Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway.

Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.

The burden of Hepatitis B virus infection in Kenya: A systematic review and meta-analysis.

Background: Chronic Hepatitis B virus (HBV) infection causes liver cirrhosis and cancer and is a major public health concern in Kenya. However, so far no systematic review and meta-analysis has been conducted to estimate the burden of disease in the country. A better understanding of HBV infection prevalence will help the government implement efficient strategies at eliminating the disease. This systematic review and meta-analysis was therefore conducted to summarize and update the available information on the burden of HBV in Kenya. Method: We systematically searched PubMed, Science Direct, Web of Science, Scopus, African Journals OnLine, and Google Scholar databases to retrieve primary studies conducted between January 1990 and June 2021 that assessed the prevalence of HBV infection in Kenya based on measurement of the Hepatitis B Surface Antigen (HBsAg). Meta-analysis was performed using the random effects model where HBsAg prevalence was estimated at a 95% confidence interval (CI) after simple pooling analysis. Potential sources of heterogeneity were also investigated. Results: Fifty studies were included in the meta-analysis with a sample size of 108448. The overall pooled prevalence estimate of HBV in Kenya was 7.8% (95% CI: 5.8-10.1). Subgroup analysis revealed the highest prevalence among patients presenting with jaundice at 41.7% (95% CI: 13.5-73.3) whereas blood donors had the lowest prevalence at 4.1% (95% CI: 2.4-6.3). Prevalence in Human Immunodeficiency Virus (HIV)-infected individuals was 8.2% (95% CI: 5.8-11.0). An estimate of the total variation between studies revealed substantial heterogeneity (I2 = 99%) which could be explained by the study type, the risk status of individuals, and the region of study. Conclusion: We present the first systematic review and meta-analysis of the prevalence of HBV in Kenya. Our results show that the burden of HBV in Kenya is still enormous. This calls for an urgent need to implement public health intervention measures and strategic policies that will bring the disease under control and lead to final elimination. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=264859, identifier: CRD42021264859.

A novel cDNA-uPA/SCID/Rag2-/- /Jak3-/- mouse model for hepatitis virus infection and reconstruction of human immune system.

Although human hepatocyte-transplanted immunodeficient mice support infection with hepatitis viruses, these mice fail to develop viral hepatitis due to the lack of an adaptive immune system. In this study, we generated new immunodeficiency cDNA-urokinase-type plasminogen activator (uPA)/SCID/Rag2-/- /Jak3-/- mice and established a mouse model with both a humanized liver and immune system. Transplantation of human hepatocytes with human leukocyte antigen (HLA)-A24 resulted in establishment of a highly replaced liver in cDNA-uPA/SCID/Rag2-/- /Jak3-/- mice. These mice were successfully infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) for a prolonged period and facilitate analysis of the effect of anti-HCV drugs. Administration of peripheral blood mononuclear cells (PBMCs) obtained from an HLA-A24 donor resulted in establishment of 22.6%-81.3% human CD45-positive mononuclear cell chimerism in liver-infiltrating cells without causing graft-versus-host disease in cDNA-uPA/SCID/Rag2-/- /Jak3-/- mice without human hepatocyte transplantation. When mice were transplanted with human hepatocytes and then administered HLA-A24-positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocyte-specific anti-HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo-reaction between transplanted human immune cells and human liver using a novel cDNA-uPA/SCID/Rag2-/- /Jak3-/- mouse. This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post-transplantation liver rejection.

The presence of vessels encapsulating tumor clusters is associated with an immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Recently, a distinct vascular pattern in hepatocellular carcinoma (HCC) called vessels encapsulating tumor-forming clusters (VETC) has received attention because of its association with poor prognosis. However, little is known about the mechanism by which VETC promotes an aggressive phenotype at the molecular level. In our study, the association between differences in stepwise signal intensity in the HB phase and molecular subtypes and somatic mutations associated with the immune microenvironment were investigated using the International Cancer Genome Consortium (ICGC) cohort (66 patients). To our knowledge, this is the first study to analyze the molecular patterns of VETC using RNA-Seq data. The VETC+ HCC group showed significantly lower overall survival and higher cumulative incidence of extrahepatic metastasis after curative hepatic resection than the VETC- HCC group. The VETC+ group exhibited molecular features indicative of lower immune activation than the VETC- group, suggesting that tumor cells in the VETC+ group were more likely to escape from the immune response, which could lead to the shorter OS (Overall survival) and higher risk of metastasis. On the other hand, gene expression levels of fibroblast growth factor receptors were upregulated in VETC+ HCC, suggesting that VETC+ HCC might benefit from lenvatinib treatment. Our results demonstrate that VETC+ HCC was associated with the suppression of tumor immune responses at the molecular level.

