Function Of Hepatitis Research Articles

Characterization of Intracellular Precore-Derived Proteins and Their Functions in Hepatitis B Virus-Infected Human Hepatocytes.

Hepatitis B virus (HBV) precore protein is not essential for viral replication but is thought to facilitate chronic infection. In addition to the secreted precore products, including the hepatitis B e antigen (HBeAg) and PreC protein, intracellular precore-derived proteins in HBV-infected human hepatocytes remain poorly characterized, and their roles, if any, remain largely unknown. Here, we detected multiple precore derivatives, including the nonprocessed precursor p25 and the processing intermediate p22, in HBV-infected human hepatocytes as well as human hepatoma cells overexpressing the HBV precore protein. Both p25 and p22 showed phosphorylated and unphosphorylated forms, which were located in different intracellular compartments. Interestingly, precore expression was associated with decreases in intracellular HBV core protein (HBc) and secreted DNA-containing virions but was also associated with an increase in secreted empty virions. The decrease in HBc by precore could be attributed to cytosolic p22, which caused HBc degradation, at least in part by the proteasome, and consequently decreased HBV pregenomic RNA packaging and DNA synthesis. In addition, cytosolic p22 formed chimeric capsids with HBc in the cell, which were further secreted in virions. In contrast, the PreC antigen, like HBeAg, was secreted via the endoplasmic reticulum (ER)-Golgi secretory pathway and was thus unable to form capsids in the cell or be secreted in virions. Furthermore, p25, as well as p22, were secreted in virions from HBV-infected human hepatocytes and were detected in the sera of HBV-infected chimpanzees. In summary, we have detected multiple intracellular precore-derived proteins in HBV-infected human hepatocytes and revealed novel precore functions in the viral life cycle. IMPORTANCE Chronic hepatitis B remains a worldwide public health issue. The hepatitis B virus (HBV) precore protein is not essential for HBV replication but may facilitate viral persistence. In this study, we have detected multiple precore protein species in HBV-infected human hepatocytes and studied their functions in the HBV life cycle. We found that the HBV precore proteins decreased intracellular HBV core protein and reduced secretion of complete virions but enhanced secretion of empty virions. Interestingly, the cytosolic precore protein species formed chimeric capsids with the core protein and were secreted in virions. Our results shed new light on the functions of intracellular precore protein species in the HBV life cycle and have implications for the roles of precore proteins in HBV persistence and pathogenesis.

Assessment of auditory function in hepatitis “C” patients managed by combination of sofosbuvir and daclatsavir

BackgroundTo evaluate the effect of daclatasvir and sofosbuvir on auditory function in hepatitis C patients. Thirty hepatitis C virus (HCV) patients were included in this study with age ranged from 22 to 55 years. All patients underwent full audiological evaluation before beginning and after completion of their treatment with combination of daclatasvir and sofosbuvir.ResultWhen the results of audiological data were compared before and after treatment, we found that; there were statistically significant differences in the pure tone audiometry (PTA) threshold and ART bilaterally mainly in the high frequency region. Transient evoked otoacoustic emission (TEOAE) reproducibility showed a statistically significant difference bilateral. However, there was no statistically significant difference in the Auditory Brainstem Response (ABR) findings.Conclusion and recommendationsThe combination therapy of daclatasvir and sofosbuvir used in patients with HCV led to deterioration in the hearing threshold in the basic audiological tests (PTA and immitancemetry). In addition, it had an adverse effect on the cochlear OHCs, with no affection on auditory nerve, brain stem functions or in sub-thalamic function, it seems that it had no effect, we cannot confirm. The study evaluate hearing at the end of treatment immediately. We recommend performing a routine monitoring of auditory functions in HCV patients treated with daclatasvir and sofosbuvir combination by specialist for early detection of auditory changes to avoid further damage to auditory system.

Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors

CD4+ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC50] <10 nM), or C61 and preS9 (EC50 <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4+ and CD8+ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4+ T cell help in viral infection with potential benefit for T cell therapy.

