Hepatic histological changes and its influencing factors of HBeAg negative chronic hepatitis B patients / 实用医学杂志

Objective To investigate hepatic histological features and its influencing factors of HBeAg-negative chronic hepatitis B patients. Methods 134 HBeAg-negative chronic hepatitis B (CHB) patients who underwent percutaneous liver biopsy were recruited in this study. The liver biopsy sections were examined after routine haematoxylin-eosin (HE) staining, and silver staining for assessment of fibrosis. The activity of liver disease was assessed by using a modified Knodell numeric histology activity index (HAI). ALT level, HBV DNA load, HBV serological markers, HBV genotype were assessed with appropriate methods. t test or analysis of variance was used to compare means. Non-parametric was done by Kruskal-Wallis test. The correlation between liver pathological change and clinical factors was analyzed by multivariate linear regression. Results Of 134 HBeAg negative CHB patients, percentages of mild (HAI 4 ~ 8), moderate (HAI 9 ~ 12), and severe hepatitis (HAI 13 ~ 18) were 26.9%, 26.1%, and 47.0%, respectively. As for hepatic fibrosis, 18.7% and 81.3% of the patients had fibrosis score < 3 and ≥3, respectively. Multivariate regression analysis showed that ALT level and hepatic fibrosis were correlated to hepatic inflammation (t = 6.687,P < 0.01; t = 3.478, P < 0.01) while age and hepatic inflammation activity were influencing factors of hepatic fibrosis (t = 3.587, P < 0.01; t =7.136, P < 0.01). However, correlation is not significant between hepatic histological change and other factors, including gender, HBVDNA, HBV genotype and HBeAb status. Conclusions In this study, hepatic histological change tend to become worse in majority of HBeAg-negative chronic hepatitis B , especially in older patients and those with high ALT level.

Long-term efficacy of individualized interferon-alpha therapy for HBeAg-negative chronic hepatitis B patients: a 2-year follow-up study / 中华传染病杂志

Objective To investigate the efficacy of individualized interferon (IFN)-alpha therapy in HBeAg-negative chronic hepatitis B patients. Methods Seventy- six Chinese HBeAg-negative chronic hepatitis B patients proven by liver biopsy were treated with 5 MU recombinant IFN-alpha 1b subcutaneously thrice every week. All the patients were followed up for at least 24 months the combined responses were defined as normalization of serum alanine transaminase (ALT) and HBV DNA＜3 log10 copy/mL. An intention-to-treat (ITT) analysis was used in this paper in which all 76 patients were included. Results Six patients were lost. Treatment duration was in the range 2-24 months with a median of 8.5 months, and combined responses were achieved at a median of 6.0 months (range 2-19 months) of treatment duration.Seventy-five-percentile of treatment duration to endpoints was 10.0 months. The combined response rate was 46.1% (35/76) at the end of treatment, 43.3% (33/76) at 12-month follow-up and 40.8% (31/76) at 24-month follow-up. The relapse rate was 20. 0% (7/35) and 25. 7% (9/35) at 12-month and 24-month follow-up, respectively. Higher necroinflammatory activity in liver biopsy predicted a good response, while gender, age, liver fibrosis, baseline ALT, aspartate aminotransferase levels and baseline HBV DNA levels were not impact factors of therapeutic effects by binary Logistic regression analysis.Conclusion Individualized prolonged IFN-alpha regimen lead to considerable sustained disease suppression in patients with HBeAg-negative chronic hepatitis B.

