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The Cancer Genome Atlas (TCGA) and other large-scale genomic data pipelines have been integral to the current understanding of the molecular events underlying renal cell carcinoma (RCC). These data networks have focused mostly on primary RCC, which often demonstrates indolent behavior. However, metastatic disease is the major cause of mortality associated with RCC and data sets examining metastatic tumors are sparse. Therefore, a more comprehensive analysis of gene expression and DNA methylome profiling of metastatic RCC in addition to primary RCC and normal kidney was performed. Integrative analysis of the methylome and transcriptome identified over 30 RCC-specific genes whose mRNA expression inversely correlated with promoter methylation, including several known targets of hypoxia inducible factors. Notably, genes encoding several metabolism-related proteins were identified as differentially regulated via methylation including hexokinase 2, aldolase C, stearoyl-CoA desaturase, and estrogen-related receptor-γ (ESRRG), which has a known role in the regulation of nuclear-encoded mitochondrial metabolism genes. Several gene expression changes could portend prognosis in the TCGA cohort. Mechanistically, ESRRG loss occurs via DNA methylation and histone repressive silencing mediated by the polycomb repressor complex 2. Restoration of ESRRG in RCC lines suppresses migratory and invasive phenotypes independently of its canonical role in mitochondrial metabolism.Implications:Collectively, these data provide significant insight into the biology of aggressive RCC and demonstrate a novel role for DNA methylation in the promotion of HIF signaling and invasive phenotypes in renal cancer.

Supplemental Table 1. Location of metastasis used for array analysis

1S: Principal component analysis (PCA) of the 44 experimental samples analyzed by array. S2. 2S: TCGA validation of up-regulated RCC associated genes. S3. TCGA validation of down-regulated RCC associated genes. Boxplots show expression level of each gene in 72 kidney normal samples and 533 kidney renal clear cell carcinoma samples. S4. Oncomine analysis of RCC associated genes. S5. Oncomine analysis of RCC associated genes. S6. Oncomine analysis of RCC associated genes. S7. Kaplan meier plots showing effect of gene expression level on overall survival of kidney renal clear cell carcinoma patients.

Background Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. Method Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. Results Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. Conclusions In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.

Background Recent progress in xenotransplantation of porcine islets to non-human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is necessary. The enzyme-linked immunospot (ELISpot) assay has been widely used to monitor antigen-specific alloreactive T-cell responses in humans; however, the utility of porcine islet-specific ELISpot assay has not yet been thoroughly evaluated for pig-to-NHPs intraportal islet xenotransplantation. Methods The optimal ELISpot assay conditions, including the number of responder and stimulator cells and the provision of costimulation, were determined. Then, ELISpot assays were conducted on serial stocks of peripheral blood mononuclear cell (PBMC) samples previously isolated from NHP recipients transplanted with porcine islets. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were used for antigen stimulation. At the same time, the ratio of CD4(+) /CD8(+) T cells and the percentage of CD4(+) FoxP3(+) T cells in the peripheral blood were evaluated. Finally, liver biopsy samples were evaluated to assess the immunopathology of the grafts. Results The optimal conditions for the ELISpot assay were defined as 2.5 × 10(5) responder cells incubated with 5.0 × 10(5) stimulator cells in 96-well, flat-bottom plates without further costimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen-specific IFN-γ-producing cells significantly increased upon overt graft rejection. However, use of third-party porcine islets as stimulators did not reflect graft rejection, suggesting that the use of donor-specific PBMCs, and not tissue (porcine islet)-specific cells, as stimulators could better serve the purpose of this assay in adult porcine islet transplantation. IFN-γ spot number was neither influenced by the peripheral blood CD4(+) /CD8(+) T-cell ratio nor the percentage of CD4(+) FoxP3(+) T cells. Finally, in cases of overt graft rejection, the number of IFN-γ spots and the graft-infiltrating T cells in biopsied liver samples increased simultaneously. Conclusion Use of PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation.

