RESUMO
Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Ânions , Antígenos Virais , Infecções por Enterovirus/prevenção & controle , Humanos , Cloreto de Sódio , SacaroseRESUMO
This study aimed to compare different types of right breast cancer radiotherapy planning techniques and to estimate the whole-body effective doses and the critical organ absorbed doses. The three planning techniques are intensity-modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT; two methods) and hybrid 3D-CRT/IMRT (three-dimensional conformal radiotherapy/intensity-modulated radiotherapy). The VMAT technique includes two methods to deliver a dose: non-continuous partial arc and continuous partial arc. A thermoluminescent dosimeter (TLD) is placed in the RANDO phantom to estimate the organ absorbed dose. Each planning technique applies 50.4 Gy prescription dose and treats critical organs, including the lung and heart. Dose-volume histogram was used to show the planning target volume (V95%), homogeneity index (HI), conformity index (CI), and other optimized indices. The estimation of whole-body effective dose was based on the International Commission on Radiation Protection (ICRP) Publication 60 and 103. The results were as follows: Continuous partial arc and non-continuous partial arc showed the best CI and HI. The heart absorbed doses in the continuous partial arc and hybrid 3D-CRT/IMRT were 0.07 ± 0.01% and 0% (V5% and V10%, respectively). The mean dose of the heart was lowest in hybrid 3D-CRT/IMRT (1.47 Gy ± 0.02). The dose in the left contralateral lung (V5%) was lowest in continuous partial arc (0%). The right ipsilateral lung average dose and V20% are lowest in continuous partial arc. Hybrid 3D-CRT/IMRT has the lowest mean dose to contralateral breast (organs at risk). The whole-body effective doses for ICRP-60 and ICRP-103 were highest in continuous partial arc (2.01 Sv ± 0.23 and 2.89 Sv ± 0.15, respectively). In conclusion, the use of VMAT with continuous arc has a lower risk of radiation pneumonia, while hybrid 3D-CRT/IMRT attain lower secondary malignancy risk and cardiovascular complications.
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During radiotherapy for head and neck tumours, the oral cavity and cheek area would be inevitably exposed to high energy radiation; thus, the material surface of the teeth, dental restorations with high atomic number, or alloy prosthodontics would generate backscatter electrons that cause the buccal mucosa adjacent to these materials to receive localized high dose enhancement, which primarily leads to side effects or oral mucositis. Based on the size of the adult oral cavity, this study aimed to use acrylic resin to create an oral phantom with two grooves on the left and right sides for placement of three molars. Moreover, the distance between the inner cheek and the side surface of the teeth could be accurately adjusted every 1âmm from 0 to 5âmm. This enhanced the dose in the buccal mucosa during head and neck radiotherapy and made the distribution measurement of the radiation dose simple and feasible at different depths (0-5âmm). Meanwhile, the study employed the film type optically stimulated luminescent dosimeter with a thickness of 0.3âmm to measure the absorbed dose inside the buccal mucosa to reduce the dose interference from radiotherapy. The study fixed three real molars in a row located at the left side of the phantom and employed 6âMV photons and intensity-modulated radiotherapy (IMRT) to treat and simulate oral cancer and measure the attenuation of the molar's backscatter dose from 0 to 5âmm in an up beam direction. The result showed that, in every 3âmm, the phantom had attenuated the enhancement of backscatter dose <3%. The irradiation dose enhancement in a single direction was twice higher than that through IMRT 7 field treatment. These measurement results were consistent with the results of previous studies.
Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Boca/efeitos da radiação , Imagens de Fantasmas , Humanos , Boca/anatomia & histologia , Mucosa Bucal/efeitos da radiação , Doses de Radiação , Dosímetros de Radiação , Radioterapia de Intensidade Modulada , Espalhamento de Radiação , Dente/efeitos da radiaçãoRESUMO
BACKGROUND: Influenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms. METHODS: Four H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated. RESULTS: The HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses. CONCLUSIONS: Our study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.
