树鼩呼肠孤病毒RT-nPCR检测方法的建立及初步应用

目的建立树鼩呼肠孤病毒(TRV)RT-nPCR检测方法,为树鼩的质量控制提供检测方法。方法从三批野外来源的具有感染临床症状的树鼩粪便中分离得到三株病毒,经电镜观察和聚丙烯酰胺凝胶电泳鉴定为哺乳动物呼肠孤病毒(MRV)。根据GenBank中已发表的MRV L1基因的保守区域,设计合成巢式引物,对所分离的三株树鼩呼肠孤病毒(TRV1、TRV2、TRV3)的RNA进行RT-nPCR扩增,优化反应条件,进行特异性、敏感性试验。应用RT-nPCR方法对25只野外来源的相同症状疑似病例样本进行检测。结果针对分离到的三株树鼩呼肠孤病毒的RNA进行RT-nPCR扩增,均得到513 bp的特异性目的片段,培养细胞及甲肝病毒、轮状病毒、单纯疱疹病毒阴性对照均未扩增出条带。敏感性试验结果显示可检测到的最小RNA模板浓度为0.01 pg/μL。25只树鼩粪便样本经RT-nPCR检测,有14只TRV阳性,其中死亡动物组10只,检出率为100%;存活动物组15只,检出率为27%。结论建立的TRV RT-nPCR检测方法特异、敏感、稳定,可用于TRV的常规检测。     

Immunomodulation of human CD19+CD25high regulatory B cells via Th17/Foxp3 regulatory T cells and Th1/Th2 cytokines

Regulatory B (Breg) cells are a special subset of immunoregulatory cells with unique phenotypes and functions. In this study, human CD19⁺CD25^high Breg cells were purified from human peripheral blood. Based on the coculture system of Breg cells and CD4⁺ T cells in vitro, Breg cells were found to promote the increase in regulatory T (Treg) cells while decreasing the number of Th17 cells. Breg cells regulate Treg cells through two processes: cell-cell contact and cytokines. TGF-βsRII, a blocker of transforming growth factor-β (TGF-β), can attenuate the effects of Treg elevation, suggesting that TGF-β is the main cytokine, while Breg cells rather than interleukin-10 (IL-10) regulate the differentiation of Treg cells. However, Th17 cells were mainly regulated by cytokines, without an obvious regulatory effect on cell-cell contacts. Breg cells may regulate Th17 cells by a pathway independent of TGF-β and IL-6. The coculture of Breg cells and CD4⁺ T cells led to changes in the cytokine spectrum, which included significant increases in IL-4, IL-6 and IL-10 but not obvious changes in IL-2, IFN-γ and TNF. The inhibitory effect of Breg cells was weakened by blocking cell-cell contacts in cultures separated with the Transwell chamber because IL-10 decreased while IL-6 increased when compared with cocultured Breg and CD4⁺ T cells. When the IL-10 inhibitor IL-10sRα was added, IL-6 and TNF levels significantly increased, while treatment with the TGF-β inhibitor TGF-βsRII did not result in similar changes, suggesting that IL-10 is an important molecule to inhibit the proinflammatory factors IL-6 and TNF in this culture system.     

Synovial C-reactive protein features high negative predictive value but is not useful as a single diagnostic parameter in suspected periprosthetic joint infection (PJI)

ObjectivesSynovial fluid C-reactive protein (syCRP) has been recently described as a new biomarker in preoperative diagnostics to identify periprosthetic joint infections (PJI). The aim of this study was to evaluate syCRP in a large cohort of patients with suspected PJI and to calculate the optimal cut-off to diagnose PJI.MethodsBetween September 2015 and June 2017, we prospectively included patients with suspected PJI, in which syCRP was additionally measured along with routine preoperative diagnostic serum and synovial biomarkers. We analysed the sensitivity and specificity of syCRP using receiver operating characteristic curves.ResultsWe included 192 cases (hip n = 80, knee n = 91, shoulder n = 21) with a final diagnosis of PJI in 26 cases (14.0%). Combined for all joints, the syCRP values were significantly higher in the PJI group than in the no PJI group (median: 13.8 vs. 0 mg/l; p < 0.001). The optimal cut-off (Youden Index: 0.71) for the PJI diagnosis combined for all joints was at a syCRP value of 2.9 mg/l with a sensitivity of 88%, a specificity of 82%, and a negative predictive value of 98%.ConclusionsSyCRP features high negative predictive value but is not useful as a single diagnostic parameter in suspected periprosthetic joint infection (PJI).     

肠道病毒71型结构蛋白和结构蛋白和非结构蛋白的分子生物学特性

  肠道病毒71型（enterovirus 71,EV71）和柯萨奇病毒A组16型感染导致的手足口病已在世界范围内引起数次大规模暴发和流行,成为婴幼儿健康的重大威胁。对EV71结构的分子生物学特征的研究有助于为EV71疫苗的研发提供重要依据,此文就EV71的几种重要结构蛋白和非结构蛋白在病毒感染过程中作用的研究现状做一综述。     

Safety and efficacy of DTaP-IPV vaccine use in healthcare workers for prevention of pertussis

Pertussis can be fatal for infants. The best way to prevent infant pertussis is to promote adult immunization. However, Tdap has not been licensed in Japan, so we investigated the effect and safety of the DTaP-IPV vaccine instead. The study examined 154 pediatric healthcare workers. Participants without effective levels of antibodies against pertussis toxin were given DTaP-IPV, reduced to 0.2 mL. In total, 48 of the 154 participants (31.2%) were seronegative for pertussis toxin. After vaccination of the seronegative participants, 40 of the 41 measured (97.5%) had acquired an effective response, and all 35 of those tested maintained a protective antibody level ten months after vaccination. Redness was observed in 14 of the 41 (34.1%) and soreness in 19 (46.3%). This study demonstrated that vaccination with reduced 0.2 mL DTaP-IPV successfully provided effective immunity. At least ten months after vaccination, all subjects maintained an adequate level of antibodies.     

