抗LPS-IgY防治烧伤小鼠肠源性内毒素血症的实验研究

目的实验研究抗LPS-IgY对烧伤肠源性内毒素血症的作用。方法标准LPS免疫鸡,采用改良水溶法(WD)从蛋黄中提取特异性抗内毒素IgY。将纯系昆明种小鼠制成30%体表面积(TBSA)Ⅲ°烧伤合并内毒素血症的动物模型,喂服抗LPS-IgY并定期检测血中LPS、二胺氧化酶(DAO)、肿瘤坏死因子(TNF-α)等相关指标。结果喂服抗LPS-IgY后,能明显降低烧伤小鼠的死亡率;明显降低血浆中LPS、DAO和TNF-α的含量;回肠黏膜的损伤程度明显减轻。结论抗LPS-IgY有效拮抗LPS,减少其移位入血,减轻回肠黏膜的损害,对烧伤小鼠肠源性内毒素血症疗效较好。     

特异性抗内毒素鸡蛋黄抗体的制备

目的：制备特异抗内毒素鸡蛋黄免疫球蛋白（Egg yolk immunoglobulin，IgY），探索防治内毒素血症的新途径。方法：用大肠杆菌J5株、内毒素（Lipopolysaccharide，LPS）及类脂A(Lipid A)作抗原免疫25周龄Roman鸡，改良水溶法提取抗体，进行紫外分光光度计检测、SDS-PAGE及Western blot免疫印迹分析，通过酶联染色反应检测其免疫学活性。结果：大肠杆菌J5株、LPS及Lipid A抗体含量分别为11.4、9.2、9.3mg/mL蛋白黄液，质量分数分别为92.3％、87.13％、90.4％，分子质量为180000u，初步鉴定其对内毒素具有特异性结合作用。结论：用大肠杆菌J5株、LPS及LipidA免疫鸡制备的IgY效价高、特异性强、产量大，将是一种防治内毒素血症的新方法。     

特异抗内毒素鸡蛋黄免疫球蛋白效价测定及理化性质研究

目的：研究特异性抗内毒素鸡蛋黄免疫球蛋白理化性质，探索一种防治肠源性感染，治疗：内毒素血症的新途径。方法：用大肠杆菌J5株(菌体表面类脂A裸露)作抗原免疫25周龄Roman鸡，制备特异性抗内毒素IgY，用酶联免疫吸附技术检测其效价及对热、酸、碱和酶的稳定性。结果：IgY抗体效价为1：25 600，能耐受巴氏消毒，对酸、碱、酶有很好的稳定性。结论：用大肠杆菌J5株免疫鸡制备的IgY效价高，理化性质稳定，适合口服应用。     

抗菌抗内毒素多肽拮抗内毒素作用的体内研究

目的：观察抗菌抗内毒素多肽对内毒素(LPS)攻击大鼠的保护作用。方法：以一个LD50的LPS(10mg/kg^-1)攻击大鼠,于LPS攻击后20秒内静脉注射抗菌抗内毒素多肽(5mg/kg^-1),在不同时相点采血,并测定大鼠血液中的TNT-α、IL-6浓度,以确定抗菌抗内毒素多肽对LPS攻击大鼠的保护作用。结果：抗菌抗内毒素多肽可明显降低LPS攻击大鼠血液中TNF-α、IL-6浓度。     

抗菌抗内毒素多肽小鼠体内拮抗内毒素作用的研究

目的：观察抗菌抗内毒素多肽对内毒素(LPS)攻击小鼠死亡的保护作用。方法：以一个LD50的LPS(20mg/kg)静脉注射攻击小鼠,分别于LPS攻击后20S内静脉注射不同剂量的抗菌抗内毒素多肽;于LPS攻击前及攻击后不同时间内分别静脉注射5mg/kg和10mg／kg的抗菌抗内毒素多肽,以观察抗菌抗内毒素肽对LPS攻击所致小鼠死亡的保护作用、抗菌抗内毒素肽对LPS攻击小鼠死亡保护作用的时效关系及抗菌抗内毒素肽对LPS攻击小鼠死亡的预防作用。     

