Improved measurement of LINE-1 sequence methylation for cancer detection

BackgroundMethylation of long interspersed nuclear element-1 (LINE-1) sequences varies among normal cells and it is often decreased in cancer genomes and white blood cells (WBC) of cancer patients. Current measurement techniques of genome-wide level are inadequate because LINE-1 methylation is distinctive at each locus. Here, we improved the detection of cancer by combining information of LINE-1 methylation pattern and level.MethodsCombined bisulfite restriction analysis (COBRA) of LINE-1, COBRA LINE-1, was used to test cancer cell lines, two oral rinse cohorts, and WBC from normal and cancer patients. COBRA LINE-1 separated LINE-1 sequences into 4 products depending on the methylation statuses of 2 CpG dinucleotides, as follows: 2 unmethylated CpGs (^uC^uC), partial methylation (^mC^uC), 1 methylated CpG (^mC), and 1 unmethylated CpG (^uC).ResultsThe association between ^mC^uC and ^uC^uC was directly correlated in normal cells (r = 0.4895, p = 0.0009) but inversely correlated in cancer (r = ? 0.8979, p = 0.0002). Oral rinse AUC values of ^uC^uC were 0.763 and 0.926 and methylation levels were 0.707 and 0.621, respectively. ^uC^uC, but not overall methylation level, differentiated cancer WBC from normal (p = 0.0082 and p = 0.4830, respectively).ConclusionLINE-1 partial methylation represents hypomethylation in normal cells but hypermethylation in cancer cells. This information improves LINE-1 methylation detection in cancer.     

Autologous serum deprivation restored IL-1 receptor antagonist production by peripheral blood mononuclear cells in patients with gastric cancer

It has been established that cancer patients have immunosuppressive substances in their sera that depress cellular immunity. Although plasma exchanges have been attempted to remove these substances and to improve immunity to cancer, little is known about its mechanism from the viewpoint of cytokine pattern. The levels of the cytokines, tumour necrosis factor-alpha, interleukin 1beta, interleukin 6, interferon-gamma and interleukin-1 receptor antagonist (IL-1ra) by peripheral blood mononuclear cells (PBMC) were determined simultaneously by the whole-blood assay and the PBMC assay in 20 patients with gastric cancer and in 10 healthy volunteers. In both assays the cytokine levels were lower in patients with cancer compared with healthy controls, with the exception of IL-1ra. In the PBMC assay, the IL-1ra level in cancer patients was significantly higher than that in controls. No statistical correlation between the cytokine levels determined by the two assays was found. We suggest that autologous serum deprivation restored and enhanced IL-1ra production, and normalized the cytokine cascade in immune response, in patients with gastric cancer.     

自体外周血单个核细胞血管腔内途径移植治疗下肢缺血性疾病的研究

目的评价动员后自体外周血单个核细胞(PBMNCs)经血管腔内途径移植治疗下肢缺血性疾病的临床疗效及影响因素。方法确诊严重的动脉硬化闭塞症(ASO)患者38例、糖尿病足(DMF)65例和血栓闭塞性脉管炎(TAO)2例,分别给予粒细胞集落刺激因子(G-CSF)300μg肌肉注射,每日2次。第3天时采集和分离PBMNCs,配成单个核细胞混悬液。经皮微球囊扩张狭窄动脉,同时将分离的PBMNCs通过导管腔内注入缺血部位。观察12个月,进行各项指标综合评估。结果PBMNCs移植后,患肢冷感、间歇性跛行等临床症状不同程度好转。跛行距离由入院时的(24.0±14.8)m逐渐提高到12个月时的(642.0±224.1)m,表现为持续延长(P<0.01)。溃疡于移植后1~3个月逐渐愈合。踝肱指数(ABI)短期明显提升,由入院时的0.60±0.07提高到1个月的0.84±0.11,12个月时略有下降,为0.70±0.07,但仍高于入院时的水平。移植前后未出现任何并发症和不良反应。结论采用动员后的PBMNCs经血管腔内途径移植治疗下肢缺血性疾病,方法简单、安全、有效,值得进一步深入研究。     

