Genes In Peripheral Blood Lymphocytes Research Articles

Specific CpG sites methylation is associated with hematotoxicity in low-dose benzene-exposed workers

Benzene is a broadly used industrial chemicals which causes various hematologic abnormalities in human. Altered DNA methylation has been proposed as epigenetic biomarkers in health risk evaluation of benzene exposure, yet the role of methylation at specific CpG sites in predicting hematological effects remains unclear. In this study, we recruited 120 low-level benzene-exposed and 101 control male workers from a petrochemical factory in Maoming City, Guangdong Province, China. Urinary S-phenylmercapturic acid (SPMA) in benzene-exposed workers was 3.40-fold higher than that in control workers (P < 0.001). Benzene-induced hematotoxicity was characterized by reduced white blood cells counts and nuclear division index (NDI), along with an increased DNA damage and urinary 8-hydroxy-2′-deoxyguanosine (all P < 0.05). Methylation levels of TRIM36, MGMT and RASSF1a genes in peripheral blood lymphocytes (PBLCs) were quantified by pyrosequencing. CpG site 6 of TRIM36, CpG site 2, 4, 6 of RASSF1a and CpG site 1, 3 of MGMT methylation were recognized as hot CpG sites due to a strong correlation with both internal exposure and hematological effects. Notably, integrating hot CpG sites methylation of multiple genes reveal a higher efficiency in prediction of integrative damage compared to individual genes at hot CpG sites. The negative dose–response relationship between the combined methylation of hot CpG sites in three genes and integrative damage enabled the classification of benzene-exposed individuals into high-risk or low-risk groups using the median cut-off value of the integrative index. Subsequently, a prediction model for integrative damage in benzene-exposed populations was built based on the methylation status of the identified hot CpG sites in the three genes. Taken together, these findings provide a novel insight into application prospect of specific CpG site methylation as epi-biomarkers for health risk assessment of environmental pollutants.

Investigating the Expression Levels of Bax and Bcl-2 Genes in Peripheral Blood Lymphocytes of Industrial Radiation Workers in the Asaluyeh Region

Investigating the Expression Levels of Bax and Bcl-2 Genes in Peripheral Blood Lymphocytes of Industrial Radiation Workers in the Asaluyeh Region

Evaluation of the relationship between de-novo DSA development and CXCR5+PD-1+CD8+ T cells and PD-1/PD-L1 mRNA expression in kidney transplant recipients.

The relationship between the Follicular Cytotoxic T cell subgroup and expression levels of PD1/PD-L1 genes and the development of donor specific antibody (DSA) is unknown. In this study, we aimed to examine CD8+CXCR5+PD-1+ follicular cytotoxic T cell levels and expression levels of PD1/PD-L1 genes in peripheral blood lymphocytes in de-novo DSA positive and negative kidney transplant recipients (KTR). In our study, expression of PD-1/ PD-L1 genes by Real-Time Quantitative PCR method and CD8+CXCR5+PD-1+ T cell expression levels by flow cytometric method were obtained from peripheral blood samples. 63 participants were included in the study (de-novo DSA positive recipients (n=22, group 1), de-novo DSA negative recipients (n=20, group 2) and healthy control (n=21, group 3). All patients had negative PRA before kidney transplantation. Expression (%) levels of target cells were evaluated by flow cytometry method. IBM SPSS Statistics for Windows Version 22 and R.3.3.2 software were used to evaluate the data. The demographic data of the groups were similar. PD-1 mRNA expression was higher in de-novo DSA positive KTR than negative (respectively, 1.03±.29/.82±.15, p: .001). CD8+CXCR5+PD-1+ T cell expression levels were found to be higher in the de-novo DSA positive group than in the negative group and similar to the healthy group (respectively, 3.06±1.98/.52±.40, p:.001, 3.06±1.98/2.78±.59, p:.62). The percentage of CD8+CXCR5+PD-1+ expressing T cells was significantly lower in the HLA-Class II+ group than other groups (HLA CI/II/ I+II, respectively, 3.63±2.72/1.65±.50/3.68±1.67, p: .04). In our study, a significant relationship was found between DSA formation and PD-1 mRNA level and CD8+CXCR5+PD-1+ follicular cytotoxic T cell in KTR.

