[Real-time dynamic recording of cerebral cortical vascular embolization and regeneration in rats].

The purpose of this study was to establish a method to record the dynamic process of vascular regeneration and remodeling in rat cerebral ischemic regions. An animal brain window model was established to continuously observe the changes of rat cortical vascular ischemia in vivo, and the model of cerebral ischemia was established by photochemical embolization. Optical coherence tomography (OCT) was performed to record the formation of vascular blockage and the injury and regeneration of small vessels during cerebral ischemia recovery. The results showed that 30 min of laser irradiation could completely block the cortical vessels in rats. Within 24-48 h after ischemia, the degree of brain injury was the greatest, and the number of blood vessels in the ischemic region reached the minimum. Then the blocked blood vessels began to be dredged, and the small blood vessels around the ischemic area began to regenerate. Small blood vessels in the superficial/deep layers of the cortex disappeared significantly after laser irradiation. During 10 d after ischemia, the blocked blood vessels were gradually dredged and recovered. On the 10th day after laser irradiation, a large number of neovascularization appeared in the superficial layer of cortex, but the deep vessels did not recover. These results indicate that the method established in this study can observe the changes of blood vessel in cerebral ischemic region continuously, which lays a foundation for further quantitative study on the dynamics of embolized blood vessels and peripheral capillaries during the recovery of cerebral ischemia.

[Effect of particulate matter 2.5 at urban centre of Hangzhou on lung impairment in rats].

OBJECTIVE: To explore the effect of ambient particle matter 2.5 (PM2.5) collected in the urban center of Hangzhou on the lung injury of rats and on the activating of endoplasmic reticulum pathway. METHODS: PM2.5 samples were collected on quartz fiber filters using a PM2.5 high-volume air sampler in the urban area of Hangzhou. The collected PM2.5 particles were extracted in ultrapure water and concentrated by vacuum freeze-drying. Twenty-four male Sprague-Dawly (SD) rats were randomly divided into 3 groups:saline control group, low dose PM2.5 exposure group (5 mg/kg BW) and high dose PM2.5 exposure groups (25 mg/kg BW). Each group received intratracheal instillation of PM2.5, once a week for 4 weeks. Twenty-four hours after the last exposure, the rats were narcotized and sacrificed, left lung was isolated and fixed with 4% paraformaldehyde for histopathological detection. The bronchoalveolar lavage fluid (BALF) was collected from the right lung. The total antioxidant capacity (T-AOC) level, the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH) in BALF were detected by chemical colorimetry. The level of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) cytokines in BALF was measured by enzyme linked immunosorbent assay (ELISA). And the protein expressions of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase receptor-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor (p-eIF2α), transcription factors C/EBP homologue protein (CHOP), inositol-requiring enzyme 1α (IRE1α) and X-box binding protein 1 (XBP1) in lung tissue were determined by Western blotting. RESULTS: Compared with control group, rats in both low dose (5 mg/kg) and high dose (25 mg/kg) PM2.5-treated groups showed obviously dose-dependent pulmonary toxicity including thickening of alveolar walls, narrowing of alveolar space, interstitial hyperplasia and inflammatory cell infiltration. Compared with control group, T-AOC level and the SOD activity in BALF in both PM2.5-treated groups were decreased dose-dependently (P<0.05), whereas the LDH activity in BALF were increased in a dose-dependent manner (P<0.05). Exposure to PM2.5 resulted in a increasing of the release of proinflammatory cytokines including TNF-α, IL-1ß and IL-6 in rat lung in a dose-dependent manner (P<0.05). The levels of GRP78, p-PERK, p-eIF2α, CHOP, IRE1α and spliced XBP1 (XBP1-S) were significantly up-regulated, whereas the level of unspliced XBP1 (XBP1-U) was down-regulated in the rat lung tissue of high-dose PM2.5 treated group. CONCLUSIONS: The PM2.5 in the urban area of Hangzhou can significantly cause lung inflammatory injury in rats. Both oxidative stress and activation of ER stress pathways may be related to such PM2.5 inhalation-induced lung inflammatory injury.

[Inhibition of hepatitis B virus S gene and C gene expression by different 10-23 DNAzymes substrate-recognition domains].

