Immune response pattern varies with the natural history of chronic hepatitis B.

BACKGROUND: Chronic hepatitis B is a highly heterogeneous disease that can be divided into four phases: Immune tolerant (IT), immune active (IA), inactive carrier (IC) and hepatitis B envelope antigen (HBeAg)-negative hepatitis (ENEG). AIM: To investigate the immune status of natural killer (NK) and T cells in different phases of chronic hepatitis B. METHODS: The frequency, phenotype and function of circulating NK cells, as well as nonantigen-specific and hepatitis B virus (HBV)-specific T cell responses were detected by flow cytometry in healthy and HBV-infected subjects. RESULTS: The ability of NK cells to produce IFN-γ was markedly attenuated in HBV-infected patients overall but was less compromised in IC patients. Patients in the IT and IA phases also displayed significantly lower TNF-α production compared to healthy subjects. NK cells were phenotypically activated in the IA and ENEG phases, as evidenced by the upregulation of NKp44 in CD56bright NK cells and CD69 in CD56dim NK cells. Furthermore, global T-cells from the ENEG phase displayed a proinflammatory cytokine profile with upregulated IFN-γ and TNF-α expression, while this profile was suppressed in IT and IA patients. Finally, core and S antigen-specific T cell responses were significantly stronger after in vitro expansion in the IC phase compared to other phases. CONCLUSION: Our findings demonstrate the changes in immune response pattern during the natural history of HBV infection. Both NK and T cells are functionally impaired in the IT and IA phases. With the spontaneous clearance of HBeAg and hepatitis B surface antigen decline, NK cell cytokine production and HBV-specific T responses are partially restored in IC phase, and the ENEG phase is dominated by nonantigen-specific T cell responses.

Decreased peripheral natural killer cells activity in the immune activated stage of chronic hepatitis B.

BACKGROUND & AIMS: The natural course of chronic hepatitis B virus (HBV) infection is characterized by different immune responses, ranging from immune tolerant (IT) to immune activated (IA) stages. In our study, we investigated the natural killer (NK) cells activity in patients at different immunological stages of chronic HBV infection. METHODS: Blood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB), including 15 patients in the immune tolerant (IT) stage, 42 patients in the immune activated (IA) stage, and 18 healthy individuals (HI). The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity. RESULTS: NK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (p<0.05). Patients in the IA stage of CHB had lower levels of NK cells activating receptor NKp30 and NKG2D expression, cytokine interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production, as compared to patients in the IT stage and HI, respectively (p<0.05). Cytotoxicity of NK cells was lower in patients in the IA stage of CHB compared to patients in the IT stage and HI, respectively (p<0.05). The level of IFN-γ but not level of TNF-α and cytotoxicity of NK cells was inversely correlated with serum HBV load in patients with CHB. Peripheral NK cells activity did not correlate with ALT level. CONCLUSION: NK cells activity was lower in CHB patients, especially in those in the IA stage.

[Characterization of HBsAbs in occult hepatitis B virus infection].