We developed a calcium phosphate (CaP)-assembled polymer nanocarrier for intracellular doxorubicin (DOX) delivery based on a mussel-inspired mineralization approach. A DOX-loaded core-shell polymer nanoparticle (DOX-NP) consisting of a poly(3,4-dihydroxy-l-phenylalanine) (PDOPA) core and a poly (ethylene glycol) (PEG) shell was utilized as a nanotemplate for CaP mineralization. The mean hydrodynamic diameter of the DOX-loaded CaP-mineralized polymer nanoparticles (DOX-CaP-NPs) was 154.3nm. Energy-dispersive X-ray spectroscopy confirmed that the DOX-CaP-NPs contained substantial amounts of Ca and P, elements found only in the CaP mineral. The loading efficiency and content of DOX, estimated by fluorescence spectroscopy, were 54.0% and 10.8wt%, respectively. The CaP deposited in the PDOPA core domain enabled the DOX-CaP-NPs to maintain a robust structure and effectively inhibit DOX release at extracellular pH, whereas at endosomal pH, the CaP core dissolved to trigger a facilitated DOX release. The DOX-CaP-NPs may serve as robust nanocarriers with a high delivery efficacy for cancer chemotherapy.

Here, we tested the performance of the mitochondrial DNA copy number (mtDNA-CN) in predicting future type 2 diabetes (n = 1108). We used the baseline clinical data (age, sex, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure) and the mtDNA-CN, hemoglobin A1c (A1C) levels and results of oral glucose tolerance test (OGTT) including fasting plasma glucose, 1-hour glucose, and 2-hour glucose levels, to predict future diabetes. We built a prediction model using the baseline data and the diabetes status at biannual follow-up of 8 years. The mean area under curve (AUC) for all follow-ups of the full model including all variables was 0.92 ± 0.04 (mean ± standard deviation), while that of the model excluding the mtDNA-CN was 0.90 ± 0.03. The sensitivity of the f4ull model was much greater than that of the model not including mtDNA-CN: the mean sensitivities of the model with and without mtDNA-CN were 0.60 ± 0.06 and 0.53 ± 0.04, respectively. We found that the mtDNA-CN of peripheral leukocytes is a biomarker that augments the predictive power for future diabetes of A1C and OGTT. We believe that these results could provide invaluable information for developing strategies for the management of diabetes.

Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activationmore » while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.« less

Although over 30 common genetic susceptibility loci have been identified to be independently associated with coronary artery disease (CAD) risk through genome-wide association studies (GWAS), genetic risk variants reported to date explain only a small fraction of heritability. To identify novel susceptibility variants for CAD and confirm those previously identified in European population, GWAS and a replication study were performed in the Koreans and Japanese. In the discovery stage, we genotyped 2123 cases and 3591 controls with 521 786 SNPs using the Affymetrix SNP Array 6.0 chips in Korean. In the replication, direct genotyping was performed using 3052 cases and 4976 controls from the KItaNagoya Genome study of Japan with 14 selected SNPs. To maximize the coverage of the genome, imputation was performed based on 1000 Genome JPT+CHB and 5.1 million SNPs were retained. CAD association was replicated for three GWAS-identified loci (1p13.3/SORT1 (rs599839), 9p21.3/CDKN2A/2B (rs4977574), and 11q22.3/ PDGFD (rs974819)) in Koreans. From GWAS and a replication, SNP rs3782889 showed a strong association (combined P=3.95 × 10(-14)), although the association of SNP rs3782889 doesn't remain statistically significant after adjusting for SNP rs11066015 (proxy SNP with BRAP (r(2)=1)). But new possible CAD-associated variant was observed for rs9508025 (FLT1), even though its statistical significance did marginally reach at the genome-wide a significance level (combined P=6.07 × 10(-7)). This study shows that three CAD susceptibility loci, which were previously identified in European can be directly replicated in Koreans and also provides additional evidences implicating suggestive loci as risk variants for CAD in East Asian.

