Hepatitis B virus infection in patients with Wilson disease: A large retrospective study.

BACKGROUND: Wilson disease (WD) is the most common genetic metabolic liver disease. Some studies have shown that comorbidities may have important effects on WD. Data on hepatitis B virus (HBV) infection in patients with WD are limited. AIM: To investigate the prevalence and clinical impact of HBV infection in patients with WD. METHODS: The clinical data of patients with WD were analyzed retrospectively, and the data of patients with concurrent WD and HBV infection were compared with those of patients with isolated WD. RESULTS: Among a total of 915 WD patients recruited, the total prevalence of current and previous HBV infection was 2.1% [95% confidence interval (CI): 1.2%-3.0%] and 9.2% (95%CI: 7.3%-11.1%), respectively. The main finding of this study was the identification of 19 patients with concurrent WD and chronic hepatitis B (CHB) infection. The diagnosis of WD was missed in all but two patients with CHB infection. The mean delay in the diagnosis of WD in patients with concurrent WD and CHB infection was 32.5 mo, which was significantly longer than that in patients with isolated WD (10.5 mo). The rates of severe liver disease and mortality in patients with concurrent WD and CHB infection were significantly higher than those in patients with isolated WD (63.1% vs 19.3%, P = 0.000 and 36.8% vs 4.1%, P < 0.001, respectively). Binary logistic regression analysis revealed a significantly higher risk of severe liver disease at the diagnosis of WD in patients with current HBV infection [odds ratio (OR) = 7.748; 95%CI: 2.890-20.774; P = 0.000)] or previous HBV infection (OR = 5.525; 95%CI: 3.159-8.739; P = 0.000) than in patients with isolated WD. CONCLUSION: The total prevalence of current HBV infection in patients with WD was 2.1%. The diagnosis of WD in CHB patients is usually missed. HBV infection is an independent risk factor for severe liver disease in WD patients. The diagnosis of WD should be ruled out in some patients with CHB infection.

Population pharmacokinetics, safety and dosing optimization of voriconazole in patients with liver dysfunction: A prospective observational study.

AIMS: Voriconazole is a broad-spectrum antifungal agent for the treatment of invasive fungal infections. There is limited information about the pharmacokinetics and appropriate dosage of voriconazole in patients with liver dysfunction. This study aimed to explore the relationship between voriconazole trough concentration (Ctrough ) and toxicity, identify the factors significantly associated with voriconazole pharmacokinetic parameters and propose an optimised voriconazole dosing regimen for patients with liver dysfunction. METHODS: The study prospectively enrolled 51 patients with 272 voriconazole concentrations. Receiver operating characteristic curves were used to explore the relationship between voriconazole Ctrough and toxicity. The pharmacokinetic data was analysed with nonlinear mixed-effects method. Dosing simulations stratified by total bilirubin (TBIL, TBIL-1: TBIL < 51 µmol/L; TBIL-2: 51 µmol/L ≤ TBIL < 171 µmol/L; TBIL-3: TBIL ≥ 171 µmol/L) were performed. RESULTS: Receiver operating characteristic curve analysis revealed that voriconazole Ctrough of ≤ 5.1 mg/L were associated with significantly lower the incidence of adverse events. A 1-compartment pharmacokinetic model with first-order absorption and elimination was used to describe the data. Population pharmacokinetic parameters of clearance, volume of distribution and oral bioavailability were 0.88 L/h, 148.8 L and 88.4%, respectively. Voriconazole clearance was significantly associated with TBIL and platelet count. The volume of distribution increased with body weight. Patients with TBIL-1 could be treated with a loading dose of 400 mg every 12 hours (q12h) for first day, followed by a maintenance dose of 100 mg q12h administered orally or intravenously. TBIL-2 and TBIL-3 patients could be treated with a loading dose of 200 mg q12h and maintenance doses of 50 mg q12h or 100 mg once daily and 50 mg once daily orally or intravenously, respectively. CONCLUSIONS: Lower doses and longer dosing intervals should be considered for patients with liver dysfunction. TBIL-based dosing regimens provide a practical strategy for achieving voriconazole therapeutic range and therefore maximizing treatment outcomes.

