Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10(7) TCID50/mL 10 days after infection when using an MOI of 10(-4). The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.

Squalene-adjuvanted H7N9 virus vaccine induces robust humoral immune response against H7N9 and H7N7 viruses.

Recent cases of avian influenza H7N9 have caused great concerns that virus may become transmittable between humans. It is imperative to develop an effective vaccine to fight against the pandemic potential of this H7N9 influenza virus to protect human from the disease. This study aims to investigate an optimized formulation for the development of H7N9 vaccines. Various doses of H7N9 inactivated whole or split-virus antigens (0.5, 1.5, or 3 µg based on hemagglutinin content) combined with squalene-based adjuvant (AddaVAX), aluminum hydroxide Al(OH)3 or without adjuvant were evaluated for the efficacy of H7N9 vaccine regiments in mice. With either H7N9 whole or split-virus based vaccines, AddaVAX-adjuvanted formulations were the most immunogenic in eliciting significant humoral immune response against H7N9 virus and exhibited strong cross-reactive response in hemagglutination inhibition (HAI) and viral-neutralization assays against H7N7 virus as well. In contrast, formulations with Al(OH)3 or without adjuvant were less immunogenic and elicited lower titers of HAI and microneutralization assays against both viruses. Dose-sparing experiments suggested that the formulation with as low as 0.004 µg of split or whole virus vaccine antigens together with 50% AddaVAX provided sufficient sero-protective HAI titers and achieved essential virus-neutralizing antibody titers against H7-subtype influenza viruses in mice. Protection experiments demonstrated that the formulation of 0.004 µg to 0.5 µg of split-virion vaccines with AddaVAX conferred full protection against viral challenge up to 100 LD50 of wild-type H7N9 virus, with 0% survival in placebo group. Taken together, our study demonstrates that squalene-based adjuvant can significantly enhance the protective efficacy of H7N9 virus vaccine and provides a useful strategy to confront the potential pandemic outbreaks of H7N9 virus.

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Feasibility of individualized treatment for hepatitis C patients in the real world.

BACKGROUND AND AIM: Individualized treatment with a combination of peg-interferon and ribavirin for patients with hepatitis C virus (HCV) infection has been validated in randomized controlled clinical trials, but its usefulness in the real world is unknown. The aim of the present study was to assess the feasibility of individualized treatment for HCV patients compared with standard therapy in a real-life clinical setting. METHODS: A total of 253 naïve patients with HCV infection who received peg-interferon and ribavirin combination treatment were analyzed and grouped into one of three clinical settings: (i) infection with genotype non-1 (HCV non-1) and treatment for standard 24 weeks (n = 105; none received an abbreviated therapy); (ii) genotype 1 (HCV-1) and standard therapy for either 24 weeks (n = 71) or 48 weeks (n = 21); and (iii) HCV-1 and individualized treatment (n = 56). The individualized therapy used was an abbreviated 24-week treatment for HCV-1 patients who achieved a rapid virological response, otherwise patients received a 48-week course of treatment. Early termination of treatment at week 16 was recommended for non-responders. RESULTS: A sustained virological response (SVR) was achieved in 83.8% of patients with HCV non-1 infection. Among the HCV-1-infected patients, 53.5% of patients who underwent standard 24-week treatment, 66.7% of patients who underwent standard 48-week treatment, and 64.3% of patients treated by individualized therapy achieved SVR. Patients infected with HCV-1 and treated by individualized therapy had a similar efficacy response compared with the standard 48-week therapy (adjusted odds ratio [OR] 0.765, 95% confidence interval [CI], 0.220-2.659, P = 0.673). Both individualized therapy (adjusted OR 2.855, 95% CI 1.189-6.855, P = 0.019) or standard 48-week treatment (adjusted OR 3.733, 95% CI 1.073-12.986, P = 0.038) had significantly higher odds of SVR compared with HCV-1 patients treated by standard 24-week course. CONCLUSION: Individualized therapy is feasible in the real world, especially for patients with HCV-1 infection.

Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif.

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alphabeta motif (CS alphabeta) consists of an alpha-helix and an antiparallel triple-stranded beta-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CS alphabeta motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CS alphabeta motif. The information is valuable to the scaffold with the CS alphabeta motif and protein engineering.

Changes in visual function during the Coriolis illusion.

