Genes In Peripheral Blood Leukocytes Research Articles

Expression level of long non-coding RNA MALAT1, GAS5, DANCR and TUG1 in peripheral blood leukocytes of patients with non-alcoholic fatty liver disease

Purpose: Comparative analysis of the expression level of long non-coding RNAs MALAT1, GAS5, DANCR, TUG1 in peripheral blood leukocytes (PBL) of healthy people and patients with NAFLD (liver steatosis, NASH of varying activity, liver cirrhosis). Materials and methods: We examined 106 patients diagnosed with NAFLD for the first time: 31 patients with liver steatosis (LS), 64 patients with weak (WA), moderate (MA) and high (HA) NASH activity and 11 patients at the stage of liver cirrhosis (LC). The control group consisted of 30 healthy donors. The mRNA level of the TUG1, DANCR, MALAT1, GAS5 genes in PBL was determined by RT-PCR. Results: A higher level of expression of the TUG1 gene was registered in the PBL of patients with NASH-WA compared to LS, and a tendency was revealed to increase the level of TUG1 mRNA in the PBL with increasing NASH activity, which indicates the possibility of using the level of TUG1 expression in the PBL as a minimally invasive diagnostic (to distinguish between LS and NASH-WA) and a prognostic marker (with the progression of NAFLD). Analysis of the expression level of lncRNA MALAT1 showed no significant differences between all studied groups. Results were obtained indicating complex dynamics of the GAS5 expression level: the level of transcripts increases during the formation of liver steatosis and then decreases during the transition to NASH. It was shown that the level of DANCR expression in the PBL of patients with NASH-WA is significantly lower than in patients with liver steatosis and NASH-MA. Conclusion: New data were obtained on the expression level of the MALAT1, GAS5, DANCR, TUG1 lncRNAs in the PBL of patients with NAFLD, indicating the possibility of using the level of TUG1 expression in the PBL as a minimally invasive diagnostic and prognostic marker in NAFLD. It has also been shown that the level of DANCR mRNA in PBL may have some diagnostic value in distinguishing between LS and NASH-WA.

Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

Impact of anthracycline-based chemotherapy on RB1 gene methylation in peripheral blood leukocytes and biomarkers of oxidative stress and inflammation in sarcoma patients.

We investigated the impact of anthracycline-based chemotherapy on methylation status of RB1 gene in peripheral blood leukocytes together with parameters of oxidative stress and inflammation in sarcoma patients. Blood samples were collected from 51 consecutive newly diagnosed sarcoma patients admitted to University Hospital Center Zagreb (Zagreb, Croatia) for first-line chemotherapy before the first cycle and post-chemotherapy. Methylation and copy number variation (CNV) of leukocyte RB1 gene were assessed using MS-MLPA probes. In addition, in blood samples, parameters of oxidative stress (ROS, MDA, SOD, and GSH) and inflammation (CRP, WBC, and NBC) were followed. In pre-chemotherapy samples, no CNVs and aberrant methylation of CpG106 promoter region of RB1 gene were detected; however, one patient had hypermethylation (by approximately 10%) of imprinted locus CpG85 in intron 2 of RB1 gene. In addition, a very good correlation of the tumor burden and CRP and tumor burden and GSH was found. The anthracycline-based chemotherapy reverts methylation of RB1 gene-imprinted locus CpG85 to normal level. Moreover, inflammation and oxidative stress parameters such as CRP, WBC, ROS, and MDA were significantly decreased in post-chemotherapy samples. This single-centered study on a cohort of consecutive sarcoma patients indicates that sarcoma patients can have aberrant germline DNA methylation and confirms the relationship of tumor burden with inflammation and oxidative stress. The applied chemotherapy protocols reverted RB1 gene methylation to normal level and decreased the level of inflammation and oxidative damage, thus indicating chemotherapy benefit to the patient's health status.

