Effect of zinc-treated Entamoeba histolytica on the human polymorphonuclear respiratory burst

One of the mechanisms that Entamoeba histolytica uses to evade host immune response is inhibition of the polymorphonuclear (PMN) leukocyte respiratory burst. In studies previously conducted in a model used in our laboratory, we observed that when treating trophozoites with different zinc concentrations certain amebic functions are inhibited while significantly limiting development of hepatic abscess in golden hamsters (Mesocricetus aureatus). We carried out an in vitro study using a chemoluminescent method to assess the effect zinc-treated amebic trophozoites exercise on respiratory burst in human PMNs. We measured response of PMNs incubated with E. histolytica trophozoites from cultures with TYI-S33 medium alone and with zinc. Zinc concentrations between 0.1 and 1.0 mM did not affect amebic trophozoite viability, and PMNs in contact with these in a zinc-free medium had an oxidative response similar to that obtained with zymosan and significantly greater (p <0.05) than that generated by cells co-incubated with amebas cultured in TYI-S33 medium alone. These results suggest that zinc alters the amebic mechanism that inhibits the oxidative function of human polymorphonuclear leukocyte.     

补体活化与中性粒细胞生产活性氧之间的反馈调节作用

目的 在试管实验中证明补体系统的活化与中性粒细胞产生的活性氧间存在相互作用的反馈关系。方法 用菊糖活化的补体作用于中性粒细胞产生活性氧,并进一步通过测定吸光度以观察补体进一步活化情况。用佛波醇肉豆蔻醋酸酯激发中性粒细胞产生的活性氧作用于补体,并进一步采用发光测定仪测定活性氧来观察活经的补体对中性粒细胞释放活性氧的促进作用。结果 试管实验证明活化的补体能刺激中性粒细胞产生活性氧,而活性氧又能进一步活     

弥猴肠缺血再灌注后生长抑素含量与中性粒细胞寿命的相关性探讨

目的探讨多器官功能障碍综合征时中性粒细胞凋亡延迟的潜在原因,以增加对全身炎症反应内源性保护机制的认识。方法对照组、实验组各5只弥猴用放射免疫分析法测定其外周血、肠黏膜生长抑素浓度;生长抑素与猕猴外周血中性粒细胞共同孵育后,观察猕猴外周血中性粒细胞的形态变化,流式细胞仪测定细胞凋亡率,电泳分析中性粒细胞DNA断裂片段,生物分子相互作用系统检测中性粒细胞上生长抑素受体(SSTR-1、SSTR-2)。结果猕猴肠缺血再灌注后,猕猴外周血及小肠黏膜中生长抑素含量均明显下降;中性粒细胞凋亡率由阻断前的15.4%±1.4%,显著降低至3.5%±0.5%(P<0.05);腹腔巨噬细胞凋亡率由阻断前的14.1%±1.7%增加至20.2%±1.8%(P<0.05);体外实验表明,生长抑素可使中性粒细胞体积缩小,细胞质密度增加,染色质固缩,细胞核变小;中性粒细胞凋亡率(50.2%±1.8%)明显高于对照组(20.0%±2.2%,P<0.01);中性粒细胞的DNA片段断裂分析电泳可见Ladder带。中性粒细胞上生长抑素受体能与生长抑素受体SSTR-1抗体、SSTR-2抗体发生结合反应,其特异结合量分别为(548±20)RU/m l及(28±21)RU/m l。结论生理状态下,生长抑素通过猕猴外周血中性粒细胞上生长抑素受体介导,诱导其凋亡;肠缺血再灌注后猕猴外周血及小肠黏膜中生长抑素含量明显下降,这使中性粒细胞凋亡延迟,从而推动了全身炎症反应综合征和多器官功能障碍综合征的发生。     

补体活化与中性粒细胞产生活性氧之间的反馈调节作用

目的在试管实验中证明补体系统的活化与中性粒细胞产生的活性氧间存在相互作用的反馈关系。方法用菊糖活化的补体作用于中性粒细胞产生活性氧,并进一步通过测定吸光度以观察补体进一步活化情况。用佛波醇肉豆蔻醋酸酯激发中性粒细胞产生的活性氧作用于补体,并进一步采用发光测定仪测定活性氧来观察活化的补体对中性粒细胞释放活性氧的促进作用。结果试管实验证明活化的补体能刺激中性粒细胞产生活性氧,而活性氧又能进一步活化补体。结论补体和中性粒细胞产生的活性氧两者间存在着相互活化的反馈调节作用。从而推测炎症过程中补体活化在炎症反应的产生中起主要作用,而在炎症反应的持续过程中,此两者起互相促进的反馈调节作用。     