Construction of an anti-hepatitis B virus preS1 antibody and usefulness of preS1 measurement for chronic hepatitis B patients: Anti-HBV PreS1 antibody.

OBJECTIVES: The preS1 region plays an essential role in hepatitis B virus (HBV) infection. We construct an antibody that binds to preS1 and a measurement system for serum preS1 in chronic HBV-infected patients. METHODS: Hybridoma clones that produce anti-preS1 antibodies were obtained by the iliac lymph node method. Epitope mapping was conducted, and an enzyme-linked immunosorbent assay (ELISA)-based method was developed. Using this ELISA system, serum preS1 levels were measured in 200 chronic HBV-infected patients. RESULTS: Eight types of hybridomas were obtained, of which antibody 3-55 using amino acids 38-47 as the epitope showed high binding affinity to preS1. Serum preS1 levels measured by ELISA using 3-55 antibody were correlated with HBsAg, HBcrAg and HBV DNA levels. Among HBeAg-negative patients without antiviral therapeutic objective (HBV DNA <3.3 log IU/mL or alanine aminotransferase ≤30 U/L), preS1 was significantly higher in subjects who had progressed to the point of requiring antiviral therapy compared to subjects who had maintained their status for the preceding three years (p<0.01). CONCLUSIONS: We constructed an antibody against preS1 and an ELISA system capable of measuring serum preS1 levels. PreS1 may serve as a novel tool to predict the need for antiviral therapy in HBeAg-negative HBV-infected patients.

In vitro analysis of hepatic stellate cell activation influenced by transmembrane 6 superfamily 2 polymorphism.

Non­alcoholic steatohepatitis (NASH) may progress via liver fibrosis along with hepatic stellate cell (HSC) activation. A single nucleotide polymorphism (SNP; rs58542926) located in transmembrane 6 superfamily 2 (TM6SF2) has been reported to be significantly associated with fibrosis in patients with NASH, but the precise mechanism is still unknown. The present study aimed to explore the role of TM6SF2 in HSC activation in vitro. Plasmids producing TM6SF2 wild-type (WT) and mutant type (MT) containing E167K amino acid substitution were constructed, and the activation of LX­2 cells was analyzed by overexpressing or knocking down TM6SF2 under transforming growth factor ß1 (TGFß) treatment. Intracellular α­smooth muscle actin (αSMA) expression in LX­2 cells was significantly repressed by TM6SF2­WT overexpression and increased by TM6SF2 knockdown. Following treatment with TGFß, αSMA expression was restored in TM6SF2­WT overexpressed LX­2 cells and was enhanced in TM6SF2 knocked­down LX­2 cells. Comparing αSMA expression under TM6SF2­WT or ­MT overexpression, expression of αSMA in TM6SF2­MT overexpressed cells was higher than that in TM6SF2­WT cells and was further enhanced by TGFß treatment. The present study demonstrated that intracellular αSMA expression in HCS was negatively regulated by TM6SF2 while the E167K substitution released this negative regulation and led to enhanced HSC activation by TGFß. These results suggest that the SNP in TM6SF2 may relate to sensitivity of HSC activation.

Circulating cytokines and angiogenic factors based signature associated with the relative dose intensity during treatment in patients with advanced hepatocellular carcinoma receiving lenvatinib.

