定量检测T细胞受体重排删除DNA环含量评价急性非淋巴细胞白血病患者胸腺近期输出功能

目的 检测急性非淋巴细胞白血病(ANLL)患者 T细胞受体重排删除DNA环(signal joint T-cell receptor excision DNA circles sjTRECs,TRECs)的含量,从而探讨患者的初始型naive T细胞水平和胸腺近期输出功能特点.方法利用实时定量PCR(TaqMan)方法,检测77例ANLL患者外周血单个核细胞(PBMC)TRECs的水平,并根据外周血中CD3阳性率计算CD3细胞中TRECs水平.14例ANLL缓解期患者和14例正常人外周血作为对照.结果 ANLL患者外周血中TRECs含量为(0.43±0.92)拷贝/1 000 PBMCs, (2.02±3.25)拷贝/1 000 CD3+细胞,明显低于正常人TRECs水平[(4.10±3.65)拷贝/1 000 PBMCs和(6.84±4.71)拷贝/1 000 CD3+细胞,P=0.0000和P=0.0001]和缓解期患者[(2.19±2.49)拷贝/1 000 PBMCs和(6.30±7.13)拷贝/1 000 CD3+细胞,P=0.0000和P=0.0005].各亚型ANLL患者的TREC水平差异无统计学意义(P＞0.05).结论 绝大多数ANLL型患者胸腺近期输出naive T细胞功能明显降低,缓解期患者的胸腺近期输出功能有一定程度恢复.     

T细胞受体删除环定量检测的意义及应用现状

T细胞受体重排删除环(TRECs)是T细胞受体基因重排过程中删除的DNA环，可作为最近从胸腺输出初始细胞的标记，其定量检测应用于胸腺近期输出功能的评价。近年来由于免疫重建研究的广泛开展，定量检测TRECs得到重视。本文拟从TRECs的产生以及定量检测在正常人和各类免疫缺陷性疾病中的应用及其意义给予综述。     

T细胞受体删除环定量检测的意义及应用现状

T细胞受体重排删除环(TRECs)是T细胞受体基因重排过程中删除的DNA环,可作为最近从胸腺输出初始细胞的标记,其定量检测应用于胸腺近期输出功能的评价.近年来由于免疫重建研究的广泛开展,定量检测TRECs得到重视.本文拟从TRECs的产生以及定量检测在正常人和各类免疫缺陷性疾病中的应用及其意义给予综述.     

胸腺近期输出功能测定在评价细胞免疫功能重建中的应用

T细胞受体重排删除环 (TRECs )是T细胞受体基因重排过程中删除的DNA环 ,可作为胸腺近期输出功能的指标 ,本文综述其在造血干细胞移植、HIV - 1感染和AIDS患者抗逆转录病毒治疗、肿瘤化疗以及细胞因子补充治疗后免疫重建评价中的应用     

胸腺近期输出功能测定在评价细胞免疫功能重建中的应用

T细胞受体重排删除环(TRECs )是T细胞受体基因重排过程中删除的DNA环,可作为胸腺近期输出功能的指标,本文综述其在造血干细胞移植、HIV-1感染和AIDS患者抗逆转录病毒治疗、肿瘤化疗以及细胞因子补充治疗后免疫重建评价中的应用.     

AML-M5患者外周血naive T细胞水平和TCR Vβ谱系利用特点

目的　了解急性髓性白血病M5亚型 (AML -M5 )患者T细胞受体重排删除DNA环 (TRECs)的含量和TCRVβ基因谱系利用和克隆性 ,从而了解AML患者的胸腺近期输出功能和TCRVβ亚家族T细胞增殖特点 .方法　利用实时定量PCR(TaqMan)方法检测 5例M5患者外周血单个核细胞TRECs的水平 ,并根据外周血中CD3阳性率计算CD3细胞中TRECs水平 .利用RT -PCR和基因扫描分析患者外周血单个核细胞的TCRVβ2 4个亚家族基因表达和克隆性 .9例正常人外周血作为对照 .结果　M5患者外周血中TRECs含量为 0 .76± 1.2 1/ 10 0 0CD3 细胞 ,明显低于正常人TRECs水平 (6 .84± 4 .71/ 10 0 0CD3 细胞 ,p <0 .0 5 ) .5例患者外周血T细胞表达不同数量Vβ亚家族 (2 - 16个 ) .基因扫描分析显示 4例病人外周血中的一些Vβ亚家族出现克隆性T细胞 ,Vβ1,Vβ15和Vβ2 1克隆性T细胞均分别见于 3例病人中 .结论　率先报道了AML -M5型患者胸腺近期输出naiveT细胞功能明显降低 ,尽管整体T细胞免疫功能低下 ,患者仍存在优势利用和克隆性增殖Vβ亚家族T细胞 ,提示其具有一定地对白血病细胞相关抗原产生特异性免疫反应的能力     

