Plasma homocysteine concentration related to diet, endothelial function and mononuclear cell gene expression among male hyperlipidaemic smokers

BACKGROUND: Elevated plasma concentration of homocysteine is an independent risk factor for development of cardiovascular diseases. MATERIALS AND METHODS: We evaluated potential links between homocysteine and atherothrombogenesis by relating the plasma concentration of homocysteine to (i) dietary antioxidants and omega-3 fatty acids (and determined influence of intervention with antioxidants or omega-3 fatty acids); (ii) markers of endothelial cell function; and (iii) peripheral blood mononuclear cell mRNA levels. RESULTS: We observed an inverse relationship between the plasma homocysteine concentration and dietary intake of vegetables, vitamin C and beta-carotene and between homocysteine and the serum concentration of folate, vitamin B12 and omega-3 fatty acids. Intervention with antioxidants or omega-3 fatty acids did not affect plasma homocysteine concentration. The plasma levels of cysteinylglycine and vitamin B12 correlated positively with circulating E-selectin and VCAM-1, respectively, whereas folate in serum and blood correlated negatively with P-selectin. A negative correlation was found between the concentrations of homocysteine and von Willebrand factor. Negative and positive correlations were found between plasma homocysteine and the mononuclear cell mRNA levels of peroxisome proliferator activated receptor delta (PPAR delta) and c-myc respectively. A negative correlation was also found between plasma homocysteine and mononuclear cell mRNA levels of the proteoglycan serglycin. Homocysteine was not correlated with serum activity of glutathione peroxidase or with the mRNA level of glutathione peroxidase in mononuclear cells. CONCLUSION: The plasma homocysteine level was negatively correlated with dietary intake of vegetables, including vitamins C and E, and serum omega-3 fatty acids, whereas supplementation with antioxidants or omega-3 fatty acids did not affect plasma homocysteine concentration. Homocysteine was not associated with circulating adhesion molecules or increased procoagulant activity, but homocysteine may alter mononuclear cell gene expression. Cysteine showed no significant correlation with these parameters.     

Increased concentrations of antibody-bound circulatory cell-free DNA in rheumatoid arthritis

BACKGROUND: Increased concentrations of cell-free DNA have been found in several disorders and have been interpreted as evidence of increased rates of cell death or turnover. Evidence from in vitro and animal experiments suggests that DNA may play a role in the pathogenesis of rheumatoid arthritis (RA). METHODS: We measured cell-free DNA in plasma and serum from patients with RA and healthy controls by use of quantitative PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA. We used protein G Sepharosetrade mark bead adsorption of plasma and elution to isolate antibody-bound DNA. RESULTS: In paired plasma and serum samples of 16 healthy controls the median GAPDH copies were 4500 genome equivalents (GE)/mL plasma (range 319-21 000) and in 26 RA patients 17 000 GE/mL plasma (2100-2 375 000, P = 0.0001). In the serum from normal controls the median GAPDH copies were 35 000 GE/mL (1700-239 000) and from RA patients 222 000 GE/mL (21 000-2 375 000, P = 0.004). A median of 81% of the cell-free DNA in RA was associated with antibody compared with 9% in healthy controls (P = 0.001). The concentrations of DNA did not vary with the type of therapy patients received. CONCLUSIONS: These results provide new evidence for a role of cell-free DNA-antibody complexes in the etiology of RA, suggest new avenues for basic research, and may prove to be relevant to diagnosis and assessment of therapy.     

