Proteasomes regulate hepatitis B virus replication by degradation of viral core-related proteins in a two-step manner.

The cellular proteasomes presumably inhibit the replication of hepatitis B virus (HBV) due to degradation of the viral core protein (HBcAg). Common proteasome inhibitors, however, either enhance or inhibit HBV replication. In this study, the exact degradation process of HBcAg and its influences on HBV replication were further studied using bioinformatic analysis, protease digestion assays of recombinant HBcAg, and proteasome inhibitor treatments of HBV-producing cell line HepG2.2.15. Besides HBcAg and hepatitis B e antigen precursor, common hepatitis B core-related antigens (HBcrAgs), the small and the large degradation intermediates of these HBcrAgs (HBcrDIs), were regularly found in cytosol of HepG2.2.15 cells. Further, the results of investigation reveal that the degradation process of cytosolic HBcrAgs in proteasomes consists of two steps: the limited proteolysis into HBcrDIs by the trypsin-like (TL) activity and the complete degradation of HBcrDIs by the chymotrypsin-like (chTL) activity. Concordantly, HBcrAgs and the large HBcrDI or HBcrDIs (including the small HBcrDI) were accumulated when the TL or chTL activity was inhibited, which generally correlated with enhancement and inhibition of HBV replication, respectively. The small HBcrDI inhibited HBV replication by assembling into the nucleocapsids and preventing the victim particles from being mature enough for envelopment. The two-step degradation manner may highlight some new anti-HBV strategies.

Furin mRNA expression in peripheral blood correlates with chronic hepatitis B virus infection.

AIM: The mechanisms underlying development of chronic hepatitis B virus (HBV) infection are related to immune tolerance, but are as yet incompletely understood. Furin has been found to be essential for maintenance of peripheral immune tolerance mediated by regulatory T cells (Treg). Such effect of furin on chronic HBV infection was investigated in this study. METHODS: Peripheral blood from 40 individuals with self-limited HBV infection, 40 patients with asymptomatic persistent HBV infection and 40 patients with chronic hepatitis B (CHB) was collected and mRNA expression levels of furin, transforming growth factor (TGF)-ß1 and the Treg-function-related forkhead transcription factor FoxP3 were detected using quantitative real-time polymerase chain reaction. CD4(+) CD25(+) FoxP3(+) Treg were detected using flow cytometry. RESULTS: Furin mRNA expression in peripheral blood was significantly higher in patients with persistent HBV infection than in individuals with self-limited infection (P < 0.01), and was much higher in CHB patients than in those with asymptomatic persistent infection (P < 0.01). Furthermore, furin mRNA was relatively higher in patients with positive hepatitis B e antigen and higher levels of serum HBV DNA (>10 000 copies/mL). In patients with CHB, furin mRNA expression was found to correlate with TGF-ß1 mRNA and FoxP3 mRNA expression using Spearman's rank correlation coefficient test. It was 5.7-times higher in CD4(+) CD25(+) T cells than in CD4(+) CD25(-) T cells and correlated with the frequency of Treg (P < 0.05). CONCLUSION: Furin mRNA expression in peripheral blood correlates with chronic HBV infection and liver damage, and seems to participate in immune inhibitory and anti-inflammatory mechanisms in HBV infection, mediated by TGF-ß1 and/or Treg.

[Hepatitis B virus-mediated effects on host expression of the proprotein convertase Furin].

OBJECTIVE: To study the effects of hepatitis B virus (HBV) infection on the expression of Furin, an important proprotein convertase, in liver cells to provide insights towards its potential as a therapeutic target for improved antiviral efficacy. METHODS: Furin expression was measured in human liver specimens (infected tissues from patients with chronic HBV hepatitis vs. normal tissues from healthy donors) and in hepatoma cell lines (HBV-infected HepG2.2.15 cells vs. uninfected parental cell lines HepG2) using quantitative real-time RT-PCR (for mRNA), western blotting and immunohistochemistry (for protein). RESULTS: Compared to the uninfected tissues and cells, the HBV-infected tissue and cells showed down-regulated expression of furin at both the mRNA and protein levels. In particular, the HepG2.2.15 cells showed -50% less furin mRNA expression than the HepG2 cells and the difference was statistically significant (P less than 0.05). CONCLUSION: HBV may suppress the host cell's expression of furin, possibly to benefit its survival and replication in the host cell.

