Immune Boosting Effect of Limosilactobacillus Reuteri in Immunocompetent C57BL/6J Mice.

Probiotics are well-known to be directly or indirectly involved in the host immune system. In this study, we analyzed the immune-boosting effects of lactic acid bacteria, including Limosilactobacillus and Lactococcus, in immunocompetent C57BL/6J mice. Three different lactic acid bacteria strains were orally administered to C57BL/6J mice for 8 weeks. Then, liver, spleen, and whole blood were harvested after sacrificing the animals. There were no significant changes in whole-body weight, weight of organs, or complete blood cell count by oral administration of lactic acid bacteria. The frequencies of CD3+, CD4+, and CD8+ T cells were significantly increased in the Limosilactobacillus reuteri MG5462 group compared to control. The frequency of NK1.1+ cells was significantly increased in the Lactococcus lactis MG5474 group compared to control. On the other hand, splenocyte proliferations and natural killer cytotoxicity did not differ between groups. In addition, the MG5462 group had a significant increase in the production of TNF-α compared to the control, which is consistent with the upregulation of T cells in the MG5462 group. Therefore, Limosilactobacillus reuteri could be a functional food additive to boost immunity by positively affecting T cell populations.

Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets.

MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an alternative animal infection model for SARS-CoV and SARS-CoV-2, aiding the development of new antiviral drugs. In this study, the MHV-A59 model was employed to investigate the potential of SARS-CoV-2 UTRs as new targets for antiviral drugs. Optimal targets within the MHV-A59 UTRs were identified using a shRNA and siRNA design tool, focusing on RNA secondary stem-loop (SL) structures in the UTRs. We then examined whether the designed RNAi constructs could inhibit MHV-A59 replication. In the 5'UTR, the stem-loop 1 (SL1) was identified as the most effective target, while in the 3'UTR, the minimal element for the initiation of negative-strand RNA synthesis (MIN) proved to be the most effective. Importantly, siRNAs targeting SL1 and MIN structures significantly reduced total RNA synthesis, negative-strand genomic RNA synthesis, subgenomic (sg) RNA synthesis, viral titer, and the plaque size of MHV-A59 compared to the control. Although not statistically significant, the combination of siSL1 and siMIN had a stronger effect on inhibiting MHV-A59 replication than either siRNA monotherapy. Interestingly, while the SL1 structure is present in both MHV and SARS-CoV-2, the MIN structure is unique to MHV. Thus, the SL1 of SARS-CoV-2 may represent a novel and promising target for RNAi-based antiviral drugs.

Clinical implications and chemo-sensitivity of adjuvant chemotherapy in patients with poorly cohesive cells-gastric cancer.

PURPOSE: Poorly cohesive cells-gastric cancer (PCC-GC) represents distinct features within the GC spectrum. The present study investigated the clinicopathologic characteristics and chemo-sensitivity for a relatively large cohort of PCC-GC patients. MATERIALS AND METHODS: A total of 268 patients diagnosed with stage II or III PCC-GC were included. GC cell lines were also analyzed for drug sensitivity to 5-fluorouracil (5-FU) and oxaliplatin in vitro. RESULTS: One hundred fifteen (42.9%) patients were stage II and 153 (57.1%) were stage III. Two hundred twenty-three (83.2%) patients received adjuvant therapy. Among these patients, 139 (62.3%) received CAPOX and 84 (37.7%) received S-1. With a median follow-up of 38.9 (1.6-137.8) months, the estimated 5-year disease-free survival (DFS) and overall survival (OS) rates were 52.3% and 61.0%, respectively. In the univariate analysis, survival was significantly better in the adjuvant chemotherapy group than in the surgery only group. In the subgroup analysis, there was no significant difference in DFS or OS between the types of adjuvant chemotherapy for either disease stage. In vitro cell line analysis, different responses to 5-FU and oxaliplatin were observed in SRC and non-SRC, where the treatment in KATOIII cell lines with oxaliplatin had less effect at a higher concentration compared to non-SRC cell lines. CONCLUSION: The current study found that adjuvant chemotherapy was not significantly associated with survival benefit for patients with resected stage II and III PCC-GC. Plus, S-1 showed numerically longer DFS and OS compared to CAPOX in PCC-GC patients, although no significant in the multivariate analysis.