Screening of molecular targets and construction of a ceRNA network for oxaliplatin resistance in colorectal cancer

Oxaliplatin resistance reduces the efficacy of chemotherapy for colorectal cancer (CRC). This study aimed to screen molecular targets of oxaliplatin resistance in CRC to construct a ceRNA network. The differentially expressed mRNA and lncRNA between the oxaliplatin-resistant and oxaliplatin-sensitive colon cancer cell lines was determined using RNA sequencing data (no. {"type":"entrez-geo","attrs":{"text":"GSE42387","term_id":"42387"}}GSE42387) from the NCBI GEO database. Gene Ontology BP (biological process) and KEGG pathway enrichment analyses were used to analyze the function and pathway enrichment of the differentially expressed mRNA and lncRNA. The lnCeDB and starBase v2.0 were used to predict miRNA, and Cytoscape software was used to build a ceRNA network. The top 5 mRNA, miRNAs, and lncRNAs with high degrees of connectivity in the ceRNA network were validated by qPCR. TCGA colon cancer clinical data was used to perform a survival analysis of patients with differential mRNA and lncRNA expression. Between the two groups, 2515 mRNAs and 23 lncRNAs were differentially expressed. We constructed a ceRNA network containing 503 lncRNA–miRNA–mRNA regulatory pairs, 210 lncRNA–miRNA pairs, 382 miRNA–mRNA pairs, and 212 mRNA co-expression pairs. The differentially expressed lncRNA, miRNA and mRNA were verified by qPCR. One lncRNA (HOTAIR) and 14 mRNAs significantly correlated with patient prognosis. The discovery of differentially expressed genes and the construction of ceRNA networks will provide important resources for the search for therapeutic targets of oxaliplatin resistance. Moreover, this resource will aid the discovery of the mechanisms behind this type of drug resistance.

Oxaliplatin resistance reduces the efficacy of chemotherapy for colorectal cancer (CRC).     

A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards

The quantitative measurement of gene expression requires consistent and reliable standards. At least two categories of standards, endogenous and exogenous, are currently used for quantitative PCR. The reliability of these two methods, however, has not been carefully compared. We hypothesized that a reliable quantitative PCR assay would be able to detect known dilutions of a given single-stranded (ss-) cDNA. By measuring VEGF ss-cDNA copy numbers or signal ratios of GAPDH/VEGF in 10x and 100x diluted samples of two original ss-cDNA preparations, an exogenous recombinant DNA standard (a VEGF-mimic plasmid) and an endogenously expressed GAPDH standard were tested for their ability to detect dilution factors. Using the recombinant DNA standard, the dilution factor was detected as 10.3 and 135.0 in 10x and 100x diluted samples of the original CaSki cell ss-cDNA, respectively. The detected dilution factors were 12.3 and 226.2, respectively, in 10x and 100x diluted ss-cDNA from U-251 MG cells. On the other hand, with the endogenous GAPDH standard, the dilution factors were detected as 2.7 and 8.0 in the same 10x and 100x dilutions of the original U-251 MG cell ss-cDNA. Using the same endogenous GAPDH standard, the detected dilution factors were both 4.8 in 10x and 100x dilutions of the original CaSki cell ss-cDNA. It was also found that the number of endogenous copies of GAPDH mRNA was about 1000 times higher than VEGF. The high internal lockup ratio of GAPDH vs VEGF copy numbers and the requirement for additional primer pairs make the use of an abundant endogenous standard an unreliable choice in quantitative or semi-quantitative PCR. In contrast, exogenous standard-based quantitative PCR was shown to be an accurate and reliable method for the quantitation of gene expression.     

Measurement of the noncomplexed free fraction of tissue inhibitor of metalloproteinases 1 in plasma by immunoassay