一氧化碳对肢体缺血再灌注患者血清所致中性粒细胞凋亡的影响及核因子κB的作用

目的 观察外源性一氧化碳（CO）对肢体缺血再灌注（IR）患者血清所致中性粒细胞（PMN）凋亡的影响及核因子（NF）κB的作用.方法 分离培养人外周血PMN,置于24-孔培养板,分为4组（n=16）：对照组,对照＋CO,IR和IR＋CO组.IR和IR＋CO组细胞应用肢体IR患者血清代替培养基孵育,其余两组应用健康志愿者血清培养.IR＋CO组和对照＋CO组细胞置含有0.025%CO和5%CO2的空气中孵育.对照组和IR组细胞始终在含5%CO2空气中孵育.应用流式细胞学技术检测PMN凋亡率,进行PMN DNA电泳观察梯状带情况,应用电泳迁移率（EMSA）检测PMN核内NF-κB活性.结果 与对照组相比,IR组PMN凋亡百分率显著降低、NF-κB活性明显增强;与IR组相比,IR＋CO组PMN凋亡百分率显著增高、NF-κB活性明显降低,出现典型DNA梯状带.结论 外源性CO可改善肢体IR患者血清所致的PMN凋亡抑制,其机制与下调NF-κB活性有关.     

单克隆抗体HIM82对中性粒细胞呼吸爆发的降调节作用

本研究用单抗HIM82对中性粒细胞（PMN）呼吸爆发的早期调控，发现HIM82（30μg／ml）对佛波酯（PMA）激活的PMN产生的O2-、H2O2分别可下调至（44．00±8．9）％（n=8，P＜0．01）和（65．16±15.41）％（n=8，P＜0．01），对因呼吸爆发产生的活性氧物质（reactiveoxygenspecies，ROS）引起质膜中性内肽酶（NEP）的纯化作用（inactivation）[活性下降（43．29±9．41）％，n＝8，P＜0．01]有防护作用[活性恢复至（6648±15.53）％，n=8，P＜0．05]。结果表明该单抗对PMA激发PMN呼吸爆发产生O2-、H2O2有明显的降调节作用，也表明质膜上相应分化抗原分子对PMN功能特别是对激活呼吸爆发的机构有重要影响。     

旁路活化补体降低PMN吞杀绿脓杆菌力的实验研究

为证明论题,本文做了3项实验:(1)人血PMN培养单层加上酵母多糖活化人血清(ZAHS)。PMN的超氧离子(O_2~-)、特殊颗粒(SG)与胞内杀菌力(ICBA)明显平行下降,于6 h最低,0.05 ml的作用最强。(2)经小鼠尾静脉注射0.5ml ZAHS/鼠,6 h后活杀。肺和血内PMN的上述指标均明显降低;肺内病变明显:急性间质炎,灶性水肿出血和萎陷,肺血屏障亚微结构损伤。(3)经抗人C_3、C_5血清(AHC_(3.5)S)体外中和的ZAHS,在体内外实验中均失去其有害作用。提示:ZAHS的有害作用与C_3、C_5碎片有关,在剂量适中且吞菌前作用时间较长的条件下方显极效。有害作用的可能机制:杀菌且能致炎的O_2~-与SG大多已于吞菌前排放在胞外,所以PMN的ICBA下降;累积于组织内的则损害PMN自身及其邻近的肺血屏障。这便于MOF与感染发生。     

Functional characteristics of neutrophils and mononuclear cells from tuberculosis patients stimulated in vitro with heat killed M. tuberculosis

BACKGROUND: The major protective immune response against intracellular bacteria, such as Mycobacterium tuberculosis, is a cell-mediated immunity involving neutrophils (PMNs) and peripheral mononuclear cells (MCs), contributing to the clearance of this microorganism and the resolution of the infection. This study was addressed to evaluate PMNs and MCs for their bactericidal function. METHODS: The sample comprised 14 tuberculosis (TB) inpatients (HIV-), and 10 healthy controls (HCo). Peripheral PMNs and MCs were separated by Ficoll-Hypaque and cultured in RPMI with or without heat-killed Mycobacterium tuberculosis (HK Mtb). Respiratory burst (RB), CD11b, IL-8 and TNFalpha receptor expression were assessed by flow cytometry in cells undergoing stimulation or not. Presence of IL-8 and TNFalpha in cell culture supernatants was determined by ELISA. RESULTS: TB patients had a lower RB response than HCo for both cell types (MCs, p <0.05, PMNs, p <0.01) regardless of HK Mtb stimulation. Compared to HCo, PMNs and MCs from TB patients presented a reduced CD11b expression, with the two subject groups showing a decrease in this marker expression following HK. Mtb was added to both cell cultures. Whereas fewer IL-8 and TNFalpha receptors were found when studying MCs and PMNs from TB patients, antigen stimulation significantly raised the expression for both cytokine receptors. Culture supernatants from MCs and PMNs of TB patients contained increased amounts of IL-8 and TNFalpha. CONCLUSIONS: The present findings may provide some explanation as to the different roles played by PMNs and MCs in TB immunopathology.     