BACKGROUND: Although lenvatinib was recently approved for treatment of advanced unresectable hepatocellular carcinoma (HCC) based on the phase III REFLECT trial, no biomarkers for management of lenvatinib treatment have been established. The aim of this study is to identify predictive biomarkers for the management of lenvatinib treatment in advanced HCC patients. METHODS: A total of 41 patients with advanced HCC were enrolled in this retrospective study. Serum levels of 22 circulating cytokines and angiogenic factors (CAFs) were measured by multiplex Luminex assay. Profiles of CAFs, clinical chemistry/hematology parameters, and clinical background were evaluated to explore biomarkers associated with clinical outcomes. RESULTS: Relative dose intensity (RDI) decreased significantly between weeks 1-2 and 3-4 (p < 0.001), and RDI during weeks 3-4 was a prominent indicator of progression-free survival (PFS). A signature based on baseline serum levels of nine CAFs associated with low RDI was identified. In a multivariate Cox regression analysis, patients with a favorable 9-CAFs signature [hazard ratio (HR) 0.42, 95% confidence interval (CI) 0.18-0.96, p = 0.040] had lower risk, and Child-Pugh grade B (HR 1.6, 95% CI 1.1-8.3, p = 0.026) and presence of macrovascular invasion (MVI; HR 2.9, 95% CI 1.0-8.3, p = 0.045) had higher risk of shorter PFS. CONCLUSION: This study demonstrates that RDI is an important predictive factor for longer PFS during lenvatinib treatment. In this hypothesis-generating exploratory analysis, we report that a CAF-signature associated with adverse events and RDI could predict PFS, which might contribute to improved management of lenvatinib treatment in HCC patients.

Signal Activation of Hepatitis B Virus-Related Hepatocarcinogenesis by Up-regulation of SUV39h1.

BACKGROUND: Hepatitis B virus (HBV) X (HBx) protein is associated with hepatocellular carcinogenesis via the induction of malignant transformation and mitochondrial dysfunction. However, the association between HBx and histone methyltransferase in carcinogenesis has not been fully clarified. In the current study, we analyzed the association between HBx and the histone methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39h1) using HBV replication models. METHODS: We constructed several HBx and SUV39h1 expression plasmids and analyzed the association between HBx and SUV39h1 with respect to HBV replication and hepatocarcinogenesis. RESULTS: SUV39h1 up-regulation was observed in HBV-infected humanized mouse livers and clinical HBV-related hepatocellular carcinoma tissues, indicating that SUV39h1 expression might be regulated by HBV infection. Through in vitro analysis, we determined that the coactivator domain of HBx interacts with the PSET (PostSET) and SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domains of SUV39h1. The expression levels of 4 genes, activating transcription factor 6, α-fetoprotein, growth arrest and DNA damage-inducible 45a, and dual-specificity phosphatase 1, known to induce carcinogenesis via HBx expression, were up-regulated by HBx and further up-regulated in the presence of both HBx and SUV39h1. Furthermore, histone methyltransferase activity, the main function of SUV39h1, was enhanced in the presence of HBx. CONCLUSIONS: We demonstrated that SUV39h1 and HBx enhance each other's activity, leading to HBx-mediated hepatocarcinogenesis. We propose that regulation of this interaction could help suppress development of hepatocellular carcinoma.

Publisher Correction: Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein's broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

Efficacy of glecaprevir and pibrentasvir treatment for genotype 1b hepatitis C virus drug resistance-associated variants in humanized mice.

Combination therapy with glecaprevir (GLE) and pibrentasvir (PIB) has high efficacy for pan-genotypic hepatitis C virus (HCV)-infected patients. However, the efficacy for patients who acquired potent NS5A inhibitor resistance-associated variants (RAVs) as a result of failure to respond to previous direct-acting antiviral (DAA) therapies is unclear. We investigated the efficacy of GLE/PIB treatment for genotype 1b HCV strains containing RAVs using subgenomic replicon systems and human hepatocyte transplanted mice. Mice were injected with serum samples obtained from a DAA-naïve patient or daclatasvir plus asunaprevir (DCV/ASV) treatment failures including NS5A-L31M/Y93H, -P58S/A92K or -P32 deletion (P32del) RAVs, then treated with GLE/PIB. HCV was eliminated by GLE/PIB treatment in mice with wild-type and NS5A-L31M/Y93H but relapsed in mice with NS5A-P58S/A92K, followed by emergence of additional NS5A mutations after cessation of the treatment. In NS5A-P32del-infected mice, serum HCV RNA remained positive during the GLE/PIB treatment. NS5A-P58S/A92K showed 1.5-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. When mice were administered various proportions of HCV wild-type and P32del strains and treated with GLE/PIB, serum HCV RNA remained positive in mice with high frequencies of P32del. In these mice, the P32del was undetectable by deep sequencing before GLE/PIB treatment, but P32del strains relapsed after cessation of the GLE/PIB treatment. GLE/PIB is effective for wild-type and NS5A-L31M/Y93H HCV strains, but the effect seems to be low for P58S/A92K and NS5A-P32del RAVs. Although NS5A-P32del was not detected, the mutation may be present at low frequency in DCV/ASV treatment failures.