Immune-Enhancing Effects of Limosilactobacillus fermentum in BALB/c Mice Immunosuppressed by Cyclophosphamide.

This study evaluates the immune-enhancing effects of Limosilactobacillus fermentum on cyclophosphamide (CP)-induced immunosuppression in BALB/c mice. In vitro, the expressions of pro-inflammatory cytokines and MAPK signaling molecules in Raw264.7 cells were analyzed by ELISA and Western blot analysis. Moreover, cell proliferation, surface receptor expression, and cytotoxicity of NK-92 cells were examined by Cell Counting Kit-8, CytoTox96 assay, and flow cytometry, respectively. To investigate the immune-enhancing effects of selected L. fermentum strains in vivo, these strains were orally administered to BALB/c mice for 2 weeks, and CP was intraperitoneally injected. Then, liver, spleen, and whole blood were isolated from each animal. Administration of single L. fermentum strains or their mixture sustained the spleen weight, the counts of white blood cells compared to non-fed group. Splenocyte proliferation and NK cytotoxicity were significantly increased in all L. fermentum-fed groups. The frequency of B220+ cells was also significantly enhanced in splenocytes isolated from L. fermentum groups. In addition, the production of cytokines (TNF-α, IFN-γ) and antibodies was recovered in splenocyte supernatants isolated from L. fermentum groups. In conclusion, L. fermentum could be a suitable functional food additive for immune-enhancing effect.

Characterization of a new CCCTC-binding factor binding site as a dual regulator of Epstein-Barr virus latent infection.

Distinct viral gene expression characterizes Epstein-Barr virus (EBV) infection in EBV-producing marmoset B-cell (B95-8) and EBV-associated gastric carcinoma (SNU719) cell lines. CCCTC-binding factor (CTCF) is a structural chromatin factor that coordinates chromatin interactions in the EBV genome. Chromatin immunoprecipitation followed by sequencing against CTCF revealed 16 CTCF binding sites in the B95-8 and SNU719 EBV genomes. The biological function of one CTCF binding site (S13 locus) located on the BamHI A right transcript (BART) miRNA promoter was elucidated experimentally. Microscale thermophoresis assay showed that CTCF binds more readily to the stable form than the mutant form of the S13 locus. EBV BART miRNA clusters encode 22 miRNAs, whose roles are implicated in EBV-related cancer pathogenesis. The B95-8 EBV genome lacks a 11.8-kb EcoRI C fragment, whereas the SNU719 EBV genome is full-length. ChIP-PCR assay revealed that CTCF, RNA polymerase II, H3K4me3 histone, and H3K9me3 histone were more enriched at S13 and S16 (167-kb) loci in B95-8 than in the SNU719 EBV genome. 4C-Seq and 3C-PCR assays using B95-8 and SNU719 cells showed that the S13 locus was associated with overall EBV genomic loci including 3-kb and 167-kb region in both EBV genomes. We generated mutations in the S13 locus in bacmids with or without the 11.8-kb BART transcript unit (BART(+/-)). The S13 mutation upregulated BART miRNA expression, weakened EBV latency, and reduced EBV infectivity in the presence of EcoRI C fragment. Another 3C-PCR assay using four types of BART(+/-)·S13(wild-type(Wt)/mutant(Mt)) HEK293-EBV cells revealed that the S13 mutation decreased DNA associations between the 167-kb region and 3-kb in the EBV genome. Based on these results, CTCF bound to the S13 locus along with the 11.8-kb EcoRI C fragment is suggested to form an EBV 3-dimensional DNA loop for coordinated EBV BART miRNA expression and infectivity.

NLRP3 Deficiency in Hepatocellular Carcinoma Enhances Surveillance of NK-92 through a Modulation of MICA/B.