Glucose ingestion-induced hyperglycemia has been known to induce inflammation, which is related to the pathogenesis of diabetic complications. To examine acute gene expression responses to physiological oral glucose ingestion in human circulating leukocytes, we conducted a microarray study of human circulating leukocytes sampled before, 1 h after, and 2 h after glucose ingestion in community-based participants without previous histories of diabetes ( n = 60). Ingestion of 75 g glucose successfully induced acute hyperglycemia (glucose concentration 91.6 ± 5.3 mg/dl for fasting and 180.7 ± 48.5 mg/dl for 1 h after glucose ingestion). Oral glucose ingestion significantly increased the expressions of 23 genes and decreased the expressions of 13 genes [false discovery rate (FDR) P value

Personalized medicine is emerging as a main paradigm for risk prediction, pre-diagnosis, and effective prevention and treatment of disease. A large number of human biospecimens and their clinical data are essential resources for the success of personalized medicine as well as other biomedical research.The National Biobank of Korea (NBK) has collected well-annotated and high quality human biospecimens, and distributes them to the Korean biomedical scientists, through the Korea Biobank Project (KBP). The ultimate goal of NBK activities is to promote biomedical research and public health. As of December-2011, the NBK has collected various human biospecimens from 525,416 participants including 325,952 Korean populations and 199,464 patients. The purpose of this paper is to introduce the KBP and quality control programs for collection of human biospecimens with high quality of NBK.

Human lymphoblastoid cell lines (LCLs) are generated by EBV-mediated B-cell transformation to provide unlimited genomic resources for human genetics and immunological studies. The LCL is a good in vitro cell model for assessing population differences in the basal expression of genes and miRNAs as well as in cellular responses to various stimulators. Recently, the utility of LCLs was extended to pharmacogenomic studies to discover genetic factors underlying individual variations in response to chemicals and environmental stresses. Although LCLs represent generally lymphoid tissue-specific biological characteristics, genomic signatures of LCLs can distinguish patients with brain-related diseases and nonlymphoid tumors from normal controls. MicroRNA is known to be an epigenetic transcriptional regulator, and its expression is induced in abnormal conditions such as perturbagen-stimulated, virus-infected, or cancer cells. The epigenetic regulation of gene expression mediated by microRNA and DNA methylation is important for understanding the pathogenesis of cancers and complex diseases as well as discovering for therapeutic targets. For integrative genomic analyses, LCLs can be utilized to generate cellular phenotypes and various genomic data (e.g., SNP, CNV, transcriptome, methylome, etc.), which can be linked to clinical information of donors. Here, we discuss miRNA-mediated gene expression in LCLs and its application to disease genomics and global transcriptional regulatory machinery studies.

The Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) is one of the major genomic resources for human genetics and immunological studies. Use of LCLs is currently extended to pharmacogenetic studies to investigate variations in human gene expression as well as drug responses between individuals. We evaluated four common internal controls for gene expression analysis of selected hematopoietic transcriptional regulatory genes between B cells and LCLs. In this study, the expression pattern analyses showed that TBP (TATA box-binding protein) is a suitable internal control for normalization, whereas GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is not a good internal control for gene expression analyses of hematopoiesis-related genes between B cells and LCLs at different subculture passages. Using the TBP normalizer, we found significant gene expression changes in selected hematopoietic transcriptional regulatory genes (downregulation of RUNX1, RUNX3, CBFB, TLE1, and NOTCH2 ; upregulation of MSC and PLAGL2) between B cells and LCLs at different passage numbers. These results suggest that these hematopoietic transcriptional regulatory genes are potential cellular targets of EBV infection, contributing to EBV-mediated B-cell transformation and LCL immortalization.

Objective: MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein–Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) and associated clinical traits. Material and Methods: Hence, we performed miRNA microarray approach with early- (p6) and late-passage (p161) LCLs. Results and Conclusion: Microarray data showed that nine miRNAs (miR-20b*, miR-28-5p, miR-99a, miR-125b, miR-151-3p, miR-151:9.1, miR-216a, miR-223* and miR-1296) were differentially expressed in most LCLs during long-term culture. In particular, miR-125b was up-regulated in all the tested late-passage LCLs. miR-99a, miR-125b, miR-216a and miR-1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long-term culture in a previous study. Linear regression analysis showed that miR-200a and miR-296-3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor’s health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV-mediated tumorigenesis.

