Regulation of interleukin-2 induced soluble Fas ligand release from human peripheral blood mononuclear cells

Adjuvant interleukin (IL)-2 immunotherapy has been used in the treatment of different malignant dieseases. However, clinical results have been rather disappointing. Therefore, further investigations on IL-2-induced mediators of cytotoxicity seem to be necessary in order to possibly create cytokine cocktails which could enhance the IL-2-induced cytotoxicity. We therefore investigated the regulation of IL-2-induced release of soluble Fas Ligand (sFasL), since this factor is known to possess anti-tumor activities. In CD3-stimulated peripheral blood mononuclear cells IL-2 induced sFasL in a dose-dependent fashion. Maximum sFasL concentrations were obtained after stimulation of MNC for 120 hrs. Inhibition of endogenous IL-12 production significantly reduced IL-2-mediated sFasL release by about 25%. In contrast, addition of IL-12 enhanced the IL-2-induced sFasL about 1,5-fold. IL-10 and IL-4 reduced the IL-2-stimulated sFasL by about 30%. Interestingly, these suppressive effects could be antagonized by the addition of IL-12. Not only exogenous IL-10 but also endogenously produced IL-10 decreased the sFasL release to that extent which had been stimulated by IL-12. Since IL-12 and IL-10 only marginally influenced the IL-2-mediated cell proliferation as well as the IL-2-induced cell death, the IL-12- and IL-10-controlled sFasL release seems to be based on an enhanced production per cell. However, the increase in cell numbers as well as the decrease of viability during cell culture might additionally contribute to the IL-2-induced increase of sFasL release. This secondary effect might explain why IL-2-mediated sFasL production is only partially controlled by regulatory cytokines such as IL-4, IL-10 or IL-12. In conclusion, addition of IL-12 might increase the efficacy of IL-2 immunotherapy by inhibition of the IL-10-mediated negative feed-back loop on IL-2-mediated sFasL release.     

慢性乙型肝炎患者树突状细胞表型的研究

目的探讨慢性乙型肝炎患者外周血来源树突状细胞（Dendritic cell，DC）数量及表型的改变，并对其与肝功能、乙肝病毒复制水平的关系进行研究。方法检测37例慢性乙肝患者和21例健康人肝功能及血清HBV DNA水平，并提取外周血单个核细胞（Peripheral blood mononuclear cell，PBMC）进行体外诱导培养，促使其发育成DC，计数其数量并检测膜表面分子的变化。分析DC数量及表型与肝功能、乙肝病毒复制水平的关系。结果与正常对照组比较，慢性乙肝患者的树突状细胞数量明显减少（P〈0．05），且其膜表面分子CD83、CD86的表达均明显降低（P〈0．05）。在慢性乙肝患者中，DC数量、DC膜表面分子CD83和CD86与血清HBV DNA之间呈负相关关系，而与肝功能之间无明显相关关系。结论慢性乙肝患者体内存在DC数量减少及成熟障碍，这种改变与肝内炎症反应程度不相关，但与乙肝病毒（Hepatitis Bvirus，HBV）的复制水平呈负相关，提示DC参与慢性乙肝患者体内HBV的清除。     

人外周血单个核细胞来源内皮祖细胞的生物学性状

目的 探讨从成人外周血单个核细胞中分离、培养内皮祖细胞（EPCs）的方法以及EPCs的生物学特征。方法 密度梯度离心法获得单个核细胞（MNCs），种植于纤连蛋白包被的培养板中。每2h去除1次未黏附细胞共2次，然后隔日换液1次，7d后计数早期集落。每例血样均分为2等份，1份在获得早期集落后进行实验；另1份持续培养至晚期集落出现进行相同实验。流式细胞技术检测细胞表面抗原表达，ELISA法测定上清液中血管内皮生长因子（VEGF）浓度。胶原凝胶细胞种植实验测定体外血管生成功能。结果 早期集落中心为圆形细胞，周边是放射状排列的纺锤形细胞，再种植不能形成第二代集落且无体外血管形成功能，细胞表面主要表达CD14和CD45，培养上清液中可测到高浓度的VEGF。晚期集落在培养21至28d间出现，再种植可形成第二代内皮细胞集落，并能在胶原凝胶中形成管腔样结构。其构成细胞与早期集落相比CD45、CD14表达显著减少（P<0.001）而CD146表达明显增加（P<0.01）。结论 人外周血单个核细胞在内皮培养条件下可形成早期和晚期集落，构成早期集落的绝大多数细胞属于单核细胞系列，可分泌VEGF但不能分化成内皮细胞，晚期集落具有内皮祖细胞的形态和生物学特征。     

