A molecular epidemiology study investigating familial clustering of hepatitis B virus infection in families with unfavorable prognoses in Northwest China.

Hepatitis B virus (HBV) infections and adverse outcome have been demonstrated to show characteristics of familial clustering. The aim of this study was to investigate the prevalence of different HBV genotypes, HBV sub-genotypes, and Pre-S mutations associated with familial HBV infection clusters with unfavorable prognoses. Families presenting with clustered HBV infections and unfavorable prognoses were enrolled in this study. Non-clustered HBV-infected individuals were used as the control group. DNA extracted from patient serum samples was used to facilitate characterization of the HBV genotypes, HBV sub-genotypes, and Pre-S mutations by phylogenetic analysis. The Pre-S/S gene was successfully amplified in 83 patients from the clustering group and 105 patients from the sporadic group. The prevalence of genotype C in the clustering group (71/83, 85.54%) was significantly higher than in the sporadic group (77/105, 73.33%) (P = 0.042). The prevalence of sub-genotype C2 in the clustering group (33/83, 39.76%) was also higher than in the sporadic group (21/105, 20%) (P = 0.003). Analyses of functional mapping of pre-S sequences showed that the prevalence of the mutation in the S promoter site (nt 3045-3189 of pre-S1 domain) was significantly increased in the clustering group compared with the sporadic group (15.7% vs. 3.8%) (P = 0.009). This study suggests that genotype C, especially sub-genotype C2, may be associated with the progression of HBV infection in familial clustering infection cohorts with unfavorable prognoses. We also observed that the natural occurrence of S promoter mutations in the clustering group was significantly prevalent.

Insulin Resistance Predicts Virological Response to Interferon-α in Chronic Hepatitis B Patients.

GOALS: To elucidate impact of insulin resistance (IR) on the response to interferon-α (IFN-α) therapy in chronic hepatitis B (CHB) patients. BACKGROUND: Metabolic factors influencing the virological response of CHB patients on IFN-α treatment are still unexplored. STUDY: Eighty CHB patients were treated with IFN-α for 48 weeks. The IR was evaluated by homeostasis model assessment of IR (HOMA-IR) before treatment. Viral load and biochemical parameters were measured at 12, 24, and 48 weeks after starting treatment, and then 24 weeks after the end of treatment. IFN-γ and tumor necrosis factor-α were tested at baseline and 12 weeks of treatment. RESULTS: Pretreatment HOMA-IR proved to be the only independent predictor of primary nonresponse, as well as the pretreatment HOMA-IR, viral load and primary nonresponse were independently associated with virological response at 24, 48 weeks of treatment and at the follow-up endpoint. The significant higher virological relapse rate in patients with IR was observed in patients with virological response at 48 weeks of treatment. The mean HOMA-IR was significantly lower in virological responders than in virological nonresponders. The secretion of IFN-γ and tumor necrosis factor-α was not induced in patients with IR at 12 weeks after IFN-α treatment. CONCLUSIONS: Our data suggest that IR is strongly associated with virological response, thus reflecting the important role played by metabolic factors in the viral kinetics during IFN-α treatment. These findings suggested clinical application of pretreatment HOMA-IR could enable treatment outcome to be predicted and treatment regimens to be determined.

Comparative study of the different activities of hepatitis B virus whole-X protein and HBx in hepatocarcinogenesis by proteomics and bioinformatics analysis.

The hepatitis B virus (HBV) whole-X gene comprises the HBV X gene and the 168-bp region immediately upstream. Although the functions of HBx in hepatocarcinogenesis are well known, the activity of the HBV whole-X protein (HBwx), with 56 additional amino acids, has not yet been explored. In this study, proteomic and bioinformatic analysis was done to determine the protein interaction profiles of HBwx and HBx and to describe their functions in carcinogenesis. A total of 203 proteins were identified that interacted with HBwx, of which 149 were unique, the rest interacting also with HBx, and 73% (148/203) of these proteins are involved in carcinogenesis. Gene ontology (GO) analysis showed that HBwx- and HBx-interacting proteins are involved in different processes, the former mainly in biosynthetic processes (glycolysis, cell-cycle functions, and protein folding), and the latter mainly in localization, viral transcription, biological adhesion and angiogenesis. Pathway networks analysis revealed that proteins interacting with HBx participate mainly in oxidative phosphorylation, localization, the cytoskeleton, and cell adhesion. In contrast, more-specific functional analysis showed that proteins interacting with HBwx are involved in apoptosis and survival, cell-cycle functions, glycolysis, and gluconeogenesis (Pathway Maps); to cellular macromolecular complex assembly, protein folding and mRNA metabolic process (GO Processes); and to regulation of protein folding in the endoplasmic reticulum and cytoplasm, transcription, cell cycle G2-M and cytoskeleton rearrangement (Process Networks). In conclusion, this study shows that HBwx functions in carcinogenesis in a way that is different from that of HBx.

