Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.     

DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith–Wiedemann syndrome

Tierling S, Souren NY, Reither S, Zang KD, Meng‐Hentschel J, Leitner D, Oehl‐Jaschkowitz B, Walter J. DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith–Wiedemann syndrome. Beckwith–Wiedemann syndrome (BWS) is one of the most prevalent congenital disorders predominantly caused by epigenetic alterations. Here we present an extensive case study of a monozygotic monochorionic male twin pair discordant for BWS. Our analysis allows to correlate BWS symptoms, like a protruding tongue, indented ears and transient neonatal hypoglycaemia, to an abnormal methylation at the KvDMR1. DNAs extracted from peripheral blood, skin fibroblasts, saliva and buccal swab of both twins, their sister and parents were analysed at 11 differentially methylated regions (DMRs) including all four relevant DMRs of the BWS region. The KvDMR1 was exclusively found to be hypomethylated in all cell types of the affected BWS twin, while the unaffected twin and the relatives showed normal methylation in fibroblasts, buccal swab and saliva DNA. Interestingly, the twins share a common blood‐specific hypomethylation phenotype most probably caused by a feto‐fetal transfusion between both twins. Because microsatellite analysis furthermore revealed a normal biparental karyotype for chromosome 11, our results point to an exclusive correlation of the observed BWS symptoms to locally restricted epimutations at the KvDMR1 of the maternal chromosome.     

Differential DNA methylation profiles of peripheral blood mononuclear cells in allergic asthmatic children following dust mite immunotherapy

Background/PurposeAllergen-specific immunotherapy (SIT) is now considered curative to allergic diseases such as asthma. Mechanistically, our previous work showed DNA hypermethylation of cytokine genes, in T-helper cells, in allergic asthmatic children treated with allergen-SIT. In this study, we extended to work to assess possible changes in the DNA methylomes of peripheral blood mononuclear cells (PBMCs), isolated from mite allergen-SIT asthmatic children, to explore further the underlying methylation changes.MethodsThirteen allergic asthmatic children who received Der p-SIT, 12 non-SIT allergic asthmatic controls, and 12 healthy controls were enrolled. Bisulfite-converted DNA from Der p-stimulated PBMCs was analyzed using Human Methylation 450 k BeadChip. Pyrosequencing and quantitative real-time PCR were used to validate the DNA methylation levels and the gene expression of individual samples.ResultsWe identified 108 significantly differentially methylated regions (DMRs) unique to Der p-treated PBMCs, with 53 probes linked to demethylated DMRs, and 55 probes linked to methylated DMRs. Three associated genes (BCL6, HSPG2, and HSP90AA1), of selected DMRs, were subjected to bisulfite pyrosequencing. Of these, BCL6 showed significant hypomethylation, while HSPG2 and HSP90AA1 were hypermethylated in SIT group, compared to the AA group. Furthermore, SIT group had significantly higher gene expression of BCL6 and lower gene expression of HSPG2. KEGG pathway analysis further revealed DMR genes involved in ECM-receptor interactions, asthma, and antigen processing and presentation pathways.ConclusionsSeveral DNA regions showed DNA methylation altered by Der p specific immunotherapy, indicating desensitization-associated methylomes. Genes belonging to these SIT-altered pathways may represent therapeutic targets for better clinical management of asthma.     

