Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8(+) T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8(+) T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8(+) T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immunotherapeutic approaches should be aimed at not only boosting a HBV-specific CD8(+) T response but also improving its function.

The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle.In the present study using a Huh7 cell line Con1 with an HCV replicon,we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling.Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells.It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site.Consistently,a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays.Thus,the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication.In addition,cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine,an inhibitor of CDKs had a similar effect to that of U0126.Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels.Further,the replication of HCV replicon in Conl cells was inhibited by IFN-α.The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs.It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

Objective:To investigate the feasibility of transplantation of neural stem cells (NSCs) modified by hypoxia-regulated nerve growth factor (NGF) gene to treat acute spinal cord injury (SCI) and observe the functional repair after SCI.Methods:Adeno-associated virus (AAV) was used as the vector to construct gene-modified NSCs. Three days after SCI attack on the animal model, the NSCs modified by hypoxia-regulated NGF were transplanted to the site of SCI as the NGF group. The GFP-modified neural stem cell group (GFP group), sham group, SCI group were set up. Hindlimb motor function was assessed by Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale, inclined plane tests and footprint analysis at 10 time points on day 1, 3, 7, 10, 14, 21, 28, 35, 42 and 60 after transplantation. The video cassette recorder (VCR) image and quantitative measurement of the height of the rat from the ground, the foot error and plantar steps were used to test the hindlimb support and flexibility of the rats. The degree of spinal cord injury in rats was roughly measured by observing the visual map of the spinal cord. The neuronal repair and morphological changes in SCI area were evaluated by Nissl staining, HE staining and immunofluorescence. CM-DiI was used to trace neural stem cells and to analyze the differentiation of NSCs by immunofluorescence.Results:Two months after transplantation of genetically modified NSCs, the BBB, inclined plane tests and footprint Analytical scores of NGF group rats were higher than those of SCI group and GFP group ( P<0.05); Through VCR image analysis, the hindlimb support and mobility of the rats in the NGF group were better than those in the SCI group and GFP group, and the difference was statistically significant ( P<0.05). Visual analysis showed that the spinal cord of the rats in each group was visually compared to the NGF group, and the spine did not show significant atrophy and color deepening, and the degree of injury was lower than that of the SCI group and GFP group; Through Nissl staining, HE staining and immunofluorescence detection, obviously positive in NeuN at the transplant site was noted at NGF group, and evidently regenerated neural structure can be seen at the morphological level. The cavity in SCI was obviously reduced, neurons and Nissl bodies were distinctly increased ( P<0.05). CM-DiI was used to track NSCs, NeuN was used to mark neurons, and GFAP was used to mark astrocytes. It was found that neural stem cells could differentiate into neurons and astrocytes. Neural stem cells in GFP group were more differentiated into astrocytes, and neural stem cells in NGF group were more differentiated into neurons. Conclusion:NSC transplantation with oxygen-regulated NGF gene mediated by adeno-associated virus can treat SCI, NSCs can differentiate into neural stem cells and astrocytes to fill the damaged cavity, NSCs secrete NGF as the carrier, playing the protective role on adjacent damaged nerve cells and reducing the death of neurons, which is expected to provide new ideas for the treatment of acute spinal cord injury, and at the same time make new attempts for the development of NGF protein drugs.

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differ-ences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononu-clear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8+ T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8+ T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8+ T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immuno-therapeutic approaches should be aimed at not only boosting a HBV-specific CD8+T response but also improving its function.

Endogenous adult neural stem cells are closely related to the normal physiological functions of the brain and many neurodegenerative diseases. Neurons are affected by factors such as extracellular microenvironment and intracellular signaling. In recent years, some specific signaling pathways have been found that affect the occurrence of neural stem cells in adult neural networks, including proliferation, differentiation, maturation, migration, and integration with host functions. In this paper, we summarize the signals and their molecular mechanisms, including the related signaling pathways, neurotrophic factors, neurotransmitters, intracellular transcription factors and epigenetic regulation of neuronal differentiation from both the extracellular and intracellular aspects, providing basic theoretical support for the treatment of central nervous system diseases through neural stem cells approach.

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5'-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5'-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5'- and 3'-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.
Cells, Cultured ; Hepacivirus ; genetics ; growth & development ; metabolism ; Humans ; Protein Biosynthesis ; RNA-Binding Proteins ; metabolism ; Virus Replication ; genetics