钌红对内毒素多种生物活性的拮抗作用

钌红对内毒素的鲎试剂凝集反应、局部Shwartzman反应、降低外周血白细胞计数和血清锌的含量等效应均有拮抗作用。电镜下可见经钌红处理过的内毒素呈无规律絮状凝聚,正常网状结构被破坏。用三硝基苯磺酸法证实钌红与内毒素结合,使其氨基含量大增。以上结果提示:钌红的多阳离子氨基可能是对内毒素多种生物活性起拮抗作用的有效结构。     

拮抗内毒素的研究进展

由创伤及感染引起的脓毒症是导致临床病人死亡的主要原因.脓毒症的本质是机体对侵入机体的病原微生物所产生的过度炎症性反应,通常而言,内毒素与免疫炎症细胞,特别是巨噬细胞上相应的受体结合,是启动机体产生防御反应及由此而形成脓毒性反应或脓毒症的关键.研究内毒素介导脓毒症的分子病理学机制及近年有关拮抗内毒素措施的研究进展很重要.     

甘氨酸对内毒素致热性的抗拮作用研究

本研究用新西兰家兔４２只，分为７组，观察不同剂量甘氨酸对内毒素致热性的灭活效果。     

抗内毒素Fab'的制备

目的 研究抗内毒素卵黄免疫球蛋白(IgY)的活性片断Fab',探讨防治内毒素血症的新途径.方法 用内毒素(LPS)作为抗原免疫25周龄德国罗曼鸡,改良水溶法提取抗内毒素IgY,胃蛋白酶切后提取Fab'片断,光密度法测抗内毒素Fab'的浓度和含量、ELISA检测抗内毒素Fab'效价、SDS-聚丙烯酰胺凝胶电泳检测其分子量及纯度. 结果 抗内毒素Fab'含量为4.2 mg/mL蛋黄液,效价为1∶51 200,纯度为92%,相对分子质量为44 000. 结论 抗内毒素Fab'产量大、效价高、特异性强.     

慢性重型肝炎患者血清抗内毒素IgG抗体动态检测分析

慢性重型肝炎患者血清抗内毒素ＩｇＧ抗体动态检测分析李跃旗，朱传琳，姚家珮，白文林，薛建亚慢性重型肝炎患者内毒素血症发生率较高，报道较多，对抗内毒素ＩｇＧ抗体的检测和其意义少见报道，为了了解慢性重型肝炎患者血清抗内毒素ＩｇＧ抗体的发生和变化情况，我们用...     

抗人层粘连蛋白Fab＇的制备

用ＤＥＡＥ纤维素ＤＥ－５２反相吸附纯化抗人层粘连蛋白（ＬＮ）ＩｇＧ，ＩｇＧ回收率达１１．６ｍｇ／ｍｌ血清。胃蛋白酶消化ＩｇＧ成Ｆ（ａｂ＇）２，再用１０ｍｍｏｌ／Ｌ２－巯基乙醇３７℃反应９０ｍｉｎ还原成Ｆａｂ＇，回收率约８１％，放免效价为１：３５００．测得纯化的ＩｇＧ、Ｆ（ａｂ＇）２及Ｆａｂ＇分子量分别为１５００００、９００００、４５０００，与理论值相近。放免检测中用Ｆａｂ＇取代抗血清可纠正对血清ＬＮ测定的误差，从而建立检测ＬＮ的准确、灵敏方法。     

The effects of endotoxins on gametes and preimplantation embryos cultured in vitro

Culture media used for human in-vitro fertilization (IVF) can be contaminated with bacterial endotoxins. All five tested types of commercially available albumin, sometimes used as a protein supplement to IVF media, were shown to contain endotoxins in varying concentrations. Endotoxins are suspected to cause embryo fragmentation and low pregnancy rates in human IVF. However, human sperm viability and the IVF of mouse oocytes and subsequent culture of the zygotes were shown to be unaffected by relatively high endotoxin concentrations. Therefore these techniques cannot be used as quality control assays to detect endotoxins in the IVF culture media.     