Traumatic brain injury-induced disruption of the circadian clock

Disturbances in the circadian rhythm have been reported in patients following traumatic brain injury (TBI). However, the rhythmic expression of circadian genes in peripheral blood leukocytes (PBL) following TBI has not yet been studied. The messenger ribonucleic acid (mRNA) expression of period 1 (Per1), Per2, Per3, cryptochrome 1 (Cry1), Cry2, brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) was quantified in PBLs from sham-operated rats and rats with acute subdural hematoma (ASDH) over a 48-h period. The rectal temperature of the animals was measured every 4 h over 2 days. The mesor, rhythm, amplitude, and acrophase were estimated using cosinor analysis. Cosinor analysis revealed that Per2, Cry1, and Bmal1 mRNAs were rhythmically expressed in the PBLs of sham-operated rats. In contrast, fluctuations in rhythmic expression were not observed following ASDH. The rectal temperature of sham-operated rats also exhibited rhythmicity. ASDH rats had a disrupted rectal temperature rhythm, a diminished amplitude, and an acrophase shift. TBI with ASDH results in dysregulated expression of some circadian genes and changes in body temperature rhythm. Further research is required to understand the pathophysiology of altered circadian networks following TBI.Key messagesFirst to investigate the mRNA expression of circadian genes in PBLs of ASDH rats.ASDH rats had disrupted rhythmicity of Per2, Cry1, and Bmal1 mRNA expression.Cosinor analysis showed that ASDH rats had a disrupted rectal temperature rhythm.

Identification of novel biomarkers related to neutrophilic inflammation in COPD.

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent chronic respiratory diseases and the fourth cause of mortality globally. Neutrophilic inflammation has a vital role in the occurrence and progression of COPD. This study aimed to identify the novel hub genes involved in neutrophilic inflammation in COPD through bioinformatic prediction and experimental validation. Both the single-cell RNA sequencing (scRNA-seq) dataset (GSE173896) and the RNA sequencing (RNA-seq) dataset (GSE57148) were downloaded from the Gene Expression Omnibus (GEO) database. The Seurat package was used for quality control, dimensions reduction, and cell identification of scRNA-seq. The irGSEA package was used for scoring individual cells. The Monocle2 package was used for the trajectory analysis of neutrophils. The CIBERSORT algorithm was used for analysis of immune cell infiltration in the lungs of COPD patients and controls in RNA-seq dataset, and weighted gene co-expression network analysis (WGCNA) correlated gene modules with neutrophil infiltration. The Mendelian randomization (MR) analysis explored the causal relationship between feature DEGs and COPD. The protein-protein interaction (PPI) network of novel hub genes was constructed, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate novel hub genes in clinical specimens. In scRNA-seq, the gene sets upregulated in COPD samples were related to the neutrophilic inflammatory response and TNF-α activation of the NF-κB signaling pathway. In RNA-seq, immune infiltration analysis showed neutrophils were upregulated in COPD lung tissue. We combined data from differential and modular genes and identified 51 differential genes associated with neutrophilic inflammation. Using MR analysis, 6 genes were explored to be causally associated with COPD. Meanwhile, 11 hub genes were identified by PPI network analysis, and all of them were upregulated. qRT-PCR experiments validated 9 out of 11 genes in peripheral blood leukocytes of COPD patients. Furthermore, 5 genes negatively correlated with lung function in COPD patients. Finally, a network of transcription factors for NAMPT and PTGS2 was constructed. This study identified nine novel hub genes related to the neutrophilic inflammation in COPD, and two genes were risk factors of COPD, which may serve as potential biomarkers for the clinical severity of COPD.

Study of the Association of Polymorphic Variants rs2295080 and rs1883965 of the <i>MTOR</i> Gene with the Development of Pulmonary Sarcoidosis

The aim of the study was to study the association of polymorphic variants rs2295080 and rs1883965 of the MTOR gene with the risk of developing pulmonary sarcoidosis. The study included 253 people (122 patients diagnosed with morphologically verified sarcoidosis with lung involvement (average age – 41.00 ± 12.56 years) and 131 healthy people (control group) (average age – 44.00 ± 14.23). The distribution of alleles and genotypes of polymorphic markers rs2295080 and rs1883965 of the MTOR gene was analyzed in the study groups. There was a statistically significant increase in the level of mRNA expression of the MTOR gene in PBL of patients with pulmonary sarcoidosis compared with the control group (p = 0.007). A decrease in the number of transcripts of this gene was noted in patients receiving therapy compared with patients without therapy (p = = 0.025). There were no statistically significant differences in the distribution of allele and genotype frequencies for polymorphic markers rs2295080 and rs1883965 of the MTOR gene in the group of patients with pulmonary sarcoidosis and in the control group: (χ2 = 0.196, d.f. = 1, p = 0.658 and χ2 = 0.637, d.f. = 2, p = = 0.728) and (χ2 = 0.034, d.f. = 1, p = 0.855 and χ2 = 0.051, d.f. = 2, p = 0.975) respectively. Conclusion: аn increased level of expression of the MTOR gene in peripheral blood leukocytes of patients with pulmonary sarcoidosis may indicate the involvement of this gene in the pathogenesis of this disease. Polymorphic markers rs2295080 and rs1883965 of the MTOR gene are not associated with the risk of developing pulmonary sarcoidosis. Probably, an increase in the expression level of the MTOR gene in patients with pulmonary sarcoidosis is due to the development of inflammation.