外周血单个核细胞中mRNA表达与类风湿关节炎活动度的相关性研究

目的 以28个关节疾病活动度评分(disease activity score in 28 joints,DAS28)为基础对不同活动度群组的类风湿关节炎(rheumatoid arthritis, RA)病人和健康人群进行分析,挑选出RA活动度相关基因,为RA的诊断治疗提供新思路与线索。方法 收集苏州大学附属第一医院风湿免疫科门诊28个不同活动度的RA病人和18个健康人的基本信息并对它们外周血单个核细胞中mRNA水平进行检测,通过生物信息学分析寻找活动度相关基因。结果 筛选出80个单纯与RA活动度相关的基因,36个与RA活动度和发病都有关系的基因。在这些基因中,12个单纯与活动度相关的基因(SOCS3、C12orf44、KIFC1、MRPS18A、GYG1、RAB35、B4GALT5、LRRC41、LILRB4、LILRA5、CR1、PDIA6)与DAS28显著相关(r值分别为0.55、0.53、0.52、0.49、0.44、0.42、0.42、0.41、0.41、0.41、0.40、0.39,P值均<0.05)且呈趋势性下降,与发病和活动度都与相关基因PPIH与DAS28显著相关(r=-0.38,P<0.05)且呈趋势性上升。生物功能聚类发现有四项功能和甲基化相关,一项功能和应激反应相关。蛋白质关联分析显示HIST1H3E、HIST1H4I、HIST1H4F、HIST1H3D和HIST1H3F这五个基因不仅出现在与甲基化相关的功能中,而且在实验、文本挖掘和共表达三个方面都存在相互关系。结论 以DAS28为基础挑选出80个单纯与RA活动度相关的基因,功能分析表明他们大多受到甲基化调控作用,部分与免疫应激反应有关。     

AHRR methylation predicts smoking status and smoking intensity in both saliva and blood DNA

Many existing DNA repositories do not have robust characterizations of smoking, while for many currently ongoing studies, the advent of vaping has rendered traditional cotinine‐based methods of determining smoking status unreliable. Previously, we have shown that methylation status at cg05575921 in whole blood DNA can reliably predict cigarette consumption. However, whether methylation status in saliva can be used similarly has yet to be established. Herein, we use DNA from 418 biochemically confirmed smokers or nonsmokers to compare and contrast the utility of cg05575921 in classifying and quantifying cigarette smoking. Using whole blood DNA, a model incorporating age, gender, and methylation status had a receiver operating characteristic (ROC) area under the curve (AUC) for predicting smoking status of 0.995 with a nonlinear demethylation response to smoking. Using saliva DNA, the ROC AUC for predicting smoking was 0.971 with the plot of the relationship of DNA methylation to daily cigarette consumption being very similar to that seen for whole blood DNA. The addition of information from another methylation marker designed to correct for cellular heterogeneity improved the AUC for saliva DNA to 0.981. Finally, in 31 subjects who reported quitting smoking 10 or more years previously, cg05575921 methylation was nonsignificantly different from controls. We conclude that DNA methylation status at cg05575921 in DNA from whole blood or saliva predicts smoking status and daily cigarette consumption. We suggest these epigenetic assessments for objectively ascertaining smoking status will find utility in research, clinical, and civil applications.     

DENV2感染外周血单个核细胞增加TNF-α基因启动子区域CpG位点的甲基化水平

 目的：检测正常人外周血单个核细胞（PBMC）感染2型登革病毒（DENV2）后肿瘤坏死因子α（TNF-α）基因启动子区域CpG位点的甲基化水平。
方法：采用亚硫酸氢盐测序PCR法检测DNA甲基化水平。结果：TNF-α基因启动子区域为-294 bp到+58 bp,覆盖11个散在CpG位点;PCR反应后取PCR产物进行琼脂糖凝胶电泳分析显示,扩增序列大小与理论预测相符合;PBMC感染DENV2 0 h和6 h 在11个甲基化位点中有2个处于甲基化状态,感染12 h有6个甲基化位点甲基化。0 h、6 h和12 h的平均甲基化率分别为103%、121%和255%,且0 h和12 h及6 h和12 h的甲基化率差异有统计学意义。结论：PBMC感染DENV2后会引起TNF-α基因启动子区域的甲基化水平增加。     

Telomere length, telomerase activity, and expression of human telomerase reverse transcriptase mRNA in growth plate of epiphyseal articular cartilage in femoral head during normal human development and in thanatophoric dysplasia