Impact of Direct Acting Antiviral Agents on kidney function in Hepatitis C Virus infected patients with chronic kidney disease

Abstract Introduction Despite the significant link between HCV and CKD progression, most of the patients with CKD infected with HCV remain untreated, because they have historically been difficult to treat due to common adverse effects associated with interferon (IFN), ribavirin, and first generation protease inhibitors. Recently, there have been major advancements in the treatment of HCV with the development of new directacting antivirals (DAAs). Objectives To evaluate the safety and efficacy of DAAs and their impact on kidney function in CKD patients. Patients and Methods We conducted a prospective observational study on 100 CKD patients stages 3-4, receiving treatment for HCV at MASRI (faculty of Medicine Ain Shams University Research Institute), with two different DAAs regimens, completed over six months follow up. Kidney function was followed during and after treatment. Results Sustained virological response (SVR) was achieved in all patients. AKI (acute kidney injury) was uncommon; it occurred in three (3%) patients, out of them, two patients showed complete recovery. Adverse events were common (43%), but serious adverse events were uncommon (2%).Improvement of eGFR (8-15 ml/min/1.73 m2) and proteinuria was found in both study groups. Conclusion DAAs were effective and welltolerated for HCV infected patients with stage 3-4 chronic kidney disease, where viral clearance caused improvement in eGFR and proteinuria.

Laboratory healthcare gap of hepatitis B care in Yogyakarta, Indonesia

Serological markers of hepatitis B virus (HBV) infection (HBsAg, anti-HBs, hepatitis B envelope antigen [HBeAg], anti-HBe, and anti-hepatitis B core [HBc] IgM/IgG) was used as the basis for diagnosis and differences in the chronicity of hepatitis B infection. This study aims to explore the laboratory healthcare gap of the patient with hepatitis B infection in our setting and whether laboratory data completeness affects the results. This study used retrospective data taken from the Medical Records of hepatitis B patients at Dr. Sardijto Hospital in 2020. The subjects in this study were hepatitis B patients with positive HBsAg. The exclusion criteria were individuals who had co-infection with the hepatotropic virus or HIV. This study was funded by Deputi Bidang Penguatan Riset dan Pengembangan, Kementerian Riset dan Teknologi/Badan Inovasi Nasional. A total of 232 patients were included in the study. All patients underwent HBsAg examination and tested positive, with 65.09% of subjects had HBV DNA examination. HBeAg examination result was only found in 18% of patients. Anti HBc examinations were only found in 2 patients. No subjects were examined for anti-HBs in this study. The proportion of subjects examined for the liver function was still low (< 50%), with the most widely checked parameters are AST and ALT while other parameters are very rare. The utilization of serological tests and hepatic function in hepatitis B patients in Yogyakarta, Indonesia is still low. Intervention and financial support are needed to improve the quality of life of patients with hepatitis B who were treated.

Impact of direct acting antiviral agents on kidney function in hepatitis C virus infected patients with chronic kidney disease

Introduction: Hepatitis C virus (HCV) infection is strongly associated with chronic kidney disease (CKD). It is an independent risk factor for developing CKD and significantly increases morbidity and mortality in CKD patients. Treatment with newer direct-acting antiviral (DAA) regimens in patients with CKD is showing conflicting results as regards safety and efficacy. Objectives: To evaluate the safety and efficacy of DAAs and their impact on kidney function in CKD patients. Patients and Methods: We conducted a prospective observational study on 100 CKD patients stages 3-4, receiving treatment for HCV at MASRI (Faculty of Medicine Ain Shams University Research Institute), with two different DAAs regimens (sofosbuvir/daclatasvir with or without ribavirin and ombitasvir/paritaprevir/ritonavir [OMV/PTV/RTV] with ribavirin), completed over six months follow up. Serum creatinine, estimated glomerular filtration rate (eGFR), and proteinuria were followed during and after treatment. Results: Sustained virological response (SVR) was achieved in all patients. Improvement of eGFR (8-15 mL/min/1.73 m2) and proteinuria was found in both study groups. Acute kidney injury (AKI) was uncommon; it occurred in three (3%) patients, out of them, two patients showed complete recovery. Adverse events were common (43%), but serious adverse events were uncommon (2%). Conclusion: DAA regimens were effective and well-tolerated for HCV infected patients with stage 3-4 CKD, where viral clearance caused improvement in eGFR and proteinuria.

Cirrhosis Hampers Early and Rapid Normalization of Natural Killer Cell Phenotype and Function in Hepatitis C Patients Undergoing Interferon-Free Therapy.

