The significance of Toll-like receptor 4 (TLR4) expression in patients with chronic hepatitis B.

PURPOSE: To investigate the importance of Toll-like receptor 4 (TLR4) expression on hepatocytes obtained from Chronic Hepatitis B patients as well as on hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines. METHODS: Expression of TLR4 in liver tissues was determined by immunohistochemistry in 75 patients with CHB and in10 healthy controls. The protein and mRNA 1eve1s of TLR4 in hepatocellular carcinoma HepG2 and HepG2.2.15 cells were measured by flow cytometry (FCM) and real-time quantitative PCR (RQ-PCR), and endotoxin triggered TNF-alpha secretion in HepG2 and HepG2.2.15 cells was evaluated by ELISA. RESULTS: TLR4 expressed mainly in the cytoplasm and some on cell membrane in hepatocytes. The staining scores of TLR4 expression in the liver tissues of patients with CHB were significantly higher than that of healthy controls. The liver tissues from patients with severe CHB expressed higher level of TLR4 than those from patients with mild CHB. Furthermore, the staining scores of TLR4 expression in the liver tissues of patients with CHB were positively correlated with the grading scores. Our results also showed that the mean fluorescence intensity and TNF-alpha secretion induced by endotoxin as well as the protein and mRNA 1eve1s of TLR4 in HepG2.2.15 cells were all significantly higher than those in HepG2 cells. CONCLUSION: TLR4 was up-regulated in the hepatocytes in patients with CHB. This indicates a potentially important interaction between TLR4 expression and the pathogenesis of CHB.

[Changes of RANTES levels in livers of patients with chronic hepatitis B: the clinical significance and the possible mechanisms].

OBJECTIVES: To study the relationship between intra-hepatic levels of regulated on activation, normal T-cell expressed and secreted (RANTES) and the disease severity and liver inflammatory degrees in patients with chronic hepatitis B and the possible mechanism of the changes of intra-hepatic levels of RANTES. METHODS: The expression of RANTES of the livers was studied using immunohistochemical stainings and morphometric quantitative measurements in liver specimens from 10 normal subjects and 64 patients with chronic hepatitis B with different degrees of liver inflammation and different clinical severity. The expressions of RANTES protein and mRNA in cell line HepG2, HepG2.2.15 and HepG2 treated with 10 ng/ml TNFa at different times were quantified by ELISA and one-step RT-PCR. RESULTS: The expression of RANTES of the livers in patients was significantly higher than that in the normal controls. Hepatic RANTES levels increased significantly and the increases were parallel to the increases of the severity of the hepatitis, from mild, moderate to severe hepatitis (the positive units were 3.7+/-1.5, 15.6+/-6.9, 24.0+/-4.0, 37.9+/-11.1, respectively) and from G0 degree to G4 degrees of liver inflammation (the positive units were 3.7+/-1.5, 15.0+/-5.7, 21.6+/-5.9, 30.3+/-8.2, 40.9+/-12.3, respectively). The expressions of RANTES protein and mRNA of HepG2.2.15 were higher than that of HepG2. RANTES protein and mRNA were induced in HepG2 by TNFa. CONCLUSION: RANTES may have an important role in the pathogenesis of chronic hepatitis B. The elevation of hepatic RANTES may be caused by hepatitis B virus and TNFa.

[Changes of Toll-like receptor (TLR) 2 and TLR4 on the peripheral blood mononuclear cells in patients with chronic hepatitis B and chronic severe hepatitis B].