<i>NOS2</i>, <i>NOS3</i>, <i>SONE</i> gene transcripts levels in peripheral blood leukocytes and their relationship with markers of endothelial dysfunction in hypertension

Objective. The aim of the study was to evaluate the level of expression of the NOS2, NOS3, SONE genes in peripheral blood leukocytes (PBL) of patients with hypertension (HTN) and to study the relationship between the level of transcripts of these genes and the content of nitric oxide metabolites and markers of endothelial dysfunction.Design and methods. The study included healthy people (25 people) and patients with HTN (stages I–II) before prescribing antihypertensive drugs (15 people) and taking cardioselective β-adrenergic receptor blockers for more than a year (metoprolol (25 mg per day) or bisoprolol (5–10 mg per day)) (20 people). The level of gene transcripts was assessed by real-time polymerase chain reaction (PCR). The level of nitric oxide metabolites was determined by the colorimetric method using the Griess reagent. The content of asymmetric dimethylarginine (ADMA), soluble forms of vascular cell adhesion molecule (sVCAM), and intercellular adhesion molecule (sICAM) in blood plasma was determined by ELISA. The content of malondialdehyde (MDA) in blood plasma was determined spectrophotometrically by color reaction with thiobarbituric acid. Statistical processing of the results was carried out using the Statgraphics Centurion XVI software package (version 16.1.11).Results. The level of nitric oxide metabolites in the blood plasma of HTN patients without antihypertensive therapy was 2,1 times higher than in healthy individuals (p = 0,001) and 1,7 times higher than in patients with HTN taking metoprolol or bisoprolol (p = 0,002). The relative content of mRNA of the NOS3 gene in PBL of individuals included in the study did not differ (p > 0,05). The level of NOS2 gene transcripts in PBL of HTN patients before the prescription of antihypertensive drugs exceeded that in healthy individuals (p = 0,0009) and in HTN patients taking metoprolol or bisoprolol (p = 0,0002). The number of SONE transcripts in the PBL of HTN patients was higher than in people with normal blood pressure (p < 0,00001 when comparing patients before the prescription of antihypertensive therapy and individuals from the control group; p = 0,04 when comparing patients with HTN taking antihypertensive drugs and normotensive subjects). The content of MDA, ADMA, sVCAM was higher in the plasma of HTN patients without antihypertensive therapy compared with people from the control group (p = 0,005, 0,003, 0,039, respectively) and patients taking metoprolol or bisoprolol (p = 0,0006, 0,019, 0,016, respectively). The content of nitric oxide metabolites positively correlated with NOS2, SONE, VCAM1 mRNA level in PBL, the content of MDA and ADMA in blood plasma (p < 0,05). A positive correlation was found between the concentration of MDA and ADMA in plasma (p = 0,03).Conclusions. An increase in the level of nitric oxide metabolites in HTN is associated with an increase in the transcriptional activity of the NOS2 gene, a disturbance of the redox balance of the body, and the development of endothelial dysfunction. The SONE gene is probably involved in the modulation of nitric oxide levels in HTN not only as an antisense transcript that destabilizes the mRNA of the NOS3 gene in vascular endothelial cells, but also indirectly, namely, through the regulation of homeostasis of immune system cells through autophagy.

Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows

Our objectives were to evaluate the effects of complete replacement of inorganic salts of trace minerals (STM) with organic trace minerals (OTM) in both pre- and postpartum diets on ovarian dynamics, estrous behavior measured by sensors, preimplantation conceptus development, and reproductive performance in dairy cows. Pregnant cows and heifers (n = 273) were blocked by parity and body condition score and randomly assigned to either STM or OTM diets at 45 ± 3 d before their expected calving. Pre- and postpartum diets were formulated to meet 100% of recommended levels of each trace mineral in both treatments, taking into consideration both basal and supplemental levels. The final target concentrations of Co, Cu, Mn, Se, and Zn were, respectively, 0.25, 13.7, 40.0, 0.3, and 40.0 mg/kg in the prepartum diet, and 0.25, 15.7, 40.0, 0.3, and 63.0 mg/kg in the postpartum diet. The STM group was supplemented with Co, Cu, Mn, and Zn sulfates and sodium selenite, while the OTM group was supplemented with Co, Cu, Mn, and Zn proteinates and selenized yeast. Treatments continued until 156 d in milk (DIM) and were assigned to individual cows using automatic feeding gates. Starting at 21 DIM, ultrasonography examinations of the ovaries were performed weekly to determine the presence of a corpus luteum and postpartum resumption of ovarian cyclicity. Cows were presynchronized with 2 injections of PGF2α at 42 and 56 DIM. Estrous behavior was monitored using electronic activity tags that indirectly measured walking activity. Cows detected in estrus after the second PGF2α were inseminated, and those not detected in estrus by 67 DIM were enrolled in a synchronization program. Cows that returned to estrus after artificial insemination (AI) were reinseminated. Pregnancy diagnosis was performed 33 d after AI, and nonpregnant cows were resynchronized. Transcript expression of interferon-stimulated genes in peripheral blood leukocytes was performed in a subgroup of cows (STM, n = 67; OTM, n = 73) on d 19 after AI. A different subgroup of cows (28 STM, 29 OTM) received uterine flushing 15 d after AI for recovery of conceptuses and uterine fluid for analyses of transcriptomics and metabolomics, respectively. In addition, dominant follicle diameter, luteal size and blood flow, and concentration of progesterone in plasma were measured on d 0, 7, and 15 relative to AI. After flushing, PGF2α was given and the dominant follicle was aspirated 2 d later to measure the concentration of trace minerals by mass spectrometry. Estrous behavior, size of the dominant follicle and corpus luteum, concentration of progesterone, time to pregnancy, and proportion of cows pregnant by 100 d of the breeding period did not differ between treatments. A greater proportion of cows supplemented with OTM had a corpus luteum detected before presynchronization (64.3 vs. 75.2%), and primiparous cows supplemented with OTM tended to resume cyclicity earlier than their STM counterparts. Cows supplemented with OTM had a greater concentration of Cu in follicular fluid than cows supplemented with STM (0.89 vs. 0.77 µg/mL, respectively). In pregnant multiparous cows, expression of receptor transporter protein 4 in peripheral blood leukocytes was 42% greater in the OTM group. Conceptuses of the 2 treatments had 589 differentially expressed transcripts, with many indicating advanced conceptus elongation and greater transcript expression of selenoproteins in the OTM group. In pregnant cows, 24 metabolites were more abundant in the uterine fluid of OTM, including spermidine, sucrose, and cholesterol. In conclusion, replacing STM with OTM caused modest improvements to resumption of ovarian cyclicity and important changes in preimplantation conceptus development, but it did not alter conception risk and pregnancy rate.

Аллельный полиморфизм генов, вовлеченных в продукцию IL-1β, и предрасположенность людей к развитию артериальной гипертензии

Background: The level of IL-1β in blood plasma is determined not only by pro-inflammatory stimuli, but also by allelic polymorphism of the IL1B and NLRP3 genes. Information on the associa-tion of allelic polymorphism of these genes with the risk of arterial hypertension (AH) is scarce and contradictory. The aim of the study: To assess the risk of developing hypertension in carriers of various allelic variants according to the polymorphic markers rs1143634 (c.3953C>T), rs16944 (c.-511T>C), rs1143627 (c.-31C>T) of the IL1B gene and rs35829419 (c.2113C>A) of the NLRP3 gene, as well as to study possible mechanisms for the inclusion of allelic polymorphism of these genes in the etiology and pathogenesis of AH. Materials and methods: 182 DNA samples from healthy people and 180 DNA samples from patients with hypertension (stages I-II) were used for genotyping rs1143634, rs16944, rs1143627 (PCR-RFLP analysis). 215 DNA samples from healthy individuals and 180 hypertensive patients were used to determine alleles and genotypes for rs35829419 of the NLRP3 gene (allele-specific PCR with TaqMan probes). Total RNA obtained from peripheral blood leukocytes (PBL) of 45 healthy people and 50 patients with AH (27 people taking metoprolol or bisoprolol for more than a year and 23 people without antihypertensive thera-py) for assessment of gene expression by real-time PCR was used. The content of pro-inflammatory proteins in the blood plasma of 40 healthy individuals was measured by ELISA. Results: Carriers of the TT genotype for the c.3953C>T marker of the IL1B gene were found to have a 3-fold increased risk of AH (OR=3,239; 95% CI: 1,858-5,649). In healthy individuals with this genotype, the con-tent of IL-1β in blood plasma and the expression of the ICAM1 gene in PBL are higher than in het-erozygotes or homozygotes for the C allele (p=0,029 and p=0,004, respectively). Individuals with the TT genotype for the c.-31C>T marker of the IL1B gene had a reduced risk of АН (OR=0,645; 95% CI: 0,481-0,866). IL1B gene expression and hsCRP levels were lower in healthy individuals with the T allele for rs1143627 (p=0,022, p=0,040, respectively). There were no differences in the distribution of allele and genotype frequencies for markers c.-511T>C of the IL1B gene and c.2113C>A of the NLRP3 gene in the studied groups. Conclusion: Polymorphic markers rs1143634 (c.3953C>T) and rs1143627 (c.-31C>T) of the IL1B gene are probably involved in the predisposi-tion of the inhabitants of Karelia to the development of arterial hypertension.

