肠艾美耳球虫张家口分离株致病性和免疫原性的初步研究

肠艾美耳球虫是兔球虫中的主要致病虫种之一,充分研究其生物学特性对兔球虫病的防治有十分重要的意义。本文对肠艾美耳球虫张家口分离株的致病性和免疫原性进行了初步研究。利用3个剂量（1×10^4、1×10^5、1×10^6）的肠艾美耳球虫张家口分离株孢子化卵囊感染35—45日龄无球虫兔,观察临床症状和体重变化,以研究其致病性;21d后,再用1×10。个肠艾美耳球虫孢子化卯囊进行攻毒,监测体重变化和卵囊排出量,评价其免疫原性。结果发现,接种1×10^4个卵囊,即可导致家兔出现临床症状,与空白对照组相比,日增重显著下降,更高感染剂量则会加重症状。攻毒后,免疫组增重未受影响,与空白对照组相比无差异,而不免疫攻毒组体重显著下降;同时免疫组的卵囊排出量显著低于不免疫攻毒组。结果表明肠艾美耳球虫张家口分离株对仔兔具有中等致病性;同时其免疫原性良好。因此可以把该株球虫作为原始株进行早熟虫株选育,并以此为基础,进行兔球虫病疫苗的开发。     

Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors

Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.     

炭疽保护性抗原第四结构域基因在重组SFV复制子载体中的表达

目的：构建炭疽毒素保护性抗原第四结构域PA4基因的重组Semliki森林病毒（Semliki forest virus，SFV）复制子载体，并观察PA4抗原在重组SFV复制子载体中的表达。方法：将PA4基因克隆到基于RNA和DNA的SFV复制子表达载体中，获得的重组SFV复制子载体直接转染．BHK21细胞，通过间接免疫荧光和Western印迹试验检测PA4在细胞中的表达；与辅助病毒载体共转染制备重组病毒颗粒，通过间接免疫荧光和Western印迹检测PA4在重组病毒颗粒感染细胞中的表达。结果与结论：成功地构建了基于RNA和DNA的PA4重组SFV复制子载体，并且在体外基于RNA和DNA的重组SFV复制子表达载体能够在细胞中有效地表达非分泌型和分泌型的PA4抗原，制备了具有感染能力并能表达PA4抗原的重组病毒颗粒，为进一步以SFV复制子作疫苗载体观察炭疽新型复制子疫苗的免疫原性奠定了基础。     

目标管理法在神经重症进修生培训中的应用

通过2年多的摸索和努力,在神经重症专科医生进修培训过程中,我们借鉴了企业式目标管理法进行临床教学,取得了一定的效果,现介绍如下。     

Diagnostic value and prognostic evaluation of Presepsin for sepsis in an emergency department

Introduction

Presepsin levels are known to be increased in sepsis. The aim of this study was to evaluate the early diagnostic and prognostic value of Presepsin compared with procalcitonin (PCT), Mortality in Emergency Department Sepsis (MEDS) score and Acute Physiology and Chronic Health Evaluation II (APACHE II) score in septic patients in an emergency department (ED) and to investigate Presepsin as a new biomarker of sepsis.

Methods

This study enrolled 859 consecutive patients with at least two diagnostic criteria for systemic inflammatory response syndrome (SIRS) who were admitted to Beijing Chao-yang Hospital ED from December 2011 to October 2012, and 100 age-matched healthy controls. Patients were stratified into four groups: SIRS, sepsis, severe sepsis, and septic shock. Plasma Presepsin and serum PCT were measured, and MEDS score and APACHE II score were calculated at enrollment. Comparisons were analyzed using the Kruskal-Wallis and Mann–Whitney U tests.

Results

On admission, the median levels of plasma Presepsin increased with sepsis severity. The areas under the receiver operating characteristic (AUC) curves of Presepsin were greater than those of PCT in diagnosing sepsis, and predicting severe sepsis and septic shock. The AUC of Presepsin for predicting 28-day mortality in septic patients was slightly lower than that of PCT, MEDS score and APACHE II score. The AUC of a combination of Presepsin and MEDS score or APACHE II score was significantly higher than that of MEDS score or APACHE II score alone in predicting severe sepsis, and was markedly higher than that of Presepsin alone in predicting septic shock and 28-day mortality in septic patients, respectively. Plasma Presepsin levels in septic patients were significantly higher in non-survivors than in survivors at 28 days’ follow-up. Presepsin, MEDS score and APACHE II score were found to be independent predictors of severe sepsis, septic shock and 28-day mortality in septic patients. The levels of plasma Presepsin were positively correlated with PCT, MEDS score and APACHE II score in every septic group.

