Rhodococcus equi-Derived Extracellular Vesicles Promoting Inflammatory Response in Macrophage through TLR2-NF-κB/MAPK Pathways

Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs produced by the virulence strain R. equi 103⁺ (103⁺-EVs) and avirulenct strain R. equi 103⁻ (103⁻-EVs). We observed that 103⁺-EVs and 103⁻-EVs are similar to other Gram-positive extracellular vesicles, which range from 40 to 260 nm in diameter. The 103⁺-EVs or 103⁻-EVs could be taken up by mouse macrophage J774A.1 and cause macrophage cytotoxicity. Incubation of 103⁺-EVs or 103⁻-EVs with J774A.1 cells would result in increased expression levels of IL-1β, IL-6, and TNF-α. Moreover, the expression of TLR2, p-NF-κB, p-p38, and p-ERK were significantly increased in J774A.1 cells stimulated with R. equi-EVs. In addition, we presented that the level of inflammatory factors and expression of TLR2, p-NF-κB, p-p38, and p-ERK in J774A.1 cells showed a significant decreased when incubation with proteinase K pretreated-R. equi-EVs. Overall, our data indicate that R. equi-derived EVs are capable of mediating inflammatory responses in macrophages via TLR2-NF-κB/MAPK pathways, and R. equi-EVs proteins were responsible for TLR2-NF-κB/MAPK mediated inflammatory responses in macrophage. Our study is the first to reveal potential roles for R. equi-EVs in immune response in R. equi-host interactions and to compare the differences in macrophage inflammatory responses mediated by EVs derived from virulent strain R. equi and avirulent strain R. equi. The results of this study have improved our knowledge of the pathogenicity of R. equi.     

Plasmids as Key Players in Acinetobacter Adaptation

This review briefly summarizes the data on the mechanisms of development of the adaptability of Acinetobacters to various living conditions in the environment and in the clinic. A comparative analysis of the genomes of free-living and clinical strains of A. lwoffii, as well as the genomes of A. lwoffii and A. baumannii, has been carried out. It has been shown that plasmids, both large and small, play a key role in the formation of the adaptability of Acinetobacter to their living conditions. In particular, it has been demonstrated that the plasmids of various strains of Acinetobacter differ from each other in their structure and gene composition depending on the lifestyle of their host bacteria. Plasmids of modern strains are enriched with antibiotic-resistant genes, while the content of genes involved in resistance to heavy metals and arsenic is comparable to plasmids from modern and ancient strains. It is concluded that Acinetobacter plasmids may ensure the survival of host bacteria under conditions of various types of environmental and clinical stresses. A brief overview of the main mechanisms of horizontal gene transfer on plasmids inherent in Acinetobacter strains is also given.     

耐镉肠杆菌的分子鉴定和抗性分析

分离到一株耐镉肠杆菌NP23,对其进行了分子鉴定和抗性研究,分析菌株在不同镉浓度条件下菌体的生长状况以及对Fe²⁺,Cu²⁺,Mn²⁺,Zn²⁺等重金属抗性,利用SDS法进行质粒消除分析。结果显示,菌株NP23是Enterobacter sp,高浓度的镉使该菌株生长延滞期变长,进入对数期后的生长量相对较低,采用SDS法进行质粒消除,初步确定NP23的镉抗性基因在基因组上,NP23对所分析的四种重金属都有一定抗性,其中对Zn²⁺和Mn²⁺的抗性较强,在3200mg/L以上。      

一种特异性检测单增李斯特菌的质粒DNA标准物质

研制了一种质粒DNA标准物质,包含目前单增李斯特菌检测常用的靶基因序列——内化素基因(Inl A)序列,可适用于扩增Inl A基因的单增李斯特菌PCR相关检测方法。采用紫外分光光度法(UV)和高分辨电感耦合等离子体质谱(HR-ICP-MS)两种方法,多家实验室联合定值的方法,对该套标准物质的均匀性、稳定性、量值以及不确定度进行详细的考察,质粒DNA标准物质的最终定值结果为94.9(±5.1)ng/μL。该质粒DNA标准物质可用于微生物实验室对单增李斯特菌PCR相关检测进行检测结果质量控制、开展方法比对和能力验证等。     

CAMBRIDGE PRIZE LECTURE DEVELOPING NEW STRAINS OF YEAST*

Genetic engineering or recombinant DNA technology is now routinely applied to the construction and development of new strains of brewing yeast. This is a direct consequence of the power of the technology which facilitates the modification, introduction and stable maintenance of specific genes in brewing yeast, without compromising the intrinsic brewing properties of the yeast itself. The way in which gene technology has been applied to the development of new strains of Bass yeast is briefly illustrated by the provision of plasmid-based systems for ensuring the stable maintenance of recombinant genes, the construction of amylolytic and β-glucanolytic yeast and the design and development of genetic systems for enhancing the value of waste brewers yeast. Commercial and regulatory issues are discussed.     