OBJECTIVE: To investigate the properties of HBsAb in occult hepatitis B virus infection and its affinity to different serotypes of hepatitis B virus surface antigen (HBsAg). METHODS: Long-term follow-up was conducted in 2 HBsAb positive patients with occult hepatitis B virus infection. HBsAg was detected using multiple diagnostic kits and the HBsAb subtype was determined by performing neutralization experiments with different serotypes of HBsAg. The viral S gene was PCR-amplified and mutation analysis was conducted. Plasmids expressing HBsAgs were constructed by inserting these PCR products into an eukaryotic expression vector and were then transfected into HepG2 cells. The cell culture supernatant and cellular extracts were detected for HBsAg respectively. Neutralization experiments were carried out in the cell culture supernatant from HBsAg plasmids transfected HepG2 cells and serum samples from these patients and others who had been confirmed to be positive for HBsAb. RESULTS: Multiple tests using various diagnostic kits showed that the 2 patients were negative for HBsAg and the three different serotypes of HBsAg (adr, adw, ay) could neutralize 82.1%-100% of HBsAb existed in the 2 patients. Sequence analysis of S gene cloned from these patients revealed that the homology to reference strain were 95.13%-97.79% and 92.04%-95.58% respectively at the nucleotide and amino acid levels. Quantitation of HBsAg showed that the expression levels of HBsAg from the two patients were 41.1% and 22.6% respectively of that of control HBsAg in cell culture supernatant and 48.1% and 59.3% respectively in cellular extract, and the supernatant/cell lysate ratios were 0.85 and 0.38 respectively. In neutralization experiments, HBsAg could be totally absorbed by control serum, whereas could only be partially neutralized by HBsAbs from the two patients (F = 353.6 and 645.2, P is less than 0.01). CONCLUSION: Both the antigenicity and the ability of HBsAg secreted outside of the cells are decreased in these HBsAb-positive patients with occult HBV infection. The HBsAbs are mainly specific for common epitopes among different serotypes of HBsAg and are probably different as compared with those produced by vaccine inoculation.

[The immunization effects of HBV core antigen and surface antigen fusion protein in mice].

OBJECTIVE: To evaluate the immunization effects of HBV core antigen and surface antigen fusion protein. METHODS: The DNA fragments encoding HBsAg 100-162 aa; HBcAg 1-78 aa and HBcAg 83-144 aa were PCR-amplified, and then cloned into pcDNA3 plasmid. The chimeric gene was subcloned into the prokaryotic vector, pRSET-B. The E.coli expressed recombinant protein purified. BALB/c mice were immunized with recombinant protein or eukaryotic expression plasmid, humoral response and cellular response were examined. RESULTS: The plasmid containing the chimeric gene of HBsAg and HBcAg induced effective anti-HBs antibody response and strong HBcAg specific lymphocyte proliferative response, but could not induce anti-HBc antibody response. Fusion protein induced strong anti-HBs and anti-HBc antibody response, and it also caused significant HBcAg specific lymphocyte proliferation. Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg can induce more effective cellular response but weaker humoral response. CONCLUSION: Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg is a more effective vaccine.

Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81.

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

[Prokaryotic expression and polyclonal antibody preparation of truncated duck hepatitis B virus core protein].

AIM: To construct a prokaryotic plasmid expressing truncated duck hepatitis B virus core protein (DHBc(1-214)), purify the recombinant protein, and to develop polyclonal antibodies against DHBc. METHODS: DHBc(1-214) was cloned into vector pRSET-B, then expressed in E.coli Rosetta(DE3) pLacI induced by IPTG. The recombinant protein was purified using Ni-NTA spin column. Polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis. RESULTS: Recombinant plasmid expressing truncated DHBc(1-214) was successfully constructed. A protein of 28,000 was expressed and purified. Polyclonal serum antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. CONCLUSION: The truncated recombinant DHBc(1-214) developed in this study is purified and shown strong antigenecity. The polyclonal antibody against DHBc protein is generated by regular immunization method, demonstrating both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of DHBV.

[Genetic evaluation of Toxicodendron vernicifluum cultivars using amplified fragment length polymorphism markers].

Accurate identification of Toxicodendron vernicifluum varieties and understanding their genetic relationships are essential for the improved varieties selection. In this study, Amplified fragment length polymorphism (AFLP) markers was used to evaluate the genetic resources of nine Toxicodendron vernicifluum cultivars from Shaanxi. Eight pairs of AFLP primers with Mse I fluorescent labeled were screened from EcoR I /Mse I primer combinations. Different pair of primers differs widely to detect genetic polymorphism of Toxicodendron vernicifluum. The polymorphic bands detected by each pair of primers ranged from 242 to 622 with average value of 350.88. Eight pairs of AFLP primers produced 4463 bands in total. Among them, 2807 bands (62.64%) were polymorphic. genetic similarity coefficient (SC) among all cultivars varied from 0.673 to 0.833, which further verified that only a little genetic diversity existed among the cultivars. It was the first time that AFLP technique was applied to identify Toxicodendron vernicifluum cultivars. The study suggested that AFLP technique is a useful tool for individual identification of Toxicodendron vernicifluum cultivars and could be applied to the Toxicodendron vernicifluum' fingerprinting work in China.

