Peripheral Blood Leukocyte Samples Research Articles

Biomarkers for Radiation Biodosimetry and Correlation with Hematopoietic Injury in a Humanized Mouse Model.

After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes invivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.

Identification of differentially expressed genes of blood leukocytes for Schizophrenia.

Schizophrenia (SCZ) is a severe neurodevelopmental disorder with brain dysfunction. This study aimed to use bioinformatic analysis to identify candidate blood biomarkers for SCZ. The study collected peripheral blood leukocyte samples of 9 SCZ patients and 20 healthy controls for RNA sequencing analysis. Bioinformatic analyses included differentially expressed genes (DEGs) analysis, pathway enrichment analysis, and weighted gene co-expression network analysis (WGCNA). This study identified 1,205 statistically significant DEGs, of which 623 genes were upregulated and 582 genes were downregulated. Functional enrichment analysis showed that DEGs were mainly enriched in cell chemotaxis, cell surface, and serine peptidase activity, as well as involved in Natural killer cell-mediated cytotoxicity. WGCNA identified 16 gene co-expression modules, and five modules were significantly correlated with SCZ (p < 0.05). There were 106 upregulated genes and 90 downregulated genes in the five modules. The top ten genes sorted by the Degree algorithm were RPS28, BRD4, FUS, PABPC1, PCBP1, PCBP2, RPL27A, RPS21, RAG1, and RPL27. RAG1 and the other nine genes belonged to the turquoise and pink module respectively. Pathway enrichment analysis indicated that these 10 genes were mainly involved in processes such as Ribosome, cytoplasmic translation, RNA binding, and protein binding. This study finds that the gene functions in key modules and related enrichment pathways may help to elucidate the molecular pathogenesis of SCZ, and the potential of key genes to become blood biomarkers for SCZ warrants further validation.

Selection for high and low antibody responses to sheep red blood cells influences cytokine and chemokine expression in chicken peripheral blood leukocytes and splenic tissue

White Leghorn chickens from a common founder population have been divergently selected for high (HAS) or low (LAS) antibody responses to sheep red blood cells (SRBC) for 49 generations resulting in 2 diverse lines for this trait. Much has been studied in these two lines; however, the impact of these selection pressures on cytokine and chemokine expression is not fully understood. The purpose of this study is to determine if selection for antibody response to SRBC impacts cytokine and chemokine expression in peripheral blood leukocytes (PBL) and spleen from HAS and LAS chickens. Total RNA was isolated from PBL and spleen after which mRNA expression of cytokines (IL4, IL6, IL10, TGF-β4) and chemokines (CXCL8, CCL4) were determined by quantitative real-time RT-PCR (qRT-PCR). The data were analyzed using Student's t test comparing HAS and LAS (P < 0.05) and are reported as corrected 40-CT. PBL and spleen samples were analyzed separately. With respect to PBL, expression of IL6 was higher (P < 0.05) in PBL isolated from LAS chickens compared to those from the HAS line whereas there were no differences (P > 0.05) in IL4, IL10, CXCL8, CCL4, or TGF-β4. The cytokine and chemokine mRNA expression profiles were different in the spleen between the two lines. IL4 and CXCL8 expression were higher (P < 0.05) in spleen samples from HAS chickens than LAS. The expression of IL6, IL10, CCL4, or TGF-β4 in the spleens did not differ (P > 0.05) between the lines. The data indicate that selection for specific antibody responses to SRBC impacts the cytokine and chemokine expression profile in PBL and spleens but in different ways in HAS and LAS. These studies provide insight into the influence that selection pressures for antibody responses have on different immune response components, specifically cytokines and chemokines typically involved in the innate response.

Alteration of the immunophenotype and cytokine profiles in patients affected by neuroendocrine neoplasms.

Neuroendocrine neoplasms (NENs) are tumors that arise from cells of the endocrine system and are most common in the gastrointestinal tract, the pancreas, and the lungs. Their incidence is rapidly increasing and the therapeutic options available are limited. Since the immune system can interfere with tumor growth and response to therapy, using flow cytometry we investigated the immunophenotype in samples of peripheral blood leukocytes from patients with pancreatic (Pan-NENs) and pulmonary NENs (Lung-NENs). Moreover, we performed a multiplex analysis of 13 key cytokines and growth factors essential for the immune response in the plasma of NEN patients and controls. Patients presented with a higher percentage of granulocytes, a lower percentage of lymphocytes, and an increase in the granulocytes to lymphocytes ratio compared to healthy donors. These alterations were more marked in patients with metastasis. Somatostatin analogs (SSAs) restored the immunophenotype of patients to that seen in healthy donors. Finally, Pan-NEN patients showed a higher plasma concentration of IP-10, MCP-1, and IL-8 compared to healthy donors, suggesting a potential role for these cytokines as diagnostic biomarkers. This study highlighted differences in the immunophenotype of patients with Pan- and Lung-NENs compared to healthy individuals; these alterations were partially restored by therapy.