Association between the methylation of CpG islands in JAK-STAT pathway-related genes and colorectal cancer

BackgroundAberrant promoter methylation of CpG islands plays an important role in carcinogenesis. However, the association between the DNA methylation of JAK-STAT pathway-related genes in peripheral blood leukocytes and colorectal cancer (CRC) susceptibility remains unclear. MethodsWe conducted a case-control study of 403 patients with CRC and 419 cancer free controls, and the DNA methylation levels of JAK2, STAT1, STAT3, and SOCS3 in peripheral blood samples from all subjects were assessed using a methylation-sensitive high-resolution melting (MS-HRM) analysis. ResultsCompared with controls, the methylation of the JAK2, STAT1 and SOCS3 genes increased the CRC risk (ORadjusted=1.96, 95% CI, 1.12–3.41, P=0.01; ORadjusted=5.37, 95% CI, 3.74–7.71, P<0.01; ORadjusted=3.30, 95% CI, 1.58–6.87, P<0.01). In the multiple CpG site methylation (MCSM) analysis, a high MCSM value denoted an increased CRC risk (ORadjusted=4.97, 95% CI, 3.34–7.37, P<0.01). ConclusionIn peripheral blood, the methylation of JAK2, STAT1, and high levels of MCSM are promising biomarkers for CRC risk.

Comprehensive bioinformatics analysis identifies LAPTM5 as a potential blood biomarker for hypertensive patients with left ventricular hypertrophy.

Left ventricular hypertrophy (LVH) is a pivotal manifestation of hypertensive organ damage associated with an increased cardiovascular risk. However, early diagnostic biomarkers for assessing LVH in patients with hypertension (HT) remain indefinite. Here, multiple bioinformatics tools combined with an experimental verification strategy were used to identify blood biomarkers for hypertensive LVH. GSE74144 mRNA expression profiles were downloaded from the Gene Expression Omnibus (GEO) database to screen candidate biomarkers, which were used to perform weighted gene co-expression network analysis (WGCNA) and establish the least absolute shrinkage and selection operator (LASSO) regression model, combined with support vector machine-recursive feature elimination (SVM-RFE) algorithms. Finally, the potential blood biomarkers were verified in an animal model. A total of 142 hub genes in peripheral blood leukocytes were identified between HT with LVH and HT without LVH, which were mainly involved in the ATP metabolic process, oxidative phosphorylation, and mitochondrial structure and function. Notably, lysosomal associated transmembrane protein 5 (LAPTM5) was identified as the potential diagnostic marker of hypertensive LVH, which showed strong correlations with diverse marker sets of reactive oxygen species (ROS) and autophagy. RT-PCR validation of blood samples and cardiac magnetic resonance imaging (CMRI) showed that the expression of LAPTM5 was significantly higher in the HT with LVH model than in normal controls, LAPTM5 demonstrated a positive association with the left ventricle wall thickness as well as electrocardiogram (ECG) parameters widths of the QRS complex and QTc interval. In conclusion, LAPTM5 may be a potential biomarker for the diagnosis of LVH in patients with HT, and it can provide new insights for future studies on the occurrence and the molecular mechanisms of hypertensive LVH.

Methylation of Immune-Related Genes in Peripheral Blood Leukocytes and Breast Cancer.

