问号钩端螺旋体侵入单核-巨噬细胞方式及其吞噬泡形成差异性研究

目的 探讨问号钩端螺旋体(简称钩体)侵入人或鼠单核-巨噬细胞方式及其吞噬泡形成差异性.方法 采用透射电镜观察问号钩体黄疸出血群赖型赖株侵入小鼠单核-巨噬样细胞J774A.1和佛波酯(PMA)激活的人单核细胞THP-1后吞噬泡形成情况.采用免疫荧光联合激光共聚焦显微镜及荧光分光光度仪等方法,观察细胞内吞抑制剂单丹磺酰尸胺(MDC)、氧化酚砷(PAO)阻断及内吞相关网格蛋白抗体封闭前后,J774A.1细胞和PMA激活的THP-1细胞内问号钩体赖株数量的变化.结果 J774A.1细胞内问号钩体存在于吞噬泡内,THP-1细胞内问号钩体无吞噬泡膜包绕.MDC和PAO能以剂量依赖方式抑制J774A.1和THP-1细胞内吞问号钩体,其中10 μmol/L以上MDC和1 μmol/L以上PAO阻断的J774A.1和THP-1细胞内问号钩体数量明显少于未阻断细胞(P＜0.05).网格蛋白抗体封闭后,J774A.1和THP-1细胞内问号钩体数量也明显减少(P＜0.05).结论 问号钩体以网格蛋白依赖性内吞途径侵入人或鼠单核-巨噬细胞.人或鼠单核-巨噬细胞内问号钩体吞噬泡形成有明显差异,这可能是人或鼠感染问号钩体后发病情况不同的原因之一.     

不同毒力的问号钩端螺旋体对Vero及J774A.1细胞的黏附和内化

目的 探讨问号钩端螺旋体对传代细胞黏附和内化的能力及其差异。方法 实验中采用非洲绿猴肾成纤维细胞(Vero)和小鼠单核巨噬样细胞(J774A．1)细胞株。采用透射电镜、扫描电镜和Fontana镀银染色法，观察问号钩端螺旋体强毒株黄疸出血群赖型56601、弱毒株波摩那群波摩那型56608黏附细胞及内化能力及其差异，采用腐生性的双曲钩端螺旋体三宝垄群patoc型Patoc I株作为对照.结果 问号钩端螺旋体黄疸出血群赖型56601株和波摩那群波摩那型56608株均能以一端或两端黏附于Vero及J774A．1细胞。钩体56601株和56608株对J774A．1的黏附率分别为49％和46．9％，对Vero细胞黏附率分别为24．2％和22．9％。钩体56601株和56608株可侵入上述2株细胞，在胞质内形成典型的吞噬泡。钩体56601株还可侵入宿主细胞核内，56608株则否。双曲钩端螺旋体二宝垄群patoc型Patoc I株不能黏附和侵入细胞。结论 两株受试的不同毒力问号钩体株均能黏附细胞，并以内化方式侵入细胞。细胞株的差异可明显影响钩体黏附和内化能力。问号钩体毒力的强弱可能与黏附能力无关，而与其侵入胞核的能力密切相关。     

