10, 11 An elevation of serum endothelin-1 has been noted in NASH, and has been positively related to the severity of liver fibrosis.12 An enhanced peripheral vasoconstrictive response to endothelin-1

has been widely reported in NASH patients and rats.12, 13 However, studies investigating Decitabine in vivo an enhanced intrahepatic vasoconstrictive response to endothelin-1 in NASH cirrhotic livers are still limited. Endocannabinoids are lipid mediators that increase in liver of diet-induced obesity models. Besides hyperleptinemia, an activated hepatic endocannabinoid system is significantly involved in the pathogenesis of NASH and cirrhosis.14, 15 Nonetheless, the relationship between hyperleptinemia, activated endocannabinoids system, aggravated hepatic steatosis, and fibrogenesis and increased IHR in NASH cirrhotic rats remains unclear. Collectively, our study aims to explore the possible contribution of hyperleptinemia to the pathogenesis of the endothelin-1 and endocannabinoids-mediated mechanisms that cause increased IHR and portal hypertension in NASH cirrhotic rats. HF/MCD, high fat/methionine-choline-deficient; learn more HSC, hepatic stellate cells; IHR, intrahepatic resistance; NASH, nonalcoholic steatohepatitis; OBRb: leptin receptor. Detailed Materials

and Methods are provided in the Supporting Information. The Zucker rats, which bear a mutation (fa) in the leptin receptor (OBRb) gene, fed HF/MCD diets were used.4-6, 13 The HF/MCD diet used consisted of 37% calories

(Cal) from fat (corn oil), 24.5% Cal from protein (lactalbumin hydrolysate), and 38.5% Cal from carbohydrate (dextrose) together with vitamins and minerals (Dyets, Bethlehem, PA) deficient in methionine and choline as recommended. The normal diet was a paired feeding protocol that controlled calorie intake using a methionine choline-sufficient diet. 上海皓元 In the first series of studies (n = 8 in each group), two groups of 3-week-old Zucker rats and two groups of age-matched lean rats were fed either the HF/MCD or normal diet for 16 weeks. This resulted in four groups: HF/MCD-Zucker rats, normal-Zucker rats, HF/MCD-lean rats, and normal-lean rats. Among the above four groups, NASH cirrhotic livers and hyperleptinemia were only observed in the HF/MCD-Zucker rats. Thus, normal-Zucker rats, HF/MCD-lean rats, and normal-lean rats that were without NASH cirrhotic livers and hyperleptinemia served as the controls for this study. In a second series of studies, the exogenous administration of mouse endotoxin free recombinant leptin (100 μg/kg/day, intraperitoneal) was given to HF/MCD+leptin-lean and normal+leptin-lean rats (n = 6) in order to directly explore the leptin-related hepatic effects in rats that have intact OBRb. In our preliminary experiments, different durations (5, 7, 10, and 13 weeks) of leptin were administrated.

The identified SNPs are located within a haplotype block, including the IL28B gene (coding for IFN-λ-3), and are strongly associated with the outcome of pegylated

(peg)-IFN/ribavirin (RBV) standard-of-care therapy. North American patients from the pharmacogenomic subcohort of the IDEAL randomized control trial (RCT)8 were tested for genetic associations with SVR by GWAS3 (Table 1). This study also included 67 patients from a second RCT comparing peg-IFN www.selleckchem.com/products/Dasatinib.html and RBV treatment outcomes between Caucasians and African Americans (AA).7 The cohort was therefore multi-ethnic, including patients of Caucasian, AA, and Hispanic ancestry. SVR was defined as undetectable HCV—RNA at 24 weeks’ post-treatment (in a minority, SVR was defined at 12 weeks’ post-treatment). All patients who achieved an SVR were included; non-responders were required to have been at least 80% adherent to therapy for inclusion.7 Of 1671 patients consenting to the pharmacogenomic analysis, 1137 were included