Prognostic significance of carboxypeptidase Q and its methylation in glioblastoma.

Background: Glioblastoma (GBM) is a highly aggressive intracranial malignant tumor. The role of carboxypeptidase Q (CPQ) in GBM remains unknown. This study was to investigate the prognostic significance of CPQ and its methylation in GBM. Methods: We collected data from The Cancer Genome Atlas (TCGA)-GBM database and analyzed the different expression of CPQ in GBM tissues and normal tissues. Then we explored the correlation of CPQ mRNA expression and DNA methylation, and confirmed the prognostic significance of them based on six additional datasets from TCGA, The Chinese Glioma Genome Atlas (CGGA) and Gene Expression Omnibus (GEO) databases. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis were utilized to investigate the biological function of CPQ in GBM. Furthermore, we determined the association of CPQ expression and immune cell infiltration, immune markers and tumor microenvironment using different bioinformatic algorithms. R (version 4.1) and GraphPad Prism (version 8.0) were used to analyze the data. Results: CPQ mRNA expression in GBM tissues was significantly higher than that in normal brain tissues. DNA methylation of CPQ was negatively correlated with its expression. Patients with low CPQ expression or higher CPQ methylation level had remarkably better overall survival (OS). The TOP20 biological processes relevant to the differentially expressed genes between high and low CPQ patients were almost all related to immunity. And the differentially expressed genes were involved in several immune-related signaling pathways. CPQ mRNA expression was outstandingly correlated with CD8+ T cells, neutrophils, macrophages and dendritic cell (DC) infiltration. Moreover, CPQ expression was meaningfully associated with the ESTIMATE score and almost all immunomodulatory genes. Conclusions: Low CPQ expression and high methylation are associated with longer OS. CPQ is a promising biomarker for predicting prognosis in patients with GBM.

Combination of Tumor Mutational Burden and Specific Gene Mutations Stratifies Outcome to Immunotherapy Across Recurrent and Metastatic Head and Neck Squamous Cell Carcinoma.

Purpose: To investigate the prognostic significance of tumor mutational burden (TMB) combined with specific prognosis-related gene mutations in immunotherapy for recurrent and metastatic head and neck squamous cell carcinoma (r/m HNSCC). Methods: One hundred thirty-two r/m HNSCC patients from the Morris and Allen cohorts had undergone immunotherapy. We constructed the immunotherapy-related gene prognostic index TP-PR combining TMB and PIK3CA, TP53, or ROS1 mutation. And we analyzed the differences in overall survival (OS) and immune cell infiltration between samples in different groups. The association of each signature's single-sample gene set enrichment analysis scores with TP-PR was tested using Spearman's correlation test. Results: The median OS of the patients with high TMB (TMB ≥10 mut/Mb) who received immunotherapy for r/m HNSCC was 2.5 times as long as that of the patients with low TMB (25 vs. 10 months). More importantly, the high TP-PR (TP-PR >0) group had better median OS (25 vs. 8 months) than the low TP-PR (TP-PR ≤0) group. CD8+ T cells and activated memory CD4+ T cells in the tissues of the patients with high TP-PR were higher than those in the patients with low TP-PR. Results showed that TP-PR stratification had a higher area under the curve (AUC) value (0.77, 95% CI 0.86-0.68) compared with TMB stratification (0.56, 95% CI 0.68-0.44). The differential gene expression in the high and low TP-PR groups mainly influenced metabolism-related signaling pathways. Conclusion: TP-PR was an effective predictor of immunotherapy outcome for r/m HNSCC, which might be better than TMB alone. Patients with high TP-PR had a better survival benefit than had the patients with low TP-PR.

Influence of a single nucleotide polymorphism in the P1 promoter of the furin gene on transcription activity and hepatitis B virus infection.