Conclusion

Presepsin is a valuable biomarker for early diagnosis of sepsis, risk stratification, and evaluation of prognosis in septic patients in the ED.     

Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences

Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.     

Enhanced immune responses using plasmid DNA replicon vaccine encoding the Hc domain of Clostridium botulinum neurotoxin serotype A

In current study, the immunogenicity of a plasmid DNA replicon vaccine (pSCARSHc) encoding the Hc domain of Clostridium botulinum neurotoxin serotype A (AHc) was investigated and compared with a conventional plasmid DNA vaccine (pcDNASHc) encoding the same antigen. In vitro, pSCARSHc incorporating Semliki Forest virus (SFV) replicon could express AHc protein and induce apoptosis of transfected cells. Comparison with the conventional plasmid DNA vaccine (pcDNASHc) yielded several interesting results. First, our self-designed pSCARSHc could induce relatively higher AHc-specific antibodies and lymphocyte proliferative responses in immunized Balb/c mice, especially at low doses. Second, while both pSCARSHc and pcDNASHc induced Th2-type immune responses, the ratio of IgG1 to IgG2a was lower in pSCARSHc groups and the Th2- and Th1-type humoral immune responses induced by pSCARSHc were also stronger than that of the pcDNASHc vaccine. Third, it was shown that the sera from pSCARSHc-vaccinated mice conferred more efficient protection than those from pcDNASHc-vaccinated mice by BoNT/A neutralization assay. Finally, mice immunized with pSCARSHc could also elicit more efficient protection against BoNT/A than pcDNASHc. These results indicate that our plasmid DNA replicon vaccine can provide strong immunogenicity and should be a potential alternative strategy to conventional DNA vaccines in developing an efficacious vaccine against C. botulinum neurotoxin serotype A.     

A Novel Aβ B-Cell Epitope Vaccine (rCV01) for Alzheimer’s Disease Improved Synaptic and Cognitive Functions in 3 × Tg-AD Mice

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive amyloid-β accumulation, loss of cognitive abilities, and synaptic alterations. Given the remarkable recovery of cognition in AD models of targeting-Aβ immunotherapy, we sought to determine the molecular correlate(s) associated with improvement. We evaluated the efficacy of a recombinant chimeric 6Aβ15-T antigen formulated with alum adjuvant as a novel Aβ B-cell epitope vaccine (rCV01) in 3 × Tg-AD mice. rCV01 elicited robust Th2-polarized Aβ-specific antibodies without autoimmune T cell responses in 3 × Tg-AD mice. The long-lasting anti-Aβ42 antibodies were associated with markedly reduced AD-like pathology, enhanced synaptic function, and improved cognitive performance in aged 3 × Tg-AD mice. This is the first report to provide one hypothesis for the improved outcomes following vaccination is a reduction in the levels of active calpain in rCV01-immunized AD mice, which is likely attributable to preventing dynamin 1 and PSD-95 degradation allowing functional recovery of cognition. rCV01 is a highly immunogenic recombinant chimeric 6Aβ15-T vaccine that shows clear neuroprotective properties in preclinical mouse models of AD and is a candidate for an effective AD vaccine.     

Protection with a recombinant Hc of Clostridium Botulinum neurotoxin serotype A from Escherichia coli as an effective subunit vaccine

A recombinant Hc of Clostridium botulinum neurotoxin serotype A (AHc) was successfully expressed in Escherichia coli for use as an antigen, and the purified AHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype A. The mice, given twice or third subcutaneous vaccinations with a dosage of 1 μg AHc mixed with Freund adjuvant, were completely protected against an intraperitoneal administration of 1,000,000 50% lethal doses (LD₅₀) of neurotoxin serotype A. Following the administration of AHc using alhydrogel adjuvant via the intramuscular route, a strong protective immune response was also elicited in the vaccinated mice. A dose-response was observed in protective efficacy with increasing AHc dosage and number of vaccinations. Mice that received two injections of ≥ 0.2 μg and three injections of ≥ 0.04 μg were completely protected when challenged with 100,000 LD₅₀ of neurotoxin serotype A. These results clearly suggest that the recombinant AHc highly expressed in Escherichia coli is very efficacious in protecting against challenge with active botulinum neurotoxin serotype A in mouse model and a good subunit candidate vaccine against botulinum neurotoxin serotype A for human use.