An improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (LAMP) assay targeting the 5' untranslated region (UTR) was developed to detect hepatitis C virus (HCV) infection. Based on an accelerating primer (AP), the present assay, named AP-LAMP, has the advantages of rapidity and sensitivity over the routine LAMP method. The possible AP-based amplification pathway during the reaction was revealed by restriction enzyme digestion and eletrophoresis. The detection limit of the AP-LAMP assay was approximately 84 IU/ml, and no cross-detection was observed. The assay was evaluated further with 126 clinical specimens, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for detection of HCV RNA.

Dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is an important pattern recognition receptor on dendritic cells (DCs), and its expression shows significant cytological and histological specificity, being interleukine-4 (IL-4) dependent. The signaling pathways through which IL-4 regulates expression of DC-SIGN are still unclear. We used phorbol 12-myristate 13-acetate- (PMA-) differentiated THP-1 cells as the in vitro model of monocyte/macrophage cells to study the signaling pathways involved in IL-4-regulated expression of DC-SIGN. We found that a high expression of DC-SIGN could be induced by IL-4 at the levels of mRNA and cell surface protein. Upregulated expression of DC-SIGN was almost completely blocked by the specific inhibitor of ERK pathway, and partly reduced by the specific inhibitors of JAK-STAT and NF-κB pathways. The activation of the three signaling pathways was directly confirmed by testing the phosphorylation of protein kinase within the cytoplasm and nucleus over time. The analysis of cis-acting elements of DC-SIGN promoter showed that the activity of DC-SIGN promoter without Ets-1 transcription factors binding site almost completely disappeared. Our results demonstrated that multiple signaling pathways are involved in IL-4 induced high expression of DC-SIGN on THP-1 cells, in which ERK pathway is the main signaling pathway and mediated by the Ets-1 transcription factors binding site.

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In this study, we investigated the mechanisms underlying the anti-inflammatory effects of honokiol in tumor necrosis factor (TNF)-a-stimulated rheumatoid arthritis synovial fibroblasts (RASFs). RASFs pre-treated with honokiol (0 -20 mM) were stimulated with TNF-a (20 ng/ml). The levels of prostaglandin E2 (PGE2), nitric oxide (NO), soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor-b1 (TGF-b1), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1a (MIP-1a) in supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and Griess assay. In addition, protein expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and phosphorylated Akt, nuclear factor kappa B (NFkB), and extracellular signal-regulated kinase (ERK)1/2 were determined by western blot. The expression of NFkBp65 was assessed by immunocytochemical analysis. TNF-a treatment significantly up-regulated the levels of PGE2, NO, sICAM-1, TGF-b1, MCP-1, and MIP-1a in the supernatants of RASFs, increased the protein expression of COX-2, iNOS, and induced phosphorylation of Akt, IkB-a, NFkB, and ERK1/2 in RASFs. TNF-a-induced expression of these molecules was inhibited in a dose-dependent manner by pre-treatment with honokiol. The inhibitory effect of honokiol on NFkBp65 activity was also confirmed by immunocytochemical analysis. In conclusion, honokiol is a potential inhibitor of TNF-a-induced expression of inflammatory factors in RASFs, which holds promise as a potential anti-inflammatory drug.

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