Clinical and histological characteristics of chronic hepatitis B with negative hepatitis B e-antigen / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the clinical and histological features of chronic hepatitis B (CHB) with negative hepatitis B e-antigen (HBeAg).</p><p><b>METHODS</b>A total of 743 in-patients with chronic hepatitis B were recruited into the study and divided into two groups according to the HBeAg status. The correlation among alanine transaminase (ALT) levels, hepatitis B virus (HBV) DNA semiquantification, and the liver histopathological data were detected.</p><p><b>RESULTS</b>Of the 743 successive in-patients, 267 (35.9%) were HBeAg-negative. The HBDAG-negative group had significantly lower serologic HBV DNA levels (63.0% of < 100 pg/ml) vs HBeAg-positive (42.6%, P < 0.001), while more sever inflammation (58.1% of inflammatory scores of histological activity index (HAIinf > or = 9) vs HBeAg-positive group (46.0%, P < 0.001) and severe fibrosis (45.3% of fibrosis scores of histological activity index (HAIfib > or = 3) vs HBeAg-positive group (27.9%, P < 0.001) of liver histology. In HBeAg-positive patients, increasing ALI levels were significantly associated with high inflammation and fibrosis scores and low HBV DNA levels. However, it was not the case in the HBeAg-negative cases. In HBeAg-positive patients, 91.3% of them had HAIinf > or = 9 and 65.7% had HAIfib > or = 3 with HBV DNA > 100 pg/ml, while 8.2% of them had HAIinf > or = 9 and 12.3% had HAIfib > or = 3 with HBV DNA < 20 pg/ml, indicating an obverse correlation between HBV DNA levels and histology scores.</p><p><b>CONCLUSIONS</b>As regards clinical and histological background, the chronic HBeAg-negative hepatitis B is a different subpopulation from the HBeAg-positive counterpart.</p>

T helper cells in patients with chronic hepatitis B virus infection / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the compositions of Th1/Th2/Th3 cells in chronic hepatitis B virus (HBV)-infected individuals by determining the expression of interleukin-4 (IL-4), inetrferon-gamma (IFN-gamma), and transform growth factor-beta (TGF-beta) in single CD4(+) T cells isolated from peripheral blood mononuclear cells (PBMCs) and the role of polarized Th cell populations in chronic HBV-infection was discussed.</p><p><b>METHODS</b>PBMCs from chronically infected HBV individuals were isolated, stimulated by PMA/Ionomycin/Monensin, and IL-4, IFN-gamma and TGF-beta production by CD4(+) T cells was determined by using fluorescence activated cell sorter (FACS) analysis.</p><p><b>RESULTS</b>The percentage of IFN-gamma-producing T cells, IL-4-producing T cells and TGF-beta-producing T cells ranged from 2.3% - 18.6%, 1.1% - 8.7% and 0.7% - 7.1% respectively in CD4(+) T cells from non-infected individuals. Most of CD4(+) T cells from PBMCs in chronically infected HBV individuals were Th0 cells. The proportion of Th1 cells increased significantly with hepatic inflammatory activity, and in the active period of chronic hepatitis B infection were higher than those in the non-active period (P < 0.05). Th2 cell percentage in CD4(+) T cells from HBV-infected individuals did not differ significantly (P > 0.05), but were higher than that from controls (P < 0.05). Th3 cell percentage in CD4(+) T cells from asymptomatic carrier (AsC) group was higher than that in the chronic hepatitis B (CHB) and control groups (P < 0.05).</p><p><b>CONCLUSIONS</b>Th1 phenotype cytokines were positively correlated with hepatic inflammatory activity in chronic hepatitis B and Th2 cells may be associated with the persistence of HBV infection. Th3 cells cooperating with Th2 cells can negatively regulate immune responses and may be associated with the immune tolerant state of chronic HBV infection.</p>

Cloning and expression and purification of hepatitis B e-antigen precursor in Escherichia coli / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification system.</p><p><b>METHODS</b>Hepatitis B virus (HBV) preC/C gene for the 25 kD HBeAg precursor was cloned into the expression vector pQE30 and the 25 kD HBeAg precursor was expressed in Escherichia coli (E. coli) and purified. Its antigenicity for 21 kD mature HBeAg was tested by western blot analysis.</p><p><b>RESULTS</b>Cloned fragments in the expression vector were sequenced and verified to be homogeneous with that of HBV (ayw subtype). Expression of the HBeAg precursor in E. coli under the transcriptional regulation of T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 25 kD. Recombinant HBeAg precursor exhibited identical potencies with 21 kD mature HBeAg that reacted with anti-HBeAg antibodies. The purification rate of the expressed HBeAg precursor was up to 89.6% and the yield of purified HBeAg precursor from this procedure was 2.4 mg/L.</p><p><b>CONCLUSION</b>25 kD HBeAg precursor exhibited biological activity and might play an important role in pathopoiesis of hepatitis B.</p>