The balance between intrahepatic IL-17(+) T cells and Foxp3(+) regulatory T cells plays an important role in HBV-related end-stage liver disease.

BACKGROUND: IL-17(+) T helper cells and Foxp3(+) regulatory T cells are CD4(+) T helper cells with reciprocally regulated differentiation and function. Their frequency and function vary in patients with chronic hepatitis B. In this study, we investigated the balance between IL-17(+) T cells and Foxp3(+) regulatory T cells and illustrated their function in the aggravation of chronic hepatitis B (CHB). RESULTS: Twenty-six patients with chronic HBV -related liver failure (CLF), thirty-one patients with acute on chronic HBV-related liver failure (ACLF) and twelve normal controls were enrolled in our study. The expressions of IL-17, Foxp3, CD4, CD8 and perforin in liver tissue were measured by immunochemistry for the evaluation of liver-infiltrating lymphocytes. The frequency of liver IL-17(+) T cells on liver inflammatory cells and their proportion in the total CD4(+) T cell population increased markedly in the ACLF group, while the frquency of Foxp3+ T cells and their proportion in the total CD4(+) T cell population did not show a significant difference in the two HBV infection groups. In addition, the ACLF group showed a dramatically higher IL-17(+) /Foxp3(+) ratio than the CLF group. CD4(+) T cells increased significantly in the liver of patients with ACLF, compared with those in the liver of patients with CLF. CONCLUSIONS: Our findings suggest that intrahepatic IL-17(+) T cells play an important role in the development of chronic HBV and that the imbalance between IL-17(+) and Foxp3(+) T cells in the liver may lead to progression of the disease but the mechanism should be further explored.

[Expression of COX-2 and PPARg in the livers of patients with acute on chronic HBV-related liver failure and their relationship with clinic parameters].

OBJECTIVE: To study the expressions of cyclooxygenase-2 (COX-2) and Peroxisome proliferator-activated receptor gamma (PPARg) in liver of patients with hepatitis B virus (HBV) related acute-on-chronic liver failure (ACLF) and their correlation with clinical parameters. METHODS: 35 patients with ACLF, 35 patients with HBV related chronic liver failure (CLF), 27 patients with chronic hepatitis B(CHB) and 15 normal control were enrolled to study the expressions of COX-2 and PPARg in the liver tissues by immunohistochemical staining, and to analyze the correlation of the COX-2 and PPARg levels in liver tissues with clinical parameters. RESULTS: COX-2 was distinctly expressed in the cytoplasm of the hepatocytes, but PPARg was mostly expressed in the nuclei of the hepatocytes and also could be seen in the cytoplasm. The expressions of COX-2 in the liver of ACLF, CLF and CHB groups increased significantly as compared with NC group (z = -5.18, -4.50, -5.32, P is less than 0.01). The levels of COX-2 in ACLF livers also increased evidently as compared with CLF groups (z = -1.98, P is less than 0.05). The expression levels of PPARg in ACLF liver tissues were much higher than the other three groups, and statistical significances existed between ACLF group and the other two groups (CLF, NC groups) (z = -2.62, -4.28, P is less than 0.01). In ACLF group, the expression of COX-2 correlated with MELD score (r = 0.337, P is less than 0.05) and the expression of PPARg correlated with HBV DNA load (r = 0.348, P is less than 0.05). Clinical data showed that the levels of AST, TBil, CHOL, PT, INR, FIB and MELD score in ACLF group were significantly different from that in CLF, CHB and NC groups. CONCLUSIONS: COX-2 expressed in liver may be a marker to reflect the degree of inflammation and injury of liver tissue. The PPARg expression of liver could be increased during chronic HBV infection and may be a protective mechanism against liver injury.