Expression of PTEN, PPM1A and P-Smad2 in hepatocellular carcinomas and adjacent liver tissues.

AIM: To investigate the expressions of PTEN, PPM1A and P-Smad2 in hepatocellular carcinoma (HCC) and their significance. METHODS: The expressions of PTEN, PPM1A and P-Smad2 in 31 HCC tissues, 25 adjacent liver tissues and 13 non-tumor liver tissues were detected by using Envision immunohistochemical technique. RESULTS: The positive expression (64.52%) and staining intensity (4.19 +/- 3.31) of PTEN in the cytoplasm of HCC were significantly lower and weaker than those in the adjacent or non-tumor liver tissues (97.37%, 7.88 +/- 0.93; 100%, 7.77 +/- 0.93, respectively) (P < 0.05), and its staining intensity in the cytoplasm of HCC, which belongs to Edmondson pathologic grades II-III and above, was also lower than that of grade I and I-II. Furthermore, its location in the nucleus or cytoplasm of liver cells was negatively correlated with the progression of liver disease (r = -0.339, P = 0.002); most of PPM1A might be only expressed in the nucleus of adjacent liver tissues, non-HCC tissues or Edmondson grade I and I-II HCC, but it was mainly expressed in the cytoplasm of HCC with Edmondson grade > or = II, weakly or negatively expressed in the nucleus (P < 0.05), and its location was negatively correlated with the progression of liver disease (r = -0.45, P = 0.0000). P-Smad2, which was mostly located in the nucleus and cytoplasm of grade I and I-II HCC, surrounding or non-tumor liver tissues, was only in the nucleus of HCC with Edmondson grade II and above (P < 0.001), and its location was positively correlated with the disease progression (r = 0.224, P = 0.016). Spearman correlation analysis revealed that P-Smad2 was significantly negatively correlated with PTEN and PPM1A (r = -0.748, P = 0.000; r = -0.366, P = 0.001, respectively); and PTEN and PPM1A were positively correlated with HCC carcinogenesis (r = 0.428, P = 0.000). CONCLUSION: The aberrant location of expression and staining intensity of PTEN, PPM1A and P-Smad2 in HCC and their relationship might have an impact on the pathogenesis of HCC.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo.

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

[Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma].

OBJECTIVES: To construct a prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1(167-360)), to express and purify the recombinant protein, and to develop the polyclonal antibody against HCCR. METHODS: HCCR-1(167-360) was amplified by RT-PCR from HepG2 cells and cloned into vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS, which was induced by IPTG. The recombinant protein was purified using Ni-NTA spin column and acrylamide gel electrophoresis. A polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis. RESULTS: Recombinant plasmid expressing truncated HCCR-1167-360 was constructed. A protein of 2.70 x 10(4) was successfully expressed and purified. High titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. CONCLUSIONS: The truncated recombinant HCCR-1(167-360) developed in this study is highly purified and shows strong antigenecity; the polyclonal antibody against this HCCR protein was generated by regular immunization method, showing both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of HCCR.

[Infection of tupaia hepatocytes with hepatitis C virus in vitro].

OBJECTIVE: Tupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro. METHODS: Serum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage. RESULTS: Both positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage. CONCLUSION: The present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.

[Distribution of hepatitis B virus genotypes in Hubei province and its clinical significance].

OBJECTIVE: To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance. METHODS: Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers. RESULTS: A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group. CONCLUSION: The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.

Alternation of AFP-mRNA level detected in blood circulation during liver resection for HCC and its significance.

