A simple approach for enhanced immune response using engineered dendritic cell targeted nanoparticles

In this study, we demonstrate a simple strategy for enhanced immune response using a two-component dendritic cell (DC) targeted antigen delivery system. One component consists of a recombinant bifunctional fusion protein (bfFp) used for DC targeting, whereas, the other component is made of biotinylated PLGA nanoparticles that encapsulate the antigen. The fusion protein (bfFp) made of a truncated core-streptavidin fused to anti-DEC-205 single chain antibody (scFv) was mixed with ovalbumin-loaded biotinylated NPs that were formulated using biotin–PEG (2000)–PLGA, and the combination, bfFp functionalized NPs was used for DC targeted antigen delivery. In vitro DC uptake studies revealed a 2-fold higher receptor-mediated uptake of bfFp functionalized NPs when compared to non-targeted NPs. Immunization of the mice with the bfFp functionalized NPs in conjunction with DC maturation stimulus (anti-CD40 mAb) enhanced OVA-specific IgG and IgG subclass responses. Splenocytes of these mice secreted significantly higher levels of Th1 (IFN-γ and IL-2) cytokines upon ex vivo restimulation with OVA. The promising outcomes of the bfFp functionalized DC targeted system support its use as a versatile vaccine delivery system for the design of monovalent or polyvalent vaccines.     

结核分枝杆菌Ag85B基因体外扩增、克隆及在耻垢分枝杆菌中的表达

目的体外扩增结核杆菌Ag85B基因,构建大肠埃希菌-分枝杆菌穿梭质粒ps3000-Ag85B,并重组耻垢分枝杆菌(Mycobacteriums megmatis mc2155)。方法采用聚合酶链式反应(PCR)方法,体外扩增结核杆菌Ag85B基因,克隆入pGEMT载体;构建亚克隆ps3000-Ag85B大肠埃希菌-分枝杆菌穿梭质粒,电转化方法将有Ag85B基因的穿梭质粒转化到耻垢分枝杆菌中。热诱导此重组耻垢分枝杆菌,用SDS-PAGE电泳观察Ag85B蛋白的表达,Western blot鉴定其生物学活性。结果PCR扩增结核杆菌Ag85B基因片段大小为990bp,构建的穿梭质粒ps3000-Ag85B酶切片段为990bp,与理论值相符。经Western blot检测,该重组耻垢杆菌表达蛋白能被结核病患者血清识别。结论Ag85B重组耻垢分枝杆菌构建成功,为下一步表达Ag85B蛋白的重组BCG(Bacilli Calmette-Guérin)疫苗的研究奠定了基础。     

Arterial Pulsatility and Circulating von Willebrand Factor in Patients on Mechanical Circulatory Support

BackgroundThe main risk factor for bleeding in patients with continuous-flow mechanical circulatory support (CF-MCS) is the acquired von Willebrand factor (VWF) defect related to the high shear-stress forces developed by these devices. Although a higher bleeding rate has been reported in CF-MCS recipients who had reduced pulsatility, the relation between pulsatility and the VWF defect has never been studied.ObjectivesThe purpose of this study was to investigate the relation between pulsatility and VWF under CF-MCS.MethodsWe assessed the effect of 2 CF-MCS on VWF multimer degradation in a mock circulatory loop (model 1). Using these devices, we investigated in a dose-effect model (model 2) 3 levels of pulsatility in 3 groups of swine. In a cross-over model (model 3), we studied the effects of sequential changes of pulsatility on VWF. We reported the evolution of VWF multimerization in a patient undergoing serial CF-MCS and/or pulsatile-MCS.ResultsWe demonstrated the proteolytic degradation of VWF multimers by high shear CF-MCS in a circulatory loop without pulsatility. We observed both in swine models and in a patient that the magnitude of the VWF degradation is modulated by the pulsatility level in the high shear-stress level condition, and that the restoration of pulsatility is a trigger for the endothelial release of VWF.ConclusionsWe demonstrated that the VWF defect reflects the balance between degradation induced by the shear stress and the endothelial release of new VWF triggered by the pulsatility. This modulation of VWF levels could explain the relationship between pulsatility and bleeding observed in CF-MCS recipients. Preservation of pulsatility may be a new target to improve clinical outcomes of patients.     

前S1抗原作为乙肝病毒复制指标的临床应用价值

目的观察前S1抗原(PreS1Ag)作为乙肝病毒复制指标的临床应用价值。方法对462名慢性乙型肝炎患者用ELISA法检测PreS1Ag和血清HBV标志物(HBV-M),用荧光定量PCR法测定HBV-DNA含量。结果462例HBsAg阳性患者中PreS1Ag阳性304人,阳性率65.8%,显著高于HBeAg阳性率(34%)(P<0.01)。HBsAg、HBeAg、HBcAb阳性组PreS1Ag阳性率为79.1%,HBsAg、HBeAb、HBcAb阳性组PreS1Ag阳性率为44.1%,两组差异有显著性(P<0.01)。PreS1Ag阳性率与HBV-DNA阳性率高度符合,阳性符合率达80%。结论前S1抗原能够敏感地反映乙型肝炎病毒复制情况,尤其可以反映HBeAg阴性患者是否有病毒复制。是对乙肝病毒标志物的有益补充,可作为临床常规检测指标。     

DNA polymorphism at the apolipoprotein B locus is associated with lipoprotein level

