胆囊收缩素对内毒素性休克大鼠SOD,MDA及吞噬细胞发光的影响

以ＳＤ大鼠ＥＳ（内毒素休克，内毒素０．８ｍｇ／１００ｇｂ．ｗ．ｉｖ）为模型，观察了ＣＣＫ－８抗ＥＳ过程中平均动脉血压（ＭＡＰ）血液超氧化物歧化酶（ＳＯＤ）脂质过氧化产物二醛（ＭＤＡ）及吞噬细胞化学发光（ＰＣＬ）的变化，实验结果：ＣＣＫ－８可使ＥＳ大鼠ＭＡＰ回升，血浆ＳＯＤ活性升高（Ｐ〈０．０１），ＭＤＡ含量减少（Ｐ〈０．０５），吞噬细胞本底发光增强，峰值增高，峰时缩短（Ｐ〈０．０５），结果表明ＣＣ     

22种抗生素对醋酸钙不动杆菌抑菌浓度的研究

用ＡＭＳ法测定了２２种抗生素对１０２株自临床标本分离的醋酸钙不动杆菌的抑菌浓度，结果显示ＣＩＰ的抑菌浓度为０．７６ｍｇ／Ｌ，ＴＯＢ、ＧＥＮ、ＩＭＩ、ＴＥＴ、ＡＭＩ和ＣＦＴ的抑菌浓度为１．７７～９．５４ｍｇ／Ｌ，ＣＦＺ、ＡＭＰ、ＣＦＳ、ＣＦＵ、ＴＩＡ和ＡＺＴ的抑菌浓度为１０．５０～１９．６２ｍｇ／Ｌ，ＰＩＰ、ＣＦＸ、ＴＩＣ、ＣＦＡ、ＣＥＰ、ＭＥＺ、ＣＦＰ和ＣＡＲ的抑菌浓度为２１．４４～３７．９３ｍｇ／Ｌ，ＮＩＴ的抑菌浓度为１８７．２８ｍｇ／Ｌ。在痰液、伤口及其它标本分离的醋酸钙不动杆菌的抑菌浓度测定结果中，多种抗生素的几何均数有显著性差异（Ｐ＜０．０５～０．００１）。     

大鼠心房特殊颗粒Ca^2＋—ATP酶特性的研究

本实验对心房特殊颗粒（ＡＳＧ）膜上Ｃａ＾２＋－ＡＴＰ酶的特性进行研究，并与肌浆网（ＳＲ）膜上的Ｃａ＾２＋－ＡＴＰ酶特性进行比较。在１０ｍｍｏｌ／Ｌ Ｃａ＾２＋溶液中，二者膜上均显示酶活性，酶活性在１ｍｍｏｌ／Ｌ Ｃａ＾２＋孵育液中均减弱，并在Ｃａ＾２＋浓度降至０．１ｍｍｏｌ／Ｌ时酶反应均不显示。在加入０．１ｍｍｏｌ／Ｌ槲皮黄酮后，ＳＲ和ＡＳＧ膜上的酶活性同时被抑制；当加入０．１ｍｍｏｌ／Ｌ寡霉素后     

中国南方汉族人群HLA—DQA1位点多态性与系统性红斑狼疮易感…

应用多聚酶链反应／序列特异寡核苷酸探针杂交（ＰＣＲ／ＳＳＯＰＨ）方法，探讨了我国东南沿海汉族人群ＨＬＡ－ＤＱＡ１等位基因多态性与系统性红斑狼疮（ＳＬＥ）的易感性关系，对５０例ＳＬＥ患者和６７例健康人对照血样本分析，表明ＳＬＥ患者具有显著高的ＤＱＡ１＊０１０２等位基因频率，ＯＲ＝４６．０４，Ｐ＜０．００１，可能是一易感等位基因，而ＤＱＡ１＊０５０１在病例与对照中呈相反结果，ＯＲ＝０．０４２，Ｐ＝０。     

