结直肠癌患者外周血Vδ2 T细胞IL-17A的表达研究1

目的:比较结直肠癌患者与正常人外周血单个核细胞来源的Vδ2 T细胞CD27、IL-17A、IFN-γ和TNF-α的表达变化。方法:抽取试验对象外周血,分离单个核细胞,采用流式细胞术分析其中CD27+Vδ2 T细胞与CD27-Vδ2 T细胞的IL-17A、IFN-γ和TNF-α的分泌表达情况。结果:结直肠癌患者外周血Vδ2 T细胞的IL-17A表达明显高于正常人[(5.99±0.80)%比(3.48±0.57)%,P=0.01;]IFN-γ与TNF-α的分泌表达具有明显正相关性(P0.0001)。结直肠癌患者和正常人CD27+Vδ2 T细胞的IL-17A表达均较CD27-Vδ2 T细胞低,而CD27+Vδ2 T细胞的IFN-γ和TNF-α分泌表达则高于CD27-Vδ2 T细胞。结论:结直肠癌患者Vδ2 T细胞的IL-17A表达是升高的,而IFN-γ与TNF-α的分泌表达之间具有显著正相关性;共刺激受体CD27影响VδT细胞的功能表达。     

黄芪糖蛋白对实验性自身免疫性脑脊髓炎小鼠的免疫调节作用

目的探讨黄芪糖蛋白(HQGP)对实验性自身免疫性脑脊髓炎(EAE)小鼠的治疗效果及可能机制。方法用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)诱导C57BL/6雌性小鼠建立EAE模型。分为黄芪糖蛋白治疗组和EAE对照组,隔天记录小鼠临床评分和体质量变化。HE染色和免疫荧光技术检测脊髓组织炎细胞浸润;MTT法检测细胞活性;Griess法检测一氧化氮(NO)释放;ELISA测定肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)分泌,γ干扰素(IFN-γ)释放;流式细胞术检测CD4+T细胞亚群变化。结果 HQGP处理可减轻EAE症状,抑制中枢神经系统炎细胞浸润。抑制脾淋巴单核细胞活性、NO和TNF-α、IL-6分泌,促进IFN-γ的分泌。增加CD4+CD25+、CD4+IL-10+、CD4+IFN-γ+T细胞亚群的比例。结论 HQGP通过调节T细胞亚群比例,抑制炎症细胞因子释放,减轻EAE炎症反应。     

IL-12有助于化疗药物处理后肿瘤患者及小鼠的细胞免疫功能重建

目的探讨白细胞介素12(IL-12)是否能够恢复化疗药物对肿瘤患者和小鼠细胞的免疫抑制作用。方法分离顺铂化疗前后肿瘤患者及正常人外周血单个核细胞(PBMC),在抗CD3抗体和抗CD28抗体共刺激条件下,加或不加IL-12。培养后,ELISA检测上清中γ干扰素(IFN-γ)和肿瘤坏死因子α(TNF-α)的水平,流式细胞术检测不同亚群T细胞IFN-γ的水平。建立顺铂毒性小鼠模型,采用以上方法检测IFN-γ、TNF-α水平。结果肿瘤患者产生的IFN-γ和TNF-α显著低于正常人;与化疗前患者相比,化疗后患者IFN-γ和TNF-α的产生明显减少,IL-12可以显著增强IFN-γ和TNF-α的产生;T细胞亚群分析的结果表明,IL-12可以恢复化疗后CD4~+T细胞和CD8~+T细胞中IFN-γ的水平。小鼠体内实验证明,化疗药物抑制小鼠T细胞的免疫功能,IL-12能完全恢复T细胞免疫功能。结论化疗药物抑制肿瘤患者和小鼠细胞的免疫应答,而IL-12能够恢复化疗药物对细胞因子的抑制作用,从而对肿瘤化疗患者重建免疫功能起到非常重要的作用。     

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.     