#2938 DE NOVO DSA IS ASSOCIATED WITH HUMAN PERIPHERAL BLOOD CXCR5+PD-1+CD8+ T CELLS AND PD-1 PD-L1 MRNA EXPRESSION IN KIDNEY TRANSPLANT PATIENTS

Abstract Background and Aims Donor-specific antibody (DSA) development is mainly responsible for chronic antibody-mediated rejection. The immunological events that cause the development of DSA is not fully elucidated. The relationship between the Follicular Cytotoxic T cell subgroup and the development of DSA is unknown. In this study we aimed to examine CD8+CXCR5+PD-1+ follicular cytotoxic T cell levels and expression levels of PD1/PD-L1 genes in peripheral blood lymphocytes in recipients who de-novo DSA positive or negative after kidney transplantation (KTX). Method In this study, the expression levels of PD-1, PD-L1 genes were determined using the Real-Time Quantitative PCR method from peripheral blood samples of 22 KTX patients with de-novo DSA positive, 20 KTX patients with DSA negative and 21 healthy control (Table 1). Our patient groups were PRA negative before transplantation. Expression (%) levels of target cells were evaluated by flow cytometry method. IBM SPSS Statistics for Windows Version 22 and R.3.3.2 software were used to evaluate the data. Results PD-1 expression level was higher in the KTX patients with de-novo DSA positive compared to the KTX patients with DSA negative group (p:0.006 CT: 0,84±0,23). According to the results of flow cytometry, CD8+CXCR5+PD-1+ cell expression (%) levels were found to be significantly higher in patients (3,06±1,98) compared to the transplant control group (0,52±0,40) (p: 0.001) (Figure 1). Conclusion It shows that there may be a direct correlation between DSA and PD-1 / PDL-1 mRNA level and CXCR5+PD1+CD8+ follicular cytotoxic T cell in transplant patients after kidney transplantation.

DNA architectural protein CTCF facilitates subset-specific chromatin interactions to limit the formation of memory CD8+ T cells

Although the importance of genome organization for transcriptional regulation of cell-fate decisions and function is clear, the changes in chromatin architecture and how these impact effector and memory CD8+ Tcell differentiation remain unknown. Using Hi-C, we studied how genome configuration is integrated with CD8+ Tcell differentiation during infection and investigated the role of CTCF, a key chromatin remodeler, in modulating CD8+ Tcell fates through CTCF knockdown approaches and perturbation of specific CTCF-binding sites. We observed subset-specific changes in chromatin organization and CTCF binding and revealed that weak-affinity CTCF binding promotes terminal differentiation of CD8+ Tcells through the regulation of transcriptional programs. Further, patients with de novo CTCF mutations had reduced expression of the terminal-effector genes in peripheral blood lymphocytes. Therefore, in addition to establishing genome architecture, CTCF regulates effector CD8+ Tcell heterogeneity through altering interactions that regulate the transcription factor landscape and transcriptome.

Gene expression profiling in peripheral blood lymphocytes for major depression: preliminary cues from Chinese discordant sib-pair study

The etiology of major depressive disorder (MDD) involves many factors such as heredity and environment. There are very few MDD-related studies in Chinese population using twin or sib-pairs for depression-control samples. Here we used the microarray approach and compared gene expression profiling of peripheral blood lymphocytes from 6 sib-pairs discordant on lifetime history of MDD. Within sib-pair differentially expressed genes are obvious fewer in the 1st, 2nd, and 5th compared with those in the 3rd, 4th, and 6th sib-pairs. Gene expression pattern of these DEGs distinguished MDD individuals from the normal one in 3rd, 4th, and 6th sib-pair but not in the 1st, 2nd, and 5th pair, suggesting heterogeneity of different sib-pairs and somewhat commonalities among the 3rd, 4th, and 6th sib-pairs. Comprehensive protein interaction network analysis revealed two key genes PTH and FGF2 in a dominant network where the majority of the genes were significantly down-regulated. PTH was significantly down-regulated in all the sib-pairs while FGF2 was in the 3rd, 4th, and 6th sib-pairs. KEGG enrichment analysis of all the DEGs in networks showed that PTH and related genes were significantly enriched in the pathway of parathyroid hormone secretion, synthesis, and action while FGF2 and related genes were significantly enriched in the pathways of cancer and specifically breast cancer. Generally reduced expression of these genes in peripheral blood lymphocytes of MDD individuals implied their functional repression associated with MDD. Pending validation in more samples, the findings in this study provided valuable cues for understanding the potential mechanism of MDD, as well as potential markers for the diagnosis and treatment of depression in the Chinese population.

