Molecular characterization of the full-length genome of the Japanese encephalitis viral strain K87P39

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain K87P39, isolated from a pool of circulating Culex tritaeniorhynchus mosquitoes in Korea. In comparison with 27 fully sequenced JEV genomes currently available, we found that the 10968-nucleotide RNA genome of K87P39 has a nine-nucleotide deletion in the 3' nontranslated variable region and that its single open reading frame has a total of eight amino acid substitutions. The K87P39 isolate is highly similar to other JEV isolates, and homology ranges from 97.9 to 89.0% at the nucleotide level, and 99.1 to 96.7% at the deduced amino acid level. Phylogenetic analyses using the full-length sequence of the 27 available JEV genomes showed that the K87P39 strain is most closely related to six Chinese SA14 derivatives and that it is distantly related to the Australian FU, Korean K94P05 and Japanese Ishikawa strains. In addition, we also found that phylogenetic relationships based on the full-length genome are highly similar to those based on the E gene, indicating that phylogenetic analysis of the E gene will be useful for studying the genetic relationships among JEV isolates. We therefore performed a more extensive E gene-based phylogenetic analysis on a selection of 70 JEV isolates available from GenBank, which represent a temporally and geographically wide variety of JEV strains.     

白细胞介素—1对大鼠下丘脑室旁核Fos癌蛋白和CRF的诱导作用

将白细胞介素-1(IL)注入大鼠侧脑室,用Fos癌蛋白抗体免疫组化法检测了下丘脑室旁核的激活神经元:大量位于含促肾上腺皮质激素释放因子(CRF)相应区域的室旁核小细胞部神经元呈Fos免疫强阳性。Fos和CRF的免疫双染色显示了许多Fos免疫阳性神经元也呈CRF免疫阳性。此外,在IL-1注射动物中,CRF的免疫阳性显著加强,提示CRF合成增加。     

大鼠下丘脑及邻近区域催产素免疫阳性神经元与垂体后叶的关系

本文运用免疫细胞化学PAP及ABC法,显示大白鼠下丘脑内OXT免疫阳性神经元,并于垂体后叶注射WGA-HRP,显示下丘脑中逆行标记细胞,结合免疫细胞化学方法,观察下丘脑及其邻近区域内HRP与OXT双标记细胞,证实下丘脑视上核、室旁核、穹窿前核和后核、血管周细胞群、下丘脑视前区、下丘脑前区及外侧区、背侧副细胞群内、室周部、第三脑室侧壁室管膜细胞下及室间孔部室管膜细胞下,均有OXT免疫阳性神经元,其中至少部分神经元可发出向垂体后叶的投射纤维。位于第三脑室侧壁室管膜下及室间孔部室管膜下的神经元,可能监测脑脊液中各种因素的变化,调节垂体后叶OXT的分泌,也可能直接通过共树突向脑脊液内释放OXT。     

人胚胎海马发育的形态学研究 Ⅴ.室管膜的发生

运用HE和Nissl染色、免疫组织化学法、透射电镜及扫描电镜，对60例6周至足月的人胚胎海马室管膜上皮变化进行了观察。发现胚胎发育过程中室管膜发生了剧烈变化。最早室管层神经上皮细胞为假复层柱状，随着未分化细胞向外迁徙，海马室管膜层神经上皮细胞迅速增殖，形成复层上皮。当室管膜层细胞停止迁徙时，室管膜开始向假复层柱状及单层柱状上皮转变。电镜观察，胚胎早期神经上皮细胞由未分化细胞构成；其特点是，细胞质内各种特化细胞器匮乏，但糖原丰富。15周左右未分化细胞开始向长突细胞及室管膜细胞分化。长突细胞电子密度高，底部有细长突起，表面有微绒毛，胞质内微丝丰富；而室管膜细胞电子密度低，底部无突起，但表面有丰富的纤毛。对长突细胞及免疫组化染色的GFAP阳性细胞进行形态和发育特征的比较，提示两者属同一类细胞。扫描电镜下，15周前室管膜表面微绒毛较多，以后纤毛逐步发育，大量密集纤毛布满于室管膜表面。此外，还能见到一类接触脑脊液神经元，这类神经元可为多极或双极，并有突起伸入室管膜上皮内。     

实时荧光定量RT-PCR监测恒河猴体内人/猴免疫缺陷病毒载量在传代过程中的变化

目的为分析中国SHIV/猕猴AIDS模型的病毒载量变化趋势,建立一种实时、灵敏、特异的针对人/猴免疫缺陷病毒的定量检测方法。方法体外转录制备RNA标准品,利用TaqManEZRT-PCR试剂盒的反应体系和针对SHIVgag保守区91个碱基的TaqMan探针和引物,建立一步法实时荧光定量RT-PCR。提取126份来自SHIV-CN97001感染恒河猴血浆病毒RNA并定量检测。结果利用梯度稀释的RNA标准品对反应体系进行优化,标准曲线下限达到2×102拷贝/ml,相关性(r>0.99)及重复性(CV=4.14%)均能达到测定要求。病毒载量的检测结果表明SHIV-CN97001在猴体内传代过程中病毒载量有先升后降的趋势,病毒载量通常在接种病毒或感染猴的全血后第14天达到高峰。血浆载量可达到105~106拷贝/ml。结论成功地建立了一步法定量SHIVRNA的实时荧光定量RT-PCR,为SHIV/恒河猴AIDS模型的建立与应用提供了灵敏的病毒载量检测方法。SHIV-CN97001的体内繁殖能力在猴体内传代过程中有所增强。     

Adenovirus-mediated intratumoral lymphotactin gene transfer potentiates the antibody-targeted superantigen therapy of cancer