Disease amelioration by mesenchymal stem cells (MSCs) has been shown to be closely related to their immunomodulatory functions on the host immune system in many disease models. However, the underlying mechanisms of how these cells affect the immune cells in vivo are not fully understood. In this study, we report findings that a small but significant number of MSCs accumulate in the secondary lymphoid organs and attenuate delayed-type hypersensitivity (DTH) response by inducing apoptotic cell death of surrounding immune cells in the draining lymph node (LN). In the migration study, i.v. infused GFP-MSCs preferentially accumulated at the boundary between the paracortical area and the germinal center in the LNs, in close proximity to various types of immune cells including T, B, and dendritic cells in a dose-dependent manner. As a result, accumulated MSCs markedly attenuated DTH response in proportion to the number of MSCs infused. During the DTH response, the infiltration of T cells in the challenged site was significantly decreased, whereas a number of apoptotic T cells were remarkably increased in the draining LN. Apoptosis was significantly induced in activated T cells (CD3+ and BrdU+), but not in the resting T cells (CD3+ and BrdU−). NO was associated with these apoptotic events. Taken together, we conclude that significant numbers of i.v. infused MSCs preferentially localize in the draining LN, where they induce apoptosis of the activated T cells by producing NO and thus attenuate the DTH response.

Objectives: The EBV-transformed lymphoblastoid cell line (LCL) is a useful resource for population-based human genetic and pharmacogenetic studies. The principal objective here was to assess expression phenotype changes during long-term subculture of LCLs, and its clinical significance. Materials and methods: We searched for genes that were differentially expressed in 17 LCLs at late (p161) passage compared to early passage (p4) using microarray assay, then validated them by real-time RT-PCR analysis. In addition, we estimated correlations between expression phenotypes of 20 LCL strains at early passage and 23 quantitative clinical traits from blood donors of particular LCL strains. Results: Transcript sequences of 16 genes including nuclear factor-κB (NF-κB) pathway-related genes (such as PTPN13, HERC5 and miR-146a) and carcinogenesis-related genes (such as XAF1, TCL1A, PTPN13, CD38 and miR-146a) were differentially expressed (>2-fold change) in at least 15 of the 17 LCL strains. In particular, TC2N, FCRL5, CD180, CD38 and miR-146a were downregulated in all 17 of the evaluated LCL strains. In addition, we identified clinical trait-associated expression phenotypes in LCLs. Conclusion: Our results showed that LCLs acquired expression phenotype changes involving expression of NF-κB pathway- and carcinogenesis-related genes during long-term subculture. These differentially expressed genes can be considered to be a gene signature of LCL immortalization or EBV-induced carcinogenesis. Clinical trait-associated expression phenotypes should prove useful in the discovery of new candidate genes for particular traits.

An efficient asymmetric synthesis of 6-aminobicyclo[2.2.1]heptane-2-carboxylic acid as a novel γ-turn mimic has been achieved. Structural analysis of the γ-amino acid derivative was carried out using 1H NMR spectroscopy and intramolecular hydrogen bonding between side chain amides confirmed the turn structure, which had been predicted by Ab initio computational study.

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4(+) Tregs have been identified, including Foxp3(+), Tr1, Th3, and Foxp3neg iT(R)35 cells. In this study, we identified a new subset of CD4(+)VEGFR1(high) Tregs that have immunosuppressive capacity. CD4(+)VEGFR1high T cells, which constitute approximately 1.0% of CD4(+) T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4(+)VEGFR1(high) T cells are distinct from known Tregs. CD4(+)VEGFR1(high) T cells suppressed the proliferation of CD4(+)CD25(-) T cell as efficiently as CD4(+)CD25(high) natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4(+)VEGFR1(+) T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4(+) VEGFR1(high) T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.

Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) promise to address the challenge posed by the limited availability of primary cells needed as a source of genomic DNA for genetic studies. However, the genetic stability of LCLs following prolonged culture has never been rigorously investigated. To evaluate genotypic errors caused by EBV integration into human chromosomes, we isolated genomic DNA from human peripheral blood mononuclear cells and LCLs collected from 20 individuals and genotyped the DNA samples using the Affymetrix 500K SNP array set. Genotype concordance measurements between two sources of DNA from the same individual indicated that genotypic discordance is negligible in early-passage LCLs (20 passages) but substantial in late-passage LCLs (50 passages). Analysis of concordance on a chromosome-by-chromosome basis identified genomic regions with a high frequency of genotypic errors resulting from the loss of heterozygosity observed in late-passage LCLs. Our findings suggest that, although LCLs harvested during early stages of propagation are a reliable source of genomic DNA for genetic studies, investigations that involve genotyping of the entire genome should not use DNA from late-passage LCLs.

Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12more » putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.« less

Maximum engraftment of transplanted islets is essential for the clinical application of a subcutaneous site. Significant barriers to the current approaches are associated with their low effectiveness, complexity and unproven biosafety. Here, we evaluated and optimized a fibrin-islet composite for effective glycemic control in a subcutaneous site whose environment is highly hypoxic due to low vascularization potential. In the setting of xenogeneic porcine islet transplantation into the subcutaneous space of a diabetic mouse, the in vivo islet functions were greatly affected by the concentrations of fibrinogen and thrombin. The optimized hydrogel-type fibrin remarkably reduced the marginal islet mass to approximately one tenth that of islets without fibrin. This marginal islet mass was comparable to that in the setting of the subcapsular space of the kidney, which is a highly vascularized organ. Highly vascularized structures were generated inside and on the outer surface of the grafts. A hydrogel-type fibrin-islet composite established early diabetic control within an average of 3.4days after the transplantation. In the mechanistic studies, fibrin promoted local angiogenesis, enhanced islet viability and prevented fragmentation of islets into single cells. In conclusion, in situ application of hydrogel-type fibrin-islet composite may be a promising modality in the clinical success of subcutaneous islet transplantation.

Biobanking became a necessity for translating genetic discoveries into clinical practice. Approaches to personalized medicine require a new model system for functional and pharmacogenomic studies of a variety of accumulating genetic variations, as well as new research environments such as biobankomics. Human lymphoblastoid cell lines (LCLs) will provide a valuable tool to meet such new demands in the biobankomics era. The National Biobank of Korea (NBK), which is leading the Korea Biobank Project, has a large collection of LCLs derived mostly from population-based cohort samples. Using a special long-term subculture collection of NBK LCLs, biological characteristics of early passage LCLs and terminally immortalized LCLs have been investigated to promote the utilization of LCLs and provide well quality-controlled LCLs for genetic and pharmacogenomic studies. As LCLs have been successfully phenotyped for cytotoxicity in response to various stimulators, including chemotherapeutic agents, environmental chemicals and irradiation, the utility of LCLs will increase in the future. Here, we discuss current and future applications of NBK LCLs for the biobankomics era.

To examine copy number variations among the Korean population, we compared individual genomes with the Korean reference genome assembly using the publicly available Korean HapMap SNP 50 k chip data from 90 individuals. Korean individuals exhibited 123 copy number variation regions (CNVRs) covering 27.2 mb, equivalent to 1.0% of the genome in the copy number variation (CNV) analysis using the combined criteria of P value (P < 0.01) and standard deviation of copy numbers (SD ≥ 0.25) among study subjects. In contrast, when compared to the Affymetrix reference genome assembly from multiple ethnic groups, considerably more CNVRs (n = 643) were detected in larger proportions (5.0%) of the genome covering 135.1 mb even by more stringent criteria (P < 0.001 and SD ≥ 0.25), reflecting ethnic diversity of structural variations between Korean and other populations. Some CNVRs were validated by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method, and then copy number invariant regions were detected among the study subjects. These copy number invariant regions would be used as good internal controls for further CNV studies. Lastly, we demonstrated that the CNV information could stratify even a single ethnic population with a proper reference genome assembly from multiple heterogeneous populations.

