铜绿假单胞菌肺感染动物模型的建立及相关炎症反应分析

目的 为了对铜绿假单胞菌引起的慢性呼吸道感染性疾病的研究,需要真实客观地反映在体内环境综合影响下该菌的致病情况,为此建立了慢性铜绿假单胞菌的肺炎动物模型,并对其引起的相关炎症反应进行分析.方法 用富含铜绿假单胞菌的琼脂糖珠灌注小鼠,建立铜绿假单胞慢性感染的动物模型,通过检测肺泡灌注液(BALF)中白细胞数、中性粒细胞百分比、白细胞介素,血清中核基质蛋白MMP-2浓度及肺组织的病理切片观测其炎症反应情况.结果 感染后动物模型肺部检出铜绿假单胞菌并出现病理改变,肺部感染各项炎症指标在2～3 d达到峰值,大致7 d恢复正常;铜绿假单胞菌肺炎模型小鼠循环系统中检测MMP-2升高,说明该炎症反应在一定程度上可引起肺部纤维化.结论 本实验成功建立了慢性铜绿假单胞菌的肺炎动物模型,可在此基础上对该菌的致病性及耐药性进行进一步研究.     

钙离子对重组创伤弧菌溶细胞素诱导THP-1细胞损伤的影响机制

目的 研究重组创伤弧菌溶细胞素( rVvhA)诱导人单核细胞白血病细胞(THP-1)的凋亡机制及其Ca2+的变化.方法 采用CCK-8法、激光共聚焦显微镜结合Fluo 3/AM法、流式细胞术结合AnnexinV -PI标记等检测rVvhA对THP-1细胞的影响,并观察胞内Ca2+浓度变化.结 果rVvhA可诱导THP-1细胞发生凋亡并引起细胞内Ca2+浓度升高,细胞内钙离子螯合剂BAPTA-AM处理组胞内钙离子升高幅度远高于细胞外钙离子螯合剂EGTA处理组.结论 rVvhA具有诱导THP-1细胞凋亡的生物学活性,并能引起细胞内Ca2+浓度升高,升高的Ca2+主要源于胞外钙离子内流.     

应用siRNA干扰技术沉默铜绿假单胞菌外排泵基因mexB的蛋白表达

目的 观察siRNA干扰质粒转入铜绿假单胞菌后,MexB蛋白表达量的变化.方法 针对mexB基因设计合成特异性siRNA分子,与pGPU6/GFP/Neo载体连接,构建pGPU6/GFP/NeosiRNA重组质粒.构建重组表达质粒pET22b+/mexB,并转化大肠杆菌BL21( DE3) plysS,诱导表达MexB蛋白,蛋白纯化后免疫家兔制备多克隆抗体.siRNA质粒分别电转化铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株,运用Western blot观察转化8、12、24h后MexB蛋白表达量的变化.结果 成功构建pGPU6/GFP/Neo-siRNA重组质粒.成功表达铜绿假单胞菌外排泵蛋白MexB,并制备多克隆兔抗.siRNA质粒对铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株的mexB基因均具有良好的沉默效果,而且沉默效果存在时间差异性.结论 siRNA质粒转化3株铜绿假单胞菌后,在8h及12 h时均可观察到MexB蛋白表达量明显减少,而在24 h时MexB蛋白表达量无改变.     

铜绿假单胞菌弹性蛋白酶抑制肺表面活性蛋白A介导的免疫吞噬作用

目的探讨肺表面活性蛋白A(SP-A)与铜绿假单胞菌(P.aeruginosa)弹性蛋白酶在铜绿假单胞菌肺部感染过程中的关系。方法铜绿假单胞菌突变菌株P.aeruginosa野毒株PAO1、弹性蛋白酶LasB突变株△lasB和回复突变株PDO240LasB鼻腔接种C3H小鼠建立肺部感染模型以观察细菌的毒力变化。将同等数量的细菌与SP-A共同孵育,通过Western blot法检测SP-A的体外降解。将预先经SP-A作用过的细菌与小鼠Raw264.7巨噬细胞孵育进行体外吞噬实验以观察细菌被吞噬的情况。结果由于弹性蛋白酶表达的缺失,铜绿假单胞菌突变菌株P.aeruginosa△lasB在通过鼻腔接种C3H小鼠建立的肺部感染模型中,与野毒株PAO1相比毒力显著降低。体外SP-A降解实验显示,△lasB基本失去了降解SP-A的能力。进一步的体外吞噬实验和聚集实验表明,△lasB在SP-A作用下更容易聚集成簇并被鼠巨噬细胞Raw264.7吞噬。结论铜绿假单胞菌弹性蛋白酶可降解SP-A,破坏SP-A介导的细菌聚集作用。     