复发转移性乳腺癌患者外周血BRCA1、CDH1、DKK1和SFRP1甲基化检测意义的研究

目的 探讨BRCA1、CDH1、DKK1和SFRP1基因甲基化与乳腺癌患者肿瘤激素受体状态、复发转移的关系.方法 利用甲基化特异性PCR法(MSP)检测115例复发转移乳腺癌患者外周血BRCA1、CDH1、DKK1和SFRP1的甲基化情况,与65例健康对照组进行比较.结果 BRCA1、CDH1和SFRP1的甲基化状态在...     

Human prostate cancer cells induce inflammatory cytokine secretion by peripheral blood mononuclear cells

A substantial number of studies provide evidence that inflammation may play a significant role in the pathogenesis of prostate cancer via increased activity of inflammatory cytokines, particularly IL-6. We have previously shown that peripheral blood mononuclear cells (PBMC) are capable of carrying out an in vitro “immunomodulatory dialog” with colon cancer cells expressed by an increased production of pro-inflammatory cytokines by PBMC. The aim of the current study was to examine the model of cell-to-cell interaction between PBMC and prostate cancer cells from two lines – androgen resistant (PC-3) and androgen-dependent (LNCaP). For that purpose, cancer cells from both lines were incubated with PBMC, and cytokine secretion by PBMC was evaluated. The results showed a cell-concentration dependent increase in secretion of the pro-inflammatory cytokine IL-6 by PBMC induced by cells from both lines, whereas generation of IL-1β and the anti-inflammatory cytokine IL-10 were found to be increased after incubation with PC-3 cells only. The secretion of IL-10 was slightly lower following incubation of PBMC with supernatants derived from PC-3 cells. The results of the study support the possibility that prostate cancer cell-induced cytokine production by PBMC, and particularly IL-6, are involved in prostate cancer development. The discrepancy between the effect of the two prostate cancer cell lines on cytokine secretion by PBMC may be due to their different androgen dependency.     

SOCS-3在脓毒症患者外周血单个核细胞中表达的研究

目的探讨细胞因子信号转导抑制因子-3（SOCS-3）在脓毒症发病机制中的作用。方法选择36例脓毒症患者,30例健康志愿者为对照组,应用RT-PCR方法测定SOCS-3在外周血单个核细胞中的表达,并和患者APACHEⅡ评分进行相关分析。结果SOCS-3在脓毒症组和对照组的外周血单个核细胞中均有表达,脓毒症组较对照组表达显著增加（P〈0．001）,SOCS-3 mRNA的表达和脓毒症A—PACHEⅡ评分呈负相关,相关系数（r）为-0．746（P〈0．01）。结论SOCS-3参与了脓毒症的发病过程,SOCS-3的表达在脓毒症患者中可能起着保护作用。     

热休克因子1在缺血性脑卒中患者外周血检测的临床意义

目的:探讨急性缺血性脑卒中患者发病48 h内热休克因子1(Hsf-1)水平及其与不同病因学亚型的关系。方法:对62例缺血性脑卒中患者进行TOAST病因学分型、用美国国立卫生研究院神经功能缺损评分(NIHSS)评估神经功能缺损,发病3个月时改良Rankin量表(mRS)评分评估预后。并与正常对照组(31例)用逆转录PCR法检测外周血单核细胞中Hsf-1mRNA的表达水平。结果:神经功能缺损轻型与重型之间差异无统计学意义(P=0.138),大动脉粥样硬化性卒中(LAA)组的Hsf-1表达高于小动脉闭塞性卒中(SAA)组,且差异具有统计学意义(P<0.01),按NIHSS进一步分层后统计学差异仍存在,预后良好组Hsf-1表达高于预后不良组且差异具有统计学意义(P=0.007)。结论:Hsf-1表达测定对缺血性卒中病因学分型有一定的参考价值,并可能成为缺血性卒中患者病情评估的参考指标之一。     