检测T细胞受体重排切除环的意义及应用研究

T细胞受体重排切除环是T细胞受体基因重排过程中切除的DNA环，可作为新近从胸腺迁出者的标记，应用于胸腺功能的评价。近年来由于免疫重建研究的缘故，检测T细胞受体切除环受到了重视。本文拟从T细胞受体切除环产生及应用的意义、方法上作一综述。     

AML-M5患者外周血naive T细胞水平和TCR Vβ谱系利用特点

目的了解急性髓性白血病M5亚型(AML-M5)患者 T细胞受体重排删除DNA环(TRECs)的含量和TCR Vβ基因谱系利用和克隆性,从而了解AML患者的胸腺近期输出功能和TCR Vβ亚家族T细胞增殖特点.方法利用实时定量PCR(TaqMan)方法检测5例M5患者外周血单个核细胞TRECs的水平,并根据外周血中CD3阳性率计算CD3细胞中TRECs水平.利用RT-PCR和基因扫描分析患者外周血单个核细胞的TCR Vβ24个亚家族基因表达和克隆性.9例正常人外周血作为对照.结果 M5患者外周血中TRECs含量为0.76±1.21/1000 CD3+细胞,明显低于正常人TRECs水平(6.84±4.71/1000 CD3+细胞,p<0.05).5例患者外周血T细胞表达不同数量Vβ亚家族(2-16个).基因扫描分析显示4例病人外周血中的一些Vβ亚家族出现克隆性T细胞, Vβ1,Vβ15和Vβ21克隆性T细胞均分别见于3例病人中.结论率先报道了AML-M5型患者胸腺近期输出naive T细胞功能明显降低,尽管整体T细胞免疫功能低下,患者仍存在优势利用和克隆性增殖Vβ亚家族T细胞,提示其具有一定地对白血病细胞相关抗原产生特异性免疫反应的能力.     

检测T细胞受体重排切除环的意义及应用研究

T细胞受体重排切除环是T细胞受体基因重排过程中切除的DNA环 ,可作为新近从胸腺迁出者的标记 ,应用于胸腺功能的评价。近年来由于免疫重建研究的缘故 ,检测T细胞受体切除环受到了重视。本文拟从T细胞受体切除环产生及应用的意义、方法上作一综述。     

080 检测T细胞受体重排切除环的意义及应用研究

T细胞受体重排切除环是T细胞受体基因重排过程中切除的DNA环，可作为新近从胸腺迁出者的标记，应用于胸腺功能的评价。近年来由于免疫重建研究的缘故，检测T细胞受体切除环受到了重视。本文拟从T细胞受体切除环产生及应用的意义、方法上作一综述。     

FK506 attenuates thymic output in patients with myasthenia gravis

Introduction

Myasthenia gravis (MG) is an antibody-mediated, T-cell-dependent autoimmune disease. The symptoms are caused by high-affinity IgG against the muscle acetylcholine receptor (AChR) at the neuromuscular junction. The production of these antibodies in B-cells depends on AChR-specific CD4⁺ T-cells and the thymus gland seems to play a significant role in the pathogenesis of MG. Altered thymic T-cell export seems to be associated with a pathological mechanism in myasthenia gravis. Tacrolimus (FK506) has recently been used to treat MG.

Material and methods

We examined the effects of tacrolimus on thymic T-cell export in patients with MG. Sixteen patients with nonthymomatous and/or thymectomized MG were treated with oral administrations of tacrolimus. To assess the effect of tacrolimus on the thymic output, we assayed the levels of T-cell receptor excision circle (TREC), a molecular marker of thymus emigrants.

