OBJECTIVE: To investigate the effects of granulocyte colony-stimulating factor (G-CSF)-mobilized autologous bone marrow stem cells on the liver regeneration of partial liver transplant. METHODS: A 50% partial liver transplantation model as established by transplanting parts of the liver of female rats to the male rats with the equal weights that had had parts of their livers resected. Then the transplanted male rats were randomly divided into 3 equal groups: G-CSF + PLTx group that was injected hypodermically with recombinant human G-CSF (rhG-CSF) daily for 5 days and then underwent liver resection and 50% partial liver transplantation (PLTx); PLTx + G-CSF group that underwent PLTx and 3 hours after the transplantation received injection of rhG-CSF daily for 5 days, and PLTx control group that underwent PLTx and 3 hours later received injection of normal saline daily for 5 days. One, 3, 5, 7, and 14 days after the PLTx blood samples and livers were collected from 6 rats from each group, and one hour before the liver was taken bromodeoxyuridine (BrdU) was injected intraperitoneally to be integrated into the synthesis of DNA in the liver cells. Immunohistochemistry was used to detect the CD34 and BrdU-positive cells. In situ hybridization was used to detect the sry (sex-determining region) gene so as to determine the origin of the proliferating cells in the transplanted liver. RESULTS: The 14-day survival rate of the G-CSF + PLTx group was 90%, significantly higher than those of the G-CSF + PLTx (60%) and PLTx group (50%) (both P < 0.05) with a significant difference between the latter 2 groups too (P < 0.05). The mitosis index of liver cells 3 days after the transplantation of the PLTx + G-CSF group was 30% +/- 5%, significantly higher than those of the G-CSF + PLTx group (24% +/- 7%) and PLTx group (24% +/- 6%) (both P < 0.05) without a significant difference between the latter 2 groups. The rate of BrdU-positive cells of the PLTx + G-CSF group was 42% +/- 6%, significantly higher than those of the G-CSF + PLTx group (38% +/- 4%) and PLTx group (34% +/- 8%) (both P < 0.05). The transplantation the mitosis index and rate of BrdU-positive cells decreased since the 7th days after transplantation in all 3 groups, however, with the same relationship among them. The numbers of CD34(+) cell around the portal area of the 2 G-CSF groups increased since the 3rd day after transplantation in comparison with the PLTx group, was the highest on the 3rd day for the G-CSF + PLTx group, and continued to increase in the PLTx + G-CSF group. Since the 3rd day after transplantation, sry-positive cells were seen in the hepatic sinusoid and portal area in the 2 G-CSF groups, and were rarely seen in the PLTx group. CONCLUSION: G-CSF treatment after 50% PLTx significantly promotes liver regeneration and ameliorates the liver damage, thus raising the survival rate.

Animals↗

PubMed2005

[Investigating the inhibitory effects of interferon-alpha on the replication of hepatitis C virus replicon].

OBJECTIVE: To investigate the inhibitory effects of interferon-alpha (IFN-alpha) on HCV replicon and evaluate the expressing levels of signal transducer and activator of transcription genes (STAT1 and STAT2) and IFN-alpha stimulated genes (ISGs) which may mediate the inhibitory effects of IFN-alpha on HCV. METHODS: Firstly HCV replicon cell culture system was established by transfecting HCV replicon RNA transcribed in vitro into Huh7 cells and screening with G418. Secondly, the established HCV replicon cells were treated with various concentrations of IFN-alpha (0, 10, 25, 50, 100, 250, 500, 750, 1000, 2500, and 5000 IU/ml) for 72 h or treated with 1000 IU/ml of IFN-alpha for different lengths of time (0, 24, 48, 72, 96 h), then the levels of HCV RNA and NS5A protein in these cells were examined by semi-quantitative RT-PCR and Western blot respectively. RESULTS: IFN-alpha could effectively inhibit HCV RNA replication. 10 IU/ml or 25 IU/ml of IFN-alpha could lead to about 68% and 75% of HCV-RNA reduction respectively. The cells treated with 1000 IU/ml IFN-alpha for 24 h or 96 h had about 75% and 88% of HCV RNA reduction compared with the cells of control, demonstrated that the inhibitory effects of IFN-alpha on HCV replicon are in dose and time dependent manners. The expressions of antiviral ISGs-PKR, 2'5'OAS, G1P3, ISG20 and ISGF3gamma are strongly induced by IFN-alpha. CONCLUSIONS: HCV replicons were sensitive to IFN-alpha treatment. The inhibitory effects of IFN-alpha on HCV RNA and NS5A are both dose and time dependents. PKR, 2'5'OAS, G1P3, ISG20 and ISGF3gamma may mediate the inhibitory effects of IFN-alpha on HCV replicon replication.

