人sCD40L-Ig重组腺病毒载体的构建、表达及功能检测

目的:构建含有分泌型人胞外区CD40L融合蛋白(sCD40L-Ig)基因的重组腺病毒载体,确定其表达和功能学意义。方法:通过PCR获得人源IgGFc和sCD40L基因并予以连接,将其插入到腺病毒穿梭质粒pAdTrack-CMV中,构建重组质粒pAdTrack-sCD40L-Ig。将其与pAdEasy-1-BJ5183菌行同源重组后,用293细胞包装,通过观察绿色荧光蛋白(GFP)和Western blot分析等方法鉴定重组腺病毒,并进行双向混合淋巴细胞反应(MLR)以确定其功能。结果:所构建的sCD40L-Ig基因的重组腺病毒,经酶切和PCR鉴定正确。原代腺病毒的滴度达到2.69×1011pfu/L,并有相对分子质量(Mr)为61×103的目的蛋白表达。MLR显示,重组腺病毒对淋巴细胞的增殖有抑制作用。结论:成功地构建了含有sCD40L-Ig基因的重组腺病毒,并对MLR有抑制作用。     

抗人CD154单抗轻重链可变区基因克隆与序列分析

目的:克隆抗人CD154抗体轻重链可变区基因,并分析其核苷酸序列,为基因工程抗体的构建奠定基础。方法:从分泌能抑制免疫反应的抗人CD154单克隆抗体杂交瘤细胞株 7E8中提取总RNA,合成cDNA第一链后,经PCR扩增获得抗人CD154单抗轻链可变区 (VL)和重链可变区 (VH)基因,分别克隆入pUC18载体,并进行序列分析。结果:①抗体的轻链可变区基因全长为 341bp,编码113个氨基酸,归属于Ig的Vκ2基因,氨基酸序列分析结果显示轻链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3位和第 93位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸;②抗体的重链可变区基因全长为 354bp,编码118个氨基酸,归属于小鼠IgVH基因,D、J区基因分别属于DSP2.9和JH2,氨基酸序列分析结果显示,重链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3和第 97位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸。结论:经核苷酸序列分析证明所克隆的基因分别为抗体的轻、重链可变区基因.     

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 (PGE2) and recruit myeloid-derived suppressor cells (MDSCs). Activated cytotoxic T lymphocytes (CTLs) induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8⁺ T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.     

原发性肺癌血中检测sFas的临床价值

目的：探讨sFas在原发性肺癌中的表达及其临床价值。方法：应用ELISA检测40例原发性肺癌标本及30例良性病变中sFas的表达情况。结果：在不同病理类型中sFas表达差异显著（P〈0．05）;在不同TNM分期中：Ⅰ期与Ⅱ期、Ⅰ期与Ⅲ期,sFas表达差异显著（P〈0．05）。在Ⅱ期与Ⅲ期表达差异不显著（P〉0．05）。结论：sFas升高有利于肺癌逃避免疫攻击,有利于其浸润与转移。     

冠心病患者外周血T淋巴细胞PD-L1的表达及意义

目的研究程序性死亡配体-1（PD-L1）在冠心病患者外周血中T淋巴细胞上的表达及意义。方法按照WHO关于冠心病诊断标准,将实验分为稳定型心绞痛（SA）组（n=23）、急性冠脉综合征（ACS）组（n=21）和正常对照组（n=18）,三组均经冠状动脉造影证实。采集三组人群的外周血,用流式细胞技术及RT-PCR技术测定T淋巴细胞PD-L1蛋白及mRNA表达情况。结果与正常对照组比较,SA组和ACS组外周血T淋巴细胞PD-L1蛋白及mRNA表达水平均显著升高（P〈0.01）;但SA组与ACS组比较差异无统计学意义（P〉0.05）。结论冠心病患者外周血T淋巴细胞PD-L1表达上调,其可能在动脉粥样硬化斑块形成过程中起作用。     

基于癌基因组图谱数据挖掘分析CC亚族趋化因子配体20在食管癌中的表达及临床意义

目的 探讨CC亚族趋化因子配体20(CCL20)在食管癌中的表达及对病程进展和预后的影响.方法 利用癌基因组图谱(TCGA)、UALCAN、GEO等相关数据库分析CCL20基因在食管癌组织与癌旁组织中的表达差异,通过Kaplan-Meier模型探讨CCL20表达水平与预后的关联性;分析CCL20与肥胖、甲基化等之间的关...     