蓝氏贾第鞭毛虫承德株分子生物学研究

目的：探讨蓝氏贾第鞭毛虫大、小型包囊存在的原因。方法：用分子生物学技术对３株贾第虫（ＣＨ２、ＣＨ３、ＣＨ４）进行ｔｉｍ基因扩增和序列测定。结果：经与ＧｅｎＢａｎｋ登记虫株比较和用ＤＮＡｓｔａｒ软件处理的结果表明,此３株贾第虫的基因型属２型（ＪＨ型）。结论：大、小型包裹在形态学上经过统计学处理显示有显著差异,但与基因型无关。     

Pro-apoptotic role of NF-κB pathway inhibition in lipopolysaccharide stimulated polymorphonuclear neutrophils

Objective To investigate the role of nuclear factor kappa B (NF-κB) pathway inhibition in lipopolysaccharide (LPS)-stimulated apoptosis of polymorphonuclear neutrophils (PMNs).Methods Rats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate (PDTC) and gliotoxin were used as NF-κB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-κB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling and DNA fragmentation. IκBα degradation was analyzed by Western blot. NF-κB DNA binding activity was detected by an electrophoretic mobility shift assay.Results (1) The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS (100μg per rat) intratracheal instillation were alleviated by PDTC (50, 100, or 200mg/kg, i. p. ) in a dose-dependent manner. (2) PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment. (3) IκBα degradation and increased NF-KB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment.Conclusion Inhibition of either NF-κB itself or the upstream signals in NF-κB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role.     

DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

目的：为了探讨来源于中国和其它国家不同地理位置贾第虫分离株之间的遗传学关系。方法：从贾第虫滋养体和包囊提取总DNA。对磷酸丙糖异构酶基因进行PCR扩增。PCR产物经限制性内切酶消化、序列测定及分析。对所得DNA序列数据用DNAstar软件处理并与登录基因库虫株的序列比较。结果：在来自我国（C1、C2、CH2、CH3）,柬埔寨（CAM）、澳大利亚（A1、A2）和美国（BP、CDC）的9株蓝氏贾第虫中,A1、A2和CAM属于第1型（WB）;CH2和CH3属于第2型（JH）;C1、C2、BP和CDC属于第3型（GS）。结论：贾第虫分离株基因型的确定可分为本虫分子系统进货和分子流行病学研究提供重要资料。     

Role of Soluble Factors in Immune and Normal Sera in the Control of Giardiasis Due to Giardia Iamblia

Hyperimmune sera(HIS),raised against crude giardia antigen,on in vitro interaction,caused more agglutination of Giardia lamblia trophozoites.Heat inactivated HIS possessed a comparable agglutinating activity as the non-inactivated controls.Non-inactivated normal(unimmunized)serum caused immobilization of Giardia trophozoite,which was checked on heat inactivation.Antibodies in immune sera are mainly responsible for agglutination,whereas the heat labile non-immune components control the mobility of the intestinal parasite.     

缺氧对大鼠心肌微血管内皮细胞与中性粒细胞粘附的影响

研究缺氧对心肌微血管内皮细胞与中性粒细胞粘附的影响。方法：采用细胞培养技术观察体外培养的心肌微血管ＥＣ与未活化的ＰＭＮ粘附，高倍镜计数粘附的ＰＭＮ数；采用免疫组化ＡＢＣ法检测缺氧微血管ＥＣ上细胞间粘附分子的表达。结果：心肌微血管ＥＣ缺氧２和６ｈ与ＰＭＮ的粘附显著增加，分别为对照组的１．４９倍和１．９６倍；进一步用抗ＩＣＡＭ－１和抗淋巴细胞功能和相关抗原单克隆抗体阻断实验发现，两种抗体在５μｇ／ｍｌ     

Effects of CD11/CD18 mediated PMN-EC adhesion on the endothelial cell damages in the early stage postburn in vitro