Results

T-cell receptor excision circle was not significantly different from those in age-matched controls before tacrolimus therapy, but they were partially decreased 4 months after tacrolimus therapy. T-cell receptor excision circle levels were significantly decreased in the thymomatous group (p < 0.05), but not in the nonthymomatous group. Tacrolimus treatment significantly attenuated TREC levels in cultured CD4^–CD8⁺ cells (p < 0.05), but total cell counts were not significantly changed.

Conclusions

These results indicate that TREC levels may become a marker of the curative effect of tacrolimus therapy for thymomatous MG, and that tacrolimus suppresses not only activating T-lymphocytes, but also naïve T-cells.     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

慢性粒细胞白血病病人TCRζ链表达特点

目的： 建立实时定量PCR方法检测TCRζ链表达水平的方法，了解慢性粒细胞白血病（CML）外周血TCRζ链表达水平。方法: 采用SYBR GreenⅠ荧光定量PCR，相对定量检测30例CML患者和30例正常人外周血的单个核细胞的TCRζ链表达情况，以β2微球蛋白基因（β2M）作为内参，根据相对定量公式：2-△△Ct计算CML病人与正常人TCRζ链表达差异倍数。结果: 成功建立SYBR GreenⅠ荧光实时定量PCR检测TCRζ链表达检测技术。18例CML患者TCRζ链出现表达低于正常人，而12例CML患者TCRζ链出现表达高于正常人。结论: CML病人中TCRζ链表达水平可分为表达下调（60%）和表达上调(40%)2组，提示部分CML病人的细胞免疫缺陷可能与其TCRζ链表达下调有关。     

实时荧光定量PCR测定胸腺近期输出功能方法的建立和应用

目的建立一种准确、快速的检测胸腺输出功能的方法。方法根据TCRδ基因序列,设计引物和探针,建立实时荧光定量PCR检测T细胞受体重排切除环(TRECs)的方法;并用于检测正常人及慢乙肝PBMCs中TRECs的含量。结果建立了测定TRECs含量的实时荧光定量PCR方法,产物与预计长度相符,序列正确;最低可扩增出5copies的模板;重复五次,Ct值的变异系数为1.06%。正常人21~45岁组的TRECs含量为(7767.4±2369.5)copies/106 PBMCs,16~20岁组为(28374.4±7820.4)copies/106 PBMCs,21~45岁慢乙肝组(6480.9±2031.2)copies/106 PBMCs,正常人21~45岁组分别与16~20岁组、同龄慢乙肝组比较差异均有统计学意义,P<0.05。结论实时荧光定量PCR检测TRECs的方法特异性强,灵敏度高,重复性好。正常人21~45岁组的胸腺输出功能低于16~21岁组,慢乙肝组低于同龄正常人。     

实时定量PCR检测正常人外周血T细胞和胸腺细胞中sjTRECs水平

目的了解正常人外周血T细胞和胸腺细胞中信号结合T细胞受体删除 DNA环(sjTRECs)的含量,从而推测正常人中幼稚T细胞的含量和胸腺的输出功能. 方法利用实时定量PCR和TaqMan方法检测11例正常人外周血单个核细胞、7例儿童和3例成人胸腺细胞DNA中sjTRECs的水平. 结果正常人中sjTRECs含量分别为8.83±4.81/1000个外周血单个核细胞;27.31±3.23/1000个成人胸腺细胞和170 .29 ±59.52/1000个儿童胸腺细胞. 结论 sjTRECs水平与年龄有关,正常人外周血中每1000个单个核细胞中约含4.5个幼稚T细胞.     

Quantitative analysis of cell-free Epstein-Barr virus DNA in the plasma of patients with peripheral T-cell and NK-cell lymphomas and peripheral T-cell proliferative diseases