AIM: To investigate whether the current manipulation of liver resection for hepatocellular carcinoma (HCC) causes dissemination of liver tumor cells into blood circulation. METHODS: Fifty patients with HCC, but without evidences of metastases, were enrolled for the study. Samples of peripheral blood before skin incision and after abdominal wall suture, and those of hepatic venous blood and portal venous blood after liver parenchyma dissection,were obtained. AFP-mRNA was extracted from the blood samples and detected with reverse transcription polymerase chain reaction assay. The changes of its expression during the operation were accessed by the ratio of fluorescence of AFP and beta-actin primers-amplified products. RESULTS: The rate of its expression before and after operation in peripheral blood, and during operation in portal venous and hepatic venous blood was 42.0 %, 34.0 %, 42.0 % and 56.0 % respectively, There was no difference between them. However, the ratio of fluorescence of AFP and beta-actin primers-amplified products from hepatic venous blood during operation is significantly higher than the other three types of samples (P<0.05). CONCLUSION: the current manipulation of liver resection for HCC can cause the releases of AFP mRNA-expressed cells from the liver, probably including tumor cells, into hepatic vein. These cells may spread into blood circulation and become the source of recurrence after operation.

FADD and TRADD expression and apoptosis in primary hepatocellular carcinoma.

AIM:To investigate the clinical features of FADD and TRADD expressions in primary hepatocellular carcinoma (HCC) and to determine their relationship with hepatic apoptosis.METHODS:FADD and TRADD expressions were detected by immunohis-tochemistry and hepatic apoptosis were determined by in situ end-labeling (ISEL).RESULTS:Ten (25.6%) cases of HCC were detected to express FADD protein. The positive rate in HCC is lower than that in non-cancerous adjacent liver tissues (62.5%) (P < 0.05). In those of grade I-II, 8(38.1%) cases were FADD positive, while only 2/18 (11.1%) cases of grade III-had detectable FADD protein (P < 0.05). No relationship was found between FADD expression and other clinical features, such as gender, age, tumor size, different-tiation or metastasis. ISEL positive cells can be seen in all cases of HCC. The hepatic apoptosis was associated with FADD expression as more apoptotic cells were detected in those cases which had moderately to strongly positive FADD, as compared with negative or weak positive FADD cases (P < 0.05). No relationship was found between FADD expression and hepatic apoptosis in non cancerous adjacent liver tissues. Fifteen of 39 (38.5%) cases of HCC were found positive for TRADD prote-in, and similar positive rate (37.5%) in non-cancerous adjacent liver tissues (P > 0.05). The expression of TRADD is correlated with HCC differentiation, as only 22.2% of moderately to highly differentiated HCC showed positive TRADD protein, while as high as 52.4% of poorly differentiated HCC had TRADD (P < 0.05). No relationship was found between TRADD expression and gender, age, tumor size or grade or metastasis, although 42.9% of HCC of grade I/II showed positive TRADD which was slightly higher than that of grade III/IV (33.3%,P>0.05). Hepatic apoptosis was not related to TRADD expression in HCC or non-cancerous adjacent liver tissues.CONCLUSION:Loss of FADD expression plays an important role in HCC carcinogenesis, and expression of TRADD also contributes to HCC development. The cell apoptosis in HCC is associated with FADD expression. However, the expression of TRADD does not correlate well with hepatic apoptosis in HCC.

Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21(WAF1) expression and hepatic apoptosis.

