八色流式细胞术检测人外周血BCG特异性效应型记忆CD4+T细胞的表型特征

目的:检测BCG刺激后,PPD+正常人外周血中细胞因子产生及其亚群.方法:分离PPD+正常人外周血单个核细胞(PBMC),BCG刺激后检测CD4+和CD8+T细胞细胞因子分泌,并用八色流式细胞术分析BCG特异性T细胞亚群.结果:BCG刺激PBMC后,主要是CD4+T细胞分泌Th1细胞因子(IFN-γ、IL-2和TNF-α),而CD8+T细胞几乎不产生细胞因子.进一步分析分泌细胞因子的细胞亚群,主要是CD4+CD45RO+ CD62L(-)CD27(-)和CD4+ CD45RO+ CD62L(-)CD27+分泌细胞因子.结论:BCG刺激PPD+正常人外周血PBMC后,主要诱导CD4+T细胞分泌细胞因子,且该细胞表现出CD4+CD45RO+ CD62L(-)效应型记忆细胞特征,可能在预防结核感染中发挥重要作用.     

卡介苗刺激后CD8~+T细胞活化、增殖及其调节

目的:探讨卡介苗(BCG)刺激后,结核菌素试验阳性(PPD+)正常人外周血单个核细胞(PBMCs)中CD8+T细胞的活化、增殖、细胞因子产生及调节性T细胞(Treg)对其调节作用。方法:体外用BCG刺激PPD+正常人PBMCs,检测CD8+T细胞的细胞因子产生、活化和增殖。纯化后获得调节性T细胞(Treg)和CD25-细胞,检测Treg对CD8+T细胞增殖的调节作用。结果:BCG诱导CD8+T细胞表达CD69和CD25等活化分子。在低剂量IL-2存在的条件下,BCG诱导CD8+T细胞发生增殖,且增殖的CD8+T细胞大部分表达Granzyme-B。体外BCG短期刺激PBMCs后,CD8+T细胞几乎不产生IFN-γ、IL-2和TNF-α。此外,调节性T细胞抑制CD8+T细胞增殖。结论:BCG诱导CD8+T细胞活化、增殖和表达颗粒酶,Treg抑制CD8+T细胞增殖。     

结核胸液中ESAT-6多肽特异性多功能CD4+T细胞数量及功能分析

目的:检测ESAT-6多肽刺激后,结核性胸膜炎患者胸液细胞中CD4+T细胞细胞因子产生及多功能CD4+T细胞频率,了解ESAT-6特异性CD4+T细胞在结核局部细胞免疫应答中的作用.方法:分离结核性胸膜炎患者胸液细胞(PFCs),ESAT-6混合多肽刺激后检测CD4+T细胞细胞因子分泌、细胞亚群、多功能性CD4+T细胞频率及细胞因子平均荧光强度.结果:BCG、ESAT-6混合多肽和ESAT-6组蛋白刺激PFCs后,主要是CD4+T细胞分泌Th1细胞因子(IFN-γ、IL-2和TNF-α),而CD8+T细胞几乎不产生细胞因子.进一步分析ESAT-6混合多肽刺激PFCs后Th1细胞的亚群组成,依据细胞因子分泌的类型及数量,该特异性Th1细胞可以分为7个不同亚群,且各亚群所占比例不同,其中多功能性T细胞亚群(同时分泌IFN-γ、IL-2和TNF-α)比例较高.细胞因子荧光强度分析表明,依据细胞因子分泌类型的增加,单个细胞水平上不同Th1亚群中各细胞因子表达的量也逐渐增加,即3+>2+>1+细胞.结论:ESAT-6混合多肽刺激结核性胸膜炎患者胸液细胞后,主要诱导CD4+T细胞分泌细胞因子,且Th1亚群中包含多功能性CD4+T细胞,该细胞在单细胞水平上分泌细胞因子的类型和数量也显著高于其他亚群.可能在结核局部感染中发挥关键保护性作用.     

