新生隐球菌GXM的纯化及其对巨噬细胞甘露糖受体表达调节的研究

目的从新生隐球菌B3501培养上清中分离和纯化荚膜多糖葡萄糖醛酸木糖甘露聚糖(GXM),观察其是否能调节巨噬细胞甘露糖受体MR的表达。方法采用乙醇沉淀荚膜多糖,十六烷基三甲基溴化铵(CTAB)特异性沉淀方法获得GXM,将GXM与巨噬细胞共孵育24 h,Western blot检测MR的表达变化情况。结果获得了毫克级的GXM,巨噬细胞与GXM孵育后甘露糖受体(mannose receptor,MR)的表达没有明显变化。结论新生隐球菌荚膜多糖GXM不影响巨噬细胞甘露糖受体MR的表达。     

小鼠脑微血管内皮细胞与新生隐球菌GXM作用后基因表达谱分析

目的探索新生隐球菌GXM能否影响脑微血管内皮细胞基因的表达,为进一步研究隐球菌嗜中枢性的分子机制提供线索。方法使用Roche Nimble Gen 12×135K小鼠基因表达谱芯片,筛选小鼠脑微血管内皮细胞系b End.3与不同浓度新生隐球菌GXM作用后差异表达的基因;结合基因本体论(Gene Ontology),使用top GO进行差异基因GO分析,结合GO语义挖掘与隐球菌侵袭中枢神经系统能力相关的差异表达基因的信息;采用荧光实时定量PCR对重要基因PIK3C2G和ADAMDEC1的表达水平变化加以验证。结果 b End.3细胞与新生隐球菌GXM作用前后基因表达对比发现,实验组GXM(90μg/m)组总共有402个基因表达上调,296个基因表达下调,GXM(180μg/m)组总共有421个基因表达上调,564个基因表达下调,细胞膜、细胞骨架、磷酸肌醇-3-激酶活化、1-磷酸肌醇-3-激酶活化、细胞紧密连接等生物过程差异表达基因较为富集;对PIK3C2G基因和ADAMDEC1基因表达水平变化进行荧光定量PCR验证,结果与芯片结果一致,基因表达水平不同程度上升,且与GXM浓度正相关。结论新生隐球菌GXM能够影响脑微血管内皮细胞基因表达,PIK3C2G基因、ADAMDEC1基因表达上调可能和隐球菌穿越血脑屏障有关。     

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞,随机分4组,每组设3个复孔。①正常对照组：常规培养,不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理,并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min,其余同NaHS+ATP组。MTT检测各组细胞活力,荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性,Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比,不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力,NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01）,但NaHS达400 μmol/L浓度时,其保护作用未进一步增加。②随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平,而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力,提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。     

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。     

新生隐球菌通过Dectin-1上调单个核细胞miR-146a的表达

目的 研究miR-146a是否参与新生隐球菌感染免疫应答过程.方法 采用RT-PCR检测了6例新生隐球菌性脑膜炎患者和6名健康个体外周血单个核细胞（PBMC）中miR-146a的表达.以热灭活新生隐球菌刺激来自健康个体的PB-MC,并加入Dectin-1抑制剂昆布多糖,采用RT-PCR检测热灭活新生隐球菌和昆布多糖对PBMC中miR-146a表达的影响.结果 新生隐球菌性脑膜炎患者PBMC中miR-146a的表达较健康个体明显增高.热灭活新生隐球菌可以上调PBMC中miR-146a的表达,昆布多糖可以削弱其上调miR-146a表达的能力.结论 热灭活新生隐球菌可以通过Dectin-1受体上调miR-146a的表达.miR-146a参与了新生隐球菌感染免疫应答过程,值得进一步研究.     

真菌GXM生物学活性及影响因素的研究进展

葡萄糖醛酸木糖甘露聚糖(GXM)是新生隐球菌(C.neoformans)荚膜的主要成分,经研究发现GXM也存在于毛孢子菌中,是阿萨希毛孢子菌(T.asahii)细胞壁的重要组成成分,与真菌的毒力和致病性密切相关。本文介绍了真菌GXM的生物学活性与影响因素,为真菌的检测及临床致病性研究提供理论依据。     