BACKGROUND: The Coriolis illusion produces spatial disorientation and is, therefore, dangerous for pilots. It is not known whether it also affects visual function (visual acuity and stereopsis). METHODS: There were 18 subjects (15 men and 3 women, mean age 24.7 yr) enrolled in the study. A spatial disorientation simulator was used to produce Coriolis stimulation. The visual acuity of the subjects was evaluated with the Rosenbaum Vision Card before and during Coriolis stimulation. Stereopsis was measured with the Titmus stereo test. Throughout the experiments, eyeball movements were observed on a television monitor. Electrooculography (EOG) and electroencephalography (EEG) were also documented. RESULTS: Before Coriolis stimulation, the visual acuity and stereopsis of all subjects were 20/20 and 40 s of arc, respectively. During the Coriolis illusion, the visual acuity of nine subjects (50%) remained 20/20, whereas the visual acuity of the others (50%) dropped by two lines. The stereopsis of most subjects (77.8%) decreased to 800 arc-seconds or less. Rhythmic nystagmus was observed, while EOG amplitudes were significantly elevated compared with those at baseline (9.41 +/- 0.26 microv2 and 8.45 +/- 0.36 microv2, respectively). EEG activity (frequency) was also greater than at baseline (13.15 +/- 0.84 Hz and 11.94 +/- 1.20 Hz, respectively; P < 0.05). CONCLUSIONS: During Coriolis stimulation, the visual acuity of the subjects remained stable, but their stereopsis was reduced. Further study is warranted.

Protein profilings in mouse liver regeneration after partial hepatectomy using iTRAQ technology.

Liver is unique in its capability to regenerate after an injury. Liver regeneration after a 2/3 partial hepatectomy served as a classical model and is adopted frequently to study the mechanism of liver regeneration. In the present study, semiquantitative analysis of protein expression in mouse liver regeneration following partial hepatectomy was performed using an iTRAQ technique. Proteins from pre-PHx control livers and livers regenerating for 24, 48 and 72 h were extracted and inspected using 4-plex isotope labeling, followed by liquid chromatography fractionation, mass spectrometry and statistical differential analysis. A total of 827 proteins were identified in this study. There were 270 proteins for which quantitative information was available at all the time points in both biologically duplicate experiments. Among the 270 proteins, Car3, Mif, Adh1, Lactb2, Fabp5, Es31, Acaa1b and LOC100044783 were consistently down-regulated, and Mat1a, Dnpep, Pabpc1, Apoa4, Oat, Hpx, Hp and Mt1 were up-regulated by a factor of at least 1.5 from that of the controls at one time point or more. The regulation of each differential protein was also demonstrated by monitoring its time-dependent expression changes during the regenerating process. We believe this is the first report to profile the protein changes in liver regeneration utilizing the iTRAQ proteomic technique.

Management and patient survival in hepatocellular carcinoma: does the physician's level of experience matter?

INTRODUCTION: The prognostic determinants of hepatocellular carcinoma (HCC) depend on tumor stage, liver function reserve, and treatments offered. The clinical impact of the physician's experience on HCC management and the survival outcome is unknown. METHODS: A total of 103 patients were managed by one high-volume physician and 249 patients by seven low-volume physicians. The experience of high-volume physician in HCC management was five times more than that of low-volume physicians. Patient survival was the single end point for this study. RESULTS: Compared to the low-volume physician group, more of the patients allocated to the high-volume physician had early stage HCC on the date of diagnosis (66/103, 64.1%; vs 119/249, 47.8%; P = 0.008), and they received curative therapies including radiofrequency ablation or liver resection (66/103, 64.1% vs 54/249, 21.7%, P < 0.001), and had significantly better survival outcome (median survival of 34 months, 95% confidence interval [CI], 17.6-50.4; vs 6 months, 95% CI, 4.3-7.7; P < 0.001) with a multivariable-adjusted hazard ratio (HR) for survival of 1.94 (95%, CI, 1.31-2.87, P < 0.001). A multivariate analysis of the pretreatment prognostic factors for these two groups identified alpha-fetoprotein (AFP) level (HR, 1.42; 95% CI, 1.01-1.99; P = 0.042), ascites (HR, 1.68; 95% CI, 1.15-2.46; P = 0.007), maximum tumor diameter (HR, 1.78; 95% CI, 1.16-2.74; P = 0.009), and portal vein thrombosis (PVT) (HR, 2.17; 95% CI, 1.49-3.17; P < 0.001) as independent factors for the low-volume physician group. However, only maximum tumor diameter (HR, 4.54; 95% CI, 1.77-11.67; P < 0.001) and PVT (HR, 5.73; 95% CI, 2.30-14.22; P = 0.002) were independent factors for the high-volume physician group. CONCLUSION: The survival of HCC patients was dependent on the level of experience of the physicians who oversaw these patients.