Hepacivirus↗

PubMed2005

Transfusion of multi-factors activated immune cells as a novel treatment for patients with chronic hepatitis B.

BACKGROUND AND OBJECTIVES: Host-virus interactions play a central role in determining the prognosis of hepatitis B virus infection. Multi-factors activated immune cells (MAICs) are autologous peripheral blood mononuclear cells activated with anti-CD3 monoclonal antibody, interleukin-2 and interferon-gamma. The present pilot clinical trial was designed to determine whether adoptively transferred MAICs inhibit the replication of hepatitis B virus and is safe for patients. STUDY DESIGN: Fourteen patients with chronic hepatitis B were enrolled in the study. A total of (1.5-4.0)x10(9) peripheral blood mononuclear cells were isolated from each patient and activated by anti-CD3 monoclonal antibody, interleukin-2 and interferon-gamma in vitro for 10 days to produce MAICs. Cell phenotypes and levels of cytokine secretion were determined during cell culture. Patients were followed up for 1 year after the transfusion of MAICs. RESULTS: After 10 days of culture, peripheral blood mononuclear cells were expanded to a mean fold of 4.68+/-1.78 and activated effectively, as demonstrated by CD25 expression and cytokine secretion. Cell activation peaked on day 4. All patients accepted the MAICs transfusion with some slight adverse events, and fulminant hepatitis and bilirubinemia were not observed. Significant HBV inhibition was observed in 8 out of 14 patients, in which 5 patients achieved complete response (defined as that serum HBV DNA levels below the detection limit, occurrence of HBeAg seroconversion and normalization of ALT were observed together after MAICs transfusion and kept for at least 6 months) and 3 patients achieved partial response (defined as that serum HBV DNA levels below the detection limit, occurrence of HBeAg disappearance and normalization of ALT were observed together in 6 months after MAICs transfusion, but kept for less than 6 months). CONCLUSION: These findings strongly suggest that MAICs transfusion, which effectively inhibits the replication of hepatitis B virus, is safe for patients.

Adolescent↗

PubMed2005

[Genotpye of HBV: research progress and clinical direction].

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Disease Susceptibility↗

PubMed2005

[Thirteen to fourteen years' follow-up of patients with hepatitis C].

OBJECTIVE: To investigate the clinical outcomes and hepatic histology of patients with hepatitis C. METHODS: Nine patients with hepatitis, 4 males and 5 females, aged 44 +/- 7, who were infected during transfusion or blood donation underwent follow-up for 13 approximately 14 years: liver histological examination by biopsy, ultrasonography, biochemical examination of alanine transaminase (ALT), apartate aminotransferase (AST), and total bilirubin (TBIL), serum anti-HCV by ELISA, and HCV RNA quantification by PCR. RESULTS: ALT were higher than the normal value at every time point and HCV RNA remained positive (3.57 x 10(8) approximately 7.21 x 10(9) copies/L) in all patients. Ultrasonography showed mild and moderate hepatitis (3 and 6 cases respectively). The modified histological activity indices (HAI) were 5.0 approximately 8.5 and the fibrosis scores were 1 approximately 4. The ALT value was higher in the moderate cases than in the mild cases, and higher in those with higher HAI. The viral load was higher in the patients infected by the virus of the genotype 1b than in the patients infected by the virus of the genotype 2. CONCLUSION: The clinical outcomes and hepatic histology of patients with hepatitis C seem not very severe.

Adult↗

PubMed2005

Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters.