Downregulation of IRAIN long non-coding RNA predicts unfavourable clinical outcome in acute myeloid leukaemia patients

Background Although it has been shown that the long non-coding RNA (lncRNA) insulin-like growth factor type 1 receptor (IGF1R) antisense imprinted non-protein coding RNA (IRAIN) is downregulated in leukaemia cell lines, its usefulness as a prognostic marker in acute myeloid leukaemia (AML) has not yet been thoroughly investigated. Here, we sought to determine whether the expression of IRAIN is associated with clinical outcome of AML patients. Subjects & Methods Using quantitative real-time polymerase chain reaction (qRT-PCR), IRAIN expression levels were assessed in peripheral blood leukocyte samples from 150 patients with AML and 50 healthy controls. Analysis was done on the relationship between IRAIN expression and clinical outcomes in AML patients. Results When compared to healthy controls, IRAIN expression was markedly reduced in AML patients (P = 0.019). IRAIN expression could distinguish French-American-British (FAB) subtypes of AML (P = 0.024). Low IRAIN expression status was associated with shorter event-free survival (EFS) in the non-t(15;17) cytogenetically abnormal AML subset (P = 0.004). IRAIN downregulation was identified as an independent adverse prognostic marker for complete remission (CR) not only in the in the non-t(15;17) cytogenetically abnormal AML subset (P = 0.006) but also in the AML-M4/M5 subgroup (P = 0.033). Conclusion Aberrantly low IRAIN expression is closely associated with lower CR rates in AML patients, particularly in non-t(15;17) cytogenetically abnormal AML and M4/M5 AML, suggesting that the determination of IRAIN expression level at diagnosis provides valuable prognostic information, serves as a promising biomarker for evaluating treatment response, and helps predicting clinical outcome of AML patients.

Long-term Temporal Stability of Peripheral Blood DNA Methylation Profiles in Patients With Inflammatory Bowel Disease

There is great current interest in the potential application of DNA methylation alterations in peripheral blood leukocytes (PBLs) as biomarkers of susceptibility, progression, and treatment response in inflammatory bowel disease (IBD). However, the intra-individual stability of PBL methylation in IBD has not been characterized. Here, we studied the long-term stability of all probes located on the Illumina HumanMethylation EPIC BeadChip array. We followed a cohort of 46 adult patients with IBD (36 Crohn's disease [CD], 10 ulcerative colitis [UC]; median age, 44 years; interquartile range [IQR] 27-56 years; 50% female) that received standard care follow-up at the Amsterdam University Medical Centers. Paired PBL samples were collected at 2 time points with a median of 7 years (range, 2-9 years) in between. Differential methylation and intra-class correlation (ICC) analyses were used to identify time-associated differences and temporally stable CpGs, respectively. Around 60% of all EPIC array loci presented poor intra-individual stability (ICC <0.50); 78.114 (≈9%) showed good (ICC, 0.75-0.89), and 41.274 (≈5%) showed excellent (ICC ≥0.90) stability, between both measured time points. Focusing on previously identified consistently differentially methylated positions indicated that 22 CD-, 11 UC-, and 24 IBD-associated loci demonstrated high stability (ICC ≥0.75) over time; of these, we observed a marked stability of CpG loci associated to the HLA genes. Our data provide insight into the long-term stability of the PBL DNA methylome within an IBD context, facilitating the selection of biologically relevant and robust IBD-associated epigenetic biomarkers with increased potential for independent validation. These data also have potential implications in understanding disease pathogenesis.