AIM:To detect the expression of caspase 3 gene in primary human hepatocellular carcinoma (HCC) and investigate its relationship top21(WAF1) gene expression and HCC apoptosis.METHODS:In situ hybridization was employed to determine caspase 3 and p21(WAF1) expression in HCC.In situ end-labeling was used to detect hepatocytic apoptosis in HCC.RESULTS:Twenty-one of 39 (53.8%) cases of HCC were found to express caspase 3 transcripts, while 46.2% of HCC failed to express caspase 3.Non-cancerous adjacent liver tissues showed more positive caspase 3(87.5%, 7/8) as compared with HCC (p < 0.05). The expression of caspase 3 is correlated with HCC differentiation, 72.2% (13/18) of moderately to highly differentiated HCC showed caspase 3 transcripts positive, while only 38.1% of poorly differentiated HCC harbored caspase 3 transcripts (italic>p < 0.05). No relationship was found between caspase 3 expression and tumor size or grade or metastasis, although 62.5% (5/8) of HCC with metastasis were caspase 3 positive and a little higher than that with no metastasis (51.6%, p> 0.05). Expression of caspase 3 alone did not affect the apoptosis index (AI) of HCC. The AI was 7.12 in caspase 3 positive tumors (n = 21), while in caspase 3-negative cases (n = 18) 6.59 (italic>p > 0.05). Expression of caspase 3 clearly segregated with p21(WAF1) positive tumors as compared with p21(WAF1) negative cases (16 of 23, 69.6% versus 5 of 16, 31.3%) with statistical significance (p = 0.017).In the cases with positive caspase 3 and negative p21(WAF1), the AI was found slightly higher, but with no statistical significance, than that with expres-sion of p21(WAF1) and caspase 3 (7.21 vs 6.98 , p>0.05).CONCLUSION:Loss of caspase 3 expression may contribute to HCC carcinogenesis, although the expression of caspase 3 does not correlate well with cell apoptosis in HCC.p21(WAF1) may be merely one of the inhibitors which can reduce caspase 3 mediated cell apoptosis in HCCs.

Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C.

AIM:To explore the status of extrahepatic hepatitis C virus (HCV) infection and replication in hepatitis C patients,and its potential implication in HCV infection and pathogenicity.METHODS:By reverse-transcriptase poly-merase chain reaction (RT-PCR),in situ hybridization (ISH) and immunohis-tochemistry, HCV RNA, HCV replicative intermediate (minus-strand of HCV RNA), and HCV antigens were detected in 38 autopsy extrahepatic tissue specimens (including 9 kidneys, 9 hearts, 9 pancreas, 5 intestines, 2 adrenal glands, 2 spleens, 1 lymph node, and 1 gallbladder) from 9 hepatitis C patients, respectively; and the status of HCV replication in extrahepatic tissues was studied.RESULTS:By RT-PCR, all 9 patients were positive for HCV RNA in kidney, heart, pancreas, and intestine, but only 6(66.7%) patients were positive for HCV replicative intermediate. HCV RNA and HCV antigens were detected in kidney, heart, pancreas, intestine, adrenal gland, lymph node, and gallbladder in 5(55.6%) and 6(66.7%) patients by ISH and immuno-histochemistry, respectively. HCV RNA and HCV antigens were not detected in these extrahepatic organs in 3(33.3%) patients, although their livers were positive for HCV.HCV replicative intermediate detected by RT-PCR was consistent with HCV RNA and HCV antigens detected by ISH and immunohistochemistry (Kappa =0.42-0.75). HCV RNA and HCV antigens were detected in myocardial cells, epithelial cells of intestinal gladular, interstitial cells of kidney, epithelial cells of tubules and glomerulus, pancreas acinar cells and epithelial cells of pancreatic duct, epithelial cells of mucous membrane sinus of gallbladder, cortex and medulla cells in adrenal gland,and mononuclear cells in lymph node. HCV RNA was also detected in bile duct epithelial cells, sinusoidal cells, and mononuclear cells in liver tissues by ISH.CONCLUSION:HCV can infect extrahepatic tissues, and many various tissue cells may support HCV replication; extrahepatic HCV infection and replication may be of concomitant state in most of patients with hepatitis C. The infected extrahepatic tissues might act as a reservoir for HCV, and play a role in both HCV persistence and reactivation of infection. HCV as an etiologic agent replicating and expressing viral proteins in extrahepatic tissues itself contributes to extrahepatic syndrome associated-HCV infection in a few patients with chronic HCV infection.