OBJECTIVE: To study the changes of TLR2 and TLR4 on peripheral blood mononuclear cells (PBMCs) and their role in the pathogenesis of chronic hepatitis B and chronic severe hepatitis B. METHODS: The expressions of TLR2 and TLR4 on 10000 CD14+ PBMCs were determined by flow cytometry in 30 healthy controls, in 31 patients with chronic hepatitis B and in 30 patients with chronic severe hepatitis B. The level of serum tumor necrosis factor alpha (TNF alpha) was determined by ELISA. The differences of expression of TLR2 and TLR4 on PBMCs and serum TNFalpha among the three groups of study subjects were determined by Student-t test. The correlations between TLR2, TLR4 and TNF alpha were determined by linear correlation test. RESULTS: The values of mean fluorescence intensity (MFI) of TLR2 on PBMCs of the healthy controls, patients with chronic hepatitis B and patients with chronic severe hepatitis B groups were 21.5+/-2.7, 39.0+/-4.1, and 47.7+/-21.4; TLR4 of those groups was 2.3+/-1.1, 3.7+/-2.3, and 6.9+/-4.1. The serum TNF alpha(ng/L) of the respective groups was 53.8+/-38.1, 164.3+/-89.9, and 359.8+/-140.0. There was a gradual increase of these values from the group of healthy controls to the group of patients with chronic hepatitis B and patients with chronic severe hepatitis B. No significant positive correlations between TLR2, TLR4 and serum TNFalpha were found. CONCLUSION: TLR2 and TLR4 may have a role in the pathogenesis of chronic hepatitis B and chronic severe hepatitis B.

[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.

[Expression of anti-HBc single-chain variable fragment mediated by recombinant replication defective adenovirus in vitro].

OBJECTIVES: To define the expression of single-chain variable fragment (ScFv) against hepatitis B virus core protein (HBc) mediated by recombinant replication defective adenovirus carrying the anti-HBc ScFv gene in vitro and to define the activity of anti-HBc ScFv combining HBcAg. METHODS: The recombinant adenoviruses carrying anti-HBc ScFv gene generated by homologous recombination in bacteria and packaged in 293 cells were transfected into HepG2 cells, and the anti-HBc ScFv was detected using SDS-PAGE and Western blot. RESULTS: Green fluorescent protein (GFP) was observed in HepG2 cells after the transfection. SDS-PAGE displayed a protein strap about 2.7 x 10(4), and the result of Western blot displayed a positive reactive strap. CONCLUSION: Anti-HBc ScFv can be expressed in cells mediated by recombinant replication defective adenovirus carrying the anti-HBc ScFv gene.

Risk factors for primary liver carcinoma in Chinese population.

AIM: To evaluate the risk factors for primary liver carcinoma (PLC) in Chinese population. METHODS: Chinese Biomedical Literature Database, China Hospital Knowledge Database and MEDLINE were searched. All the related literatures were screened, and the risk factors for PLC in Chinese population were studied. Heterogeneity was evaluated by odds ratio (OR) q test. Combined OR and its 95% confidence interval (95%CI) were calculated, the association between the investigated risk factors and PLC was determined. Validity and bias of the findings were evaluated by sensitivity analysis and funnel plot analysis respectively. RESULTS: Fifty-five of one hundred and ninety identified studies were accepted according to the inclusive criteria. Ten factors related to PLC were demonstrated by sensitive analysis and funnel plot analysis. They were cirrhosis (OR = 11.97, P = 0.000), HBV infection (OR = 11.34, P = 0.000), HCV infection (OR = 4.28, P = 0.000), family history of liver cancer (OR = 3.49, P = 0.000), unstable emotion (OR = 2.20, P = 0.000), depressed characters (OR = 3.07, P = 0.000), aflatoxin (OR = 1.80, P = 0.000), alcoholic (OR = 1.88, P = 0.000), intake of musty food (OR = 1.87, P = 0.000) and drinking contaminated water from pond (OR = 1.77, P = 0.003). CONCLUSION: The main risk factors for PLC in China are liver diseases, family history of liver carcinoma, poor psychic status, aflatoxin, and some unhealthy behaviors.

[Clinical investigation of famciclovir in chronic hepatitis B patients irresponsive to alpha interferon treatment].