Clustering Cases of Chlamydia psittaci Pneumonia in Coronavirus Disease 2019 Screening Ward Staff.

We describe 4 cases of Chlamydia psittaci pneumonia among medical staff in a coronavirus disease 2019 (COVID-19) screening ward, as well as the experience of dealing with this nosocomial infection event. Atypical pneumonia, in addition to COVID-19, should be considered when clustering cases occur, even during a COVID-19 pneumonia pandemic.

Cefotiam Treatment in Children: Evidence of Subtherapeutic Levels.

BACKGROUND: Cefotiam, a second-generation cephalosporin, is a broad-spectrum antibiotic with good antibacterial action against both gram-negative and gram-positive bacteria. It is used widely in clinical practice, although bacterial drug resistance makes its clinical use problematic. The authors hypothesized that subtherapeutic concentrations of cefotiam leads to bacterial resistance. The present study was conducted to evaluate whether the standard cefotiam dosing regimen resulted in a subtherapeutic concentrations in children. METHOD: Data were prospectively collected from pediatric patients with suspected or confirmed community-acquired pneumonia who were receiving cefotiam at the standard dosing regimen (40-80 mg/kg, 2 or 3 times daily). A blood sample was collected after 70%-100% of the dosing interval, and plasma concentrations were determined by high-performance liquid chromatography using an ultraviolet detector. RESULTS: The data from 88 patients (age, 3.0 ± 2.8 years; weight, 15.4 ± 8.3 kg) were used for analysis. The average of cefotiam concentrations was 0.06 mcg/mL (range: <0.05-0.79 mcg/mL). Most patients (n = 72, 81.8%) had concentrations below 0.1 mcg/mL; only 2 patients had concentrations higher than 0.4 mcg/mL. CONCLUSIONS: The standard dosing regimen for cefotiam resulted in extremely low plasma concentrations in children; such low concentrations may lead to antimicrobial drug resistance. Thus, an increase in cefotiam dosage in children to 80 mg/kg 4 times daily is recommended (maximum dose on the label).

Intravoxel incoherent motion derived liver perfusion/diffusion readouts can be reliable biomarker for the detection of viral hepatitis B induced liver fibrosis.

BACKGROUND: Recent two studies reported that intravoxel incoherent motion (IVIM) analysis can separate healthy livers and viral hepatitis B (VHB) induced liver fibrosis. However, in these two studies the starting b value for bi-exponential decay analysis was b =10 and 15 s/mm2 respectively. The current study has two primary aims. The first is to further confirm the diagnostic value of IVIM in detecting liver fibrosis. The second is to test whether by sampling very low b value densely, then b =0 s/mm2 image could be included to improve IVIM's diagnostic performance. METHODS: This was a prospective study with data acquired at the Third Xiangya Hospital of Central South University, Changsha, China. Healthy volunteers and patients suspected of VHB induced liver fibrosis with liver biopsy performed, as well as hepatocellular carcinoma patients scheduled for surgery, were recruited. All the hepatocellular carcinoma patients had liver fibrosis. After exclusions based on pre-defined criteria for image data quality, for IVIM analysis this study included 20 healthy volunteers; 4 chronic VHB patients with biopsy showing no liver fibrosis; 11 stage-1 liver fibrosis patients, 10 stage-2 liver fibrosis patients, 2 stage-3 liver fibrosis patients, and 5 stage-4 liver fibrosis patients. In the liver fibrosis patients, 1, 19, and 8 cases had inflammation grade-0, grade-1, and grade-2 respectively. The reference IVIM bi-exponential decay curve fitting analysis was segmented fitting performed with b =2 s/mm2 image as the starting point and a threshold-b of 60 s/mm2. This reference fitting method was compared with threshold-b of 40 s/mm2, full fitting, fitting starting from b =0, 5, and 10 s/mm2 respectively. The potential correlation between IVIM readouts and liver function was assessed for the liver fibrosis patients. RESULTS: Based on the smaller coefficient of variation (CoV) for the volunteer group and the smaller patient/volunteer ratios [= (mean measurement for patient groups)/(mean measurement for healthy volunteers)], the comparison of fitting methods favored the reference approach starting from b =2 s/mm2 with a threshold-b of 60 s/mm2. The IVIM measures of four patients without liver fibrosis resembled those of healthy subjects. PF offered the best diagnostic value for separating healthy livers and fibrotic livers, and a threshold of PF =0.1406 separated all fibrotic livers and healthy livers with an exception of one hepatocellular carcinoma patient (fibrosis grade-2/inflammation grade-2). The correlation between fibrosis grading and inflammation grading was weakly positive; while compared with fibrotic livers with inflammation grade-1, fibrotic livers with inflammation grade-2 showed a trend of higher Dfast. A weak correlation is shown with lower PF and lower Dfast associated with lower total protein, lower albumin; higher alanine transaminase, higher aspartate transaminase; higher total bilirubin, and higher direct bilirubin. CONCLUSIONS: Segmented-fitting with threshold-b =60 s/mm2 and starting from non-zero very low b value outperforms other methods. IVIM has high sensitivity in detecting liver fibrosis, and PF and Dfast have potential correlation with serum liver function biomarkers. IVIM measures and liver fibrosis grading are not in a linear relationship.

