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To investigate the effect of thrombocytopenia on the migration patterns of adoptive dendritic cell(DC) in vivo.The mouse model of thrombocytopenia was established by intraperitoneal administration of anti-CD41 mAb MWReg30. Mouse bone marrow(BM)-derived DC were injected into thrombocytopenia mouse by footpad infusion and intravenous infusion. The DC migration and distribution pattern were detected by bioluminescence imaging.More than 80% platelets were cleared in the experimental group which was infused with anti-CD41 antibody. At 72 h after injection, the percentage of injected DC that migrated from footpad to popliteal lymph nodes(PLNs) and inguinal lymph nodes(ILNs) were (0.32±0.02)% and (0.02±0.01)% in experimental group, and (0.27±0.15)% and (0.02±0.02)% in control group, respectively. Statistic data showed that there was no statistical difference between these 2 groups (P0.05). The issue distribution pattern of intravenously injected DC between experimental group and control group were not distinctly different, and large amounts of injected DC accumulated in the spleen, liver draining lymph-nodes lungs and liver.Thrombocytopenia has not a distinct effect on the migratory capacity and tissue distribution of DC by either footpad or intravenous injection.

In this study, we further investigated a previously developed aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells. We found that this C6-8 aptamer specifically binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and that it specifically labeled multiple tumor-cell lines as effectively as hnRNP A2/B1 monoclonal antibodies. When conjugated with fluorescent carbon nanodots (CDots) it could freely enter multiple living tumor cell lines (HepG2, MCF-7, H1299, and HeLa), whose growth it inhibited by targeting hnRNP A2/B1. Similar inhibitory effects were observed when the GFP-HepG2 hepatocarcinoma cells treated with C6-8-conjugated CDots were implanted in nude mice. Our work provides a new aptamer for targeting/labeling multiple tumor cell types, and its nanoparticle conjugates bring further advantages that increase its potential for use in cancer diagnosis and therapy.

Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.

Background/Aims Interferon beta (IFN-β) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-β activation in mouse liver by noninvasive molecular imaging. Methods Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-β. Luciferase activity was used as an indicator of the IFN-β promoter activity in vitro and in vivo. The expression level of IFN-β in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. Results A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-β in the sera was not induced by MHV-3. Moreover, IFN-β activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-β production in the sera. Therefore, a novel visual method to monitor IFN-β promoter activity was established in the current study. Conclusion This novel sensitive method can be used for not only assessing IFN-β activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-β production.

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

AIM: To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). METHODS: The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. RESULTS: HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-β promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. CONCLUSION: Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.

The lack of robust small animal models has been an obstacle to the screening of Hepatitis C virus (HCV) NS3/4A protease inhibitors in vivo. Here, we described a reporter assay system for in vivo noninvasive imaging of NS3/4A serine protease activity using split firefly luciferase complementation strategy. The reporter construct ANluc(NS5A/B)BCluc constitutes the split N- and C-terminal fragments of luciferase, fused to interacting peptides, pepA and pepB, respectively, with an intervening HCV NS3/4A cleavage motif of NS5A/B. We proved that the reporter molecule could be proteolytically cleaved by NS3/4A at the NS5A/B motif in cells and living animals. Association of pepA and pepB brought inactive fragments of luciferase into close proximity, thereby restoring bioluminescence activity. The increase in luciferase activity was proportional to the dose of active NS3/4A protease. The ANluc(NS5A/B)BCluc reporter also could be used to detect the activity of NS3/4A-specific shRNA and IFN-alpha. Therefore, the reporter assay system using split firefly luciferase complementation strategy should prove useful for evaluating NS3/4A protease activity in cells and living animals.

Background The development of small molecule inhibitors of hepatitis C virus (HCV) core protein as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, lack of a robust and convenient small animal model has hampered the assessment of in vivo efficacy of any antiviral compound. Methodology/Principal Findings The objective of this work was to develop a novel method to screen anti-core protein siRNA in the mouse liver by bioluminescence imaging. The inhibitory effect of two shRNAs targeting the highly conserved core region of the HCV genome, shRNA452 and shRNA523, was examined using this method. In the transient mouse model, the effect of shRNA-523 was detectable at as early as 24 h and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48 h post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6 h and 12 h after injection respectively, and the inhibitory effect persisted for 1 day after a single injection while shRNA-Scramble did not seem to have an effect on the luciferase activity in vivo. Conclusions/Significance Thus, we developed a simple and quantitative assay for real-time monitoring of HCV core protein inhibitors in mice.