OBJECTIVES: To evaluate the efficacy and safety of famciclovir on the decreasing levels of serum HBV-DNA and ALT and HBeAg/antiHBe seroconversion in chronic hepatitis B patients irresponsive to 3 months treatment with alpha interferon. METHODS: Two hundred and nineteen patients with chronic HBV infection, defined as positive HBsAg, HBeAg and HBV DNA, were enrolled and randomly half-and- half put into famciclovir and placebo groups. The two groups received either famciclovir 500 mg tid or a placebo treatment for 24 weeks, and then were followed-up for another 24 weeks with no treatment. RESULTS: At the end of 24 weeks, the log value of HBV DNA dropped from 6.54+/-1.26 to 5.70+/-2.03 in the famciclovirt group and were elevated from 6.30+/-1.32 to 6.51+/-1.65 in the placebo group (P < 0.01). The rate of cases with persistence HBV DNA dropped 2 log of quantity in the famciclovir group and was 28.28% (28/99); it was 9.47% (9/95) in the placebo group (P < 0.01). Those with persistence negative HBV DNA was 28.28% (28/99) in the flamciclovir treated group and 14.74% (14/95) in the placebo group (P < 0.05). Those persistently being HBeAg negative were 7.69% (7/91) in the famciclovir treated group and 3.33% (3/90) in the placebo group (P > 0.05). The HBeAg/antiHBe seroconversion was 4.40% (4/91) in the famciclovir group and 2.22% (2/90) in the placebo group (P > 0.05). The percentage of cases with normal of ALT level was 15.15% in the famciclovir group and 6.35% in the placebo group (P < 0.05). CONCLUSION: Famciclovir is effective in inhibiting HBV DNA replication and in decreasing serum ALT levels. The rate of HBeAg/antiHBe seroconversion in the famciclovir treated group was similar to that of the placebo group. Famciclovir was well tolerated without severe adverse effects during our treatment.

Effects of c-myb antisense RNA on TGF-beta1 and beta1-I collagen expression in cultured hepatic stellate cells.

AIM: To investigate the effects of c-myb antisense RNA on cell proliferation and the expression of c-myb, TGF-beta1 and beta1-I collagen in cultured hepatic stellate cells (HSC) from rats. METHODS: Recombinant retroviral vector of c-myb antisense gene (pDOR-myb) was constructed, and then transfected into retroviral package cell line PA317 by means of DOTAP. The pseudoviruses produced from the resistant PA317 cells were selected with G418 to infect HSCs isolated from rat livers. The cell proliferation was measured by 3-[4, 5-Dimethylthiazolzyl]-2, 5-diphenyl tetrazo-dium bromide(MTT) method. The expression of c-myb, alpha (1)-I collagen and TGF-beta1 mRNA, and c-myb protein in HSCs was detected with semi-quantitive reverse transeription-polymerase chain reaction (RT-PCR) and Western-blot respectively. RESULTS: HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, the c-myb protein expression, cell proliferation,and alpha (1)-I collagen and TGF-beta1 mRNA expression were repressed significantly compared with their corresponding control groups (P<0.01). CONCLUSION: c-myb plays a key role in activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation, alpha (1)-I collagen and TGF-beta1 mRNA expression, suggesting that inhibition of c-myb gene expression might be a potential way for the treatment of liver fibrosis.

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.

Prevalence of hepatitis G virus infection and homology of different viral strains in Southern China.