Examination of ATM, BRCA1, and BRCA2 promoter methylation in patients with pancreatic cancer

BackgroundPancreatic cancer is a lethal disease with a poor 5-year survival rate. Pathogenic germline variants in the coding regions of ATM, BRCA1, and BRCA2 are found in up to 4.8% of pancreatic cancer patients. Germline promoter methylation and gene silencing arising from a germline variant or through other mechanisms have been described as a cause of tumor suppressor gene inactivation. MethodsWe measured the level of promoter methylation of the ATM, BRCA1, and BRCA2 genes in peripheral blood lymphocytes from 655 patients with pancreatic cancer using real-time PCR. ResultsNo evidence of germline promoter methylation of any of these genes was found. Promoter methylation levels were minimal with no patient having promoter methylation greater than 3.4%, 3.3%, and 7.6% for ATM, BRCA1 and BRCA2, respectively, well below levels found in patients who have inherited promoter methylation (∼50%). ConclusionsWe found no evidence of germline promoter methylation for the pancreatic susceptibility genes ATM, BRCA1 and BRCA2 in patients with pancreatic cancer. This study reveals that constitutive germline methylation of promoter CpG islands is rare in pancreatic cancer.

Consumption of anthocyanin-rich beverages affects Nrf2 and Nrf2-dependent gene transcription in peripheral lymphocytes and DNA integrity of healthy volunteers

Recently, we demonstrated that the consumption of a bolus of bilberry extract modulates the transcription of Nrf2-regulated genes in peripheral blood lymphocytes (PBL) of healthy volunteers, accompanied by decreased DNA-damage. In the present study, we addressed the question whether consumption of consumer-relevant amounts of anthocyanin-rich beverages can achieve similar effects. The impact of three different anthocyanin-rich beverages on Nrf2-dependent gene transcription as well as and the status of DNA-damage in whole blood was investigated. After a polyphenol-reduced diet, five healthy male subjects consumed a bolus (700 mL) of respective test beverages with blood sampling up to 8 h after intake. All beverages affected the transcription of Nrf2, HO-1 and NQO-1, but showed different potencies and persistence of effects. Consumption of red fruit juice significantly reduced total DNA strand breaks (with formamidopyrimidine-DNA-glycosylase-(fpg) treatment) after 8 h in blood samples of the volunteers, suggesting antioxidant and DNA protective effects, albeit transcript levels of Nrf2-dependent genes had reached the basal state. The amount of basic DNA strand breaks (damage without oxidative DNA strand breaks) remained unchanged during the monitoring period. In contrast, a beverage prepared from grape skin extract significantly increased basic and total DNA strand breaks 2 h after intake, underlining the necessity of further investigations regarding composition, safety and consumer´s acceptance of respective products to exclude undesired adverse effects. Consumption of a bolus of anthocyanin-rich beverages affected Nrf2 and Nrf2-dependent gene transcription in human PBL and DNA integrity, which is indicative for systemic effects.

Identifying the role of ASPN and COMP genes in knee osteoarthritis development

BackgroundOsteoarthritis (OA) is a common cause of musculoskeletal disability among elders and is characterized by late-onset degeneration of articular cartilage. OA affects various joints, commonly hand, knee, and hip, with clinical features that are unique to each joint. This study was initiated to identify and evaluate the role of the ASPN and COMP genes in the development of knee OA.MethodsA case–control study was carried out involving 500 cases with knee OA (diagnosed by the American College of Rheumatology) and an equal number of healthy controls. Blood was drawn for genomic DNA isolation. PCR-RFLP and TaqMan assay methods were used to identify the SNPs. mRNA and protein expression of genes were carried out in peripheral blood lymphocytes (PBLs) by RT-PCR and Western immunoblotting. The data obtained were analyzed for the statistical significance between control and case groups.ResultsThe variant genotype of ASPN and COMP genes was found to be present at a relatively higher frequency in cases than controls. RT-PCR and immunochemical studies revealed increased mRNA and protein expression of such gene in PBLs isolated from cases of knee OA as compared to healthy control.ConclusionThe allelic alteration in ASPN and COMP genes in knee OA cases points to the role of these genes in the development of knee OA. Further, increased mRNA and protein expression of ASPN and COMP in peripheral blood samples of patients with the disease suggest that expression profile of candidate gene could be used as a biomarker for predicting the development and progression of knee OA.