The recognition of human leukocyte antigen (HLA) molecules by specific receptors is a crucial step in the regulation of natural killer (NK) cell function. Killer cell immunoglobulin-like receptor (KIR) 3DS1 is one of the activating receptors of NK cell and is implicated in slowing disease progression in HIV infection. KIR3DS1 play an important role in the outcome of multiple diseases associated with viral infections. In contrast to the inhibitory receptor, much less is known about the ligands of KIR3DS1. In order to achieve a better understanding of the biology of KIR3DS1 and its ligand systems, it is necessary to identify the ligands of KIR3DS1. In this work, we utilized recombinant HLA-B2705 molecules and DsbA-KIR3DS1 fusion protein to monitor the interaction between HLA-B2705 complexes and DsbA-KIR3DS1 using BIAcore 3000 SPR sensor and found that the specific binding between KIR3DS1 and HLA-B2705 existed and the affinity was 6.95x10(-6) mol//L. So we concluded that HLA-B2705 is a possible ligand of KIR3DS1.

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

To evaluate the role of adiponectin in regulating tumor necrosis factor alpha (TNF alpha) production and preventing fulminant autoimmunological damage of hepatocytes following concanavalin A (Con A) injection into mice.Three days after recombinant plasmids pAA-neo-mAd were injected into the mice via the tail veins, Con A was injected into the mice. Mice transfected with empty pAA-neo vector served as controls. The serum levels of alanine aminotransferase (ALT), TNF alpha and adiponectin were detected, and histological examination of livers was carried out at different time points after the Con A injection. All results were subjected to statistical analyses.Histological examinations showed that the damage in livers of mice with high serum adiponectin levels was milder than that of the controls. The serum levels of ALT and TNF alpha were both lower than those of the controls (P less than 0.01, respectively). Statistical analyses showed the serum levels of ALT was negatively related to the levels of adiponectin in the sera (r=-0.5034).Adiponectin is effective in protecting hepatocytes from Con A-induced immunological injury. The mechanism of this protective effect may be caused by inhibiting the synthesis and/or release of TNF alpha.

AIM: To establish nested-PCR methods for the detection of SENV-D and SENV-H and to investigate the epidemiology of SEN virus in China. METHODS: According to published gene sequences, primers from the conserved region were designed. Then, 135 samples from healthy voluntary blood donors and 242 samples from patients with various forms of liver disease were detected by nested-PCR of SENV-D/H. Some PCR products were cloned and sequenced. RESULTS: By sequencing, the specificity of genotype-specific PCR was confirmed. SENV-D/H DNA was detected in 31% of the blood donors, which was higher than those in America and Italy (2%), and in Japan and Taiwan (15%-20%). The prevalence of SENV-D/H viremia was significantly higher in patients with hepatitis B and hepatitis C than in blood donors (59%-85% vs 31%, P < 0.05). The prevalence among patients with non-A-E hepatitis was significantly higher than among blood donors (68% vs 31%, P < 0.01), and equivalent to that among patients with hepatitis B and C. CONCLUSION: Nested-PCR with genotype-specific primers could serve as a useful SENV screening assay. SENV has the same transmission modes as HBV and HCV. The high prevalence in patients with non-A-E hepatitis may attribute to the transmission modes, and SENV may not serve as the causative agents.

To express stably human CD81 gene on mouse hepatoma cell line Hepa 1-6 using liver specific promoter.RNA were isolated from human HepG2 cells which could be infected with hepatitis C virus. RT-PCR was carried out using human CD81 gene specific primers. Amplified fragments were cloned into pGEM-T vector. Albumin promoter and enhancer which were liver tissue specific were ligated to the 5'end of human CD81 gene and SV40 polyA sequence was fused with 3'end of CD81. The fused CD81 gene was inserted into eukaryotic expression vector pcDNA3 to construct a recombinant vector pcDNA3-Alb p-CD81 which was then transfected into Hepa 1-6 cells through lipofectamine mediation. Human CD81 mRNA transcription and its protein expression were detected by RT-PCR and FACS, respectively.Sequence analysis showed that the cloned gene segment was human CD81 gene sequence. After transfection, transcripted human CD81 mRNA was obtained and human CD81 molecules were expressed stably on Hepa 1-6 cells.The obtained positive cell clones which stably express HCV receptor human CD81 lay the foundation for further study on interactions between HCV envelope proteins and human CD81, screening of HCV infection blocking drugs and development of HCV infection mouse model.

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1–6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.