巨噬细胞对新生隐球菌B3501标准株的吞噬作用

目的检测巨噬细胞对新生隐球菌活力的影响。方法新生隐球菌标准株B3501与小鼠巨噬细胞系J774细胞共孵育后,检测其出芽率,并通过电镜观察B3501在J774细胞内的超微结构。结果被吞噬的B3501超微结构完好,J774细胞对B3501菌株的吞噬指数在5.67%±1.29%~8.76%±3.09%,而B3501菌在J774细胞内的出芽率较高,可达46.85%±6.63%,出芽率随共孵育时间延长而下降,但4hrs组和8hrs组无明显差别(P>0.05)。超微结构观察显示细胞内的新生隐球菌细胞壁完整。结论虽然巨噬细胞存在着胞内和胞外的抗隐球菌活性,但新生隐球菌仍可在其细胞内外存活并繁殖。     

巨噬细胞对新生隐球菌主要毒性基因表达的影响

目的研究巨噬细胞对新生隐球菌B3501标准株的主要毒性基因表达影响。方法将对数生长期的J774．16巨噬细胞分别与新生隐球菌野生株B3501共孵育4h,收集被J774．16吞噬的B3501作为实验组,提取实验组和在37℃条件下5％二氧化碳单独培养的对照组B3501的RNA,采用实时荧光定量PCR技术,检测J774．16细胞内和对照组B3501的CNLAC1、CAP60、URE1、NMT表达的差异。结果实验组中新生隐球菌的CAP6,CNLAC1,NMT及URE1基因的mRNA在每百万看家基因（GAPDH基因）中的平均含量分别为（2．698±0．084）×10^4,（1．806±0．322）×10^4,（2．267±0．074）×10^4和（4．041±0．271）×10^4;而对照组这4种毒性因子基因含量分别为：（1．139±0．183）×10^6,（9．324±5．028）×10^3,（1．326±0．028）×10^6和（1．307±0．001）×10^6,均远比实验组高,其中以NMT最为明显。结论新生隐球菌被巨噬细胞吞噬后,主要的毒性因子基因表达下降,其中以NMT最为明显,而CNLAC1下降幅度最小。     

新生隐球菌毒性因子研究进展

新生隐球菌为环境中的真菌,是引起免疫损伤患者脑膜脑炎的主要病原体。新生隐球菌有毒株能够快速适应宿主环境的诸多变化,改变基因/蛋白的表达,利用多种策略在宿主防御和治疗药物的压力下可塑性地适应和生存,并在宿主不同的组织器官中顽强生存,某些免疫系统完好的宿主也不能幸免。新生隐球菌应用伪装躲避识别、逃避固有免疫和适应性免疫应答、改变细胞内转运等手段使病原体穿越天然屏障在脑中生长/持续感染。了解其中的毒性因子在持续感染中的作用,有助于揭示新生隐球菌的致病机理。     

新生隐球菌在巨噬细胞内寄生的机制

新生隐球菌是一种可以引起易感患者发生脑膜脑炎的真菌。最近的体内研究认为,新生隐球菌是一兼性的细胞内致病病原苗,这与体外研究结果是一致的。对新生隐球菌在巨噬细胞内寄生机制的研究,有助于理解它的致病机制,包括对其休眠和持续感染机制的认识,同时也有利于研究宿主有效防御反应和评价治疗药物的效果。     

禾谷顶囊壳白化突变株的建立和白化性状的遗传

禾谷顶囊壳（Ｇａｕｍａｎｎｏｍｙｃｅｓ　ｇｒａｍｉｎｉｓ　Ｓａｃｃ．）ｖ．Ａｒｘ　＆Ｏｌｉｖｉｅｒ）是重要的植物病原真菌,可引起禾本科作物全蚀病,其中小麦变种（Ｇ．ｇｒａｍｉｎｉｓ．ｖａｒ．ｔｒｉｔｉｃｉ）分布最广,为害最重。禾谷顶囊壳有性生殖为同宗配合。１９９６～１９９８年,作者利用紫外线诱变技术获得了禾谷顶囊壳的白化突变菌株,井对白化突变性状的遗传规律进行了研究,取得了明确的结果,从而为开展该菌的遗传学研究创造了条件。１材料与方法１．１供试菌种供试菌株２０个,分别由陕西、甘肃、宁夏部分地区小…     

新生隐球菌Cap59缺陷株ura5突变株的筛选方法

改进筛选新生隐球菌Cap59荚膜缺陷株ura5突变株的方法。采用硫酸二乙酯化学诱导新生隐球菌Cap59荚膜缺陷株,利用5-氟乳清酸(5-FOA)反筛选法筛选ura5尿嘧啶合成基因突变株。用新方法筛选到2株Cap59荚膜缺陷株ura5突变株。建立了一种筛选新生隐球菌荚膜缺陷株ura5突变株的简易方法。     

新生隐球菌与血管内皮细胞相互作用的研究进展

隐球菌性脑膜脑炎的发病率在逐年上升,而隐球菌与血管内皮细胞的相互作用是其侵犯其他深部组织的前提,能够在血管内皮细胞内繁殖并造成其损伤是隐球菌致病的重要因素。本文就两者的相互作用作一综述。     