Screening of prognostic risk microRNAs for acute myeloid leukemia.

Objectives This study aimed to investigate the risk miRNAs (microRNAs) for AML (acute myeloid leukemia) prognosis and related regulatory mechanisms. Methods MiRNA and gene expression data, as well as clinical data of 176 patients were first downloaded from TCGA. Then miRNAs and genes significantly affecting the survival time based on KM survival curve were identified using Log Rank test. Next, COX proportional-hazard regression analysis was performed to screen the risk miRNAs (P-value < 0.05). Common genes from survival analysis and predicted by miRWalk were used to construct the miRNA regulatory network with the risk miRNAs. Finally, a protein-protein interaction (PPI) network was constructed, as well as functional annotation and pathway enrichment analysis. Results The survival analysis revealed 33 miRNAs and 1,377 genes significantly affecting the survival time. HR values of nine miRNAs (up-regulated hsa-mir-606, 520a, 137, 362, 599, 600, 202, 639and down-regulated 502) were either >1 or <1. The miRNA regulatory network contained 477 nodes and 944 edges. The top ten genes of the constructed PPI network were EGFR, EIF4G1, REL, TOP1, COL14A1, HDAC3, MRPL49, PSMA2, TOP2A and VCAM1 successively. According to pathway enrichment analysis, 6 KEGG pathways and 6 REACTOME pathways were obtained respectively. Conclusion Up-regulated hsa-mir-520a, 599, 606, 137 and 362 may increase the prognostic risk for AML patients via regulating the expression of corresponding target genes, especially COL14A1, HDAC3, REL, EGFR, PSMA2, EIF4G1, MRPL49 and TOP1.

The optimal threshold of serum ceruloplasmin in the diagnosis of Wilson's disease: A large hospital-based study.

BACKGROUND AND AIMS: A ceruloplasmin (CP) concentration <200 mg/L is conventionally considered as one of the major diagnostic criteria for Wilson's disease (WD). However, the diagnostic accuracy of this threshold has never been investigated in a sufficiently large group of patients. This study aims to present the results of serum CP measurements in various patients and to identify the optimal cutoff value of CP for the diagnosis of WD. MATERIALS AND METHODS: We identified patients whose CP levels were evaluated from January 1, 2016 to December 31, 2016 using a laboratory information database. Data related to CP measurement were retrieved. We carefully reviewed patients' electronic medical records to correct errors and to obtain other necessary data. Data related to WD were retrieved from a special document containing medical records of patients with WD, which were created, modified, and maintained by authors. RESULTS: CP level was determined in 4048 patients (WD, 297; non-WD, 3751). The mean serum CP level in patients with WD was 50.6±44.2 mg/L, which was significantly lower than that in non-WD patients (293.2±117.3 mg/L, p<0.001). Only 1.0% of patients with WD had CP ≥200 mg/L. The sensitivity and specificity of CP for the diagnosis of WD were 99.0 and 80.9%, respectively, for the conventional cutoff value <200 mg/L and 95.6 and 95.5%, respectively, for the cutoff value <150 mg/L; the latter provided a higher diagnostic accuracy for WD. 53.0% of patients with liver failure, 37.7% of patients with nephrotic syndrome, and 23.0% of patients age 1 to 6 months had serum CP <200 mg/L. Patients who were pregnant and those with malignant tumors, and infectious and inflammatory diseases had significantly higher mean serum CP levels. CONCLUSION: The optimal cutoff value of CP for the diagnosis of WD in China is 150 mg/L, with a sensitivity of 95.6% and specificity of 95.5%, thereby providing the highest diagnostic accuracy for WD.

