The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic hepatitis B.

UNLABELLED: In the past 50 years there have been considerable efforts to identify the cellular receptor of hepatitis B virus (HBV). Recently, in vitro evidence from several groups has shown that the sodium-taurocholate cotransporting polypeptide (NTCP, which is encoded by SLC10A1 and transports bile acids into hepatic cells in enterohepatic recirculation) is a strong candidate. In particular, in vitro the p.Ser267Phe variation of SLC10A1 results in loss of HBV receptor function. We tested the role of NTCP as a receptor for HBV in chronic hepatitis B patients using a genetic association study. We selected SLC10A1 variants from 189 exomes. We used Sanger sequencing to follow up the association of the various SLC10A1 variants in a Han Chinese cohort of 1899 chronic hepatitis B patients and 1828 healthy controls. We further investigated the potential impact of the p.Ser267Phe variant on NTCP function using structural analysis. The p.Ser267Phe variant was associated with healthy status (P = 5.7 × 10(-23) , odds ratio = 0.36) irrespective of hepatitis B virus surface antibody status (P = 6.2 × 10(-21) and 1.5 × 10(-10) , respectively, when the cases were compared with hepatitis B virus surface antibody-positive and -negative controls). The variation was also associated with a lower incidence of acute-on-chronic liver failure (P = 0.007). The estimated heritability explained by this single variation was ∼3.2%. The population prevented fraction was around 13.0% among the southern Chinese. Our structural modeling showed that the p.Ser267Phe variant might interfere with ligand binding, thereby preventing HBV from cellular entry. CONCLUSION: The p.Ser267Phe NTCP variant is significantly associated with resistance to chronic hepatitis B and a lower incidence of acute-on-chronic liver failure. Our results support that NTCP is a cellular receptor for HBV in human infection.

Imbalance of interleukin-17-producing CD4 T cells/regulatory T cells axis occurs in remission stage of patients with hepatitis B virus-related acute-on-chronic liver failure.

BACKGROUND AND AIM: Although regulatory T cells (Treg) and interleukin-17-producing CD4 T cells (Th17) have been demonstrated to play opposing roles in inflammation-associated diseases, their frequency and balance in different stages of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) remain unknown. METHODS: Fourteen patients with HBV-associated ACLF were studied and defined into different stages according to disease activity. Circulating Th17 cells and Treg cells were analyzed by flow cytometry, and the cytokines were quantitated by enzyme-linked immunosorbent assay. Results were correlated with temporal changes in viral load, disease progression and compared with 30 chronic hepatitis B (CHB) subjects and 18 healthy subjects. RESULTS: We showed a significantly higher frequency of circulating Th17 cells in the remission stage of ACLF when compared with the progression stage, the CHB group, or normal controls. However, the frequency of circulating Treg cells was significantly lower in the remission stage of ACLF when compared with the progression stage or the CHB group. The increase in Th17 cells and concomitant decrease in Treg cells created an imbalance in the remission stage of ACLF patients, which negatively correlated with disease progression. In addition, we showed that ACLF patients in the remission stage had an altered profile of cytokines that regulated the induction of Th17 cells and Treg cells. CONCLUSIONS: ACLF patients in the remission stage had an imbalance of Th17 to Treg cells, which could be used as a prognostic marker to predict disease progression. This imbalance could play a role in the immunopathogenesis of HBV-related ACLF.

[Safety evaluation and quality control of MSCs from hepatitis B patient in vitro].

OBJECTIVE: To get mesenchymal stem cells (MSCs) from hepatitis B patient and to valuate the safety and quality after long-term culture in vitro. METHODS: The cells obtained directly from bone marrow and cultured in Mesen Pro medium supplemented with FGF, and the morphology of MSCs was observed. Surface antigens of the MSCs were analyzed by flow-cytometry. The bacteria, virus, endotoxin and residual serum of cell suspension were detected. The MSCs and perpheral blood T lymphocytes were co-cultured in 48 well plates for 72 h and the T lymphocyte proliferation was measured by using MTT reduction method and the effect of MSCs on T lymphocyte transformation stimulated by PHA was also observed. The oncogenicity of MSCs was verified by the tumorigenesis test in sofo agar. The genetic stability of MSCs was examined by karyotype analysis. RESULT: The MSCs from hepatitis B patient could be passaged to many generations and had strong abilities of proliferation. They expressed stem cell-surface antigens and maintained normal karyotype, prevented the pollution of bacteria and viruses, inhibited the immune response of allogenic T lymphocytes and no oncogenicity found. CONCLUSION: The MSCs have proliferative potentials, can be passaged in long-term cultures in Mesen Pro medium without oncogenicity, can maintain normal karyotype, can inhibit the immune response of T lymphocytes and can alleviate the grafe-versus diseases. The MSCs can be served as a new type of cells in cell and gene therapy.