Classification of genotyping hepatitis B virus with multiplex PCR / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a simple and practicable method to identify the different genotypes of hepatitis B virus (HBV).</p><p><b>METHODS</b>Based on the alignment of 114 complete nucleotide sequence for HBV DNA of different genotypes, the specific sequence of each genotype was found. Six primer sets were designed for each of the six genotypes according to the genotype-specific sequence, and used separately for PCR. The genotype of HBV was identified according to the positive result of PCR. Three primer sets for B, C and D genotypes were added into a single tube for PCR reaction, and HBV was genotyped according to the length of the amplified DNA.</p><p><b>RESULTS</b>There was no difference in the genotyping result of PCR by single or multiplex primers, which was identical to the PCR-RFLP method.</p><p><b>CONCLUSIONS</b>This multiplex PCR method is simple, precise, sensitive, and easy to use.</p>

Diagnostic value of ultrasonic examination in patients with different stages of liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To discuss the diagnostic value of ultrasonic examination in patients with early liver cirrhosis and the relation with different stages of liver fibrosis.</p><p><b>METHODS</b>In the series, 263 patients with chronic hepatitis B were under taken liver biopsy and ultrasonic examination of type B for determination of liver cirrhosis images, width of the main portal vein and the splenic vein, tumefaction of the spleen. Data were analysed statistically.</p><p><b>RESULTS</b>Sixties of 263 patients were diagnosed as early liver cirrhosis. The diagnostic sensitivity, specificity, misdiagnostic rate, missed diagnostic rate, and Jonden's index of ultrasonic examination for early liver cirrhosis were 52.5%, 88.3%, 11.7%, 47.5%, and 0.508, respectively. The width of the main portal vein with liver fibrosis of S1, S2, S3, and S4 were 10.93 mm +/- 1.25 mm, 11.35 mm +/- 1.06 mm,11.29 mm +/- 1.52 mm, and 11.4 8mm +/- 1.25 mm, respectively with statistic difference between S4 and S1 (P=0.03). The width of the spleen vein of S1, S2, S3, and S4 were 6.518 mm +/- 2.033 mm, 7.190 mm +/- 1.569 mm, 7.444 mm +/- 1.805 mm and 8.406 mm +/- 2.227 mm, respectively with statistic difference between S4 and S2 (P=0.035). The incidence of tumefaction of the spleen was increased with the degree of liver fibrosis.</p><p><b>CONCLUSIONS</b>The diagnostic sensitivity of ultrasonic examination for early liver cirrhosis is low. The width of the main portal vein, the spleen vein and the incidence of tumefaction of the spleen are related with the degree of liver fibrosis. The regeneration node of liver cirrhosis may contribute to the development of portal hypertension.</p>

Tissue tropism of the TTV in experimentally infected rhesus monkeys / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To determine whether the transfusion-transmitted virus (TTV) is hepatotropic.</p><p><b>METHODS</b>Total DNA was extracted from various tissues of 5 experimentally infected Rhesus monkeys during the viremic period. A dot hybridization was done with viral double stranded DNA probes or single antisense probes.</p><p><b>RESULTS</b>The double-stranded probe was hybridized with DNA from the liver, bone marrow, spleen,stomach, small intestine and colon. The single-stranded antisense probe was hybridized with DNA from the liver, small intestine and bone marrow of all 5 monkeys, but not with that from other tissues.</p><p><b>CONCLUSIONS</b>As the viral genome was of negative polarity, the plus-stranded fragment identified in our study might be a replicative intermediate, and was only demonstrated in the liver, small intestine, and bone marrow by dot blot hybridization with single-stranded antisense probes. It is suggested that the TTV replicates in the liver, bone marrow and small intestine, and TTV might be hepatotropic.</p>