A strong association has been uncovered between DNA variation at the apolipoprotein B (apoB) locus (detectable with the restriction endonuclease XbaI) and apoB level. The findings are suggestive of associations also between this DNA polymorphism and total cholesterol as well as fasting triglyceride levels, confirming recent results reported by British workers. The data suggest that lipid/apolipoprotein associations with the XbaI polymorphism are primarily caused by an effect on apoB level. In the present and in a previously reported study we found a strong association between the XbaI polymorphism and the homospecific Ag antigenic variation in low density lipoprotein (LDL) which had previously exhibited associations with lipid levels. The present data indicate that the apoB/lipid associations of the Ag and XbaI polymorphisms may reflect the same phenomenon. The associations reported could reflect variation in an apoB domain close to Ag as well as to the XbaI restriction site that is of importance for lipid binding by apoB. Alternatively, the association of apoB level with the XbaI polymorphism (which reflects a silent third base mutation in a threonine codon) could reflect phenomena related to codon usage.     

细胞因子IL-18基因对Ag85A DNA疫苗诱导产生抗体水平的影响

目的 :研究表达含白介素 (IL) 18基因的质粒 ,对结核分枝杆菌 (MTB)H37Rv Ag85A基因疫苗诱导免疫应答的影响。方法 :从正常人外周血单核细胞 (PMBCs)中提取RNA ,用RT PCR扩增IL 18cDNA ,并克隆入载体pGEM Teasy中。测序证实后 ,亚克隆真核表达载体pcDNA3.1的BamHⅠ和EcoRⅠ酶切位点。将分别表达人IL 18基因的真核表达质粒pcIL18与MTBpcAg85A基因疫苗联合肌注免疫BALB c小鼠 ,共免疫 3次 ,每次间隔 2周。每次免疫后 2周采血 ,分离血清 ,用ELISA检测小鼠血清抗Ag85A抗体的滴度。结果 :用RT PCR成功地从人PMBCs的RNA中扩增IL 18cDNA ,测序结果正确。用BamHⅠ和EcoRⅠ酶切鉴定证实 ,目的基因已插入载体pcDNA3.1中 ,阳性克隆命名为pcIL18。联合应用pcIL18,三次免疫后血清抗Ag85A抗体的滴度明显较单独应用pcAg85A增高。结论 :pcIL18与pcAg85A基因疫苗联合免疫 ,可显著增强Ag85A抗原的特异性体液免疫应答。pcIL18+pcAg85A基因联合免疫是否具有增强Ag85A抗原特异性细胞免疫的作用有待进一步研究     

结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察

目的:构建编码结核分枝杆菌Ag85A分泌蛋白重组真核表达质粒,研究其与hIL-12联合免疫小鼠后的细胞免疫应答。方法:(1)构建质粒:采用PCR法从H37Rv菌株中扩增Ag85A编码基因,用限制性内切酶消化后,插入克隆载体PMD20-T中,经酶切鉴定与序列测定证实后,以亚克隆法构建于真核表达载体PCDNA3.1的相应酶切位点。(2)动物实验:50只C57BL/6N小鼠随机分为:①Ag85A基因疫苗+hIL-12质粒组(联合免疫组);②重组Ag85A基因疫苗组;③卡介苗BCG组(阳性对照);④空载体组(阴性对照);⑤PBS组(空白对照)。基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106CFU BCG免疫1次,约0.3 ml/只。第三次免疫小鼠后28天,处死各组小鼠,分离脾细胞,ELISA法检测脾细胞培养上清液中IFNγ-、IL-2、IL-4水平;乳酸脱氢酶释放法检测脾细胞杀伤活性;分离的脾细胞经TB-PPD刺激后,XTT比色法检测脾淋巴细胞增殖活性。结果:(1)成功构建结核分枝杆菌Ag85A基因DNA疫苗。(2)联合免疫组能诱导较强烈的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-r和IL-2水平显著高于Ag85A基因疫苗组,与BCG组相当,IL-4分泌减少;特异性CTL杀伤活性明显增强;淋巴细胞增殖活性也明显高于其他组别。结论:hlL-12表达质粒能够增强结核分枝杆菌Ag85A基因DNA疫苗所诱导的小鼠免疫应答。     

结核分枝杆菌Ag85A蛋白的原核表达、纯化和免疫反应性

目的通过原核表达获得结核分枝杆菌Ag85A蛋白。方法用PCR从结核分枝杆菌H37Rv菌株中扩增出编码Ag85A的fbpA基因,克隆入原核表达载体pProEXHTb,产生重组质粒pPro85A后,转化至大肠杆菌感受态细胞BL21并诱导大量表达。用镍纯化系统纯化重组Ag85A蛋白,用不同分枝杆菌感染的小鼠血清通过ELISA确定其免疫反应性。利用PCR技术鉴定fbpA基因在不同分枝杆菌的分布。结果32 ku的Ag85A蛋白获得高效表达和纯化。表达Ag85A蛋白的fbpA基因在结核分枝杆菌H37Rv、H37Ra、BCG、草分枝杆菌、土地分枝杆菌、耻垢分枝杆菌和次要分枝杆菌中均有表达,但在牝牛分枝杆菌中未表达。结核病患者和结核分枝杆菌毒株H37Rv感染小鼠血清所产生的抗Ag85A抗体滴度最高。结论重组Ag85A蛋白已成功表达纯化,并保留了免疫反应性。     

乳洁灵胶囊治疗乳腺病284例

笔者以理气活血为主，兼泻肝火，补肝肾疏通乳络，解郁软坚，清热解毒，自拟乳洁灵胶囊治疗乳腺疾病２８４例，总有效率达９０．２％。