UNLABELLED: Hepatitis B e antigen (HBeAg) is a viral strategy of immune response evasion associated with hepatitis B virus (HBV) persistence. Spontaneous HBeAg seroconversion is usually accompanied by liver disease remission. Unfortunately, this goal is difficult to achieve and requires expensive and time-consuming treatment. Furin, a proprotein convertase, is involved in HBeAg maturation and is therefore a potential therapeutic target or indicator for predicting disease progression and antiviral response. Here we demonstrate that healthy Han Chinese from southern China (an endemic area of HBV infection) harbor a common single nucleotide polymorphism (SNP; -229 C/T) in a 1268-bp region of the P1 promoter of the furin gene [FES upstream region (Fur)]. A luciferase reporter gene assay showed that transcription activity is about 3 times higher in allele T carriers than in allele C carriers of this SNP. Allele T includes a suboptimal transcription factor NF-E2 [i.e., nuclear factor (erythroid-derived 2)]-binding motif according to bioinformatics and studies using site-directed mutagenesis. We also observed that individuals carrying allele T were more likely to become persistently infected. When persistently infected patients were divided into subgroups according to recent guidelines and HBeAg-defective virus infection was taken into account, patients with allele T or genotype TT had a decreased likelihood of HBeAg seroconversion or an increased likelihood of progressing to HBeAg-negative chronic hepatitis B or liver cirrhosis if accompanied by HBeAg-defective virus infection. CONCLUSION: The common SNP in the P1 promoter of the Fur gene affects furin transcription activity and HBV infection outcome, possibly by increasing furin messenger RNA expression, and this suggests that furin is a potential therapeutic target and that this SNP is a potential predictor of disease progression or therapeutic response.

[Study on a putative, proprotein convertase-cleaved product of HBV core protein in vitro].

OBJECTIVE: To investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product. METHODS: Recombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution. RESULTS: HBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15,000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein. CONCLUSION: HBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.

Expression of SOCS-1 in the liver tissues of chronic hepatitis B and its clinical significance.

AIM: To study the expression of suppressor of cytokine signaling-1 (SOCS-1) in the liver tissues of chronic hepatitis B (CHB) and the clinical significance of this expression. METHODS: The expression of SOCS-1 in liver tissues of 45 cases of CHB was investigated by immunohistochemical staining, and its correlations with inflammation grades and fibrosis stage were analyzed by SPSS statistics software. RESULTS: The result showed SOCS-1 expressing could be observed in the liver tissue of CHB. The expression of SOCS-1 was mainly distributed near the portal area in the liver tissue of mild inflammation CHB group, and was diffusely distributed in the liver tissue of moderate and severe inflammation groups. SOCS-1 positive stains mainly appear in the hepatocytes, only a few of liver interstitial cells were involved. Inside the hepatocyte, SOCS-1 positive stains are mainly distributed in the plasma. Some of the staining was observed on the membrane. The inclusion bodies in the plasma of hepatocytes were observed occasionally. There were both obvious correlations between the expression of SOCS-1 and the inflammatory grade, and that between the expression of SOCS-1 and the fibrosis stage. CONCLUSION: The distribution of SOCS-1 in the liver tissue of CHB is variable. This expression was correlated with the inflammation grade and fibrosis stage.

Comparison of biological characteristics of marrow mesenchymal stem cells in hepatitis B patients and normal adults.

AIM: To establish a culture system of marrow mesenchymal stem cells (MSCs) from hepatitis B patients and normal adults and to compare their biological characteristics. METHODS: MSCs were isolated from bone marrow in 34 male hepatitis B patients and 15 male normal adults and cultivated in vitro. Their biological characteristics including surface markers, shapes and appearances, growth curves, first passage time and passage gene-rations were compared. RESULTS: Cultivation achievement ratio of hepatitis B patients was lower than that of normal adults, no statistical significance (82.35% vs 100%, P > 0.05). Compared with MSCs of normal adults, MSCs of hepatitis B patients presented a statistical lower growth curve, longer first passage time (13.0 +/- 1.6 d vs 11.4 +/- 1.5 d, P < 0.05), fewer passaging generation numbers (10.5 +/- 1.4 generations vs 12.3 +/- 1.7 generations, P < 0.05), though both shared same appearances, shapes and surface markers. MSCs in hepatitis B patients would expand, spread out and age more easily and there were more refractive particles in the cytoplasm. CONCLUSION: MSCs from hepatitis B patients can be cultured in vitro. Although their appearance, shape and surface marker are similar to those of MSCs from normal adults, there are differences in their biological characteristics.

[Optimization of cultivation methods of MSCs from hepatitis B patients and investigation of their biological characteristics].