Mendelian susceptibility to mycobacterial diseases: state of the art

BackgroundMendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to infections caused by intracellular pathogens, such as mycobacteria, due to impaired IFN-γ immunity. To date, 18 different genes associated with MSMD have been reported.ObjectivesThis review describes recent discoveries, a 2020–2021 update, in MSMD through the introduction of three novel genetic disorders, namely, AR IFN-γ, T-bet, and ZNFX1 complete deficiency, as well as molecular mechanisms underlying multifocal osteomyelitis in patients with this condition.SourcesPubMed databases were searched for reports of MSMD since January 2020. Relevant articles and their references were screened.ContentThe review covers a general overview, known genes, classifications, symptoms, and treatments for MSMD. MSMD is classified into two groups: isolated MSMD and syndromic MSMD. Among the 18 genes responsible, 13 cause isolated MSMD, which is characterized by selective predisposition to one or more mycobacterial and related infections, and 8 cause syndromic MSMD, which involves the combination of the mycobacterial disease infectious phenotype with additional clinical phenotypes. Among the three genetic etiologies described herein, AR IFN-γ deficiency is classified as isolated MSMD, whereas AR T-bet and ZNFX1 deficiency are classified as syndromic MSMD. Multifocal osteomyelitis is a representative symptom of MSMD, and a high frequency of multifocal osteomyelitis is reported in MSMD patients due to impaired IFN-γ responses, such as with AD IFN-γR1, AD IFN-γR2, or AD STAT1 deficiency. Impaired inhibition of osteoclast differentiation and bone resorption owing to a poor response to IFN-γ has been shown to be in association with multifocal osteomyelitis in MSMD.ImplicationsOver the past decade, genetic dissection by next-generation sequencing techniques has contributed to the understanding of the molecular bases of human immunity to mycobacteria. However, genetic etiologies are lacking for half of MSMD cases. Further studies will be needed to elucidate the pathogenesis of MSMD.     

Concentrations of Soluble CD95 and CD8 Antigens in the Plasma and Levels of CD8+CD95+, CD8+CD38+, and CD4+CD95+ T Cells Are Markers for HIV-1 Infection and Clinical Status

Apoptosis mediated via the CD95 (FAS/APO-1) receptor is thought to play a role in the depletion of CD4+ T cells in HIV infection. In the present study expression of the CD95 antigen on lymphocyte subsets and the plasma level of soluble CD95 (sCD95) were determined in HIV-1-infected adults. The expression of CD95 was increased on CD8 cells in all groups of HIV+ individuals, while increased expression of CD95+ cells on CD4 cells was limited to individuals with CD4 counts of <200 mm³. The proportion of CD4+ that expressed CD95 was inversely correlated with the percentage of CD4+ PBL. The concentration of sCD95 was significantly higher in the plasma of HIV-infected individuals than in normal controls. The level of sCD95 in HIV-infected subjects showed no correlation with the percentage of PBL expressing CD95, indicating that the increased level of sCD95 did not reflect release from CD95+ PBL. The plasma sCD95 concentration was significantly correlated with the percentage of CD8+ cells and, particularly, with CD8+CD38– cells. A striking inverse correlation was found between the sCD95 plasma concentration and the proportion of CD4+CD95+ cells out of the total CD4+ population. There was no correlation between the serum level of sCD95 and that of soluble CD8 (sCD8), both of which were increased in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals was correlated with the percentage of CD95+ and CD8+CD38+ cells. The present study indicates that plasma sCD95 may be one of the factors that regulate apoptotic death of lymphocytes in HIV infection.     

AITD甲状腺细胞Fas、FasL的单抗免疫组化测定

测定凋亡相关蛋白Fas、FasL在自身免疫性甲状腺疾病(AITD)病人甲状腺细胞中的表达,以研究细胞凋亡在AITD发病中的作用.本研究采用甲状腺粗针穿刺取甲状腺组织,然后用单克隆抗体免疫组织化学法和图像分析检测甲状腺细胞中Fas、FasL的表达,于电镜下观察结果.Graves病(GD)组Fas、FasL染色强度显著低于对照组(P＜0.01).桥本氏甲状腺炎(HT)组两者染色强度显著高于对照组(P＜0.01).这说明Fas、FasL这两种凋亡相关蛋白在HT病人甲状腺细胞中表达显著增强,而在GD病人中表达较少,进一步证实了细胞凋亡在AITD发病机制中具有重要作用.     

Inhibition of TCR triggering by a spectrum of altered peptide ligands suggests the mechanism for TCR antagonism

Understanding the parameters involved in T cell activation has been complicated by the discovery of partial T cell agonists. Altered peptide ligands (APL) have recently shown that different subsets of T cell responses can be selectively activated by certain peptides, which define a hierarchy of T cell activation. For cytotoxic T cells, this hierarchy ranges from sensitizing target cells for lysis through proliferation to effector cell induction. The degree of TCR down-regulation mediated by APL-MHC interactions correlates well with the induction of specific T cell effector functions. This suggests that the potential agonist response induced by a given peptide occurs at different triggering thresholds. To examine the relative agonist and antagonist functions of different peptides, we have investigated the ability of lymphocytic choriomeningitis virus glycoprotein-derived APL to induce or inhibit a range of effector functions in naive CD8⁺ T cells. By this, we have defined a hierarchy of peptides that display a range of properties from strong agonist to no agonist function. At each level, peptides that were ranked lower in this hierarchy were able to interfere or antagonize the induction of effector functions by higher ranking peptides. We have therefore shown that this spectrum of peptides ranging from strong to no agonist function has an inverse gradient from strong antagonist to no antagonist function. Moreover, the ability of the different peptides to inhibit TCR internalization correlated with their ranking within the hierarchy. These findings support the model that antagonists are effectively preventing TCR oligomerization and functional TCR triggering.