Identification of novel mutations among Iranian NPC1 patients: a bioinformatics approach to predict pathogenic mutations

BackgroundNiemann-Pick disease type C (NPC) is a rare lysosomal neurovisceral storage disease caused by mutations in the NPC 1 (95%) or NPC2 (5%) genes. The products of NPC1 and NPC2 genes play considerable roles in glycolipid and cholesterol trafficking, which could consequently lead to NPC disease with variable phenotypes displaying a broad spectrum of symptoms.MaterialsIn the present study 35 Iranian NPC unrelated patients were enrolled. These patients were first analysed by the Filipin Staining test of cholesterol deposits in cells for NPC diagnostics. Genomic DNA was extracted from the samples of peripheral blood leukocytes in EDTA following the manufacturer's protocol. All exon–intron boundaries and coding exons of the NPC1gene were amplified by polymerase chain reaction (PCR) using appropriate sets of primers. Thereafter, the products of PCR were sequenced and analysed using the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The variants were reviewed by some databases including the Human Gene Mutation Database (HGMD) (http://www.hgmd.cf.ac.uk/ac/index.php) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar (. Moreover, all the variants were manually classified in terms of the American College of Medical Genetics and Genomics (ACMG) guideline.ResultsThe sequence analysis revealed 20 different variations, 10 of which are new, including one nonsense mutation (c.406C > T); three small deletions, (c.3126delC, c.2920_2923delCCTG, and c.2037delG); and six likely pathogenic missense mutations, (c.542C > A, c.1970G > A, c.1993C > G, c.2821 T > C, c.2872C > G, and c.3632 T > A). Finally, the pathogenicity of these new variants was determined using the ACMG guidelines.ConclusionThe present study aimed to facilitate the prenatal diagnosis of NPC patients in the future. In this regard, we identified 10 novel mutations, and verified that the majority of them occurred in six NPC1 exons (5, 8, 9, 13, 19, and 21), that should be considered with a high priority for Iranian patients' cost-effective evaluation.

Mutations in the tumor suppressor gene p53 in cattle are associated with enzootic bovine leukosis

Enzootic bovine leukosis (EBL) is a B-cell lymphoma caused by the bovine leukemia virus (BLV). Although an association between EBL and mutations in the bovine tumor suppressor gene TP53 (bTP53) has been suggested, the substantive incidence rate of bTP53 mutations in EBL cattle is still unclear. In this study, we investigated the complete sequence (exons 2–11) of bTP53 in tissue and peripheral blood leukocyte (PBL) samples obtained from 154 EBL cattle and 117 cattle without EBL (non-EBL cattle) to elucidate the correlation between bTP53 mutations and EBL. The detection frequencies of non-synonymous (NS) and deletion mutations in bTP53 in EBL cattle were significantly higher than those in non-EBL cattle in both tissue and PBL samples (p < 0.05). Among these mutations in EBL cattle, 73.7 % (42/54) were homologous to those of human TP53 (hTP53), which were previously detected in various tumors. It has been reported that 95.2 % (40/42) of these hTP53 mutations induced complete or partial loss of the transactivating function of its encoding protein, P53. Moreover, the BLV proviral load in tissue samples was significantly higher in cattle harboring bTP53 NS and deletion mutations than in cattle without these mutations in both EBL and BLV-infected non-EBL cattle (p < 0.05). Although the activity of the mutant variants of bP53 must be further investigated, our findings revealed that bTP53 mutations are involved in tumorigenesis in BLV-infected cells and EBL-associated carcinogenesis.

Gene expression in peripheral blood mononuclear cells in impulsive aggression: Intermittent explosive disorder compared with non-aggressive healthy and psychiatric controls

Evidence of chronic, systemic, low levels of inflammation is present in several stress-related psychiatric conditions including schizophrenia, depression, and intermittent explosive disorder (IED). We analyzed leukocyte gene expression (mRNA) to quantify the activity of pro and anti-inflammatory signaling pathways. Work performed in non-aggressive populations has uncovered a Conserved Transcriptional Response to Adversity (CTRA) characterized by an upregulation of pro-inflammatory gene transcription in chronically stressed individuals. We used pathway-based bioinformatic analyses of genome-wide transcriptional profiles of peripheral blood leukocyte samples from IED study participants (N = 45) and controls [healthy (n = 45) and psychiatric (n = 34)], with analyses focusing on the pro-inflammatory transcription control pathway mediated by the NF-kB family of transcription factors (typically upregulated in CTRA) and the antiviral transcription control pathway mediated by anti-viral response (IRF) family transcription factors (typically downregulated in CTRA). Compared with both healthy and psychiatric controls, individuals with IED had upregulated transcriptional activity of the antiviral response (IRF), but no evidence of pro-inflammatory NF-kB activation. Analyses implicated CD4 + T cells, CD8 + T cells, and B lymphocytes in IED-related transcriptional alterations, but showed no significant indication of monocyte involvement. This suggests that the inflammatory profile of IED differs substantially from that observed previously in other stress-related disorders, and may involve a pathogen-driven adaptive immune etiology.