抗LPS mAb Fab片段cDNA的克隆及其在CHO细胞中的表达

目的 构建抗LPS单克隆抗体Fab段的真核表达载体,并在CHO细胞中表达。方法 以逆转录PCR方法扩增抗LPU mAb重链Fd和轻链cDNA,分别克隆入载体pGEM-Teasy。经测序分析后,再分别亚克隆入载体pcDNA3,构建成重组载体pcDNA3-Fd和pcDNA3-LC,并以PCR方法扩增pcDNA3-LC中包括CMV启动子、轻链序列和BGH Poly(A)在内的轻链表达序列。经适当的限制性内切酶酶切后,插入到质粒pcDNA3－Fd中Fd表达序列的前部,构建成表达载体pcDNA3-Fab,以脂质体包裹转染CHO细胞。经G418筛选,用ELISA、免疫荧光和Western blot鉴定阳性克隆。结果 用ELISA和免疫荧光法,筛选出4株高表达Fab的细胞株。Western blot显示,表达产物的Mr为50000。同时,ELISA结果显示,该Fab具有良好的结合LP5的活性。结论 成功地在CHO细胞中表达抗LPS mAb的Fab段,为进一步鉴定其抗LPS的作用,使之成为诊断和治疗革兰氏阴性细菌感染的工具奠定了基础。     

LPS inhalation challenge: a new tool to characterize the inflammatory response in humans

Inhaling bacterial endotoxin and its derivative LPS can induce a distinct inflammatory response, varying among hosts. Experimental LPS-inhalation is an established procedure in inflammation research. We evaluated experimental LPS-inhalation in 20 young healthy volunteers to determine the safety and the reproducibility of markers of inflammation and clinical findings (symptoms, lung function, exhalative NO, and body temperature). LPS was increased every 30 min up to cumulative 100 μg, the protocol was repeated after 2, 4, and 6 weeks. During 71 provocations, 13 episodes of clinical complaints were observed in 10 subjects. Those were a total of 11 local reactions (15.5%, e.g., cough), and six systemic reactions (8.5%, e.g., fatigue). All adverse events resolved spontaneously within 10 h. Changes of FEV₁ and eNO showed no significant differences between the four visits. In the majority of our subjects (88.2% on visit 1–3, 76.5% on visit 4), a rise in body temperature (>0.5°C) was recorded and normalised latest after 24 h. On the first and the last visit, serum concentrations of CrP and LBP increased significantly and correlated well with each other (r = 0.71; P < 0.001). LPS-challenge is a safe and tolerable tool to investigate inflammatory response in humans and could lead to better characterization of patients with chronic inflammatory disease. Richard Kitz and Markus A. Rose contributed equally and should therefore, be considered as first authors.     

交叉保护性抗LPS多克隆抗体的制备与LPS多表位模拟肽的筛选

目的 获得具交叉保护性的抗细菌脂多糖(LPS)多克隆抗体与模拟LPS多表位的噬菌体展示环肽克隆。方法制备具交叉反应性的兔抗鼠伤寒沙门菌抗血清，鉴定抗血清对大肠杆菌和铜绿假单胞菌攻击小鼠的交叉保护性。以亲和纯化的多克隆抗体为靶，亲和筛选噬菌体随机环七肽库。双夹心ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆。结果兔抗血清能与多种来源的LPS反应，对用大肠杆菌和铜绿假单胞菌攻击的小鼠有显著的保护性。用亲和层析纯化的多克隆抗体为靶分子进行3轮筛选，随机挑选46个克隆，其中20个克隆显示与多抗结合。鼠伤寒沙门菌LPS可抑制阳性噬菌体克隆与多抗结合，所有克隆的IC50(达到50％抑制率的LPS浓度)为125ng/ml。挑选其中10个克隆测序并推导氨基酸序列，其中5个克隆具X-QFYP-X-A保守序列；3个克隆具LFTFAHY序列；2个克隆具YQYYPAA序列，所有序列非极性氨基酸含量平均值为80．0％。结论获得能与多种LPS反应且具交叉保护作用的兔多克隆抗体，筛选得到可模拟鼠伤寒沙门菌LPS多表位噬菌体展示环七肽。     

The effects of Porphyromonas gingivalis LPS and Actinobacillus actinomycetemcomitans LPS on human dendritic cells in vitro, and in a mouse model in vivo

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.     