Human hepatocellular carcinoma (HCC) is the most common and even worse at prognosis. The patients with HCC which accompanied by other diseases, such as cirrhosis, can be limited in various treatments, such as chemotherapy, not HCC patients without other diseases. NLRP3 inflammasome plays an important role in the innate immune response, but emerging evidence has indicated that the NLRP3 inflammasome is implicated in all stages of cancer development. Various cells express NLRP3 protein through the autocrine or paracrine signaling in their environment, but NK cells do not. The expanding evidence shows that patients who suffer from liver cancers have a low frequency of natural killer (NK) cells, and the function of these cells is also impaired. Thus, we examined how the expression of NLRP3 in HCC cells affects cancer surveillance by NK cells in a state of a co-culture of both cells. When the expression of NLRP3 in HCC cells was ablated, MICA/B on the surface of HCC cells was upregulated through the lowered expression of matrix metalloproteinase. The expression of MICA on the surface of HCC cells interacted with the NKG2D receptor on NK-92 cells, which led to NK cytotoxicity. Furthermore, in a xenograft mice model, NLRP3 KO HCC cells delayed tumor development and metastasis as well as increased the sensitivity to NK cell cytotoxicity. Taken together, NLRP3 KO in HCC could enhance NK immunosurveillance through an interaction of NKG2D-MICA.

Cisplatin Resistance in Epstein-Barr-Virus-Associated Gastric Carcinoma Acquired through ATM Methylation.

Epstein-Barr-virus-associated gastric carcinoma (EBVaGC), first reported in 1992, currently accounts for 10% of all gastric carcinoma worldwide. EBVaGC has unique DNA hypermethylation phenotypes that allow for higher proportions of DNA methylation than any other gastric cancer. CpG islands in the gene promoter region are one of the major regions in which DNA methylation controls gene transcription. Despite cisplatin-based chemotherapy being one of the standard treatment regimens for advanced gastric cancer, including EBVaGC, cisplatin alone or in combination with 5-fluorouracil has been limited by its less potent anticancer activity and the occurrence of cisplatin resistance. Accordingly, the current study evaluated the anticancer activities of a combination of cisplatin and 5-Azacytidine (5-AZA) against EBVaGC. Our findings showed that cisplatin upregulated the DNMT3A gene, whereas shRNA-targeted removal of DNMT3A mRNA contributed to cisplatin-mediated EBV lytic reactivation. Moreover, the removal of DNMT3A mRNA upregulated the ATM gene through DNA demethylation on the ATM promoter. Furthermore, CRISPR/Cas9-targeted removal of the ATM gene resulted in significantly reduced cell susceptibility and EBV lytic reactivation by a combination of cisplatin and DNMT3A inhibitor 5-AZA. Finally, 5-AZA exhibited a synergistic effect with cisplatin in anti-EBV and anti-EBVaGC activities by increasing drug susceptibility and EBV lytic reactivation. The aforementioned results suggest that cisplatin combined with DNA methylation inhibitors could be a novel therapeutic approach for EBVaGC.

Quercetin Synergistically Inhibit EBV-Associated Gastric Carcinoma with Ganoderma lucidum Extracts.

Mycotherapy has been shown to improve the overall response rate during cancer treatment and reduce some chemotherapy-related adverse events. Ganoderma lucidum is a traditional mushroom used for pharmaceutical purposes. G. lucidum extracts (GLE) showed potential antitumor activities against several cancers. These tumor inhibitory effects of GLE were attributed to the suppression of the proliferation and metastasis of cancer cells. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is defined as the monoclonal proliferation of carcinoma cells with latent EBV infection. The inhibitory effects of GLE against EBVaGC are questionable. The aim of this study was to investigate GLE as potential antitumor agents and a counterpart of quercetin (QCT) for the cotreatment in suppressing EBVaGC development. Therefore, this study conducted antitumor assays using a EBVaGC xenograft mice model and found that GLE could suppress tumor development. These inhibitory effects were significantly augmented by the low concentration of the quercetin (QCT) cotreatment in the xenograft mice. The addition of GLE in low concentrations synergistically reinforced QCT-induced apoptosis and EBV lytic reactivation. GLE contains various polysaccharides and triterpenes, such as ganoderic acid. Interestingly, the addition of ganoderic acid A (GAA) could produce similar bioactive effects like GLE in QCT-mediated antitumor activity. The GAA addition in low concentrations synergistically reinforced QCT-induced apoptosis and EBV lytic reactivation. GAA was sufficiently effective as much as GLE. Therefore, our results suggested that QCT-supplemented GLE could be a potential food adjunct for the prevention of EBVaGC development.

