SHIV及其在AIDS疫苗和药物研究中的应用

艾滋病已成为21世纪威胁人类健康的最主要的疾病,最有效的预防措施,就是研制安全有效的疫苗。疫苗安全性和有效性评价,以及疫苗组合的选择和免疫程序的策略,需要在合适的动物模型中进行分析。SIV/恒河猴模型曾被认为是最有效的研究模型。但是,SIV和HIV之间基因的差异,使得这个模型存在很大局限性。研究人员还曾经致力于HIV-1/黑猩猩模型,但伦理学等方面的问题导致该模型也不能被广泛利用[1]。嵌合猿猴/人免疫缺陷病毒(Chimeric simian/human immunodeficency virus,SHIV),是以猿猴免疫缺陷病毒(Simian immunode ficiency virus,SIV…     

AIDS猕猴模型在HIV疫苗研究中的应用

对HIV疫苗的研究一直是国际上艾滋病方面研究的热点和难点。动物模型则为疫苗研究必不可缺少的重要工具,缺乏合适的动物模型很大程度上制约了AIDS疫苗的研究。目前在国际上SIV或SHIV感染的猕猴模型为最常用的AIDS研究模型,受猕猴背景及病毒特性等多种因素的影响,使得以上两种模型在HIV疫苗研究中仍存在一定的局限性。为了更好地发挥猕猴模型在HIV疫苗研究中的巨大潜力,开发理想的AIDS猕猴模型已成为目前HIV疫苗研究的首要任务。本文简要介绍了AIDS疫苗的研发策略、研发概况以及SIV/SHIV猕猴模型在HIV疫苗中的应用,并对其中存在的问题及其应用前景进行了探讨。     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

表达SIV Gag/Env基因的DNA疫苗、重组腺病毒和重组痘苗病毒三载体疫苗联合免疫小鼠的细胞免疫研究

获得性免疫缺陷综合征即艾滋病是人类面临的严重公共卫生威胁,目前常用的药物疗法仍存在一定的缺陷,尚不能彻底治愈AIDS并阻断HIV的传播。将治疗型疫苗用于HIV感染的治疗具有一定的发展潜力,但缺乏适宜的HIV感染动物模型阻碍了治疗型HIV疫苗的研制。SIV能够感染非人灵长动物并引起类似AIDS的猴免疫缺陷病,因而常被用作研究HIV及其疫苗的替代动物模型。为了对SIV疫苗在猴感染模型中治疗SIV感染的效果进行评价,我们分别构建了表达SIV gag和env基因的DNA疫苗、重组腺病毒和重组痘苗病毒疫苗,并联合使用这三种疫苗免疫小鼠,对包括不同抗原组合、不同免疫次序及间隔的免疫策略进行探索和优化。IFN-γ酶联免疫斑点和小鼠体内杀伤试验的结果显示,通过三载体疫苗联合免疫,能够在小鼠体内诱导出强度较高、持续时间较长的SIV Gag/Env特异性细胞免疫反应。并且,重复免疫后仍可以诱导较高水平的免疫反应。该结果为在SIV感染猴模型中评价多载体疫苗序贯和重复免疫治疗SIV感染的研究奠定了基础,也为治疗型HIV疫苗的研究提供了参考。     

重组HIV-AEgp145和SIV-envT基因的rAAV8在小鼠体内的免疫原性研究

病毒性载体疫苗是一种有前途的艾滋病疫苗.为了构建以重组腺相关病毒8型(recombinant adeno associated virus type 8,rAAV8)为载体,表达HIV或SIV包膜蛋白的艾滋病疫苗.同时在小鼠体内对其免疫原性进行评价,为下一步研究奠定基础.本研究分别构建了表达HIVAE亚型和SIVmac239株包膜蛋白(不含胞内区)的rAAV8-AEgp145和rAAV8-SIVenvT两种重组病毒,并通过PCR和WesternBlot方法对重组病毒进行了体外鉴定.将两种重组病毒分别接种BALB/c小鼠,应用ELISA和ELISPOT方法检测小鼠的HIV/SIV特异性抗体滴度和细胞免疫应答强度.结果显示,rAAV8能够在293T细胞中高效表达HIV AEgp145和SIVenvT基因.小鼠接种两种重组病毒3-5W后,均能检测到gp120特异性抗体和env特异性细胞免疫应答,并且在16-20W后反应强度仍显著高于对照组.以上结果提示,携带HIVAEgp145和SIVenvT基因的rAAV8载体能够在小鼠体内诱导中等强度并且持续时间较长的特异性体液和细胞免疫应答.     