Activatedpolymorphonuclearneutrophils(PMNs)havebeenshowntoproducedamagesonvascularendothelialcells(ECs)throughthegener-ationofoxygen-derivedfreeradicalsandthere-leaseofproteases;andPMN-ECadhesionisessen-tialtomediateECdamages['j.TheintegrinCD11/CD18consistsof3heterodimericsubunits,eachofwhichcomprisesadistinctAchain(CDl1a,CDl1bandCD1lc)thatisnoncovalentlyassociatedwitlhacommonBchain(CD18).TheimportanceoftheCD1l/CD18integrininPMNadhesionandemigrationinsystemicvasculaturehasbeencom-…     

不同间歇低氧暴露内皮细胞与多形核白细胞共培养下核转录因子KBp65表达水平的研究

目的 探讨不同间歇低氧暴露的内皮细胞和多形核白细胞（ PMN） 共培养后内皮细胞中核转录因子κB p65（ NF-κB p65） 的水平变化。方法 18 只雄性Wistar 大鼠随机分为常氧组和间歇低氧组（ IH 组） 。IH 组暴露于间歇低氧环境中, 氧浓度波动于5. 4% ～20. 7% , 低氧频率为30 次/h,8 h/d,6 周后处死大鼠, 腹主动脉取血, 分离纯化PMN。PMN分别与常氧暴露内皮细胞、IH 暴露内皮细胞（ 低氧频率12 次/h, 共4 h） 共培养4 h。细胞按暴露情况分为常氧内皮＋ 常氧PMN 组, IH 内皮＋常氧PMN组, 常氧内皮＋ IHPMN 组, IH 内皮＋ IHPMN 组。Western blotting 测定内皮细胞中NF-κB p65 浓度, GAPDH 为内参, 以NF-κB p65 /GAPDH 标准化NF-κB p65 的表达量。结果 IH 内皮＋常氧PMN组与常氧内皮＋IHPMN组NF-κB p65 表达量分别为2. 49 ±0. 39 和2. 14 ±0. 33, 两组间差异无统计学意义（ P 〉0. 05） , 但两组均较常氧内皮＋ 常氧PMN 组显著升高（ 0. 68 ±0. 20, P 〈0. 05） 。IH 内皮＋IHPMN组NF-κB p65 表达水平为4. 17 ±1. 48, 较其余三组显著升高（ P 〈 0. 05） 。结论 间歇低氧可分别致内皮细胞和PMN发生炎症反应, 而当两种细胞同时暴露于间歇低氧条件时炎症反应最为严重。上述两种细胞间的相互作用和引发的炎症反应可能是低氧内皮细胞损伤和睡眠呼吸暂停综合征并发心血管合并症的重要原因。     

The intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia

目的：探讨蓝氏贾第鞭毛虫（Giardia lamblia）磷酸丙糖异构酶基因种内差异。方法：提取虫体总DNA,对所有虫株磷酸丙糖异构酶（tim）基因部分片段进行PCR扩增。测定序列后,用简约法和NJ法构建系统树进行系统发育分析。结果：共有124个位点存在变异（占所有测定序列中的23％）,且大多数为发生在密码子的同义突变。两种构树方法所得二树的分枝结构相似,均将受试的16株蓝氏贾第虫分为明显的两组。结论：宿主及地理因素对蓝氏贾第虫群体的遗传多样性影响不大。在DNA分子进货水平上,自然选择的影响十分显。可将tim基因作为蓝氏贾第虫群体遗传结构一个十分有效的遗传标记。     

Changes in insulin sensitivity, secretion and glucose effectiveness during menstrual cycle

BACKGROUND: Several clinical conditions suggest an effect of sex steroids on glucose homeostasis in women. Studies examining this phenomenon have yielded controversial results. METHODS: To investigate the effect of the menstrual cycle on insulin sensitivity, glucose effectiveness and acute insulin response to glucose using the tolbutamide-modified intravenous glucose tolerance test (IVGTT) during the follicular (day 8 +/- 1) and luteal (day 23 +/- 1) phases of the menstrual cycle, the authors recruited 12 healthy regularly menstruating women. All had fasting glucose concentration of < 100 mg/dl [corrected] (89.7 +/- 6.2) with no family history of diabetes mellitus; their body mass indices were < 25 kg/m2 (22.41 +/- 1.44 kg/m2). RESULTS: The mean insulin sensitivity (Si) values decreased during the cycle. Insulin sensitivity (Si x 10(-4)/min.mU/ml) was higher in the follicular phase (5.03 +/- 0.72) and decreased in the luteal phase (2.22 +/- 0.45) (p < 0.001). Glucose effectiveness (Sg min-1) did not change as a function of the phase of the menstrual cycle. Sg estimates were 0.0229 +/- 0.00323 in the follicular phase, and 0.0225 +/- 0.00319 (p = NS) in the luteal phase, respectively. Acute insulin response (AIRg mU/ml) was 276.4 +/- 27.8 in the follicular phase. An adaptive increase (304.4 +/- 51.1) in response to the insulin resistance during the luteal phase was observed, but this increase was not statistically significant (p = NS). CONCLUSIONS: Knowledge of the variations in insulin sensitivity that occur during the normal menstrual cycle provides a basis of comparison for studies of other clinical conditions. Also, this phenomenon should be considered if the determination of insulin resistance is the purpose of certain epidemiological studies.     