Telomeres are important in chromosome structure and function, protecting against their degradation. However, few studies have examined telomeres in growth plates within articular cartilage during normal development. We investigated frozen sections that were obtained from 57 reference autopsy cases (aged from 16 weeks of gestation to 91 years) and from 2 patients with thanatophoric dysplasia. In the reference cases, telomere length was significantly longer in growth plates obtained from the 10 cases that were aged from 16 weeks of gestation to 10 years than in those from 47 of the adult cases (aged 20 to 91 years). In fetal, neonatal, and child cases, telomerase activity was significantly higher in the hypertrophied zone (HZ) in growth plates than in the other 3 zones. The hTERT mRNA staining intensity (staining area) was stronger (larger) in HZ and the proliferating zone than in the calcified zone and resting zone. In thanatophoric dysplasia, telomere length and telomerase activity were short and low, respectively, compared with those of normal growth plates at an equivalent age, and expression of hTERT mRNA was negative or weakly positive in all 4 zones within growth plates. These results suggest that telomere length and telomerase activity have significant effects in the growth plates of articular cartilage, particularly at developmental ages from fetus to child. We speculate that short telomere length and low telomerase activity may be important for chondrocyte differentiation in rhizomeric shortening of the limbs in thanatophoric dysplasia.     

Immunoassay and molecular methods to investigate DNA methylation changes in peripheral blood mononuclear cells in HIV infected patients on cART

This study aimed to investigate the influence of antiretroviral therapy on methylation markers, in a group of HIV infected, heavily treated patients. Immune and molecular methods were used to investigate potential changes in methylation profile in DNA isolated from peripheral blood mononuclear cells collected from antiretroviral-experienced HIV infected patients and healthy controls. The percentage of 5-methylcytosine was inversely correlated with proviral DNA and active replication while DNMT1 (p = 0.01) and DNMT3A (p = 0.004) independently correlated with active viral replication. DNMT3A expression increased with total treatment duration (p = 0.03), number of antiretroviral drugs ever used (p = 0.003), and cumulative exposure to protease inhibitors (p = 0.02) even in currently HIV undetectable patients.     

Oxidative stress,telomere shortening,and DNA methylation in relation to low‐to‐moderate occupational exposure to welding fumes

Evidence suggests that exposure to welding fumes is a risk factor for lung cancer. We examined relationships between low‐to‐moderate occupational exposure to particles from welding fumes and cancer‐related biomarkers for oxidative stress, changes in telomere length, and alterations in DNA methylation. We enrolled 101 welders and 127 controls (all currently nonsmoking men) from southern Sweden. We performed personal sampling of respirable dust and measured 8‐oxodG concentrations in urine using a simplified liquid chromatography tandem mass spectrometry method. Telomere length in peripheral blood was measured by quantitative polymerase chain reaction. Methylation status of 10 tumor suppressor genes was determined by methylation‐sensitive high‐resolution melting analysis. All analyses were adjusted for age, body mass index, previous smoking, passive smoking, current residence, and wood burning stove/boiler at home. Welders were exposed to respirable dust at 1.2 mg/m³ (standard deviation, 3.3 mg/m³; range, 0.1–19.3), whereas control exposures did not exceed 0.1 mg/m³ (P < 0.001). Welders and controls did not differ in 8‐oxodG levels (β = 1.2, P = 0.17) or relative telomere length (β = ?0.053, P = 0.083) in adjusted models. Welders showed higher probability of adenomatous polyposis coli (APC) methylation in the unadjusted model (odds ratio = 14, P = 0.014), but this was not significant in the fully adjusted model (P = 0.052). Every working year as a welder was associated with 0.0066 units shorter telomeres (95% confidence interval ?0.013 to ?0.00053, P = 0.033). Although there were no clear associations between concentrations of respirable dust and the biomarkers, there were modest signs of associations between oxidative stress, telomere alterations, DNA methylation, and occupational exposure to low‐to‐moderate levels of particles. Environ. Mol. Mutagen. 56:684–693, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.     

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.     

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。     

Detecting Epstein-Barr virus DNA from peripheral blood mononuclear cells in adult patients with systemic lupus erythematosus in Taiwan

Epstein-Barr virus (EBV) has been found by many serology studies to be associated with systemic lupus erythematosus (SLE). However, the results of DNA studies have been conflicting. Therefore, instead of antibody to EBV, we studied the association between EBV DNA and SLE. In this case-control study in Taiwan, we enrolled 87 SLE patients and 174 age- and sex-matched controls. Peripheral blood mononuclear cells of SLE patients and matched controls were tested for EBV DNA by polymerase chain reaction (PCR) and Southern blot. Of the 87 SLE patients, 71 (81.6%) were found to be positive for EBV DNA, while 85 (48.9%) of the 174 controls (odds ratio 4.64, 95% confidence interval 2.50–8.62, P<0.0001) were positive. While the EBV DNA-positive rate did not decline with age in SLE patients (P>0.05), it did decline with age in controls (P<0.05). Furthermore, based on a real-time quantitative PCR study, we have found a significant difference between EBV viral load in SLE and controls (P=0.008). Therefore, in our molecular study of DNA level, we found evidence for the association of EBV infection and SLE, suggesting that EBV contributes, if not to the development of SLE, then to disease perpetuation.     