Assuntos
Imunização , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/química , Influenza Humana/prevenção & controle , Vírus Reordenados/imunologia , Animais , Embrião de Galinha , Cães , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Vírus Reordenados/genéticaRESUMO
BACKGROUND: Influenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Since 1950, chicken embryonated eggs have been used as the main method for producing seasonal influenza vaccines. However, this platform has the main drawback of a lack of scale-up flexibility, and thus, egg-based vaccine manufacturers cannot supply sufficient doses within a short period for use for pandemic prevention. As a result, strategies for reducing the manufacturing time and increasing production capacity are urgently needed. Non-virion vaccine methods have been considered an alternative strategy against an influenza pandemic, and the purpose of maintaining an immunogenic capsule structure with infectious properties appears to be met by the virus-like particle (VLP) platform. RESULTS: An influenza H7N9-TW VLP production platform using insect cells, which included the expression of hemagglutinin (HA), NA, and M1 proteins, was established. To scale up H7N9-TW VLP production, several culture conditions were optimized to obtain a higher production yield. A high level of dissolved oxygen (DO) could be critical to H7N9-TW VLP production. If the DO was maintained at a high level, the HA titer obtained in the spinner flask system with ventilation was similar to that obtained in a shake flask. In this study, the HA titer in a 5-L bioreactor with a well-controlled DO level was substantially improved by 128-fold (from 4 HA units (HAU)/50 µL to 512 HAU/50 µL). CONCLUSIONS: In this study, a multigene expression platform and an effective upstream process were developed. Notably, a high H7N9-TW VLP yield was achieved using a two-step production strategy while a high DO level was maintained. The upstream process, which resulted in high VLP titers, could be further used for large-scale influenza VLP vaccine production.
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TiO(2) nanowire (NW)/nanoparticle (NP) composite films have been fabricated by hybridizing various ratios of hydrothermal anatase NWs and TiO(2) NPs for use in dye-sensitized solar cells (DSSCs). Scanning electron microscopy (SEM) images reveal that uniform NW/NP composite films were formed on fluorine-doped tin oxide (FTO) substrates by the dip-coating method. The NWs are randomly but neither vertically nor horizontally oriented within the composite film. The TiO(2) NP DSSC possesses superior performance to those of the NW/NP composite and the pure NW cells, and the efficiency of the NW/NP composite DSSC increases on increasing the NP/NW ratio in the composite anode. All types of DSSC possess the same dependence of performance on the anode thickness that the efficiency increases with the anode thickness to a maximum value, then it decreases when the anode is thickened further. Electrochemical impedance spectroscopy analyses reveal that the NP DSSCs possess larger effective electron diffusion coefficients (D(eff)) in the photoanodes and smaller diffusion resistances of I(3)(-) in electrolytes compared to those in the NW/NP and the NW DSSCs. D(eff) decreases when NWs are added into the photoanode. These results suggest that the vertical feature of the NWs within the anodes is crucial for achieving a high electron transport rate in the anode.
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The objective of this study was to review the prevalence and prognostic impact of pretreatment thrombocytosis in patients with oral squamous cell carcinoma (OSCC). Medical records of 253 patients with OSCC were retrospectively reviewed. The relationship between the platelet count, TNM stage, histologic differentiation, recurrence rate, metastasis, and survival were analyzed. Pretreatment thrombocytosis was found in 61 (24.1%) of 253 patients and was associated with aggressive tumour growth (p=0.015), lymph node metastasis (p=0.009), and distant metastasis (p=0.001). Association with tumour recurrence fell just short of statistical significance (p=0.058). Patients with preoperative thrombocytosis had a poorer survival than those with normal platelet counts (p=0.004). Multivariate analysis using Cox's proportional hazards model indicated that thrombocytosis was an independent predictor of shorter survival (RR 2.48, 95% CI 1.30- 4.73, p=0.006).