DNA-天坛痘苗复合型艾滋病疫苗小鼠体内生物分布研究

目的考察DNA-天坛痘苗复合型艾滋病疫苗在小鼠体内生物分布情况。方法采用定量PCR方法分别对复合疫苗免疫后DNA疫苗的分布情况以及复合疫苗组分HIV-痘病毒载体疫苗单独免疫后的生物分布情况进行测定。结果复合疫苗免疫后5d和30d,主要是在注射部位检测到质粒DNA残留,在免疫后第56d,各脏器均未检测到质粒DNA残留。HIV-痘病毒载体疫苗单独免疫后第5d,仅在给药部位检测到DNA残留,免疫后第36d,各脏器均未检测到DNA残留。结论复合疫苗免疫机体后短期只在注射部位分布,且随时间延长减少至消失,不会长期在机体内存留。     

汉滩病毒核酸疫苗滴鼻免疫小鼠表达动力学研究

目的 构建汉滩病毒DNA疫苗，考查小鼠鼻内接种DNA疫苗后的基因组织定位及表达动力学，为进一步研究汉滩病毒DNA疫苗粘膜免疫的疗效作前期准备。方法 采用PCR法从PJSA1175-S质粒扩增汉滩病毒的S基因片段后装入PcDNA3．1B(-)载体，并以此候选DNA疫苗滴鼻免疫BALB／c小鼠，在不同时间、不同部位取其组织抽提基因组DNA和组织总RNA，用PCR及RT-PCR检测质粒的分布和表达动力学。结果 构建了汉滩病毒DNA疫苗PcDNA3．1B-S1.3。免疫接种该疫苗第1天后在小鼠鼻咽、肺、肾、肝、脾、小肠等组织中均有质粒分布；第1周内上述组织中均可检测到特异mRNA，第2周起小肠、肝及肾中特异mRNA消失。结论成功构建了PcD-NA3．1B-S1.3DNA疫苗，构建的疫苗滴鼻免疫后在小鼠体内得到广泛的分布及表达。     

质粒DNA的鼻腔吸收和生物分布

近来 ,在基因疫苗和基因治疗领域 DNA疫苗鼻腔途径接种受人关注。本文报道了质粒 DNA鼻内接种的吸收动力学和程度 ,以及 DNA质粒在淋巴结和各器官中的分布。　　作者以编码 β半乳糖苷酶基因的质粒DNA为研究模型质粒。将模型质粒 DNA鼻内接种 6～ 8周龄 BALB/c小鼠 ,每只小鼠接受 50μg质粒 DNA,于接种后不同时间取血清、器官、鼻组织、肌肉和淋巴结 ,用定量聚合酶链反应 (PCR)测定质粒 DNA含量。　　结果表明 ,质粒 DNA鼻内接种后 5分钟被吸收进入系统循环 ,此时的血清浓度为1 50± 83 pg/ml,接种后 90分钟血清浓度达到高峰 ,…     

腺病毒载体HIV疫苗在C57BL/6小鼠体内的生物分布评价

目的:建立腺病毒载体HIV疫苗检测的实时荧光定量PCR方法,并评价疫苗在C57BL/6小鼠体内的生物分布。方法:以gag基因片段为检测对象,建立绝对定量PCR方法。C57BL/6小鼠单次肌内注射5×109vp HIV疫苗,给药后d 3,15,30,60,85收集组织脏器,用建立的荧光定量PCR方法定量检测HIV疫苗。结果:检测方法线性范围为1×102～1×107copies.μL-1,特异性强,重复性较好。HIV疫苗主要分布在注射位点,淋巴结、脾脏和肝脏也有少量分布;疫苗的量随时间延长逐渐减少。结论:肌内注射HIV疫苗后导致广泛的分布,分布的量呈时间依赖性减少,没有生殖细胞转移的危险,HIV疫苗在小鼠体内是安全的。     

DNA疫苗的不同构象在体内外表达差异研究

目的:研究DNA疫苗构象对体内和体外表达的影响。方法:分别构建绿色荧光蛋白表达质粒(pcDNA3.1-EGFP)和萤火虫荧光素酶表达质粒(pDRVISV1.0-Fluc),对其存在的3种拓扑形式(包括超螺旋、线性、闭环切刻等)和反复冻融状态对表达效率的影响进行分析,前者用脂质体介导转染293FT细胞,用流式细胞仪检测EGFP的表达情况;后者通过接种BALB/c小鼠,用活体成像技术监测荧光素酶的表达情况。结果:对pcDNA3.1-EGFP质粒,流式细胞仪检测其转染293FT细胞48 h后显示,4种处理组的质粒表达EGFP的效率和荧光强度有所差异,依次为新鲜制备质粒(89.76%,2682.68)>反复冻融处理质粒(76.35%,2313.48)>闭环切刻质粒(68.45%,1245.95)>线性质粒(38.28%,929.5)。对pDRVISV1.0-Fluc质粒,采用小鼠活体成像技术持续观察接种后28 d的体内荧光素酶的荧光信号强度情况,结果显示:接种后0.2 d观察到荧光,之后强度持续开始增强,至第5 d达到平台期,维持至18 d。质粒各处理组的荧光强度组间有显著差异,依次为新鲜制备质粒(108.6)>反复冻融处理质粒(108.4)>线性质粒(108.3)>切刻质粒(108.0)。2种质粒不同构象的体内体外表达中,均是新鲜制备质粒的表达量最高,因其中超螺旋为其主要构象。结论:质粒中超螺旋比例高时其转染效率及表达量最高,提高超螺旋比例是DNA疫苗质量控制的关键。     