Background: Chronic hepatitis C virus (HCV) infection impairs natural killer (NK) cell phenotype and function. Whether restoration of NK cells occurs after successful interferon (IFN)-free therapies remains a controversial issue.Aim: To analyze how HCV-related liver cirrhosis impacts changes in NK cells prior and post-IFN-free therapies.Methods: NK cell analysis by multicolor flow cytometry was performed in HCV-infected patients with (n = 17) and without (n = 14) cirrhosis at baseline, week 4 during therapy, and weeks 12 and 48 after the end of therapy (FU12 and FU48, respectively). Non-HCV cirrhotic patients (n = 12) and healthy individuals (n = 12) served as controls.Results: At baseline, HCV cirrhotic patients presented an altered distribution of NK subsets (CD56dim and CD56bright) with higher expression of NKp46, HLA-DR, NKp30, KIR2DL2/L3, NKG2A, and CD85j receptors compared to healthy controls. All frequencies normalized by FU48, except for CD85j+ cells. Likewise, substantial alterations were detected in NK cell function assessed by (i) signal transducer and activator of transcription 1 (STAT1) and phosphorylated levels of STAT1 and STAT4, (ii) degranulation (CD107a), (iii) cytotoxicity [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)], and (iv) cytokine production [IFN-γ and tumor necrosis factor-α (TNF-α)]. Of note, NK cell function at FU48 remained partially impaired. In contrast, non-cirrhotics showed normal baseline frequencies of HLA-DR-, NKG2A-, and CD85j-expressing NK cells. Importantly, altered baseline frequencies of NK cell subsets and NKp46+ CD56dim cells, as well as NK cell function, were rapidly and completely restored.Conclusions: NK cell phenotype alterations persist after HCV eradication in cirrhotic patients, while their function is only partially restored, compromising immune restoration and immunosurveillance.

Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins

Hepatitis C virus (HCV) is an enveloped virus and encodes two envelope glycoproteins, E1 and E2. E1 and E2 are transmembrane type I proteins with a N-terminal ectodomain and C-terminal anchor. During their synthesis, E1 and E2 ectodomains are targeted in the endoplasmic reticulum lumen where they are modified by N-linked glycosylation. After their synthesis, E1 and E2 assemble as a non-covalent heterodimer. The N-linked glycosylation is based on the recognition of specific asparagine residue in the context of the consensus sequence Asn-X-Ser/Thr. E1 contains potentially 4 or 5 N-linked glycosylation sites and E2 up to 11. Recent data indicated that some glycans of glycoproteins E1 and E2 play a major role in protein folding and heterodimer formation. Some N-linked glycans of E2 were involved in interactions with CD81, a putative cellular receptor for HCV. It appeared that N-linked glycans of E1 and E2 played an important role of in the viral entry.

Characteristics of Impaired Dendritic Cell Function in Patients With Hepatitis B Virus Infection.

Dendritic cells (DCs) are antigen-presenting cells with a central role in host immune response. This study analyzed gene expression and DC function in hepatitis B virus (HBV) patients, functions impaired because of HBV, and identified the genes related to these functions. Peripheral blood mononuclear cells from 64 HBV patients and 19 healthy controls were analyzed. Peripheral blood DCs were stained with antibodies against human leukocyte antigen-DR/Lin-1/CD123/CD11c and separated into plasmacytoid DCs (pDCs) and myeloid DCs by fluorescence-activated cell sorting. Using an interferon-gamma enzyme-linked immunospot assay, we analyzed antigen-specific response in HBV-infected patients. Regarding DC function, we analyzed antigen-presenting capacity, cell migration capacity, phagocytic capacity, and cytokine production capacity. DC gene expression was analyzed by microarray to identify genes related to DC function. No difference was found in the number of DCs in peripheral blood between healthy participants and HBV patients. In cell-surface marker analysis, CD80, CD83, CD86, CD40, and C-C motif chemokine receptor 7 expression levels in pDCs were related to the HBV-specific T-cell response. DCs from HBV patients exhibited decreases in antigen-presenting capacity, migration capacity, and cytokine production capacity. In gene expression analysis, immune-related genes with greatly reduced expression levels in chronic hepatitis B patients were identified. Of these genes, interleukin (IL)-6 signal transducer (IL6ST) expression level positively correlated with DC surface marker expression level. Adjustment of IL6ST expression level in DCs and treatment with oncostatin M resulted in recovery of DC function. Conclusion: IL6ST expression was identified as one cause of decline in DC function in HBV patients. Adjustment of IL6 family cytokine signaling may be useful for recovering reduced DC function in HBV infection.