Valsartan reduces interleukin-1beta secretion by peripheral blood mononuclear cells in patients with essential hypertension

BACKGROUND: Chronic low-grade inflammation response may contribute to the pathology of essential hypertension. Angiotensin II (Ang II) may be partly responsible for this process. Our early studies showed that individuals with essential hypertension had increased interleukin-1beta (IL-1beta) secretion by peripheral blood mononuclear cells (PBMCs). In this study, we investigated whether treatment with valsartan, an angiotensin receptor blocker, lowered IL-1beta secretion by PBMCs in patients with essential hypertension. METHODS: Twenty-four patients with essential hypertension were randomized to treatment with valsartan (80 mg/day, group B) or matching routine therapy group (group A) for 2 weeks. PBMCs were isolated by gradient centrifugation. IL-1beta concentrations in supernatant from PBMCs were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with routine therapy group, patients treated with valsartan had decreased secretion of IL-1beta in PBMCs after stimulated by lipopolysaccharide (2857+/-643 vs. 2146+/-508 pg/ml, P<0.05). CONCLUSIONS: We suggest a direct anti-inflammatory effect of valsartan and a pro-inflammatory effect of Ang II in patients with essential hypertension.     

胃癌患者外周血单个核细胞HLA基因表达研究

目的探讨胃癌患者外周血单个核细胞(PBMC)HLA-A、HLA-B、HLA-DP、HLA-DR、HLA-G基因的表达水平及对胃癌的临床应用价值。方法设计HLA-A、HLA-B、HLA-DP、HLA-DR、HLA-G基因引物和实时荧光PCR相对定量法反应体系,在ABI7500实时荧光定量PCR仪上检测43例胃癌患者和22例健康人PBMC内基因表达水平并分析其差异。结果(1)胃癌患者HLA-A、HLA-B、HLA-DP、HLA-DR、HLA-G的mRNA相对表达量分别为0.71±0.26、0.61±0.37、0.91±0.33、0.90±0.38、0.77±0.27。健康组分别为1.23±0.60、1.17±0.60、1.07±0.68、1.12±0.55、0.98±0.37。与健康组相比,胃癌患者HLA-A、HLA-B、HLA-G表达水平降低,差异有统计学意义。HLA-DP和HLA-DR则无明显差异。(2)在不同分化的胃癌患者中,HLA-A、HLA-B、HLA-DP、HLA-DR和HLA-G的mRNA表达水平无明显差异。胃癌Ⅰ期+Ⅱ期的HLA-G表达水平高于Ⅲ期+Ⅳ期,差异有统计学意义。不同分期的HLA-A和H...     

Beta-endorphin concentrations in the peripheral blood mononuclear cells of migraine and tension-type headache patients

Levels of beta-endorphin in peripheral blood mononuclear cells have been studied as a new approach to investigating opioid tone in migraine and tension-type headache. Sixty-one patients with migraine without aura, 39 with migraine with aura and 23 with episodic tension-type headache were compared with 37 healthy controls. Peripheral blood samples were taken from patients not enduring headache attacks and not undergoing prophylactic treatment. A significant reduction in peripheral blood mononuclear cell beta-endorphin concentrations was observed in migraine patients with and without aura, but not in tension-type headache patients. Altered transmitter modulation to peripheral blood mononuclear cells may be the cause of this alteration, which could be part of a more diffuse opioid system derangement in migraine subjects.     