Analysis of the HEXA, HEXB, ARSA, and SMPD1 Genes in 68 Iranian Patients.

Lysosomal storage diseases (LSDs) are known as genetic disorders with an overall prevalence of 1 per 7700 live births. Sphingolipidosis, which is a subgroup of LSDs, is resulted from mutations in the coding genes of specific enzymes of sphingolipid hydrolases. The current study aimed to provide additional knowledge on the genotype of sphingolipidoses disease among Iranian patients affected by the disease. In this research, we studied 68 unrelated Iranian patients diagnosed with one kind of sphingolipidoses from 2014 to 2019. Thereafter, genomic DNA was isolated from their peripheral blood leukocytes samples in EDTA in terms of the manufacturer's protocol. All the coding exons and exon-intron boundaries of the related genes were sequenced and then analyzed using the NCBI database. Finally, they were reviewed using some databases such as the Human Gene Mutation Database (HGMD) and ClinVar ( https://www.ncbi.nlm.nih.gov/clinva ). By studying 22 MLD patients, 18 different variations of the ARSA gene were found, one of which was new including, named as c.472T > G p. (Cys158Gly). Out of 15 Sandhoff disease (SD) patients, 11 different variations of the HEXB gene were found. Correspondingly, the c.1083-2delA was not reported earlier. By investigating 21 Iranian patients with Tay-Sachs disease (TSD), one new variant was found as c.622delG. The study of 10 Niemann-Pick disease A/B (NPDA/B (patients has led to the identification of 9 different SMPD1 gene variations, among which 3 variations were novel mutations. The results of the present study can be expanded to the genotypic spectrum of Iranian patients with MLD, SD, TSD, and NPD diseases and also used to innovate more effective methods for the detection of genetic carriers as well as diagnosing and counseling of Iranian patients affected with these disorders.

Methodological basics for differential detection of EBV1/EBV2 and HHV6A/HHV6B

Among a whole variety of EBV- and HHV6-linked diseases only infectious mononucleosis is subject to official statistical reporting in Russia that substantially complicates objective assessment of etiological structure, incidence rate, characteristics of developing epidemic process. Currently, the data on the genetic EBV heterogeneity, even at the level of the main types (EBV1 and EBV2), as well as HHV6A and HHV6B, prevalence and clinical importance are mainly limited to foreign research publications. Few publications assessing this issue are available in Russian scientific papers. At the same time, examining circulation of virus genetic types (variants) and use of such data in implementing epidemiological surveillance after some other infections have been commonly practiced. One of the key issues is the level of developed laboratory support for molecular genetic monitoring. The goal of the study was to improve methodological basics for differential detection of HHV6A/B and the major EBV types. There were used samples of peripheral blood leukocytes collected from children aged 1–15 years with acute (n = 50) and asymptomatic infectious mononucleosis (n = 29). The detection and quantification of EBV and HHV6 DNA was performed by using real-time PCR. For differential determination of EBV1/EBV2 and HHV6A/HHV6B, an optimized one-round PCR with electrophoretic agarose gel detection amplification products was used. The data from our own study showed that frequency of detected EBV and HHV6 DNA in acute infectious mononucleosis patients comprised 74 and 72% compared to control group reaching 35 and 74%, respectively. It was found that among the examined children of Nizhny Novgorod Region, EBV1 and HHV6B prevailed in the viral population that agrees with existing insights about their geographical distribution in the adjacent territories. EBV2 was found in a single sample only in the control group. HHV6A was not detected in any of the groups. The methodological approach optimized in this study allows to separately detect HHV6A/HHV6B and the main EBV types according to a unified laboratory protocol, whereas combining it with additional stage of DNA enrichment increases the diagnostic sensitivity of PCR analysis, minimizes proportion of discordant and false negative results. Such an integrated approach can be applied for diagnostic, epidemiological and research purposes.