Influence of mutation in HBV precore region on the expression of HLA-I in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the influence of host cellular HLA-I molecules expression by HBV precore region mutants.</p><p><b>METHODS</b>The plasmids of the wild type of HBV precore region and the mutants of A83 and A83/A86 were constructed, and then transected into HepG2 cells. The biological activity of HBV precore gene in the host cells was identified. The expression of HLA-I molecules was detected by flow cytometric analysis.</p><p><b>RESULTS</b>DNA segments similar with HBV precore gene size and HBeAg were detected by PCR and ELISA, respectively. The fluorescence intensity of HLA-I on host cells was different: wild type being 1.3; A83, 17.6; and A83/A86, 7.3.</p><p><b>CONCLUSIONS</b>HBV precore gene hot-spot mutants in vitro can increase the expression of HLA-I molecules on host cells.</p>

Clinical and histological features of fibrosing cholestatic hepatitis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the clinical and histological characteristics of fibrosing cholestatic hepatitis (FCH) and the therapeutic effect of lamivudine.</p><p><b>METHODS</b>By retrospective analysis, 17 cases developed severe jaundice in 794 renal-transplanted recipients, and of them, FCH was clinically suggested in 11 and confirmed by liver biopsy in 6 cases.</p><p><b>RESULTS</b>The prevalence of chronic HBV infection in renal transplantation patients was 9.3%, of whom the FCH occurred in 22.9%. In 6 liver-biopsied cases, the onset was within 1.5-22 months. Two cases remitted who had early received lamivudine and 4 cases who were treated with the drug before transplantation did not develop the disease. All patients received large amounts of multiple immuno-suppressors after transplantation. About one fifth of HBV-infected cases gradually developed cholestatic hepatitis and some of them rapidly proceeded to hepatic failure. All had very high serum level of HBV DNA. The histology revealed unique lesion combination. The hepatocytes had widespread ballooning change and some ground-glass appearance. There were liver cytolysis and focal cell loss, bile stasis, periportal fibrosis, while only mild lymphocytic infiltration.</p><p><b>CONCLUSIONS</b>Fibrosing cholestatic hepatitis may happen following renal transplantation. Lamivudine has marked therapeutic effect for FCH.</p>

Cloning and implication of eukaryotic expression vectors containing hepatitis B virus genomes with partial deletion in the core promoter / 中华传染病杂志

Objective In order to further study the influence of a mutant on viral replication and transfection, a eukaryotic vector with mutation of 20/21 bp deletion (1748/ 1747 to nt 1767) in core promoter region and precore stop mutation (nt.1896) was constructed. Methods A linearized genome containing the entire HBV 3.5kb mRNA transcriptional units (P3.8Ⅰ vector) and initiating from the basic core promoter upstream sequences was used as a tool, the objective eukaryotic vectors were constructed by the molecular cloning and PCR based site directed mutagenesis in vitro. The capability of progeny virus production and transcription were examined with Southern blot and Northern blot analysis respectively, after transfection of the recombinant HBV plasmids into HepG2 cells by using liposome. Results The eukaryotic vectors were constructed successfully and their sequences were confirmed by clone sequencing. Both Southern and Northern blotting of DNA and RNA extracted from the transfected cells showed markedly reduced mutant activity to produce progeny virus, to transcript both 3.5kb precore/pregenome mRNA and 2.1kb preS/S mRNA. Conclusions The levels of replication and transcription are markedly reduced in the mutant compared with those in wild type HBV.