Doxorubicin nmr in the final analysis after consideration of adherence and genotyping quality control criteria. The study was performed using the Illumina 610-Quad BeadChip (Illumina, San Diego, CA, USA). Seven SNPs demonstrated genome-wide significance for an association with SVR. The top association SNP, rs12979860, was associated with a twofold to threefold increase in the SVR rate in all three ethnic groups (overall cohort, P = 1.37 × 10−28). rs12979860 is a bi-alleleic SNP (C/T) with three possible genotypes (CC, CT, TT), where

the CC genotype is associated with an increased SVR rate. Six other SNPs on a common haplotype block were also associated with SVR at the genome-wide level. These SNPs were in linkage disequilibrium with the discovery SNP, and their effects were largely explained by rs12979860. Another important observation was that the frequency of the good-response IL28B allele was lower in AA compared MCE to Caucasians (AA: C allele frequency = 64% vs Caucasians = 89%), explaining over half of the difference in the SVR rate between the AA and Caucasian patients.3 In this paper, another random multi-ethnic cohort was genotyped, which identified the good-response allele to be present at even higher rates in Asians compared to Caucasians, The global distribution of IL28B genotype frequency has since been mapped in greater detail, and a very high frequency of the good-response allele noted in patients of Asian ancestry, consistent with the higher SVR rates that have been observed historically in Asian populations.9 The different frequency of the good-response alleles between ethnic populations might therefore explain much of the racial differences in IFN treatment response. The IL28B genotype was confirmed to be the most powerful pretreatment predictor of SVR in a subsequent intent-to-treat analysis of the IDEAL pharmacogenomics cohort.

001). No significant differences in rebleeding after 6 weeks were observed between patients with and without HCC (19% versus 17%; P = 0.714; Table 3). However, overall failure

of secondary prophylaxis was more frequent in patients with HCC than controls (32% versus 21%; P = 0.05). 17-AAG mouse Expectedly, lack of secondary prophylactic measures was associated with secondary prophylaxis failure (data not shown; P < 0.001). Similarly, PVT was associated with secondary prophylaxis failure (none, 25%; benign, 21%; malignant, 35%; P < 0.001). During follow-up, 3 patients from each group received LT. Most patients without HCC died of decompensated liver disease (40 of 49), whereas those with HCC died of decompensated liver disease (34 of 109), tumoral disease (7 of 109), or a combination of both (61 of 109). Seven patients from each group had nonhepatic deaths. Transplant-free survival was significantly shorter in patients with HCC (median survival of 5 Veliparib price months versus over 38 months in patients without HCC; log rank: P < 0.001; Fig. 1A). This difference

was maintained in each Child-Pugh class (log rank: P < 0.001; Fig. 1B-D). Previous decompensation was significantly associated with survival in the overall group; however, in patients with HCC, no significant differences were observed according to this variable (Fig. 2). Survival curves of patients with HCC according to BCLC classification is shown in Supporting Fig. 1. To simplify the statistical analysis and according to these survival curves, patients were divided in two groups of BCLC classification (0, A, and B and C and D). Expectedly, patients with BCLC 0, A, and B had better survival rates (median survival: 17.3 months; IQR, 9.6-36.1) than patients with BCLC C and D (1.5 months; IQR, 0.3-3.7), and both groups presented a 上海皓元医药股份有限公司 worse outcome than patients without HCC (median survival: >60 months; Fig. 3). Given the uneven distribution of well-known prognostic markers of rebleeding and death, multivariate analysis was performed

to evaluate the adjusted effect of HCC on survival (Table 4A). Even when considering the other variables, HCC and lack of secondary prophylaxis remained independent predictors of death. Stratified analysis was performed to evaluate specifically the effect of use of secondary prophylaxis in patients according to BCLC. In patients with BCLC 0, A, and B, most had secondary prophylaxis. However, lack of secondary prophylaxis was associated with death (log rank: P < 0.001) with a median survival of 0.9 months in patients without prophylaxis (2 of 57; 4%), compared to 22 months in patients with prophylaxis (55 of 57; 96%). Similarly, in patients with BCLC C and D, and despite their dismal prognosis, lack of secondary prophylaxis was also associated with death (log rank: P < 0.001) with a median survival of 0.7 months (24 of 71; 34%), compared to 3 months in patients who had secondary prophylaxis (47 of 71; 66%; Fig. 4).

