Induction of CD8+ T-cell responses against subunit antigens by the novel cationic liposomal CAF09 adjuvant

Vaccines inducing cytotoxic T-cell responses are required to achieve protection against cancers and intracellular infections such as HIV and Hepatitis C virus. Induction of CD8+ T cell responses in animal models can be achieved by the use of viral vectors or DNA vaccines but so far without much clinical success. Here we describe the novel CD8+ T-cell inducing adjuvant, cationic adjuvant formulation (CAF) 09, consisting of dimethyldioctadecylammonium (DDA)-liposomes stabilized with monomycoloyl glycerol (MMG)-1 and combined with the TLR3 ligand, Poly(I:C). Different antigens from tuberculosis (TB10.3, H56), HIV (Gag p24), HPV (E7) and the model antigen ovalbumin were formulated with CAF09 and administering these vaccines to mice resulted in a high frequency of antigen-specific CD8+ T cells. CAF09 was superior in its ability to induce antigen-specific CD8+ T cells as compared to other previously described CTL-inducing adjuvants, CAF05 (DDA/trehalose dibehenate (TDB)/Poly(I:C)), Aluminium/monophosphoryl lipid-A (MPL) and Montanide/CpG/IL-2. The optimal effect was obtained when the CAF09-adjuvanted vaccine was administered by the i.p. route, whereas s.c. administration primed limited CD8+ T-cell responses. The CD4+ T cells induced by CAF09 were mainly of an effector-memory-like phenotype and the CD8+ T cells were highly cytotoxic. Finally, in a mouse therapeutic skin tumor model, the HPV-16 E7 antigen formulated in CAF09 significantly reduced the growth of already established subcutaneous E7-expressing TC-1 tumors in 38% of the mice and in a corresponding prophylactic model 100% of the mice were protected. Thus, CAF09 is a potent new adjuvant which is able to induce CD8+ T-cell responses against several antigens and to enhance the protective efficacy of an E7 vaccine both in a therapeutic and in a prophylactic tumor model.     

Study of different routes of immunization using outer membrane vesicles of Neisseria meningitidis B and comparison of two adjuvants

Outer membrane vesicles (OMVs) of Neisseria meningitidis contain important antigens to trigger an immune response against meningococci and have been studied as vaccines compounds. The immune response to a vaccine may be affected by its constitution and route of administration. Therefore, Swiss mice were immunized by different routes with OMVs of N. meningitidis B with dimethyl dioctadecyl ammonium bromide in bilayer fragments (DDA-BF) or aluminum hydroxide (AH) as adjuvants. The adjuvants and different routes were compared regarding the immune responses by ELISA, western blot, delayed type hypersensitivity (DTH) and histopathologic analysis. The antigenic preparation generated humoral and cellular immune responses. In quantitative analyzes, in general, AH was superior to DDA-BF. However, analysis such as IgG avidity index, bactericidal activity and immunoblot, revealed no important differences regarding the adjuvant or route of immunization. Regarding the parameters tested, it was not possible to define a superiority between the adjuvants and routes of immunization proposed by this study.     

生物技术药物中残留DNA检测方法的比较

生物技术药物是所有以生物质为原料的生物活性物质及其人工合成类似物通过现代生物技术制得的药物,包括细胞因子、重组蛋白、抗体、疫苗和寡核苷酸等,临床上已开始广泛应用,为制药工业带来了革命性变化.但是生物技术药物中残留DNA可能存在安全性问题,因此应尽可能将产品中残留DNA的水平降到最低.新版美国药典将推荐实时定量PCR法作为生物技术药物中宿主残留DNA检定的唯一标准方法.该法的技术优势在于序列特异性高、灵敏度高、重现性好,还可以实现定量检测,使得结果更为精确,从而为生物技术药物企业在工艺研究和成品质量控制方面提供了可靠的检测手段.此综述对4类检测方法进行了比较,重点比较两种实时定量方法.     

VCAM-1 specific PEGylated SAINT-based lipoplexes deliver siRNA to activated endothelium in vivo but do not attenuate target gene expression

In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNFα-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development.     

Toxicity of imine–iminium dyes and pigments: electron transfer,radicals, oxidative stress and other physiological effects

Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer–reactive oxygen species–oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer–reactive oxygen species–oxidative stress arising from imine–iminium. Copyright © 2014 John Wiley & Sons, Ltd.     

食品中非法添加和滥用化学染料的分析研究

建立了食品中碱性黄、碱性嫩黄、碱性橙II、酸性橙II、罗丹明B、对位红、苏丹类1-4号11种有机染料的高效液相色谱分析方法。在0.2-10μg/mL的浓度范围内线性相关系数良好,相关系数范围为0.9996-0.9999,检测限0.05μg/mL。该方法涉及的化学试剂少,操作简便,结果准确。对阳性样品进一步采用液质联用技术确证。     

不同商品规格川贝母总生物碱含量测定的方法研究

目的 建立酸性染色法用于测定川贝母中总生物碱的含量.方法 采用三氯甲烷-甲醇(4∶1)混合溶液提取川贝母总生物碱;采用酸性染料染色法,以西贝母碱为指标成分,测定总生物碱的含量.结果 4个规格川贝母样品中总生物碱含量均符合2010年版《中国药典(一部)》的规定.结论 该方法不需要复杂仪器、操作简便,可作为川贝母的质量检测方法.     

Receptor-Mediated Gene Delivery to HepG2 Cells by Ternary Assemblies Containing Cationic Liposomes and Cationized Asialoorosomucoid

A new cholesterol-carborane conjugate (BCH) has been synthesized as a potential targeting agent for boron neutron capture therapy (BNCT) of cancers. The compound is extremely water insoluble and was formulated in two liposomal formulations to determine if the compound could be adequately taken up by 9L rat glioma cells in cell culture. Several factors potentially affecting the cellular uptake were evaluated, such as concentration of BCH in the incubation medium, incubation time, cell confluence, and the addition of polyethylene glycol (PEG) phospholipids to the liposomal formulation. The studies indicated that the cellular uptakes of BCH in the conventional and PEG liposomal formulations were 49.1 and 45.9 μg boron/g cells, respectively. Therefore, this compound, formulated in both liposomal formulations, delivered sufficient levels of boron to cancer cells in vitro, indicating that BCH is a promising approach for use in BNCT. The uptake appeared to depend upon BCH concentration in the media as well as the confluence of the cells. The greater boron uptake by nonconfluent cells indicated that active growth of cells was a factor in the uptake of this compound.     

A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400 nm and zeta potential of 40 mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41 ± 0.38 log₁₀ CFU) than traditional vaccine Bacillus Calmette–Guerin (BCG) (6.01 ± 0.33 log₁₀ CFU) and PBS control (6.53 ± 0.26 log₁₀ CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.     

氨基胍对内毒素活化小鼠巨噬细胞L-arginine转运及CAT-2表达影响的实验研究

目的:通过氨基胍(AG)干预内毒素活化小鼠巨噬细胞RAW264.7模型,观察NO的产生、左旋精氨酸(L-Arg)转运以及碱性氨基酸转运体-2(CAT-2)mRNA的表达,旨在研究氨基胍对L-Arg跨膜转运及CAT-2表达的影响.方法:实验分为四组,即对照组,LPS对照组,AG组,AG+LPS组.接种RAW264.7细胞...