Expression of Bcl-2 and Bax genes in peripheral blood lymphocytes of depressed and nondepressed individuals.

Background:Involvement of the immune system is one of the issues raised in the pathophysiology of depression. BCL2 and BAX genes are related to immune system regulation. We investigated the BCL2 and BAX expression as a probable mechanism of immune system involvement in depression.Materials and Methods:This case–control study was conducted on 28 patients with major depression (case) and 28 nondepressed individuals (control) within the age range of 18–55 years in the Isfahan University of Medical Sciences. Clinical interviews, based on the Diagnostic and Statistical Manual of Mental Disorders, were conducted to detect depression, and Beck's Depression Inventory was used to measure the severity of depression in the individuals. In addition, a real-time polymerase chain reaction was employed to compare the level of Bax and Bcl-2 gene expression in peripheral blood lymphocytes. The multivariate covariance analysis was used to explore the correlation between BCL2 and BAX gene expression and to control the effect of duration and severity of depression.Results:The results showed that none of the variables including group membership, the duration of depression, and the severity of depression were not significantly correlated with the expression of BCL2 and BAX genes. Furthermore, there was no statistically significant relationship between the Bax and Bcl-2 genes expression in case and control groups (P > 0.05).Conclusion:Depression may have no impact on Bax and Bcl-2 gene expression in patients with major depression. Studies with larger sample size are recommended.

The Effect of Dopamine on Gene Expression of Alpha-synuclein and Transcription Factors GATA-1, GATA-2, and ZSCAN21 in Parkinson’s Disease

At present, it is believed that disregulation of alpha-synuclein (SNCA) metabolism and its aggregation is associated with the pathogenesis of Parkinson’s disease (PD). It is thought that the death of dopaminergic neurons in PD can be mediated by the effect of dopamine on the process of alpha-synuclein oligomerization, primarily by direct oxidation of the protein. At the same time, the effect of dopamine on the regulation mechanism of the SNCA gene expression is not excluded. It is known that transcription factors GATA-1, GATA-2, and ZSCAN21 can participate in the regulation of SNCA expression. In the present study, we evaluated the effect of exogenous dopamine (100 μM) on the mRNA level of the SNCA, GATA-1, GATA-2, and ZSCAN21 genes and the level of total alpha-synuclein in cultured peripheral blood lymphocytes (PBL) of patients with PD (n = 18) and individuals of the control group (n = 14) using real-time PCR and ELISA, subsecuently. A decrease in the SNCA expression level under the action of dopamine was first found in PBL of patients with PD (P = 0.013), of the control group (P = 0.004), and of the combined group that included patients with PD and control individuals (P = 0.001). When assessing alpha-synuclein level, a tendency toward its decrease in PBL of individuals from the control group (P = 0.068), as well as of the combined group of PD patients and control individuals (P = 0.059), was revealed by comparing the cells treated and untreated with dopamine. An increase in the mRNA level of the ZSCAN21 gene in PBL of control group individuals (P = 0.022), as well as of the GATA-1 gene in PBL of the group of patients with PD (P = 0.019) cultured in the presence of dopamine, was shown. An increase in the mRNA levels of the genes GATA-1, GATA-2, and ZSCAN21 in PBL under the treatment with dopamine was also observed in the combined group of patients with PD and control individuals (P = 0.027, 0.029, and 0.002 for GATA-1, GATA-2, and ZSCAN21, respectively). Thus, the obtained data show the effect of dopamine on the mRNA level of the SNCA, GATA-1, GATA-2, and ZSCAN21 genes in cultured PBL of PD patients and control group individuals, suggesting a possible effect of dopamine on the regulation of SNCA expression.

Expression of Bax and Bcl2 Genes in Peripheral Blood Lymphocytes of Patients with Differentiated Thyroid Cancer.