BACKGROUND: We previously found differences in total concentrations of tissue inhibitor of metalloproteinases 1 (TIMP-1) in plasma from donors and cancer patients. Because TIMP-1 can exist in more than one molecular form, a new immunoassay to specifically detect free TIMP-1 was developed and concentrations were determined in plasma from healthy donors and colorectal cancer (CRC) patients. METHODS: We established and validated an immunoassay for the specific measurement of free TIMP-1 that uses a polyclonal anti-TIMP-1 antibody for capture and a monoclonal anti-TIMP-1 antibody that binds only free TIMP-1 for detection of antigen. Plasma samples from healthy donors and CRC patients were assayed for free TIMP-1. Total TIMP-1 was measured by our previously published assay. RESULTS: The mean (SD) concentrations of free TIMP-1 were similar in citrate [55.5 (11.5) microg/L] and EDTA plasma [58.9 (13.3) microg/L] from 76 donors (r(2) = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) microg/L] to total TIMP-1 [83.8 (19.8) microg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r(2) = 0.19; total, r(2) = 0.27; P <0.0001), increasing 50% over an age span of 45 years. Free and total TIMP-1 were significantly increased in CRC patients (P <0.0001), whereas the ratio of free to total TIMP-1 (mean, 0.58) was significantly lower than in donors. CONCLUSIONS: Most of the TIMP-1 in donor plasma is present in its free form, and free TIMP-1 increases with age. Free and total TIMP-1 are increased in CRC patient plasma, but the ratio of free to total TIMP-1 is significantly lower in these patients than in donors.     

丹参酮ⅡA抑制醛固酮生物合成的作用

背景醛固酮是左心室肥厚重要的致病因子,有证据提示中药丹参提取物能够干预醛固酮的生物合成. 目的探讨丹参酮ⅡA对心肌醛固酮合成相关基因表达的作用. 设计随机对照观察. 单位华中科技大学同济医学院附属同济医院. 材料实验于2002-11/2004-03在华中科技大学同济医学院附属同济医院急诊科实验室完成,选用12周龄雄性自发性高血压SHR大鼠20只,随机分为高血压组、丹参酮ⅡA组,每组10只. 方法丹参酮ⅡA组经尾静脉注射丹参酮ⅡA溶液1.5 mg/(kg·d),高血压组给予相当容积的蒸馏水.给药12周后断头处死大鼠,留取心肌标本,通过反转录-聚合酶链反应,以GAPDH基因扩增引物表为内参照,分别测定心肌醛固酮合成相关基因CYP11B1及CYP11B2 mRNA表达. 主要观察指标心肌醛固酮合成相关基因CYP11B1及CYP11B2 mRNA表达水平. 结果20只大鼠被纳入实验,并全部进入结果分析,无脱失值.①两组大鼠心肌CYP11B1及CYP11B2基因表达的定性分析以100bp Plus Lad-der为Marker,分别在440 bp,461 bp及336 bp处可见清晰的扩增条带,DNA测序证实为CYP11B1,YP11B2及GAPDH的编码基因片段.②两组大鼠心肌CYP11B1及CYP11B2基因表达的定量分析丹参酮ⅡA组CYP11B1及CYP11B2的mRNA表达水平明显低于高血压组(0.924±0.121,1.343±0.132,P＜0.05;1.017±0.119,1.675±0.126,P＜0.01). 结论丹参酮ⅡA可能通过直接下调心脏局部醛固酮合成相关基因CYP11B1及CYP11B2 mRNA表达,抑制心脏局部醛固酮的生物合成,从而发挥抗高血压左室肥厚的效应.     

Circulating cell-free nucleic acids as biomarkers in colorectal cancer screening and diagnosis

Screening methods for the most frequent diagnosed malignant tumor, colorectal cancer (CRC), have limitations. Circulating cell-free DNA (cfDNA) analysis came into focus as a potential screening test for CRC. Detection of epigenetic and genetic alterations of cfDNA as DNA methylation or DNA mutations and related ribonucleic acids may improve cancer detection based on unique, CRC-specific patterns. In this review the authors summarize the CRC-specific nucleic acid biomarkers measured in peripheral blood and their potential as screening markers. Detection of DNA mutation has inadequate sensitivity; however, methylated DNA can be established with higher sensitivity from CRC plasma samples. The ribonucleic acid based miRNA studies represented higher sensitivity for CRC as compared with mRNA studies. Recently, isolation of cfDNA has become automated, highly reproducible and a high throughput method. With automated possible diagnostic tools, a new approach may be available for CRC screening as liquid biopsy.     