001). No significant differences in rebleeding after 6 weeks were observed between patients with and without HCC (19% versus 17%; P = 0.714; Table 3). However, overall failure

of secondary prophylaxis was more frequent in patients with HCC than controls (32% versus 21%; P = 0.05). I-BET-762 ic50 Expectedly, lack of secondary prophylactic measures was associated with secondary prophylaxis failure (data not shown; P < 0.001). Similarly, PVT was associated with secondary prophylaxis failure (none, 25%; benign, 21%; malignant, 35%; P < 0.001). During follow-up, 3 patients from each group received LT. Most patients without HCC died of decompensated liver disease (40 of 49), whereas those with HCC died of decompensated liver disease (34 of 109), tumoral disease (7 of 109), or a combination of both (61 of 109). Seven patients from each group had nonhepatic deaths. Transplant-free survival was significantly shorter in patients with HCC (median survival of 5 Selleck R788 months versus over 38 months in patients without HCC; log rank: P < 0.001; Fig. 1A). This difference

was maintained in each Child-Pugh class (log rank: P < 0.001; Fig. 1B-D). Previous decompensation was significantly associated with survival in the overall group; however, in patients with HCC, no significant differences were observed according to this variable (Fig. 2). Survival curves of patients with HCC according to BCLC classification is shown in Supporting Fig. 1. To simplify the statistical analysis and according to these survival curves, patients were divided in two groups of BCLC classification (0, A, and B and C and D). Expectedly, patients with BCLC 0, A, and B had better survival rates (median survival: 17.3 months; IQR, 9.6-36.1) than patients with BCLC C and D (1.5 months; IQR, 0.3-3.7), and both groups presented a MCE worse outcome than patients without HCC (median survival: >60 months; Fig. 3). Given the uneven distribution of well-known prognostic markers of rebleeding and death, multivariate analysis was performed

to evaluate the adjusted effect of HCC on survival (Table 4A). Even when considering the other variables, HCC and lack of secondary prophylaxis remained independent predictors of death. Stratified analysis was performed to evaluate specifically the effect of use of secondary prophylaxis in patients according to BCLC. In patients with BCLC 0, A, and B, most had secondary prophylaxis. However, lack of secondary prophylaxis was associated with death (log rank: P < 0.001) with a median survival of 0.9 months in patients without prophylaxis (2 of 57; 4%), compared to 22 months in patients with prophylaxis (55 of 57; 96%). Similarly, in patients with BCLC C and D, and despite their dismal prognosis, lack of secondary prophylaxis was also associated with death (log rank: P < 0.001) with a median survival of 0.7 months (24 of 71; 34%), compared to 3 months in patients who had secondary prophylaxis (47 of 71; 66%; Fig. 4).

e., activation, as well as decreased the amount of occludin and its associate ZO-1 (Fig. 1A). The decrease of occludin and

ZO-1 was reversed RXDX-106 datasheet by the p38 MAPK-specific inhibitor SB203580 but not by the p42/44 MAPK inhibitor PD98059 (not shown). In contrast, alterations in other TJ proteins (claudin-5 and ZO-2) were not reversed by SB20358 (not shown). To further corroborate the effect of p38 MAPK, we inhibited p38 MAPK expression by using siRNA. We found that blocking p38 MAPK up-regulation effectively mitigated the reduction of occludin and ZO-1 (Fig. 1B,C). These results showed that p38 MAPK is important for occludin regulation. To investigate the effect of p38 MAPK on the transcription and expression of occludin, we transfected bEnd3 cells with p38 MAPK cDNA for 18 hours, then treated the cells with or without SB203580 at 1 μM for 3 hours. Using RT-PCR, we found that overexpressing p38 MAPK resulted in significant