THU-147-Improvement of neutrophil function in hepatitis C patients under direct acting anti-viral treatment is associated with hemolysis parameters

THU-147-Improvement of neutrophil function in hepatitis C patients under direct acting anti-viral treatment is associated with hemolysis parameters

The impact of interferon therapy on the sexual function of hepatitis C male patients

Objective To evaluate the impact of interferon therapy on male sexual function. Patients and methods One hundred hepatitis C patients were included in the study. Fifty two patients were receiving interferon and 48 patients were not receiving interferon. All participants underwent history taking including International Index of Erectile Function (IIEF-5) score, general and local examination, assessment of hormonal profile, and pharmacopenile duplex Doppler ultrasonography. Results The IIEF-5 score of the noninterferon-treated group (18 ± 6.5) was significantly higher than the interferon-treated group (12 ± 4.5) (P = 0.022). Forty (76.9%) patients on interferon reported low sexual desire compared with 22 (45.8%) patients of noninterferon-treated group (P = 0.001). Total testosterone among interferon-treated patients (188 ± 0.32 ng/dl) was significantly lower than noninterferon-treated patients (270 ± 0.52 ng/dl) (P = 0.001). Similarly, free testosterone level among interferon-treated patients (3 ± 0.22 ng/dl) was significantly lower than noninterferon-treated group (7 ± 2.3 ng/dl) (P = 0.05). Estradiol levels among interferon-treated patients (80 ± 3.3 pg/ml) were significantly higher than noninterferon-treated patients (58 ± 2.3 pg/ml) (P = 0.01). However, prolactin level showed no significant difference between the interferon-treated (13 ± 1.3 ng/ml) and the noninterferon-treated groups (12 ± 1.5 ng/ml) (P = 0.59). Thirty eight (73%) patients on interferon showed vasculogenic erectile dysfunction compared with 32 (66.7%) patients not receiving interferon (P = 0.11). Twenty two (42.2%) patients on interferon showed veno-occlusive dysfunction which was significantly higher than noninterferon group (16.6%) (P = 0.03). Eighteen (37.5%) patients of the noninterferon group showed mixed vasculogenic erectile dysfunction compared with eight (15.4%) patients on interferon (P = 0.05). Conclusion Interferon had negative impact on libido, IIEF-5 score, and hormonal profile but did not affect penile hemodynamics.

Hepatitis B virus inhibits the expression of complement C3 and C4, in vitro and in vivo.

The immune system serves an important function in Hepatitis B virus (HBV) infection, and the complement system is a major component of innate immunity. However, the regulatory effect of HBV on complement proteins has not yet been fully elucidated. The present study focused on investigating the impact of HBV on the expression of complement proteins C3 and C4. A total of 226 patients with a clinical diagnosis of HBV infection were enrolled in the study, including 153 with chronic hepatitis B (CHB) and 73 with hepatocellular carcinoma (HCC), whereas 116 healthy individuals were included as a control group. Immunoturbidimetric detection was performed to determine the levels of complement C3 and C4 in the serum of the patients with HBV and the control group. The results revealed that the mean ± standard deviation C3 and C4 content was 1.223±0.237 and 0.226±0.052 g/l for the control group, 0.687±0.150 and 0.145±0.070 g/l for the patients with CHB, and 0.829±0.332 and 0.174±0.088 g/l for the patients with HCC, respectively. The levels of complement C3 and C4 in the patients with CHB or HCC were significantly lower than the control group (P<0.05). The HBV infectious clone pHBV1.3 was used to transfect Huh7 cells; Huh7 cells transfected with the pBlue-ks empty vector were used as the blank control. The changes in mRNA and protein expression of complements C3 and C4 were detected by RT-PCR and western blotting. When compared with the control cells, the Huh7 cells transfected with pHBV1.3 exhibited reduced C3 and C4 mRNA and protein expression levels. It was concluded that HBV can inhibit the expression of complement C3 and C4 in vitro and in vivo, which may lay the foundation for revealing the pathogenesis of HBV.