Context:Thyroid cancer is the most common endocrine malignancy worldwide. Iodine-131 is used in the treatment of thyroid cancer with dosage of 100 mCi. In the medical applications of ionizing radiation besides the advantages such as diagnosis and treatment of diseases, the risks arising from exposure should be considered as well.Aims:The present study aimed to evaluate the changes in expression levels of apoptotic Bax and Bcl-2 and the ratio of Bax/Bcl-2, in the peripheral blood lymphocytes (PBLs) of patients with differentiated thyroid cancer (DTC).Settings and Design:This study was conducted on fifty thyroid cancer patients who had undergone surgery and were under treatment with 100 and 150 mCi doses. Subjects and Methods: Blood samples were taken from the patients, one before iodine treatment and another 48 h after therapy. Bax and Bcl-2 gene expression levels were measured by using real-time reverse transcriptase polymerase chain reaction.Statistical Analysis Used:The data were analyzed using one-way analysis of variance followed by samples t-test and independent samples t-test.Results:Significant changes were observed in the percentage of apoptotic cells, in groups, after radioiodine therapy compared with before treatment. The ratio of Bax/Bcl-2 in both groups showed a significant increase (P < 0.001). The relative expression level of Bax gene showed a significant increase in comparison with the control group.Conclusions:Iodine therapy reduced expression of Bcl-2 and a significant expression of Bax and finally increased the ratio of Bax/Bcl-2. Iodine therapy led to apoptosis in the PBLs of patients with DTC. Therefore, it can be suggested that this method can be useful for monitoring and detecting destructive effects of ionizing radiation in nuclear medicine patients.

Expression of Genes and Their Polymorphism Influences the Risk of Knee Osteoarthritis.

Introduction Genetic factors including the level of expression of the fingerprint of genes involved in the development of bones and cartilage such as GDF-5 or ESR-α or CALM-1 are known to be strong determinants of the osteoarthritis (OA) in Caucasian and Oriental populations. Because of high prevalence of OA in Indian population and availability of limited genetic data, we determined whether similar genetic factors are involved in Indians as well. Methods A case control study was carried out involving 500 patients of knee OA and equal number of healthy controls. Genotyping analyses in whole blood, mRNA, and protein expressions in peripheral blood lymphocytes (PBLs) were performed using established protocols. Results Our results showed a significantly decreased level of mRNA and protein expressions for GDF-5, ESR-α, and CALM-1 genes in PBLs of OA cases when compared to healthy controls. The frequency of variant genotypes of these genes was also increased significantly in cases of OA compared to controls. Conclusion Our results demonstrated that the decrease in expression of GDF-5, ESR-α, and CALM-1 in PBLs and association of polymorphism in these genes may be important in predicting the severity and thereby the progression of OA in Indian population.

Aberrant methylation of nucleotide excision repair genes is associated with chronic arsenic poisoning

Objective: To define whether aberrant methylation of DNA repair genes is associated with chronic arsenic poisoning.Methods: Hundred and two endemic arsenicosis patients and 36 healthy subjects were recruited. Methylight and bisulfite sequencing (BSP) assays were used to examine the methylation status of ERCC1, ERCC2 and XPC genes in peripheral blood lymphocytes (PBLs) and skin lesions of arsenicosis patients and NaAsO2-treated HaCaT cells.Results: Hypermethylation of ERCC1 and ERCC2 and suppressed gene expression were found in PBLs and skin lesions of arsenicosis patients and was correlated with the level of arsenic exposure. Particularly, the expression of ERCC1 and ERCC2 was associated with the severity of skin lesions. In vitro studies revealed an induction of ERCC2 hypermethylation and decreased mRNA expression in response to NaAsO2 treatment.Conclusion: Hypermethylation of ERCC1 and ERCC2 and concomitant suppression of gene expression might be served as the epigenetic marks associated with arsenic exposure and adverse health effects.

