Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor.

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

Milk and dairy consumption and risk of bladder cancer: a meta-analysis.

OBJECTIVE: To explore potential relations between the intake of milk or dairy products and the risk of bladder cancer. METHODS: Eligible studies published up to May 2011 were retrieved via both computer searches and manual review of references. Random-effects models were used to calculate summary relative risk estimates (SRRE) based on high-contrast to low-intake values. Sensitivity and influence analyses were conducted, and heterogeneity among study results was explored through stratified analyses by study design, gender, geographic region, year of publication, or whether or not adjustment for several confounders (ie, age, gender, body mass index, smoking, and total energy intake). RESULTS: We extracted data from 14 studies on milk (involving 4879 cases) and 6 studies on dairy products (3087 cases). The total study population was up to 324,241 individuals. Overall, there was no significant association between milk intake and bladder cancer (SRRE 0.89, 95% CI 0.77-1.02). However, an inverse association was found in the United States (SRRE 0.88, 95% CI .79-.99). In addition, no significant association was observed between consumption of dairy products and risk of bladder cancer (SRRE 0.95, 95% CI .71-1.27), though an inverse association was detected in the Japanese population (SRRE 0.56, 95% CI .40-.80). CONCLUSION: There appears to be enough evidence to support the null hypothesis. The overall result was not statistically significant. The findings of this meta-analysis are not supportive of an independent relationship between the intake of milk or dairy products and the risk of bladder cancer. However, these findings are based on limited research. Further efforts should be made to confirm these findings.

[Immobilization of streptavidin-tagged bioactive hTNF-alpha on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice].

OBJECTIVE: To investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-alpha on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice. METHODS: A total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-alpha, SA-GFP, and SA-hTNF-alpha treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-alpha fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions. Immunohistochemistry was performed to detect the retention time of SA-hTNF-alpha fusion protein in the biotinylated mouse bladder mucosa and the distribution of CD4(+) and CD8(+) lymphocytes in the mucosa and tumor tissues, with the tumor growth and mouse survival also observed. The cytotoxiciy of the tumor-specific lymphocytes was evaluated. The mice responding well to the treatment were re-challenged by MB49 and monitored for survival. RESULTS: SA-hTNF-alpha could be efficiently and stably immobilized on the bladder mucosal surface for as long as 7 days. On day 60 after MB49 implantation, 18 out of 22 SA- hTNF-alpha-treated mice survived, with 9 appearing tumor-free, but all the mice in PBS control group died. Five out of 9 tumor-free mice in SA-hTNF-alpha group showed resistance to a re-challenge with intravesical MB49. The numbers of CD4(+) and CD8(+) lymphocytes were significantly greater in SA-hTNF-alpha group than in the other groups (P<0.05). The cytotoxicity of the tumor-specific lymphocytes was significantly stronger in SA-hTNF-alpha group than in the other groups (P<0.05). CONCLUSION: SA-hTNF-alpha immobilized on the biotinylated mucosal surface of the bladder wall can significantly inhibit the tumor growth and promote the survival of the mice bearing orthotopic superficial bladder cancer.

[A simple and efficient method for establishing a mouse model of orthotopic MB49 bladder cancer].

OBJECTIVE: To establish a simple and efficient method for establishing a mouse model of orthotopic superficial bladder cancer. METHODS: C57BL/6 mice were anesthetized with sodium pentobarbital and catheterized with modified IV catheter (24 G). The mice were intravesically pretreated with HCl and then with NaOH, and after washing the bladders with phosphate-buffered saline (PBS), 100 microl (1 x 10(7)) MB49 cells were infused and allowed to incubate in the bladder for 2 h followed intravesical mitomycin C (MMC) administration. The tumor formation rate, survival, gross hematuria, and bladder weight were determined as the outcome variables, and the pathology of the bladders was observed. RESULTS: Instillation of MB49 tumor cells resulted in a tumor formation rates of 100% in all the pretreated groups while 0% in the control group without pretreatment. MMC significantly reduced the bladder weight as compared to PBS. CONCLUSION: We have successfully established a stable, reproducible, and reliable orthotopic bladder cancer model in mice.

