[Differential liver histopathological features of chronic HBV infection patients with normal and mildly elevated serum ALT].

To study the liver histopathological features that are distinctive between chronic hepatitis B virus (HBV) infection patients who have normal serum alanine aminotransferase (ALT)/asparatate aminotransferase (AST) and those with mildly elevated serum ALT/AST. One-hundred-and-thrity-four chronic HBV infection patients with normal serum ALT/AST and 165 chronic HBV infection patients with mildly elevated serum ALT/AST were included in the study. Liver biopsies were performed and used to assess the histological changes by hematoxylin-eosin and reticular fiber staining; mild to severe scoring for inflammation was made as grade G0-G4 and for fibrosis stage as S0-S4. HBV DNA levels were detected by fluorescent quantitative PCR. HBV serological markers were examined by chemiluminescence. The mildly elevated serum ALT/AST group had more male patients than the normal serum ALT/AST group. In the normal serum ALT/AST group, 50.0% (67/134) of the patients had moderate histological changes and only 3.0% (4/134) had severe changes (G3-4 and/or S3-4). In the mildly elevated ALT/AST group, 65.7% (174/265) of patients had moderate histological changes and 16.2% (43/265) had severe changes (G3-4 and/or S3-4). Hepatic inflammation and fibrosis were significantly more severe in the mildly elevated serum ALT/AST group than in the normal ALT/AST group (x2 = 26.386, P less than 0.01; x2 = 15.299, P less than 0.01). In the normal ALT/AST group, the severity of inflammation and fibrosis were positively correlated with age (rs = 0.620, P less than 0.01; rs = 0.347, P less than 0.01). In the mildly elevated ALT/AST group, the severity of inflammation and fibrosis were negatively correlated with age (rs = -0.807, P less than 0.01; rs = -0.557, P less than 0.01). In both groups, the severity of inflammation and fibrosis were negatively correlated with HBV DNA levels (rs = -0.215, P less than 0.01, rs = -0.527, P less than 0.01, rs = -0.951, P less than 0.01; rs = -0.715, P less than 0.01) and were not positively correlated with HBeAg. The majority of the chronic HBV infection patients with normal serum ALT/AST and those with mildly elevated serum ALT/AST had moderate liver pathological changes. All patients with low HBV DNA levels were closely followed-up, regardless of HBeAg-positive status.

[Relationship between liver pathological characteristics and serum HBeAg and HBV DNA in 1057 patients with chronic hepatitis B].

OBJECTIVE: To study the relationship between liver pathological changes and serum HBeAg and HBV DNA in 1057 patients with chronic hepatitis B. METHODS: Liver puncture biopsy for histopathological examinations were performed in 1057 patients with chronic hepatitis B. The quantitative analysis of serum HBV DNA by fluorogenic quantitative PCR and HBeAg by chemoluminescence were also conducted. RESULTS: The inflammatory grade and fibrosis stage were higher in HBeAg-negative patients (G4 and S4 were 7.83% and 12.17% respectively) than in HBeAg-positive patients (G4 and S4 were 3.39% and 5.44% respectively). The inflammatory grade and fibrosis stage were higher in HBeAg-positive patients with low-level HBV DNA (G3G4 was 45.64% and S3S4 was 30.20% for HBV DNA104-105), whereas they were higher in HBeAg-negative patients with high-level HBV DNA (G3G4 was 54.55% for HBV DNA106-107 and S3S4 was 42.85% for HBV DNA108-109). CONCLUSION: There were some correlation between the liver pathological changes and serum HBeAg and HBV DNA levels in patients with chronic hepatitis B. It is important to perform the liver pathological examination and antiviral therapy as early as possible in patients with HBeAg-negative chronic hepatitis B.

Procuration and identification of bacteria in paraffin-embedded liver tissues of hepatocellular carcinoma by laser-assisted microdissection technique.

This study was aimed at procuring directly and identifying the bacteria which had been found in paraffin-embedded liver tissues of hepatocellular carcinoma (HCC) patients. In our previous studies, Helicobacter spp. had been detected by polymerase chain reaction (PCR) and observed by histology in the liver tissues of HCC patients but had never been cultured successfully. To obtain and identify the uncultured bacteria, laser microdissection and pressure catapulting (LMPC) techniques were applied. Following microdissection from the liver tissue sections, these bacteria were examined by PCR using Helicobacter genus-specific 16S rRNA primers and sequence analysis. Amplified products of 16S rRNA were positive in all six microdissected samples with bacteria, and showed 99%-100% similarity with Helicobacter pylori by sequence analysis. Another H. pylori-specific 26 kDa gene (encoding one 26 kDa protein as H. pylori-specific antigen) was also tested by PCR. Four of six samples were positive. Therefore, Helicobacter spp. detected by PCR in the liver tissues of HCC patients in our previous studies are actually the bacteria observed by histology and identified as H. pylori by further sequence analysis. The laser-assisted microdissection technique can be extensively applied for identification of bacteria in tissue samples in bacteriology research.

[Clinical characteristics of the patients with dengue fever seen from 2002 to 2006 in Guangzhou].