一氧化氮在培养的大鼠心肌细胞缺氧—复氧损伤中的作用

目的：观察一氧化氮（ＮＯ）对心肌细胞缺氧－复氧损伤（ＨＲＩ）的作用。方法：培养的大鼠心肌细胞，培养液中分别预先加入ＮＯ前体Ｌ－精氨酸（Ｌ－Ａｒｇ）、ＮＯ供体ＳＩＮ－１或硝普钠（ＳＮＰ）、ＮＯＳ抑制剂Ｌ－ＮＮＡ或ＮＯＳ诱导剂脂多糖（ＬＰＳ），经缺氧１２０ｍｉｎ，复氧６０ｍｉｎ处理后，检测细胞存活率，乳酸脱氢酶（ＬＤＨ）漏出量，亚硝酸盐（ＮＯ２）含量及细胞诱导型ＮＯ合酶（ｉＮＯＳ）活性等指标的改变。结果：①与常氧组比较，缺氧－复氧ＨＲ降低细胞存活率（２３％，Ｐ＜０．０１），增加ＬＤＨ漏出（６２倍，Ｐ＜０．０１），ｉＮＯＳ活性（７７％，Ｐ＜０．０１），ＮＯ２含量（６１７％，Ｐ＜０．０１）。②ＨＲ前预先加入ＳＩＮ－１、ＳＮＰ或Ｌ－Ａｒｇ，均引起ＬＤＨ漏出进一步增高（Ｐ＜０．０１），细胞存活率进一步降低（Ｐ＜０．０１或Ｐ＜０．０５）。③Ｌ－ＮＮＡ２ｍｍｏｌ／Ｌ，单独应用对细胞损伤的影响无统计学意义，与Ｌ－Ａｒｇ联合应用，则减弱Ｌ－Ａｒｇ的细胞损伤作用。④ＬＰＳ１μｇ／ｍＬ，增加ｉＮＯＳ活性（２６倍，Ｐ＜０．０１）和ＬＤＨ漏出（５６％，Ｐ＜０．０１）。结论：ＮＯ加重心肌细胞ＨＲＩ，是心肌细胞ＨＲＩ的损伤因子     

rr－烯醇化酶单克隆抗体双位点夹心ELISA的建立和临床应用

在自制的８株烯醇化酶（ＮＳＥ）ＭｃＡｂ中，经方阵排列滴定筛选出无交叉反应且效价较高的２株ＭｃＡｂ，用辣根过氧化物酶（ＨＲＰ）为标记物，建立了ＮＳＥ－ＭｃＡｂ双位点夹心ＥＬＩＳＡ。经直线回归方程、取代试验、阻断试验检验，该标准曲线近似直线关系，可测定标本中ＮＳＥ的范围为５～５０μｇ／Ｌ。对２３例小细胞肺癌（ＳＣＬＣ）（Ⅰ和Ⅱ期４例、Ⅲ期７例、Ⅳ期１２例）．３２例非小细胞肺癌（ＮＳＣＬＣ），２２例肺良性疾病（ＢＰＤ）和３６例健康体检者血清ＮＳＥ进行了测定，ＳＣＬＣ的阳性率为８２．６％（１９／２３），其中Ⅰ和Ⅱ期为５０％（２／４）；Ⅲ期为８５．７％（６／７），Ⅳ期为９１．７％（１１／１２）；ＮＳＣＬＣ的阳性率为３．１％（１／３２），ＢＰＤ阳性率为４．５％（１／２２）。ＳＣＬＣ的显著高于ＮＳＣＬＣ和ＢＰＤ（Ｐ＜０．０１）；ＳＣＬＣ的Ⅳ和Ⅲ期显著高于Ⅰ和Ⅱ期（Ｐ＜０．０１）。２３例ＳＣＬＣ经综合治疗后，完全缓解组和部份缓解组血清ＮＳＥ水平非常显著低于进展组和无改变组（Ｐ＜０．０１）。本文研究结果证明，ＮＳＥＭｃＡｂ双位点夹心ＥＬＩＳＡ具有灵敏、特异和简便快速的特点，对ＳＣＬＣ的血清学诊断有较高的灵敏度和特异性，对ＳＣ     

抗心磷脂抗体与神经精神性狼疮

用酶联免疫吸附试验检测神经精神性狼疮（ＮＰＬＥ）。非ＮＰＬＥ狼疮患者，ＳＬＥ脑血管疾病患者及正常对照者血清及脑脊液中抗心磷脂抗体（ＡＣＬ抗体）。结果提示ＮＰＬＥ组ＡＣＬ抗体阳性率在血清中为９０．０％，脑脊液为３３．０％，非ＮＰＬＥ狼疮组ＡＣＬ阳性率在血清中为４４．０％，脑脊液为８．３％，非ＳＬＥ脑血管疾病组ＡＣＬ阳性率在血清中为５０．０％，脑脊液为４０．０％，正常人组ＡＣＬ阳性率在血清及脑脊液中均     