IFN-α磷酸化STAT1和STAT4诱导人NK细胞产生IFN-γ并增强其杀伤活性

目的研究IFN-α对CD56+NK细胞的细胞因子分泌、杀伤功能和细胞内信号传导的作用。方法分离健康人PBMC分别与培养液、IFN-α和IL-12培养,利用酶联免疫吸附法(ELISA)检测培养上清中IFN-γ的水平。同时采用流式细胞仪在单个细胞水平上分析IFN-α诱导IFN-γ产生的细胞亚群以及该细胞亚群对肿瘤细胞的杀伤作用和信号传导机制。结果经IFN-α刺激后,PBMC能产生低剂量的IFN-γ。细胞亚群分析的结果表明,IFN-α诱导CD56+NK细胞产生IFN-γ,但对CD4+T和CD8+T细胞无明显作用。IFN-α促进NK细胞的杀伤功能。促进STAT1和STAT4磷酸化。结论 IFN-α通过磷酸化STAT1和STAT4,增加NK细胞IFN-γ分泌和增强杀伤功能。     

甲状腺与外周血T淋巴细胞亚群比例差异

目的观察正常甲状腺与外周血T淋巴细胞亚群,包括调节性T细胞(regulatory T cells,Treg)、辅助性T细胞1(T helper 1 cells,Th1 cells)和辅助性T细胞2(T helper 2 cells,Th2 cells)的分布,Treg细胞中干扰素-γ(interferon-γ,IFN-γ)、白介素-4(interleukin-4,IL-4)及其mRNA的表达。方法 20名良性甲状腺肿物患者、3名胸腺手术及3名脾脏切除术患者,术前当天留取外周空腹静脉血,术中分别留取甲状腺、胸腺及脾脏标本,分离单个核细胞,流式细胞术检测标本组织及外周血T淋巴细胞亚群差异。磁珠分离法分离甲状腺及外周血CD25+T细胞,RT-PCR检测Treg中IFN-γ和IL-4 mRNA的表达。NKT细胞受体激动剂α-Galcer刺激Treg 7 d后,RT-PCR检测Treg中IFN-γ、IL-4、CD1d、Vα24及Vβ11 mRNA的表达。结果 1)甲状腺组织Th1、Th2细胞比例低于外周血(P0.001),而Th0、Treg细胞比例高于外周血(P0.001)。2)在甲状腺内,几乎所有的CD4+CD25+Foxp3+细胞可以同时分泌IL-4、IFN-γ,RT-PCR提示其IL-4、IFN-γmRNA表达明显高于外周血,而在外周血中,CD4+CD25+Foxp3+细胞并不表达IL-4或IFN-γ。3)在胸腺和脾脏中,未检测到同时表达IL-4、IFN-γ的CD4+CD25+Foxp3+细胞。4)甲状腺内Treg在α-GalCer刺激前后均不表达NKT细胞的表面标志CD1d、Vα24及Vβ11 m RNA,而IL-4和IFN-γmRNA的表达在刺激前后无明显差异。结论正常甲状腺与外周血T淋巴细胞亚群分布存在差异,甲状腺内Treg特异性的同时表达IL-4及IFN-γ。     

老年人T淋巴细胞PD-1高表达在炎症状态下对其功能的影响

目的探讨在LPS模拟的炎症环境中,老年人T淋巴细胞功能与程序性细胞死亡受体-1(PD-1)表达的相关性。方法选取我院健康体检人员110例,其中年轻人55例(65岁),老年人55例(≥65岁)。流式细胞术检测外周血T淋巴细胞亚群(CD45-Per CP/CD3-APC/CD8-FITC)数目及其PD-1(CD279-PE)表达水平。分离外周血单个核细胞,分为对照组、LPS刺激组、LPS+抗PD-1组,CCK-8法和流式凋亡检测术检测细胞活性及凋亡影响,ELISA法检测培养上清中TNF-α、IL-6、IL-10、IL-2和IFN-γ表达水平。结果与年轻人相比,老年人各T淋巴细胞亚群绝对值均显著下降,PD-1表达水平升高(P0.05)。老年人LPS+抗PD-1组较LPS组细胞活性升高,凋亡率下降(P0.05),年轻人LPS+抗PD-1组与LPS组之间的细胞活性和凋亡率差异均无统计学意义(P0.05)。老年人LPS组TNF-α表达水平显著低于年轻人,IL-10/TNF-α比值高于年轻人(P0.05)。结论炎症状态下,老年人T淋巴细胞功能减低与PD-1高表达密切相关。免疫应答时淋巴细胞抑制性细胞因子分泌增多,更易处于免疫抑制状态,与老年人较年轻人更易罹患感染性疾病密切相关。     