Serum Procalcitonin Correlates with Renal Function in Hepatitis B Virus-Related Acute-on-Chronic Liver Failure

Background/Aims: To investigate the relationship between elevated serum procalcitonin (PCT) and renal function in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). Methods: HBV-ACLF patients (n = 201) presenting to the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, from January 2013 to November 2016 were categorized into three groups according to serum PCT levels: (i) normal group (n = 74) had PCT of ≤ 0.5 ng/mL; (ii) elevated group (n = 85) had PCT in the range 0.5–1.0 ng/mL; and (iii) highly elevated group (n = 42) had PCT of > 1.0 ng/mL. Thirty-five cases received standard care after admission. Serum PCT levels and renal function were determined during a two-week follow-up. Results: Significant increases in serum creatinine (Cr) were recorded in male and female patients in the elevated group and highly elevated group compared with the normal group (P < 0.05). In addition, serum Cr levels in male and female patients were significantly higher in the highly elevated group than in the elevated group (P < 0.05). The glomerular filtration rate (GFR) was significantly lower in the highly elevated group (P < 0.05) and this group had the highest risk of altered Cr (45.9% in males; 80% in females) and abnormal GFR (37.5%). Serum PCT levels correlated significantly with all renal function parameters including homocysteine (Hcy), GFR, Cr, blood urea nitrogen, uric acid, and cystatin C at baseline and during treatment. Univariate and multivariate analyses indicated that serum PCT was a strong predictor of renal dysfunction. Conclusion: Serum PCT is closely related to renal dysfunction in HBV-ACLF.

FRI-242 - Changes in markers of liver function in hepatitis C virus genotype 1b Asian patients with compensated cirrhosis treated with ombitasvir/paritaprevir/ritonavir plus dasabuvir with ribavirin in the ONYX-II study

FRI-242 - Changes in markers of liver function in hepatitis C virus genotype 1b Asian patients with compensated cirrhosis treated with ombitasvir/paritaprevir/ritonavir plus dasabuvir with ribavirin in the ONYX-II study

Positive association between serum apolipoprotein M levels and hepatitis B virus DNA load in HBeAg-negative chronic hepatitis B.

BackgroundHepatitis virus B (HBV) has infected millions of people worldwide. Notably, such infections can be associated with hepatic complications. Levels of apolipoprotein M (apoM), a component of high-density lipoprotein (HDL), are known to be significantly elevated in patients with chronic hepatitis B (CHB). The aim of this study was to investigate the relationship between HBV DNA load in serum and serum apoM levels in patients with CHB.MethodsA total of 73 HBeAg-negative CHB patients, 50 HBeAg-positive CHB patients, and 79 non-CHB controls were included in the study cohort. The age and body mass index (BMI) of the study participants were matched. Serum levels of apoM and the HBV antigens HBsAg and HBeAg were measured by enzyme-linked immunosorbent assay (ELISA) analysis. Serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), cholesterol, and triglycerides (TG) were assessed using an automatic biochemical analyzer. Serum HBV DNA levels were quantified by real-time PCR analysis. Data were analyzed by Spearman’s rank correlation coefficient, Pearson correlation coefficient, and multivariate linear regression model (continuous variables), or Student’s t-test (mean differences).ResultsBoth the HBeAg-negative CHB and HBeAg-positive CHB patient groups exhibited elevated serum levels of apoM. Moreover, serum apoM levels were positively correlated with serum HBV DNA levels in HBeAg-negative CHB patients (r = 0.394, p < 0.001). Conversely, there was no significant relationship between apoM and HBV DNA levels in the HBeAg-positive CHB group (r = 0.197, p = 0.170). The median log copies/mL value for HBV DNA (4.00) was considered the cutoff point for the HBeAg-negative CHB group. Notably, a significant number of patients with HBV DNA levels above the cutoff point also had higher serum apoM levels (63.38 ± 29.84 vs. 41.41 ± 21.84; p = 0.001).ConclusionsOur findings reveal that the correlation between serum apoM levels and viral loads may depend on HBeAg status, as serum apoM levels were positively correlated with HBV DNA levels in HBeAg-negative CHB patients. These results suggest that HBeAg may play a role in apoM-related lipid metabolism and anti-inflammatory functions in hepatitis B patients. Thus, our findings may facilitate the clinical management of HBV infection.