Interleukin-2 production in peripheral blood mononuclear cells of patients with gastrointestinal tumors

Peripheral blood mononuclear cells of patients with different tumors of the gastrointestinal tract (esophagus, stomach, colon, rectum), with Crohn's disease and healthy controls were analyzed for their capacity to produce mitogen induced interleukin-2 (I1-2). Tumor patients could be divided into two groups, one group exhibiting a nearly normal and a second group showing a significantly decreased production of I1-2. Most of the patients with a carcinoma of the colon were found in the low-producer group. Patients with Crohn's disease did not significantly differ from the relevant controls. Tumor patients with proven metastasis produced less I1-2 as compared to those with localized disease although the differences are not significant. When I1-2 containing supernatants of patients with tumors were additionally analyzed for the presence of prostaglandin E2, an inverse relationship could be demonstrated. Supernatants showing a suppressed activity of I1-2 exhibited increased amounts of PGE2, indicating a modulation of I1-2 production by prostaglandin E2.     

Beta-endorphin levels are reduced in peripheral blood mononuclear cells of cluster headache patients

Opioid system hypofunction has been postulated in cluster headache (CH) on the basis of reduced opioid levels found in the cerebrospinal fluid (CSF). In this study beta-endorphin levels were monitored in the peripheral blood mononuclear cells of 65 episodic CH patients (32 in remission and 33 in cluster period) and 50 healthy controls. Beta-endorphin levels were significantly lower than controls in CH patients experiencing both phases of the illness (ANOVA, p < 0.0001). The persistence of this abnormality during pain-free remission suggests a primary alteration in the regulation of beta-endorphin in peripheral blood mononuclear cells. We speculate that these findings reflect reduced CNS levels of beta-endorphin in CH.     

老年冠心病患者外周血粘附细胞产生白细胞介素1的研究

目的 探讨老年冠心病人外周血粘附细胞产生白细胞介素1(IL-1)的变化及其与动脉粥样硬化的关系。方法 采用细胞培养生物学活性检测及放免分析的方法测定冠心病人外周血粘附细胞产生IL-1的活性。结果 老年冠心病人外周血粘附细胞产生IL-1的活性明显高于正常老年人。结论 老年冠心病人IL-1活性的增高可通过对血管平滑肌细胞、内皮细胞、补体以及在炎症损伤中的作用对动脉粥样硬化的发生产生重要影响。     

Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in peritoneal dialysis patients.

OBJECTIVE: The aim of the present study was to examine the association between infection with Chlamydia pneumoniae and symptomatic atherosclerosis in peritoneal dialysis (PD) patients. DESIGN: Cross-sectional study. SETTING: Peritoneal Dialysis Unit of Kingston General Hospital. PATIENTS: Fifty-five prevalent PD patients. OUTCOME MEASURES: (1) Infection with C. pneumoniae diagnosed by detection of DNA in peripheral blood mononuclear cells (PBMCs) using polymerase chain reaction. (2) Symptomatic atherosclerosis involving the coronary, cerebral, or peripheral circulation. RESULTS: The DNA of C. pneumoniae was detected in PBMCs in 33 patients (60.0%). Atherosclerosis was present in 16 of 33 (48%) PBMC C. pneumoniae DNA-positive patients, and in 10 of 22 (45%) PBMC C. pneumoniae DNA-negative patients (p = 0.83). Using multiple logistic regression and controlling for a number of known cardiovascular risk factors, PBMC C. pneumoniae DNA status was not predictive of atherosclerosis. The only significant independent predictors of atherosclerosis were diabetes and age. CONCLUSIONS: In prevalent PD patients, a high prevalence of symptomatic atherosclerosis and of Chlamydia pneumoniae DNA in PBMCs were seen; however, the results of the present study do not support the presence of an association between infection with C. pneumoniae and atherosclerosis.     