suppression of messenger RNA (mRNA) levels of occludin and ZO-1 but not claudin-5 and ZO-2 (Fig. 2A,C). The effect was reversed with SB203580. Importantly, up-regulating p38 MAPK did not change the MMP-9 mRNA level in the brain EC (Fig. 2A,C). With western blotting, we found that occludin and ZO-1 were significantly reduced by p38 MAPK up-regulation, and the reduction was partly restored with the p38 MAPK inhibitor SB203580 (Fig. 2B,D). In selleck screening library contrast, claudin-5 and ZO-2 were not affected. Because p38 MAPK is associated with IκBα degradation and NFκB activation,31, 32 we investigated the

status of IκBα protein and NFκB in bEnd3 cells in which p38 MAPK cDNA was overexpressed. The bEnd3 cells were transfected with p38 MAPK cDNA then treated with 10 nM of NFκB activation inhibitor. We found that p38 MAPK up-regulation reduced IκBα, occludin, and ZO-1 levels. Importantly, we demonstrated that p38 MAPK activation induces IκBα degradation, which was reversed by treatment with the p38 MAPK inhibitor 上海皓元医药股份有限公司 SB203580 (Fig. 2E). Moreover, we found that after administering NFκB inhibitor the degradation of IκBα became enhanced in p38 MAPK-up-regulated cells, and the decrease of occludin and ZO-1 was reversed (Fig. 2E). Overall, our results indicate that p38 MAPK induces the degradation of IκBα, which leads to the release of NFκB activation that regulates occludin and ZO-1 expression in mouse brain EC cells. MMP-9 has been shown to transactivate EGFR.33, 34 Ligation of EGFR results in the activation of MAPK cascades23 and potentially in modulating TJ proteins.35 We thus speculated that EGFR activation might be important in MMP-9-induced alteration of occludin. We first confirmed whether MMP-9 could transactivate EGFR in bEnd3 cells. As shown in Fig. 3A,B, when bEnd3 cells were exposed to MMP-9, EGFR was phosphorylated (p-TyrEGFR), i.e., activated, as determined by western blotting.

Intrahepatic lipid accumulation plays a pathogenic role in liver injury in response to chronic ethanol exposure.[1] Lipin-1 plays an important role in regulating lipid metabolism by way of its cytoplasmic and nuclear effects.[5-7] In the present study, we provide evidence demonstrating that hepatic lipin-1 deficiency led to dramatically pronounced changes in terms of steatosis, inflammation, and fibrosis in response to chronic ethanol administration compared

to WT mice. This suggests that the 3-MA solubility dmso induction in lipin-1 in alcoholic fatty liver disease may play a protective role by limiting inflammation, promoting efficient lipid storage, and/or controlling the transcriptional regulation of fatty acid

catabolism. Correlating closely with the rapid onset and progression of steatosis and inflammation, hepatic PGC-1α abundance was found to be severely diminished in ethanol-fed lipin-1LKO mice leading to reduced expression of several PGC-1α target genes encoding fatty acid oxidation enzymes, decreased rates of hepatic fatty acid oxidation, reduced generation of ketone bodies, and impaired VLDL secretion. These findings can be interpreted to suggest that the loss nuclear lipin-1 leads to these deleterious effects since lipin-1 is known to regulate these processes at the transcriptional level.[5-7, 10] Indeed, lipin-1α overexpression suppressed alcohol-induced TG accumulation potentially by transcriptionally activating fatty acid catabolism. However, Proteases inhibitor it is possible that loss of lipin-1 enzymatic activity may somehow be affecting signaling pathways that lead to PGC-1α deactivation. For example, adipocyte-specific lipin-1 deletion led to impaired cAMP signaling in

