[Effects of TFDP3 on regulating the autophagy and apoptosis of LNCaP cells].

AIM: To investigate the effect of TFDP3 on prostate cancer cell line LNCaP by transgenic method, and to explore the effect of TFDP3 on regulating the autophay and apoptosis by co-regulation with E2F1. METHODS: LNCaP cells were transfected with pcDNA3.1-TFDP3, pCMV-E2F1-HA or pcDNA3.1 empty vector.The expression of TFDP3, E2F1 and LC3B were detected by real-time PCR after transfection for 24 h. Western blotting was used to monitor the changes in autophagy-associated protein LC3B, Apoptosis of transfected cells were analyzed by flow cytometry. RESULTS: The results showed that activation of TFDP3 upregulates the expression of autophagy genes-microtubule-associated protein-1 light chain-3B (LC3B), and E2F1 antagonizes TFDP3-induced autophagy, and TFDP3 can inhibit E2F1-induced apoptosis. CONCLUSION: TFDP3 upregulates the expression of autophagy gene LC3B and inhibits E2F1-induced apoptosis, and may play an important role in prostate cancer.

Evaluation of a new rapid influenza A diagnostic test for detection of pandemic (H1N1) 2009 and seasonal influenza A virus.

BACKGROUND: Rapid influenza A diagnostic tests (RIDTs) play an important role in the clinical setting, especially in the influenza post-pandemic era with three influenza A viruses in circulation. OBJECTIVES: Determine the sensitivity and specificity of a new RIDT (FluA Dot) by comparison with BD Directigen EZ FluA+B and CDC rRT-PCR. STUDY DESIGN: Two sets of experiments were conducted to determine the performance of the new test. (1) Serial dilutions of eight pandemic (H1N1) 2009 (pH1N1) isolates, five seasonal H3N2 isolates, five seasonal H1N1 isolates and three recombinant nucleoproteins were tested by FluA Dot assay, Directigen EZ FluA+B test and CDC real-time RT-PCR. (2) Using CDC rRT-PCR as the gold standard, the clinical sensitivity and specificity of the FluA Dot and Directigen EZ FluA+B were evaluated in nasopharyngeal swab (NPS) specimens of 807 patients presenting with influenza-like illness. RESULTS: The average analytical sensitivity of FluA Dot (0.06 ng/mL for recombinant nucleoproteins and 2.16 ± 0.85 log 10 TCID(50) for viruses) was approximately 10-fold higher than Directigen EZ FluA+B (1-2 ng/mL for recombinant nucleoproteins and 3.54 ± 0.81 log 10 TCID(50) for viruses), and was approximately 10-fold lower than the CDC rRT-PCR (1.09 ± 0.69 log 10 TCID(50) for viruses). Among 807 NPS specimens tested, the sensitivities and specificities of FluA Dot were 91.1% (95%CI: 86.7-94.4%)/99.7% (95%CI: 98.7-99.9%), and the Directigen EZ FluA+B were 71.9% (95%CI: 65.7-77.6%)/99.8%(95%CI: 99.0-99.9%). CONCLUSION: The new test (FluA Dot) exhibit higher sensitivity than Directigen EZ FluA+B both in pH1N1 and seasonal influenza A detection. The promising RIDT can play important roles in influenza diagnosis and therapy.

Anti-HCV reactive volunteer blood donors distribution character and genotypes switch in Xi'an, China.

HCV is prevailed in the world as well as in China. Blood transfusion is one of the most common transmission pathways of this pathogen. Although data of HCV infection character were reported during the past years, anti-HCV reactive profile of China donors was not fully clear yet. Furthermore, infection progress was found related to the HCV genotype. Different genotype led to different efficacy when interferon was introduced into HCV therapy. Here we provided character data of HCV infection in China blood donors from the year of 2000 to 2009. The infection rate in local donors was lower than general population and descended from 0.80% to 0.40% or so in recent years. About 83% HCV strains were categorized into genotypes 1b and 2a. But 1b subtype cases climbed and 2a subtype cases decreased. The current study threw more light on HCV infection of blood donors in China, at least in the Northern region.

Immune response to fused core protein of hepatitis C virus and truncated tetanus toxin peptides in mice.

Because no vaccine or effective therapy is available, thousands of people with HCV have died in recent years. Cytotoxic T lymphocytes (CTLs) play a critical role in the host cellular immune response against HCV. CTL epitopes in HCV core protein have been identified and used in vaccine development. T helper epitopes could promote cytokine secretion and antibody production to fight HCV. Tetanus toxin, an immunogen with many T helper epitopes, was once used in HBV therapeutic vaccine design. Here, eukaryotic and prokaryotic expression vectors were constructed to express truncated fragments of tetanus toxin and core genes of HCV. HLAA2.1 transgenic mice were inoculated with a recombinant plasmid vehicle with these two heterogenic gene fragments, and this augmented the titres of antibody against HCV. Antigen-specific lymphocyte proliferation, Th1 and Th2 cytokine levels and the number of lysed cells were markedly increased in the combined immunization group compared to controls. These findings provide new insights into a potential role for T helper epitopes from tetanus toxin combined with protein from the HCV core gene, which has numerous CTL epitopes. This design strategy may aid in the development of new vaccines against HCV.

Hepatitis B virus genotypes and evolutionary profiles from blood donors from the northwest region of China.