Serum levels of soluble Fas ligand in patients with silicosis

Certain patients with silicosis have been reported to exhibit immunological abnormalities such as the appearance of antinuclear antibodies and the occurrence of autoimmune diseases. Fas ligand (FasL) is a type II membrane protein which induces apoptosis by binding to its membrane receptor, Fas. FasL is converted to a soluble form by a metalloproteinase-like enzyme. We have already found serum soluble Fas (sFas) levels in silicosis patients as well as in patients with systemic lupus erythematosus (SLE) to be significantly higher than those in healthy volunteers. To examine further the role of the Fas/FasL system in silica-induced immunological abnormalities, we investigated serum soluble FasL (sFasL) levels in silicosis patients with no clinical symptoms of autoimmune diseases, using ELISA for sFasL. Although the serum sFasL levels in patients with SLE were significantly higher than those in healthy volunteers and showed a slight positive correlation with serum sFas levels, those in silicosis patients exhibited no significant difference from those in healthy volunteers, and there was no correlation with serum sFas levels. However, sFasL levels were elevated in silicosis patients with slight dyspnoea or normal PCO2 among various clinical parameters of silicosis. It may be speculated that the immunological disturbances presented by the abnormalities of apoptosis-related molecules in silicosis patients do not occur with a similar degree of respiratory involvement. Further studies are required to clarify which kinds of factors are involved in silicosis patients who exhibit immunological abnormalities.     

钙离子载体对外周血单核细胞来源的树突状细胞的影响

目的：探讨钙离子载体（calcium ionophore,CI)对外周血单核细胞来源的树突状细胞（DC）的影响。方法：分离分离献血者外周血单核细胞。分别加入重组人粒/单集落刺激因子（rhGM-CSF)100μg/L，rhGM-CSF100μg/L CI10μg/L及rhGM-CSF CI各100μg/L，体外培养40h后，于光镜及电镜下观察细胞的形态，流式细胞仪检测细胞的表面标志，MTT比色法检测上述分子对自体T细胞的刺激增殖作用。结果：外周血单核细胞在GM-CSF CI各100μg/L的条件下培养40h，就可看到典型的DC形态，其表面CD14分子表达减少。HLA-DR，CD40，CD83及CD86分子的表达明显增高，且具有明显刺激自体T细胞增殖的能力。结论：CI有显著加速GM-CSF诱导的外周血单核细胞向DC转化的作用。     

Detection and quantitation of human papillomavirus DNA in peripheral blood mononuclear cells from blood donors

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Also, HPV has been associated with anogenital cancer, oropharyngeal cancer, genital warts, and other dermatological diseases. HPV infects epithelial cells and their replication is closely linked to epithelial differentiation. The presence of HPV DNA in peripheral blood mononuclear cells (PBMC) has been reported in some patients with head and neck cancer, cervical cancer, and other genital diseases. However, the presence of HPV DNA in blood in asymptomatic subjects is still unresolved. The objective of this study was to evaluate the presence of HPV DNA in PBMC from asymptomatic blood donors. Blood samples were collected from 207 healthy Chilean blood donors. Genomic DNA was extracted from PBMC and HPV DNA detection was performed by real-time quantitative polymerase chain reaction assays with GP5+/6+ primers. HPV typing was carried out by genetic sequencing of a 140 to 150 bp fragment of the L1 gene. HPV DNA was detected in 6.8% (14/207) of blood donors. Single HPV infections were detected in seven blood donors. High-risk HPV was found in 6.3% (13/207) of cases: nine blood donors were infected with HPV-16, five with HPV-18, two with HPV-51, and one case was infected with either 32, 33, 45, 59, 66, 70, or 82. The median viral load value was 21.3 copies/mL blood or 13.4 HPV (+) cells per 10 ⁴ PBMC. These results show that HPV DNA is present in PBMC from healthy blood donors and it suggests that blood could be a new route of HPV dissemination.     

慢性心力衰竭患者与正常人外周血单核细胞基因的表达差异

目的应用cDNA芯片技术研究慢性心力衰竭（CHF）患者和正常对照组外周血单核细胞（PBMC）基因表达差异。方法抽提PBMC总RNA并纯化mRNA,反转录合成单链、双链cDNA后,体外转录合成cRNA。分别用cy3-dCTP和cy5-dCTP标记cRNA。将基因芯片和杂交探针变性后杂交、洗涤,并分析cy3、cy5两种荧光信号的强度和比值。结果CHF患者和正常对照组比较,共有123个表达差异相关基因,其中有102个表达上调,21个表达下调。涉及黏附分子、热休克蛋白、信号传导、细胞周期、凋亡等。结论CHF与正常人PBMC基因表达存在显著差异。进一步深入研究这些基因在CHF发生发展中的作用,对推动CHF发生的分子机制及防治研究可能有重要意义。     