Peroxiredoxin V保护HT22小鼠海马神经细胞抵抗NO诱导的细胞凋亡

Peroxiredoxin V(Prx V)是过氧化物酶peroxiredoxins家族中的一员,在神经细胞中含量丰富,具有通过清除细胞内活性氧(reactive oxygen species,ROS)和过氧亚硝酸盐抑制氧化应激诱导的细胞凋亡的作用。过量的一氧化氮(nitric oxide,NO)具有较强的神经毒性,可引起小胶质细胞炎性反应,诱导神经细胞凋亡从而引发神经退行性疾病,而且可诱导神经小胶质细胞Prx V的表达,参与小胶质细胞的活性调控过程。但是,NO诱导的海马神经细胞凋亡过程中Prx V的作用尚不清楚。该研究利用硝普化钠(sodium nitroprusside dihydrate,SNP)作为NO供体,检测了NO诱导的HT22小鼠海马神经细胞的凋亡及对Prx V蛋白表达的影响。结果显示,SNP诱导的HT22细胞凋亡呈现时间、浓度依赖性;并特异性地抑制了Prx V的表达,致使细胞内ROS水平升高,激活线粒体依赖的经典凋亡途径,导致HT22细胞的凋亡。该研究结果揭示,NO通过抑制细胞内Prx V的表达导致细胞内ROS水平升高,最终诱导HT22细胞发生凋亡的机制,为保护NO诱导的神经细胞凋亡提供了新的理论依据。     

新生隐球菌胞外蛋白水解酶对脑微血管内皮细胞的作用

目的初步探讨新生隐球菌分泌的胞外蛋白水解酶在新生隐球菌穿越血脑屏障致病过程中的作用。方法在含有成熟的脑微血管内皮细胞的培养皿中,分别加入胞外蛋白水解酶相关成分及其特异性抑制剂后,利用相差显微镜动态观察微血管内皮细胞形态学的改变;应用免疫组织细胞化学技术检测基质金属蛋白酶-9（MMP-9）、微管相关蛋白（Tau-LRP）和低密度脂蛋白受体相关蛋白（LDL—LRP）表达的变化。结果①加入丝氨酸蛋白酶1h后可观察到内皮细胞开始收缩,面积变小,细胞间隙增宽,细胞收缩有时间依从性,至10h时仅为处理前的20％;加入丝氨酸蛋白酶＋抑肽酶后细胞形态学无明显变化（P〉0．05）。②加入隐球菌浓缩上清液1h后内皮细胞开始收缩,至6h时为原来的20％;加入菌株浓缩上清液＋抑肽酶后细胞形态学无明显变化（P〉0．05）。③丝氨酸蛋白酶使内皮细胞的MMP-9、Tau．LRP、LDL—LRP的表达上调,与对照组比较,有显著统计学差异（P〈0．01）。结论新生隐球菌分泌的胞外蛋白水解酶可能通过上调MMP-9和（或）Tau—LRP、LDL—LRP的表达,诱导内皮细胞基质降解和细胞自身微管结构及紧密连接发生变化,最终导致血脑屏障通透性增加,菌体细胞穿越血脑屏障而致病。     

蒺藜中甾体皂苷对新生隐球菌生物膜形成的抑制作用

目的 观察蒺藜甾体皂苷类化合物TTS-12对新生隐球菌生物膜形成的影响,探讨其可能的作用机制.方法 光镜观察TTS-12对新生隐球菌生物膜生长形态的影响;MTT法观察TTS-12对新生隐球菌生物膜形成的影响;实时定量RTPCR观察不同浓度TTS-12对新生隐球菌细胞生物膜关键基因PMT4表达的影响.结果 经TTS-12处理的新生隐球菌生物膜结构更疏松,TTS-12可剂量依赖性地降低新生隐球菌生物膜生长动力学指标及PMT4基因表达水平(P＜0.01).结论 TTS-12可抑制新生隐球菌生物膜的形成.通过降低新生隐球菌PMT4基因表达可能是其抑制新生隐球菌生物膜的形成作用机制之一.     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Glucuronoxylomannan-mediated interaction of Cryptococcus neoformans with human alveolar cells results in fungal internalization and host cell damage

Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.     

Capsular polysaccharides galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce macrophage apoptosis mediated by Fas ligand

The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF-alpha, NO and iNOS expression, while GXM predominantly induced TGF-beta secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.     