产肠毒素大肠杆菌基因工程疫苗在SD大鼠中的免疫原性

目的观察产肠毒素大肠杆菌(Enterotoxigenic E.coli,ETEC)基因工程疫苗在SD大鼠中的免疫原性。方法将含ETEC抗原成分热不稳定肠毒素B亚单位(Heat-labile enterotoxin Bsubunit,LTB)的双歧杆菌疫苗给SD大鼠灌胃4次,同时设对照组,以PBS平行灌胃。采用ELISA法检测大鼠粪便上清液IgA及血清IgG抗体水平;免疫组化法检测大鼠空肠中sIgA抗体的表达;肠绊试验检测肠毒素的活性。结果免疫疫苗的SD大鼠粪便上清液中产生了IgA抗体,血清中产生了特异性的IgG抗体,肠道内产生了特异性分泌型sIgA抗体;免疫组化结果显示,随着免疫次数的增加,抗体分泌量明显增加;双歧杆菌疫苗免疫的大鼠能够抵抗LT抗原的侵袭,而对照组大鼠则出现明显的肠道积液现象。结论构建的双歧杆菌疫苗具有良好的免疫效果,可保护SD大鼠免受ETEC的感染。     

人源化叶酸受体α抗体质控方法的建立

目的建立人源化叶酸受体α抗体的质控方法。方法应用基于表面等离子体共振(Surface plasmon resonance,SPR)技术的BIAcore3000系统测定人源化叶酸受体α单抗与重组人叶酸受体α的亲和力,从而反映该抗体的功能;采用ELISA法测定其抗原结合力;SDS-PAGE和SEC-HPLC测定其纯度;紫外分光光度法测定其蛋白含量;毛细管等电聚焦电泳法测定等电点;其他各项指标检测按《中国药典》三部(2005版)要求进行。结果人源化叶酸受体α单抗成品的平均相对百分效价为105%,RSD为6.0%。单抗成品及参考品与叶酸受体的结合力均存在量效关系,且符合四参数方程式:Y=(A-D)/[1+(X/C)B]+D,其曲线相关系数在0.98以上,成品经3次测定,相对抗原结合力平均值为97%,RSD为25%。还原SDS-PAGE分析显示,单抗成品的IgG重链和轻链百分比为98.2%;非还原SDS-PAGE分析显示,完整IgG百分比为96.7%;SEC-HPLC分析显示,单抗成品单体为99.1%,聚合体为0.9%。其他各项指标均符合《中国药典》三部(2005版)要求。结论已初步建立了人源化叶酸受体α抗体的质控方法,为该制品的质量控制奠定了基础。     

乙型肝炎基因工程疫苗的反应及效果观察

应用重组痘苗病毒表达的乙肝基因疫苗接种了100名7～9岁儿童。采用0、1、2月免疫程序,3个不同剂量组(20,10及5μg)均产生较高的抗体阳转率:3个月时大于96％、9个月时均达100％,免后2年仍在85％以上。9月时抗体水平达高峰,其抗体GMT分别为540.4(20μg),556.8 (10μg)及392.3m(?)u/ml(5μg)。没有观察到严重的副反应。与酵母重组疫苗(Merck产品)和血源疫苗的免疫效果比较无明显区别。表明重组痘苗病毒表达的乙肝疫苗是安全、有效的。     

伤寒-鼠伤寒基因重组菌株Vi4072与伤寒Ty2株和枸橼酸杆菌的Vi抗原比较

将来源于枸橼酸杆菌编码产生Vi抗原的ViaB基因片段,通过基因重组技术引入减毒的鼠伤寒杆菌而组建的Vi4072重组株与伤寒沙门氏菌Ty2株和枸橼酸杆菌的Vi抗原,通过琼脂双向扩散试验、被动血凝试验、对BALB/c小鼠的免疫力试验以及对Vi抗原分子的化学元素含量、红外光谱、超导核磁C谱、H谱分析测试。结果表明上述三种菌株产生的Vi抗原其血清学特性、免疫保护作用及化学分子结构等基本相同。且Vi4072重组株表达Vi抗原量高于伤寒Ty2株。