Abnormal DNA methylation contributes to breast cancer (BC). Immune-related genes play crucial roles in BC development and progression. This study aims to investigate the effect of methylation of immune-related genes in peripheral blood leukocytes (PBLs) on BC risk. GSE51032 and GSE104942 datasets were used to identify significantly differentially methylated CpG sites (DMCs) of immune-related genes. A case-control study was conducted using MethylTarget sequencing to validate the relationship between the methylation levels of the screened genes and BC risk. We also evaluated the association between methylation haplotypes of screened genes and BC risk. Moreover, we sorted the blood leukocytes into T cells, B cells, and monocytes to detect the difference of DNA methylation in different cell subtypes. A total of five DMCs were screened from GEO datasets, including cg01760846 (PSMC1), cg07141527 (SPPL3), cg15658543 (CARD11), cg21568368 (PSMB8), and cg24045276 (NCF2). In the case-control study, there were significant associations between methylation of the CpG sites in the five genes and BC risk. Methylation haplotype burdens of PSMC1, CARD11, and PSMB8 were associated with reduced BC risk. Moreover, there were heterogeneities in the methylation levels of the genes in different cell subtypes. In conclusion, methylation of PSMC1, SPPL3, CARD11, PSMB8, and NCF2 in PBLs were associated with BC risk. The five-gene methylation could be the potential biomarkers for predicting BC risk.

DNA Hypomethylation of the MPO Gene in Peripheral Blood Leukocytes Is Associated with Cerebral Stroke in the Acute Phase

Dysregulation of the oxidant-antioxidant system contributes to the pathogenesis of cerebral stroke (CS). Epigenetic changes of redox homeostasis genes, such as glutamate-cysteine ligase (GCLM), glutathione-S-transferase-P1 (GSTP1), thioredoxin reductase 1 (TXNRD1), and myeloperoxidase (MPO), may be biomarkers of CS. In this study, we assessed the association of DNA methylation levels of these genes with CS and clinical features of CS. We quantitatively analyzed DNA methylation patterns in the promoter or regulatory regions of 4 genes (GCLM, GSTP1, TXNRD1, and MPO) in peripheral blood leukocytes of 59 patients with CS in the acute phase and in 83 relatively healthy individuals (controls) without cardiovascular and cerebrovascular diseases. Wefound that in both groups, the methylation level of CpG sites in genes TXNRD1 and GSTP1 was ≤5%. Lower methylation levels were registered at a CpG site (chr1:94,374,293, GRCh37 [hg19]) in GCLM in patients with ischemic stroke compared with the control group (9% [7%; 11.6%] (median and interquartile range) versus 14.7% [10.4%; 23%], respectively, p<0.05). In the leukocytes of patients with CS, the methylation level of CpG sites in the analyzed region of MPO (chr17:56,356,470, GRCh3 [hg19]) on average was significantly lower (23.5% [19.3%; 26.7%]) than that in the control group (35.6% [30.4%; 42.6%], p<0.05). We also found increased methylation of MPO in smokers with CS (27.2% [23.5%; 31.1%]) compared with nonsmokers with CS (21.7% [18.1%; 24.8%]). Thus, hypomethylation of CpG sites in GCLM and MPO in blood leukocytes is associated with CS in the acute phase.

OPG/RANKL/RANK gene methylation among alcohol-induced femoral head necrosis in northern Chinese men

Background and purposeAlcohol-induced osteonecrosis of the femoral head (ONFH) is a complex and heterogeneous disease. Genetic factors and epigenetic modifications are one of the pathogenesis of the disease. However, the influence of epigenetic factors on the disease has not been systematically studied. Our research aims to determine the methylation changes of alcohol-induced ONFH.MethodsAn analytical cross-sectional study of a Chinese male population (50 alcohol-induced ONFH patients and 50 controls). The EpiTYPER of the Sequenom MassARRAY platform was used to detect the DNA methylation status of 132 cytosine-phosphate-guanine (CpG) sites in the OPG/RANKL/RANK gene promoter region.ResultsIn the whole study group, the chi-square test was used to analyze the methylation rate between the two groups, and six CpG sites were found to be different, among which OPG1_CpG_2, OPG3_CpG_4, RANK1_CpG_6, RANK3_CpG_10, RANKL2_CpG_21, and RANKL2_CpG_46 in the case group were higher than those in the control group, while OPG4_CpG_2 was lower than that in the control group. The results showed that in patients with alcohol-induced ONFH, 146 CpG sites were examined for differences in methylation levels compared with healthy controls, 32 of which were not detected, and 23 of the remaining 114 sites showed differences in methylation levels compared with alcohol-induced ONFH patients. Receiver operator characteristic (ROC) curve analysis demonstrated the methylation levels of OPG/RANKL/RANK could efficiently predict the existence of alcohol-induced ONFH.ConclusionOur study of Chinese men suggests that several CpG sites in the OPG/RANKL/RANK gene in peripheral blood leukocytes of patients with alcohol-induced ONFH are in an abnormal methylation state (hypermethylation tended to be more frequent).