OBJECTIVE: To optimize cultivation methods of bone marrow mesenchymal stem cells (MSCs) from hepatitis B patients and to investigate their biological characteristics. METHODS: Growth curves of hepatitis B patients MSCs cultivated with five culture media and two inoculation methods were compared; the shapes, appearances, surface markers and bionomics of the cultivated MSCs were studied. RESULTS: Inoculating the cells obtained directly from bone marrow aspirations was not as successful as using the marrow cells after their density gradient centrifugations (76% vs 88%), but the differences in the results were not statistically significant (P more than 0.05). The successful cultivation rates using five culture media were different and the differences were statistically significant (P less than 0.01). The autoserum medium was most successful, fatal bovine serum (FBS) medium was next successful and the non-patient serum medium was the least successful. The growth curves of the cultivations using the different media were parallel to this. Changing the whole culture media every 2 or 3 days was better than changing half of the media. The shapes, appearances, surface markers and the growth characteristics of MSCs from the hepatitis B patients were almost the same as MSCs from the normal adult. CONCLUSION: The best cultivation method of MSCs from hepatitis B patients is: separating marrow cells using density gradient centrifugal separation, cultivating them using an autoserum culture medium, and completely changing the medium every 2-3 days. The biological characteristics of MSCs from the hepatitis B patients using the above methods are almost the same as those from normal adults.

[MxA gene-88 G/T polymorphism influences the outcomes of HBV infection].

OBJECTIVE: To study the relationship between a G/T substitution at position -88 of myxovirus resistance-1 gene (MxA) and the self-limiting or chronic infection of HBV. METHODS: Blood samples from 100 patients with self-limiting HBV infection (positive anti-HBs and anti-HBc) and from 340 patients with chronic HBV infection were collected. MxA-88 G/T polymorphism was typed using a protocol based on competitively differentiated-polymerase chain reaction. For statistical analysis, odds ratio and chi-square test were used. RESULTS: The detective rate of G/G genotype (low expression genotype) of MxA-88 G/T was 50.2% (221/440), those of T/T genotype (high expression genotype) and G/T heterozygous genotype were 5.5% (24/440) and 44.3% (195/440). Compared to patients with chronic infection, patients with self-limiting infection had lower frequency of G/G genotype (41.0% vs 52.9%, P < 0.05) or G allele (62.5% vs 75.9%, P < 0.01) and had higher frequency of T/T genotype (16.0% vs 2.4%, P < 0.01) or T allele (37.5% vs 24.1%, P < 0.01), but there was no significant difference in the G/T heterozygous genotype. CONCLUSIONS: MxA gene -88 G/T polymorphism influences the natural outcomes of HBV infection to some extent. This SNP of MxA gene may be used as a clinical prognostic marker of HBV infection.

[Changes of antigen-specific cytotoxic T lymphocytes during acute flare-ups in chronic hepatitis B patients].

OBJECTIVE: To detect HBV antigen specific cytotoxic T lymphocyte (CTL) changes in patients during acute flare-ups and to study their association with flare-ups and aggravations into grave hepatitis by quantitative analysis of HLA-A2* restricted HBcAg-specific CTL cells. METHODS: The frequency of HBcAg-specific CTL cells in the peripheral blood mononuclear cells (PBMC) from 29 patients with persistent infection with HBV were quantified by flow cytometry using one HLA-A2*HBV peptide pentamers complex (Pro5TM MHC Pentamers). RESULTS: There was a statistical difference of HBcAg specific CTLs between the patients with acute exacerbations (1.4%+/-0.8%) and the patients with immune tolerance (0.6%+/-0.4%) (t = 2.180, P = 0.01-0.05); There was no significant difference between the grave hepatitis group (1.3%+/-1.0%) and the chronic hepatitis group (1.4%+/-0.8%) regarding frequencies of antigen specific CTL (t = 0.215, P = 0.833-0.05). The level of antigen specific CTLs in PBMC in the 6 cases of chronic hepatitis B with acute exacerbations maintained a relatively high level (more than 0.7%) within the 12 week follow-up period. CONCLUSION: HBcAg-specific CTLs may play an important role in hepatic flare-ups in patients with chronic HBV infection, but there was no direct relationship between antigen- specific CTLs and grave hepatitis.

Simultaneous detection of two major lamivudine-resistant mutants using competitively differentiated-PCR.