Abstract 577: A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer

Abstract Introduction: Analysis of cell-free DNA (cfDNA) has recently emerged as a non-invasive modality for guiding cancer diagnostics and treatment decisions. However, efforts have predominantly focused on the analysis of single nucleotide variants (SNVs) and insertions/deletions (indels). Despite the clinical significance of many larger variants such as structural variations (SVs) and copy number alterations (CNAs), detecting them in cfDNA remains a challenge. Given the lack of existing tools we seek to develop an integrated bioinformatic pipeline for SV and CNA detection in cfDNA following targeted hybrid-capture next-generation sequencing (NGS), along with standard SNV and indel analysis. Methods: SVs were first detected using Manta, Lumpy and Delly in plasma cfDNA in comparison with matched peripheral blood leukocyte (PBL) DNA samples from cancer patients, then combined to identify consensus SVs and genotyped throughout samples from patients and healthy individuals. Next, consensus SVs were called somatic events if they were supported by split reads and discordant read pairs in cfDNA samples from patients but not in matched PBL or healthy donor cfDNA samples. For CNA analysis, the ratio of read depth between patient-derived plasma cfDNA and a panel of healthy controls was calculated across genomic bins using the CNVkit tool, followed by bias correction and recentralization using CNA negative control genes to account for read coverage imbalances in targeted NGS. Last, SNV and indel analysis was integrated from the CAPP-Seq pipeline. Results: We applied our pipeline to targeted hybrid-capture NGS data from 48 patients across two independent cohorts of metastatic castration resistant prostate cancer (mCRPC). The targeted panel covered the full-length AR gene body and a hotspot region of TMPRSS2-ERG fusion break points. Consistent with earlier whole genome studies, we confirmed known CNAs and SVs in tumor suppressors, oncogenes and regulatory elements including AR gene and AR enhancer duplications (22/48, 46% of patients), TMPRSS2-ERG gene fusions (9/48, 19%), PTEN and TP53 loss (8/48, 17%). Notably, our pipeline outperformed FACTERA which did not detect any TMPRSS2-ERG gene fusions or AR/enhancer tandem duplications. Subsequent analysis showed high concordance between plasma cfDNA and matched tumor biopsies, and our pipeline recapitulated the landscape of SVs and CNAs in an in silico cfDNA simulation from tumor biopsies. Finally, we showed that alterations of the AR/enhancer locus detected by our pipeline were strongly associated with treatment resistance, patient progression-free and overall survival in mCRPC. Conclusion: We developed a unified pipeline for detection of SVs, CNAs and small mutations in cfDNA targeted sequencing with potential applications in monitoring cancer progression and predicting patient treatment response. Citation Format: Ha X. Dang, Jace Webster, Pradeep S. Chauhan, Steven H. Hartman, Wenjia Feng, Elisa M. Ledet, Haley Ellis, Patrick J. Miller, Ellen B. Jaeger, Sydney A. Caputo, Peter K. Harris, A. Oliver Sartor, Russell K. Pachynski, Aadel A. Chaudhuri, Christopher A. Maher. A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 577.

Characterization and strong risk association of TLR2 del -196 to -174 polymorphism and Helicobacter pylori and their influence on mRNA expression in gastric cancer.

BACKGROUNDToll-like receptor-2 (TLR2) is responsible for recognizing Helicobacter pylori (H. pylori) and activating the immune response. Polymorphisms in TLR2 may modulate gastric carcinogenesis.AIMTo evaluate whether the TLR2 19216T/C (rs3804099) and TLR2 -196 to -174 ins/del (rs111200466) polymorphisms contribute to gastric carcinogenesis in the Brazilian population, and to determine the influence of both polymorphisms and H. pylori infection on TLR2 mRNA expression.METHODSDNA was extracted from 854 peripheral blood leukocyte or gastric tissue samples [202 gastric cancer (GC), 269 chronic gastritis (CG), and 383 control/healthy (C)] and genotyped by allele-specific PCR or restriction fragment length polymorphism (RFLP)-PCR. Quantitative polymerase chain reaction by TaqMan® assay was used to quantify TLR2 mRNA levels in fresh gastric tissues (48 GC, 36 CG, and 14 C).RESULTSRegarding the TLR2 -196 to -174 polymorphism, the ins/del and del/del genotypes were associated with a higher risk of GC by comparison with the C in all of the analyzed inheritance models (codominant, dominant, recessive, overdominant and log-additive; P < 0.0001). Similarly, an increased risk was observed when comparing the GC and CG groups [codominant (P < 0.0001), dominant (P < 0.0001), recessive (P = 0.0260), overdominant (P < 0.0001) and log-additive (P < 0.0001)]. In contrast, TLR2 19216T/C was associated with a protective effect in the GC group compared to the C group [dominant (P = 0.0420) and log-additive (P = 0.0300)]. Regarding the association of polymorphisms with H. pylori infection, individuals infected with H. pylori and harboring the TLR2 -196 to -174 ins/del polymorphism had an increased risk of gastric carcinogenesis [codominant (P = 0.0120), dominant (P = 0.0051), overdominant (P = 0.0240) and log-additive (P = 0.0030)], while TLR2 19216T/C was associated with a protective effect [codominant (P = 0.0039), dominant (P < 0.0001), overdominant (P = 0.0097) and log-additive (P = 0.0021)]. TLR2 mRNA levels were significantly increased in the GC group (median RQ = 6.95) compared to the CG group (RQ = 0.84, P < 0.0001) and to the normal mucosa group (RQ = 1.0). In addition, both H. pylori infection (P < 0.0001) and the presence of the polymorphic TLR2 -196 to -174del (P = 0.0010) and TLR2 19216 C (P = 0.0004) alleles influenced TLR2 mRNA expression.CONCLUSIONThe TLR2 -196 to -174 ins/del and TLR2 19216 T/C polymorphisms are strongly associated with GC. TLR2 mRNA expression levels are upregulated in neoplastic tissues and influenced by both the presence of H. pylori and variant genotypes.