烧伤病人痂下水肿液对中性粒细胞膜表面 CD11/CD18表达的影响

目的 了解烧伤病人痂下水肿液（STF）对中性粒细胞（PMN）活化及CD11/CD18表达的影响。方法 应用流式细胞术动态检测严重烧伤病人STF 刺激健康人PMN后PMN膜表面CD11a/CD18和CD11b/CD18的表达以及STF与PMN同孵育后培养液中髓过氧化物酶（MPO）变化。结果 健康人PMN在STF刺激1 h后，培养液中MPO活性即迅速升高并在24 h内保持此高水平，同时，PMN膜表面CD11a/CD18和CD11b/CD18也迅速升高达峰值并在24 h内保持此高水平。结论 STF可活化PMN并刺激PMN表达CD11a/CD18和CD11b/CD18，STF对烧伤后PMN致组织损害可能起重要作用。     

晚期糖基化终产物刺激内皮细胞分泌单核细胞趋化蛋白-1信号传导途径

目的　研究晚期糖基化终产物 (AGE)修饰蛋白对人内皮细胞分泌单核细胞趋化蛋白 1(MCP 1)的影响及其作用的信号传导途径。方法　将培养的人脐静脉内皮细胞 (HUVEC)和人内皮细胞株ECV30 4与不同浓度AGE修饰的人血清白蛋白 (AGE HSA)或牛血清白蛋白 (AGE BSA)共同培养。用Western印迹及酶联免疫吸附法 (ELISA)检测MCP 1蛋白合成及分泌 ,用逆转录PCR(RT PCR)法检测MCP 1mRNA表达 ,用流式细胞术观察细胞内氧化应激 ,用免疫沉淀 激酶活性测定法分析细胞p38丝裂素活化蛋白激酶 (p38 MAPK)活性。结果　AGE HSA和AGE BSA以时间和剂量依赖的方式上调内皮细胞MCP 1mRNA和蛋白的表达并诱导细胞氧化应激和活化p38 MAPK。 5 0 μg/mlAGE HSA与HUVEC共同培养 12h ,使细胞上清中的MCP 1浓度由 4 8 3pg/μg± 0 6pg/μg蛋白上升至 14 8 1pg/μg± 12 6pg/μg蛋白 (P <0 0 1)。5 0 μg/mlAGE HSA与HUVEC共同孵育 30min ,使p38 MAPK的磷酸化活性升高 91%± 14 % (P <0 0 1)。抗氧化剂或p38通路特异阻断剂SB 2 0 35 80能够阻断AGE修饰蛋白诱导的MCP 1表达。结论　AGE修饰蛋白能够通过p38 MAPK信号传导途径上调内皮细胞分泌MCP 1,这一作用是经氧化应激机制介导。     

Establishment of a C57BL/6N mouse model of giardiasis

建立人源蓝氏贾第鞭毛虫C57BL/ 6N小鼠动物感染模型。方法　用分离自河北和江苏两地蓝氏贾第鞭毛虫感染者的包囊 (河北株 ,CD和徐州株 ,XZ)分别感染两组C57BL/6N小鼠 ,收集受染鼠粪便 ,用蔗糖梯度离心法分离纯化包囊 ,计算排囊数量 ,分析排囊规律 ;观察小肠病理组织学改变。结果　C57BL/ 6N小鼠对此 2株蓝氏贾第鞭毛虫具有很高的易感性 ,接受具有活力的 1× 1 0 4 个包囊 /只的 3 6只小鼠 ,均获得感染 ,其中呈间歇性排囊者为 3 2只 ( 88 9% ) ,连续性排囊者 4只 ( 1 1 1 % )。排囊潜伏期为接种后第3d ,高峰期为第 6d。 2小时所排粪便含包囊数最高可达 2 3× 1 0 7个 /g粪。小肠粘膜出现表面分泌物增加 ,间质充血、水肿 ,炎性细胞浸润 ,粘膜坏死脱落等病理变化。结论　C57BL/ 6N小鼠可作为建立蓝氏贾第鞭毛虫感染的良好动物模型