Comparison of intracellular responses between HBV genotype A and C infection in human hepatocyte chimeric mice.

BACKGROUND AND AIMS: The clinical course and responsiveness to antiviral treatments differs among hepatitis B virus (HBV) genotypes. However, the cause of these differences is unclear. In the present study, we compared mRNA expression profiles in human hepatocyte chimeric mice infected with HBV genotypes A and C. METHODS: Fifteen chimeric mice were prepared and divided into the following three groups: uninfected control mice, HBV genotype A-infected mice, and HBV genotype C-infected mice. Human hepatocytes were collected from these mouse livers and gene expression analyses were performed using next-generation RNA sequencing. RESULTS: Although similar pathways were influenced by HBV infection, including inflammation mediated by chemokine and cytokine signaling, p53, and integrin signaling pathways, expression levels of up-regulated genes by HBV genotype A or C infection were quite different. In HBV genotype A-infected hepatocytes, 172 genes, including KRT23 and C10orf54, were significantly more highly expressed than in HBV genotype C-infected cells, whereas 10 genes, including SPX and IER3, were expressed at significantly lower levels. Genes associated with the p53 pathway and the inflammation mediated by chemokine and cytokine signaling pathway were more highly expressed in cells with HBV genotype A infection, whereas genes associated with CCKR signaling map and oxidative stress response were more highly expressed in cells with HBV genotype C infection. CONCLUSION: Several differences in gene expression with respect to HBV genotype A and C infection were detected in human hepatocytes. These differences might be associated with genotypic difference in the clinical course or responsiveness to treatment.

Endoplasmic reticulum-mediated induction of interleukin-8 occurs by hepatitis B virus infection and contributes to suppression of interferon responsiveness in human hepatocytes.

The events in the immune response to hepatitis B virus (HBV) remain unclear. We analyzed the direct influence of HBV on gene expression in human hepatocytes under immunodeficient conditions using a human hepatocyte chimeric mouse model. HBV-infected or non-infected chimeric mouse livers were collected, and gene expression profiles were compared. Since IL-8 was the most significantly up-regulated gene at 8 weeks after HBV infection, we focused on IL-8 and found that HBx and the large HBs (L-HBs) protein induce transcription of IL-8 via endoplasmic reticulum stress. This stress induces IL-8 transcription via NFAT activation and contributes to suppression of interferon responsiveness in HBV-infected human hepatocytes. In the present study, we identified a novel regulatory mechanism in which the L-HBs protein activates IL-8 via endoplasmic reticulum stress, suggesting a key role for IL-8 in the immune response to HBV and a potential new target for antiviral treatments of HBV infection.

CTL-associated and NK cell-associated immune responses induce different HBV DNA reduction patterns in chronic hepatitis B patients.

The activation of hepatitis B virus (HBV)-related hepatitis is associated with both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). We analyzed the association between the immune response and changes in the proportion of Pre-S deletion variants. We quantified Pre-S deleted HBV (HBV-del) and wild-type HBV (HBV-wt) DNA levels in sera obtained from HBV-infected mice and chronic hepatitis B patients. In chronic hepatitis B patients, the HBV-del proportion usually increased during or after ALT elevation but did not occur during all ALT elevations. To clarify this difference in the immunological responses, we performed in vivo analyses using HBV-infected human hepatocyte chimeric mice. Although HBV-del proportions did not change in mice with NK cell-associated hepatitis or in mice treated with entecavir, the proportions sharply increased in mice with CTL-associated hepatitis. Furthermore, the number of patients in which HBV-del proportions were greater than 5% was significantly higher in chronic hepatitis B patients than in asymptomatic carriers (P = 0.023). We identified associations between virological response in chronic hepatitis B patients and two different immune responses. The proportion of HBV-del variants could be a useful biomarker for distinguishing between chronic hepatitis and asymptomatic carriers.

Limitations of daclatasvir/asunaprevir plus beclabuvir treatment in cases of NS5A inhibitor treatment failure.