内毒素对内皮细胞促凝及抗凝功能的影响

革兰阴性杆菌所致重症感染常引起弥散性血管内凝血和多器官功能衰竭。其主要病理生理机制之一是内毒素直接或间接作用于内皮细胞，受损的内皮细胞释放多种促凝因子、趋化因子、调节因子、粘附因子和血小板活化因子等。在促进炎症反应、启动补体系统的同时，进一步加剧凝血系统的活化；天然抗凝系统，包括抗凝血酶、蛋白c和组织因子途径抑制物，则受到不同程度的抑制；纤溶系统虽然在一定程度上被活化，但不能抵御内毒素血症时促凝和抗凝系统的失衡，其结果促进了高凝状态的发生。     

脂多糖对人脐静脉血管内皮细胞的直接损伤作用

血管内皮细胞 (ｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＥＣ)炎症反应是感染性休克向不良方向演变的重要原因 ,有关激活剂 (ＬＰＳ、ＩＬ 1)导致ＥＣ活化的研究是败血症病理反应的中心课题。内毒素引起微循环ＥＣ损伤是内毒素性组织损伤的重要环节之一。本实验探讨了ＬＰＳ对体外培养脐静脉ＥＣ的直接损伤作用。1　材料与方法1 1　材料 :ＲＭＰＩ 16 40培养基、ＬＰＳ(Ｓｉｇｍａ产品 )1 2　细胞培养及鉴定 :内皮细胞传 4代。ＰＭＮ的分离采用Ｐｅｒｃｏｌｌ’ｓ分离液 5 0 0ｇ离心 2 0ｍｉｎ取白细胞层用培养基稀释成2× 10 6 ｍＬ备用。1 3　…     

Effect of various E. coli LPS chemotypes on apoptosis and activation of human neutrophils

We studied the effects of various Escherichia coli LPS chemotypes on the production of reactive oxygen species and regulation of apoptosis in human neutrophils. A correlation was found between the increase in chemiluminescence (Re-LPS<Ra-LPS<S-LPS) and lengthening of the polysaccharide chain in endotoxins. Shortening of the polysaccharide chain was associated with more pronounced inhibition of neutrophil apoptosis under the influence of endotoxins, which correlated with the increase in hydrophobicity of these chemotypes. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 136–138, August, 2006     

Bacterial lipopolysaccharide (LPS)-specific antibodies in commercial human immunoglobulin preparations: superior antibody content of an IgM-enriched product

The anti-LPS antibody content of commercial intravenous immunoglobulins was examined by quantitative ELISA using LPS preparations from Escherichia coli, Klebsiella and Pseudomonas aeruginosa O serotypes occurring most frequently in Gram-negative septicaemia. Three IgG products from different manufacturers and one IgM-enriched product were tested. Mean antibody levels were significantly higher in the IgM fraction of the IgM-enriched product compared with ‘pure’ IgG products, indicating that natural antibodies against bacterial LPS belong primarily to the IgM class. Immunoblotting studies showed that antibody specificities were directed mainly against O side chain epitopes. Antibodies against rough mutant LPS representing various chemotypes were detected in IgG but not in IgM products. The virtual absence of antibodies against Vibrio cholerae LPS indicated that human anti-LPS antibodies result from continuous environmental exposure to Gram-negative pathogens. These data support the further development of IgM-enriched preparations for prophylaxis and treatment of Gram-negative nosocomial infections.