铜绿假单胞菌感染豚鼠后生物被膜形成的研究

目的 建立体内铜绿假单胞菌生物被膜模型，研究体内细菌生物被膜的组织学及细菌学特征。方法 通过吸入法使铜绿假单胞菌以气雾剂的形式吸入豚鼠肺内并生长定植，分别观察不同时期肺组织内细菌生物被膜的特征。结果 定植在肺内的铜绿假单胞菌以肉芽肿结节的形式存在，外周包绕类上皮细胞和成纤维细胞。结节内细菌被被膜基质所包绕并彼此连结，中间镶嵌宿主炎性细胞。接种后3周，肺内仍可见结节并培养出铜绿假单胞菌。结论 用吸入法可建立较稳定的铜绿假单胞菌肺感染生物被膜，其以肉芽肿结节的形式存在，宿主的反应细胞参与了生物被膜的形成。     

SOCS3过表达对铜绿假单胞菌慢性肺部感染小鼠Th17免疫反应的影响

目的探讨铜绿假单胞菌慢性肺部感染小鼠CD4~+T淋巴细胞SOCS3过表达对Th17免疫反应的作用。方法构建铜绿假单胞菌慢性肺部感染小鼠模型,并分离培养小鼠脾脏的CD4~+T淋巴细胞。构建SOCS3慢病毒过表达载体。在体外通过慢病毒基因转染的方法实现CD4~+T淋巴细胞SOCS3基因过表达。对SOCS3过表达细胞给予IL-23刺激,采用Western blot检测细胞中的p-STAT3水平,real-time PCR检测细胞中的RORγt水平,流式细胞分析IL-17+细胞数量,ELISA检测细胞培养液中的IL-17水平。结果铜绿假单胞菌慢性肺部感染小鼠CD4~+T淋巴细胞SOCS3过表达后,IL-23刺激诱导的p-STAT3蛋白水平、RORγt mRNA水平、IL-17~+细胞数量以及培养液中的IL-17水平均较对照组明显降低。结论体外研究表明SOCS3过表达对铜绿假单胞菌感染小鼠的Th17免疫反应具有抑制作用。     

抗铜绿假单胞菌 PcrV 蛋白单克隆抗体的筛选和功能验证

目的:获得能应用于治疗铜绿假单胞菌(Pseudomonas aeruginosa)感染的抗PcrV 蛋白全人源单克隆抗体。方法:以铜绿假单胞菌PAO1 基因组为模板,PCR 扩增PcrV 基因并构建重组表达载体pET-28a-PcrV,转化至大肠杆菌BL21(DE3),利用IPTG 诱导表达,并通过镍离子螯合树脂亲和纯化PcrV 蛋白。利用噬菌体抗体库筛选PcrV 结合的噬菌体克隆并进行序列测定,以测序正确的阳性克隆质粒为模板,PCR 扩增该抗体的重链可变区基因和轻链可变区基因并构建重组表达载体转染至293E 细胞,收取培养细胞上清,并利用Protein A 树脂亲和纯化抗体。利用ELISA 实验检测抗体亲和力,并通过小鼠铜绿假单胞菌感染肺炎模型验证抗体活性。结果:获得重组蛋白PcrV,通过噬菌体展示技术筛选并制备了一株全人源抗PcrV单克隆抗体YG5。ELISA 实验结果表明YG5 抗体具有较高的亲和力(EC50 =61 ng/ ml),进一步的实验结果表明,YG5 能有效抵抗铜绿假单胞菌感染,降低小鼠的死亡率。结论:靶向铜绿假单胞菌PcrV 蛋白的全人源单克隆抗体YG5 能够有效地抑制铜绿假单胞菌致病性,降低其对机体感染,有可能成为抗铜绿假单胞菌感染的治疗性抗体。     

噬菌体治疗实验性小鼠耐亚胺培南铜绿假单胞菌感染的研究

目的探讨噬菌体治疗耐药性铜绿假单胞菌感染的疗效.方法以BALB/c小鼠为实验动物,建立耐药性铜绿假单胞菌全身感染模型,应用体外试验所筛选的宽噬噬菌体进行治疗.结果噬菌体在感染复数（multiple of infection,MOI）≥0.01时能有效杀灭铜绿假单胞菌,显著提高小鼠生存率.延迟治疗3 h仍有40%的生存率.结论通过对小鼠生存率、噬菌体在体内分布及机体对噬菌体的免疫反应等指标的观察分析,噬菌体制剂简捷高效,对机体正常组织无毒副作用,从而为临床治疗全身或局部铜绿假单胞菌感染提供了新途径.     