BACKGROUND: The level of circulating EBV DNA is a prognostic marker in patients with some EBV-associated malignant diseases. OBJECTIVES: To investigate the presence and nature of Epstein-Barr virus (EBV) DNA in the plasma and to evaluate the correlation of plasma concentrations of EBV DNA with the EBV genomic status in peripheral blood T-cells and neoplastic cells and with the clinical outcome of patients with peripheral T-cell and NK-cell lymphomas (PTCL) and peripheral T-cell proliferative diseases (PTPD). STUDY DESIGN: EBV DNA in the plasma of 45 patients and 45 controls was measured using real-time PCR. The presence of the EBV genome in the isolated peripheral blood lymphocytes (CD3+ and CD3- cells) was analysed by PCR. Detection of EBV-encoded early RNA (EBER) in corresponding tumor tissues was carried out using in situ hybridization. DNase I digestion was applied to plasma samples to detect naked EBV DNA. RESULTS: Cell-free EBV DNA was detected in 32/38 (84%) of PTCL patients and 5/7 (71%) of PTPD patients, but not in the controls. Patients with EBV genome in peripheral blood CD3+ cells and EBV genome (EBER) in the tumor cells, compared to those without these findings, had significantly higher plasma EBV DNA levels. The majority of circulating EBV DNA molecules was naked form. The plasma EBV DNA levels were not related to survival. CONCLUSIONS: The concentration of EBV DNA in the plasma was not a prognostic marker in PTCL and PTPD patients.     

Rapid migration of thymic emigrants to the colonic mucosa in ulcerative colitis patients

Inflammatory bowel disease (IBD) is associated with imbalances of the local intestinal immune responses, with dysregulated CD4⁺ T cells contributing to the chronic inflammation. Having demonstrated altered T cell maturation in the thymus in two different mouse models of colitis, we set out to investigate whether abnormalities in T cell maturation is present in patients with ulcerative colitis (UC) or Crohn's disease (CD). Specimens were obtained from peripheral blood (CD; n = 14, UC; n = 22), colon and small intestinal specimens (CD; n = 6, UC; n = 13). As controls, peripheral blood specimens were obtained from healthy volunteers, patients with adenocarcinomas (n = 18) and colonic specimens from patients with adenocarcinomas (n = 14). Recent thymic emigrants were estimated by analysis of the normalized ratio of T cell receptor excision circles (TRECs) by real‐time polymerase chain reaction (PCR). The frequency of naive‐ and proliferating T lymphocytes and markers of extrathymic T cell maturation in the mucosa was analyzed by flow cytometry and real time‐PCR. TREC levels in peripheral blood T lymphocytes were similar between IBD patients and controls. In contrast, UC patients demonstrated significantly increased levels of TRECs both in intraepithelial and lamina propria lymphocytes from the colonic mucosa compared to patients with adenocarcinomas and CD. However, markers for extrathymic T cell maturation in the mucosa were not different between controls and IBD patients. The increased TREC levels in mucosal but not peripheral blood lymphocytes in UC patients in the absence of increased extrathymic maturation in situ in the mucosa together demonstrate that recent thymic emigrants are recruited rapidly to the inflamed mucosa of these patients.     

急性单核细胞白血病病人外周血TCR Vα基因谱系和T细胞克隆性增殖特点

目的：了解急性单核细胞白血病（AML-M5）病人TCR Vα29个亚家族的分布及其克隆性。方法: 利用RT-PCR分别扩增8例M5病人外周血单核细胞的TCR Vα29个亚家族基因的CDR3。阳性的PCR产物进一步经荧光素标记和基因扫描分析产物的CDR3长度，了解T细胞克隆性。9例健康人作为对照。结果: RT-PCR分析显示正常人表达绝大多数Vα亚家族基因，而8例M5病人外周血仅可以检测到1-10个Vα亚家族基因，出现频率最高的是Vα3（6/8例，75%），其次为Vα12（5/8例，62.5%），有15个Vα亚家族表达缺失（Vα1、4、5、7、9、14-18、20、21、26、28和Vα29）。基因扫描分析显示：8例AML-M5病人中有6例存在克隆性增殖T细胞，其中，以Vα12亚家族出现克隆性增殖T细胞频率（3/5例）最高，有2例仅存在单一的克隆性增殖Vα3亚家族T细胞。正常人外周血各Vα亚家族T细胞主要呈多克隆性。结论: M5病人外周血Vα亚家族T细胞存在明显的选择性选用及克隆性增殖特点，这可能是机体T细胞受M5细胞刺激而引起机体产生相应的特异性免疫反应，同时T细胞TCR Vα亚家族分布及克隆性增殖情况具有个体特异性。     

Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the α/β T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the α/β T-cell repertoire in autoimmune and infectious diseases.