Hypermethylation in Gene Promoters Are Induced by Chronic Exposure to Benzene, Toluene, Ethylbenzene and Xylenes.

Gas station attendants are occupationally exposed to benzene, toluene, ethylbenzene and xylene (BTEX) compounds and thus more susceptible to the biological effects of this mixture present in gasoline, especially due to the carcinogenicity of benzene. Furthermore, the harmful effects of BTEX exposure may be potentiated by genetic and epigenetic inactivation of critical genes. The objective was to evaluate such gene-BTEX interactions accessing the promoter methylation status of p14ARF, p16INK4A and GSTP1 in peripheral blood leukocyte samples. The 59 exposed and 68 unexposed participants from Rio de Janeiro, Brazil, were included. The promoter methylation status was accessed by methylation-specific PCR (MSP) and GSTP1 Ile105Val polymorphism was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) technique. Both p14ARF and p16INK4A were significantly hypermethylated in exposed subjects compared to unexposed (p = 0.004 and p<0.001, respectively). Additionally, p16INK4A hypermethylation in the exposed group was correlated with chromosomal abnormalities (CAs) (p = 0.018), thus highlighting the influence of the gene-environment interactions on genome instability. Noteworthy, p16INK4A methylation was significantly associated with miscarriage among female attendants (p = 0.047), in which those who reported miscarriage exhibited hypermethylation in at least 2 of the 3 genes analyzed. The GSTP1 heterozygote genotype, which could affect the metabolism of benzene detoxification, was found in both groups but was more frequent in those occupationally exposed. No significant association was observed between GSTP1 genotypes and methylation status. Together, these findings indicate that gas station attendants with the aforementioned epigenetic and genetic profiles may be at greater risk of occupational BTEX exposure-induced genome instability, which could require concerted efforts to establish more preventive actions and constant biomonitoring in gas station attendants.

Prevalence of equine herpesvirus 2 (EHV-2) in equine ocular disease

Equine gammaherpesvirus 2 (EHV-2) has been linked to keratitis and keratoconjunctivitis but has also been isolated in horses showing no signs of disease. The aim of the current study was to assess the importance of EHV-2 infection in the aetiopathogenesis of ocular disease, where the applied treatment failed. Seventy-eight horses with nonhealing ocular disease were examined at the Equine Clinic of the University of Veterinary Medicine and Pharmaceutical Sciences, Brno, Czech Republic, between the years 2009 and 2016. In total, 96 conjunctival swabs were taken and, starting from 2014, peripheral blood leukocytes (PBLs) were also examined in 42 patients. Positive EHV-2 results were detected in 53 ocular swab samples (54.64%) and in 22 PBL samples (51.16%). The horses were divided into three groups according to age, up to 3 years, from 3 to 15 years and older than 15 years. Depending on the clinical presentation, horses were also divided into nonulcerative or ulcerative keratitis, keratouveitis, keratoconjunctivitis, and corneal degeneration groups. The group of young horses had a significantly higher ocular swab positivity compared to the middle group (P = 0.01). Increased bilateral ocular occurrence with decreasing age was observed, although it was not significant (P = 0.04). Significant correlation was confirmed between PBL samples and ocular swabs (P = 0.01). This correlation was even higher in cases of bilateral infection. No significant differences were detected when comparing the groups according to the clinical presentation. This study describes the prevalence of EHV-2 in different age group horses with non-healing keratopathies.