OBJECTIVE: To study the clinical characteristics of the patients with dengue fever (DF) seen from 2002 to 2006 in Guangzhou in order to prevent and treat dengue fever better. METHODS: Clinical data from 1342 inpatients with DF seen from 2002 to 2006 were retrospectively analyzed. The dengue virus was isolated by C6/36 cell culture and genotyped by reverse transcriptase-polymerase chain reaction and gene sequence analysis. RESULTS: The average age of the patients was 34.4 years, without sex difference in distribution. Most of the patients had obvious toxemic symptoms including fever (100 percent), headache (85.9 percent), myalgia (64.5 percent), bone soreness (46.6 percent) and skin rash (65.9 percent). Leukopenia, thrombocytopenia, elevated alanine aminotransferase, elevated aspartate aminotransferase and hypokalemia were found in 66.0 percent, 61.3 percent, 69.0 percent , 85.7 percent and 28.4 percent of patients, respectively. DF-IgM could be detected in 90 percent of patients. The virus was identified as dengue virus type-I. CONCLUSIONS: The epidemic of DF was caused by dengue virus- I from 2002 to 2006 in Guangzhou. Most of the patients had classic DF clinical manifestation with high percentage of hepatic injury. Few patients progressed to dengue hemorrhagic fever.

Hepatitis B virus is inhibited by RNA interference in cell culture and in mice.

BACKGROUND AND AIMS: For chronic hepatitis B virus (HBV) infection the effects of current therapies are limited. Recently, RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we studied the effects of HBV-specific 21-bp short hairpin RNAs (shRNAs) targeted to the surface antigen (HBsAg) region and the core antigen (HBcAg) region both in a cell culture system and in a mouse model for HBV replication. METHODS: HBsAg and hepatitis B e antigen (HBeAg) in the media of the cells and in the sera of the mice were analyzed by time-resolved immunofluorometric assay, intracellular HBcAg by immunofluorescence assay, HBsAg and HBcAg in the livers of the mice by immunohistochemical assay, HBV DNA by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and HBV mRNA by semi-quantitative reverse transcriptase PCR (RT-PCR). RESULTS: Transfection with the shRNAs induced an RNAi response. Secreted HBsAg was reduced by >80% in cell culture and >90% in mouse serum, and HBeAg was also significantly inhibited. Immunofluorescence detection of intracellular HBcAg revealed 76% reduction. In the liver tissues by immunohistochemical detection, there were no HBsAg-positive cells and >70% reduction of HBcAg-positive cells for shRNA-1. And for shRNA-2 the detection of HBsAg and HBcAg also revealed substantial reduction. The shRNAs caused a significant inhibition in the levels of viral mRNA relative to the controls. HBV DNA was reduced by >40% for shRNA-1 and >60% for shRNA-2. CONCLUSIONS: RNAi is capable of inhibiting HBV replication and expression in vitro and in vivo and thus may constitute a new therapeutic strategy for HBV infection.

[Inhibition of hepatitis B virus replication and expression by RNA interference in vivo].

OBJECTIVE: To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting HBV C gene region on hepatitis B virus (HBV) in vivo. METHODS: An animal model of HBV infection was developed hydrodynamically, and pcDNA3.1-HBV and siRNA were together injected into the tail vein of the BALB/c mice. HBsAg was analyzed by time-resolved immunofluorometric assay, HBV DNA was analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR, and viral specific proteins (HBsAg and HBcAg) in the mice livers were assayed using immunohistochemical staining. RESULTS: In the mice, the siRNA effectively inhibited HBV replication and expression compared with the controls. The inhibitive effect of siRNA on HBV lasted at least 3 days. CONCLUSION: These results demonstrate that RNAi can substantially inhibit HBV replication and expression in vivo.

[Inhibition of hepatitis B virus replication and expression by RNA interference in vitro].

OBJECTIVE: To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that target HBV S gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. METHODS: HepG2.2.15 was used as target cell. The plasmid expressing small interfering RNA was transfected into the cultured cells via liposome metafectene, HBsAg and HBeAg were analyzed by time-resolved immunofluorometric assay, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV S-mRNA was detected by semi-quantitative RT-PCR. RESULTS: The plasmid expressing siRNA was successfully constructed. The S region siRNAs could effectively inhibit both antigens secretion and HBV replication compared with controls, HBsAg levels decreased by 75%, 82%, 89%; HBeAg levels decreased by 32%, 38%, 43%; HBV DNA production decreased by 30%, 43%, 49%; The HBV mRNA species was reduced by 30%, 70%, 90% when transfected with 1 microg, 2 microg, 4 microg HBV S-siRNA, respectively. CONCLUSION: These results demonstrate that RNAi can substantially inhibit HBV replication and the antigens expression in the infected cells. These inhibitive effect of siRNA on HBV was dose-dependent and sequence-specific.

[Inhibition of hepatitis B virus replication by RNA interference in vitro].

OBJECTIVE: To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that targets HBV core gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. METHODS: HepG2 2.2.15 was used as target cells. The plasmid and liposome metafectene were cotransfected into the cultured cells, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR. RESULTS: The plasmid expressing siRNA was successfully constructed. The two constructed siRNAs could effectively inhibit HBV replication, and their inhibitive effect on HBV was dose-dependent. CONCLUSION: These results showed that siRNA could substantially inhibit HBV replication in the infected cells