问号钩端螺旋体内化过程中细胞内游离Ca2+水平变化及细胞凋亡

目的建立观察问号钩端螺旋体(简称钩体)黏附的双荧光染色法,探讨不同毒力钩体对细胞内游离Ca2+水平及细胞凋亡的影响.方法以问号钩体黄疸出血群赖型56601株和双曲钩体三堡垄群patoc型PatocⅠ株抗血清为一抗、羊抗兔IgG荧光素F(ab)2和罗丹明F(ab)2片段为二抗的双荧光染色法,分别检测56601株和PatocⅠ株钩体对Vero、J774A.1细胞的黏附作用.采用fluo-3/AM胞内Ca2+特异荧光标记激光共聚焦技术,检测问号钩体56601株和波摩那群波摩那型56608株、双曲钩体PatocⅠ株作用的J774A.1细胞胞内游离Ca2+水平的变化.采用FITC-annexinⅤ/PI荧光标记流式细胞术,检测紫外线灭活前后的56601株钩体诱导Vero和J774A.1细胞凋亡的情况.结果所建立的双荧光染色法能清晰地观察到强毒力的56601株钩体对Vero和J774A.1细胞的黏附,无毒力的PatocⅠ株钩体则否.正常J774A.1细胞胞内游离Ca2+基础值为(105.0±7.0)%,PatocⅠ株钩体作用细胞的荧光强度变化百分数一直波动于(102.2±5.2)%.56601株钩体感染J774A.1细胞胞内游离Ca2+浓度迅速增高,呈现为双峰型曲线,其荧光强度变化百分数分别为(747.5±35.7)%和(804.6±40.8)%.56608株钩体感染J774A.1细胞胞内游离Ca2+浓度呈现为缓慢的单一坡型升高,其最大荧光强度变化百分数为(402.4±23.6)%,明显小于56601株钩体,差异有统计学意义(P<0.01).紫外线灭活前后56601株钩体作用Vero细胞的凋亡率分别为84.5%和78.2%,J774A.1细胞凋亡率分别为34.5%和30.9%.结论所建立的双荧光染色法可用于观察钩体的黏附.细胞胞内游离Ca2+水平与所感染的钩体菌株毒力成正相关.有毒力的钩体接触细胞时即可诱导凋亡发生,启动细胞凋亡信号通路的配体分子可能位于钩体表面.     

问号钩端螺旋体在体外诱导细胞凋亡及相关信号通路的研究

目的 了解问号钩端螺旋体诱导不同宿主细胞凋亡的作用及相关胞内信号传导通路.方法 建立问号钩体黄疸出血群赖型赖株小鼠单核-巨噬样细胞J774A.1、人脐静脉内皮细胞EVC304和人Ⅱ型肺泡上皮细胞A549感染模型.采用FITC-Annexin V/PI荧光标记流式细胞术检测细胞凋亡或坏死情况.分别采用荧光比色法和Western blot检测感染的J774A.1细胞caspase-3,-8,-9活性和凋亡相关蛋白FADD(Fas-associated death domain)表达水平.结果 问号钩体赖株感染1～6 h后,36.70%～63.70%的J774A.1细胞可H{现明显的早期凋亡,感染12 h时转变为晚期凋亡或坏死为主(53.68%).78.52%问号钩体赖株感染的A549细胞仪出现晚期凋亡或坏死.问号钩体赖株感染的EVC304细胞无细胞凋亡或坏死现象.感染的J774A.1细胞caspase-3和-8最大活性分别为(1453.41±36.07)和(1402.15±59.09)Fu,是未感染细胞的16.38和29.99倍.感染的J774A.1细胞caspase-9虽略有升高为(89.42±5.08)Fu,但明显低于caspase-3和-8(P<0.001).随着感染时间的延长,感染的J774A.1细胞FADD蛋白表达量逐步增加.结论 问号钩体诱导宿主细胞凋亡的效应町因细胞种类不同而有明显差异,FADD→caspase-8→caspase-3是介导问号钩体感染J774A.1细胞凋亡的主要信号通路.     