AIM: To investigate the prevalence of hepatitis G virus (HGV) infection and to analyse the homology of different HGV strains in Southern China. METHODS: A total of 1993 sera from different groups in Guangdong, Hong Kong, and Yunnan were detected by reverse transcription polymerase chain reaction (RT-PCR). The nucleotide sequences of 5'untranslated region (5'UTR) derived from 20 strains and NS5 region from 3 strains were determined. RESULTS: The positive rate of HGV RNA was 0.89 % in community population, 2.57 % in blood donors, 17.86 % in intravenous drug abusers, 14.13 % in patients with hemodialysis, 13.66 % in those with hepatocellular carcinoma, 25.30 % in non A-E hepatitis, 7.22 % in hepatitis B, 12.73 % in hepatitis C, 41.67 % in patients received bone marrow transplantation, respectively. The homology was 90.40-100 % in 5'UTR among different strains, while that of NS5 region was 93.3-94 % in nucleotide sequence, and 97-99.2 % in amino acid sequence. CONCLUSION: These results showed that there was a high incidence of HGV infection in patients from Southern China, being treated for bone marrow transplantation, hepatocellular carcinoma and those on haemodialysis. Furthermore, there was also a high frequency of co-infection of HGV with HBV, HCV, non A-E viral hepatitis and that among intravenous drug abusers. The study also showed that sequence variation in different strains was associated with geographical factors but there was no significant difference in 5'UTR in circulating viruses between different patient groups. Finally, by sequential analysis of viral species present in individual patients over a three months period there was no evidence of sequence variation in the 5' UTR.

Liver fibrosis in chronic viral hepatitis: an ultrasonographic study.

AIM: To select valuable ultrasonographic predictors for the evaluation of hepatic inflammation and fibrosis degree in chronic hepatitis, and to study the value of ultrasonography in the evaluation of liver fibrosis and compensated liver cirrhosis in comparison with serology and histology. METHODS: Forty-four ultrasonographic variables were analyzed and screened using color Doppler ultrasound system in 225 patients with chronic viral hepatitis and compensated liver cirrhosis. The valuable ultrasonographic predictors were selected on the basis of a comparison with histopathological findings. The value of ultrasonography and serology in the evaluation of liver fibrosis degree and the diagnosis of compensated liver cirrhosis was also studied and compared. Meanwhile, the influencing factors on ultrasonographic diagnosis of compensated liver cirrhosis were also analyzed. RESULTS: By statistical analysis, the maximum velocity of portal vein and the degree of gall-bladder wall smoothness were selected as the valuable predictors for the inflammation grade (G), while liver surface, hepatic parenchymal echo pattern, and the wall thickness of gall-bladder were selected as the valuable predictors for the fibrosis stage (S). Three S-related independent ultrasonographyic predictors and three routine serum fibrosis markers (HA, HPCIII and CIV) were used to discriminate variables for the comparison of ultrasonography with serology. The diagnostic accuracy of ultrasonography in moderate fibrosis was higher than that of serology (P<0.01), while there were no significant differences in the general diagnostic accuracy of fibrosis as well as between mild and severe fibrosis (P<0.05). There were no significant differences between ultrasonography and serology in the diagnosis of compensated liver cirrhosis. However, the diagnostic accuracy of ultrasonography was higher in inactive liver cirrhosis and lower in active cirrhosis than that of serology (both P<0.05). False positive and false negative results where found when the diagnosis of compensated liver cirrhosis was made by ultrasonography. CONCLUSION: There are different ultrasonographic predictors for the evaluation of hepatic inflammation grade and fibrosis stage of chronic hepatitis. Both ultrasonography and serology have their own advantages and disadvantages in the evaluation of liver fibrosis and compensated liver cirrhosis. Combined application of the two methods is hopeful to improve the diagnostic accuracy.

A 3-year clinical trial of lamivudine in treatment of patients with chronic hepatitis B.