结核分枝杆菌H37Ra诱导核苷酸结合寡聚结构域2基因敲除(NOD2~(-/-))小鼠的1型辅助T(Th1)细胞型免疫应答

目的探讨1型辅助T(Th1)细胞的细胞因子肿瘤坏死因子α(TNF-α)和γ干扰素(IFN-γ)及多功能T细胞在感染结核分枝杆菌H37Ra的核苷酸结合寡聚结构域2基因敲除(NOD2~(-/-))小鼠中的作用。方法 NOD2~(-/-)小鼠与野生型C57BL/6小鼠气管分别滴入MTB减毒株H37Ra建立肺部感染模型并设立生理盐水对照组,每组10只。4周后,取肺组织进行HE染色及病理评分;ELISA检测肺组织匀浆中TNF-α和IFN-γ含量;流式细胞术检测脾细胞中多功能CD4~+ T/CD8~+ T细胞的比例。结果 NOD2~(-/-)小鼠感染H37Ra后肺组织炎症加重,肺组织TNF-α和IFN-γ含量增加。与生理盐水组相比,两种小鼠感染后, CD4~+/CD8~+T细胞中TNF-α~+、 IFN-γ~+单阳性细胞及TNF-α~+IFN-γ~+细胞均显著增加;与C57BL/6小鼠感染组相比, NOD2~(-/-)小鼠感染组体内TNF-α~+ CD4~+ T细胞、 IFN-γ~+ CD4~+T细胞及IFN-γ~+ CD8~+ T细胞显著增加。结论 H37Ra可诱导NOD2~(-/-)小鼠的Th1细胞型免疫应答反应。     

IL-12恢复化疗药物对人细胞免疫功能的抑制作用

目的探讨IL-12是否能够恢复化疗药物对人抗原特异性和非特异性细胞的免疫抑制功能及其机制。方法人PBMCs在抗CD3和抗CD28共刺激条件下,加或不加化疗药物和IL-12,不同的方法检测细胞因子IFN-γ和TNF-α的产生及其mRNA的表达。利用流式细胞术或Western blotting检测T细胞不同亚群细胞因子IFN-γ和转录因子T-bet、p-STAT-1、p-STAT-4的表达。利用卡介苗(BCG)刺激PPD+PBMCs,检测化疗药物及IL-12对抗原特异性细胞IFN-γ和TNF-α的表达影响。结果化疗药物对人IFN-γ和TNF-α的产生有显著的剂量依赖性抑制作用,同时IL-12对其抑制作用的恢复也呈显著的剂量依赖性;BCG刺激PPD+PBMCs实验证明,化疗药物抑制抗原特异性细胞产生IFN-γ和TNF-α,IL-12能够恢复其免疫抑制功能。进一步研究发现,化疗药物通过下调STAT-1和STAT-4的磷酸化以及T-bet的表达影响IFN-γ和TNF-α的产生,IL-12通过上调STAT-4的磷酸化恢复了免疫的抑制作用。结论化疗药物抑制人的细胞免疫应答,而IL-12通过上调STAT-4的磷酸化完全恢复其抑制作用,从而对肿瘤化疗病人重建免疫功能(包括抗感染和抑制癌细胞的复发)起到非常重要的作用。     

活动性结核病患者外周血单个核细胞中结核分枝杆菌抗原特异性多能CD4+和CD8+T淋巴细胞的特征研究

目的 研究活动性结核病患者外周血单个核细胞中结核分枝杆菌抗原特异性多能T淋巴细胞细胞因子的分泌特征.方法 利用γ干扰素释放试验和多色流式细胞术分析了13例活动性结核病患者、11例肺部感染性/肿瘤疾病患者以及14例健康对照者外周血中结核分枝杆菌抗原特异性(ESAT-6和CFP-10)CD4+Th1和CD8+Tc淋巴细胞表达细胞因子IFN-γ、TNF-α和IL-2的情况.结果 与肺部感染性/肿瘤疾病组和健康对照组相比:(1)活动性结核病组具有较低比例的分泌TNF-α+的CD4+Th1细胞、较高比例的分泌IFN-γ+和IFN-γ+TNF-α+IL-2+的CD4+Th1细胞;(2)活动性结核病组具有较高比例的分泌IFN-γ+TNF-α+IL-2+的CD8+Tc细胞.结论 实验结果提示活动性结核病患者中表达IFN-γ+TNF-α+IL-2+的多能CD4+Th1及CD8+Tc细胞,可能在区别活动性结核病与肺部感染性/肿瘤疾病方面具有一定的临床参考价值.     