Cryptococcus neoformans capsular glucuronoxylomannan induces expression of fas ligand in macrophages

The major component of capsular material of Cryptococcus neoformans is glucuronoxylomannnan (GXM), a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. The results reported here show that 1) soluble purified GXM induces a prompt, long-lasting, and potent up-regulation of Fas ligand (FasL) on macrophages, 2) the up-regulation of FasL is related to induced synthesis and increased mobilization to the cellular surface, 3) this effect is largely mediated by interaction between GXM and TLR4, 4) FasL up-regulation occurs exclusively in GXM-loaded macrophages, 5) macrophages that show up-regulation of FasL induce apoptosis of activated T cells expressing Fas and Jurkat cells that constitutively express Fas, and 6) anti-Fas Abs rescue T cells from apoptosis induced by GXM. Collectively our results reveal novel aspects of the immunoregulatory properties of GXM and suggest that this nontoxic soluble compound could be used to dampen the immune response, to promote or accelerate the death receptor, and to fix FasL expression in a TLR/ligand-dependent manner. In the present study, we delineate potential new therapeutic applications for GXM that exploit death receptors as key molecular targets in regulating cell-mediated cytotoxicity, immune homeostasis, and the immunopathology of diseases.     

Cryptococcus neoformans capsular polysaccharide component galactoxylomannan induces apoptosis of human T-cells through activation of caspase-8

The major virulence factor of Cryptococcus neoformans is its polysaccharide capsule composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoproteins. A variety of immunomodulating activities have been described for GXM and mannoproteins but little is known about possible interactions of GalXM with the immune system. In the present article, we investigate the effect of purified soluble GalXM on human T lymphocytes. The results indicate that, GalXM (i) can affect selected immune responses; (ii) causes significant impairment of T cell proliferation and increases interferon-gamma and interleukin-10 production; and (iii) induces apoptosis of T lymphocytes through activation of caspase-8 that terminates with fragmentation of DNA. These results are the first to suggest a role for GalXM in C. neoformans virulence by demonstrating that it can target human T cells, and that it may impair the development of an effective specific T cell response.     

Direct inhibition of T-cell responses by the Cryptococcus capsular polysaccharide glucuronoxylomannan

The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections.     

Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Cryptococcus flavescens, a strain originally identified as C. laurentii, was isolated from the cerebrospinal fluid of an AIDS patient, and the soluble capsular polysaccharide of the yeast was investigated. Glucuronoxylomannan (GXM) was obtained from C. flavescens under conditions similar to those used to obtain C. neoformans polysaccharide. However, the GXM differed from C. neoformans polysaccharide in the decreased O-acetyl group content. The structure of GXM was determined by methylation analysis, partial acid hydrolysis, NMR analyses, and controlled Smith degradation. These analyses indicated that GXM has the following structure: an alpha-(1-->3)-D-mannan backbone with side chains of beta-D-glucuronic acid residues bound to the C-2 position of the mannose residue. The C-6 position of the mannose is substituted with D-man-beta-(1-->4)-D-xyl-beta-(1--> disaccharide. Furthermore, the existence of side chains containing more than two xylose residues was suggested. This mannosylxylose side chain is a novel structure in polysaccharides of C. neoformans and other Cryptococcus species.     

Self-aggregation of Cryptococcus neoformans capsular glucuronoxylomannan is dependent on divalent cations

The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.     

Glucuronoxylomannan of Cryptococcus neoformans exacerbates in vitro yeast cell growth by interleukin 10-dependent inhibition of CD4+ T lymphocyte responses

Glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is the most important virulence factor of this fungus. We analyzed the molecular events related to protective immune responses against a non-encapsulated strain of C. neoformans, mediated by murine splenic CD4(+) T lymphocytes in vitro, and the impact of GXM addition upon these events. Both the lymphoproliferation of CD4(+) T cells and the control of fungus growth were dependent on B7 co-stimulation. Addition of GXM did not modify CD4(+) T cell proliferation, but exacerbated infection in cultures obtained from normal and infected hosts. GXM enhanced the secretion of IL-10 and IL-4, while it reduced the production of pro-inflammatory cytokines TNF-alpha and IFN-gamma. The blockade of IL-10 activity with neutralizing antibodies increased TNF-alpha production and reduced yeast cell growth. The findings suggest that GXM exacerbates infection by down-regulating cell-mediated protective immune response and that IL-10 is implicated in yeast evasion.     

Role for Golgi reassembly and stacking protein (GRASP) in polysaccharide secretion and fungal virulence

Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP orthologue had attenuated virulence in an animal model of cryptococcosis, in comparison with wild-type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP orthologue mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.