Construction of mutant gene of HBV and investigating its biological significance / 中华传染病杂志

Objective To study the biological significance of the common mutant preC/C gene in clinical HBV in China. Methods Site directed mutagenesis based on the unique enzyme site elimination was used to construct eukaryocyte expression vector with mutant HBV/C gene(V60、G87、L97). Expression vectors with wild and mutant preC/C gene were transferred into HepG2 cell. Culture supernatant was detected by ELISA for HBeAg. Results Result of DNA sequencing showed that the constructed mutant HBV preC/C gene had only one specific site variation compared with the wild type sequence. Goal DNA fragment was detected positive in the HepG2 cells transferred with wild and mutant preC/C gene. A value of HBeAg in the supernatant of the cells harboring L97 variant was higher than that of the wild and other variant strains( P

ANALYSIS OF X GENE MUTATIONS IN HEPATITIS B VIRUS GENOME POSITIVE FOR ANTI-HBE BETWEEN ASYMPTOMATIC CARRIER AND PATIENTS WITH CHRONIC ACTIVE HEPATITIS / 解放军医学杂志

To elucidate whether the mutations in X region of hepatitis B virus (HBV) might be responsible for the different clinical profiles in cases positive for antibody to hepatitis B e antigen. The nucleotide sequences of X gene regions in serum HBV were examined in 14 asymptomatic carriers (AsC) and 14 chronic active hepatitis (CAH) patients with antibody to hepatitis e antigen. The results showed that 12 of 14 AsCs (85.7%) had insertions, deletions or point mutations in nucleotide sequence of X region resulting in truncation of the X protein by creating frame shift mutation or a new stop codon, whereas no patient with CAH had those X gene mutations( P

Relationship between the expression of B7-1 in liver tissue and the effect of interferon-alpha treatment in patients with chronic hepatitis B / 中国实用内科杂志

Objectives To evaluate the relationship between the expression of B7-1 in liver tissue and the effect of interferon-alpha(?-IFN) treatment in patients with chronic hepatitis B(CHB).Methods The expression of B7-1 in liver biopsy specimens from 68 CHB patients was studied with immunohistochemistry before ?-IFN treatment.Results B7-1 was expressed in 45(66 2%) liver tissues among 68 patients with CHB,but none in 5 normal controls.The total response ratio of ?-IFN in patients with B7-1 positive was 66 7%(30/45),which was significantly higher than 39 1%(9/23)in the patients with B7-1 negative (? 2=7 20,P

PRELIMINARY STUDY ON BIOLOGICAL SIGNIFICANCE OF HBV POST-TRANSCRIPTIONAL REGULATORY ELE- MENTS POINT MUTATION / 解放军医学杂志

To explore the relationship between HPRE mutations and noncytolytic anti-HBV infection, the objective eukaryotic expression vectors were constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identification was performed using PCR and sequencing analysis. The results showed that eukaryotic expression vectors containing HPRE segment and mutating point were constructed successfully as confirmed by sequencing analysis. The activity of CAT gene obviously increased in the T to C mutation at nt 1504 of HPRE and no alteration in the C to T(G) at nt 1508. The mutation at nt 1508 of HPRE may escape the suppression role of IFN-?on HPRE. These results suggested that the mutation of HPRE might be affected the function of HPRE and influence the regulative function of IFN-? on HPRE, but not of 1FN-? nor of TNF-?.

Relationship between the expression of B7-1 in liver tissue and the effect of interferon-alpha treatment in patients with chronic hepatitis B / 中国实用内科杂志

Objectives To evaluate the relationship between the expression of B7-1 in liver tissue and the effect of interferon- alpha(α- IFN) treatment in patients with chronic hepatitis B(CHB).Methods The expression of B7 -1in liver biopsy specimens from 68 CHB patients was studied with immunohistochemistry before α-IFN treatment.Results B7-1 was expressed in 45(66.2%) liver tissues among 68 patients with CHB,but none in 5 normal controls.The total response ratio of α- IFN in patients with B7-1 positive was 66.7%(30/45),which was significantly higherthan 39.1%(9/23)in the patients with B7-1 negative(x2 =7.20,P <0.01).B7-1 expression was closely corelat-ed with the histological activity grade(HAI) and serum alanine transaminase(ALT) level.Conclusions The level of B7-1 expression in liver tissue may be regarded as an effective parameter for predicting α-IFN response in patients with CHB.