The possible interaction of CDA14 and protein elongation factor 1alpha.

The CDA14, a 45kD protein, is currently annotated as PTX1-like protein or ERGIC 32. Over expressing CDA14 can slow PC11 cell proliferation rate. In HepG2 cells, it had been demonstrated that CDA14 is involved in protein transportation. The knowledge about the protein is very limited and not clarified. The CDA14 and its homologous proteins form a family and are restricted to eukaryotes. In the family, there are no homologous sequences with resolved three-dimensional structure and their functions are difficult to predict. Transcriptional expression of CDA14 in three hepatoma cell lines, Hu7, HCC and HepG2, was lower than normal liver tissue and liver carcinoma tissue. In this study, functional proteomic techniques were utilized in searching the interacting counterpart of CDA14. Several proteins involved in protein translation and folding were selectively precipitated with CDA14 and identified mass spectrometry. Interaction of CDA14 and elongation factor 1alpha was confirmed by Western blotting and confocal microscopy. Elongation factor 1alpha is a multiple function protein and involved in several biological mechanisms, including protein synthesis, cell proliferation, apoptosis and tumorigensis. Over-expression of CDA14 down regulated the proliferation of HepG2 cells. These results suggest that CDA14 participated in the elongation factor 1alpha regulated mechanisms.

Predictors of alpha-fetoprotein elevation in patients with chronic hepatitis C, but not hepatocellular carcinoma, and its normalization after pegylated interferon alfa 2a-ribavirin combination therapy.

BACKGROUND AND AIM: Elevated serum alpha-fetoprotein (AFP) levels are noted in patients with chronic hepatitis C (CHC) without hepatocellular carcinoma (HCC). The change in AFP levels after treatment with pegylated interferon and ribavirin (Peg-IFN/RBV) combination therapy is still unknown. The aim of this study was to investigate the predictors of elevated serum AFP in patients with CHC, and its change after Peg-IFN/RBV therapy. METHODS: A total of 123 patients, intended to receive pegylated interferon alfa-2a plus ribavirin therapy, were enrolled. Eighty-three patients had complete treatment and received follow up for and additional 24 weeks. The factors that may affect the elevation of pretreatment AFP and the normalization of post-treatment AFP were determined. RESULTS: The mean AFP level was 18.5 +/- 63.0 ng/mL (range, 1.3-676.0 ng/mL); 41 (33.3%) of the 123 patients had elevated serum AFP (more than 10 ng/mL) at baseline. A multivariate logistic regression analysis disclosed that older age (odds ratio [OR], 1.093; 95% confidence interval [CI], 1.015-1.177; P = 0.018), more advanced METAVIR fibrosis stage (OR, 5.237; 95% CI, 1.244-22.037; P = 0.024), a higher aspartate aminotransferase (AST) level (IU/L) (OR, 1.020; 95% CI, 1.008-1.033; P = 0.001), and lower platelet count (x10(9)/L, OR, 0.985; 95% CI, 0.968-0.994; P = 0.003) were independent determinants of pretreatment AFP elevation. After treatment, 72 of 83 (86.7%) cases were found to have normal post-treatment AFP levels (<10 ng/mL) at the end of follow up (EOF). Post-treatment negativity of the chronic hepatitis C virus (HCV)-RNA (OR, 10.014; 95% CI, 1.000-100.329; P = 0.050) and the post-treatment platelet count (x10(9)/L) (OR, 1.025; 95% CI, 1.001-1.050; P = 0.040) were associated with normal AFP at EOF. AFP progressively decreased with significant differences starting from the 12th week after treatment to the end of treatment, and was lowest at the EOF date for the sustained viral response (SVR) group. On the contrary, the non-SVR group did not have an AFP change during and after treatment. CONCLUSION: Older age, low platelet count, higher AST levels, and advanced fibrosis predisposed chronic hepatitis C patients without HCC to have elevated serum AFP levels. After Peg-IFN/RBV combination therapy, a higher platelet count and HCV viral eradication were determinants of normal AFP at EOF. Serial AFP levels decreased after treatment, presenting in a time-dependent manner, specifically for the SVR group.