CpG-ODN联合抗CD40抗体活化小鼠调节性B细胞抑制CD4+T细胞增殖

目的分析CpG寡脱氧核苷酸(CpG-ODN)联合抗CD40抗体刺激后的调节性B细胞(Breg)对其免疫调节功能的影响。方法用流式细胞分选仪分选小鼠脾脏CD5+CD1 dhighBreg与CD5-CD1 dlowB细胞亚群,CpG-ODN联合抗CD40抗体激活24 h,再与纯化的CD4+T细胞共培养,流式细胞术检测活化后的Breg及对照CD5-CD1 dlowB细胞亚群白细胞介素10(IL-10)的表达,以及共培养抗CD3抗体联合抗CD28抗体激活的CD4+T细胞的增殖与γ干扰素(IFN-γ)的水平。结果 CpG-ODN联合抗CD40抗体激活的CD5+CD1 dhighBreg亚群与CD4+T细胞共培养时能明显抑制活化的CD4+T细胞的增殖,同时IFN-γ的水平降低;而CpG-ODN联合抗CD40抗体刺激后的对照B细胞亚群则不能抑制CD4+T细胞的增殖与IFN-γ分泌。CD5+CD1 dhigh Breg亚群在CpG-ODN联合抗CD40抗体刺激24 h,IL-10的表达水平明显高于CD5-CD1 dlowB细胞亚群。结论体外CpG-ODN联合抗CD40抗体刺激后的CD5+CD1 dhighBreg亚群通过分泌IL-10抑制CD4+T细胞的活化。     

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.     

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.     

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.     

Intestinal helminthiasis in school children in Haiti in 2002

A survey on intestinal helminths in school children was conducted in Haiti in 2002. This first nationwide study involving the entire country was stratified by department according to urban and rural zones using the cluster method. Focusing on elementary school children (n=5792; age range 3 to 20 years), it involved 26 urban and 49 rural schools randomly selected. Stools were preserved in formalin and examined by the Ritchie technique. Thirty-four per cent of stools (1981/5792) tested positive for intestinal helminths with the following parasites identified: Ascaris lumbricoides (27.3%), Trichuris trichiura (7.3%), Necator americanus (3.8%), Hymenolepsis nana (2%), Taenia sp. (0.3%) and Strongyloides stercoralis (0.2%). The helminth prevalence was higher in rural (38.4%) compared to urban areas (30%). There was no significant difference in prevalence by sex and age. The importance of geohelminths changed from one department to another with the highest prevalence found in the Southern department of Grande Anse (73.7%) and the lowest prevalence in the Center department (20.6%). Five out of the country's nine departments had a similar prevalence varying from 25.5% to 28.2%. Intestinal helminthic polyparasitism was observed in a percentage of infested school children comprise between 3.4% and 28.6% according in relation to the geographical area. A program to fight against geohelminths in school children should be initiated as a public health priority. Albendazole is the drug of choice. Frequency of drug distribution should be based on the prevalence of geohelminths in each department.     

Experience in adult population in dengue outbreak in Delhi

A dengue outbreak has recently hit the Indian capital. We studied the clinical profile of adult patients. Five hundred and sixty patients of dengue infection were admitted in a specially created ward according to the criteria laid down by WHO. Haematemesis (28.28%), epistaxis (26.78%) and malena (14.28%) were some of the common presentations. Similarly lymphadenopathy, especially cervical (30.89%), palatal rashes (26.96%) and hepatomegaly (23.75%) were the most commonly encountered findings on physical examination. Most of the cases were of dengue fever with haemorrhage and only 2.5% cases were classified under dengue haemorrhagic fever or dengue shock syndrome. The average hospital stay was 3.4 days but only 9.8 hours in the eleven patients who died, suggesting their late arrival in preterminal situation giving little time for resuscitation. Thrombocytopenia was not a feature and only 12.85% patients had platelet count less than 70,000/cmm. Most of the patients who were admitted with thrombocytopenia, showed normalization in their platelet counts in next few days. Serological examination demonstrated evidence of recent dengue infection in 41.17% patients. Few patients required blood or platelet concentrate transfusion. Eleven patients died, three due to DIC, one of intracranial haemorrhage and seven due to massive gastric haemorrhage. Rest of the patients recovered completely. Thus we can conclude that recent outbreak in Delhi was of dengue fever with haemorrhage and mortality was very low in patients who came early to the hospital.     

Changes in the hypothalamic nuclei in in experimental allergy

Summary In rabbits subjected to prolonged sensitization and in which the Arthus phenomenon was induced there was a marked reaction of the hypothalamic nuclei. Staining by Gomori's method indicated a cellular swelling, loss of granules, and protoplasmic vacuolization in the supraoptic nucleus. There was a considerable increase in the size of the cross-sectional area of the cells. The same effects were much less well shown in the paraventricular nucleus. These results show that marked signs of increased neurosecretion developed in the animals at the height of the Arthus phenomenon.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten Éksperimental'noi biologii i Meditsiny, Vol. 55, No. 4, pp. 110–113, April, 1963.     

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.