DNA methylation of hypertension-related genes is influenced by the MTHFR 677TT genotype and riboflavin supplementation

AbstractIntroduction:The C677T polymorphism in the folate metabolising enzyme methylenetetrahydrofolate reductase (MTHFR) is associated with hypertension. Riboflavin acts as a cofactor for MTHFR in one-carbon metabolism which generates methyl groups for utilisation in important biological reactions such as DNA methylation. Supplementation with riboflavin has previously been shown to lower blood pressure in individuals with the MTHFR 677TT genotype. The mechanism regulating this gene-nutrient interaction is currently unknown but may involve aberrant DNA methylation which has been implicated hypertension.Objectives:The aims of this study were to examine DNA methylation of hypertension-related genes in adults stratified by MTHFR C677T genotype and the effect of riboflavin supplementation on DNA methylation of these genes in individuals with the MTHFR 677TT genotype.Materials and Methods:We measured DNA methylation using pyrosequencing in a set of candidate genes associated with hypertension including angiotensin II receptor type 1 (AGTR1), G nucleotide binding protein subunit alpha 12 (GNA12), insulin-like growth factor 2 (IGF2) and nitric oxide synthase 3 (NOS3). Stored peripheral blood leukocyte samples from participants previously screened for the MTHFR C677T genotype who participated in targeted randomised controlled trials (1.6mg/d riboflavin or placebo for 16 weeks) at Ulster University were accessed for this analysis (n = 120).Results:There were significant differences in baseline average methylation between MTHFR CC and TT genotypes at NOS3 (p = 0.026) and AGTR1 (p = 0.045) loci. Riboflavin supplementation in the TT genotype group resulted in altered average methylation at IGF2 (p = 0.025) and CpG site-specific alterations at the AGTR1 and GNA12 loci.Conclusion:DNA methylation at genes related to hypertension were significantly different in individuals stratified by MTHFR genotype group. Furthermore, in MTHFR 677TT genotype individuals, there were concurrent alterations in DNA methylation at genes linked to hypertension in response to riboflavin supplementation. This is the largest study to date to demonstrate an interaction between DNA methylation of hypertension-related genes and riboflavin supplementation in adults with the MTHFR 677TT genotype. Further work using a genome-wide approach is required to better understand the role of riboflavin in altering DNA methylation in these genetically at-risk individuals.

Leukocyte Telomere Length in Patients with Radiographic Knee Osteoarthritis.

Relative mean telomere sequence amount was determined by quantitative PCR (qPCR) of peripheral blood leukocyte (PBL) samples obtained at recruitment (n = 310) from individuals from the Osteoarthritis (OA) Initiative consortium. Knees were radiologically evaluated according to the Kellgren-Lawrence (KL) score, ranging from 0 to 4, considering a KL grade ≥ 2 as radiographic evidence of OA (n = 124). Telomere size decreased as baseline KL score increased, being significantly shorter in subjects with KL ≥2 (Mann-Whitney U-test, P < 0.0001). PBL telomere size was also associated with age, hypertension, body mass index (BMI) and waist circumference. Nevertheless, logistic regression analysis showed that PBL telomere size was a consistent risk factor for concurrent knee OA, independent of these health parameters. Shorter PBL telomeres may indicate a premature aging status which enhances chondrocyte senescence and degenerative joint disease. Environ. Mol. Mutagen. 60:298-301, 2019. © 2018 Wiley Periodicals, Inc.