Rapid, specific and sensitive methods without advanced equipment are required urgently in developing countries in order to detect or monitor lamivudine-resistant mutants routinely before or in the course of the therapy. A protocol is described for the detection of two major YMDD mutations simultaneously through modifying a previous competitively differentiated-PCR (CD-PCR) by revising the strategy, increasing the number of competitively differentiated primers, increasing the number of labeled haptens, optimizing the amplification system and analyzing its products by enzyme immunoassay. Special care was taken to promote the sensitivity, specificity and the ability of the protocol to detect mutation in mixture of the mutants and wild strain. YMDD mutants in clinical serum samples were detected simultaneously, specifically and rapidly only with assistance of the equipment used widely in highly prevalent areas of hepatitis B virus infection.

Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation.

AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10 micromol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 10(3) copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

High level of hepatitis B virus DNA after HBeAg-to-anti-HBe seroconversion is related to coexistence of mutations in its precore and basal core promoter.

AIM: G1896A mutation in precore or A1762T/G1764A mutations in basal core promoter are suspected to be responsible for patients with detectable level of HBV DNA in serum after seroconversion from HBeAg to anti-HBe. However, G1896A variant has impaired, while A1762T/G1764A variant may have intact replication ability. They themselves or their coexistence status may play different roles in such meaningless seroconversion. For these reasons, the significances of these two types of mutations were comparatively investigated in this study. METHODS: One hundred and sixty-five sera with positive anti-HBe and HBV DNA were collected from different patients. Mutations of G1896A and A1762T/G1764A among these serum samples were detected using competitively differentiated PCR. HBV DNA was demonstrated using real-time quantitative PCR. RESULTS: G1896A and/or A1762T/G1764A mutations were detected in 89.1% (147/165) out of patients with detectable HBV DNA in serum after HBeAg-to-anti-HBe seroconversion. The positive rate of G1896A variants was significantly higher than that of A1762T/G1764A mutations (77.6% vs 50.3%, chi2 = 26.61, P<0.01). The coexistence positive rate of these two types of mutations was 38.8% (64/165). Coexistence mutations were found in 77.1% (64/83) out of sera with A1762T/G1764A mutations, and in 50.0% (64/128) out of sera with G1896A mutation. Compared with variants with G1896A mutation only, the coexistence mutations were predominant in patients with high level of serum HBV DNA, and related to higher total bilirubin, lower serum albumin and progressive liver diseases. CONCLUSION: The coexistence of G1896A mutation and A1762T/G1764A mutations is very common, and responsible for the major cases with high level of HBV DNA in serum and progressive liver diseases after HBeAg-to-anti-HBe seroconversion. This coexistence mutation variant may have higher pathogenicity and replication ability.

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of alpha determinant region.

AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice. METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo. RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the alpha determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106+/-26.7 vs 1 814.4+/-776.3, P<0.01, t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs 6/10, 4.56+/-3.52 mIU/mL vs 27.60+/-17.3 mIU/mL, P = 0.02, t = 2.7). CONCLUSIONS: HBsAg with deleted second-loop of the alpha determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.

Construction of exogenous multiple epitopes of helper T lymphocytes and DNA immunization of its chimeric plasmid with HBV pre-S2/S gene.

AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB were generated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Bam HI), 5' terminal of S gene (HincII, Xba I) and 3' terminal of S gene (Acc I) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits. RESULTS: The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the co-inoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7+/-69.1 mIU/mL vs 27.6+/-17.3 mIU/mL, P<0.01, t = -6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54+/-7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA II antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.

Novel assay of competitively differentiated polymerase chain reaction for screening point mutation of hepatitis B virus.

AIM: Point mutation, one of the commonest gene mutations, is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple. For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice. METHODS: Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3' end except for last 2 base pairs and a different non-complemented region in 5' end were designed. Thus, competitive amplification might be carried out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube. The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR. RESULTS: CD-PCR was successfully established. It could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 10(2)-10(8)copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 10(2)-10(4)copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of detectable viremia than that with a higher level of detectable viremia (P=0.0192). CONCLUSION: CD-PCR is more specific since it is less influenced by the amount of initial templates and the cross amplification between mutant- and wild-type amplified products. It is also simple and time-saving. Thus, CD-PCR might be useful in routine gene typing and point mutation screening. HBV G1896A or other more important mutations have to be routinely detected in patients with a detectable level of viremia after HBeAg/antibody conversion in clinical practice.