细胞周期及其调控基因对问号钩端螺旋体诱导单核-巨噬细胞凋亡的影响

目的 了解不同细胞周期及其调控基因对问号钩端螺旋体(简称钩体)诱导人或鼠单核-巨噬细胞凋亡的影响.方法 采用细胞周期染色试剂盒及流式细胞仪检测问号钩体黄疸出血群赖型赖株感染前后小鼠单核-巨噬样细胞株J774A.1和人单核细胞株THP-1的细胞周期及其变化.采用细胞周期阻滞剂及流式细胞仪建立细胞周期同步化的J774A.1和THP-1细胞并进行鉴定.采用AnnexinV/PI凋亡检测试剂盒及流式细胞仪检测问号钩体赖株感染后细胞周期同步化与非同步化J774A.1和THP-1细胞早期凋亡、晚期凋亡/坏死率.采用实时荧光定量RT-PCR检测问号钩体赖株感染前后J774A.1和THP-1细胞的细胞周期及凋亡基因p21、p27、p53、c-myc和cycA mRNAs水平变化.结果 未感染钩体的正常J774A.1和THP-1细胞均分别处于G1、S和G2/M期,感染后均以G1期细胞为主,但S期THP-1细胞有所增加,J774A.1细胞则否(P＜0.05).J774A.1和THP-1细胞可分别被不同细胞周期阻滞剂阻滞在G1、S、G2/M或M期.G1期J774A.1和THP-1细胞感染后均无明显的早期凋亡现象,M期细胞早期凋亡、晚期凋亡/坏死率均明显升高(P＜0.05),G1期THP-1细胞晚期凋亡/坏死率明显升高(P＜0.05),J774A.1细胞则否.J774A.1和THP-1细胞感染后,p21 mR-NA水平均明显高于未感染细胞(P＜0.05),J774A.1细胞c-myc和p27 mRNAs水平、THP-1细胞cycAmRNA水平也高于未感染细胞(P＜0.05).结论 不同细胞周期及其调控基因对问号钩体诱导人或鼠单核-巨噬细胞凋亡有明显影响,但存在细胞种类差异性.     

问号钩端螺旋体ompA基因分析及其重组表达产物的免疫学鉴定

目的 了解我国15群15株问号钩端螺旋体(简称问号钩体)参考标准株携带ompA基因情况,重组表达OmpA(rOmpA)并鉴定rOmpA的免疫原性和免疫保护性.方法 采用酚-氯仿法提取问号钩体基因组DNA,PCR扩增全长ompA基因,T-A克隆后测序.构建问号钩体黄疸出血群赖型56601株ompA基因的原核表达系统,采用SDS-PAGE及Bio-Rad凝胶}冬{像分析系统检测rOmpA表达情况及其产鼍.rOmpA免疫家兔以获得抗血清,采用免疫扩散试验检测抗血清效价.采用Western blot检测rOmpA与其抗血清和问号钩体56601株全菌抗血清的免疫反应性,显微镜凝集试验(MAT)检测rOmpA抗血清对15株问号钩体的交叉凝集情况.分别采用问号钩体黏附J774A.1细胞模型和豚鼠感染模型,了解rOmpA兔抗血清黏附阻断及rOmpA免疫保护作用.结果 15株问号钩体均含有序列保守的ompA基因,双曲钩体Patocl株则否.rOmpA表达量约占细菌总蛋白的20%.rOmpA能诱导家兔产生抗体,其抗血清免疫扩散效价为1:4.兔抗血清及问号钩体56601株全菌抗血清均能与rOmpA产生阳性Western blot信号.rOmpA抗血清对15株问号钩体的MAT效价为1:20～1:320.1:10～1:160稀释的rOmpA抗血清均能阻断问号钩体黏附J774A.1细胞,100μg和200μg rOmpA对豚鼠的免疫保护率分别为50.0%和75.0%.结论 ompA基因仅存在于不同血清群致病性问号钩体基因组中.rOmpA具有较好的抗原性,多种免疫学方法 检测显示,有可能作为通用型问号钩体基因上程疫苗的候选抗原.     

问号钩端螺旋体经线粒体相关信号通路诱导巨噬细胞凋亡的研究

目的 确定线粒体相关信号转导途径在问号钩端螺旋体(简称钩体)诱导小鼠单核-巨噬细胞凋亡过程中的作用.方法 建立问号钩体黄疸出血群赖株诱导小鼠单核-巨噬样细胞株J774A.1凋亡模型.采用透射电镜观察感染细胞线粒体病变情况,JC-1染色法检测感染细胞线粒体膜电位变化,荧光探针DCFH-DA检测感染细胞内活性氧(ROS)水平.采用试剂盒检测感染细胞caspase-8和caspage-9活性变化.流式细胞术检测感染细胞凋亡情况以及cagpage阻断剂阻断凋亡的效果.采用Western blot检测线粒体内和胞质中的细胞色素c(cytc)以及凋亡诱导因子(Air)、核酸内切酶G(EndoG)和Smac水平.应用免疫荧光染色法检测AIF和EndoG从细胞质至核内的转位.结果 问号钩体赖株可诱导J774A.1细胞凋亡.感染细胞的线粒体有明显病变,线粒体膜电位降低且胞内活性氧水平升高.感染细胞caspage-8活化,caspase-9则否,但caspase阻断剂不能完全阻断细胞凋亡.感染细胞AIF和EndoG从线粒体释放至胞质并转位至细胞核内.未检测到感染细胞胞质内CytC水平升高及Smac的释放.结论 线粒体可通过非caspase途径的AIF和EndoG参与问号钩体诱导单核一巨噬细胞凋亡的过程.     