荧光定量RT—PCR实时检测心脏移植患者外周血单核细胞16种免疫排斥基因整体表达变化的初步研究

目的探讨外周血单核细胞免疫排斥相关基因的检测对心脏移植排斥反应的诊断价值。方法采取心脏移植术患者外周血,应用实时荧光定量RT-PCR技术,观察心脏移植术后不同时期外周血单个核细胞中与免疫排斥相关多个基因系统的mRNA表达水平,并与术前和正常组的基因表达水平对照。结果心脏移植术前外周血单个核细胞16种与免疫排斥相关候选基因的mRNA的相对表达量与正常对照组比较差异无统计学意义（P〉0．05）。初步筛选出与机体免疫排斥状态有更大相关性的7种基因,术后3个月内心脏功能稳定组较术前和正常对照组ITGA4、FKB、ILlR-2mRNA表达水平上调,PF4、ITGAM、TGFβ1、RHOU表达水平降低。这与临床观察的移植术后1～3个月免疫排斥概率最大相吻合。结论检测外周血单个核细胞基因mRNA表达水平的荧光定量RT—PCR方法简便快速、特异、重复性好、可信度高,但在检测心脏移植排斥反应方面值得进一步深入研究。     

Anticoagulants affect matrix metalloproteinase 9 levels in blood samples of stroke patients and healthy controls

ObjectivesMatrix metalloproteinase-9 (MMP-9) represents a promising marker for acute stroke management. In clinical studies MMP-9 has been quantified by ELISA using differing protocols. We aimed to establish a valid protocol by evaluation of preanalytics.Design and methodsBlood from stroke patients (n = 28) and healthy controls (n = 28) was drawn into tubes containing different anticoagulants (EDTA, citrate, lithium-heparin (heparin) and heparin with proteinase inhibitors) and processed after 0, 60 and 240 min. MMP-9 plasma protein and mRNA from mononuclear leukocytes were determined.ResultsIn regard to anticoagulants used, samples showed different MMP-9 protein baseline values and kinetics. Stable MMP-9 protein concentrations were only measured from EDTA samples. Particularly in samples with proteinase inhibitors protein and mRNA concentrations increased over time. Kinetics did not differ between patients and controls.ConclusionsPreanalytics plays a key role for determination of MMP-9. EDTA seems to be a valid anticoagulant for MMP-9 protein measurement.     

肺癌患者血浆及外周血单个核细胞Lunx mRNA检测的临床意义

目的检测肺癌患者血浆及外周血单个核细胞肺特异性X基因(Lunx)mRNA,探讨两者对肺癌辅助诊断的临床意义。方法采用荧光定量聚合酶链反应(PCR)检测肺癌患者、肺良性疾病患者、肺外肿瘤患者及健康人血浆及外周血单个核细胞Lunx mRNA。结果肺癌组患者血浆Lunx mRNA阳性率显著高于肺良性疾病组(χ2=113.10,P<0.01)、肺外肿瘤组(χ2=125.34,P<0.01)和健康组(χ2=100.33,P<0.01);Ⅲ～Ⅳ期肺癌患者血浆Lunx mRNA阳性率高于Ⅰ、Ⅱ期肺癌患者(χ2=7.07,P<0.05)。肺癌组患者外周血单个核细胞LunxmRNA阳性率显著高于肺良性疾病组(χ2=32.79,P<0.01)、肺外肿瘤组(χ2=44.44,P<0.01)和健康组(χ2=44.44,P<0.01);Ⅲ～Ⅳ期肺癌患者外周血单个核细胞Lunx mRNA阳性率显著高于Ⅰ、Ⅱ期肺癌患者(χ2=24.52,P<0.01)。血浆Lunx mRNA检测对肺癌辅助诊断的敏感性高于单个核细胞检测的敏感性(χ2=36.46,P<0.01),血浆检测的阴性预测值高于外周血单个核细胞检测的阴性预测值(χ2=16.37,P<0.01)。结论血浆与外周血单个核细胞Lunx mRNA检测均可用于肺癌的辅助诊断,前者敏感性较后者高。