For investigating the expression of cancer/testis (CT) antigens in patients with hepatocellular carcinoma (HCC) in China, and evaluating the correlations between the expression of these CT antigens and clinical parameters, we collected tumors and adjacent non-cancerous tissues of 43 HCC patients from Beijing and 30 HCC patients from Guangxi province. Expression of the mRNA of 14 CT antigens was evaluated by reverse transcription PCR (RT-PCR). The correlation between CT antigen expression and clinical parameters was statistically analyzed. The mRNA expression frequencies of CT antigens in tumor tissue were: MAGE-A1, 69.9%; MAGE-A3, 47.9%; MAGE-A4, 20.0%; MAGE-A10, 36.7%; SSX-1, 67.4%; SSX-2, 35.6%; SSX-4, 48.8%; SSX-5, 30.2%; NY-ESO-1, 42.5%; MAGE-B1, 52.0%; MAGE-B2, 60.0%; MAGE-C1, 48.0%; MAGE-C2, 68.0%; and SCP-1, 33.3%. However, in adjacent tissues, no CT antigen mRNA expression was detected, except SSX-1 in 9.3% patients. In each HCC tissue, the expression of a minimum of one, two, or three CT antigens was in the range of 80-90, 70-80 or 50-70%, respectively. MAGE-A3 mRNA expression differed between the HCC patients in Beijing and Guangxi (P=0.002). The average age of the HCC patients bearing CT antigen positive tumors was higher than that of the HCC patients bearing CT antigen negative tumors. The expression of MAGE-A3, SSX-1, SSX-2, SSX-4, MAGE-B2, MAGE-C1, and MAGE-C2 correlated significantly with older age (P<0.05). Moreover, the expressions of MAGE-A4 and SCP-1 were related to alpha-fetoprotein abnormality (P<0.05), and the expression of NY-ESO-1 was related to early tumor stage (P<0.05). There was no correlation observed between the expression of CT antigens and the sex, HBV infection or tumor size.

Adult↗

PubMed2005

In vitro resistance to interferon of hepatitis B virus with precore mutation.

AIM: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-alpha), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS: HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS: We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind III and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION: These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Antiviral Agents↗

PubMed2005

Nonstructural 5A gene variability of hepatitis C virus (HCV) during a 10-year follow up.

BACKGROUND: Seven patients with chronic hepatitis C virus (HCV) infection were followed up for 10 years in order to analyze the molecular evolution of the HCV nonstructural 5A (NS5A) gene. METHODS: Serum samples were obtained from seven patients with anti-HCV who were HCV-RNA positive. The HCV NS5A region, including the interferon sensitivity-determining region (ISDR), protein kinase R binding domain (PKR-BD), and V3 was amplified and cloned, and then sequenced. The nucleotide sequences of the region at the beginning and at the end of the 10 years were aligned with the CLUSTAL W program, version 1.8, and the corresponding amino-acid sequences were deduced. RESULTS: The three serine residues at positions 2197, 2201, and 2204, suggested to be important for hyperphosphorylation of NS5A, were highly conserved in different patients and were within the quasispecies of each patient. The wild-type ISDR or minimally mutated strain was dominant in all the patients and the number of quasispecies decreased over time. CONCLUSIONS: Our findings showed that the functional domains of NS5A were generally conserved over an interval of 10 years. Quasispecies distribution was variable and distinctly clustered over time during the natural course of HCV infection. These results may contribute to better understanding of HCV natural infection.

Adult↗

PubMed2005

Expression of MAGE-A1 mRNA is associated with gene hypomethylation in hepatocarcinoma cell lines.

BACKGROUND: A correlation between melanoma-associated antigen (MAGE) A1 mRNA expression and genome-wide hypomethylation has been observed in some carcinomas, but this relationship is not known in hepatocarcinoma. METHODS: Total RNA and genomic DNA were prepared from ten human hepatocarcinoma cell lines in which the genetic characteristics are stable. MAGE-1 mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR), and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern-blot assay. The methylation status of the MAGE-A1 gene promoter was measured by enzyme digestion and PCR. RESULTS: MAGE-A1 mRNA was detected in the hepatocarcinoma cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405, which showed moderate to low levels of cell differentiation. In contrast, MAGE-A1 mRNA expression was not detected in the hepatoma cell lines HepG2215, HepG2, QGY-7701, and Huh7, which showed moderate to high levels of differentiation. The level of demethylation in MAGE-A1 mRNA-positive cell lines was much higher than that in MAGE-A1 mRNA-negative cell lines (P=0.02). CONCLUSIONS: The results suggest that MAGE-A1 mRNA expression in human hepatoma cell lines is associated with hypomethylation of the genome and the MAGE-A1 promoter domain. This study will be helpful to reveal the expression mechanisms of MAGE-like tumor antigens in cancer cells.

Antigens, Neoplasm↗

PubMed2005

Genome-wide expression profiling of the response to azole, polyene, echinocandin, and pyrimidine antifungal agents in Candida albicans.