Hepatitis B virus whole-X and X protein play distinct roles in HBV-related hepatocellular carcinoma progression.

BACKGROUND: The role of HBV X protein (HBx) in the development of hepatocellular carcinoma (HCC) has been well studied. However, little is known about the molecular functions of HBV whole-X protein (HBwx), a protein fused with HBx and upstream 56 amino acid, in HCC. In current study, the molecular functions of HBwx in HCC pathogenesis has been investigated, as well as comparison between HBwx and HBx. METHODS: Expression of HBwx and HBx in 50 HCC tissues was examined by immunohistochemistry. Their tumor-forming abilities were evaluated by an animal model and colony formation assay. Migration and invasion were detected by transwell assay and subcellular localization was tracked by GFP fluorescence. Cell proliferation, cell cycle and apoptosis were detected by CCK8 and FCM. Protein level was determined by Western blotting. RESULTS: HBwx was present in 72 % (36/50) of the liver tumor tissues and mainly expressed in the nucleus and deposited in the cytoplasm surrounding karyotheca. HBwx showed a promoting effect on tumorigenesis and growth in vivo and in vitro as well as cell migration and invasion, whilst such effect is compromised compared with that of HBx. Further analysis demonstrated differences in cell proliferation, cell cycle and cell apoptosis between cells expressing HBwx and those expressing HBx. Additionally, it was confirmed that RKIP-p-ERK pathway was involved in HBwx-related tumor formation. CONCLUSION: HBwx, with the extra 56 amino acids, is closely related with hepatocarcinogenesis, while displays different biological functions from HBx.

Effect of the HBV whole-X gene on the expression of hepatocellular carcinoma associated proteins.

BACKGROUND: The hepatitis B virus (HBV) pre-X gene resides upstream of the HBV X gene, and together they form the HBV whole-X gene. Although it has been evident that the HBV whole-X protein is involved in the development of hepatocellular carcinoma, its biological role and molecular mechanism remain largely unknown. METHODS: In this study, we subcloned the HBV whole-X gene and constructed a HBV whole-X expressing vector. After transfection of the HBV whole-X gene into HL-7702 cells, the profile of the differential cellular protein composition in the cells was analyzed by using two-dimensional electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: The results showed that 18 major proteins were differentially expressed in the cells transfected with or without the HBV whole-X gene. The expression of these genes was further confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. CONCLUSION: Our findings provide a new insight into the investigation of the pathological role that the HBV whole-X gene plays in the development of hepatocellular carcinoma and may lead to the design of novel strategies for the treatment of this disease.

Natural Killer Group 2A Expressed on Both Peripheral CD3-CD56+NK Cells and CD3+CD8+T Cells Plays a Pivotal Negative Regulatory Role in the Progression of Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

To explore the role of surface receptors natural killer group 2A (NKG2A) and natural killer group 2D (NKG2D) on CD3+CD8+T cells and CD3-CD56+NK cells in the progression of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF), we measured the expression of NKG2A and NKG2D on the surface of these 2 types of circulating cells by flow cytometry in 3 groups. One group consists of 36 patients with chronic hepatitis B (CHB), another one consists of 22 patients with HBV-related ACLF, and the last one has 12 normal controls (NC). The experimental result indicated that there was no significant difference in the proportion of CD3+CD8+T cells in total lymphocytes between the 3 groups. However, the percentage of CD3-CD56+NK cells in ACLF group was evidently higher than that in the CHB group (P < 0.05). In addition, the expression of NKG2D on CD3+CD8+T cells in the ACLF group was significantly lower than that in the CHB group (P < 0.05), but there were no statistically significant differences in its percentages on CD3-CD56+NK cells between the 3 groups. The expression of NKG2A on CD3+CD8+T cells in the ACLF group was significantly higher than that in the NC group (P < 0.05), and on NK cells was significantly higher than that in the CHB group (P < 0.05) and NC group (P < 0.01). The increase in ratios of NKG2A to NKG2D on CD3+CD8+T cells and CD3-CD56+NK cells in the ACLF group was significantly more than that in the CHB group and NC group. The results indicate that the imbalance between NKG2A and NKG2D may contribute to the progression of HBV-related ACLF mediated by CD3-CD56+NK cells and CD3+CD8+T cells. Compared with NKG2D, NKG2A expressed on both peripheral CD3-CD56+NK cells and CD3+CD8+T cells plays a more pivotal negative regulatory role in the progression of HBV-related ACLF.