Changes in mRNA expression of p53 and related downstream genes in peripheral blood lymphocytes in workers occupationally exposed to arsenic

To investigate the changes in mRNA expression of p53 and related downstream genes in peripheral blood lymphocytes in workers occupationally exposed to arsenic as well as its influencing factors, and to analyze the mechanism of genetic toxicity of arsenic. With cluster random sampling, 79 workers from an arsenic smelting plant were selected as exposure group, and another 24 people without occupational exposure to arsenic were selected as control group. The relative mRNA expression of p53 and related downstream genes in the peripheral blood lymphocytes of the two groups was determined by quantitative realtime PCR. The levels of inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by hydride generation-atomic absorption spectrometry. The exposure group had significantly higher levels of iAs, MMA, and DMA than the control group (P<0.01); the exposure group had significantly higher relative mRNA expression (2(-ΔΔCt)) of p53 and four related downstream genes in peripheral blood lymphocytes than the control group (P<0.05); the relative mRNA expression of p53 and related downstream genes was positively correlated with each other (P<0.01), with a correlation coefficient greater than 0.4; the levels of arsenic compounds in urine were positively correlated with the relative mRNA expression of p53 and some of its downstream genes (P<0.05). The changes in mRNA expression of p53 and related downstream genes are closely related to the metabolic transformation of inorganic arsenic in workers occupationally exposed to arsenic, and it also plays an important role in genetic toxicity and carcinogenic effect in people exposed to arsenic.

The Key Proteins of Dopaminergic Neurotransmission of Human Peripheral Blood Lymphocytes: Changed mRNA Level in Alcohol Dependence Syndrome.

The expression of dopamine receptor (DRD), Nurr1 transcription factor (NR4A2), and α-sinucleine (SNCA) genes in peripheral blood lymphocytes is evaluated. The results indicate that alcohol dependence is associated with high expression of SNCA and DRD4 (signifi cantly higher than in the control group) and is not associated with changes in the work of NR4A2 and DRD3 genes. The levels of DRD3 and DRD4 mRNA form a positive linear correlation (p≤0.05). The expression of SNCA and DRD4 genes can serve as an important peripheral marker of alcohol dependence development, which is essential for antipsychotic therapy.

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection.

The profile of T-cell receptor beta-chain variable (TRBV) genes usually skews in subjects with virus infection or cancer. The gene melting spectral pattern (GMSP) can be used to determine the profile of the TRBV gene family. To explore the portrait of the TRBV family in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection (AHI), peripheral blood mononuclear cells (PBMCs) were separated and further sorted into CD4+ and CD8+ T-cell subsets. The molecular features of the TRBV complementary determining region 3 (CDR3) motifs were determined using GMSP analysis. When aGMSP profile showed a single peak, the monoclonally expanded TRBV gene was cloned and sequenced. Skewed expansions of multiple TRBV genes were observed among the CD4+ and CD8+ T-cell subsets and the PBMCs. The frequency of monoclonally expanded TRBV genes in the CD8+ T-cell subset was significantly higher than that of the CD4+ T-cell subset and the PBMCs. Compared to other members of the TRBV gene family, TRBV11, BV15 and BV20 were predominantly expressed in the repertoire of peripheral blood lymphocytes in recovered AHI subjects. The relatively conserved amino acid motifs of TRBV5.1 and BV20 CDR3 were also detected in the CD4+ and CD8+ T-cell subsets. These results demonstrate the presence of multiple biased TRBV families in recovered AHI subjects. TRBV11, BV15 and BV20, especially from the CD8+ T-cell subset, may be relevant to the pathogenesis of subjects with AHI. The preferentially selected TRBV5.1 and BV20 with the relatively conserved CDR3 motif may be potential targets for personalized treatments of chronic HBV infection.

Single-nucleotide Polymorphism and Haplotypes of TNIP1 Associated with Systemic Lupus Erythematosus in a Chinese Han Population

To determine the association of systemic lupus erythematosus (SLE) with single-nucleotide polymorphisms (SNP) in the TNIP1 gene and compare the expression of this gene in cases and controls from a Chinese Han population in this replication study. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to genotype 19 SNP in TNIP1 in Chinese Han patients with SLE (n = 341) and controls (n = 356). Genotypes were analyzed by codominant, dominant, and recessive models. Analysis of allele frequencies and linkage disequilibrium was also performed. Western blotting and qRT-PCR were used to measure the expression of these genes in peripheral blood mononuclear cells of SLE cases and controls. Seven SNP loci were significantly associated with SLE in our population (p < 0.05 for all comparisons). Two TNIP1 gene haplotypes (ATTGCGC and GTCCTAT) were associated with SLE (p = 0.0246 and p = 0.0024, respectively). Western blotting and qRT-PCR results provide evidence that patients with SLE had significantly reduced expression of TNIP1/ABIN-1 relative to controls. Analysis of SNP in the TNIP1 gene and expression of this gene in peripheral blood lymphocytes indicated these SNP were associated with the occurrence of SLE in Han Chinese patients. Future studies should examine the roles of these SNP in the pathogenesis of SLE.