急性髓系白血病患者外周血单个核细胞Toll样受体9以及血清肿瘤坏死因子和凋亡基因FAS的表达

背景：Toll样受体9(TLR9)、肿瘤坏死因子a、Fas可能共同参与白血病的发生发展过程。 目的：测定TLR9在急性髓系白血病患者外周血单个核细胞中的表达及血清肿瘤坏死因子α、Fas水平。 方法：从急性髓系白血病患者与正常对照组中分离出外周血单个核细胞,采用反转录-聚合酶链反应法检测外周血单个核细胞中TLR9 mRNA的表达水平,采用酶联免疫吸附试验法检测血清肿瘤坏死因子α、Fas水平。 结果与结论：在急性髓系白血病患者初治组和难治复发组中,外周血单个核细胞中TLR9 mRNA表达高于正常对照组(P < 0.01),化疗后完全缓解组与正常对照组差异无显著性意义(P > 0.05);各病例组血清肿瘤坏死因子α、Fas水平显著高于正常对照组(P < 0.01)。TLR9 mRNA的表达与血清肿瘤坏死因子α、Fas水平均呈正相关。     

Up-regulation of Fas ligand (FasL) mRNA expression in peripheral blood mononuclear cells (PBMC) after major surgery

FasL, which is expressed mainly on activated lymphocytes, can induce apoptosis (programmed cell death) of cells which express Fas. Fas/FasL interaction is primarily beneficial in maintaining immunological and physiological homeostasis by eliminating unnecessary cells. Dysregulation of the interaction, however, leads to tissue damage. We investigated how Fas/FasL levels changed after major surgery. The major aim of this study was to elucidate the involvement of the Fas/FasL system in postoperative inflammation. The investigation involved 10 patients admitted to the intensive care unit after surgery. Although the percentage of Fas⁺ cells and the amount of Fas expression tended to increase, there was no significant difference between pre- and post-operative samples. In contrast, the levels of FasL mRNA were dramatically up-regulated after operation. Post-operative C-reactive protein (CRP) levels increased and correlated well with FasL levels (r = 0.91, P < 0.01). Lymphocyte counts decreased after operation and were inversely proportional to FasL levels (r = 0.58, P < 0.05). These results suggest that the enhanced FasL expression is likely to be related to systemic inflammatory responses induced during the perioperative period. FasL up-regulation may be involved in the aggravation of tissue damage, including lymphocytopenia, in the early post-operative period.     

肿瘤坏死因子相关凋亡诱导配体在外周血单个核细胞中的表达

目的 :研究肿瘤坏死因子相关凋亡诱导配体 (TRAIL)在外周血单个核细胞 (PBMC)中的表达及其意义。方法 :用免疫组织化学的方法检测PBMC中TRAIL的表达情况。结果 :淋巴细胞分离液得到的PBMC中 ,淋巴细胞占 (97.4 8± 3.4 3) % ,单核细胞占 (3.15± 0 .83) % ,TRAIL免疫反应阳性的淋巴细胞占总数的 (2 6 .31± 3.18) % ,呈TRAIL免疫反应阳性的单核细胞占总数的(1.0 4± 0 .13) % ,TRAIL免疫反应阳性物质分布于胞质内 ,核呈阴性反应。结论 :正常人外周血分离的淋巴细胞及单核细胞有TRAIL表达。     

Circadian clock gene expression in human peripheral blood mononuclear cells

Circadian clock genes have been identified in humans but information regarding their expression has remained very limited. However from a basic point as well as in a diagnostic and therapeutic perspective, it is important to evaluate molecular clock gene expression. Peripheral blood mononuclear cells represent an ideal material to investigate non-invasively the human clock at the molecular level. Several studies including ours reported rhythmic expression of clock genes in these cells, with significant intersubject variability of expression. In addition, our results reveal the existence of different chronotypes of clock gene expression patterns and suggest specific regulatory mechanisms in these human cells as compared to other peripheral tissues.     

Interleukin-4 gene expression in human peripheral blood mononuclear cells

Interleukin-4 (IL-4) mRNA was detected in normal human peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A by Northern blot analysis. The signal was undetectable in PBMC before the stimulation, but became detectable 3 hrs after the stimulation and reached a maximum in 3-6 h and disappeared gradually thereafter. Immunosuppressive drugs such as ciclosporin, hydrocortisone and prednisolone inhibited the IL-4 mRNA expression dose dependently. Interferon-gamma did not show any inhibitory effect on IL-4 gene expression.     

