疫苗诱导的特异性细胞杀伤活性检测方法的建立

目的建立一种基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,以完善疫苗及基因治疗药物的评价方法。方法用羧基荧光素二乙酸盐琥珀酰亚胺酯(Carboxyfluorescein diacetate,succinimidyl ester,CFSE)标记淋巴细胞,利用肿瘤坏死因子(Tumor necrosis factor,TNFα)模拟杀伤淋巴细胞,用碘化丙啶(Propidium iodide,PI)染色,流式细胞仪检测,确定CFSE和PI的浓度及作用时间。利用已确知有较强细胞免疫作用的治疗性乙型肝炎疫苗免疫小鼠,分离特异性淋巴细胞并用特异性肽刺激,分离未免疫小鼠的淋巴细胞作为靶细胞,并用特异性抗原肽致敏,并从靶细胞的标记、效应细胞培养时间,效靶作用时间、效靶比几方面进行优化,确定试验方法的操作流程。结果采用CFSE/PI双标记能有效分离实验所需各组群,细胞分为CFSE+PI-、CFSE+PI+、CFSE-PI+、CFSE-PI-4个组群,可区分活细胞和凋亡细胞。CFSE标记靶细胞的时间为6 h;效应细胞培养时间为72 h;效靶作用时间为6 h;效靶比可使用100∶1和50∶1。结论建立了基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,该方法可有效和精确地评价CTL杀伤效应,完善了疫苗及基因治疗药物的评价方法。     

The relation between growth of four microbes on six different plasterboards and biological activity of spores

Microbial growth on water-damaged building materials is commonly associated with adverse health effects in the occupants. We examined the growth of Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus, isolated from water-damaged buildings, on six different brands of plasterboards. The microbial growth was compared with the biological activity of the spores, that is the potential to induce cytotoxicity and proinflammatory mediators in RAW264.7 macrophages. These results showed that the microbial growth on plasterboard depended on both the microbial strain and the brand of plasterboard used. The biological activity of spores appeared to be regulated by different growth conditions on plasterboards so that good microbial growth was associated with a low bioactivity of the spores, whereas the spores collected from plasterboard supporting only weak growth usually were biologically active. Cytotoxicity of either S. chartarum or A. versicolor did not correlate with any particular growth conditions or induced inflammatory responses. Instead, there were positive correlations between cytotoxicity and levels of induced proinflammatory cytokines for P. spinulosum and S. californicus. These data suggest that both the microbial growth on plasterboard and the resulting bioactivity of spores vary and might be affected by changing the growth conditions provided by the plasterboards.     

Choline- versus imidazole-based ionic liquids as functional ingredients in topical delivery systems: cytotoxicity,solubility, and skin permeation studies

Background: Poor drug solubility represents a problem for the development of topical formulations. Since ionic liquids (ILs) can be placed in either lipophilic or hydrophilic solutions, they may be advantageous vehicles in such delivery systems. Nonetheless, it is vital to determine their usefulness when used at concentrations were cell viability is maintained, which was considered herein.

Method: Five different ILs were prepared—three imidazole-based ILs: [C2mim][Br], [C4mim][Br], and [C6mim][Br]; and two choline-based ILs: [Cho][Phe] and [Cho][Glu]. Their cytotoxicity in human keratinocytes (HaCat cells), their influence in drug solubility and in percutaneous permeation, using pig skin membranes, was evaluated.

Results: Caffeine and salicylic acid were used as model actives. Choline-based ILs proved to be more suitable as functional ingredients, since they showed higher impact on drug solubility and a lower cytotoxicity. The major solubility enhancement was observed for caffeine and further solubility studies were carried out with this active in several concentrations of the choline-based ILs (0.1; 0.2; 0.5; 1.0; 3.0 and 5.0%, w/w) at 25?°C and 32?°C. Solubility was greatly influenced by concentrations up to 0.5%. The choline-based ILs showed no significant impact on the skin permeation, for both actives. The size of the imidazole-based ILs alkyl chain enhances the caffeine solubility and permeation, but also the ILs cytotoxicity. Stable O/W emulsions and gels were prepared containing the less toxic choline-based ILs and caffeine.

Conclusions: Our results indicate that the choline-based ILs were effective functional ingredients, since, when used at nontoxic concentrations, they allowed a higher drug loading, while maintaining the stability of the formulations.     

Bioactive,luminescent erbium-doped hydroxyapatite nanocrystals for biomedical applications

Diagnosis and imaging play an essential key role in primary detection, screening, and image-guided smart nanomedicine for healthcare solutions. This study illustrates the successful fabrication of luminescent lanthanide (erbium)-doped hydroxyapatite (Er-HAp) by one step facile wet-chemical precipitation method. The chemical compositions, morphology, optical, and biological properties were systematically characterized using relevant different structural, compositional analytical instrumentation and cytotoxicity assays. After erbium doping, synthesized luminescent nano-structured materials exhibited elongated morphology, with well dispersed <50 nm size distribution. The photoluminescence (PL) study confirmed three emission bands assigned to ⁴F_3/2 → ⁴I_15/2 (purple), ⁴F_7/2 → ⁴I_15/2 (blue), and ⁴S_3/2 → ⁴I_15/2 (green) transition states, respectively. In vitro bioactivity and optical imaging studies conducted in osteoblast like MG-63 cells confirmed the nontoxic luminescent behavior of the synthesized nanomaterials.     

Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents

In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, in vitro and in vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC₅₀=0.78±0.01 μM), HT29 (IC₅₀=0.92±0.15 μM) and K562 (IC₅₀=47.25±1.24 μM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G₁/G₀ phase and decreased cell population in G₂/M phase. The excellent in vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50 mg kg⁻¹ dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents.     