丹参酮Ⅱ—A对豚鼠单个心室肌细胞跨膜电位及L—型钙电流的影响

本实验采用膜片钳全细胞记录方法，观察了丹参酮Ⅱ－Ａ（ＤＳＴ）对豚鼠单个心室肌细胞跨膜电位及Ｌ－型钙电流以的影响，用蛋白酶（０．２ｍｇ／ｍｌ０循环消化的方法获得了７０～８０％耐钙心肌细胞，实验表明，ＤＳＴ能显著缩短豚鼠心肌单细胞的动作电位时程（ＡＰＤ），呈剂量依赖关系（ｒ＝－０．９９）至静息电位（ＲＰ）无何影响，ＤＳＴ１０μｍｏｌ．Ｌ＾－１对超射（ＯＳ）和动作电位幅度（ＡＰＡ）无显著影响，ＤＳＴ２０     

冠心病患者血浆氧化修饰低密度脂蛋白与血清铁蛋白关系的研究

对５３例冠心病患者及３０例正常人血浆氧化修饰低密度脂蛋白（ＯＸ－ＬＤＬ）与血清铁蛋白（ＳＦ）进行了测定，结果显示：冠心病患者血浆ＯＸ－ＬＤＬ和ＳＦ水平平均显著高于正常对照组（Ｐ〈０．００１和Ｐ〈０．０５），且两者呈显著正相关（ｒ＝０．４８５，Ｐ〈０．００１），冠心病患者血浆ＯＸ－ＬＤＬ水平升高可能与体内铁贮存增加有关。     

IL－10mRNA在SLE患者外周血单个核细胞上的表达

采用随机引物标记法将ＩＬ－１０ｃＤＮＡ标记地高辛甙元（Ｄｉｇｏｘｉｇｅｎｉｎ，ＤＩＧ）作为探针，用点杂交检测正常人和ＳＬＥ患者ＰＢＭＣ表达ＩＬ－１０ｍＲＮＡ量，经图象分析和计算机处理发现ＳＬＥ患者ＩＬ－１０ｍＲＮＡ表达量明显高于正常对照组（０．３３５５±０．０９２／０．１３６２±０．０５３５；Ｐ＜０．００１）。同时我们用ＰＨＡ和ＬＰＳ体外激活正常人ＰＢＭＣ，发现激活的ＰＢＭＣ表达ＩＬ－１０ｍＲＮＡ的量显著高于未经激活的对照组（０．４３８２±０．０７０７／０．１３６２±０．０５３５；Ｐ＜０．００１）。由此推论ＳＬＥ患者体内单个核细胞在体内已被激活，且通过产生ＩＬ－１０来调节患者免疫应答，这一作用可能与ＳＬＥ患者病情的发展有一定的关系。     

High sensitivity pyrogen testing in water and dialysis solutions

BACKGROUND: The dialysis patient is confronted with hundreds of litres of dialysis solution per week, which pass the natural protective barriers of the body and are brought into contact with the tissue directly in the case of peritoneal dialysis or indirectly in the case of renal dialysis (hemodialysis). The components can be tested for living specimens or dead pyrogenic (fever-inducing) contaminations. The former is usually detected by cultivation and the latter by the endotoxin-specific Limulus Amoebocyte Lysate Assay (LAL). However, the LAL assay does not reflect the response of the human immune system to the wide variety of possible pyrogenic contaminations in dialysis fluids. Furthermore, the test is limited in its sensitivity to detect extremely low concentrations of pyrogens, which in their sum result in chronic pathologies in dialysis patients. The In vitro Pyrogen Test (IPT) employs human whole blood to detect the spectrum of pyrogens to which humans respond by measuring the release of the endogenous fever mediator interleukin-1beta. Spike recovery checks exclude interference. The test has been validated in an international study for pyrogen detection in injectable solutions. METHODS: In this study we adapted the IPT to the testing of dialysis solutions. RESULTS: Preincubation of 50 ml spiked samples with albumin-coated microspheres enhanced the sensitivity of the assay to detect contaminations down to 0.1 pg/ml LPS or 0.001 EU/ml in water or saline and allowed pyrogen detection in dialysis concentrates or final working solutions. CONCLUSIONS: This method offers high sensitivity detection of human-relevant pyrogens in dialysis solutions and components.     

Rapid detection of Gram-negative bacteriuria by Limulus amoebocyte lysate assay.