阿片类物质对猴免疫缺陷病毒感染的CEM×174细胞内p53合成的调节作用(英文)

已经证明阿片类物质如吗啡能够刺激猴免疫缺陷病毒 ( SIV)的复制 ,并最终加速细胞死亡 ,但是其机理却研究很少 .为探讨吗啡和甲硫氨酸脑啡肽对细胞内 p53合成的作用 ,用 SIV感染CEM× 1 74细胞 ,同时分析 SIV感染时病理过程的机理 .在 CEM× 1 74细胞感染 SIV后不同时间 ,p53含量逐渐增加 ,但 1 0 -7mol/L的吗啡仅在起始阶段对其有促进作用 .在 SIV感染组加入吗啡或甲硫氨酸脑啡肽进行时间曲线实验时 ,p53含量较低 .加入 1 0 -8～ 1 0 -6mol/L吗啡 8h,正常细胞 p53含量仅有轻微改变 .但在 SIV感染情况下 ,则呈现剂量依赖性的大量增加 .相反 ,1 0 -8- 1 0 -6mol/L甲硫氨酸脑啡肽在 8h时能增加正常细胞 p53合成达 60 % .在 SIV感染时 ,SIV本身能够促进 p53的含量 .尽管各组 p53仍然高于对照组 ,但甲硫氨酸脑啡肽对其不再起作用 .结果提示甲硫氨酸脑啡肽对正常细胞 p53含量有明显影响 ,而吗啡 8h增加 SIV感染细胞的 p53含量可能是其加速爱滋病病理过程的机理之一 .     

IL-12基因对HIV-1核酸疫苗诱导的免疫应答的影响

研究白细胞介素-12(IL 12)基因对HIV-1核酸疫苗诱导免疫应答的影响,以探求治疗性HIV-1核酸疫苗的新策略.将pCI-neoGAG联合白细胞介素-12基因或者pCI-neoGAG单独免疫Balb/c小鼠,通过ELISA检测免疫小鼠的特异性抗体和IFN-γ,通过MTT实验检测免疫小鼠脾淋巴细胞增殖实验,通过乳酸脱氢酶(LDH)实验检测小鼠特异性细胞毒性T淋巴细胞(CTL)反应.与pCI-neoGAG免疫组比较,pCI-neoGAG联合白细胞介素-12基因免疫组小鼠血清的抗HIV-1p24抗体滴度降低,有显著性差异(P＜0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合白细胞介素-12基因免疫组小鼠血清的IFN-γ升高,有显著性差异(P＜0.01);pCI-neoGAG联合白细胞介素-12基因免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组,有显著性差异(P＜0.01).因此,白细胞介素-12基因基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-12基因对体液免疫有抑制作用.     

人免疫缺陷病毒/艾滋病细胞免疫疫苗研究进展

由于人免疫缺陷病毒(HIV)具有变异快、亚型多、攻击免疫系统等特殊的生物学特点,HIV/艾滋病疫苗至今尚未研制成功。20多年来,艾滋病疫苗研究主要采用中和抗体为主和细胞免疫为主等两种策略,然而目前仍没有实质性突破。诱发广谱有效的强CD8+T细胞反应是研制有效HIV疫苗的重要策略。以次要保护性抗原为靶抗原、优化目的基因表达、多抗原联合使用策略,为研究HIV细胞免疫疫苗引入了新的思路。综合分析这些进展,对于重新思考艾滋病疫苗的研究策略可能会有所帮助。     

猴/人嵌合免疫缺陷病毒应用的研究进展

猴/人类免疫缺陷病毒(SHIV)自20世纪80年代末开始构建,并被广泛应用于人类免疫缺陷病毒Ⅰ型(HIV-1)疫苗的攻毒实验和抗HIV中和抗体的活性评价.随着研究的深入,SHIV构建涉及的HIV-1病毒亚型也越来越多,如HIV-1 B、HIV-1 C、HIV-1 E等;同时,对SHIV致病性、细胞嗜性和应用的研究也有长足的进步,促进了HIV疫苗研究,并为HIV致病性研究提供了宝贵经验.     

平顶猴在HIV/AIDS动物模型中的应用及研究进展

非人灵长类动物模型在HIV-1致病机制研究以及抗AIDS药物和疫苗研发中具有重要作用。由于缺乏HIV-1直接感染的动物,SIV/SHIV猕猴模型是目前AIDS研究中应用最为广泛的动物模型。虽然SIV/SHIV猕猴模型与人AIDS具有一定的相似性,但SIV/SHIV与HIV-1间的遗传差异较大,致使SIV/SHIV猕猴模型存在很多局限性。创建合适的非人灵长类动物模型仍然是HIV/AIDS研究中的热点和难点。平顶猴是目前唯一可以被HIV-1感染的旧大陆猴,在HIV-1静脉传播和性传播模型研究中具有许多优势。该文综述了SIV、HIV、SHIV和HSIV通过静脉和黏膜途径感染平顶猴的特征,并简要介绍了病毒在平顶猴细胞中复制的分子机制以及建立平顶猴AIDS模型的限制因素和前景。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.