Construction of hepatitis B virus C gene retroviral vector and its expression in immortalized human peripheral blood B cell lines / 中华传染病杂志

Objective In order to study the biological significance of HBV/C gene variant in vitro.Methods Retroviral vector pXT1 was used to construct the HBV/C gene expression vector and the recombinant plasmid pXT1-HBV/C was used to transfect immortalized human peripheral blood B cell lines to express HBcAg steadily in the host cells.Results plasmid pXT1-HBV/C was detected positive by PCR as well as enzyme digestion with Bgl Ⅱ and Xho Ⅰ.Meanwhile.transfected cells was detected to contain HBV/C gene by PCR and HBcAg was expressed in 47.4%of the ceils by means of flow cytometry.Conclusion Retroviral expression vector with HBV/C gene can transfeet into eucaryotic cells effectively and express the goal gene steadily.The recombinant cells may be used in the systematic studies on the biological significance of HBV/C gene variant.

THE ESTABLISHMENT AND PRELIMINARY USE OF D-GALACTOSAMINE CYTOTO- XICITY MODEL OF PRIMARILY CULTURED HEPATOCYTES / 解放军医学杂志

A cytotoxicity model of primary cultured hepatocytes with D-galactosamine (D-Gal) has been established. The ALT value of culture medium was used as the marker of cell injury. Using this model, the in vitro antihepatotoxic effects of hepatic stimulator substance (HSS), isolated from regenerating adult rat liver, as well as Potenlin and 15-amino acid solution were observed. The results showed that although HSS could stimulate DNA synthesis in serum-free culture of adult rat hepatocytes, it had no effect to reduce ALT value in vitro. However the latter could be reduced by Potenlin or 15-amino acid solution in high concentrations. The described model may be useful for preliminary screening of antihepatotoxic activity of drugs.

ANTI-HBV EFFECT OF GALACTOSIDES MODIFIED LAMIVUDINE TARGETED TO LIVER / 解放军医学杂志

The aim of the present study was to investigate the inhibitory effect of galactosides modified lamivudine (LA) on hepatitis B virus (HBV) and examine the liver targeting ability of lamivudine modified by galactosides in vitro and in vivo (mice). Lamivudine nanoparicles modified by galactosides (LAP GSLN) were prepared and delivered into 2.2.15 cells. After 10 days, hepatitis virus B e antigen (HBeAg) expression in 2.2.15 cells was detected by ELISA, and immune fluorescence levels of HBV DNA in the medium were examined by quantitative polymerase chain reaction (PCR). The cytotoxicity of LAP GSLN on 2.2.15 cells was observed as well. In the in vivo experiment, ten male mice were randomly divided into 2 groups: lap GSLN group (i.v.injection of LAP GSLN) and LA group (i.v.injection of LA). Lamivudine levels in serum, hepatic, renal, pulmonary, and splenic tissues were detected by reversed phase high performance liquid chromatography (RP HPLC). On the 6th day, the expression of HBeAg was found inhibited by LPA GSLN. HBV DNA replication was also inhibited by LAP GSLN on the 4th day. Hepatic LAP GSLN concentrations in LAP GSLN group were 3.3 fold higher that of the LA group. The above results suggested galactosides modified lamivudine could effectively inhibit the antigen expression and DNA replication of HBV, and it showed a high liver targeting ability in vivo .

CERTAIN ASPECTS REGARDING CHRONIC SUBCLINICAL HEPATITIS B VIRUS INFECTION / 解放军医学杂志

Infection rate of HBV in the high-risk population, such as dentists and the patients undertaking hemodialysis, was not higher than those in general population. The occurrence of the subclinical infection state might be resulted from hypo-responsiveness of humoral and cellular immunity to HBsAg. Of 257 cases of liver biopsy, 44% of them were diagnosed as chronic hepatitis of various categories. No relationship was found between the expression patterns of viral antigens and the inflammatory activity in the liver. The infection state was quite stable. HBeAg/anti-HBe seroconversion was not a turning point from replicative to nonreplicative phase of the virus. The individuals with intrahepatic integrated HBV DNA might be the genome carriers with sero-negative HBsAg. The above results illustrate the characteristics of hyporesponsive HBV infection in hyperendemic area in China.