OBJECTIVE: To explore the inhibition effects of 10-23 DNAzymes with different substrate-recognition domains targeting hepatitis B virus (HBV) S gene and C gene expression in 2.2.15 cells. METHODS: 10-23 DNAzymes with different substrate-recognition domains specific to HBV S gene open reading frame (ORF) A(157)UG and HBV C gene ORF A(1816)UG were designed and synthesized, respectively. Different 10-23 DNAzymes were transfected into 2.2.15 cells which is a stable HBV producing cell line. HBsAg and HBeAg secreted into culture media were detected by radioimmunoassay (RIA) and HBV DNA levels were measured by real-time PCR. 3-(4, 5-dimethylthiagol-2-yl)-2, 5-drphnyl tetrazolium bromide (MTT) assays were performed to evaluate cytotoxicity. RESULTS: HBsAg and HBeAg expressions were reduced by various DNAzymes (0.1 - 2.5 micromol/L) with different substrate-recognition domains after transfection. The antiviral effects of DNAzymes were apparent until 72 h post-transfection. The inhibition rates of the DNAzymes at the same dose on HBsAg and HBeAg in the same period of post-transfection were as the following: DrzBS-9 > DrzBS-8 > DrzBS-7; DrzBC-9 > DrzBC-8 > DrzBC-7. Among all the DNAzymes used, DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent, with HBsAg and HBeAg reduced 95% and 92% 48 h post-transfection at the dose of 2.5 micromol/L, respectively. The inhibition effects on HBV DNA by various DNAzymes with different substrate-recognition domains were of no significance. There were no evident cytotoxic effects of these DNAzymes in the range from 0.1 to 2.5 micromol/L. CONCLUSION: 10-23 DNAzymes with different substrate-recognition domains targeting HBV S gene and C gene mRNA possessed specific inhibition effects in 2.2.15 cells, and DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent.

[Cleavage of in vitro transcripts of hepatitis B virus C gene by 10-23 DNA enzyme].

OBJECTIVE: To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly. RESULT: Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively. CONCLUSION: 10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.

In vitro cleavage of hepatitis B virus C mRNA by 10-23 DNA enzyme.

BACKGROUND: 10-23 DNA enzyme is one kind of deoxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2.2.15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A(1816)UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. RESULTS: Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km, Kcat and Kcat/Km for Drz-HBV-C-9, were 1.4 X 10(-9) mol, 1.6 min-1 and 1.1 X 10(9) mol(-1) x min(-1), respectively. CONCLUSIONS: 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.

Effect of IFNalpha-2a on Fas expression and apoptosis rate of peripheral blood cytotoxic T cells in patients with hepatitis B.

BACKGROUND: Interferon(IFN) with antiviral and immunomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNalpha-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells(CTLs) in patients with hepatitis B. METHODS: Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNalpha-2a treatment. RESULTS: Before IFNalpha-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNalpha-2a treatment, Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNalpha-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS: Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNalpha-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.

HBV genotype characterization and distribution in patients with HBV-related liver diseases in Zhejiang Province, P.R. China: possible association of co-infection with disease prevalence and severity.

BACKGROUND: There are 8 well-documented genotypes of hepatitis B virus (HBV) at this time point. Genotyping can be accomplished based on a partial sequence of hepatitis B virus (HBV) genome such as the pre-S or S gene. Several methods have been developed and used for HBV genotyping including direct sequencing, restriction fragment length polymorphism, line probe assay and enzyme-linked immunoassay. Recently, a novel, rapid and cost-effective genotyping method based on PCR amplification assay using type-specific primers that can identify all six major genotypes has been developed. This study was undertaken to characterise HBV genotypes and investigate the association between the prevalence of different genotypes and the severity of HBV-induced liver diseases. METHODS: Serum samples from carriers of HBV and patients with HBV-related liver diseases from Zhejiang Province were screened for viral serological markers using commercially available radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) kits. Serum HBV DNA load was determined by real-time detection PCR. A type-specific primer based the nested-PCR method was employed in the HBV genotyping. The genotype results obtained were confirmed by direct sequencing of nested PCR amplicons of the pre-S region. Ten samples of each genotype (B and C) were sequenced. RESULTS: The survey on a cohort of 125 HBV carriers in and around Hangzhou City, Zhejiang Province showed the existence of HBV genotypes A (0.8%), B (48%), C (40.8%), D (0.8%), mixed B and C (9.6%) and an absence of E and F genotypes. Distribution of HBV genotypes in patients with liver diseases revealed a statistically insignificant higher prevalence of genotype B in mild chronic hepatitis (CH). Among the three genotypes B, C and mixed B/C infections 11 (73.3%), 3 (20%) and 1 (6.7%), (P<0.05), respectively in subjects with moderate CH, genotype B was significantly predominant. The infection patterns for genotypes B, C and B/C mixed in (i) liver cirrhosis (LC) 4 (23.5%), 10 (58.8%) and 3 (17.7%) and (ii) hepatocellular carcinoma (HCC) 2 (28.6%), 5 (71.4%) and 0 (0.0%) respectively revealed a marked association of C genotype with liver disease; however, the association was statistically insignificant (P>0.05). Differences in positive rate of HBeAg for the three genotypes B, 16 (30.8%), C, 27 (51.9%), and mixed B/C, 9 (17.3%) were significant (P<0.05), with genotype C showing predominance. CONCLUSIONS: These findings show an interesting distribution of HBV A-D genotypes in Zhejiang Province. Furthermore, our results indicate a novel and markedly high prevalence of mixed B/C genotype infections in subjects with severe CH and LC, and a possible association of mixed B/C infections with the severity of liver diseases in this region of Mainland China.