Molecular mechanisms of HBeAg in persistent HBV infection.

PURPOSE: The aim of this study is to investigate the T-lymphocyte subpopulation and expression of programmed cell death-1 (PD-1), toll-like receptor (TLR)3, TLR4, and interferon (INF)-γ to illustrate the relationship between hepatitis B e antigen (HBeAg) and persistent hepatitis B virus (HBV) infection. METHODS: Blood was taken from normal subjects into anticoagulation tubes to separate peripheral blood mononuclear cells (PBMCs). The PBMCs were divided into four groups and cultured with various concentrations of HBeAg for 72 h. Changes in the T-cell subset were analyzed through cell counting by flow cytometry, and expression of TLR3, TLR4, and PD-1 was assessed by flow cytometry and Western blot. The concentration of IFN-γ was analyzed using enzyme-linked immunospot (ELISPOT) experiments. RESULTS: PBMCs were stimulated with various concentrations of HBeAg for 72 h and assayed by flow cytometry to determine CD4+ and CD8+ cell counts. The relative frequencies of CD4+ and CD8+ subpopulations and the CD4+/CD8+ ratio decreased compared with the control group, and T-cell impairment was significantly associated with higher HBeAg load. TLR3, TLR4, and PD-1 protein expression was assessed using flow cytometry and Western blotting. Expression of TLR3, TLR4, and PD-1 increased with increasing concentration of HBeAg. ELISPOT experiments were used to determine the concentration of IFN-γ. IFN-γ production in treatment groups was lower than in the control group. Comparing IFN-γ production in treatment groups, IFN-γ production in PBMCs stimulated with high dose of HBeAg was lower than for those stimulated with low-dose HBeAg. CONCLUSIONS: HBeAg can inhibit proliferation of lymphocytes, increase TLR3, TLR4, and PD-1 expression, and decrease IFN-γ production. This may be one of the molecular mechanisms of HBV immune tolerance.

Recognition of the human antibody-mediated platelet destruction in adult ITP patients by C-reactive protein.

Immune thrombocytopenia purpura (ITP) is characterized by destruction of circulating platelets and the presence of antiplatelet IgG antibodies, which opsonize platelets for splenic clearance resulting in low levels of circulating platelets, and the disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors also play a role. Although the main cause of ITP remains unclear, but its relationship with some infection was demonstrated, including viral or bacterial infections. C-reactive protein (CRP), a member of the pentraxin family, is a major acute-phase protein in humans and is a clinical marker of infection. We aimed to investigate the correlation between the levels of CRP and the presence of antiplatelet IgG antibodies in adults with newly diagnosed ITP. CRP levels and platelet counts were measured in the blood samples from a 60 ITP patient (with confirmed anti-GPIIb/IIIa antibodies), 60 infection patients (all without anti-GPIIb/IIIa antibodies) and 60 normal individuals. The bleeding score, recover time of intravenous immune globulin (IVIg) therapy and the number of megakaryocytes in bone marrow were recorded in ITP patients. The platelet count, bleeding score, recover time of intravenous immune globulin (IVIG) therapy and the number of megakaryocytes in bone marrow and CRP concentrations were compared in ITP group using Spearman's correlation coefficient. We examined the influence of intraperioneal CRP administration on antibody-mediated platelet destruction in mice. There were no statistical differences in gender, age and body mass index among the three groups (P>0.05). Though CRP levels are significantly elevated in ITP patients and infection patients (P<0.05), the platelet count was markedly lower only in ITP patients. We found that CRP was inert toward platelets without antiplatelet antibodies in this study. There are a significant correlation between CRP levels and platelet counts, bleeding severity and the number of megakaryocytes in bone marrow aspiration (r=-0.5079, r=0.5498, r=0.4172, P<0.001, respectively). Moreover, a significant correlation was observed between the recovery time of platelet count and CRP levels (r=-0.5569, P<0.001). In mice, platelet count was lower in Anti-CD41 (0.75 µg)+, CRP (200 µg) group as compared with Anti-CD41 (0.75 µg)+, CRP(-) group and Anti-CD41 (0.75 µg)-, CRP (200 µg) group (P<0.05). In summary, this study indicated that CRP levels are significantly elevated in ITP patients all with confirmed anti-GPIIb/IIIa antibodies, which is able to predict the clinical bleeding severity of ITP patients. The slower CRP levels reduction after IVIg treatment predicted slower platelet count recovery in ITP.