that cell type,[15] and this pathway seems to be very important MCE公司 for regulation of PGC-1α in liver. Altogether, our results demonstrate that genetic ablation of hepatic lipin-1 aggravates experimental alcohol-induced steatohepatitis in mice. A marked increase in hepatic PAP activity has long been known to occur in alcoholic fatty liver in animals and humans.[2-4, 9] We have previously shown that lipin-1 is strongly induced in ethanol-induced fatty liver in mice and we sought to determine whether loss of lipin-1 would attenuate the increased PAP activity and intrahepatic triglyceride accumulation in response to ethanol feeding. Interestingly, our present study showed that removal of lipin-1 from the liver effectively abolished the increase in hepatic PAP activity caused by ethanol, but dramatically exacerbated ethanol-induced fatty liver in mice. Studies have demonstrated that fld mice display liver steatosis partly due to increased hepatic lipin-2-mediated PAP activity.

These studies highlight the complexity of targeting insulin sensitivity to tackle the primary obstacles encountered in treating type 2 diabetes and obesity, namely insulin and leptin resistance. Further studies clarifying the cross-talk between liver metabolism and central regulation of appetite and energy expenditure are clearly warranted. The authors thank Dr. P. Bhathal,

find more (an experienced liver histopathologist, Melbourne Pathology, Melbourne, Australia) for reviewing the H&E-stained sections of the liver. We also thank Dr. Anne Johnston for editing of the manuscript. Additional Supporting Information may be found in the online version of this article. “
“Most direct-acting antivirals (DAAs) that are being developed as therapy against hepatitis C virus target the NS3/4A protease, the NS5A

protein, and the NS5B polymerase. The latter enzyme offers different target sites: the catalytic domain for nucleos(t)ide analogues as well as a number of allosteric sites for nonnucleos(t)ide inhibitors. Two NS3/4A protease inhibitors have been approved recently, and more than 40 new NS3/4A, NS5A, or NS5B inhibitors are in development. These agents can achieve very high cure see more rates when combined with pegylated interferon-β and ribavirin and

show promising clinical results when administered in all-oral combinations. In addition to the more canonical drug targets, new alternative viral targets for small-molecule drug development are emerging, such as p7 or NS4B and viral entry. Future research will need to define well-tolerated and cost-effective DAA combinations that provide the highest rates of viral eradication in all patients (including those with advanced liver disease), MCE公司 the broadest spectrum of action on viral genotypes showing minimal or no clinical resistance, and the shortest treatment duration. (Hepatology 2013) For more than a decade, the standard treatment of chronic hepatitis C virus (HCV) infection has been based on the combination of pegylated interferon-β (PEG-IFN) and ribavirin (RBV) administered for 24 or 48 weeks. These regimens eradicate infection in 40% to 50% of treated patients with genotype 1 (HCV-1) infection and 80% of treated patients with genotype 2 (HCV-2) and 3 (HCV-3) infection. In addition, PEG-IFN/RBV regimens are poorly tolerated and contraindicated in a high percentage of patients. In order to address the shortcomings of PEG-IFN/RBV therapy, new direct-acting antivirals (DAAs) are being developed that target specific HCV functions.

SVR could be attained in 79% and 41–44% for TT and GT/GT genotype, respectively. There were few studies addressing whether IL28B genotype would influence the on-treatment viral kinetics, SVR, or relapse rate in patients receiving retreatment yet in Asian patients.19 Our study just provided clear information regarding the distribution and impact of IL28B genotype on the outcomes of receiving 48-week PEG-IFN/RBV retreatment in Asian relapsers. RVR is the key predictor for SVR. In treatment-naïve CHC genotype 1 patient,[31] RVR ensures SVR (84% vs 41% for with vs without RVR). In those achieving RVR, however, IL28B rs12979860 genotype did not influence