Novel Therapeutic Approaches for Epstein-Barr Virus Associated Gastric Cancer.

Epstein-Barr virus (EBV)-associated gastric cancer (GC) (EBVaGC) is classified as one of four GC subtypes by comprehensive molecular characterization. Though the mechanism of tumorigenesis by EBV infection has not yet been fully clarified, EBV infection might contribute to the malignant transformation of GC cells by involving various cellular processes and signaling pathways. EBVaGC has shown the following distinct characteristics in contrast to other subtypes: extreme DNA hypermethylation, recurrent phosphatidylinositol 4,5-biphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) mutations, overexpression of programmed cell death ligand 1/2 (PD-L1/2), and occasional immune cell signaling activation. Therefore, using these molecular features as guides, targeted agents need to be evaluated in clinical trials for EBVaGC. Accordingly, this review uses the best available evidence to focus on novel therapeutic approaches using the distinct pathologic characteristics of EBVaGC patients.

Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression.

Interleukin (IL)-6 plays a crucial role in the progression, invasion, and metastasis of breast cancer. Triple-negative breast cancer (TNBC) cell line MDA-MB-231 is known for its aggressive metastasis. Epithelial to mesenchymal transition (EMT) is a critical process in cancer metastasis. The positive correlation between IL-6 and EMT in tumor microenvironment is reported. We found significantly upregulated IL-6 expression in MDA-MB-231 cells. A blockade of IL-6 expression decreased levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), and cell cycle-related molecules, including cyclin-dependent kinases (CDKs) and cyclins in MDA-MB-231 cells. A short-hairpin RNA (shRNA)-mediated blockade of IL-6 expression inhibited migration and N-cadherin expression and induced E-cadherin expression in MDA-MB-231 cells. Growth rate was slower for the tumors derived from IL-6 shRNA-treated MDA-MB-231 cells than for those derived from control shRNA-treated MDA-MB-231 cells. The expression of pSTAT3, phosphorylated extracellular signal-regulated kinase (pERK), PI3K, pAkt, snail, vimentin, and N-cadherin was significantly lower in tumors from IL-6 shRNA-treated MDA-MB cells. In addition, apigenin treatment significantly inhibited the growth of MDA-MB-231-derived xenograft tumors along with the protein expressions of pSTAT3, pERK, IL-6, PI3K, pAkt, and N-cadherin. Our results demonstrate that the anti-invasive effect of apigenin in MDA-MB-231-derived xenograft tumors is mediated by the inhibition of IL-6-linked downstream signaling pathway.

Exon 9 Mutation of PIK3CA Associated With Poor Survival in Patients With Epstein-Barr Virus-associated Gastric Cancer.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer (GC) is known to harbor a significant enrichment of of phosphatidylinositol 4, 5-biphosphate 3- kinase catalytic subunit alpha isoform (PIK3CA). Therefore, this study investigated the clinical relevance and prognostic role of PIK3CA mutations in patients with EBV-GC. MATERIALS AND METHODS: After reviewing 1,318 consecutive cases of surgically resected GC, 120 patients were identified as EBV-positive using EBV-encoded RNA in situ hybridization. PIK3CA mutations were identified in formalin-fixed and paraffin-embedded surgical specimens from 112 patients with EBV-GC with available tumor tissue samples. Real-time polymerase chain reaction was used to evaluate hot-spot mutations of exons 1, 4, 7, 9, and 20 of PIK3CA. RESULTS: Among the 112 patients, the frequency of PIK3CA mutations was 25.0% (n=28), and among the 28 patients harboring a PIK3CA mutation, most mutations were identified in exon 9 (n=21, 18.8%). The presence of PIK3CA mutation was also correlated with a higher T category (p<0.001) and N category (p<0.001), as well as the presence of perinueral invasion (p<0.001) and venous invasion (p<0.001). In a univariate analysis, PIK3CA mutation showed no association with overall survival (OS) (p=0.184) or disease-free survival (DFS) (p=0.150). Patients harboring exon 9 PIK3CA mutations exhibited a significantly shorter OS (p=0.023) and DFS (p=0.013) than the patients lacking an exon 9 PIK3CA mutation, yet without statistical significance in the multivariate analysis. Notably, exon 9 E542K mutation of PIK3CA was associated with the worst DFS (p=0.011). CONCLUSION: The current data show that PIK3CA mutations appear to play an important role in carcinogenesis and tumor aggressiveness in EBV-GC, and also support the concept that exon 9 mutation of PIK3CA is a prognostic indicator for predicting patient outcomes and a rationale for therapeutic targeting in EBV-GC.