慢性乙肝患者树突状细胞对Th1/Th2分化的影响

探讨慢性乙肝患者树突状细胞(dendritic cells,DC)对CD4+Th细胞亚群分化的影响。分离慢性乙肝患者外周血单个核细胞(PBMC),以rhIL-4(50 ng/ml)、rhGM-CSF(10 ng/ml)和rhTNF-α(100 u/ml)诱导培养DC。以流式细胞仪检测DC表面CD1a、CD83、CD80、CD86、HLA-DR分子表达情况。MTT法检测DC刺激同种异体淋巴细胞增殖能力。免疫磁珠分离外周血CD4+T细胞亚群,PMA+Ionomycin刺激后胞内荧光染色,流式细胞仪检测辅助性T细胞(helper T cell,Th)内特征性细胞因子IFN-γ/IL-4以判断Th1/Th2分化。ELISA法检测DC或Th细胞培养上清中IL-6、IL-12、IFN-γ和IL-4的含量。结果:慢性乙肝患者的DC表达CD1a、CD83、CD80、CD86、HLA-DR分子水平明显低于正常人(P<0.01);培养至第7天,慢性乙肝患者DC分泌的IL-12水平低于正常人(P<0.01),而分泌的IL-6水平增高(P<0.05)。与正常人相比,慢性乙肝患者外周血中Th1细胞占CD4+T细胞的百分比较低(P<0.01),其Th细胞培养上清中IFN-γ的量也较低(P<0.01)。患者DC与同种异体的健康人Th细胞共培养,刺激Th1型细胞因子IFN-γ产生的能力低于正常人(P<0.01)。慢性乙肝患者体内DC功能的异常可能导致了外周血Th1细胞分化不足。     

Ag85A DNA疫苗加强免疫显著提高卡介苗初免小鼠的抗结核T细胞免疫应答

目的 构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法 以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗;接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多因子胞内流式细胞术(intracellular staining)分析免疫小鼠的Mtb抗原特异性效应细胞免疫水平与分泌IFN-γ/TNF-α/IL-2的多功能CD4+T细胞频率及其强度以及CD8+T细胞免疫应答.结果 与BCG免疫及DNA单独免疫组相比,Ag85A DNA加强免疫不仅能显著提高小鼠IFN-γ+TNF-α+IL-2+多功能T细胞,IFN-γ+IL-2+、IL-2+TNF-α+双功能T细胞与IL-2+单功能T细胞的频率以及IL-2的分泌能力,还能显著诱导小鼠产生更多分泌IFN-γ和IL-2的CD8+T细胞.结论 本研究成功构建了表达Mtb免疫优势抗原Ag85A的DNA疫苗并分析了其免疫原性,证实了BCG初免-DNA加强的免疫策略可同时显著增强实验小鼠的Mtb抗原特异性CD4+T和CD8+T细胞应答水平,有利于提高BCG的免疫原性,为增强BCG逐渐下降的抗结核保护效果提供新思路.     

结核性胸液T细胞组成和细胞因子表达的探讨

目的:探讨结核性胸液中CD3+ T、CD3+ CD4+ T、CD3+ CD8+ T细胞和CD4+/CD8+比值及细胞因子表达。方法:分离外周血单个核细胞和胸液细胞,流式细胞仪检测CD3+、CD3+ CD4+、CD3+ CD8+和CD4/CD8比值。ELISA法检测胸液上清中IFN-γ、IL-2和TNF-α的水平。流式细胞仪检测CD3+ CD4+和CD3+ CD8+ T细胞Th1细胞因子IL-2、TNF-α、IFN-γ的产生。结果:与外周血相比,胸液细胞CD3+ CD4+ T和CD3+ T细胞比例显著增加。ELISA结果表明,胸液上清中存在大量Th1型细胞因子。流式细胞仪分析结果表明,IL-2、TNF-α和IFN-γ主要由CD3+ CD4+ T细胞产生,而CD3+CD8+T细胞较少或不分泌细胞因子。结论:结核病人胸液中CD3+和CD3+ CD4+ T细胞比例增加,胸液中存在大量Th1细胞因子,且主要来源于CD3+ CD4+ T细胞亚群,可能在抗结核免疫中发挥重要作用。     

