OBJECTIVETo evaluate the replication and encapsidation of HBV mutants with the truncated C gene.

METHODSThe HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome.

RESULTSThe C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay.

CONCLUSIONThe C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

Cell Line, Tumor ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Mutation ; Plasmids ; genetics ; Transfection ; Virus Replication

The aim was to investigate the cytolytic activity of cytotoxic T lymphocytes (CTL) induced by dendritic cells (DC) derived from human cord blood in vitro. Human cord blood mononuclear cells (CBMNC) were cultured in vitro with addition of various cytokines. DC was confirmed by morphology, immune phenotype and capacity of stimulating MLR (mixed lymphocyte reaction). CTL were generated through the co-culture of autologous T lymphocytes and DC. (51)Cr-release assays were performed for the measurement of cytotoxicity of CTL. The results showed that distribution of the subgroups of T lymphocytes in CBMNC was similar to that in adult peripheral blood. The percentage of CD1a-expressing cells in CBMNC was very low, merely (0.41 +/- 0.09)%. During culture, DC became larger and more irregular in shape. Spiny dendrites or multiple cell processes in morphology emerged on the surface of DC. Among the cell populations at 15 days of culture, there were (28.4 +/- 3.55)% of CD1a-expressing cells, (63.67 +/- 23.33)% of CD86-positive, (8.7 +/- 1.49)% of CD83-positive and (32.5 +/- 1.53)% of HLA-DR-positive cells. DC derived from CBMNC is capable of stimulating the proliferation of allogeneic lymphocytes in MLR. CTL derived from autologous T lymphocytes induced by dendritic cells pulsed with lysates of HL-60 cells, possessed specific cytolytic effects against HL-60 cells. In conclusions, relatively high percentage of CD1a-expressing cells can be generated in culture system of this study. DC derived from cord blood is able to induce the production of anti-leukemic CTL in vitro.
Antigens, CD1 ; analysis ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Fetal Blood ; immunology ; Humans ; Immunophenotyping ; Leukemia ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo explore the effect and mechanism on HBV replication in C gene truncated mutant.

METHODSProtein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot.

RESULTSThe recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C.

CONCLUSIONC gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication

Cell Line ; Genetic Therapy ; Hepatitis B ; therapy ; Humans ; Mutation ; Transfection ; Viral Core Proteins ; genetics ; Virus Replication

Objective To explore the possible role of parvalbumin (PV)-positive interneuron in the pathogenesis of increased susceptibility to epileptic seizures in FMR1 gene knockout (FMR1 KO)mice. Methods Immunohistochemistry was employed to determine the expression of PV in CA1 and CA3 regions of the hippocampus, the striate cortex, the temporal auditory cortex and the piriform cortex of FVB strain FMR1 KO mice and wild type (WT) controls at the age of 2, 4 and 6 w. Western blotting was used to detect the level of PV in the cerebral cortex and hippocampus of the above mice. Results The numbers of PV-positive interneuron in CA1 and CA3 regions of the hippocampus, the striate cortex,the temporal auditory cortex and the piriform cortex of FMR1 KO mice at the age of 2 and 4 w were significantly decreased as compared with those in the age-matched WT mice (P<0.05). The level of PV in the cerebral cortex and hippocampus in FMR1 KO mice at the age of 2 and 4 w was also significantly decreased than that in the age-matched WT mice (P<0.05). Conclusion Decreased numbers of PV-positive interneuron and level of PV might induce the increased susceptibility to epileptic seizures inFMR1 KO mice.