Combined daclatasvir (DCV)/asunaprevir (ASV) plus beclabuvir (BCV) treatment shows a high virological response for genotype 1b chronic hepatitis C patients. However, its efficacy for patients for whom previous direct-acting antiviral (DAA) therapy failed is not known. We analysed the efficacy of DCV/ASV/BCV treatment for HCV-infected mice and chronic hepatitis patients. Human hepatocyte chimaeric mice were injected with serum samples obtained from either a DAA-naïve patient or a DCV/ASV treatment failure and were then treated with DCV/ASV alone or in combination with BCV for 4 weeks. DCV/ASV treatment successfully eliminated the virus in DAA-naïve-patient HCV-infected mice. DCV/ASV treatment failure HCV-infected mice developed viral breakthrough during DCV/ASV treatment, with the emergence of NS5A-L31V/Y93H HCV resistance-associated variants (RAVs) being observed by direct sequencing. DCV/ASV/BCV treatment inhibited viral breakthrough in NS5A-L31V/Y93H-mutated HCV-infected mice, but HCV relapsed with the emergence of NS5B-P495S variants after the cessation of the treatment. The efficacy of the triple therapy was also analysed in HCV-infected patients; one DAA-naïve patient and four prior DAA treatment failures were treated with 12 weeks of DCV/ASV/BCV therapy. Sustained virological response was achieved in a DAA-naïve patient and one of the DCV/ASV treatment failures through DCV/ASV/BCV therapy; however, HCV relapse occurred in the other patients with prior DCV/ASV and/or sofosbuvir/ledipasvir treatment failures. DCV/ASV/BCV therapy seems to have limited efficacy for patients with NS5A RAVs for whom prior DAA treatment has failed.

Prevalence of NS5A resistance associated variants in NS5A inhibitor treatment failures and an effective treatment for NS5A-P32 deleted hepatitis C virus in humanized mice.

Patients with chronic hepatitis C virus (HCV) infection who have failed to respond to direct-acting antiviral (DAA) treatment often acquire drug resistance-associated variants (RAVs). The NS5A-P32 deletion (P32del) RAV confers potent resistance to NS5A inhibitors; therefore, patients who acquire this deletion are likely to fail to respond to DAA re-treatment. We investigated the prevalence of N55A-P32del in patients who failed to respond to prior NS5A inhibitor treatment using direct sequencing and analyzed the efficacy of DAA combination treatment in the presence of NS5A-P32del RAVs using human hepatocyte transplanted mice. NS5A-P32del was detected in one of 23 (4.3%) patients who had failed to respond to prior NS5A inhibitor treatment. Although four weeks of NS3/4A protease inhibitor glecaprevir plus NS5A inhibitor pibrentasvir treatment effectively suppressed HCV replication in wild-type HCV-infected mice, serum HCV RNA never became negative in P32del HCV-infected mice. When P32del HCV-infected mice were treated with four weeks of glecaprevir plus pibrentasvir combined with the NS5B polymerase inhibitor sofosbuvir, serum HCV RNA became negative, and the virus was eliminated from the liver in three out of four mice. We conclude that the combination of sofosbuvir and glecaprevir plus pibrentasvir may be an effective new treatment option for patients with NS5A-P32del.

The association between serum cytokine and chemokine levels and antiviral response by entecavir treatment in chronic hepatitis B patients.

BACKGROUND: Although nucleoside/nucleotide analogue therapy is thought to suppress chronic hepatitis B (CHB) via regulation of inflammatory cytokines/chemokines, the mechanism is still unclear. In this study, serum cytokine/chemokine levels were measured in CHB patients treated with entecavir, and the association with antiviral response was analysed. METHODS: A total of 78 Japanese patients with CHB were enrolled, and serum cytokine/chemokine levels were measured at baseline and at 12, 24 and 48 weeks of entecavir treatment using the MULTIPLEX kit. RESULTS: Antiviral response to entecavir treatment was significantly associated with hepatitis B surface antigen (HBsAg) titre and serum interferon-gamma-inducible protein 10 (IP-10) level (12w; P=0.0002; OR=0.020 [95% CI 0.002, 0.156], P=0.003; OR=0.042 [95% CI 0.005, 0.336], respectively). HBe-positive patients whose serum macrophage-derived chemokine (MDC) level was lower (<582.83 pg/ml) and IP-10 level was higher (≥1,323.13 pg/ml) achieved hepatitis B e antigen (HBeAg) loss earlier than those who remained HBeAg-positive (P=0.044). HBsAg reduction by entecavir treatment was significantly associated with higher initial tumour necrosis factor-alpha (TNF-α) level (≥15.20 pg/ml) and higher alanine aminotransferase level (≥73 IU/l; P=0.009; OR=18.460 [95% CI 2.044, 166.709], P=0.022; OR=7.709 [95% CI 1.341, 44.327], respectively). CONCLUSIONS: Results of the present study indicate that changes in cytokine/chemokine levels following entecavir therapy are associated with response to antiviral therapy in CHB patients. Monitoring of serum cytokine/chemokine levels could be useful for predicting reduction of HBV DNA and HBsAg and HBe seroconversion.

