Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant

Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.     

The recombinant Hc subunit of Clostridium botulinum neurotoxin serotype A is an effective botulism vaccine candidate

Vaccination with recombinant His-tagged isoforms of the Clostridium botulinum Hc domain of neurotoxin serotype A (rAHc) have effectively protected against challenge with active botulinum neurotoxin serotype A. To establish a formulation suitable for human use, rAHc was expressed in Escherichia coli without a His-tag and purified by sequential chromatography on ion-exchange and hydrophobic-interaction resins. Purified rAHc was used to vaccinate mice and survival was evaluated following challenge with active toxin. rAHc-vaccinated mice were protected against an active toxin challenge in mouse models of disease and a dose–response relationship was observed between the dose of rAHc administered and protection. Vaccination with rAHc in the presence or absence of adjuvants was also tested following intramuscular or subcutaneous vaccination to determine the optimal route of vaccination in the context of active toxin challenge. The data presented in the report suggested that rAHc administered with or without adjuvants functioned effectively over time in protecting mice against challenge with neurotoxin suggesting that this form of rAHc may be developed into a human vaccine candidate designed for the prevention of botulism.     

猫脑缺血坏死后Lac波1.2ppm处出现一未知氢波的观察

目的 在猫持续性局灶脑缺血模型上对某一未知氢波（Peak of unknown,Pu)进行证实，并推测其未来的应用价值。方法 猫脑缺血后以DWI及猫立体定向图谱为缺血后不同区域氢谱定位，并在缺血后不同时间进行氢谱检查。观察乳酸，N-乙酰天冬氨酸，肌酐，胆碱及Pu的变化。结果 猫局灶脑缺血后，缺血受累区在乳酸出现及NAA减少的前提下。缺血后2d至7d内，Pu波持续存在。结论 Pu波的出现与脑缺血坏死有着密切的关系。可能是脑组织坏死后产生的固有物质，它可能作为脑组织坏死的特异波而有着非常重要的临床诊断应用价值。     

Pyrrole alkaloids from Bolbostemma Paniculatum

Three pyrrole alkaloids were isolated from Bolbostemma paniculatum. Their structures were elucidated as 4-(2-formyl-5-methoxymethylpyrrol-1-yl)butyric acid methyl ester (1), 2-(2-formyl-5-methoxymethylpyrrol-1-yl)-3-phenylpropionic acid methyl ester (2) and α-methyl pyrrole ketone (3) by spectroscopic techniques. Among them, 1 and 2 are new compounds.     

Evaluation of a recombinant Hc of Clostridium botulinum neurotoxin serotype F as an effective subunit vaccine

A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of C. botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene. Subsequently, the purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). After the administration of FHc protein mixed with Freund adjuvant via the subcutaneous route, a strong protective immune response was elicited in the vaccinated mice. Mice that were given two or three vaccinations with a dosage of 1 or 10 μg of FHc were completely protected against an intraperitoneal administration of 20,000 50% lethal doses (LD₅₀) of BoNT/F. The BoNT/F neutralization assay showed that the sera from these vaccinated mice contained high titers of protective antibodies. Furthermore, mice were vaccinated once, twice, or three times at four different dosages of FHc using Alhydrogel (Sigma) adjuvant via the intramuscular route and subsequently challenged with 20,000 LD₅₀ of neurotoxin serotype F. A dose response was observed in both the antibody titer and the protective efficacy with increasing dosage of FHc and number of vaccinations. Mice that received one injection of 5 μg or two injections of ≥0.04 μg of FHc were completely protected. These findings suggest that the recombinant FHc expressed in E. coli is efficacious in protecting mice against challenge with BoNT/F and that the recombinant FHc subunit vaccine may be useful in humans.     

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab′)₂ fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab′)₂ antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab′)₂ antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.     

兔球虫PCR检测方法的改进和应用

兔球虫病由艾美耳属球虫寄生于消化道而引起,对养兔业的危害极大。由于兔艾美耳球虫的11个虫种在致病性上有较大差异,因此进行准确的虫种鉴定对兔球虫病的诊断和防治有十分重要的意义。为提高和扩大PCR方法检测粪便样品中艾美耳球虫的灵敏度和应用范围,我们利用全基因组扩增技术对提取自粪便样品的球虫基因组进行预扩增,提高PCR反应初始模板含量,再利用兔艾美耳球虫ITS-1序列种特异性引物对WGA产物进行PCR扩增,以鉴定虫种。结果显示,经全基因组扩增后的样品的PCR检测能够在单个卵囊的水平上进行虫种鉴定。利用该方法,我们对10份卵囊含量较少的田间兔粪样品进行了PCR检测,对其中的虫种进行了的鉴定。检测结果显示,与卵囊形态鉴定相比,该方法不仅与能确定形态的形态学检测结果一致,还能够检测出形态学不易判断的虫种,体现了更高的准确性。该技术的应用可提高田间样品检测的敏感性和准确度,为兔球虫临床感染情况提供实践指导。