Suppression of HSP27 increases the anti­tumor effects of quercetin in human leukemia U937 cells.

Quercetin, a natural flavonoid, inhibits the growth of leukemia cells and induces apoptosis. Heat shock protein 27 (HSP27) has been reported to promote the development of leukemia by protecting tumor cells from apoptosis through various mechanisms. The present study investigated the effects of small hairpin (sh)RNA-mediated HSP27 knockdown on the anti­cancer effects of quercetin in U937 human leukemia cells. Cells were transfected with recombinant lentiviral vector pCMV­G­NR­U6­shHSP27 (shHSP27), which expressed shRNA specifically targeting the HSP27 gene, alone or in combination with quercetin. The results showed that shHSP27 and quercetin synergistically inhibited U937 cell proliferation and induced apoptosis by decreasing the Bcl2-to-Bax ratio. Furthermore, this combined treatment significantly suppressed the infiltration of tumor cells and the expression of angiogenesis­associated proteins HIF1α and VEGF. Compared with shHSP27 or quercetin alone, shHSP27 plus quercetin markedly decreased the protein expression of cyclinD1 and thus blocked the cell cycle at G1 phase. The Notch/AKT/mTOR signaling pathway is important in tumor aggressiveness; quercetin plus shHSP27 significantly decreased Notch 1 expression and the phosphorylation levels of the downstream signaling proteins AKT and mTOR. The inhibitory effects of quercetin plus shHSP27 on this pathway may thus have been responsible for the cell cycle arrest, inhibition of proliferations and infiltration as well as enhancement of apoptosis. Therefore, these findings collectively suggested that suppression of HSP27 expression amplified the anti­cancer effects of quercetin in U937 human leukemia cells, and that quercetin in combination with shHSP27 represents a promising therapeutic strategy for human leukemia.

shRNA-mediated silencing of sorcin increases drug chemosensitivity in myeloma KM3/DDP and U266/ADM cell lines.

Sorcin is a penta-EF hand calcium binding protein, which is involved in the resistance to chemotherapeutics in cancer cells, and is overexpressed in various cancer cells. However, tumor relapse combined with the development of drug resistance remains a significant problem. Here, we demonstrated that silencing of Sorcin in chemotherapy resistance myeloma U266/ADM and KM3/DDP cell lines resulted in reduced cell proliferation, cell cycle arrest and cell apoptosis. Sorcin siRNA successfully silenced Sorcin mRNA and protein expression. Silencing of Sorcin also significantly reduced the mRNA and protein expression levels of MDR1, MRP1, GST-π, Survinvin, Livin, Bcl-2, Cyclin-D1, phospho-Src, C-myc, p21, NF-κB and phospho-AKT, while p53 expression and caspase-3 and caspase-8 activity significantly increased when compared with control group. Silencing of Sorcin significantly increased the sensitivity of KM3/DDP cells to cisplatin and the sensitivity of U266/ADM to adriamycin, compared to cells untransfected and transfected with negative control shRNA. In addition, intracellular accumulation of Rhodamine 123 significantly increased in KM3/DDP and U266/ADM cells. In summary, our studies indicate that drug resistance can be effectively reversed in cisplatin-resistance and adriamycin-resistant myeloma cells through delivery of siRNAs targeting Sorcin. Assessment of potential as a target for human myeloma treatment is clearly warranted.

Prospective evaluation of the diagnostic accuracy of hepatic copper content, as determined using the entire core of a liver biopsy sample.