Adenosine Induces EBV Lytic Reactivation through ADORA1 in EBV-Associated Gastric Carcinoma.

Cordyceps species are known to contain numerous bioactive compounds, including cordycepin. Extracts of Cordyceps militaris (CME) are used in diverse medicinal purposes because of their bioactive components. Cordycepin, one of the active components of CME, exhibits anti-proliferative, pro-apoptotic, and anti-inflammatory effects. Cordycepin structurally differs from adenosine in that its ribose lacks an oxygen atom at the 3' position. We previously reported that cordycepin suppresses Epstein⁻Barr virus (EBV) gene expression and lytic replication in EBV-associated gastric carcinoma (EBVaGC). However, other studies reported that cordycepin induces EBV gene expression and lytic reactivation. Thus, it was reasonable to clarify the bioactive effects of CME bioactive compounds on the EBV life cycle. We first confirmed that CME preferentially induces EBV gene expression and lytic reactivation; second, we determined that adenosine in CME induces EBV gene expression and lytic reactivation; third, we discovered that the adenosine A1 receptor (ADORA1) is required for adenosine to initiate signaling for upregulating BZLF1, which encodes for a key EBV regulator (Zta) of the EBV lytic cycle; finally, we showed that BZLF1 upregulation by adenosine leads to delayed tumor development in the EBVaGC xenograft mouse model. Taken together, these results suggest that adenosine is an EBV lytic cycle inducer that inhibits EBVaGC development.

Synthesis and Evaluation of Manganese(II)-Based Ethylenediaminetetraacetic Acid-Ethoxybenzyl Conjugate as a Highly Stable Hepatobiliary Magnetic Resonance Imaging Contrast Agent.

In this study, we designed and synthesized a highly stable manganese (Mn2+)-based hepatobiliary complex by tethering an ethoxybenzyl (EOB) moiety with an ethylenediaminetetraacetic acid (EDTA) coordination cage as an alternative to the well-established hepatobiliary gadolinium (Gd3+) chelates and evaluated its usage as a T1 hepatobiliary magnetic resonance imaging (MRI) contrast agent (CA). This new complex exhibits higher r1 relaxivity (2.3 mM-1 s-1) than clinically approved Mn2+-based hepatobiliary complex Mn-DPDP (1.6 mM-1 s-1) at 1.5 T. Mn-EDTA-EOB shows much higher kinetic inertness than that of clinically approved Gd3+-based hepatobiliary MRI CAs, such as Gd-DTPA-EOB and Gd-BOPTA. In addition, in vivo biodistribution and MRI enhancement patterns of this new Mn2+ chelate are comparable to those of Gd3+-based hepatobiliary MRI CAs. The diagnostic efficacy of the new complex was demonstrated by its enhanced tumor detection sensitivity in a liver cancer model using in vivo MRI.

Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus.