Natural killer cell phenotype modulation and natural killer/T-cell interplay in nucleos(t)ide analogue-treated hepatitis e antigen-negative patients with chronic hepatitis B.

Natural killer (NK) and hepatitis B virus (HBV)-specific T cells are functionally impaired in chronic hepatitis B (CHB). Understanding to what extent nucleos(t)ide analogue (NUC) therapy can improve T- and NK-cell responses is important in the perspective of immunomonitoring strategies for a safe and earlier NUC withdrawal and of novel combination therapies based on modulation of antiviral immunity. To gain further insights into T/NK-cell interplay, we studied NK-cell phenotype and function in hepatitis B e antigen-negative chronic HBV patients either untreated (25) or NUC treated (36 hepatitis B surface antigen [HBsAg](+) and 10 HBsAg(-)/hepatitis B surface antibody [anti-HBs](+)). Interferon-gamma, interleukin-2, and tumor necrosis factor alpha (TNF-α) production by HBV-specific T cells was also analyzed in NUC-treated patients. NK cells from chronic naïve patients showed an "inflammatory" phenotype defined by increased expression of TNF-related apoptosis-inducing ligand (TRAIL), CD38, and Ki67 that significantly declined upon viremia suppression and alanine aminotransferase normalization induced by NUC therapy. Reversion to a quiescent NK-cell phenotype was associated with restoration of the HBV-specific T-cell function. T- and NK-cell responses showed an inverse correlation, with an opposite behavior in individual NUC-treated patients. NK-cell depletion as well as TRAIL and NKG2D pathway blockade induced a significant improvement of the HBV-specific T-cell function. NK cells can express regulatory activity on T cells in NUC-treated patients with prevalent inhibition of CD4 T cells, likely needed to limit persistent T-cell activation. NK-cell phenotype is modulated by NUC therapy and its reversion to quiescence mirrors efficient HBV-specific T-cell responses. Thus, changes of NK-cell phenotype may predict acquisition of antiviral control before anti-HBs seroconversion and represent the groundwork for future studies aimed at assessing whether NK phenotyping can be translated into the clinical practice to guide NUC suspension.

Hypoxia impairs anti-viral activity of natural killer (NK) cells but has little effect on anti-fibrotic NK cell functions in hepatitis C virus infection

Natural killer (NK) cells have been shown to exert anti-viral as well as anti-fibrotic functions in hepatitis C virus (HCV) infection. Previous studies, however, analyzed NK cell functions exclusively under atmospheric oxygen conditions despite the fact that the liver microenvironment is hypoxic. Here, we analyzed the effects of low oxygen tension on anti-viral and anti-fibrotic NK cell activity. Peripheral (n=34) and intrahepatic (n=15) NK cells from HCV(+) patients as well as circulating NK cells from healthy donors (n=20) were studied with respect to anti-viral and anti-fibrotic activity via co-culture experiments with HuH7 replicon cells and hepatic stellate cells, respectively. Anti-viral activity of resting NK cells from healthy controls was not affected by hypoxia. However, hypoxia significantly reduced the response of healthy NK cells to cytokine stimulation. In contrast to healthy controls, we observed resting and cytokine activated peripheral NK cells from HCV patients to display a significantly decreased anti-viral activity when cultured at 5% or 1% oxygen, suggesting HCV NK cells to be very sensitive to hypoxia. These findings could be confirmed when intrahepatic NK cells were tested. Finally, we show that anti-fibrotic NK cell activity was not affected by low oxygen tension. Our results show that anti-viral function of NK cells from HCV(+) patients is critically affected by a hypoxic microenvironment and, therefore, indicate that in order to obtain an accurate understanding of intrahepatic NK cell anti-HCV activity, the laboratory modelling should take into account the liver specific levels of oxygen.

Osteopontin promotes dendritic cell maturation and function in response to HBV antigens.