UNLABELLED: Hepatic copper determination is an important test for the diagnosis of Wilson's disease (WD). However, the method has not been standardized, the diagnostic accuracy has not been evaluated prospectively, and the optimal cut-off value remains controversial. Accordingly, we aimed to prospectively evaluate the diagnostic accuracy of hepatic copper content, as determined using the entire core of a liver biopsy sample. Patients for whom a liver biopsy was indicated were consecutively enrolled. Hepatic copper content was determined with atomic absorption spectroscopy. All assays were performed using careful quality control by a single technician. WD diagnosis was based on WD score or its combination with clinical follow-up results. A total of 3,350 consecutive patients underwent liver biopsy. Six hundred ninety-one patients, including 178 with WD, underwent two passes of liver biopsy with hepatic copper determination. Mean hepatic content in WD patients was 770.6 ± 393.2 µg/g dry weight (wt). Sensitivity, specificity, and positive and negative predictive values of hepatic copper content for WD diagnosis in the absence of primary biliary cirrhosis (PBC) or primary sclerosing cholangitis at the cut-off value of 250 µg/g dry wt. were 94.4%, 96.8%, 91.8%, and 97.8%, respectively. The most useful cut-off value was 209 µg/g dry wt, with a sensitivity and specificity of 99.4% and 96.1%, respectively. A total of 23.3% of patients without WD and PBC had hepatic copper content >75 µg/g dry wt. CONCLUSION: A liver biopsy sample of more than 1 mg dry wt may reliably reflect hepatic copper content and should be used for hepatic copper determination. Hepatic copper determination is a very valid procedure for the diagnosis of WD, and the most useful cut-off value is 209 µg/g dry wt.

Role of Nrf2 in chronic liver disease.

Nuclear erythroid 2-related factor 2 (Nrf2) is a central regulator of antioxidative response elements-mediated gene expression. It has a significant role in adaptive responses to oxidative stress by interacting with the antioxidant response element, which induces the expression of a variety of downstream targets aimed at cytoprotection. Previous studies suggested oxidative stress and associated damage could represent a common link between different forms of diseases. Oxidative stress has been implicated in various liver diseases, including viral hepatitis, nonalcoholic fatty liver disease/steatohepatitis, alcoholic liver disease and drug-induced liver injury. Nrf2 activation is initiated by oxidative or electrophilic stress, and aids in the detoxification and elimination of potentially harmful exogenous chemicals and their metabolites. The expression of Nrf2 has been observed throughout human tissue, with high expression in detoxification organs, especially the liver. Thus, Nrf2 may serve as a major regulator of several cellular defense associated pathways by which hepatic cells combat oxidative stress. We review the relevant literature concerning the crucial role of Nrf2 and its signaling pathways against oxidative stress to protect hepatic cell from oxidative damage during development of common chronic liver diseases. We also review the use of Nrf2 as a therapeutic target to prevent and treat liver diseases.

[A diagnostic model for alcoholic liver disease based on a generalized regression neural network].

OBJECTIVE: To study the feasibility and rationale of using a generalized regression neural network model integrated with multiple disease indicators for diagnosing alcoholic liver disease (ALD). METHODS: ALD indicators were identified by reviewing the clinical testing results of 40 ALD patients from the literature and 135 patients from the Second Xiangya Hospital of Central South University, who were also classified by physician experts upon clinical consultation. Seven indicators were selected as diagnosis indexes and applied to a general regression neural network diagnostic model. Thirty-four of the reported patients and 120 of the clinical patients were selected for use as training samples to establish the indicator recognition pattern for the model, and the remaining six and 15 patients from the two respective groups were selected for use as testing samples to determine the model's diagnostic ability. RESULTS: The model provided a correct diagnosis of ALD sub-classification for 94.1% (32/34) of the reported patients and 100% (120/120) of the clinical patients in the training set. The correct diagnosis rates achieved with the training sets were 100% for both the reported patient group (6/6) and the clinical patient group (15/15), indicating that the results of the diagnostic model were in good agreement with the ALD classifications generated by the clinical expert consultations. CONCLUSION: The general regression neural network model based on multiple indicators of ALD is capable of providing accurate and comprehensive diagnosis of ALD and may be feasible for clinical applications.