2型糖尿病肾病患者外周血单个核细胞组织因子活性检测的意义

目的探讨2型糖尿病肾病患者外周血单个核细胞组织因子促凝活性（TF-PCA）的改变及其临床意义。方法 63名2型糖尿病患者按照尿白蛋白/肌酐比共分为三组：比值〈30mg/g为正常蛋白尿组（n=22）,30mg/g≤比值〈300mg/g为微量白蛋白尿组（n=26）,比值≥300mg/g为大量白蛋白尿组（n=15）;同时选择21名健康体检者作为对照组。采用一期凝固法测定外周血单个核细胞TF-PCA。常规检测空腹血糖、糖化血红蛋白、超敏CRP（Hs-CRP）、尿酸（UA）、胱抑素C（CYSC）,并进行相关分析。结果糖尿病组外周血TF-PCA随尿蛋白水平升高而升高,同时单个核细胞TF-PCA与空腹血糖、Hs-CRP、UA等呈正相关,相关系数分别为0.419、0.293、0.232（P〈0.05）。结论 2型糖尿病患者外周血单个核细胞TF-PCA明显升高,且与多种因素相关,TF-PCA升高可能与DN的发病机制、病程进展相关。     

Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins

Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%–5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%–16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T1D; however, findings do not support the involvement of large DNAm differences at single CpG sites alone in T1D.     

Gene expression profiling in peripheral blood mononuclear cells from lupus patients with active and inactive disease

Systemic lupus erythematosus (SLE) is characterized by periods of flare and remission. The search for parameters associated with disease activity has been an area of intense investigation. To identify genes that best differentiate patients with active from those with inactive disease, the expression pattern of 375 genes was analyzed in peripheral blood mononuclear cells (PBMC) from 12 patients with active and 14 patients with inactive disease. Using the "nearest shrunken centroids" method, 29 genes were found to best discriminate the two groups. Among these genes, 14 were upregulated and 15 were downregulated in patients with active compared to those with inactive disease. Fourteen of these genes also correlated with SELENA-SLEDAI with correlation coefficients ranging from 0.4 to 0.7. Most of these genes have not been previously associated with disease activity and belong to a variety of families such as adhesion molecules, proteases, TNF superfamily, and neurotrophic factors. Using a cross-validation method, the error rate for classifying samples in the two groups was 30%. These results highlight the potential use of microarray data in identifying genes associated with disease activity in SLE, which could become potential biomarkers or future therapeutic targets.     

A method for production and determination of histamine releasing activity from human peripheral blood mononuclear cells

Histamine releasing factors, i.e. cytokines capable of inducing histamine release from basophils or mast cells, have been suggested to be involved in the pathogenesis of, for example, allergic late-phase reactions. Here we describe a controlled method for production and determination of histamine releasing activity (HRA) from human peripheral blood mononuclear cells (MNC). MNC were incubated with concanavalin A (Con A) for 2 h and cultured for another 40 h in fresh serum free medium. The culture supernatants were concentrated 19–25 fold by ultrafiltration (molecular weight cut-off: 3000 Da). The preparations of HRA induced dose- and Ca²⁺-dependent histamine release from leukocytes. Supernatants of parallel cultures of unstimulated MNC did not induce histamine release. The HRA was neither due to exogenous histamine releasing compounds (e.g. Con A) nor to residual histamine in the preparations of HRA. The kinetics of HRA induced histamine release (half-maximal release after >40 min) were slower and more protracted than those of anti-IgE induced histamine release. However, based on a comparison between HRA induced histamine release from leukocytes and purified (97%) basophils, this did not appear to be due to an indirect effect on the basophils. Finally, neither the production of nor the response to HRA was dependent on the allergic status of the donor.