问号钩端螺旋体经线粒体相关信号通路诱导巨噬细胞凋亡的研究

目的 确定线粒体相关信号转导途径在问号钩端螺旋体(简称钩体)诱导小鼠单核-巨噬细胞凋亡过程中的作用.方法 建立问号钩体黄疸出血群赖株诱导小鼠单核-巨噬样细胞株J774A.1凋亡模型.采用透射电镜观察感染细胞线粒体病变情况,JC-1染色法检测感染细胞线粒体膜电位变化,荧光探针DCFH-DA检测感染细胞内活性氧(ROS)水平.采用试剂盒检测感染细胞caspase-8和caspage-9活性变化.流式细胞术检测感染细胞凋亡情况以及cagpage阻断剂阻断凋亡的效果.采用Western blot检测线粒体内和胞质中的细胞色素c(cytc)以及凋亡诱导因子(Air)、核酸内切酶G(EndoG)和Smac水平.应用免疫荧光染色法检测AIF和EndoG从细胞质至核内的转位.结果 问号钩体赖株可诱导J774A.1细胞凋亡.感染细胞的线粒体有明显病变,线粒体膜电位降低且胞内活性氧水平升高.感染细胞caspage-8活化,caspase-9则否,但caspase阻断剂不能完全阻断细胞凋亡.感染细胞AIF和EndoG从线粒体释放至胞质并转位至细胞核内.未检测到感染细胞胞质内CytC水平升高及Smac的释放.结论 线粒体可通过非caspase途径的AIF和EndoG参与问号钩体诱导单核一巨噬细胞凋亡的过程.     

问号钩端螺旋体鞘磷脂酶类溶血素基因功能分析及其感染细胞后转录水平变化

目的 了解问号钩端螺旋体(简称钩体)鞘磷脂酶类溶血素基因sph1～sph4产物溶血活性及其感染细胞后转录水平的变化.方法 以致病性问号钩体黄疸出血群赖型赖株、波摩那群波摩那型罗株和非致病性双曲钩体三宝垄群Patoc型Patoc Ⅰ株基因组DNA为模板,采用PCR扩增全长sph1～spl4基因片段,扩增产物T-A克隆后测序.构建sph1～sph4基因原核表达系统,采用SDS-PAGE检测目的 重组蛋白rSph1～rSph4的表达情况,Ni-NTA亲和层析柱提纯rSph1～rSpM.采用绵羊血平板对rSph1～rSph4溶血活性进行鉴定,实时荧光定量RT-PCR检测问号钩体赖株感染J774A.1细胞前后sph1～sph4基因转录水平的变化.结果 问号钩体赖株和罗株基因组DNA中均能扩增sph1～sph4基因,双曲钩体Patoc Ⅰ株则否.与报道的相应基因序列比较,所克隆的sph1～sph4基因核苷酸序列相似性均为100%.所构建的原核表达系统能分别表达目的蕈组蛋白rSph1～rSph4.rSph1～rSph4均有溶血活性,其中以rSph2溶血活性最强.问号钩体赖株感染J774A.1细胞后,sph1～sph4基因转录水平均上调,其中sph2和sph4基因mRNA水平上调更为明显.结论 sph1～sph4基因仅存在于致病性问号钩体中,其表达产物有溶血活性.问号钩体赖株感染细胞后sph1～sph4基因转录水平的上调,提示此类鞘磷脂酶类溶血素可能在问号钩体感染宿主过程中有重要作用.     