BACKGROUND: Lamivudine was approved for the treatment of chronic hepatitis B in China in 1999; however the long-term result has not yet been reported in detail. This clinical trial was to evaluate the long-term efficacy and safety of 3-year lamivudine treatment for chronic hepatitis B and the impact of emergence of YMDD mutation of hepatitis B virus (HBV). METHODS: This multi-center, randomized, double-blind, placebo controlled trial began from 1996 to 1999. A total of 429 patients with serum HBsAg, HBeAg and HBV DNA positive were randomized to receive either lamivudine 100 mg daily (322 patients) or placebo (107) for the first 12 weeks. All patients were given subsequently open labelled lamivudine 100 mg/d for a total of 156 weeks. RESULTS: After 12-week lamivudine therapy, the levels of serum HBV DNA decreased rapidly. The negativity of HBV DNA (<1.6 pg/ml) at week 12 was 92.2% in the lamivudine group, whereas it was only 14.1% in the placebo group (P<0.01). After 1-year lamivudine treatment, 72.7% of the patients showed undetectable serum HBV DNA (<1.6 pg/ml). At the end of 3 years, serum HBV DNA continued to be substantially suppressed with a median level below a detectable level in patients with non-YMDD variant HBV, which was increased to 86 mEq/ml (bDNA method, equivalent hybridization method 10 pg/ml) in patients with YMDD mutation. At the end of 1, 2 and 3 years, the rates of HBeAg loss were 9.5%, 16.8% and 20.0% respectively and the rates of HBeAg/anti-HBe seroconversion were 8.3%, 11.5% and 17.3%. The rates of HBeAg loss and seroconversion were correlated with the baseline level of ALT. In patients with a baseline level of alanine transaminase (ALT)>2 x upper limit of normal (ULN) and ALT >5xULN, the rates of HBeAg loss were 42.2% and 66.7%, and the rates of seroconversion were 34.4% and 61.1% respectively (P<0.01) at the end of year 3. The levels of ALT at year 3 remained normal in 58.8% of patients whose baseline level of ALT was elevated, and in 79.1% of patients whose level of ALT was normal before treatment. YMDD mutations occurred in 12.1%, 49.7% and 70.5% of patients respectively at year 1, 2 and 3. In patients with YMDD mutation, the levels of HBV DNA were increased slightly with mild to moderate elevation of ALT level. HBeAg loss and seroconversion were 20.0% and 15.1% in patients with YMDD mutation at the end of year 3, which were lower than those in non-variant patients (P<0.01). Adverse drug reactions or events varied generally from mild to moderate. In 2 patients serious adverse events (fatigue and abdominal distension) were related to medication. ALT flares (ALT>5xULN) occurred in 17 patients: 10 were YMDD mutants and 7 were non-mutants; all of them were relieved. No death occurred in the period of 3 years. CONCLUSION: Sustained inhibition of HBV replication and clinical improvement could be obtained after 3-year lamivudine therapy of good tolerance and safety.

The levels of serum fibrosis marks and morphometric quantitative measurement of hepatic fibrosis.

OBJECTIVE: To study the relationship between the serum levels of hyaluronic acid (HA), procollagen type III (PCIII), collagen type IV (CIV) and the histological degree of hepatic fibrosis evaluated by image analysis, and the clinical significance of serum HA, PCIII, CIV in the diagnosis of hepatic fibrosis in patients with chronic viral hepatitis. METHODS: The concentrations of serum HA, PCIII, CIV in 151 patients with chronic viral hepatitis were measured by radioimmunoassay. Liver biopsies were performed in all the patients. Histological sections of 4 microm thickness were stained with Masson's trichrome for fibrosis assessment. Morphometric quantitative measurements for hepatic fibrosis assessment in the 4 microm sections were performed using a fully automated image analysis system. Serum levels of HA, PCIII, and CIV were analyzed at different stages of liver pathology and compared with the morphometric quantitative measurements of hepatic fibrosis. RESULTS: The serum levels of HA, PCIII, CIV all elevated gradually with the progression of the disease, and all reached the highest in patients with liver cirrhosis. There was a significant difference in the levels of these 3 components between liver cirrhosis group and the other groups (P<0.05). They all increased steadily with the histological stages of hepatic fibrosis, and reached the highest levels in stage IV. The serum levels of HA, PCIII, CIV were all positively correlated with the histological stages of liver sections and the morphometric measurement (P<0.001). The coefficients with stages were 0.694, 0.493, 0.552 (P<0.001), respectively and with surface density of total collagen on liver biopsy sections by image analysis were 0.715, 0.595, 0.573 (P<0.001), respectively. CONCLUSION: The serum levels of HA, PCIII, CIV were in consistent with the degree of hepatic fibrosis, and the determination of these marks is valuable for detecting hepatic fibrosis.