Methylation of multiple genes in hepatitis C virus associated hepatocellular carcinoma

We studied promoter methylation (PM) of 11 genes in Peripheral Blood Lymphocytes (PBLs) and tissues of hepatitis C virus (HCV) associated hepatocellular carcinoma (HCC) and chronic hepatitis (CH) Egyptian patients. The present study included 31 HCC with their ANT, 38 CH and 13 normal hepatic tissue (NHT) samples. In all groups, PM of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A, O6MGMT was assessed by methylation-specific PCR (MSP). APC and O6-MGMT protein expression was assessed by immunohistochemistry (IHC) in the studied HCC and CH (20 samples each) as well as in a different HCC and CH set for confirmation of MSP results. PM was associated with progression from CH to HCC. Most genes showed high methylation frequency (MF) and the methylation index (MI) increased with disease progression. MF of p14, p73, RASSF1A, CDH1 and O6MGMT was significantly higher in HCC and their ANT. MF of APC was higher in CH. We reported high concordance between MF in HCC and their ANT, MF in PBL and CH tissues as well as between PM and protein expression of APC and O6MGMT. A panel of 4 genes (APC, p73, p14, O6MGMT) classifies the cases independently into HCC and CH with high accuracy (89.9%), sensitivity (83.9%) and specificity (94.7%). HCV infection may contribute to hepatocarcinogenesis through enhancing PM of multiple genes. PM of APC occurs early in the cascade while PM of p14, p73, RASSF1A, RARB, CDH1 and O6MGMT are late changes. A panel of APC, p73, p14, O6-MGMT could be used in monitoring CH patients for early detection of HCC. Also, we found that, the methylation status is not significantly affected by whether the tissue was from the liver or PBL, indicating the possibility of use PBL as indicator to genetic profile instead of liver tissue regardless the stage of disease.

Expression profiling of selected genes of toxication and detoxication pathways in peripheral blood lymphocytes as a biomarker for predicting toxicity of environmental chemicals

To develop a rapid and sensitive tool for determining gene expression profiles of peripheral blood lymphocytes (PBL) as a surrogate for predicting toxicity associated with environmental exposures, studies were initiated using Taqman Low Density Array (TLDA), a medium throughput method for real time PCR (RT-PCR), for selected genes involved in toxication and detoxication processes. Total RNA was prepared from PBL and liver samples isolated from young rats treated with inducers of drug metabolizing enzymes, e.g. phenobarbital (PB, 80mg/kg i.p. X5 days) or methylcholanthrene (30mg/kg, i.p. X5 days) or ethanol (0.8ml/kg, i.p. X1 day). TLDA data showed that PBL expressed drug metabolizing enzymes (DMEs), though the level of expression was several folds lower when compared to liver. Treatment with different inducers of DMEs produced a similar pattern of an increase in the expression of various phase I and phase II DMEs and their respective transcription factors in liver and PBL. While treatment with MC increased the expression of MC inducible cytochrome P450 (CYP) 1A1, 1A2, 1B1, 2A2 & 3A1 and their associated transcription factors in PBL, an increase in the expression of CYP2B1, 2B2, 2C11 & 3A1 and their transcription factor was observed in PBL after PB treatment. Similarly, treatment of ethanol increased the expression of CYP2E1 and 3A1 along with transcription factors in PBL. These inducers were found to increase the expression of various phase II enzymes such as glutathione S-transferases, GSTs (GSTM1, GSTA1, GSTP1 and GSTK1), NQO1, Ephx1 and Sod1, genes involved in inflammation and apoptosis (p53, BCl2, Apaf1 and Caspase9) in both PBL and liver. The data suggests that the low-density array of selected genes in PBL has the potential to be developed as a rapid and sensitive tool for monitoring of individuals exposed to environmental chemicals as well as in clinical studies.