Masseter response to long-term experimentally induced anterior crossbite in Sprague-Dawley rats

ObjectiveTo detect the long-term response to unilateral anterior crossbite (UAC) in masticatory muscles and in molecular biomarkers of peripheral blood leukocytes. DesignFifty-six six-week-old Sprague-Dawley rats were used. The gene-fold changes in peripheral blood leukocytes were detected by the microarray analysis to compare the rats that received 20-week UAC treatment with age-matched controls (n = 4). Muscle atrophy-related gene Fbxo32 was selected based on the data of the microarray analysis verified by using real-time PCR. The remaining 36 rats were randomly separated in the UAC and control groups at 12 and 20 weeks (n = 12). The protein expression of Fbxo32 and the muscle injury and myogenesis-related markers, αB-crystallin and desmin, were detected in the masseter and lateral pterygoid muscles by western blot assay. ResultsIn the 20-week UAC group, the masseter muscle weight was lower than that in the age-matched control group, and the expression level of Fbxo32 gene in peripheral blood leukocytes was increased according to the microarray analysis confirmed by real-time PCR detection. The increased protein expression levels of Fbxo32 were detected in the masseter in the 20-week UAC group, and the protein expression levels of desmin and αB-crystallin were decreased at this time point. No similar changes were detected in the lateral pterygoid muscle. ConclusionsMasseter atrophy is induced by long-term stimulation of UAC. The increased expression of the Fbxo32 gene in peripheral blood leukocytes may be a candidate biological marker of masseter atrophy.

Changes in peripheral inflammation-related gene expression by postprandial glycemic response in healthy Japanese men

ObjectivesContinuous postprandial hyperglycemia is associated with the onset of cardiovascular disease. In recent years, the mRNA expression of inflammation-related genes in peripheral blood leukocytes has been shown to be induced by an increase in blood glucose levels. The aim of this study was to investigate differences in the expression of inflammation-related genes in peripheral blood leukocytes in response to an increase in blood glucose from individuals who consumed two kinds of breakfast meals with different glycemic indexes (GIs). MethodsTwenty healthy Japanese men 40 to 70 y of age were given low- or high-GI meals for breakfast for 14 d. Clinical examinations were performed on days 7 and 14. Their blood glucose levels and insulin concentrations were measured from before breakfast ingestion to 120 min after. Additionally, using the blood obtained before and 120 min after breakfast, the mRNA expression levels of inflammation-related genes in peripheral leukocytes were measured. ResultsThe blood glucose levels were significantly lower in the low-GI meal intake group at 30, 60, and 120 min after breakfast than in the high-GI meal intake group. The intake of high-GI meals for 6 d led to an increase in the mRNA levels of interleukin-1β, S100A4, and CD18 compared with the period of low-GI meals. ConclusionThe intake of a low-GI breakfast for 1 wk in healthy Japanese men resulted in lower postprandial blood glucose and insulin levels, which were accompanied by a reduced expression of inflammation-related genes in peripheral blood leukocytes.

ASSOCIATION OF rs4537545 (C&gt;T) ALLELE POLYMORPHISM IN THE IL6R GENE WITH THE DEVELOPMENT OF RHEUMATOID ARTHRITIS

Objective: to analyze the association of the polymorphic marker rs4537545 (C&gt;T) in the IL6R gene with the development of rheumatoid arthritis (RA) in the population of the Republic of Karelia. Subjects and methods. The investigation included 190 samples of DNA extracted from the whole blood of healthy individuals and 158 samples from patients with RA. Genotyping was performed using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. The expression level of IL6 and IL6R genes in peripheral blood leukocytes (PBLs) from healthy individuals was assessed by real-time PCR. The plasma content of interleukin-6 (IL-6) in healthy donors was measured by enzyme-linked immunosorbent assay (ELISA). Results and discussion. An association of the polymorphic marker rs4537545 (C&gt;T) in the IL6R gene with the development of rheumatoid arthritis was found in the population of the Republic of Karelia. The risk of RA in the persons carrying the T-allele of rs4537545 in the genotypes was 2.1 times higher than that in the C-allele carriers (odds ratio (OR), 2.103; 95% confidence interval, 1.032-4.287). The genotypes were ascertained to have an effect on the level of IL6R gene in PBL and on the plasma content of IL-6 in the healthy donors. Conclusion. The polymorphic marker rs4537545 (C&gt;T) in the IL6R gene is involved in the genetic predisposition of humans to RA development through modulation of the level of transcriptional activity of the IL6R gene and the content of IL-6.