With Cytometric Bead Assay, the Interleukin-10/HBV DNA Ratio Is an Early Predictor for Response to Interferon-α Treatment in Chronic Hepatitis B.

Both host immunity and hepatitis B virus (HBV) contribute to the therapeutic response to interferon (IFN)-α treatment in chronic hepatitis B (CHB) based on the immune modulation function to eliminate virus, while neither viral load nor cytokine alone could reflect the complex interaction between host immune response and virus. This study aimed at exploring a parameter of combined immunological and virological indexes to predict the response profile to IFN-α treatment in patients with CHB. Biochemical (alanine transaminase), virological (HBV DNA load, HBsAg, HBeAg), and immunological [IFN-γ, tumor necrosis factor (TNF), interleukin (IL)-2, IL-4, IL-6, and IL-10] indexes were dynamically monitored (at baseline, 2, 4, 8, 12, 24, 36, 48, and 72 weeks) in 41 patients who received a 48-week IFN-α treatment. First, we found that responders displayed an elevation in both Th1 and Th2 type cytokines from baseline to 4 weeks. What is more, the HBV DNA load at the baseline and IL-10 at 4 weeks showed significant differences between responders and nonresponders and were highly associated with the response to IFN-α treatment. More importantly, IL-10/HBV DNA was identified as a positive predictor for response to IFN-α treatment (odds ratio=5.785) by logistic regression analysis. We show here that the IL-10/HBV DNA ratio, a parameter of combined immunological and virological factors, can be useful in predicting the response to IFN-α treatment in CHB.

Restoring the Treg cell to Th17 cell ratio may alleviate HBV-related acute-on-chronic liver failure.

AIM: To investigate the role of T helper 17 cells (Th17) and regulatory T cells (Treg) in hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF). METHODS: We enrolled 79 patients with HBV infection into the study, 50 patients with HBV-related ACLF and 29 patients with chronic hepatitis B (CHB), from the First Affiliated Hospital of Medical College from January 2009 to June 2012. The ACLF patients were diagnosed according to the criteria recommended by The 19(th) Conference of the Asian Pacific Association for the Study of the Liver in 2009. Twenty healthy individuals with a similar gender and age structures to the two patient groups were also included as the normal controls (NC). Of the 50 ACLF patients, 28 were subsequently classified as non-survivors: 19 patients died from multi-organ failure, 3 underwent liver transplantation, and 6 discontinued therapy during follow-up because of financial reasons. The remaining 22 ACLF patients whose liver and anticoagulation function recovered to nearly normal levels within the next 6 mo were classified as survivors. The number of circulating Treg and Th17 cells was determined upon diagnosis and during the 8th week of follow-up through flow cytometry. RESULTS: The percentage of circulating Treg cells in the ACLF group was significantly higher than that in the CHB group (5.50% ± 1.15% vs 3.30% ± 1.13%, P < 0.01). The percentages of circulating Th17 cells in the ACLF and the CHB groups were significantly higher than that in the NC group (6.32% ± 2.22% vs 1.56% ± 0.44%, P < 0.01; 3.53% ± 1.65% vs 1.56% ± 0.44%, P < 0.01). No significant difference in Treg cell to Th17 cell ratio was observed between the ACLF group and the CHB group (0.98 ± 0.44 vs 1.12 ± 0.64, P = 0.991), whereas those in the two HBV infection groups were significantly lower than that in the NC group (1.85 ± 1.22; both P < 0.01). The percentage of Treg cells in the survivors during the 8(th) week of follow-up was significantly lower than that during peak ACLF severity [total bilirubin (TBIL) peak] (3.45% ± 0.97% vs 5.18% ± 1.02%, P < 0.01). The percentage of Th17 cells in survivors during the 8(th) week of follow-up was significantly lower than that during the peak TBIL (2.89% ± 0.60% vs 5.24% ± 1.46%; P < 0.01). The Treg cell to Th17 cell ratio during the 8(th) week of follow-up was significantly higher than that during the TBIL peak (1.22 ± 0.36 vs 1.10 ± 0.54; P < 0.05). CONCLUSION: Restoring the Treg cell to Th17 cell ratio during the follow-up phase of ACLF could maintain the immune system at a steady state, which favours good prognosis.