铜绿假单胞菌活菌对呼吸道上皮细胞表达IL-8的影响

目的:探讨铜绿假单胞菌活菌与人呼吸道上皮细胞的相互关系,细菌对呼吸道上皮炎症反应的影响。方法:采用PAO1及ATCC 27853两株铜绿假单胞菌,在体外与培养的呼吸道上皮细胞株A549及无血清培养的人支气管上皮原代细胞相互作用,收集细胞培养上清,ELISA检测上清IL-8浓度。结果:两株绿脓杆菌均能诱导呼吸道上皮细胞IL-8分泌增加,在细菌刺激下,A549细胞IL-8分泌比对照高出5倍(P<0.05),原代上皮细胞IL-8分泌比对照高出8倍(P<0.05)。结论:铜绿假单胞菌呼吸道感染的过程中,细菌与上皮细胞的直接作用可能是呼吸道炎症反应的重要原因。铜绿假单胞菌刺激上皮细胞炎症的分子机制和信号传导值得进一步探讨。     

10-23脱氧核酶抑制铜绿假单胞菌耐药基因VIM2作用的初步研究

目的 探讨10-23脱氧核酶(DRz)抑制铜绿假单胞菌耐药基因VIM2的表达及其在逆转铜绿假单胞菌耐药中的作用.方法 根据计算机模拟的 VIM2 mRNA 的二级结构设计合成 5 条VIM2特异的10-23DRz(DRz1～DRz5),在无细胞反应体系中鉴定10-23DRz对VIM2 mRNA的切割活性后,将其导入铜绿假单胞菌,利用E-test法检测亚胺培南对处理前后铜绿假单胞菌的MIC值.结果 DRz3、DRz4和DRz5在无细胞反应体系中可对VIM2 mRNA进行有效切割,且其活性具有高度特异性.与对照相比,1O-23DRz可以降低亚胺培南对铜绿假单胞菌的MIC值.结论 实验中所设计的10-23DRz能在细胞外高效特异性的切割VIM2 mRNA;在细胞内能协同业胺培南抑制铜绿假单胞菌耐药.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> quorum sensing signal molecule <Emphasis Type="Italic">N</Emphasis>-(3-oxododecanoyl) homoserine lactone on mast cells

Quorum sensing system is a cell-to-cell communication system that plays a pivotal role in virulence expression in bacteria. Recent advances have demonstrated that the Pseudomonas aeruginosa quorum sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC₁₂-HSL), exerts effects on mammalian cells and modulates host immune response. Mast cells (MCs) are strategically located in the tissues that are constantly exposed to external stimulus. Therefore, it is very much possible that 3OC₁₂-HSL may interact with MCs. Little is known, however, about specific effects of 3OC₁₂-HSL on MCs. To address this, we investigated the influence of 3OC₁₂-HSL on cell viability, apoptosis, intracellular calcium and cytokine release in MCs. We found that at high concentrations (100 μM), 3OC₁₂-HSL inhibited proliferation and induced apoptosis in P815. The 3OC₁₂-HSL treatment significantly increased intracellular calcium release in both P815 and HMC-1. We also observed that 3OC₁₂-HSL-induced histamine release and degranulation in HMC-1 cells. Furthermore, 3OC₁₂-HSL-induced IL-6 production at lower concentrations (6.25–12.5 μM) but steadily reduced IL-6 production at high concentration (50–100 μM). These data demonstrate that P. aeruginosa 3OC₁₂-HSL affects MCs function.     

Mast cells play a crucial role in Staphylococcus aureus peptidoglycan-induced diarrhea

Bacterium-induced diarrhea results in 2 to 2.5 million deaths in the world each year. The mechanism needs to be further understood. Staphylococcus aureus infection has a close relation with diarrhea; its cell wall component peptidoglycan (PGN) has strong biological activity on immune cells and possibly plays a role in S. aureus-induced diarrhea. The present study showed that oral PGN-induced diarrhea in mice in a dose-dependent manner. Intestinal epithelial cells absorbed PGN via the intracellular pathway. Intestinal mast cells were activated after PGN gavage. Toll-like receptor (TLR)2 expression was detected in mast cells in the intestine as well as in the murine mast cell line p815 cells. Blocking TLR2 or nucleotide-binding oligomerization domain (NOD)1 with related antibodies or RNA interference abolished PGN-induced p815 cell activation. The mast cell mediator histamine and serotonin had synergistic effects in PGN-induced diarrhea. In summary, oral PGN can induce diarrhea in mice, and TLR2 and NOD1 mediate the PGN-induced mast cell activation that plays a critical role in diarrhea induction. Blockade of TLR2 or NOD1 or treating mice with a mast cell stabilizer can efficiently inhibit PGN-induced-diarrhea, providing potential therapeutic significance.     