HCV genotype 2a strain JFH-1 replicates and produces viral particles efficiently in human hepatocellular carcinoma (huh) 7.5 cells, which provide a stable in vitro cell infection system for the hepatitis C virus (HCVcc system). Natural killer (NK) cells are large lymphoid cells that recognize and kill virus-infected cells. In this study, we investigated the interaction between NK cells and the HCVcc system. IL-10 is a typical immune regulatory cytokine that is produced mostly by NK cells and macrophages. IL-21 is one of the main cytokines that stimulate the activation of NK cells. First, we used anti-IL-10 to neutralize IL-10 in a coculture of NK cells and HCVcc. Anti-IL-10 treatment increased the maturation of NK cells by enhancing the frequency of the CD56+dim population in NK-92 cells. However, with anti-IL-10 treatment of NK cells in coculture with J6/JFH-1-huh 7.5 cells, there was a significant decrease in the expression of STAT1 and STAT5 proteins in NK-92 cells and an increase in the HCV Core and NS3 proteins. In addition, rIL-21 treatment increased the frequency of the CD56+dim population in NK-92 cells, Also, there was a dramatic increase in the expression of STAT1 and STAT5 proteins in rIL-21 pre-stimulated NK cells and a decrease in the expression of HCV Core protein in coculture with J6/JFH-1-huh 7.5 cells. In summary, we found that the functional activation of NK cells can be modulated by anti-IL-10 or rIL-21, which controls the expression of HCV proteins as well as HCV RNA replication.

Phylogenetic comparison of Epstein-Barr virus genomes.

Technologies used for genome analysis and whole genome sequencing are useful for us to understand genomic characterization and divergence. The Epstein-Barr virus (EBV) is an oncogenic virus that causes diverse diseases such as Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma (HL), and gastric carcinoma (GC). EBV genomes found in these diseases can be classified either by phases of EBV latency (type-I, -II, and -III latency) or types of EBNA2 sequence difference (type-I EBV, type-II EBV or EBV-1, EBV-2). EBV from EBV-transformed lymphoblastoid cell line (LCL) establishes type-III latency, EBV from NPC establishes type-II latency, and EBV from GC establishes type-I latency. However, other important factors play key roles in classifying numerous EBV strains because EBV genomes are highly diverse and not phylogenetically related to types of EBV-associated diseases. Herein, we first reviewed previous studies to describe molecular characteristics of EBV genomes. Then, using comparative and phylogenetic analyses, we phylogenetically analyzed molecular variations of EBV genomes and proteins. The review of previous studies and our phylogenetic analysis showed that EBV genomes and proteins were highly diverse regardless of types of EBV-associated diseases. Other factors should be considered in determining EBV taxonomy. This review will be helpful to understand complicated phylogenetic relationships of EBV genomes.

Intratumoural PD-L1 expression is associated with worse survival of patients with Epstein-Barr virus-associated gastric cancer.

BACKGROUND: This study investigated the clinical relevance and prognostic impact of the overall expression of programmed cell death protein ligand-1 (PD-L1) and programmed cell death protein ligand-2 (PD-L2), in patients with Epstein-Barr virus-associated gastric cancer (EBVaGC). METHODS: After reviewing 1318 consecutive cases of surgically resected or endoscopic submucosal dissected gastric cancers, the expression status of PD-L1 and PD-L2 in 120 patients with EBVaGC identified by EBV-encoded RNA in situ hybridisation was retrospectively analysed using immunohistochemistry (IHC). For each IHC marker, positivity was separately in intraepithelial tumour cells (iTu-) and immune cells in the tumour stroma area (str-). RESULTS: Among 116 eligible patients, 57 (49.1%) and 66 patients (56.9%) were determined as iTu-PD-L1-positive and str-PD-L1-positive, respectively, whereas 23 (21.6%) and 45 patients (38.8%) were determined as iTu-PD-L2 positive and str-PD-L2 positive, respectively. Intraepithelial tumour cell PD-L1 positivity was found to be significantly associated with lymph node (LN) metastasis (P=0.012) and a poor disease-free survival (DFS) (P=0.032), yet not overall survival (P=0.482). In a multivariate analysis, iTu-PD-L1 positivity was independently associated with a poor DFS (P=0.006, hazard ratio=12.085). In contrast, str-PD-L2-positivity was related to a lower T category (P=0.003), absence of LN metastasis (P=0.032) and perineural invasion (P=0.028). Intraepithelial tumour cell and str-PD-L2 positivity showed a trend towards an improved DFS, although not significant (P=0.060 and P=0.073, respectively). CONCLUSIONS: Intraepithelial tumour cells PD-L1 expression can be used to predict a poor outcome in patients with EBVaGC and can represent a rational approach for PD-1/PD-L pathway-targeted immunotherapy.

