppressed NR0B2 expression in HepG2 cells mediated through binding of KLF6 to the NR0B2 promoter region. Conclusion: Here, we show an association between KLF6 expression and the clinical course and overall survival in PSC patients. Mechanistically, we identified a direct interaction of KLF6 with the FXR target gene NR0B2