Caspase-11非经典炎症小体对问号钩端螺旋体诱导J774A.1细胞炎性细胞因子分泌水平的影响

目的:了解caspase-11非经典炎症小体对问号钩端螺旋体(钩体)诱导J774A.1细胞分泌炎性细胞因子的影响。方法:采用钩体56601株感染小鼠单核-巨噬细胞株(J774A.1)建立细胞模型,应用real-time RT-PCR检测J774A.1细胞caspase-11、IL-1β、IL-1α和IL-18 mRNA水平,采用ELISA定量检测J774A.1细胞上清液中caspase-11、IL-1β、IL-1α和IL-18水平。结果:Real-time RT-PCR检测结果显示,钩体感染J774A.1细胞1、2、4、8、12和24 h后,caspase-11 mRNA水平分别为未感染细胞的5.12、14.21、8.94、14.06、18.58和0.93倍,caspase-11阻断后分别下降至0.10、0.07、0.10、0.09、0.07和0.45倍( P<0.05);钩体感染后,IL-1β、IL-1α和IL-18 mRNA水平均显著上调,caspase-11阻断后IL-1β mRNA水平分别下降至0.05、0.03、0.02、0.05、0.06和0.02倍( P<0.05);IL-1α mRNA分别下降至0.14、0.07、0.15、0.10、0.03和0.06倍( P<0.05);IL-18 mRNA分别下降至0.08、0.10、0.16、0.18、0.10和0.07倍( P<0.05)。ELISA检测结果显示,钩体感染J774A.1细胞后细胞上清液中caspase-11、IL-1β、IL-1α和IL-18水平均显著上调,caspase-11阻断后caspase-11分别下降至43.07、41.64、51.96、86.56、105.36和129.95 pg/ml( P<0.05);IL-1β分别下降至15.01、14.19、68.02、31.20、173.13和104.98 pg/ml( P<0.05);IL-1α分别下降至12.14、15.40、38.01、21.97、24.48和27.09 pg/ml( P<0.05);IL-18分别下降至96.27、102.21、85.34、116.28、155.36和114.03 pg/ml( P<0.05)。 结论:Caspase-11非经典炎症小体参与介导问号钩体诱导小鼠单核-巨噬细胞IL-1β、IL-1α和IL-18的分泌。     

The formation and function of the neutrophil phagosome

Neutrophils are the most abundant circulating leukocyte and are crucial to the initial innate immune response to infection. One of their key pathogen-eliminating mechanisms is phagocytosis, the process of particle engulfment into a vacuole-like structure called the phagosome. The antimicrobial activity of the phagocytic process results from a collaboration of multiple systems and mechanisms within this organelle, where a complex interplay of ion fluxes, pH, reactive oxygen species, and antimicrobial proteins creates a dynamic antimicrobial environment. This complexity, combined with the difficulties of studying neutrophils ex vivo, has led to gaps in our knowledge of how the neutrophil phagosome optimizes pathogen killing. In particular, controversy has arisen regarding the relative contribution and integration of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived antimicrobial agents and granule-delivered antimicrobial proteins. Clinical syndromes arising from dysfunction in these systems in humans allow useful insight into these mechanisms, but their redundancy and synergy add to the complexity. In this article, we review the current knowledge regarding the formation and function of the neutrophil phagosome, examine new insights into the phagosomal environment that have been permitted by technological advances in recent years, and discuss aspects of the phagocytic process that are still under debate.     

Inside the phagosome: A bacterial perspective

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.     

On regulation of phagosome maturation and antigen presentation

Phagocytosis has essential functions in immunity. Here we highlight the presence of a subcellular level of self-non-self discrimination in dendritic cells that operates at the level of individual phagosomes. We discuss how engagement of Toll-like receptor signaling controls distinct programs of phagosome maturation. An inducible mode of phagosome maturation triggered by these receptors ensures the selection of microbial antigens for presentation by major histocompatibility class II molecules during the simultaneous phagocytosis of self and non-self.     