系统性红斑狼疮外周血单个核细胞CD40L的表达增高

目的:了解系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)的白细胞分化抗原40配体(CD40L)表达,探讨其在发病中的作用。方法:分离SLE患者和正常人PBMCs,采用流式细胞术,检测其在正常状况和应用植物凝集素(PHA)及地塞米松(Dex)后,CD40L的表达水平,并进行比较;分析SLE患者CD40L的表达水平和狼疮活动指数(SLEDAI)的相关性。结果:活动期SLE患者PBMCs的CD40L阳性细胞百分率(%)明显高于对照组,且高于静止期SLE患者;应用PHA处理24h后,3组PBMC表达CD40L均明显增加,但活动期SLE患者增加更明显;应用地塞米松后,SLE患者(活动期和静止期)PBMCs的CD40L表达明显减少,对照组无明显改变;SLE患者(活动期和静止期)CD40L的表达水平和SLEDAI均呈明显正相关。结论:CD40L在SLE患者PBMCs的表达增加,和疾病活动度有关;其受PHA和Dex调控,在SLE发病和病程中起重要作用。     

肺癌患者外周血单核细胞端粒酶活性表达与分析

目的通过测定肺癌患者、良性肺病患者和健康人外周血端粒酶活性(TA),探讨其对肺癌的诊断价值。方法采用PCR-TRAP-ELISA法检测不同病理类型、不同临床分期的肺癌患者42例以及良性肺病患者20例和正常人15例外周血单个核细胞(PBMNC)端粒酶活性,并测定肺癌患者等的血CEA水平,进行相关分析。结果42例肺癌患者TA升高的阳性率达69.05%(29/42),OD值为0.45±0.37;20例良性肺病患者只有2例TA升高呈阳性,OD值为0.11±0.06,而15例正常人全部呈阴性,OD值为0.08±0.03。肺癌组血TA升高的阳性率及OD值均高于良性肺病组和正常对照组,差异有显著性(P<0.005)。PCR-TRAP-ELISA法检测肺癌组TA特异度为90%,灵敏度为69.05%,优于CEA检测(P<0.01)。肺癌I期TA阳性率为25%(1/4),而IV期阳性率为100%(8/8),TA高低与临床分期呈明显正相关(r=0.585,P=0.001)。结论1.PCR-TRAP-ELISA法检测外周血单个核细胞TA升高的阳性率明显高于CEA检测,可联合或替代CEA应用于肺癌的诊断及鉴别诊断;2.外周血TA可作为分子指标,辅助TNM分期,用于指导肺癌的治疗及预后判断等。     

多发性硬化患者外周血单个核细胞SP3基因mRNA表达缺失研究

目的 研究我国多发性硬化(multiple sclerosis,Ms)患者外周血单个核细胞基因SP3(specificprotein 3)mRNA表达缺失的特点及其与临床表型的关系.方法 采集56例MS患者外周血单个核细胞,提取总RNA,设计SP3特异性引物,采用逆转录PCR方法观察SP3基因mRNA表达情况,并与其他疾病组、健康对照组进行比较.结果 56例MS患者中23例SP3为阴性结果,SP3表达缺失率为41.1%;健康对照组35例有5例为阴性结果,缺失率为8.6%;其它疾病对照组27例有4例为阴性结果,缺失率为14.3%.MS组与两对照组之间的差异有统计学意义(P＜0.01).SP3表达缺失组与表达组急性期的改良残废程度量表评分差异无统计学意义,稳定期则差异有统计学意义(P＜0.05).结论 通过对MS患者SP3基因mRNA表达的研究,观察到中国人MS存在该基因mRNA表达缺失.SP3与MS临床表现及其免疫学发病机制之间可能存在一定的相关性.     

Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome

BACKGROUND: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. AIMS: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. METHODS: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. RESULTS: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. CONCLUSION: These results suggest that patients with CFS have reproducible alterations in gene regulation.     

Interleukin-4 and interferon-gamma production by peripheral blood mononuclear cells from food-allergic patients

The aim of this study was to investigate whether patients with food allergy had a cytokine imbalance of interleukin (IL)-4 and interferon-gamma (IFN-γ) production. Diagnostic procedures including skin prick tests, determination of food-specific serum IgEs, and positive double-blind, placebo-controlled, food challenges identified 15 adult patients. They were compared with 15 age- and sex-matched healthy subjects. Peripheral blood mononuclear cells (PBMC) were incubated for 24, 48, and 72 h in the presence of phytohemagglutinin plus phorbol myristate acetate. After mitogen stimulation, culture supernatants from patients with food allergy contained significantly less IFN-γ but increased IL-4 when compared with healthy controls. Secretion of IL-4 was maximal at 24 h and IFN-γ secretion was maximal at 72 h. There was no correlation between cytokine secretion in vitro and serum IgE level. These findings demonstrated that an imbalance of IL-4 and IFN-γ production is present in food allergy, as documented in other allergic diseases, but other mechanisms are probably also involved.