HCV-induced caspase-8 activity modulates caspase-9 cleavage.

<p>Huh7.5.1 cells were infected with HCVcc in the absence or presence of caspase-8 inhibitor (20 µM) for 72 h before harvest. The expression levels of truncated Bid (t-Bid) and cleaved caspase-9 were analyzed by Western blotting with antibodies specific to t-Bid and cleaved caspase-9. α-tubulin was used as an internal loading control. The relative intensity of t-Bid and cleaved caspase-9 normalized to α-tubulin was analyzed by ImageJ. Samples: MC, media control.</p

HCV infection activates DR4/DR5-dependent caspase cascade.

<p>(A) Western blot analysis of Bax and cleaved caspase-8, caspase-9, and caspase-3 in HCV-infected cells. Huh7.5.1 cells infected with HCVcc in the absence or presence of recombinant TRAIL (100 ng/ml) were used for Western blot analysis with antibodies against the indicated proteins. β-actin was used as an internal loading control. Samples: MC, media control. (B and D) Silencing DR4 and DR5 prevent caspase-9 cleavage and caspase-3/7 activity induced by HCV infection. Huh7.5.1 cells transfected with non-targeting (NT) or gene-specific (ST) siRNA pools targeting DR4 and DR5, respectively, were infected with HCVcc. At 3 days post-infection, the mRNA levels of DR4 and DR5 were analyzed by real-time qRT-PCR (B) (mean±SD; n = 3; *p<0.05, **p<0.01) and whole cell lysates were analyzed by Western blotting with antibodies specific to the indicated proteins (C). (D) The activity of caspase-3/7 was measured according to manufacturer's instructions (mean±SD; n = 3; *p<0.005).</p

HCV infection stimulates DR4 and DR5 expression.

<p>(A-C) Quantitative analyses of DR4 and DR5 gene expression in HCV-infected cells. Huh7.5.1 cells were infected with HCVcc. At 3 days post-infection, cells were used for analysis of DR4 and DR5 gene expression. (A and B) Intracellular mRNA level of DR4 and DR5 genes were analyzed by real-time qRT-PCR. Human GNB2L1 was used to normalize changes in DR4 and DR5 gene expression (mean±SD; n = 3; *p<0.001). (C) The protein expression levels of DR4 and DR5 were analyzed by Western blotting with anti-DR4 and DR5 antibodies. The expression of HCV core protein was analyzed by Western blotting with anti-HCV core antibody. β-actin was used as an internal loading control. The relative intensity of DR4 and DR5 expression normalized to β-actin was analyzed by ImageJ.</p

HCV-induced DR4/DR5-mediated caspase-8 activity affects PARP cleavage.

<p>(A) Western blot analysis of cleaved PARP in HCV-infected cells. Whole cell lysates extracted from HCV-infected Huh7.5.1 cells grown in the absence or presence of recombinant TRAIL (100 ng/ml) were analyzed by Western blotting with antibodies specific to the indicated proteins. (B) Huh7.5.1 cells transfected with non-targeting (NT) or gene-specific (ST) siRNA pools targeting DR4 and DR5, respectively, were infected with HCVcc. At 3 days post-infection, whole cell lysates were analyzed by Western blotting with antibodies specific to the indicated proteins. (C) HCV-infected Huh7.5.1 cells stimulated with recombinant TRAIL (100 ng/ml) were treated with Z-VAD-FMK (pan inhibitor; 40 µM), Z-IETD-FMK (caspase-8 inhibitor; 40 µM), and Z-LEHD-FMK (caspase-9 inhibitor; 40 µM), respectively, for 72 h before harvest. The expression level of PARP was analyzed by Western blotting with anti-cleaved PARP antibody. (D) Western blot analysis showing dose-dependent effects of recombinant TRAIL on PARP cleavage in HCV-infected cells. HCV-infected Huh7.5.1 cells were treated with recombinant TRAIL for 72 h at the indicated dose points. Whole cell lysates were analyzed by Western blotting with anti-cleaved PARP antibody. Samples: MC, media control. (A-D) β-actin was used as an internal loading control.</p

A prospective comparative assessment of the accuracy of the FibroScan in evaluating liver steatosis

<div><p>Background/aims</p><p>Recent studies have demonstrated the utility of the FibroScan^® device in diagnosing liver steatosis, but its usefulness has not been thoroughly appraised. We investigated the usefulness of the controlled attenuation parameter (CAP) in detecting and quantifying liver steatosis.</p><p>Methods</p><p>A prospective analysis was applied to 79 chronic liver disease patients who underwent a liver biopsy, a FibroScan investigation, ultrasonography, and hepatic steatosis index (HSI). The presence and degree of steatosis as measured by the FibroScan device, ultrasonography and HSI were compared with the results for the liver biopsy tissue.</p><p>Results</p><p>There was substantial concordance between the liver biopsy results and the CAP as evaluated by the kappa (κ) index test for detecting liver steatosis (κ_CAP = 0.77, <i>P</i><0.001; κ_{ultrasonography} = 0.60, <i>P</i><0.001; κ_HSI = 0.47, <i>P</i><0.001). The areas under the receiver operating characteristic curve (AUROCs) of the CAP, ultrasonography, and HSI were 0.899 [95% confidence interval (CI) = 0.826–0.972)], 0.859 (95% CI = 0.779–0.939), and 0.766 (95% CI = 0.655–0.877), respectively. The optimal CAP cutoff value for differentiating between normal and hepatic steatosis was 247 dB/m, which produced sensitivity and specificity values of 91.9% and 85.7%, respectively, as well as a positive predictive value of 85.0% and a negative predictive value of 92.3%.</p><p>Conclusion</p><p>The CAP produces results that are highly concordant with those of a liver biopsy in detecting steatosis. Therefore, the CAP is a noninvasive and reliable tool for evaluating liver steatosis, even in the early stages.</p></div

The optimal cut off value of the CAP for differentiating between normal and hepatic steatosis.

<p>The optimal cut off value of the CAP for differentiating between normal and hepatic steatosis.</p

ROC curves and AUROC for CAP, ultrasonography and HSI for detecting hepatic steatosis.

<p>ROC, Receiver operating characteristics (ROC); AUROC, and area under ROC; CAP, Controlled attenuation parameters, HSI, hepatic steatosis grade.</p

Summary of previous studies on the accuracy and cutoff values of CAP in discriminating steatosis.

<p>Summary of previous studies on the accuracy and cutoff values of CAP in discriminating steatosis.</p

Factors associated with steatosis on liver biopsy.

<p>Factors associated with steatosis on liver biopsy.</p

Clinical characteristics of the patients.

<p>Clinical characteristics of the patients.</p