Serum HMGB1 concentrations at 4 weeks is a useful predictor of extreme poor prognosis for advanced hepatocellular carcinoma treated with sorafenib and hepatic arterial infusion chemotherapy.

BACKGROUND: Biomarkers predicting the response to the anticancer treatment and prognosis in patients with advanced hepatocellular carcinoma (HCC) are required. Recently, high mobility group box 1 (HMGB1) was reported to promote HCC progression and be associated with poor prognosis for patients with HCC. The purpose of this study was to assess serum HMGB1 concentrations before and during sorafenib treatment or hepatic arterial infusion chemotherapy (HAIC) and to explore the ability of serum HMGB1 concentrations to predict prognosis. METHODS: Serum HMGB1 concentrations were measured in 71 and 72 patients with advanced HCC treated with sorafenib and HAIC, respectively, to assess their usefulness for prediction of the response to the treatment and prognosis. RESULTS: Multivariate analysis identified high HMGB1 at 4 weeks (P = 0.001), high α-fetoprotein (AFP) at baseline (P = 0.025), tumor liver occupying rate (P = 0.009) and modified RECIST (mRECIST, P < 0.0001) as independent predictors of poor overall survival in sorafenib treatment. High HMGB1 at 4 weeks (P = 0.025), vascular invasion to the hepatic vein (Vv) (P = 0.009), mRECIST (P < 0.0001) and Child-Pugh B (P = 0.004) were identified as independent predictors of poor overall survival in HAIC treatment. The concentrations of HMGB1 at baseline and 4 weeks were not correlated with conventional tumor markers and progressive disease assessed by mRECIST at 8 weeks. CONCLUSIONS: These results suggest that serum HMGB1 at 4 weeks after the start of treatment might be a useful biomarker with added value to the conventional tumor marker and radiologic responses to predict poor overall survival in patients with advanced HCC treated with sorafenib or HAIC.

Persistent Loss of Hepatitis B Virus Markers in Serum without Cellular Immunity by Combination of Peginterferon and Entecavir Therapy in Humanized Mice.

Nucleot(s)ide analogues and peginterferon (PEG-IFN) treatment are the only approved therapies for chronic hepatitis B virus (HBV) infection. However, complete eradication of the virus, as indicated by persistent loss of hepatitis B surface antigen (HBsAg), is rare among treated patients. This is due to long-term persistence of the HBV genome in infected hepatocytes in the form of covalently closed circular DNA (cccDNA). In this study, we investigated whether administration of a large dose of a nucleoside analogue in combination with PEG-IFN can achieve long-term loss of HBsAg in human hepatocyte chimeric mice. Mice were treated with a high dose of entecavir and/or PEG-IFN for 6 weeks. High-dose combination therapy with both drugs resulted in persistently negative HBV DNA in serum. Although small amounts of HBV DNA and cccDNA (0.1 and 0.01 copy/cell, respectively) remained in the mouse livers, some of the mice remained persistently negative for serum HBV DNA at 13 weeks after cessation of the therapy. Serum HBsAg and hepatitis B core-related antigen (HBcrAg) continued to decrease and eventually became negative at 12 weeks after cessation of the therapy. Analysis of the HBV genome in treated mice showed accumulation of G-to-A hypermutation and CpG III island methylation. Persistent loss of serum HBV DNA and loss of HBV markers by high-dose entecavir and PEG-IFN combination treatment in chimeric mice suggests that control of HBV can be achieved even in the absence of a cellular immune response.