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.

Promoting hepatic growth factor in the treatment of heavy type hepatitis and severe chronic hepatitis: a multicenter clinical study.

BACKGROUND: The mortality rate of heavy type hepatitis is high. No special treatment is available except general treatment. This multicenter clinical study was designed to observe the safety and efficacy of promoting hepatic growth factor (PHGF) in the treatment of heavy type hepatitis and severe chronic hepatitis. METHODS: 347 patients with heavy type hepatitis and 324 with severe chronic hepatitis were subjected to administration of 120 microg of PHGF per day for 4 weeks on the basis of general treatment. Those who were being effectively treated would last additional 2 to 4 weeks. Blood routine, urine routine, blood urea nitrogen (BUN), blood creatinine (Cr), blood ammonia, alpha fetoprotein (AFP), electrolyte, alanine transaminase (ALT), aspartate transaminase (AST), serum total bilirubin (TBIL), serum direct bilirubin (DBIL), prothrombin time activity (PTA), total protein (TP) and albumin (ALB) were detected in the patients before treatment, 2 weeks after treatment, and at the end of the treatment. Any side-effect would be recorded. RESULTS: In the patients with severe chronic hepatitis, the total effective rate of the treatment was 88.9%. The levels of ALT, AST and TBIL decreased significantly (P<0.001), whereas those of PTA and ALB increased significantly (P<0.001), and the level of AFP increased slightly. In patients with heavy type hepatitis, the total effective rate of this treatment was 78.4%, and patients at different stage showed different results. The total effective rates of patients with early, medium and terminal stage heavy type hepatitis were 89.9%, 84.8% and 27.5%, respectively. No severe side-effect was shown. CONCLUSION: PHGF is effective and safe in the treatment of patients with heavy type hepatitis and severe chronic hepatitis. But it should be administered early in patients with heavy type hepatitis so as to get better curative effects.

[CpG oligodeoxynucleotide enhances humoral and cellular immune responses against HBsAg in mice].

OBJECTIVE: To induce stronger humoral and cell mediated immune response against HBsAg and seek more effective methods to treat hepatitis B virus infection. METHODS: 21-mer phosphorothioate oligodeoxynucleotide (ODN) and its control ODN were synthesized and used as an adjuvant of HBsAg and commercial hepatitis B vaccine to enhance their immune responses against HBsAg. Four groups of 33 mice received 2 doses of the mixtures (1.67 microg HBsAg: 16.5 microg CpG ODN) at an interval of 15 days. Group A: HBsAg only, 7 mice; group B: HBsAg + CpG ODN, 10 mice; group C: commercial hepatitis B vaccine, 6 mice; group D: commercial hepatitis B vaccine + CpG ODN, 10 mice. Serum anti-HBs levels and cytotoxic T lymphocyte response (CTL) of splenocytes were assayed 15 days after the last vaccination. RESULTS: The mean absorbent values of blood anti-HBs in the 4 groups were 0.109, 0.435, 0.422 and 0.575, respectively. The rates of CTL response were 8.5%, 37.0%, 1.5%, and 28.0%, respectively. CONCLUSIONS: (1) CpG ODN markedly improves humoral (anti-HBs) and cellular immune response (CTL) induced by HBsAg immunization. (2) There is a synergistic interaction between aluminum and CpG ODN in enhancing the humoral and cellular immune responses against HBsAg. Therefore, CpG ODN might be used to treat chronic hepatitis B virus infection after its safety and effectiveness have been confirmed.

[The long-term efficacy of lamivudine in chronic hepatitis B: interim analysis of 3-year's clinical course].