治疗性HIV腺病毒载体疫苗的生物分布

目的C57BL/6小鼠肌肉注射治疗性HIV腺病毒载体疫苗后,研究病毒载体在小鼠体内的生物分布情况,从而预测可能的毒性靶器官,考察疫苗在动物体内的代谢情况,并为疫苗的临床研究提供重要的参考资料。方法每只小鼠肌肉注射给予5×109vp。分别在给药后第3、15、30、60和85天进行组织脏器取材。用Qiagen DNA提取试剂盒进行组织脏器基因提取,用经过确认的实时定量PCR方法检测各组织脏器中病毒载体的分布情况。结果(1)病毒载体主要分布在注射位点,且随着时间的延长,注射位点中病毒载体的分布逐渐减少;(2)淋巴结、脾脏、肝脏和骨髓中有相对较少的病毒载体分布,且不断的消除,但是与注射位点中的病毒载体的分布相比非常少,随着时间的延长,可以完全消除;(3)所有动物的性腺(睾丸/子宫,附睾/卵巢)、肾脏、肺脏、脑、心脏和血液中,在不同的时间点都没有检测到病毒载体的分布。结论治疗性HIV腺病毒载体疫苗肌肉注射给药后,能引起系统毒性的可能性较小,从疫苗的生物分布角度,治疗性HIV腺病毒载体疫苗是安全的。     

gp55DNA疫苗保护猪抵抗古典猪瘟

在欧洲和亚洲的许多国家 ,古典猪瘟病毒 (CSFV)在商品猪群中引起较高的发病率和死亡率。而保护性抗原 gp5 5具有高度构象依赖性 ,从而使灭活病毒或由重组细菌产生的蛋白质失去保护力。本文报道注射含有编码 gp5 5基因片段的 DNA疫苗可为猪群提供强的保护性免疫力。　　将 CSFV gp5 5的 DNA片段插入质粒载体 p CI。构建重组质粒 p CI- gp5 5。同样 ,将编码 A/PR/8/34流感病毒血凝素 (HA)的基因片段插入质粒载体 p CI,构成 p CI- HA。在第 1项试验中 ,以 8周龄猪为研究对象 ,试验组接种 p CI- gp5 5 ,部位分别在半腱肌、腹股沟浅…     

加米霉素注射液在猪体内残留消除研究

目的 为了探讨自制加米霉素注射液在猪体内的残留消除规律，验证休药期。方法 本试验将选取体质健康的28 头猪，随机分为2 组，对照组4 头，试验组24 头。试验组猪按6mg/kg B.W. 单次肌注射加米霉素，对照组不做处理。在注射后 第0.5、7、14、21、28 和35d 时，采集肌肉、肝脏、肾脏、皮脂及注射部位肌肉。UPLC-MS/MS 法测定组织中的加米霉素残留量， 并用WT 1.4 软件计算休药期。结果 加米霉素在猪肌肉、肝脏、肾脏、皮脂和注射部位肌肉的休药期分别是9.68、11.62、17.37、 24.35 和26.75d。结论 为保证兽药使用安全、食品安全，推荐加米霉素注射液在猪的休药期为27d。     

DNA疫苗安全性探讨

DNA疫苗安全性问题主要来自DNA疫苗元件与疫苗工程菌，包括DNA疫苗与细胞基因组整合、接种后诱导宿主产生免疫耐受和自身免疫疾病、质粒DNA非目的基因编码蛋白所带来的负作用、抗性基因的转移问题、疫苗中内毒素和抗生素及其他有害物质残留问题、宿主菌体内DNA复制与纯化过程中基因稳定性问题。     

麻疹病毒DNA疫苗接种在食蟹猴中诱导特异性体液和细胞免疫应答

麻疹病毒在发展中国家仍然造成很高的发病率和死亡率。人们多方研究以求改进疫苗效果 ,除佐剂亚单位疫苗和重组病毒载体疫苗外 ,以编码麻疹病毒蛋白的 DNA质粒免疫动物的研究亦获相当进展。如接种编码麻疹病毒蛋白 DNA质粒的小鼠产生了特异性体液和细胞免疫应答。基因枪注射恒河猴诱生了麻疹病毒特异性中和抗体 ,并减少了猴体被攻毒后产生的麻疹病毒血症。本文作者观察了食蟹猴经 DNA质粒免疫后特异性体液和细胞免疫应答情况。　　试验中使用的 Helios TM基因枪注射系统可以直接把小量 DNA送入上皮细胞内。把编码麻疹病毒蛋白的 DNA…     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.