Undernutrition in Pregnant Rats Induces Glucose Intolerance with Enhanced Expression of Inflammation-Related Genes in Peripheral Leukocytes of the Offspring.

Impaired glucose tolerance (IGT) induces chronic inflammation and subsequent development of complications triggered by arteriosclerosis. Moreover, undernutrition in pregnant rodents can induce IGT in their offspring. Here, we assessed whether undernutrition in pregnant rats would induce chronic inflammation in their offspring by measuring the expression levels of inflammation-related genes in peripheral blood leukocytes. Pregnant Wistar rats were divided into two groups: the control group received an American Institute of Nutrition Rodent diet (AIN-93G) ad libitum, and the undernutrition group had their diet restricted by 50% (w/w) compared with the control group from day 10 of pregnancy until birth of the offspring. Subsequently, mothers and pups were allowed to access the AIN-93G diet freely. At day 35 after birth, male pups were fasted for 4 h and subsequently orally administered with glucose solution (2 g/kg body weight). Blood glucose area under the curve (AUC) after glucose loading was significantly greater in the undernutrition group than the control group. The mRNA levels for inflammatory cytokines were increased by glucose loading especially in the undernutrition group. Expressions of genes encoding S100A9 and cell adhesion molecule CD11b were increased by glucose loading in the undernutrition group. Thus, undernutrition of pregnant rats during mid to late gestation induced the expression of inflammation-related genes in peripheral blood leukocytes of their offspring, with the development of IGT and impaired insulin secretion.

The Role of Inducible NOS2 Gene Polymorphism in the Development of Essential Arterial Hypertension.

The risk of essential arterial hypertension was assessed in carriers of the NOS2 gene variants (rs1800482 (-954G>C), rs3730017 (C>T)). In subjects carrying C allele (rs1800482), the risk for essential arterial hypertension developing was higher by 1.7 times (OR=1.712, 95%CI 1.07-2.74), while the presence of T-allele (rs3730017) had a protective effect (OR=0.304, 95%CI 0.192-0.482). In patients with essential arterial hypertension, the presence of the C allele (rs1800482) was associated with a higher content of NO metabolites in the blood plasma. A positive correlation was found between the plasma content of nitrites and nitrates and the level of transcripts of VCAM1, ICAM1 genes in peripheral blood leukocytes. We found the influence of the C allele carriership on the expression VCAM1 and ICAM1 genes in patients with essential hypertension. It was hypothesized that this polymorphic site in the NOS2 gene can be involved in the development of endothelial dysfunction and essential arterial hypertension through modulation of NO level under condition of inflammation.

Immune function in Trachemys scripta following exposure to a predominant brevetoxin congener, PbTx-3, as a model for potential health impacts for sea turtles naturally exposed to brevetoxins.

Many species of marine life in southwestern Florida, including sea turtles, are impacted by blooms of the toxic dinoflagellate, Karenia brevis. Sublethal exposure to toxins produced by K. brevis has been shown to impact sea turtle health. Since all sea turtles in the Gulf of Mexico have protected status, a freshwater turtle, Trachemys scripta, was used as a model for immune system effects following experimental exposure to a predominant brevetoxin congener in K. brevis blooms, PbTx-3. Exposure to PbTx-3 was oral or intratracheal and health effects were assessed using a suite of immune function parameters: innate immune function (phagocytosis, plasma lysozyme activity), adaptive immune function (lymphocyte proliferation), and measures of oxidative stress (superoxide dismutase (SOD) and glutathione-S-transferase (GST) activity in plasma). Inflammation was also measured using plasma protein electrophoresis. In addition, differential expression of genes in peripheral blood leukocytes was determined using suppression subtractive hybridization followed by real-time PCR of specific genes. The primary immune effects of sublethal brevetoxin exposure in T. scripta following PbTx-3 administration, appear to be an increase in oxidative stress, a decrease in lysozyme activity, and modulation of immune function through lymphocyte proliferation responses. Plasma protein electrophoresis showed a decreased A:G ratio which may indicate potential inflammation. Genes coding for oxidative stress, such as thioredoxin and GST, were upregulated in exposed animals. That sublethal brevetoxin exposures impact immune function components suggests potential health implications for sea turtles naturally exposed to toxins. Knowledge of physiological stressors induced by brevetoxins may contribute to the ultimate goal of developing directed treatment strategies in exposed animals for reduced mortality resulting from red tide toxin exposure in sea turtles.