Immune mechanisms induced by an HSV-1 mutant strain: Discrepancy analysis of the immune system gene profile in comparison with a wild-type strain

Herpes simplex virus is a prevalent pathogen of humans of various age groups. The fact that no prophylactic or therapeutic vaccine is currently available suggests a significant need to further investigate the immune mechanisms induced by the virus and various vaccine candidates. We previously generated an HSV-1 mutant strain, M3, with partial deletions in ul7, ul41 and LAT that produced an attenuated phenotype in mice. In the present study, we performed a comparative analysis to characterize the immune responses induced by M3 versus wild-type HSV-1 in a mouse model. Infection with wild-type HSV-1 triggered an inflammatory-dominated response and adaptive immunity suppression and was accompanied by severe pathological damage. In contrast, infection with M3 induced a systematic immune response involving full activation of both innate and adaptive immunity and was accompanied by no obvious pathological changes. Furthermore, the immune response induced by M3 protected mice from lethal challenge with wild-type strains of HSV-1 and restrained virus proliferation and impaired latency. These data are useful for further HSV-1 vaccine development using a mutant strain construction strategy.     

成年树鼩骨骼系统CT三维可视化模型建立及分析

目的 建立树鼩骨骼系统的三维可视化模型，为树鼩的骨骼系统疾病诊断提供依据。方法 使用日本东芝Aquillon one 320 排螺旋CT进行扫描，100 kV，80 mA，球管转数每转0.35 s，螺距1.35，图像为0.5 mm，层间隔为0.5 mm，使用骨算法重建。在并行计算环境中采用Vitrea软件包，选择Musculoskeletal CT选项，采用容积成像（VR）、多平面成像（MPR）、曲面成像（CPR）技术进行三维重建。结果 重建后的可视化模型结构明显、清楚，可以真实地在计算机中重现出树鼩的骨骼系统三维模型。在此模型中，在头骨背面可见五条隆起的嵴，侧面观可见四个大孔：位于前端的外鼻孔、前颌骨后上方的眶下孔、由颧弓所范围着的眼窝和鼓泡外侧的外耳道。此外，可见一些较小的头骨孔，如视神经孔、颌下神经孔。利用该成像技术可定量测定各主要骨骼的长度、前后径、左右径和身长、尾长等数据。尤其是还获得了以往通过解剖难以测量准确的数据：如腭长、臼齿列长等较细微的数据，以及解剖不易获得脊椎前后径及左右径的数据。同时还利用三维可视化技术发现了树鼩存在有骨盆形态不对称、胸廓增大、骨折、跟腱周围滑膜囊钙化的骨骼异常情况。结论 所建立的CT三维可视化技术可确定树鼩骨骼系统的特殊特征，对树鼩骨骼系统无侵入性的生态分类、鉴定、进化分析均有重要意义。     

Vesicular stomatitis virus-based vaccines expressing EV71 virus-like particles elicit strong immune responses and protect newborn mice from lethal challenges

Enterovirus 71 (EV71) belonging to the Picornaviridae family is considered the most frequently detected causative agent in hand-foot-and-mouth disease (HFMD) and is a serious threat to public health in the Asia-Pacific region. There are currently no approved vaccines or effective drugs for EV71. In this study, using recombinant vesicular stomatitis virus (rVSV) expressing viral VP1 protein (mVP1) of EV71 as a control, we generated two types of rVSVs that can form EV71 virus-like particles (VLPs). First, we co-infected two rVSVs singly expressing P1 (mP1) and 3CD (m3CD) of EV71. Second, we inserted P1 and 3CD into one VSV backbone to generate an rVSV expressing P1 and 3CD together (mP1-3CD). When P1 and 3CD were expressed in the cells either co-infected with mP1 and m3CD (mP1/m3CD) or infected with mP1-3CD, P1 was cleaved by 3CD and produced VP1, VP3, and VP0 to form VLPs. Furthermore, mice immunized with mP1/m3CD or mP1-3CD showed higher humoral and cellular immunity responses than mice immunized with mVP1. Finally, the rVSVs expressing the EV71 proteins were evaluated in mice to determine their potential to protect against a lethal EV71 virus challenge, and among all the rVSVs, the mP1-3CD was shown to be the most promising vaccine candidate for EV71 protection.     

A novel inactivated enterovirus 71 vaccine can elicit cross-protective immunity against coxsackievirus A16 in mice

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that mainly affects infants and children. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major pathogens of HFMD. Two EV71 vaccines were recently licensed in China and the administration of the EV71 vaccines is believed to significantly reduce the number of HFMD-related severe or fatal cases. However, a monovalent EV71 vaccine cannot cross-protect against CA16 infection, this may result in that it cannot effectively control the overall HFMD epidemic. In this study, a chimeric EV71, whose VP1/210–225 epitope was replaced by that of CA16, was constructed using a reverse genetics technique to produce a candidate EV71/CA16 bivalent vaccine strain. The chimeric EV71 was infectious and showed similar growth characteristics as its parental strain. The replacement of the VP1/210–225 epitope did not significantly affect the antigenicity and immunogenicity of EV71. More importantly, the chimeric EV71 could induce protective immunity against both EV71 and CA16, and protect neonatal mice against either EV71 or CA16 lethal infections, the chimeric EV71 constructed in this study was shown to be a feasible and promising candidate bivalent vaccine against both EV71 and CA16. The construction of a chimeric enterovirus also provides an alternative platform for broad-spectrum HFMD vaccines development.