新生隐球菌感染小鼠肺泡巨噬细胞相关细胞因子与疾病病程相关性研究

目的通过检测新生隐球菌感染小鼠巨噬细胞相关细胞因子的表达水平,探讨其在感染小鼠疾病病程的作用。方法应用单侧小鼠鼻孔接种感染新生隐球菌建立小鼠吸入感染隐球菌模型,在感染后第1、4、7、11、14、18、21天,PAS染色观察小鼠肺组织病理变化,并通过RT-PCR检测相应时间点小鼠巨噬细胞内相关细胞因子(IL-6、IL-8、TGF-β、TNF-α)的表达。结果小鼠吸入感染隐球菌后,PAS染色发现第4天肺内散在分布隐球菌,第7天可见肉芽肿形成,第11天大量炎性细胞浸润,第14天见肉芽肿内大量隐球菌,第18天隐球菌分布至全肺,第21天肺组织大量坏死;RT-PCR结果显示TGF-β和IL-6的表达在感染后14天达到最高值,然后逐渐降低,其中TGF-β升高幅度更为明显。结论在新生隐球菌感染小鼠中,TGF-β参与了机体的抗真菌免疫,在调节炎症反应方面有重要作用。     

新生隐球菌Cap59缺陷株ura5突变株的筛选方法*

改进筛选新生隐球菌Cap59荚膜缺陷株ura5突变株的方法。采用硫酸二乙酯化学诱导新生隐球菌Cap59荚膜缺陷株 ,利用 5 氟乳清酸 (5 FOA)反筛选法筛选ura5尿嘧啶合成基因突变株。用新方法筛选到 2株Cap59荚膜缺陷株ura5突变株。建立了一种筛选新生隐球菌荚膜缺陷株ura5突变株的简易方法。     

Role of laccase in the biology and virulence of Cryptococcus neoformans

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.     

An ectophosphatase activity in Cryptococcus neoformans

There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.     

Molecular typing of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul

In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by Cryptococcus neoformans. This pathogen is a ubiquitous environmental basidiomycetous encapsulated yeast, commonly found in soil and avian excreta. The present study investigates further the population structure of clinical and environmental C. neoformans isolates from south Brazil. One hundred five clinical and 19 environmental (pigeon excreta and Eucalyptus spp.) isolates from the Brazilian state Rio Grande do Sul were characterized based on morphological, biochemical, molecular and serological data. The majority of the clinical and environmental isolates analyzed belonged to C. neoformans var. grubii serotype A (89.5 and 52.6%, respectively), were mating type alpha (98.1 and 94.7%, respectively) and were phospholipase-positive (94.3 and 73.7%, respectively). PCR-fingerprinting with the microsatellite-specific primer M13 and the minisatellite-specific primer (GACA)(4) grouped the majority of the isolates into the molecular type VNI (89.5 of the clinical and 52.6% of the environmental isolates). Our results add considerable new information to the few available data on ecology, molecular biology and epidemiology of C. neoformans in the southern region of Brazil.     

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised; the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25°C and 37°C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.     

新型隐球酵母铜应答转录因子Cuf1与荚膜的生物合成相关性

[目的]新型隐球酵母是人类条件致病真菌,主要感染免疫缺陷患者.该酵母最显著的特征是细胞外包被着多糖荚膜,这一重要致病因子的调控机制复杂.本文研究旨在阐述编码铜依赖转录因子的CUF1基因对其荚膜生物合成的负调控作用.[方法]以野生型菌株为对照,对CUF1缺失的突变菌株进行菌落形态观察、荚膜墨汁染色的显微观察、细胞聚沉试验以及荚膜定量分析.[结果]与野生型菌株相比,△cuf1突变株产生的菌落更粘,显微镜下亦可明显观察到荚膜更厚.同样数量的细胞,突变株聚沉平衡后体积更大.此外,荚膜粗提物定量称重分析也证明突变株产生了更多的荚膜.并且外源铁可以回复△cuf1突变株荚膜过量产生的表型.[结论]铜应答转录因子1(Cuf1)对荚膜的生物合成具有负调控作用.Cuf1可能通过铁的高亲和吸收途径调控铁吸收而实现该作用的.     

真菌miRNA的发现

microRNAs (miRNAs)在植物和动物中大量存在,但是否在真菌中存在一直是个未解之谜.本研究组在担子菌新型隐球酵母Cryptococcus neoformans中发现了miRNA.两个miRNA,miR1和miR2,长度分别是22nt和18nt,前体是70nt,和动物miRNA相近.通过报告基因,证实miRl/2具有沉默功能.真菌miRNA的发现为研究其进化、功能等提供有用知识.     

新生隐球菌对角质形成细胞活力的作用研究

目的研究新生隐球菌体外对角质形成细胞活力的影响。方法将新生隐球菌父代标准株与子代荚膜缺陷株于体外分别与角质形成细胞分别共培养,同时设立热灭活的菌体、空白对照,再分别设立菌体与细胞直接接触与不接触共培养相互对照,分别作用0.5 h、1 h和2 h后,采用流式细胞仪检测隐球菌角质形成细胞的调亡率。结果随着时间延长,与空白对照组及热灭活组比较,实验组角质形成细胞的凋亡率逐渐增加。无荚膜株与父代有荚膜株比较,无荚膜株对细胞活力的影响在作用后1 h、2 h明显低于有荚膜株。2种菌株不直接接触培养使细胞的凋亡率明显下降;不直接接触的有荚膜株与热灭活的菌体之间比较差异不显著。结论虽然有荚膜株与无荚膜株隐球菌均可以使角质形成细胞活性明显降低,但荚膜可以显著增强菌体对细胞活力的影响;角质形成细胞活力的降低主要是通过与菌体接触培养后产生的,诱导细胞调亡需要菌体与细胞的直接接触。