SVR (CC vs CT/TT = 85% vs 76–100%, respectively). In contrast, in those who failed to achieve RVR, SVR rate would significantly increase if favorable IL28B genotype existed Sirolimus in vivo (CC vs CT/TT = 66% vs 24–31%, respectively). Our study consistently provided data about the impact of IL28B genotype and RVR on SVR in relapsers receiving retreatment. RVR is statistically significant in achieving SVR (86% vs 32% for with vs without RVR, respectively; P < 0.0001). Once achieving RVR, there was no difference between these two IL28B genotypes in achieving SVR (SVR, 85% vs 100%

for TT and GT, respectively). buy Dabrafenib In the absence of RVR, favorable IL28B genotype would positively influence SVR (48% vs 10% for TT and GT, respectively; P = 0.0048). One goal of our study was to predict the response earlier during retreatment. If patient is prone to eradicate the virus, we should encourage them to complete the course of therapy. If they have low possibility for viral clearance, we might consider stopping or changing the medical regimen in order to avoid unnecessary adverse effects. Accordingly, we tried to combine IL28B genotype and RVR or cEVR to determine SVR. We found

that the SVR rate was 85% (PPV = 85%) if there was favorable IL28B genotype and RVR. SVR would be only 10% if there was unfavorable IL28B genotype and without medchemexpress achieving RVR (NPV = 90%). If the cEVR was adopted as a predictor, the prediction capacity was not superior to the combination of RVR and IL28B genotype (SVR, TT with cEVR vs GT without cEVR = 76 vs 0%). Combination of IL28B genotype with RVR thus seemed to be an ideal early marker for continuing or stopping the therapy. There were some limitations in our study. First, some clinical information in the first course of treatment was not available, especially the viral kinetics. Second, there was no liver biopsy data in the retreatment group, and it was impossible to analyze the association between the stage of liver fibrosis and chance of SVR.

See the Supporting Materials and Methods for details regarding DNA sequencing, HGF immunoassay, flow cytometry, and cell viability analysis. The Student t test was used to compare data between two groups. Analysis of variance was used to evaluate Selleckchem DAPT the difference among multiple groups. A prior report demonstrated that MHCC97-L and MHCC97-H cell lines have low and high metastatic

potential, respectively.25 Given the current hypothesis proposed by Thiery31 and Bernards and Weinberg32 that links a mesenchymal phenotype to metastasis, we investigated whether metastatic HCC cells have mesenchymal features in comparison with nonmetastatic Huh7 and Hep3B cells.33 In terms of morphology, MHCC97-L and MHCC97-H cells demonstrated a fibroblast-like appearance, whereas Huh7 and Hep3B cells displayed a cobblestone appearance (Fig. 1A). In terms of gene expression, MHCC97-L and MHCC97-H cells demonstrate low E-cadherin expression, consistent with a mesenchymal phenotype, and high expression of

E-cadherin repressor Zeb2 compared with Huh7 and Hep3B cells (Fig. 1B). There was no significant difference in expression of Snail, Twist, and Zeb1 between the four cell lines (data not shown). Protein expression confirmed a mesenchymal phenotype in MHCC97-L and MHCC97-H cells, with decreased E-cadherin expression and increased fibronectin expression (Fig. 1C). The mesenchymal phenotype of MHCC97-L and MHCC97-H cells correlates with strong expression and constitutive phosphorylation of c-Met (Fig. 1B,C). Several published reports have indicated that PLX3397 cost mutations in the c-Met gene correlate with activation in multiple different cancers.20-22 Therefore, we investigated whether the activation of c-Met in MHCC97-L and MHCC97-H cells was due to a mutation. Sequencing of the c-Met gene demonstrated none of the reported