Clinical features of chronic hepatitis B patients with YMDD mutation after lamivudine therapy.

OBJECTIVE: To study the clinical features of chronic hepatitis B (CHB) patients with tyrosine-methionine-aspartate-aspartate (YMDD) mutation after lamivudine therapy. METHODS: This investigation was a retrospective study of 63 CHB patients with YMDD mutation during lamivudine therapy. Clinical data, including period and types of YMDD mutation; hepatitis B virus (HBV) DNA levels and alanine aminotransferase (ALT) levels before and after YMDD mutation were measured. YMDD mutation in the HBV DNA polymerase gene was determined using polymerase chain reaction (PCR) and direct sequencing. HBV DNA quantification was determined using real-time PCR. Relevant serum markers of HBV were measured. The follow-up period was 12 months after YMDD mutation. RESULTS: YMDD mutation occurred 7-44 months (median, 21.5 months) after the start of lamivudine therapy. The majority of the cases (42/63, 66.6%) had YMDD mutants detected between 12 and 24 months. Four types of YMDD mutation were observed in this study, rtL180M/M204V mutation was the predominant type (26/63, 41.3%). A proportion of patients (16/63, 25.4%; 12/63, 19.1%) had higher HBV DNA levels and ALT levels (after mutation vs before mutation), respectively. CONCLUSION: The majority of patients with YMDD mutants had similar or lower HBV DNA levels and ALT levels compared with baseline values. This subset of patients might have benefited from the continued lamivudine therapy. The patients with increased ALT and HBV DNA levels (breakthrough hepatitis) should benefit from the addition of a newer nucleotide analogue (e.g. adefovir).

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance.

OBJECTIVES: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. METHODS: One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. RESULTS: One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. CONCLUSIONS: HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.

Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes.

OBJECTIVE: This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. METHODS: PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h. RESULTS: After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. CONCLUSION: This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells.

AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.

Recent advances in DNA vaccine of hepatitis virus.

Nucleic acid vaccine or DNA vaccine is a hopeful vaccine to prevent and treat viral hepatitis. Problems exist in different DNA vaccines for HBV or HCV. Optimal animal model should be established study vaccine against hepatitis. Apart from the strategy to enhance the efficiency of DNA vaccine, combined use of cytokines or chemokines, different routes of inoculation, design of optimal vector, ISS insertion in the plasmid vectors, etc to enhance the efficiency of DNA vaccine are reviewed.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens.

OBJECTIVE: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased immune responses. METHODS: Four recombinant plasmids constructed included the coding regions for the core protein (pC) and for the core, E1 and E2 together (pCE1E2), IL-12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. RESULTS: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE1E2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC=18.65%+/-5.71%, pCE1E2=20.07%+/-11.11%, pC+pIL-12=60.11%+/-17.37%, pCE1E2+pIL-12=67.48%+/-15.57%, respectively. The average A values of anti-HCV were pC=0.415+/-0.127, pCE1E2=0.358+/-0.096, pC+pIL-12=0.210+/-0.086, pCE1E2+pIL-12=0.258+/-0.125. CONCLUSION: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.

Influence of HLA class II molecules on the outcome of hepatitis B virus infection in population of Zhejiang Province in China.

OBJECTIVE: To investigate the correlation between HLA class II molecules and the different outcome of viral hepatitis B. METHODS: Thirty patients with chronic hepatitis B and 56 subjects who had spontaneously recovered from HBV infection in Zhejiang were enrolled in this investigation. HLA class II molecules types and alleles were determined by PCR-ssp. RESULTS: HLA-DR12 was found in 21 of the 56 subjects (38%) recovered from hepatitis B, compared to 3 of the 30 patients with chronic hepatitis B [10%, relative risk (rr), 0.19; Pcorr<0.025]. The frequency of the allele of HLA-DR12 DRB1*1201 was higher in the subjects recovered from hepatitis B infection (32%) than in the patients with chronic hepatitis B (3%; rr, 0.07; Pcorr<0.005). On the contrary, more HLA-DR9 was detected in the patients with chronic hepatitis B (43%) than in the subjects who recovered from hepatitis B infection (18%; rr, 3.52; Pcorr<0.025). HLA-DQ9 was also detected with a higher frequency in the patients with chronic hepatitis B (43%) than in the subjects who recovered from hepatitis B (20%; rr, 3.13; Pcorr<0.05). CONCLUSIONS: The HLA class II molecule DR12 and its allele DRB1*1201 are associated with protection against chronic hepatitis B in Zhejiang Province, China. HLA-DR9 and DQ9 are associated with chronicity of HBV infection.

