多种小分子干扰RNA联合抑制乙型肝炎病毒的体外研究

小分子干扰RNA(siRNA)能够在哺乳动物细胞中引起包括病毒基因在内的基因沉默。为了研究多种siRNA联合应用在体外抑制乙型肝炎病毒(HBV)复制中的效果,本研究设计了12种针对不同HBV靶点的siRNA,转染可稳定分泌HBV颗粒的HepG22.2.15细胞,并采用了酶联免疫法检测上清液中HBsAg和HBeAg的含量,实时定量PCR法检测细胞中HBV的DNA含量。结果发现这12种分子均能在不同程度上抑制病毒复制。进一步研究表明它们对HBV的抑制作用在一定程度上存在剂量效应和协同作用,单分子siRNA在80nmol/L处对HBsAg和HBeAg的抑制率分别可达到约80%和60%,而多分子siRNA组合在20nmol/L处对HBsAg就能达到90%的抑制率,但对HBeAg表达的抑制率提高不明显。单分子siRNA在80nmol/L处对HBVDNA复制的抑制率可达到90%以上,而多分子siRNA组合在20nmol/L处对DNA含量就能达到约90%的抑制率。本研究的结果为进一步开发新的联合应用多种siRNA治疗HBV的途径打下了基础。     

Serotype distribution of invasive Streptococcus pneumoniae in Canada during the introduction of the 13-valent pneumococcal conjugate vaccine, 2010

A baseline serotype distribution was established by age and region for 2058 invasive Streptococcus pneumoniae isolates collected during the implementation period of the 13-valent pneumococcal conjugate vaccine (PCV13) program in many parts of Canada in 2010. Serotypes 19A, 7F, and 3 were the most prevalent in all age groups, accounting for 57% in <2 year olds, 62% in 2-4 year olds, 45% in 5-14?year olds, 44% in 15-49?year olds, 41% in 50-64?year olds, and 36% in ≥65?year olds. Serotype 19A was most predominant in Western and Central Canada representing 15% and 22%, respectively, of the isolates from those regions, whereas 7F was most common in Eastern Canada with 20% of the isolates. Other prevalent serotypes include 15A, 23B, 12F, 22F, and 6C. PCV13 serotypes represented 65% of the pneumococci isolated from?<2 year olds, 71% of 2-4 year olds, 61% of 5-14 year olds, 60% of 15-49 year olds, 53% of 50-64 year olds, and 49% of the?≥65?year olds. Continued monitoring of invasive pneumococcal serotypes in Canada is important to identify epidemiological trends and assess the impact of the newly introduced PCV13 vaccine on public health.     

羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖(Sargassum Fusiforme Polysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western blot法测定Caspase-3酶原的变化; RT-PCR检测Caspase-3 mRNA表达。结果显示:SFPS作用lovo细胞24,36,48和72h的IC_(50)分别为375,355,178和60mg/L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征:细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5-300mg/L作用24h后,显示有凋亡细胞特有的DNA梯状条带;而500mg/L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。     

Elevated plasma levels of soluble TNF receptors are associated with morbidity and mortality in patients with acute lung injury

Ventilator-induced lung injury (VILI) is an inflammatory process that can be attenuated by lung protective ventilation strategies. Our objectives to further investigate the pathogenesis of ALI and VILI and the mechanism of lung protection in these syndromes were: 1) to determine if plasma measurements of soluble TNF receptor I (sTNFRI) and II (sTNFRII) would predict the development of ALI and mortality in a small single center trial; 2) to test the predictive value of these markers and of TNF-alpha in a larger, broader group of patients with ALI; 3) to test the hypothesis that low tidal volume ventilation (LTVV) would be associated with a decrease in plasma levels of TNF-alpha, sTNFRI, and sTNFRII. In the single center study, sTNFRI and II levels were higher in patients at risk for and with ALI, but they did not predict the development of the syndrome. In the multicenter trial sTNFRI and II were strongly associated with mortality (OR 5.76/1 log10 increment in receptor level; 95% CI 2.63-12.6 and OR 2.58; 95% CI 1.05-6.31, respectively) and morbidity measured as fewer nonpulmonary organ failure-free and ventilator-free days. The LTVV strategy was associated with an attenuation of plasma sTNFRI levels. In vitro, stimulated A549 cells release sTNFRI but not sTNRFII. In conclusion, plasma levels of sTNFRI and II can serve as biomarkers for morbidity and mortality in patients with ALI. Furthermore, LTVV is associated with a specific decrease in sTNFRI levels. This suggests that one beneficial effect of LTVV may be to attenuate alveolar epithelial injury.     

