The Effect of Enzymatically Polymerised Polyphenols on CD4 Binding and Cytokine Production in Murine Splenocytes

High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, anti-HIV, and anti-cancer activities. The purpose of this study was to evaluate the immunomodulating activities of enzymatically polymerised polyphenols, and to clarify the underlying mechanisms of their effects. The cytokine-inducing activity of the enzymatically polymerised polyphenols derived from caffeic acid (CA), ferulic acid (FA), and p-coumaric acid (CoA) was investigated using murine splenocytes. Polymerised polyphenols, but not non-polymerised polyphenols, induced cytokine synthesis in murine splenocytes. Polymerised polyphenols induced several cytokines in murine splenocytes, with interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most prominent. The underlying mechanisms of the effects of the polymerised polyphenols were then studied using neutralising antibodies and fluorescent-activated cell sorting (FACS) analysis. Our results show that polymerised polyphenols increased IFN-γ and GM-CSF production in splenocytes. In addition, the anti-CD4 neutralised monoclonal antibody (mAb) inhibited polymerised polyphenol-induced IFN-γ and GM-CSF secretion. Moreover, polymerised polyphenols bound directly to a recombinant CD4 protein, and FACS analysis confirmed that interaction occurs between polymerised polyphenols and CD4 molecules expressed on the cell surface. In this study, we clearly demonstrated that enzymatic polymerisation confers immunoactivating potential to phenylpropanoic acids, and CD4 plays a key role in their cytokine-inducing activity

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Effect of various polyphenols on cell viability in murine splenocytes.

<p>C57BL/6 splenocytes were stimulated with various polyphenols (0–100 µg/mL). After 48 h, relative living cell numbers were assessed by the MTT method. The vertical axis represents the percentage living cell numbers of the splenocytes, and was obtained on the basis of the ratio to the control cells. The values represent the mean ± standard deviation, n = 3.</p

Effect of polymerised polyphenols on cytokine production in murine splenocytes.

<p>C57BL/6 splenocytes were stimulated with various polyphenols (0–100 µg/mL). After 48 h, supernatants were collected and concentrations of cytokines; (A) IL-1β, (B) IL-4, (C) IL-6, (D) IFN-γ, and (E) GM-CSF were measured by ELISA. The values represent the mean ± standard deviation, n = 3. Significant difference from untreated splenocytes: ***<i>p</i><0.001.</p

The competitive binding of polyphenols and anti-CD4 antibody to cell-surface CD4.

<p>Fresh splenocytes were pre-incubated with various polyphenols. After blocking the Fc receptors, splenocytes were incubated with anti-CD3e-PE, anti-CD4-APC, anti-CD8a-FITC, or isotype-matched control mAbs. The interaction between cell-surface receptors and monoclonal antibodies was examined using FACS. Grey shading represents the control cells with control antibody; grey lines represent the control cells with functional antibody; black continuous lines represent the monomer phenylpropanoic acids-treated cells with functional antibody; and black dashed lines represent the polymerised polyphenols-treated cells with functional antibody. The data shown are representative of 3 independent experiments.</p

Contribution of T cell population to the polymerised polyphenol-induced cytokine production from murine splenocytes.

<p>Total splenocytes and CD3e⁺ cell-eliminated splenocytes were stimulated with various polymerised polyphenols (100 µg/mL). After 48 h, the supernatants were collected, and the concentrations of (A) IFN-γ and (B) GM-CSF were measured by ELISA. The values represent the mean ± standard deviation, n = 3. Significant difference from untreated splenocytes: *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Involvement of CD4 in cytokine production induced by polymerised polyphenols from splenocytes.

<p>Splenocytes were pre-incubated with 1 µg/mL of (A) anti-CD4 mAb or (B) anti-CD8a mAb, and each control isotype (rat IgG2b and rat IgG2a; respectively) for 1 h, and then exposed to various polymerised polyphenols (0–100 µg/mL). After 48 h of incubation, the supernatant was collected, and the concentrations of IFN-γ and GM-CSF were determined by ELISA. The values shown represent the mean ± standard deviation, n = 3. Significant difference from isotype control: *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Regional genetic differences among Japanese populations and performance of genotype imputation using whole-genome reference panel of the Tohoku Medical Megabank Project

Abstract Background Genotype imputation from single-nucleotide polymorphism (SNP) genotype data using a haplotype reference panel consisting of thousands of unrelated individuals from populations of interest can help to identify strongly associated variants in genome-wide association studies. The Tohoku Medical Megabank (TMM) project was established to support the development of precision medicine, together with the whole-genome sequencing of 1070 human genomes from individuals in the Miyagi region (Northeast Japan) and the construction of the 1070 Japanese genome reference panel (1KJPN). Here, we investigated the performance of 1KJPN for genotype imputation of Japanese samples not included in the TMM project and compared it with other population reference panels. Results We found that the 1KJPN population was more similar to other Japanese populations, Nagahama (south-central Japan) and Aki (Shikoku Island), than to East Asian populations in the 1000 Genomes Project other than JPT, suggesting that the large-scale collection (more than 1000) of Japanese genomes from the Miyagi region covered many of the genetic variations of Japanese in mainland Japan. Moreover, 1KJPN outperformed the phase 3 reference panel of the 1000 Genomes Project (1KGPp3) for Japanese samples, and IKJPN showed similar imputation rates for the TMM and other Japanese samples for SNPs with minor allele frequencies (MAFs) higher than 1%. Conclusions 1KJPN covered most of the variants found in the samples from areas of the Japanese mainland outside the Miyagi region, implying 1KJPN is representative of the Japanese population’s genomes. 1KJPN and successive reference panels are useful genome reference panels for the mainland Japanese population. Importantly, the addition of whole genome sequences not included in the 1KJPN panel improved imputation efficiencies for SNPs with MAFs under 1% for samples from most regions of the Japanese archipelago