Cloning and expression of cDNAs from hepatitis E virus structural gene.

OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection. METHODS: A 492 base cDNA was collected from 5-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E.coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E. RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100. CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant.

OBJECTIVE: To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines. METHODS: BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA; splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro. RESULTS: The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone, but there was not significantly different (P>0.05). Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-Gamma in supernatant of splenocytes cul-tured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05). CONCLUSION: The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

[Construction of HBV S gene recombinant and its immunity induced in mice]

OBJECTIVE: To investigate the feasibility of HBV DNA vaccines. METHODS: HBV S gene was obtained by PCR and the PCR product was cloned into pcDNA3. The recombinant was screened by antibiotics, identified by digestion and confirmed by sequencing. The plasmid was then transfected into mammalian cell COS-7 for transient expression. Then the recombinant was injected into mice and the immune responses induced in mice were investigated. RESULTS: The sequence of HBV S gene was correct and HBsAg could be detected in cells transfected with pcDNA3-S. After immunization, the positive rate in mice immunized with pcDNA3-S and pCMV-S was 70%(7/10) and 80% (8/10). The mean levels of anti-HBs were (32.14+/-13.79)mIU/ml and (28.50+/-11.87)mIU/ml respectively. There was no statistically significant difference between them P 0.05 . The mean levels of anti HBs in the control group and blank groups were both less than 10 mIU/ml. In mice immunized with pcDNA3-S and pCMV-S the results were (35.40+/-4.85)% and (38.20+/-7.69)% when E/T was 20:1, or (23.95+/-3.98)% and (24.55+/-3.59)% when E/T was 10:1, again showing no difference statistically (P>0.05). The specific CTL cytotoxicity rate of control and blank groups was both less than 5%. CONCLUSION: A specific humoral and cellular immune response can be induced in mice by intramuscular injection of pcDNA3-S.

[Detection and analysis of the new kind of G743C and G743A point mutation of HBV P gene in hepatitis B virus infected patients resistant to lamivudine].

OBJECTIVE: To explore the point mutation in hepatitis B virus polymerase (HBV P) gene in HBV-infected patients resistant to lamivudine. METHODS: HBV P gene was amplified by PCR and the products was sequenced to analyze the YMDD mutation. Then the variants were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with the following restriction enzymes: Fok I, Ssp I, Alw441 and were separated by 8.0% polyacrylamide gel electrophoresis. RESULTS: Comparing with the sequences of standard HBV genome, there were 16 patients with G743C mutation and 1 patient with G743A mutation, and the codon ATG turned to ATC and ATA, YMDD motif changed into YIDD. But this kind of YIDD mutation was not proved by PCR-RFLP assay in the 17 patients. CONCLUSIONS: The G743C and G743A mutations in HBV P gene, resulting in YMDD motif changed into YIDD, are detected only by direct sequencing, not by PCR-RFLP. The new kind of G743C and G743A point mutations in HBV P gene is important for the detection of HBV P gene YMDD mutation.

[Serum levels of sFas, sICAM-1, IL-18 in patients with chronic hepatitis C and their clinical significance]

OBJECTIVE: To investigate the serum levels of soluble Fas antigen (sFas), soluble intercellular adhesion molecules-1 (sICAM-1), interleukin-18 (IL-18) in patients with chronic hepatitis C and to study their roles in pathogenesis of chronic hepatitis C. METHODS: Serum sFas, sICAM-1, IL-18 levels were measured in 30 cases of chronic hepatitis C before and after treatment of interferon-alpha by enzyme-linked immunosorbent assay (ELISA), serum titer of HCV-RNA was detected by quantitative PCR and serum ALT activity was also detected. RESULTS: Serum levels of sFas sICAM-1 IL-18 in chronic hepatitis C patients were significantly higher than those in normal controls (P<0.01), showing correlation with serum HCV-RNA titer (r=0.915, r=0.795, r=0.757, respectively, P<0.01), Serum levels of sICAM-1, IL-18 showed correlation with serum ALT level(gamma=0.952, gamma=0.969, respectively, P<0.01), but no relationship was observed between serum sFas and serum ALT level(P>0.05). Serum levels of sFsa sICAM-1 IL-18 markedly decreased in responsive patients while no change was observed in patients with no response after treatment. CONCLUSION: Soluble Fas, soluble ICAM-1, IL-18 may participate in the pathogenesis of chronic hepatitis C and show correlation with the severity of histological inflammation and viral titer.

[Development of a system for quick screening of efficient HBx-siRNA].

OBJECTIVE: To develop a system for quick screening of efficient siRNA targeted HBx mRNA. METHODS: Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis. RESULT: (1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388. CONCLUSION: Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.