Bacterial superantigens are extremely potent activators of murine and human T lymphocytes. To engineer superantigens for cancer immunotherapy, staphylococcal enterotoxin A (SEA) was genetically fused to the Fab region of the human colon carcinoma-reactive monoclonal antibody (mAb) C215. Fusion protein C215Fab-SEA can trigger cytotoxic T cells against C215 antigen positive tumor cells and induce tumor-suppressive cytokines. However, the antitumor effect of C215Fab-SEA is often not satisfactory because of T cell deletion after activation and failure to induce potent CTL activity after repeated administration. Lymphotactin (Lptn) is a potent chemoattractant for T cells and NK cells. To improve the therapeutic efficacy of fusion protein C215Fab-SEA we investigated in this study the antitumor responses elicited by combination of C215Fab-SEA and adenovirus-mediated intratumoral Lptn gene transfer in the preestablished C215 antigen expressing B16 melanoma murine model. More significant inhibition of tumor growth and prolonged survival time were observed in tumor-bearing mice that received combined therapy of C215Fab-SEA and Ad-Lptn than those of mice treated with C215Fab-SEA or Ad-Lptn alone. The highest CTL activity of tumor-bearing mice was induced after combined therapy. Intratumoral coadministration of C215Fab-SEA and Ad-Lptn augmented splenic NK activity of tumor-bearing mice most markedly. Our data demonstrate that the in vivo antitumor effect of C215Fab-SEA immunotherapy is potentiated significantly by combination with intratumoral Lptn gene transfer through more efficient induction of specific and nonspecific antitumor immune responses.     

Structure and immersion behavior of plasma-sprayed apatite-matrix coatings

The microstructure and properties of a series of plasma-sprayed coatings from sinter-granulated powders fabricated from SiO2, CaO, P2O5 and Na2O-containing HA composite powders on Ti-6Al-4V substrate were reported. The immersion behavior of these coatings in a simulated body fluid (SBF) was also investigated. The results showed that sinter-granulated apatite-matrix powders were irregularly shaped and appeared quite similar. XRD patterns showed that during fabrication of the powders, P2O5 and SiO2 enhanced the decomposition of HA structure, while CaO and Na2O did not. Reasonably high bond strengths (45-50 MPa) were obtained from all coatings. The plasma spray process itself enhanced the decomposition of apatite and chemical reactions among different phases. When immersed in SBF, the intensities of such phases as alpha- and beta-TCP in all coatings decreased with immersion time and an apatite precipitation took place on all coating surfaces. The immersed SiO2- and CaO-containing HA (HSC) coating had the highest rate of apatite precipitation among all coatings. The variations in calcium ion concentration in simulated body fluid indicated that the HSC-immersed solution reached its maximal Ca concentration the earliest, while the HSCP (HA, SiO2, CaO and P2O5)-immersed solution reached its maximum the latest.     

Lifestyle Risk Score: handling missingness of individual lifestyle components in meta-analysis of gene-by-lifestyle interactions

Recent studies consider lifestyle risk score (LRS), an aggregation of multiple lifestyle exposures, in identifying association of gene-lifestyle interaction with disease traits. However, not all cohorts have data on all lifestyle factors, leading to increased heterogeneity in the environmental exposure in collaborative meta-analyses. We compared and evaluated four approaches (Naïve, Safe, Complete and Moderator Approaches) to handle the missingness in LRS-stratified meta-analyses under various scenarios. Compared to “benchmark” results with all lifestyle factors available for all cohorts, the Complete Approach, which included only cohorts with all lifestyle components, was underpowered due to lower sample size, and the Naïve Approach, which utilized all available data and ignored the missingness, was slightly inflated. The Safe Approach, which used all data in LRS-exposed group and only included cohorts with all lifestyle factors available in the LRS-unexposed group, and the Moderator Approach, which handled missingness via moderator meta-regression, were both slightly conservative and yielded almost identical p values. We also evaluated the performance of the Safe Approach under different scenarios. We observed that the larger the proportion of cohorts without missingness included, the more accurate the results compared to “benchmark” results. In conclusion, we generally recommend the Safe Approach, a straightforward and non-inflated approach, to handle heterogeneity among cohorts in the LRS based genome-wide interaction meta-analyses.Subject terms: Genetics, Risk factors     

BDNF对体外培养的大鼠脊髓前角神经元内突触素Ⅰ与突触囊泡素表达的影响

探讨BDNF对体外培养的大鼠脊髓前角神经元内突触素I与突触囊泡素(SYN)表达的影响。取孕14d大鼠子宫内胎鼠的脊髓腹侧部分神经元,体外有血清培养。在培养7d后,随机分成对照组、BDNF组和抗BDNF组。BDNF组培养液中加入BDNF(20ng/ml),抗BDNF组培养液中加入BDNF抗体(20μg/ml),对照组加入等量Hanks液。3d后在倒置显微镜下计数三组神经元存活数,并用NF200、MAP2、NSE的免疫组化反应对神经细胞进行鉴定。行突触素I与SYN免疫组化反应,对部分细胞行突触素ImRNA原位杂交反应,运用图像分析系统对突触素I与SYN免疫组织反应阳性产物以及突触素I原位杂交反应阳性产物作光密度分析。结果发现有血清培养时各组脊髓前角神经元的存活数无显著差异(P>0.05);BDNF组突触素I与SYN免疫反应阳性产物的平均光密度值高于其它两组,抗BDNF组最低(P<0.01)。BDNF组突触素ImRNA阳性产物的平均光密度值明显高于其它两组,抗BDNF组突触素ImRNA阳性产物的平均光密度值最低(P<0.01)。本研究结果提示BDNF对有血清培养时脊髓前角神经元的存活没有明显影响,但BDNF可明显上调培养的脊髓前角神经元内突触素I与SYN的表达。