[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.

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.

[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.

Interleukin-10 promoter polymorphisms are associated with the mode and sequel of HBeAg seroconversion in patients with chronic hepatitis B virus infection.

BACKGROUND: The influence of interleukin-10 (IL-10) gene promoter polymorphisms on the mode and sequel of HBeAg seroconversion (a favorable event usually) in patients with chronic Hepatitis B virus (HBV) infection has not been clarified. PATIENTS AND METHODS: IL-10 genotyping and haplotype analyses of 340 HBsAg carriers and 100 volunteers with self-limiting HBV infection from southern China, a high prevalent area of HBV were performed according to the single nucleotide polymorphisms in its promoter (-1,082, -819 and -592) using a competitively differentiated PCR. RESULTS: High-producer genotype (GG at -1,082) or haplotype (GCC) was rarely found in patients from southern China (<1%). Intermediate-producer haplotype (ACC) was closely associated with chronic liver disease (P=0.004); compared with this, low-producer genotype (AA at -592) and haplotype (ATA) were closely associated with asymptomatic carriers (P=0.035 and 0.035). Intermediate-producer genotype (AC at -592) and haplotype (ACC) were closely associated with covert seroconversion of HBeAg (P=0.0086 and 0.0013) and progressive sequel after HBeAg seroconversion (P=0.013 and 0.0008), while, low-producer genotype (AA at -592) and haplotype (ATA) were closely associated with overt seroconversion of HBeAg (P=0.0023 and 0.0061) and silent sequel after HBeAg seroconversion (P=0.0009 and 0.001). CONCLUSIONS: IL-10 gene promoter polymorphisms significantly influence the mode and sequel of HBeAg seroconversion in patients with chronic HBV infection.

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.

An isothermal amplification system based on the tandem-repeated DNA probe.

The hybridization methods and polymerase-based amplification methods are usually employed to detect pathogens and gene expressions quantitatively in clinical practices nowadays. However, the sensitiveness of the former and the specificity of the latter are not yet satisfied. To solve this problem, some promising comprehensive methods have been developed recently. Here we reported a new comprehensive method, a tandem repeated DNA probe-based amplification (TRPBA) system. To establish the TRPBA, TR48, an artificial tandem repeated DNA probe with 48 repeats of a 50 base pair unit was constructed. It could be efficiently amplified by Bst DNA polymerase at 61 degrees C for only 1 h. The products were analyzed either by direct gel electrophoresis or by gel electrophoresis after the digestion with restriction endonuclease HincII. The sensitiveness was as few as 100 copies per test, which was comparable with PCR-based techniques. The TR48 splicing with the DNA fragment of hepatitis B virus used as probe could successfully develop TRPBA to detect hepatitis B virus DNA. The TRPBA can be used in the future to detect many other genes or microorganisms simply by splicing TR48 with their DNA fragments. Thus, TRPBA might be useful because of its sensitiveness and simpleness.

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.

[Initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B].

OBJECTIVES: To probe into the initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B (CHB). METHODS: Forty-four CHB patients with positive HBeAg and HBV DNA were treated with interferon. Serum ALT and viral markers levels of HBsAg, HBeAg, anti-HBc and HBV DNA were examined regularly. Liver biopsy was carried out just before the treatment. RESULTS: The rate of HBeAg seroconversion was 75% at the sixth month, and 68.2% after one year of follow up. The rate of damage sensitive stage of hepatocytes was 47.7%. The average onset time was (3.14+-1.49) weeks after the treatment, and lasted for (8.24+-3.52) weeks. The ALT level raised (1.73+-1.13) times. The occurrence of damage sensitive stage of hepatocytes was indicator for good curative effect (Fisher exact probability, P=0.028). Damage sensitive stage of hepatocytes was more often developed in patients with moderate inflammation, overexpression of HBcAg in liver and higher level of HBeAg in blood stream before treatment. HBeAg and anti-HBc levels in peripheral blood decreased in the onset period of damage sensitive stage of hepatocytes. CONCLUSIONS: The initiative factors of the damage sensitive stage of hepatocytes may be: HBeAg decreasing in peripheral blood induced by interferon may dismiss immune lutation of HBeAg and anti-HBc to cytotoxic T lymphocyte (CTL), which recognize HBcAg as target, thus activates the cytotoxicity of HBV-infected hepatocytes mediated by CTL.

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.

[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.