The regulation of phagosome maturation in Dictyostelium

Macropinocytosis (fluid uptake) and phagocytosis (particle uptake) are processes that result in the formation of intracellular membrane enclosed vacuoles termed macropinosomes and phagosomes, respectively. Macropinosomes and phagosomes are modified by fission and fusion reactions with the endo-lysosomal pathway that eventually transform these vacuoles into a lysosomal environment. Many human bacterial pathogens, including species of Mycobacteria, Legionella, and Chlamydia, are thought to survive by disrupting the normal membrane trafficking events that usually result in the formation of phago-lysosomes and death of the microorganism. In addition, a number of important pathogens facilitate homotypic phagosome fusion in order to generate an intracellular environment conducive for survival. A greater understanding of the regulation of phagosomal maturation and fusion will be critical in designing new therapies to treat infections caused by intracellular pathogens. The genetically tractable phagocyte, D. discoideum, has proven extremely useful in dissecting the signaling pathways regulating macropinocytosis, phagocytosis, phagosomal maturation and phagosome–phagosome fusion. A body of knowledge has accumulated and demonstrates important roles for Rab GTPases, the cytoskeleton, phosphoinositide metabolism and pH regulation in regulating phagosome maturation. This review will summarize the current state of knowledge.     

Histamine uptake by human endometrial cells expressing the organic cation transporter EMT and the vesicular monoamine transporter-2

Cellular reuptake of monoamines, which is mediated by cell membrane transporters, is followed by accumulation in vesicles by vesicular monoamine transporters (VMAT). The aim of this study was to demonstrate the presence of functional monoamine transporters with high affinity for histamine in human endometrial tissue, since histamine has been implicated as a paracrine signal during endometrial decidualization and embryo implantation. In situ hybridization with (35)S-labelled cRNA probes was used for detection of the organic cationic transporter-2 (OCT-2), the extraneuronal monoamine transporter (EMT), and VMAT-2 in cryosections of normal human endometrial tissue. To identify functional transporters for histamine in endometrial cells, we incubated primary cultures of stromal cells and cultures of attached glands with (3)H-labelled histamine. Cultures were pretreated with either corticosterone, a specific inhibitor of EMT, or reserpine, a specific inhibitor of VMAT-2. EMT mRNA was localized in the stroma with peak expression in the secretory phase, whereas OCT-2 mRNA was expressed by few cells in the stroma throughout the cycle. VMAT-2 mRNA was localized in the stroma during the proliferative phase and in the epithelium during the secretory phase. Thus, EMT and VMAT-2, which both have high affinity for histamine, are strongly expressed in endometrial cells. Both corticosterone and reserpine significantly reduced the uptake of (3)H-histamine in stromal cells during the proliferative as well as the secretory phase. This indicates the presence of functional EMT and VMAT-2 transporter proteins throughout the cycle, even though their periods of maximal mRNA expression were limited. The results of uptake experiments with glandular epithelial cells confirmed not only the presence of functional VMAT-2 transporter protein in the secretory phase but also the absence of a histamine-specific plasma membrane transporter throughout the cycle. Thus, endometrial tissue contains both plasma membrane and vesicular membrane monoamine transporters with high affinity for histamine. They can potentially influence the reproductive process by the uptake of extracellular histamine and subsequent release on demand.     

Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle

BACKGROUND: Prostaglandin E2 (PGE2) has been identified as the key ovulatory PG in the primate follicle. Follicular PGE2 levels increase just before the expected time of ovulation, suggesting that the midcycle LH surge induces the expression of enzymes involved in PGE2 synthesis. METHODS: To identify the specific form(s) of prostaglandin E synthase (PGES) expressed by the primate periovulatory follicle, we examined granulosa and theca cell expression of the three microsomal (m) and cytosolic (c) forms of PGES (mPGES-1, mPGES-2 and cPGES) identified to date. Monkey granulosa cells and whole monkey ovaries were obtained from animals receiving exogenous gonadotropins to stimulate multiple follicular development; monkeys then received an ovulatory dose of HCG to initiate periovulatory events. RESULTS: Expression of mPGES-1 mRNA and protein by granulosa cells of periovulatory follicles increased in response to HCG administration, peaking just before the expected time of ovulation. Immunocytochemistry showed that mPGES-1 protein was present in both granulosa and theca cells of monkey periovulatory follicles. Monkey granulosa cells also expressed mPGES-2 and cPGES mRNA, but mRNA levels did not change in response to HCG administration. Isolated monkey theca cells expressed both mPGES-1 and cyclooxygenase-2 mRNA, and produced PGE2 in vitro. Human granulosa-lutein cells obtained from women undergoing treatment for infertility expressed mRNAs for mPGES-1, mPGES-2 and cPGES. CONCLUSIONS: These data indicate that mPGES-1 is a gonadotropin-regulated PG synthesis enzyme expressed by granulosa cells of primate periovulatory follicles and suggest that mPGES-1 may be the primary PGES responsible for the increased follicular PGE2 levels necessary for primate ovulation.     

Phosphatidylinositol 3-kinases and their roles in phagosome maturation

Phagosome maturation is a highly organized and sequential process that results in the formation of a microbicidal phagolysosome. This results in crucial contributions to innate and adaptive immunity through pathogen clearance and antigen presentation. Thus, it is important to understand the regulatory networks that control the extent and nature of phagosome maturation. PI3Ks are lipid kinases that catalyze the phosphorylation of the 3' position of the inositol ring. This enzyme family is divided into three classes based on structure and substrate preferences. Previously, only the class III PI3K, hVps34, was thought to contribute to phagosome maturation. Recent evidence, however, suggests important contributions by class I PI3Ks in bringing about the diverse phagosome maturation phenotypes. Class I PI3Ks have also been implicated in the activation of Rab GTPases that function in maturation, such as Rab14. In addition, recent studies have illuminated the overlap between phagosome maturation and autophagy, which itself is regulated by multiple classes of PI3K. Taken together, a picture of phagosome maturation is emerging in which multiple classes of PI3Ks are involved in modulating maturation phenotypes. This review summarizes the known contributions of PI3Ks to phagosome maturation. Special emphasis is placed on the impact of PI3Ks on different maturation outcomes stemming from the engagement of diverse phagocytic receptors and on Rab and Ca(2+) signaling cascades.     

Neutrophil NADPH oxidase is reduced at the Anaplasma phagocytophilum phagosome

The intracellular organism Anaplasma phagocytophilum causes human granulocytic ehrlichiosis and specifically infects and multiplies in neutrophilic granulocytes. Previous reports have suggested that, for its survival, this bacterium suppresses the neutrophil respiratory burst. To investigate the mechanism of survival, we first assessed the kinetics of A. phagocytophilum entry into neutrophils by using double-labeling confocal microscopy. At 30, 60, 120, and 240 min of incubation, 25, 50, 55, and 70% of neutrophils contained bacteria, respectively. The neutrophil respiratory burst in the presence of A. phagocytophilum was assessed by a kinetic cytochrome c assay and by measurement of oxygen consumption. Neutrophils in the presence of A. phagocytophilum did not produce a significant respiratory burst, but A. phagocytophilum did not inhibit the neutrophil respiratory burst when phorbol myristate acetate was added. Immunoelectron microscopy of neutrophils infected with A. phagocytophilum or Escherichia coli revealed that NADPH oxidase subunits gp91(phox) and p22(phox) were significantly reduced at the A. phagocytophilum phagosome after 1 and 4 h of incubation. In neutrophils incubated simultaneously with A. phagocytophilum and E. coli for 30, 60, and 90 min, gp91(phox) was present on 20, 14, and 10% of the A. phagocytophilum phagosomes, whereas p22(phox) was present in 11, 5, and 4% of the phagosomes, respectively. Similarly, on E. coli phagosomes, gp91(phox) was present in 62, 64, and 65%, whereas p22(phox) was detected in 54, 48, and 48%. We conclude that A. phagocytophilum does not suppress a global respiratory burst and that, under identical conditions in the same cells, A. phagocytophilum, but not E. coli, significantly reduces gp91(phox) and p22(phox) from its phagosome membrane.