The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone

The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acyl-homoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl-homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl-homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl-homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems.     

Effects of bacterial N-acyl homoserine lactones on human Jurkat T lymphocytes-OdDHL induces apoptosis via the mitochondrial pathway

Diverse Gram-negative bacteria communicate with each other by using diffusible N-acyl-homoserine lactone (AHL) signaling molecules to coordinate gene expression with cell population density. This mechanism termed ‘quorum sensing’ is involved in the regulation of physiological functions as well as multiple virulence determinants. It becomes more and more evident, that bacteria communicate not only with each other but also with their host. Up to now, little is known about this interkingdom communication. The AHL quorum sensing molecule N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL) from Pseudomonas aeruginosa has been shown to influence the immune system of the host. The role and potential influence of other AHL molecules from other bacteria have so far not been determined. In this paper, we investigated the role of 7 different AHLs on apoptosis of human Jurkat T lymphocytes. We found, that among all homoserine lactones tested, only OdDHL rapidly induced apoptosis which was accompanied by the breakdown of the mitochondrial transmembrane potential (ΔΨ_m). Since overexpression of anti-apoptotic Bcl-2 completely abrogated the apoptotic effect, we presume that OdDHL induces apoptosis by activation of the intrinsic mitochondrial apoptosis pathway. The reason that bacteria induce apoptosis is largely unknown. We suspect that through apoptosis an anti-inflammatory response is triggered.     

Production of tumour necrosis factor by mastocytoma P815 cells

P815, a transformed mouse mastocytoma cell line, produced and released cytotoxic factors after stimulation with phorbol 12-myristate 13-acetate (PMA), but not with lipopolysaccharide (LPS), calcium ionophore A23187 and IgE receptor triggering. The cytotoxic activity was reduced 60% by antibodies to mouse tumour necrosis factor (TNF). In addition, we demonstrated that TNF mRNA was already expressed under normal culture conditions and that it increased after stimulation with PMA. Although it was unknown whether factors other than TNF were lymphotoxin or some other unknown factors, it has been suggested that mast cells have cytotoxic activity, and that they contribute to inflammatory response through the release of TNF, which has a wide range of biological activity.     

Demonstration that platelet-activating factor is capable of activating mast cells and inducing a chemotactic response

Platelet-activating factor (PAF) is generated in a variety of inflammatory conditions in which mast cells accumulate. However, little is known about the ability of PAF to influence mast cell function directly. In this study we examine the ability of PAF to activate mast cells and regulate mast cell chemotaxis. PAF was found to induce intracellular calcium mobilization and chemotactic responses in both murine and human mast cells. PAF induced transient increases in intracellular Ca2+ concentrations with a 50% effective dose of 1 nM and induced significant migratory responses at PAF concentrations of 1 nM to 1 microM in the human leukaemia mast cell line (HMC-1). Using signal transduction inhibitors, both PAF-induced calcium mobilization and migration of mast cells were shown to require activation of pertussis toxin-sensitive G proteins. PAF-induced calcium and chemotactic responses were cross-desensitized by C5a. Together, these data demonstrate that PAF is capable of activating distinct signalling pathways in mast cells associated with calcium mobilization and cell migration; and that PAF may thus contribute to the regulation of mast cell responses and hyperplasia at sites of inflammation.     

Mitochondria Distinguish Granule-Stored from de novo Synthesized Tumor Necrosis Factor Secretion in Human Mast Cells

Background: Mast cells are immune cells derived from hematopoietic precursors that mature in the tissue microenvironment. Mast cells are critical for allergic, immune and inflammatory processes, many of which involve tumor necrosis factor (TNF). These cells uniquely store TNF in their secretory granules. Upon stimulation, mast cells rapidly (30 min) secrete β-hexosaminidase and granule-stored TNF through degranulation, but also increase TNF mRNA and release de novo synthesized TNF 24 h later. The regulation of these two distinct pathways is poorly understood. Methods: Human LAD2 leukemic mast cells are stimulated by substance P. TNF secretion and gene expression were measured by ELISA and real-time PCR, and mitochondrial dynamics was observed in live cells under confocal microscopy. Cell energy consumption was measured in terms of oxygen consumption rate. Results: Here, we show that granule-stored TNF is preformed, and its secretion from LAD2 mast cells stimulated by substance P (1) exhibits higher energy consumption and is inhibited by the mitochondrial ATP pump blocker oligomycin, (2) shows rapid increase in intracellular calcium levels, and (3) exhibits reversible mitochondrial translocation, from a perinuclear distribution to the cell surface, as compared to de novo synthesized TNF release induced by lipopolysaccharide. This mitochondrial translocation is confirmed using primary human umbilical cord blood-derived mast cells stimulated by an allergic trigger (IgE/streptavidin). Conclusion: Our findings indicate that unique mitochondrial functions distinguish granule-stored from newly synthesized TNF release from human mast cells, thus permitting the versatile involvement of mast cells in different biological processes.     