As alkylating agents, cyclophosphamides (CTX) are used to treat various cancers and, ironically, to boost immune responses. In the present study, we attempted to elucidate the mechanism responsible for the immunomodulatory effect of high-dose CTX in an established tumor model. A single injection of high-dose CTX increased the survival rate of immunocompetent, but not immunodeficient, mice. Notably, 10 days after CTX injection, the number of CD44(high) memory T cells significantly increased, without a selective decrease in the actual number and percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the proportion of Tregs among CD4+ T cells decreased due to expansion of memory and other CD4+ T cell subtypes. This outcome was accompanied by an increase in IL-15 mRNA and up-regulation of IL-15 receptors in the CD44+CD8+ T cell compartment. We postulate that the CTX-induced change in T cell balance may increase anti-tumor immunity.

An efficient asymmetric synthesis of 6-aminobicyclo[2.2.1]heptane-2-carboxylic acid as a novel gamma-turn mimic has been achieved. Structural analysis of the gamma-amino acid derivative was carried out using (1)H NMR spectroscopy and intramolecular hydrogen bonding between side chain amides confirmed the turn structure, which had been predicted by Ab initio computational study.

Much research evidence supports the hypothesis that chronic, low-grade inflammation related to innate immunity may play an important role in the pathophysiology of type 2 diabetes mellitus (T2DM). Runt-related transcription factor 2 (RUNX2; MIM# 600211) acts as a scaffold that controls the integration, organization, and assembly of nucleic acids. To examine whether the novel promoter variant in RUNX2 is associated with the risk of T2DM and related phenotypes, RUNX2-742GT was genotyped in 378 T2DM patients and 382 normal controls recruited in the Korean T2DM Study. Statistical analysis revealed that RUNX2-742GT was associated with serum triglyceride level (TG) in nondiabetic controls, although it was not associated with the risk of T2DM. Individuals who carry T/T, T/G, and G/G genotypes had the highest (2.061 +/- 0.20), intermediate (2.01 +/- 0.19), and the lowest (1.97 +/- 0.18) levels of log [TG (mmol/l)] (P = 0.007), respectively. Our data on this important variant of RUNX2 suggest that lipid metabolism might be affected by genetic polymorphisms in the promoter region.

The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes.

Array CGH has been applied to detect chromosomal aberrations in cancer and genetic diseases. Epstein-Barr virus (EBV)-infected B lymphocytes are transformed to continuously proliferating lymphoblastoid cell lines (LCLs), which are a very common genome resource for human genetic studies. We used bacterial artificial chromosome (BAC) array CGH to assess a chromosomal aberration of LCLs in EBV-induced B-cell transformation. At early passages, LCLs exhibited a greater copy number variation in 1p36.33 compared to primary B-cells. Quantitative polymerase chain reaction (PCR) confirmed the increase in the copy number in 1p36.33. Because a segment of 1p36.33 is nearly identical to a part of the mitochondrial DNA, this increase was attributed to an increase in the copy number of mitochondrial DNA. The expression levels of mitochondrial biogenesis-related genes were elevated in the LCLs, which is consistent with the increased copy numbers of mitochondrial DNA, suggesting that increased mitochondrial biogenesis is indicative of the progression of EBV-mediated B-cell transformation. In addition, our array CGH of LCLs revealed potential copy number polymorphisms of chromosomal segments among Korean populations. Taken together, these findings suggest that LCLs in the early passages preserve the chromosomal integrity of primary B-cells at the cytogenetic level during EBV-transformed B-cell immortalization, except for a copy number variation in 1p36.33 due to increased mitochondrial DNA copy numbers. Thus, analyses of array CGH profiles of diseases should take into account the potential for copy number variation of 1p36.33.

We report on pH-responsive core-shell polymer micelles with catechol-Fe(3+) coordinated core cross-links, which provide robustness to drug-loaded polymer micelles and allow the facilitated intracellular release of loaded anticancer drugs in response to an endosomal acidic pH.