PurposeDendritic cells (DCs) play critical roles in promoting innate and adaptive immunity in microbial infection. Functional impairment of DCs may mediate the suppression of viral-specific T-cell immune response in chronic hepatitis B (CHB) patients. Osteopontin (OPN) is involved in several liver diseases and infectious diseases. However, whether OPN affects DC function in hepatitis B virus (HBV) infection is unknown.MethodsTwenty CHB patients and 20 healthy volunteers were recruited. OPN secreted by DCs was compared. Peripheral blood mononuclear cells cultured with OPN antibody were examined to study the costimulatory molecular expression and interleukin (IL)-12 production of DCs after HBV antigenic stimulation. OPN-deficient mice were used to investigate the influence of OPN on DC maturation and function after HBV antigenic stimulation in vitro and in vivo. Exogenous OPN was administrated to further verify the functioning of DCs from CHB patients upon HBV antigenic stimulation.ResultsWe found that OPN production of DCs from CHB patients was significantly lower than those from healthy volunteers. The absence of OPN impaired IL-12 production and costimulatory molecular expression of DCs upon stimulation with HBV antigens. Defective DC function led to reduced activation of Th1 response to HBV antigens. In addition, OPN deficiency in DCs reduced the HBV antigen-induced inflammatory response in the liver of mice. Importantly, OPN administration significantly promoted the maturation of DCs from CHB patients in vitro.ConclusionThese findings suggested that OPN could improve the maturation and functioning of DCs in the immune response to HBV antigens, which might be useful to further improve the effect of DC vaccine.

Hepatitis B virus X protein co-activates pregnane X receptor to induce the cytochrome P450 3A4 enzyme, a potential implication in hepatocarcinogenesis

Hepatitis B virus X protein is a key regulator of hepatocarcinogenesis. The pregnane X receptor is a xenobiotic nuclear receptor that plays a role in the regulation of drug-metabolizing enzymes including the cytochrome P450 3A4, an enzyme important for the bioactivation of the liver carcinogen aflatoxin B1. To identify novel host factor that interacts with hepatitis B virus X protein and the functional interaction between hepatitis B virus X protein and pregnane X receptor in hepatocarcinogenesis. Co-immunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation were utilized to assess the interaction between hepatitis B virus X protein and pregnane X receptor. The functional relevance of hepatitis B virus X protein-pregnane X receptor interaction was investigated in cell cultures and hepatocellular carcinoma samples. We observed that hepatitis B virus X protein and pregnane X receptor co-localize in hepatic cells. Pregnane X receptor interacted with hepatitis B virus X protein via the ligand-binding domain of pregnane X receptor. Functionally, hepatitis B virus X protein increased the transcriptional activity of pregnane X receptor. Pregnane X receptor was able to recruit hepatitis B virus X protein to the CYP3A4 gene promoter. In clinic samples, the expression of pregnane X receptor was high in hepatitis B virus-associated liver cirrhosis and stage I hepatocellular carcinoma, but low in state II and stage III hepatocellular carcinoma. We revealed a novel function of hepatitis B virus X protein in co-activating pregnane X receptor. The increased expression of pregnane X receptor and its target gene CYP3A4 are potential biomarkers for the early stage of hepatitis B virus-associated hepatocarcinogenesis.

The Effects of High-Dose Qinggan Huoxue Recipe on Acute Liver Failure Induced by D-Galactosamine in Rats

Qinggan Huoxue Recipe is a traditional Chinese medicine, which has been usually used to improve liver function in hepatitis. In order to investigate the effects of high-dose Qinggan Huoxue Recipe on acute liver failure and explore the potential mechanism, we had built acute liver failure models in rats by intraperitoneal injection of D-galactosamine (D-GalN). High-dose Qinggan Huoxue Recipe was delivered by gavage. After treatment, the blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), albumin (ALB), cholinesterase (CHE), and prothrombin time (PT) were determined. The pathological score of liver tissue was recorded. Proliferating cell nuclear antigen (PCNA) immunohistochemistry staining and fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) of high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), and Caspase-3 were performed. The survival curve was also depicted. Our results demonstrated that high-dose Qinggan Huoxue Recipe could significantly improve liver function and increase survival rates in rats with acute liver failure. These effects were supposed to be mediated by suppressing inflammatory reaction and apoptosis.