肺结核患者外周血单个核细胞中lncRNA NEAT1的检测及临床意义

目的探讨肺结核患者外周血单个核细胞(PBMC)中长链非编码RNA(lncRNA)核富含丰富的转录本1(NEAT1)的表达及其临床意义。方法采用实时荧光定量PCR(RT-PCR)技术检测65例肺结核病患者(结核组)及30例健康体检者(健康对照组)PBMC中lncRNA NEAT1的表达量,分析其与结核病发生发展以及转归的相关性。结果 RT-PCR检测结果显示lncRNA NEAT1在肺结核患者PBMC中的相对表达水平是健康对照组的4.90±0.72倍,差异有统计学意义(P0.01)。LncRNA NEAT1在初治和复治病例中表达差异无统计学意义(P0.05);lncRNA NEAT1在肺部有无空洞及空洞数量分组之间表达差异有统计学意义(P0.05),即lncRNA NEAT1随空洞累及范围的增加而升高。肺结核患者PBMC中lncRNA NEAT1表达量随着治疗时间逐渐下降,恢复至正常水平。结论肺结核患者PBMC中lncRNA NEAT1表达升高,且与肺结核的发展和转归有关,监测PBMC中lncRNA NEAT1表达或可以评估结核病患者预后情况。     

肿瘤病人T细胞核仁嗜银蛋白和辅助性T细胞亚群变化

目的:探讨肿瘤病人外周血单个核细胞(PBMC)中T细胞核仁嗜银蛋白(AgNORs)变化和辅助性T(Th)细胞亚群变化的关系及其临床意义。方法PBMC经多克隆T细胞活化剂刺激,以图像分析法检测AgNORs区面积与细胞核仁面积的百分比(1.S％):PBMC被离子霉素和佛波醇酯活化5h后,以酶联免疫斑点法(ELISPOT)检测IFNY、和IL-4产生细胞的频度(％):以离子霉素加佛波醇酯刺激24h,用ELISA试刑盒检测培养上清液中的IFNY、和IL-4水平。结果 肿瘤病人(n=86)和正常人(n=44),PBMC中活化T细胞的AgNORs表达分别为4.9±1.21.S％和.7.82±1.28 1.S％,肿瘤组非常显著下降,P<0.05:肿瘤组PBMC中1112细胞额度显著上升,Th1／Th2比值显著下降:同时,肿瘤组PBMC产生IFN和IL-4水平发生相应变化,变化方式与Th亚群变化相似:在肺癌组中,转移的与未转移的,复发的与未复发的相比,Th1细胞数、Th1／Th2之比和IFNY、产生水平非常显著下降:在肿瘤组AgNORs表达与Th1细胞频度、Th1／Th2比值、IFNY产生和肿瘤病人生活状态的Kamofsky评分正相关。结论 现有病例观察结果表明,肿瘤病人PBMC小T细胞表达AgNORs下降,与患者Th1细胞功能下降正相关。     

大黄素对糖尿病肾病患者外周血单个核细胞MCP-1的影响

目的探讨大黄素对糖尿病肾病患者外周血单个核细胞MCP-1的影响.方法选取32例非胰岛素依赖型糖尿病伴肾损害患者和健康对照24例,PBMC提取采用Ficoll梯度密度离心法,细胞被分成4组,观察4种不同浓度的大黄素(0、10、50、100μg/ml)对糖尿病肾病患者外周血单个核细胞MCP-1的影响,MCP-1测定采用ELISA法.结果糖尿病肾病外周血单个核细胞MCP-1水平高于正常对照(78.40±35.67vs 29.30±17.31pg/ml,P＜0.05),且与24h尿蛋白定量、尿白蛋白水平呈正相关关系(r=0.633,P＜0.05;r=0.701,P＜0.05;).大黄素(30～100μg/ml)均明显降低了糖尿病肾病患者外周血单个核细胞MCP-1水平(78.40±35.67 vs 61.00±28.90,P＜0.05;78.40±35.67 vs 41.00±18.00,P＜0.01;78.40±35.67vs 21.00±6.90,P＜0.01),大黄素浓度越大,抑制效应越明显(P＜0.01).结论糖尿病肾病尿MCP-1水平是增高的,大黄素能抑制糖尿病肾病患者外周血单个核细胞MCP-1水平,大黄素下调MCP-1的表达可能是其降低蛋白尿、改善肾功能的重要环节.