Serum hyaluronic acid, procollagen type III and IV in histological diagnosis of liver fibrosis.

OBJECTIVE: To assess the significance of serum hyaluronic acid (HA), procollagen type III (PCIII), collagen type IV (CIV) in the histological diagnosis of liver fibrosis. METHODS: The concentrations of serum HA, PCIII, CIV in 253 patients with chronic liver diseases were measured by radioimmunoassay. Liver biopsies were performed in all patients at the same time. The liver was pathologically evaluated by a pathologist according to a scoring system. Combined with the results of liver pathological diagnosis, the accuracy of serum HA, PCIII, CIV in diagnosing patients with hepatic fibrosis (staging >/=S2) or cirrhosis (S4) was assessed using the receiver operating curve (ROC). RESULTS: The cutoff values of serum HA, PCIII and CIV for identifying patients with hepatic fibrosis (>/=S2) or cirrhosis (S4) were determined. The cutoff values of serum HA, PCIII and CIV for detecting patients with fibrosis (stage >/=S2) were 90 micrograms/L, 90 micrograms/L, 75 micrograms/L, respectively; their sensitivity (Se) was 80.4%, 82%, 63.1%; their specificity (Spe) was 70.2%, 60.8%, 83.8%; their positive predictive values (PPV) were 86.7%, 83.5%, 90.4%; their negative predictive values (NPV) were 59.8%, 58.4%, 48.4%, respectively. The cutoff values for detecting patients with liver cirrhosis were 210 micrograms/L for HA, 96.2% for Se, 85.3% for Spe, 65.4% for PPV, 98.8% for NPV; 150 micrograms/L for PCIII, 76.4% for Se, 68.7% for Spe, 40.4% for PPV, 91.3% for NPV; 90 micrograms/L for CIV, 80% for Se, 75.8% for Spe, 47.8% for PPV, 93.2% for NPV, respectively. CONCLUSIONS: Serum HA, PCIII and CIV can be determined for an accurate diagnosis of hepatic fibrosis in various stages. HA is the best for screening liver cirrhosis.

[Effect of exogenous epitopes of helper T lymphocyte on humoral immunity of HBV S gene DNA immunity].

OBJECTIVE: To study the effect of exogenous epitope of helper T lymphocyte (HTL) on humoral immunity of HBV S gene DNA immunity. METHODS: Two universal HTL epitopes, amino acid residue (aa) 830-843 of the tetanus toxoid (TTE) and artificial epitope (PADRE), and 3 unique epitopes, aa1-20 of tubercle bacteria hot shock protein 65 (TBE), aa54-65 of rubella protein E2-4 (ME) and aa35-48 of trachoma hot shock protein 60 (CE) were chosen. Eukaryotic expression vectors were constructed by inserting single or multiple exogenous epitopes in HBV S gene just after the initial code of translation. BALB/c mice were inoculated with 100 micro g of recombinant DNA per mouse, and given boost inoculation for 3 times with 3-week interval. Mouse blood were collected one month just after the third boost inoculation. Anti-HBs was detected using Abbott test kits. RESULTS: HBV S eukaryotic gene expression vectors, pHB and 6 exogenous HTL epitope HBV S gene vectors, pHB-TBE, pHB-PADRE, pHB-TTE, pHB-MTE2, pHB-MTE3 and pHB-MTE5 were constructed successfully with anti-HBs level (IU/L) of 10 +/- 5, 5 +/- 5, 49 +/- 7, 29 +/- 6, 16 +/- 8, 23 +/- 7 and 28 +/- 8 respectively. Among 3 single epitopes, TTE and PADRE had obviously effect on promoting the anti-HBs response of HBV S gene, while TBE had no promoting effect. All of the 3 multiple epitopes were shown the effect of immune promoting. CONCLUSION: Some exogenous HTL epitopes had obviously effect on promoting the anti-HBs response of HBV S gene. Multiple epitopes also had humoral immunity promoting effect, but there was no synergic effect among their own HTL epitopes. PADRE might be an important candidate for new efficient HB vaccine. The multiple epitope cluster consisted form 5 exogenous epitopes might be an important candidate for the reinoculating HB vaccine or therapy HB vaccine.