Natural killer cells and tumor metastasis.

Natural killer (NK) cells are cytotoxic lymphocytes that recognize tumor cells or stressed cells through 'missing-self' signals, such as altered or absent expression of MHC class I molecules. The function of NK cells is regulated by the activation or inhibition of receptors present on their surface. The activation of NK cells results in cytotoxic activity on target cells through release of toxic granules and inflammatory cytokines. However, NK cells infiltrating tumors have been frequently shown to exhibit a skewed phenotype that includes decreased antitumor activity and enhanced protumor activities, such as angiogenesis and metastasis. In fact, many studies have reported that tumor microenvironments induce a protumor phenotype in NK cells. Here, we review the biological properties of NK cells in the context of tumorigenesis and tumor progression, with a specific focus on the interactions between NK cells and critical tumor microenvironments, such as epithelial-to-mesenchymal transition, matrix metalloproteinases, and tumor-associated chronic inflammation in tumor metastasis.

DNA hypermethylation induced by Epstein-Barr virus in the development of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a recently recognized disease entity defined by the presence of EBV in gastric carcinoma cells. EBV infection causes major epigenetic alterations in the EBV genome and its cellular host genome, suggesting that EBV acts as a direct epigenetic driver for EBVaGC. One of the major epigenetic events in the viral and cellular genomes to control transcription is DNA hypo- or hyper-methylation. Particularly, local and global hypermethylation have been reported in EBVaGC. It is therefore important to understand the molecular mechanisms of DNA hypermethylation during EBVaGC carcinogenesis. To understand the functional roles of DNA methylation and suggest therapeutic target candidates for EBVaGC, we reviewed recent literature reporting DNA hypermethylation in EBVaGC. We summarized the identified candidate genes that are markedly hypermethylated in EBVaGC, which can potentially be targets for chemotherapies with demethylating agents.

Programmed Cell Death 1 (PD-1) and Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) in Viral Hepatitis.

Virus-specific cluster of differentiation 8 (CD8+) cytotoxic T cells (CTL) recognize viral antigens presented on major histocompatibility complex (MHC) class I chains on infected hepatocytes, with help from CD4+ T cells. However, this CTL response is frequently weak or undetectable in patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are receptors in the CD28 family of costimulatory molecules, providing inhibitory signals to T cells. The overexpressions of PD-1 and CTLA-4 in patients with viral infection have been shown to associate with functional impairment of virus-specific T cells. In acute viral hepatitis, PD-1 and CTLA-4 are up-regulated during the symptomatic phase, and then down-regulated after recovery. These findings suggest that PD-1 and CTLA-4 have protective effects as inhibitory molecules to suppress cytotoxic T cells which induce harmful destruction of viral infected hepatocytes in self-limited viral hepatitis. In chronic viral hepatitis, the extended upregulations of PD-1 and CTLA-4 are associated with T cell exhaustion and persistent viral infection, suggesting positive correlations between expression of immune inhibitory factors and the chronicity of viral disease. In this review, we summarize recent literature relating to PD-1, CTLA-4, and other inhibitory receptors in antigen-specific T cell exhaustion in viral hepatitis, including hepatitis A, B, C, and others.

Alteration of DNA Methylation in Gastric Cancer with Chemotherapy.

Epigenetic alterations such as DNA methylation, histone acetylation, and chromatin remodeling can control gene expression by regulating gene transcription. DNA methylation is one of the frequent epigenetic events that play important roles in cancer development. Cancer cells can gain significant resistance to anticancer drugs and escape programmed cell death through major epigenetic changes, including DNA methylation. To date, several research groups have identified instances of both (i) hypermethylation of tumor suppressor genes, and (ii) global hypomethylation of oncogenes. These changes in DNA methylation status could be used as biomarkers for the diagnosis and prognosis of cancer patients undergoing chemotherapies or other clinical therapies. Herein, we describe genes for which methylation is dependent upon anticancer drug resistance in patients with gastric cancer; we then suggest a significant epigenetic target to focus on for overcoming anticancer drug resistance.