OBJECTIVE: To evaluate the long-term efficacy and safety of 3-year lamivudine treatment for chronic hepatitis B and the impact of emergence of YMDD mutation of hepatitis B virus (HBV). METHODS: This multi-center, randomized, double-blind, placebo-controlled trial began in 1996. A total of 429 patients with serum HBsAg, HBeAg and HBV DNA positive were randomized to receive either lamivudine 100 mg daily (n = 322) or placebo (n = 107) in a 3:1 ratio for the first 12 weeks. Thereafter, all patients were offered open label lamivudine 100 mg/d for a total of 156 weeks. RESULTS: After 12 weeks of lamivudine treatment, serum HBV DNA levels decreased rapidly; at week 12 the negativity of HBV DNA (< 1.6 pg/ml) was 92.2%, whereas it was only 14.1% (P < 0.01) in the placebo group. After 1 year of lamivudine treatment, in 72.7% of the patients serum HBV DNA was undetectable (< 1.6 pg/ml). At the end of 3 years, serum HBV DNA continued to be substantially suppressed; the median level was below detectable level in non-YMDD variant patients and was increased to 10 pg/ml in YMDD variant patients. At the end of 1, 2 and 3 years, the HBeAg loss rates were 9.5%, 16.8% and 20.0% respectively; and the HBeAg/anti-HBe sero-conversion rates were 8.3%, 11.5% and 17.3% respectively. The rates of HBeAg loss and seroconversion correlated with baseline ALT levels, in patients with baseline ALT > 2ULN and ALT > 5ULN, the loss of HBeAg was 42.2% and 66.7%, sero-conversion rates were 34.4% and 61.1% respectively (P < 0.01) at the end of year 3. ALT levels at year 3 remained normal in 58.8%, and below baseline in 79.1% of the patients whose ALT were abnormal before treatment. YMDD mutations developed in 12.1%, 49.7% and 70.5% of the patients respectively at year 1, 2 and 3. HBV DNA levels were increased slightly or moderately and accompanied with elevation of ALT. HBeAg loss and sero-conversion could be achieved in YMDD variant patients to 20.0% and 15.1% at the end of year 3, but lower than that in non-variant patients (P < 0.01). The adverse drug reactions or events were generally mild to moderate, 2 patients were reported to have serious events related to the study medication. ALT flares (ALT > 5ULN) occurred in 17 patients, 10 with YMDD variants and 7 with non-variants, but all resolved. No deaths were reported in the 3 year treatment period. CONCLUSION: Sustained HBV replication and clinical improvement could be obtained by 3-year long-term Lamivudine therapy with good tolerance.

[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.

[Reciprocal priming-boosting role of HBsAg and DNA vaccines].

OBJECTIVE: To evoke more effective humoral and cell-mediated immunization against hepatitis B virus (HBV) infection. METHODS: HBsAg-primed mice were boosted with HBs-DNA vaccine, and HBs-DNA-primed mice were boosted with HBsAg vaccine. Anti-HBs level was assayed by ELISA and cytotoxic T lymphocyte (CTL) response was tested by lactic acid dehydrogenase (LDH) releasing method two weeks after the boosted immunization. RESULTS: Anti-HBs level and CTL responsive rate at the effector/target cell ratio of 100:1 were 0.38 and 36% in HBsAg/HBs-DNA vaccination group, 0.32 and 27% in HBs-DNA/HBsAg vaccination group, 0.48 and 1.5% in HBsAg/HBsAg vaccination group, 0.24 and 68% in HBs-DNA/HBs-DNA vaccination group, respectively. CONCLUSION: Priming with HBs-DNA vaccine followed by boosting with conventional HBsAg vaccine results in greater antibody response (F = 21.19, P < 0.05), and CTL response after HBsAg vaccination can be improved by boosting with HBs-DNA vaccine (F = 165.59, P < 0.05). It brings to better efficacy by combining HBsAg vaccine with HBs-DNA vaccine.

[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.