[Effect of adenovirus-mediated TK/GCV gene therapy in combination with TNF-alpha against murine bladder cancer cells in vitro].

OBJECTIVE: To investigate the cell-killing effect of adenovirus-mediated TK-ganciclovir (GCV) gene therapy in combination with tumor necrosis factor-alpha (TNF-alpha) against murine bladder carcinoma cells in vitro. METHODS: Murine bladder carcinoma MB49 cells were transfected with the adenoviral vector containing TK gene and green fluorescent protein (GFP) gene. The transfection efficiency was observed and the TK gene expression in the transfected cells was detected by RT-PCR. The survival rate of MB49 cells in response to TNF-alpha treatment and that of the TK gene-transfected cells after treatment with GCV and GCV+TNF-alpha were determined by MTT assay. The apoptosis of the cells after the treatments was analyzed by flow cytometry. RESULTS: In cells transfected with TK gene, the cell inhibition rate increased gradually with the increment of GCV and TNF-alpha concentration. GCV in combination with TNF-alpha resulted in significantly increased killing efficiency of the cells as compared with GCV or TNF-alpha treatment alone, and the effect of the combined treatment was enhanced as the TNF-alpha concentration increased. GCV treatment (50 microg/ml) alone produced a cell killing rate of (24.39-/+1.10)%, and when combined with 5 microg/ml TNF-alpha, the rate was increased to (40.05-/+0.97) %, and further to (65.47-/+0.67) % when TNF-alpha concentration increased to 20 microg/ml. Flow cytometry revealed obvious apoptosis of the cells 8 h after treatments with TK/GCV, TNF-alpha, or TK/GCV+TNF-alpha, and the combined treatment resulted in the highest cell apoptotic rate. CONCLUSION: TK/GCV in combination with TNF-alpha can enhance the effect of suicide gene therapy against murine bladder carcinoma cells and effectively induce apoptosis of the cells.

[Lethal effect of adenovirus-mediated HSV-TK gene in combination with hydroxycamptothecin on human bladder cancer in vitro].

OBJECTIVE: To evaluate the lethal effect of adenovirus-mediated HSV-TK-ganciclovir (GCV) gene therapy in combination with hydroxycamptothecin (HCPT) on hunman bladder carcinoma cell line T-24 cells. METHODS: Human bladder carcinoma cell line T-24 was transfected with adenovirus expression vector containing TK gene and green fluorescent protein (GFP) gene, and the transfection efficiency was observed and TK expression detected by PCR. After successful cell transfection indicated by GFP expression, GCV and hydroxycamptothecin are respectively added into the cell culture with normal T-24 cells serving as the blank control group. The growth inhibition rate of hunman bladder carcinoma cells in response to HCPT treatment for 72 h and the cell survival rate of 24 h, 48 h and 72 h after transfection with different protocols were observed by MTT assay. The apoptosis of the cells treated with GCV (0.5 mg/ml)+HCPT (10 mg/L) for 4 h was observed by flow cytometry. RESULTS: The cell inhibition rate increased gradually with increment of HCPT concentration, from 14% at HCPT concentration of 0.01 mg/L to 60% at 50 mg/L, but for a concentration above 100 mg/L, the inhibition rate did not exhibit further increase (P=0.216). GCV alone and GCV in combination with HCPT both resulted in significantly decreased survival rate of human bladder carcinoma cells (P=0.00), and the killing efficiency of the cells by GCV+HCPT protocol increased obviously with increment of HCPT concentration and prolongation of the action time. The cells treated with 0.5 mg/ml GCV alone for 72 h retained a cell survival rate of 34.6%, which was lowered to only 8.07% with combined treatment with GCV (0.5mg/ml) and HCPT (10 mg/L). Typical apoptotic peak before M1 phase of the cells appeared 4 h after treatment with GCV+10 mg/ml HCPT, which resulted in a apoptosis rate of 52.93%. CONCLUSION: HSV-TK/GCV in combination with HCPT can enhance the lethal effect of suicide gene therapy against human bladder carcinoma cells and effectively induce apoptosis of the cells.