Abstract 4668: Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma

Abstract Introduction T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors having a shared antigen specificity but different amino acid or nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we evaluate evidence for T cell convergence within tumor biopsy research samples from a set of 85 subjects with melanoma. Methods Total RNA from 85 tumor biopsy research samples (non-FFPE) was extracted for use in long-amplicon TCRB chain sequencing (mean amplicon length of 330bp covering CDR1, 2 and 3) via the Ion AmpliSeq Immune Repertoire Assay Plus, TCRB. To evaluate T cell convergence within each biopsy, we searched for instances where TCRB chains were identical in amino acid space (shared variable gene identity and CDR3 amino acid sequence) but had distinct nucleotide sequences owing to N-addition and exonucleotide chewback within the V-D and D-J junctions of the CDR3. To provide context, we evaluated evidence for T cell convergence with T cell repertoires derived from healthy donor peripheral blood leukocytes (PBL). Results Sequencing of melanoma biopsies yielded an average of 6029 clones per sample. 11 of 85 samples yielded fewer than 100 clones and were eliminated from downstream analysis. Convergent T cell receptors were identified in 68/74 (92%) of tumor infiltrating T cell repertoires having greater than 100 detected clones. The frequency of convergent rearrangements was approximately 50-fold greater in the melanoma-infiltrating T cell repertoire than healthy PBL samples (p&amp;lt;.001). Conclusions These data suggest that T cell convergence may be a common feature of the melanoma infiltrating T cell repertoire. Convergence was more frequently observed within the TME than T cell repertoires derived from healthy PBL, consistent with elevated antigen-driven T cell selection within the TME. The extent to which convergence is a feature of the TME in other cancers is not yet known. T cell receptor convergence may be driven by T cell responses to tumor neoantigen within the TME. In such case, in silico identification of convergent T cell receptors by long-amplicon sequencing may serve as an approach for rapid identification of antigen-specific T cell receptors for future therapeutic use. For research use only. Citation Format: Timothy J. Looney, Sean Glenn, Sarabjot Pabla, Jeff Conroy, Carl Morrison, Alice Zheng, Lauren Miller, Elizabeth Linch, Denise Topacio, Geoff Lowman, Fiona Hyland, Mark Anderson. Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4668.

Genome-wide analysis reveals that altered methylation in specific CpG loci is associated with childhood obesity.

Over the past decades, the epidemic of childhood obesity has greatly increased, and it has recently become a global public health concern. Methylation, serving as a crucial regulator of the gene-environment interaction, has exhibited a strong association with obesity. In this study, we aimed to evaluate the relationship between DNA methylation and childhood obesity, and further uncover the potential association of aberrantly methylated genes with obesity. DNA samples of peripheral blood leukocytes from three obese subjects (mean BMI: 21.67) and 4 age/sex matched controls (mean BMI: 14.92) were subjected to Infinium Human Methylation 450 Bead Array analysis. A total of more than 4 85 000 methylation sites were identified across the genome, and 226 methylated CpGs (DMCpGs) were differentially methylated between these two groups. Subsequent Gene Ontology (GO) and KEGG Pathway analyses showed that these DMCpGs were mainly engaged in immunity and lipoprotein metabolism, indicating their physiological significance. Further verification of the candidate CpG sites within the HDAC4, RAX2, APOA5, CES1, and SLC25A20 gene loci, were performed using bisulfite sequencing PCR (BSP) in a cohort of 42 controls and 39 obese cases. The results revealed that methylation levels within HDAC4 and RAX2 loci were positively associated with obesity, while the methylation levels of loci within APOA5 and CES1 loci were negatively correlated with obesity. Thus, alterations in methylation of CpG sites of specific genes may contribute to childhood obesity, which provide novel insights into the aetiology of obesity.

Association between mitochondrial DNA content and baseline serum levels of HBsAg in chronic hepatitis B infection.

Recent studies have demonstrated a potential link between mitochondrial DNA (mtDNA) content and cirrhosis or hepatocellular carcinoma (HCC). However, there are few studies evaluating mtDNA content as a noninvasive marker of chronic hepatitis B infection (CHB). In this study, we conducted a case-control study to determine mtDNA content in peripheral blood leukocyte (PBL) samples from 76 CHB cases naïve to antivirus therapy and 96 healthy controls, and then evaluated the association between mtDNA content and baseline serum concentration of HBV markers. Consequently, CHB cases had significantly higher mtDNA content than healthy controls (1052.85 vs 618.98, P < 0.001). Pearson's correlation analysis revealed that mtDNA content was negatively correlated with the baseline levels of hepatitis B surface antigen (HBsAg) (r = -0.291, P = 0.011) in CHB patients. In a trend analysis, a statistically significant association was detected between lower mtDNA content and increasing levels of HBsAg (P = 0.015). In conclusion, our study provides the first epidemiological evidence that mtDNA content of CHB cases naive to antivirus therapy is significantly higher than healthy controls and the levels of mtDNA content is negatively associated with HBsAg. mtDNA content may serve as a potential noninvasive biomarker of CHB which may need more researches to validate.