Hepatitis B virus (HBV) is prevalent in China and screening of blood donors is mandatory. Up to now, ELISA has been universally used by the China blood bank. However, this strategy has sometimes failed due to the high frequency of nucleoside acid mutations. Understanding HBV evolution and strain diversity could help devise a better screening system for blood donors. However, this kind of information in China, especially in the northwest region, is lacking. In the present study, serological markers and the HBV DNA load of 11 samples from blood donor candidates from northwest China were determined. The HBV strains were most clustered into B and C genotypes and could not be clustered into similar types from reference sequences. Subsequent testing showed liver function impairment and increasing virus load in the positive donors. This HBV evolutionary data for China will allow for better ELISA and NAT screening efficiency in the blood bank of China, especially in the northwest region.

Inhibition of prostate cancer by suicide gene targeting the FCY1 and HSV-TK genes.

Prostate cancer is one of the most prevalent tumors. The switch of androgen signal dependence makes therapy more complex. Although reports on introduction of a single suicide gene exist, double suicide gene therapy has not been reported yet. In the current study, two suicide genes were constructed in the pIRES plasmid driven by PSMA promoter. 5-FC and GCV combination in vitro led to a higher growth inhibition on prostate cancer compared to a single pro-drug. Retarded xenograft tumor growth was observed in castrated nude mice after double suicide gene activation. Furthermore, decreased metastasis was observed with double suicide gene treatment. These findings suggest that specific double suicide gene strategy could be a potential option for the therapy of prostate cancer.

Survivin gene RNA interference inhibits proliferation, induces apoptosis, and enhances radiosensitivity in HeLa cells.

OBJECTIVE: Survivin is a new member of the inhibitors of apoptosis (IAPs) family. It is upregulated in various malignancies including human cervical carcinomas. Reduction of this molecule has resulted in chemosensitization, but it is uncertain whether it can lead to radiosensitization. We observed the effect of survivin gene RNA interference (RNAi) on the proliferation, apoptosis, and radiosensitivity of the human cervical carcinoma cells HeLa. STUDY DESIGN: Human cervical carcinoma cells (HeLa) were transfected with the specific siRNA expression vector (pSilencer2.1-s2) designed to target survivin mRNA. A corresponding site-mutated vector was constructed as a negative control (pSilencer2.1-NC). The expression of survivin mRNA and its protein among the stable transfected cells and the untransfected ones was detected by semi-quantitative RT-PCR and Western blotting respectively. The cell growth was examined by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle distribution and cell apoptosis were measured by flow cytometry. The changes in cell radiosensitivity were observed by clonogenic survival assay. RESULTS: Three stable transfected cell lines: HeLa-s2 (with pSilencer2.1-s2), HeLa-NC (with pSilencer2.1-NC), and HeLa-U6 neo (with empty vector pSilencer2.1-U6 neo) were established. The expression levels of survivin gene mRNA and protein in HeLa-s2 were significantly lower than in HeLa-NC, HeLa-U6 neo, and those untransfected HeLa cells. The expression inhibitory rates were 62.8% and 60.1%. The cell proliferation of HeLa-s2 was inhibited, and the highest inhibitory rate was 57.8+/-2.1%. The changes in cell cycle distribution in HeLa-s2 compared with the other three cell lines were obvious, many cells were blocked in the G(0)/G(1) phase 72.7+/-3.1% (P<0.05), reduced sharply in the G(2)/M phase (5.1+/-2.9)% (P<0.05), and also the apoptotic rate was 29.2+/-1.4%, obviously increasing (P<0.05). At the same dose of radiation, the cloning efficiency of HeLa-s2 declined notably (P<0.05); the cell survival curve showed a significant decrease in D(0) and D(q), which were 3.15 and 1.21, respectively (P<0.05), and the radiation enhancement ratios were 2.01 (a ratio of D(0)) and 1.77 (a ratio of D(q)). CONCLUSIONS: Survivin gene RNAi not only could inhibit the proliferation of human cervical carcinoma cells (HeLa), but also could significantly enhance the radiosensitivity of those cells through the reduction of its mRNA and protein. Therefore, an RNAi-targeted survivin gene strategy would be a potential approach to radiosensitization therapy in human cervical carcinomas.

In vitro and in vivo prodrug therapy of prostate cancer using anti-gamma-Sm-scFv/hCPA fusion protein.

BACKGROUND: Raising selectivity to tumor cells is a major challenge for most chemotherapy drugs. One of approaches to realizing this goal is antibody-directed enzyme prodrug therapy (ADEPT). This study was done to investigate the curative effect of a new ADEPT system for the treatment of prostate cancer. METHODS: Methotrexate (MTX) prodrugs were synthesized and anti-seminoprotein (SM) single-chain antibody/human carboxypeptidase-A fusion protein (scFv/hCPA) was prepared. Therapeutic effects of this ADEPT system were evaluated. RESULTS: The synthesis of prodrugs was successful and the prodrugs were confirmed no cytotoxicity, but hydrolysis with tumor-targeted scFv/hCPA fusion protein gave 1,000-fold higher cytotoxicity than MTX-alpha-Phe only. Cell cycle assays showed that tumor cells were arrested in the S phase after ADEPT treatment; furthermore, tumors were inhibited significantly in scFv/hCPA and MTX-alpha-Phe treated mice. CONCLUSIONS: Our results suggest that targeted activation cytotoxicity against established prostate cancer by scFv/hCPA mediated ADEPT is tumor-specific and has no systemic toxicity in vitro and in vivo.