[Dynamic changes in PD-1, TLR3, and TLR4 surface expression on peripheral blood mononuclear cells in chronic hepatitis C patients undergoing peg-IFNalpha-2a plus ribavirin combination therapy].

OBJECTIVE: To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA. RESULTS: The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05). CONCLUSION: Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.

[Hepatitis C virus strain JFH1 down-regulates expression of growth arrest and DNA damage-inducible gene 45a in human hepatoma Huh7.5.1 cells].

OBJECTIVE: To investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells. METHODS: HCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing. RESULTS: The HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01). CONCLUSION: HCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.

[The therapeutic effect of Anluohuaxian capsule combined with adefovir dipivoxil on patients with chronic hepatitis B and influence on hepatic histology].

UNLABELLED: To observe the efficacy of adefovir dipivoxil(ADV) in combination with Anluohuaxian capsule in the treatment of chronic hepatitis B (CHB) patients. METHODS: 72 cases with CHB were randomly divided into two groups. 36 cases of treatment group were given ADV combined with Anluohuaxian capsule for 48 weeks. 36 cases of control group were given ADV. The levels of serum ALT, AST, Alb, TBil, HA, LN, CIV, HBV DNA and hepatic tissue were compared before and after being treated. RESULTS: After 48 weeks treatment,the liver function, serum fibrosis index and histology of treatment group and control group all have improved. After treatment, the two groups in the levels of ALT(t=0.746, P=0.342), AST (t=0.369, P=0.713), TBil (t=0.146, P=0.684), Alb(t=0.148, P=0.883), liver tissue inflammation mobility scoring (t=1.666, P=0.100) and HBV DNA negative rate (x2=0.141, P=0.708) were no evident difference.The level of HA, LN, CIV were significantly lower in treatment group(101.58+/-30.11, 147.89+/-41.72, 38.75+/-9.50) compared with control group(182.25+/-117.59, 181.50+/-56.96, 74.92+/-31.14) (P less than 0.05). After the treatment, the liver tissue fibrosis scoring was significantly lower in treatment group (10.61+/-2.37) compared with before the treatment (12.28+/-3.16) (P less than 0.05).There was no difference found between after the treatment (11.36+/-2.93) and before the treatment (12.17+/-3.01) in control group (P more than 0.05). CONCLUSIONS: The results show that the treatment with ADV in combination with Anluohuaxian capsule can play promoting antifibrotic effect and significant improved liver histology of chronic hepatitis B patients.

The oncogenic role of NS5A of hepatitis C virus is mediated by up-regulation of survivin gene expression in the hepatocellular cell through p53 and NF-κB pathways.

Approx. 4% of patients experiencing chronic infection of human HCV (hepatitis C virus) ultimately develop HCC (hepatocellular carcinoma). The NS5A (non-structural protein 5A) encoded by HCV has been reported to have an oncogenic role during HCV infection, but the precise mechanism remains largely unclear. The aim of this study is to investigate the signal transduction pathways that mediate the role of NS5A in hepatocarcinogenesis. HepG2 cells were transfected with a plasmid expressing HCV NS5A protein. Subsequently, cell proliferation was analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and cell counting, apoptosis was analysed by Hoechst 33342 staining, and the gene expression profile was identified by microarray and subsequently validated by RT-PCR (reverse transcription-PCR). The protein levels of survivin, p53, NOS2A (nitric oxide synthase 2A), cyclin D1 and NF-κB (nuclear factor κB) were monitored by Western blotting. Our results showed that transfection of HCV NS5A expression plasmid significantly down-regulated the expression of nine genes and up-regulated the expression of ten genes among the 104 genes detectable by the microarray associated with signalling transduction. The increased expression of survivin mRNA and protein, down-regulated p53 protein levels and increased NOS2A, cyclin D1 and NF-κB protein levels were further identified. Our results suggested that HCV NS5A protein can enhance survivin transcription by increasing p53 degradation and stimulating NOS2A expression as well as NF-κB relocation to the nucleus. The functions of survivin in anti-apoptosis and regulation of cell division might mediate the role of NS5A in HCV-induced HCC.