The Limulus amoebocyte lysate (LAL) test was evaluated for rapid detection of gram-negative bacteriuria in an adult patient population. Time to gelation of a standard LAL preparation was used as a measure of significant (greater than 10(5) bacteria per ml) gram-negative bacteriuria, and the results of 190 LAL assays were compared with quantitative urine cultures. Initially, 33 of 36 urine specimens containing greater than 10(5) gram-negative bacteria per ml were detected by LAL assay. The three false-negative LAL tests were the result of urine pH levels below the pH minimum for LAL gelation; neutralization of these urine specimens resulted in positive LAL assays and 100% correlation with culture results. All 36 bacteriuric urine specimens were LAL positive within 15 min, with the majority of assays (86.1%) being positive after only 10 min of incubation at 37 degrees C. These data compared favorably with gelation times of 15 min when 1 X 10(5) to 2 X 10(5) gram-negative bacteria per ml were added to sterile urine. Two urine samples obtained from male patients with culture-proven gonococcal urethritis yielded positive LAL assays. The LAL assay was shown to correctly differentiate 96.2% of urine specimens as containing less than 10(5) or greater than 10(5) gram-negative bacteria per ml. The results of this study have shown that the LAL test can be used as a rapid, simple, and reliable screening procedure for the diagnosis of clinically significant gram-negative bacteriuria.     

Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions. In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans.     

The detection of endotoxin by in vitro production of endogenous pyrogen: Comparison with limulus amebocyte lysate gelation

The sensitivities of leukocyte endogenous pyrogen (EP) production and limulus amebocyte lysate (LAL) gelation to endotoxin from E. coli (minimum i.v. pyrogenic dose 4 ng/kg in rabbits) were determined. Concentrations of 0.5–1.0 ng/ml could be detected by LAL. The minimum endotoxin concentration which generated detectable EP from 2 × 10⁶ monocytes was 10-fold lower (0.05–0.1 ng/ml). At an endotoxin concentration of 0.4 ng/ml the minimum number of monocytes required for detectable EP production was 5 × 10⁵. It is concluded that the LAL gelation test cannot safely be used to exclude significant endotoxin contamination in a cellular system where EP production is being measured. The same conclusion applies even more forcibly to the in vitro production of lymphocyte activating factor (LAF, interleukin-1), since it appears that LAF and EP are identical and sub-pyrogenic amounts of EP are easily detectable in the LAF assay.     

Inflammatory response to pyrogens determined by a novel ELISA method using human whole blood

Presence of pyrogens on implants, medical devices, drugs and biological materials compromise on the biosafety and poses a major health hazard in therapeutics. Detection of pyrogenic contamination has so far been done with either in vivo rabbit pyrogen assay or Limulus Amoebocyte Lysate (LAL) methods, each of which having their distinct advantages and disadvantages. An indigenously developed ELISA method quantifying the pro-inflammatory response triggered by pyrogens on human whole blood is demonstrated for its versatility to detect the pyrogenic response to gram-negative, gram-positive bacteria, chemical and biological pyrogens. The method was used to test and quantitate the pyrogen levels in polymeric biomaterials. Unlike the existing pyrogen test procedures, this assay is adapted to detect all pyrogens, besides yielding faster, sensitive and quantifiable data, thereby reduce/replace animal experimentation. The method also provided insight into the possible correlation between variable blood profile among individuals and their role in determining inflammatory response to different pyrogenic stimuli.     

New microassay for quantitation of endotoxin using Limulus amebocyte lysate combined with enzyme-linked immunosorbent assay.

A combined method of the Limulus amebocyte lysate (LAL) test and the enzyme-linked immunosorbent assay for quantitation of endotoxin was developed based on our observation that the antigenicity of coagulogen, a major protein in LAL, was lost when LAL reacted with endotoxin as shown by immunoblotting. Determination of the residual coagulogen by an enzyme-linked immunosorbent assay system with monoclonal antibody against coagulogen revealed that the loss of the antigenicity of coagulogen was proportional to the concentration of endotoxin. An inverse linear curve was established between the endotoxin concentration and absorbance. Standard curves of the LAL enzyme-linked immunosorbent assay with different detection limits (from 0.1 to 100 pg of the control standard endotoxin per ml) were obtained from one batch of commercial LAL by adjusting incubation time and dilution of LAL. The reaction curves of various endotoxins were parallel to one another, whereas the kinetics differed from that of (1-3)-beta-D-glucan. The LAL enzyme-linked immunosorbent assay is a highly reproducible microassay, using only 10 microliter of test sample and LAL reagent; because the color and turbidity of plasma samples do not interfere with the assay, it is well suited for quantitation of endotoxins in clinical specimens.     

Endotoxin contamination in wound dressings made of natural biomaterials

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.     

Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.     

International validation of pyrogen tests based on cryopreserved human primary blood cells

Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.     

Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.