H2O2 impairs inflammatory mediator release from immunologically stimulated RBL-2H3 cells through a redox-sensitive, calcium-dependent mechanism

OBJECTIVES: In this study we examined the effects of the inflammatory agent hydrogen peroxide (H2O2) on IgE-mediated mast cell responses. MATERIALS AND METHODS: Release of preformed granular mediators and newly synthesised TNF-alpha were measured in the RBL-2H3 mast cell line stimulated through IgE receptors (FcepsilonRI) in the presence of varying concentrations of H2O2. The sensitivity of the intracellular calcium response to H2O2 exposure was investigated. RESULTS: We found that H2O2 treatment impaired the release of preformed and newly synthesised mediators. H2O2 treatment simultaneously led to a profound inhibition of the calcium response. Calcium fluxes from both intra- and extracellular sources were impaired. H2O2 action was dependent on the intracellular redox state. Receptor activation directly stimulated intracellular H2O2 production. CONCLUSION: While in many cells H2O2 induces potent inflammatory responses we show that it can be an anti-inflammatory agent by not only inhibiting the release of preformed mediators but also by affecting the secretion of newly synthesized TNF-alpha. Inhibition is a consequence of the profound effect on intracellular calcium levels. The activation of an intracellular oxidative burst by FcepsilonRI aggregation and the sensitivity of intracellular responses to redox-altering agents point to an important regulatory mechanism of mast cell responses in inflammatory tissues.     

Direct effects of conjugated linoleic acid isomers on P815 mast cells in vitro

Conjugated linoleic acid (CLA) is a dietary fatty acid which causes extensive remodeling and mast cell recruitment in the mouse mammary gland. Two CLA isomers, 9,11- and 10,12-CLA, have differing effects in vivo, with only 10,12-CLA increasing mast cell number. The purpose of this project is to test the hypothesis that CLA acts directly on the mast cell. The P815 mastocytoma cell line was assayed for the effects of CLA on mast cell number, proliferation, apoptosis, and differentiation. Both CLA isomers decreased viable mast cell number, with no effect on membrane integrity, or cell cycle distribution. 10,12-CLA induced an increase in apoptosis, assessed by Annexin-FITC binding. Both isomers increased mast cell granularity, and secretion of MMP-9. The complex effects of CLA isomers on mast cells in the mammary gland are distinct from direct effects on mast cells in vitro, and may require interactions between multiple cell types present in vivo.     

The Pseudomonas aeruginosa quorum-sensing molecule N-3-(oxododecanoyl)-L-homoserine lactone inhibits T-cell differentiation and cytokine production by a mechanism involving an early step in T-cell activation

The Pseudomonas aeruginosa quorum-sensing molecule N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to have immunomodulatory activity in several systems, although the mechanism of that activity remains to be fully characterized. We demonstrate here, using a defined in vitro model of antigen responses by T-cell receptor (TCR)-transgenic mouse splenic CD4 T cells, that the effect of OdDHL on activation and cytokine production is complete within 4 h of antigen or mitogen stimulation and does not depend on the insertion of OdDHL in the cell membrane, despite a previous report that immunosuppression by homoserine lactones required a minimum acyl chain length of 11 carbons (S. R. Chhabra, C. Harty, D. S. W. Hooi, M. Daykin, B. W. Bycroft, P. Williams, and D. Pritchard, J. Med. Chem. 46:97-104, 2003). We also demonstrate that while OdDHL can have toxic effects on nonlymphoid leukocytes, it does not induce significant cell death in T cells at the concentrations (< or =10 microM) used in these experiments. In addition, we show that primary and secondary antigen-specific cytokine responses are equally susceptible to inhibition by OdDHL and that the compound inhibits the differentiation of both Th1 and Th2 cells. However, the precise balance of cytokine production by CD4 T cells stimulated in the presence of OdDHL varies with both the antigen concentration and its affinity for the transgenic TCR. Thus, conflicting reports of the nature of the immunosuppression by OdDHL may be due in part to the differences in antigen affinity and concentration in different models.