Antifungal agents exert their activity through a variety of mechanisms, some of which are poorly understood. We examined changes in the gene expression profile of Candida albicans following exposure to representatives of the four currently available classes of antifungal agents used in the treatment of systemic fungal infections. Ketoconazole exposure increased expression of genes involved in lipid, fatty acid, and sterol metabolism, including NCP1, MCR1, CYB5, ERG2, ERG3, ERG10, ERG25, ERG251, and that encoding the azole target, ERG11. Ketoconazole also increased expression of several genes associated with azole resistance, including CDR1, CDR2, IFD4, DDR48, and RTA3. Amphotericin B produced changes in the expression of genes involved in small-molecule transport (ENA21), and in cell stress (YHB1, CTA1, AOX1, and SOD2). Also observed was decreased expression of genes involved in ergosterol biosynthesis, including ERG3 and ERG11. Caspofungin produced changes in expression of genes encoding cell wall maintenance proteins, including the beta-1,3-glucan synthase subunit GSL22, as well as PHR1, ECM21, ECM33, and FEN12. Flucytosine increased the expression of proteins involved in purine and pyrimidine biosynthesis, including YNK1, FUR1, and that encoding its target, CDC21. Real-time reverse transcription-PCR was used to confirm microarray results. Genes responding similarly to two or more drugs were also identified. These data shed new light on the effects of these classes of antifungal agents on C. albicans.

Amphotericin B↗

PubMed2005

[Lamivudine treatment of patients with decompensated HBV cirrhosis and mutation in YMDD motif].

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Amino Acid Motifs↗

PubMed2005

[Subgenomic replicon model of hepatitis C virus and it's application].

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Genome, Viral↗

PubMed2005

[Expression of MAGE-A1 in human hepatoma cell lines associated with genic hypomethylation].

OBJECTIVES: To investigate the correlation between MAGE-A1 mRNA expression and genic demethylation in hepatoma cell lines. METHODS: Total RNA and genomic DNA were prepared from 10 human hepatoma cell lines. MAGE-1 mRNA expression was determined with RT-PCR and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern blot assay. The genomic DNA was digested by HpaII, then the promoter of MAGE-A1 gene was amplified with primers CDS21, EDP4 and CDS20. EDP4 and the PCR products were further hybridized with a probe to detect the methylation in the promoter of the MAGE-A1 gene. HLA-A locus was typed using SSP kit. RESULTS: In cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405, MAGE-A1 mRNA expression was positive and cell differentiation was moderate or low. In cell lines of HepG2 2.2.15, HepG2, QGY-7701 and Huh7, MAGE-A1 mRNA expression was negative and cell differentiation was well or moderate. The level of genomic demethylation in MAGE-A1 mRNA positive cell lines was much higher than that in MAGE-A1 mRNA negative cell lines (t = 2.896, P = 0.02). The methylation analysis showed that methylation in the promoters of MAGE-A1 gene of HepG2 2.2.15, HepG2, QGY-7701 and Huh7 was high, and that methylation in those of SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405 was low. CONCLUSION: The results suggest that MAGE-A1 mRNA expression in the human hepatoma cell lines is associated with genic hypomethylation.

Antigens, Neoplasm↗

PubMed2005

[Analysis of prognosis of patients with decompensated liver cirrhosis using the criterion of the Model of End-Stage Liver Disease].

OBJECTIVE: To investigate the relationship between the Model for End-Stage Liver Disease (MELD) with Child-Pugh scoring, and the prognosis of patients with decompensated liver cirrhosis. METHODS: 110 patients with decompensated liver cirrhosis were graded with MELD formula and with Child-Pugh. The death rate was observed within three months. RESULTS: 31 patients died within 3-months. The mortality of patients whose MELD scores were between 10 approximately 19, 20 approximately 29, and > or = 30 was higher than those with MELD < or = 9 (The mortality of those with MELD less than 9, 10 approximately 19, 20 approximately 29, or > or = 30 was 11.76%, 38.18%, 64.71%, 75.00% respectively). The mortality of patients whose MELD scores were > or = 18 was higher than those with MELD < 18 (The mortality of those with MELD < 18, MELD > or = 18 was 26.58%, 58.06% respectively. chi2 = 9.643). The mortality of Child A, B, C was 14.89%, 42.55%, 75% respectively. CONCLUSION: Both MELD and Child-Pugh scores can accurately predict the short-term prognosis of patients with decompensated liver cirrhosis.

Aged↗