T cell receptor transgenic mouse on RAG knock out background does not have regulatory T cells (Tregs). We could induce adaptive Tregs in one of such mouse strain Marilyn by HY-antigen-mismatched islet transplantation. We transplanted male C57BL/6 islet to streptozotocin-induced diabetic female Marilyn mouse. Marilyn mouse is HY(minor histocompatibility antigen in Y chromosome)-specific TCR transgenic mouse on RAG2 knock out C57BL/6 background. Naïve Marilyn mouse rejects male skin. But, male islets were accepted by Marilyn mouse and induced adaptive Tregs. After acceptance of islet grafts, we grafted male C57BL/6 skin to confirm the tolerance to male antigen. In that skin graft, induced Tregs could prevent rejection to male’s skin. We could also detect Tregs after female islet transplantation. But, they were donor-origin Tregs, and those Tregs could not protect the male skin from rejection. In conclusion, we can make de novo induction of Tregs by islet transplantation, and those Tregs are antigen-specific Tregs.

Background Recent efforts have been made to link complex human traits and disease susceptibility to DNA copy numbers. The leptin receptor (LEPR) has been implicated in obesity and diabetes. Mutations and genetic variations of LEPR gene have been discovered in rodents and humans. However, the association of DNA copy number variations at the LEPR gene locus with human complex diseases has not been reported. In an attempt to study DNA copy number variations associated with metabolic traits and type 2 diabetes mellitus (T2DM), we targeted the LEPR gene locus in DNA copy number analyses. Results We identified DNA copy number variations at the LEPR gene locus among a Korean population using genome-wide SNP chip data, and then quantified copy numbers of the E2 DNA sequence in the first two exons overlapped between LEPR and LEPROT genes by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method. Among the non-diabetic subjects (n = 1,067), lower E2 DNA copy numbers were associated with higher fasting glucose levels in men (p = 1.24 × 10-7) and women (p = 9.45 × 10-5), as well as higher total cholesterol levels in men (p = 9.96 × 10-7). In addition, the significant association between lower E2 DNA copy numbers and lower level of postprandial 2hr insulin was evident only in non-diabetic women, whereas some obesity-related phenotypes and total cholesterol level exhibited significant associations only in non-diabetic men. Logistic regression analysis indicated that lower E2 DNA copy numbers were associated with T2DM (odds ratio, 1.92; 95% CI, 1.26~2.96; p < 0.003) in our nested case-control study. Interestingly, the E2 DNA copy number exhibited a negative correlation with LEPR gene expression, but a positive correlation with LEPROT gene expression. Conclusions This work suggests that a structural variation at the LEPR gene locus is functionally associated with complex metabolic traits and the risk of T2DM.

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naïve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naïve and pre-activated T cells in which IL-10 and IDO play important roles.

Background CXCR3 is a chemokine receptor that plays important roles in mediating chemotactic signals and modulating the activation of lymphocytes. We have previously conducted a case-control study by using a candidate gene approach to investigate the association of CXCR3 polymorphisms with the risk of asthma. Results from the epidemiologic study showed that a common nucleotide variant in the CXCR3 intron (rs2280964G>A) was associated with disease susceptibility (1006 cases and 384 control subjects; odds ratio, 0.81; 95% CI, 0.69-0.94; P = .007). Objective The aim of our study was to evaluate the epidemiologic study and provide functional evidence for the association of rs2280964G>A with asthma by investigating the effects of intronic variant on chemokine-mediated phenotypes of human-derived T cells. Methods We used cell line–based in vitro and human primary T cell–based ex vivo studies to examine the functional consequences of the intronic polymorphism, focusing on the regulation of gene expression, splicing, and immune responsiveness toward activating signals. Results We present functional evidence indicating that the rs2280964A allele significantly correlates with decreased CXCR3 gene expression, which would lead to variation in immune cell responses to chemokine-cytokine signals in vitro and ex vivo that includes a decrease in chemotactic activity. Conclusion These findings, in conjunction with those of our previous epidemiologic studies, might implicate a functional link between a common nucleotide variant of a chemokine receptor gene, CXCR3 , and a cause for a complex-trait disease, asthma.