儿童癫痫患者T淋巴细胞活化并产生促炎因子

目的检测儿童癫痫患者外周T淋巴细胞活化状态以及细胞因子水平变化。方法选取20名儿童癫痫患者和19名健康对照儿童,采集外周血,分离外周血单个核细胞(PBMC),用细胞膜表面标记抗体流式细胞术检测T淋巴细胞表面共刺激分子CD69、CD25和细胞毒性T淋巴细胞相关蛋白4(CTLA4)的表达,用细胞内因子染色结合流式细胞术检测T细胞细胞因子γ干扰素(IFN-γ)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和IL-17A的表达,利用细胞内因子染色结合流式细胞术检测调节性T淋巴细胞(Treg)表达IL-10的情况。结果与对照组相比,儿童癫痫患者外周血中T淋巴细胞表面高表达共刺激分子CD69、CD25和CTLA4,活化的CD4+T细胞高表达IFN-γ、TNF-α、IL-6和IL-17 A。在儿童癫痫患者高表达IL-10的Treg数目增加。结论儿童癫痫患者外周T淋巴细胞活化并产生细胞因子。     

Development in in vitro toxicology

There are three principal pressures driving the development of in vitro toxicology: (1) the need for more efficient testing systems to cope with the large number of xenobiotics currently being developed; (2) public pressure to reduce animal experimentation; and (3) a need for a better understanding of the mechanisms of toxicity. Within this, in vitro toxicology is focused on local, systemic, and target-organ toxicity. It is becoming increasingly apparent that a step or decision-tree approach using input of a variety of experimental data (physicochemical properties, biokinetics, cytotoxicity) provides the most efficient system for predicting toxicity. Examples of the use of in vitro toxicity systems for prediction of systemic toxicity and target-organ (liver) toxicity are presented.Originally presented at ECCP 93.     

Seasonal variations in acute toxoplasmosis in pregnant women in Slovenia

Between December 1999 and December 2004, 40 081 pregnant women were examined for toxoplasmosis with Toxo-IgG, Toxo-IgM enzyme immunoassay. Women with positive results were then retested with the Toxo-IgG avidity assay for recent toxoplasmosis. Recent acute toxoplasmosis in pregnant women was found to be significantly more frequent (p < 0.01) during winter than summer. The incidence of acute toxoplasmosis during winter-spring was also significantly more frequent (p < 0.025) than summer-autumn. This phenomenon should be taken into account when formulating preventive measures for toxoplasmosis, especially for pregnant women.     

Age-related changes in polyamines in memory-associated brain structures in rats

Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines and have important roles in maintaining normal cellular function, regulating neurotransmitter receptors and modulating learning and memory. Recent evidence suggests a role of putrescine in hippocampal neurogenesis, that is significantly impaired during aging. The present study measured the polyamine levels in memory-related brain structures in 24- (aged), 12- (middle-aged) and 4- (young) month-old rats using liquid chromatography/mass spectrometry and high performance liquid chromatography. In the hippocampus, the putrescine levels were significantly decreased in the CA1 and dentate gyrus, and increased in the CA2/3 with age. Significant age-related increases in the spermidine levels were found in the CA1 and CA2/3. There was no difference between groups in spermine in any sub-regions examined. In the parahippocampal region, increased putrescine level with age was observed in the entorhinal cortex, and age did not alter the spermidine levels. The spermine level was significantly decreased in the perirhinal cortex and increased in the postrhinal cortex with age. In the prefrontal cortex, there was age-related decrease in putrescine, and the spermidine and spermine levels were significantly increased with age. This study, for the first time, demonstrates age-related region-specific changes in polyamines in memory-associated structures, suggesting that polyamine system dysfunction may potentially contribute to aged-related impairments in hippocampal neurogenesis and learning and memory.     

休克过程中肾上腺髓质素变化的研究进展

Adrenomedullin (AM) is a new peptidergic regulator of vascular function. AM serves as a hormone, which has many biological properties, plays an important role in the many pathophysiological processes, especially shock. This review will highlight the structure, biological properties of AM and the relationship between AM and shock.     

Secular change in menarche in women in Madrid

The age at menarche was estimated by recollection in 1617 women between the ages of 18 and 60 in Madrid and a nearby suburb, Pinto. The population of Pinto is working-class and the Madrid group, taken from residential neighbourhoods , belongs to the upper middle class. In both groups we found a diminution in average age at menarche, from 14.04 to 13.02 years in Madrid and from 14.55 to 13.16 years from about 1935 to about 1965 in Pinto. These changes have been more intense in the group which is less well-off economically, where living conditions have varied much more drastically.     

Pitfalls in TRAP assay in routine detection of malignancy in effusions

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.