Epistatic Gene-Based Interaction Analyses for Glaucoma in eMERGE and NEIGHBOR Consortium

Primary open angle glaucoma (POAG) is a complex disease and is one of the major leading causes of blindness worldwide. Genome-wide association studies have successfully identified several common variants associated with glaucoma; however, most of these variants only explain a small proportion of the genetic risk. Apart from the standard approach to identify main effects of variants across the genome, it is believed that gene-gene interactions can help elucidate part of the missing heritability by allowing for the test of interactions between genetic variants to mimic the complex nature of biology. To explain the etiology of glaucoma, we first performed a genome-wide association study (GWAS) on glaucoma case-control samples obtained from electronic medical records (EMR) to establish the utility of EMR data in detecting non-spurious and relevant associations; this analysis was aimed at confirming already known associations with glaucoma and validating the EMR derived glaucoma phenotype. Our findings from GWAS suggest consistent evidence of several known associations in POAG. We then performed an interaction analysis for variants found to be marginally associated with glaucoma (SNPs with main effect p-value <0.01) and observed interesting findings in the electronic MEdical Records and GEnomics Network (eMERGE) network dataset. Genes from the top epistatic interactions from eMERGE data (Likelihood Ratio Test i.e. LRT p-value <1e-05) were then tested for replication in the NEIGHBOR consortium dataset. To replicate our findings, we performed a gene-based SNP-SNP interaction analysis in NEIGHBOR and observed significant gene-gene interactions (p-value <0.001) among the top 17 gene-gene models identified in the discovery phase. Variants from gene-gene interaction analysis that we found to be associated with POAG explain 3.5% of additional genetic variance in eMERGE dataset above what is explained by the SNPs in genes that are replicated from previous GWAS studies (which was only 2.1% variance explained in eMERGE dataset); in the NEIGHBOR dataset, adding replicated SNPs from gene-gene interaction analysis explain 3.4% of total variance whereas GWAS SNPs alone explain only 2.8% of variance. Exploring gene-gene interactions may provide additional insights into many complex traits when explored in properly designed and powered association studies.     

Adenovirus-mediated expression of pig α(1,3) galactosyltransferase reconstructs Gal α(1,3) Gal epitope on the surface of human tumor cells

INTRODUCTIONGal a(1, 3) Gal (gal epitope) is a carbohydrate epitope, which is produced in large amounton the cells of pigs, mice and New World monkey(monkey of South America) by the glycosylationenzyme G alal 1 ) 4G IcNAc3- a- D- galactosyltransferase[or(1, 3)GT; EC2.4.1.511111. This enzyme is active in the Golgi appaxatus of cells and transfers galactose from the sugandonor uridine diphoSphate galactose (UDP-galactose) to the acceptor Nacetyllactosamine residue (Galaal-4GlcNAc-R…     

Immunogenicity is not improved by increased antigen dose or booster dosing of seasonal influenza vaccine in a randomized trial of HIV infected adults

Introduction

The risk of poor vaccine immunogenicity and more severe influenza disease in HIV necessitate strategies to improve vaccine efficacy.

Methods

A randomized, multi-centered, controlled, vaccine trial with three parallel groups was conducted at 12 CIHR Canadian HIV Trials Network sites. Three dosing strategies were used in HIV infected adults (18 to 60 years): two standard doses over 28 days, two double doses over 28 days and a single standard dose of influenza vaccine, administered prior to the 2008 influenza season. A trivalent killed split non-adjuvanted influenza vaccine (Fluviral™) was used. Serum hemagglutinin inhibition (HAI) activity for the three influenza strains in the vaccine was measured to assess immunogenicity.

Results

297 of 298 participants received at least one injection. Baseline CD4 (median 470 cells/µL) and HIV RNA (76% of patients with viral load <50 copies/mL) were similar between groups. 89% were on HAART. The overall immunogenicity of influenza vaccine across time points and the three influenza strains assessed was poor (Range HAI ≥40 = 31–58%). Double dose plus double dose booster slightly increased the proportion achieving HAI titre doubling from baseline for A/Brisbane and B/Florida at weeks 4, 8 and 20 compared to standard vaccine dose. Increased immunogenicity with increased antigen dose and booster dosing was most apparent in participants with unsuppressed HIV RNA at baseline. None of 8 serious adverse events were thought to be immunization-related.

Conclusion

Even with increased antigen dose and booster dosing, non-adjuvanted influenza vaccine immunogenicity is poor in HIV infected individuals. Alternative influenza vaccines are required in this hyporesponsive population.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00764998","term_id":"NCT00764998"}}NCT00764998     

Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome

It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-alpha, IFN-gamma, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.