mutations, as identified through alignment analysis (Supporting Materials and Methods, data not shown). Because sequencing demonstrated no previously reported mutation, we hypothesized that the activation was due to autocrine secretion of HGF. MCE公司 An analysis of secreted proteins in conditioned media failed to demonstrate any HGF secreted by MHCC97-L and MHCC97-H cells (Supporting Materials and Methods, data not shown). Thus, the precise mechanism of c-Met activation in MHCC97-L and MHCC97-H cells remains unknown. Constitutive activation or HGF-stimulated tyrosine phosphorylation of c-Met is blocked by PHA665752, a small molecular compound that functions as a selective inhibitor of c-Met phosphorylation26 in gastric, lung, and pancreatic cancer cells.27 Using PHA665752, we investigated the effect of tyrosine kinase inhibition on the activation of c-Met and downstream signaling pathways in human HCC. As shown in Fig. 2, PHA665752 treatment eliminated c-Met phosphorylation at multiple tyrosine residues (Y1234/Y1234 and Y1349) and reduced downstream phosphorylation of Akt and Erk (P44/42) in c-Met–positive MHCC97-L and MHCC97-H cells.

As previously described,9 to define HCV infection status, we first tested for HCV antibody by the HCV version 3.0 enzyme-linked immunosorbent assay Test System (Ortho-Clinical Diagnostics,

Raritan, NJ). Participants who were positive by HCV enzyme immunoassay were considered to have been infected with HCV and those with sufficient archived plasma (n = 2,073) were tested for HCV viremia by a branched-chain DNA assay (bDNA) (VERSANT HCV RNA 3.0 Assay, analytic sensitivity 2.5 × 103 copies/mL; Bayer-Diagnostics, Tarrytown, NY). Those positive for HCV RNA were considered to have chronic HCV infection and those with a negative result were considered to have resolved HCV infection. Methods of testing for HIV-1 and HBV infection status in these subjects have been described.9 Total nucleic acid was isolated from 500 μL of serum (Roche MagNa EPZ-6438 mw Pure LC Total Nucleic Acid Isolation Kit-Large Volume; Roche Diagnostics Corporation, Indianapolis, IN), and reverse transcription (RT) was performed. Polymerase chain reaction (PCR)

was carried out in a reaction mixture containing 3 μL of complementary DNA, 10 μL of HotStar Taq Master Mix Tamoxifen supplier (Qiagen, Valencia, CA), and 1 μL of each of the following primers: forward 5′- TGGGGTTCTCGTATGATACCC-3′ and reverse 5′-CCTGGTCATAGCCTCCGTGAA-3′, to amplify the 5′-NS5B (nonstructural 5B protein) region. PCR product was purified with Exosap-IT (USB Corporation, Cleveland, OH) and combined with 2.0 μL of Big MCE公司 Dye terminator (ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit v3.1; Applied Biosystems, Foster City, CA) and 100 pmol of primer forward (5′-NC or 5′-NS5B). The sequencing reaction was carried out for 30 cycles, and electrophoresis was performed on an ABI Prism 3730 XL instrument (Genewiz, South Plainfield, NJ). Raw sequence data were analyzed by Sequencher 4.8 Gene codes to trim ambiguous sequences. To query HCV genotype, sequences were compared to an HCV database operated by the Los Alamos National Laboratory (available at: http://hcv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html) using BLAST. Viral genotype

call was based on the highest score and lowest e-value, using the NS5B sequence, unless those results were negative or missing, in which case genotype was based on the 5′NC region. DNA was extracted from cryopreserved lymphocytes using a modified salt precipitation-extraction method (Gentra Systems, Minneapolis, MN) or from granulocytes using a silica membrane-binding method using Qiagen DNA purification columns (Qiagen). The NCI Core Genotyping Facility performed genotyping for IL28B rs12979860 using an optimized TaqMan assay (available at: http://variantgps.nci.nih.gov/cgfseq/pages/snp500.do). All analyses were cross-sectional and based on a single study visit. We determined median HCV RNA levels (log10 copies/mL) overall and among subgroups.