冠状动脉支架辐射灭菌后生物相容性试验

评价聚酯聚乙烯复合膜/聚乙烯(冠状动脉支架系统)经辐射灭菌后的生物相容性,展望并促进辐射灭菌在高分子生物材料灭菌应用的可行性.按照国际标准化组织(International organization for standardization,ISO) 111737标准进行聚酯聚乙烯复合膜/聚乙烯(冠状动脉支架系统)初始污染菌检测,并依据ISO11137标准方法完成了辐照灭菌的剂量设定.设定初始污染菌数为332.49cfu/件,验证剂量为9.53kGy(SAL10-2),最低灭菌剂量为23.1kGy(SAL10-6),对经最低灭菌剂量辐照灭菌合格后的产品进行,包括人血细胞染色体遗传毒性试验、细胞毒性试验、致敏试验、皮内刺激试验、血液相容性试验等生物相容性评价.辐照灭菌后的聚酯聚乙烯复合膜/聚乙烯(冠状动脉支架系统)材料无细胞毒性、无遗传毒性(不会引起染色体畸变),不会致敏和引致皮内刺激,血液相容性良好,产品的包装材料材质和性能未改变.经设定剂量辐照灭菌后的聚酯聚乙烯复合膜/聚乙烯(冠状动脉支架系统)生物相容性好,辐照灭菌作为医用高分子生物材料灭菌方法值得推广应用.     

抗人淋巴细胞球蛋白制剂的质量比较

抗人淋巴细胞球蛋白(ATG/ALG)是一种有效的免疫抑制剂。目前对ATG制剂的活性及质量体外检测方法均以细胞毒和花环抑制试验为主。本文用上述方法检定了兔抗人淋巴细胞球蛋白(R-ATG)和马抗人淋巴细胞球蛋白(E-ATG)。结果表明:两种制剂的细胞毒滴度和花环抑制试验基本相同。应用~3H—胸腺嘧啶渗入试验研究ATG对PBMC促有丝分裂活性,结果表明:R-ATG的活性高于E-ATG。ATG体外促有丝分裂活性可作为预测ATG临床疗效的一个参数。     

The molecular shape and the field similarities as criteria to interpret SAR studies for fragment-based design of platinum(IV) anticancer agents. Correlation of physicochemical properties with cytotoxicity

Molecular shape similarity and field similarity have been used to interpret, in a qualitative way, the structure-activity relationships in a selected series of platinum(IV) complexes with anticancer activity. MM and QM calculations have been used to estimate the electron density, electrostatic potential maps, partial charges, dipolar moments and other parameters to correlate the stereo-electronic properties with the differential biological activity of complexes. Extended Electron Distribution (XED) field similarity has been also evaluated for the free 1,4-diamino carrier ligands, in a fragment-based drug design approach, comparing Connolly solvent excluded surface, hydrophobicity field surface, Van der Waals field surface, nucleophilicity field surface, electrophilicity field surface and the extended electron-distribution maxima field points. A consistency has been found when comparing the stereo-electronic properties of the studied series of platinum(IV) complexes and/or the free ligands evaluated and their in vitro anticancer activity.     

Histamine reduction by Maillard reaction with glucose

Histamine, well known as a toxic biogenic amine, is found in a variety of foods. Reducing its concentration and toxicity is desirable. In this study, the glucose/histamine Maillard reaction was proposed as a novel tool for histamine control. Effects of temperature, heating time, initial pH value, NaCl concentration, initial histamine concentration and initial glucose concentration on percentage removal of histamine in the glucose/histamine Maillard reaction model were investigated. The results showed that histamine reduction was affected by these variables, and could be almost eliminated under appropriate conditions. Fluorescence intensity and ultraviolet–visible spectroscopy analyses were used to characterize the glucose/histamine Maillard reaction. Cytotoxicity assay revealed that the glucose/histamine Maillard reaction significantly reduced the toxicity of histamine (P < 0.05). Furthermore, histamine concentrations in canned tuna samples were significantly reduced by thermal treatment with glucose (P < 0.05). This study demonstrates that the glucose/histamine Maillard reaction is a promising method for histamine control.     

Ozonated Olive Oil with a High Peroxide Value for Topical Applications: In-Vitro Cytotoxicity Analysis with L929 Cells

Previous studies have shown that ozonated vegetable oils have been used topically for healing of cutenous wounds. The aim of this study is to evaluate the dose dependent use of ozonated olive oil with high peroxide value (OZ) on the viability of cells for preventing side effects in topical applications. To the best of our knowledge, there are no reports investigaing the effect of peroxide value of ozonated olive oil associated with its cytotoxic activity on mouse non-neoplastc fibroblast cell lines (L929). Therefore, the present study was carried out by using OZ alone and/or in combination with glycerol and olive oil. In our study OZ was prepared by using pure olive oil. Both olive oil and glycerol are non-toxic and can be mixed with OZ uniformly. The cytotoxic activity of samples against L929 fibroblasts was assessed using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. The peroxide value of synthesized OZ was found to be in the range of 2700–2900 mEq O₂/kg oil. The OZ/olive oil group did not show any cell death at all concentrations tested (p > 0.05) however OZ/glycerol group showed statistically significant reductions in viability at higher concentrations (p = 0.004–0.006) compared to the control group. Conclusively, using OZ/olive oil with a peroxide value of 2700–2900 mEq O₂/kg oil for short-term incubation was non-cytotoxic to the L929 fibroblast cell line.