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1.  Genetic Polymorphisms in SLC23A2 as Predictive Biomarkers of Severe Acute Toxicities after Treatment with a Definitive 5-Fluorouracil/Cisplatin-Based Chemoradiotherapy in Japanese Patients with Esophageal Squamous Cell Carcinoma 
Objective: Definitive chemoradiotherapy (CRT) with 5-fluorouracil (5-FU) and cisplatin (CDDP) is one of the standard therapies for esophageal squamous cell carcinoma (ESCC); however, inter-individual variations in clinical outcomes have yet to be investigated. In the present study, single nucleotide polymorphisms (SNPs) in SLC23A2 gene were retrospectively evaluated in 49 Japanese patients with ESCC who were treated with a definitive 5-FU/CDDP-based CRT, and the predictive values for the clinical response, severe acute toxicities, and long-term survival were assessed.
Methods: A course consisted of the continuous infusion of 5-FU at 400 mg/m2/day for days 1-5 and 8-12, the infusion of CDDP at 40 mg/m2/day on days 1 and 8, and radiation at 2 Gy/day on days 1 to 5, 8 to 12, and 15 to 19, with a second course being repeated after a 2-week interval. The SLC23A2 SNPs rs2681116, rs13037458, rs1715364, rs4987219, and rs1110277 were evaluated.
Results: The rs2681116 and rs13037458 had a tendency to predict the clinical response (p=0.144 and 0.085, respectively) and long-term survival (p=0.142 and 0.056, respectively). The rs4987219 and rs1110277 correlated with severe acute leukopenia (p=0.025) and stomatitis (p=0.019), respectively.
Conclusions: Further investigations with a larger number of patients or an in vitro study are needed to confirm the predictive values of genetic polymorphisms in SLC23A2.
PMCID: PMC3936025  PMID: 24578608
esophageal squamous cell carcinoma; chemoradiotherapy; biomarker; SLC23A2; polymorphism.
2.  Quantification of Pregenomic RNA and Covalently Closed Circular DNA in Hepatitis B Virus-Related Hepatocellular Carcinoma 
Pregenomic RNA (pgRNA) is generated from covalently closed circular DNA (cccDNA) and plays important roles in viral genome amplification and replication. Hepatic pgRNA and cccDNA expression levels indicate viral persistence and replication activity. This study was aimed to measure hepatic pgRNA and cccDNA expression levels in various states of hepatitis B virus (HBV) infection. Thirty-eight hepatocellular carcinoma (HCC) patients, including 14 positive for hepatitis B surface antigen (HBsAg) and 24 negative for HBsAg but positive for anti-hepatitis B core (anti-HBc) antibody, were enrolled in this study. In HBsAg-negative but anti-HBc-positive group, HBV-DNA was detected in 20 of 24 (83%) noncancerous liver tissues for at least two genomic regions based on polymerase chain reaction (PCR) analysis. pgRNA and cccDNA expression levels in occult HBV-infected patients were significantly lower than those in HBsAg-positive patients (P < 0.001). pgRNA and cccDNA in cancerous tissues were also detected without significant difference from those in noncancerous tissues. In conclusion, cccDNA and pgRNA are detected and represented HBV replication not only in noncancerous but also in cancerous liver tissues. In addition, the replication is shown in not only patients with HBsAg-positive but also occult HBV-infected patients, suggesting the contribution to HCC development.
PMCID: PMC3880697  PMID: 24455286
3.  Predominant mucosal IL-8 mRNA expression in non-cagA Thais is risk for gastric cancer 
AIM: To study gastric mucosal interleukine-8 (IL-8) mRNA expression, the cytotoxin-associated gene A (cagA) mutation, and serum pepsinogen (PG) I/II ratio related risk in Thai gastric cancer.
METHODS: There were consent 134 Thai non-cancer volunteers who underwent endoscopic narrow band imaging examination, and 86 Thais advance gastric cancer patients who underwent endoscopic mucosal biopsies and gastric surgery. Tissue samples were taken by endoscopy with 3 points biopsies. The serum PG I, II, and Helicobacter pylori (H. pylori) immunoglobulin G (IgG) antibody for H. pylori were tested by enzyme-linked immunosorbent assay technique. The histopathology description of gastric cancer and non-cancer with H. pylori detection was defined with modified Sydney Score System. Gastric mucosal tissue H. pylori DNA was extracted and genotyped for cagA mutation. Tissue IL-8 and cyclooxygenase-2 (COX-2) mRNA expression were conducted by real time relative quantitation polymerase chain reaction. From 17 Japanese advance gastric cancer and 12 benign gastric tissue samples, all were tested for genetic expression with same methods as well as Thai gastric mucosal tissue samples. The multivariate analysis was used for the risk study. Correlation and standardized t-test were done for quantitative data, P value < 0.05 was considered as a statistically significant.
RESULTS: There is a high non cagA gene of 86.8 per cent in Thai gastric cancer although there are high yields of the East Asian type in the positive cagA. The H. pylori infection prevalence in this study is reported by combined histopathology and H. pylori IgG antibody test with 77.1% and 97.4% of sensitivity and specificity, respectively. The serum PG I/II ratio in gastric cancer is significantly lower than in the non-cancer group, P = 0.045. The serum PG I/II ratio of less than 3.0 and IL-8 mRNA expression ≥ 100 or log10 ≥ 2 are significant cut off risk differences between Thai cancer and non-cancer, P = 0.03 and P < 0.001, respectively. There is a significantly lower PGI/II ratio in Japanese than that in Thai gastric cancer, P = 0.026. Serum PG I/II ratio at cut off less than 3.0 and IL-8 mRNA expression Raw RQ > 100 or log10 > 2 are significantly difference between Thai cancer group when compared to non-cancer group, P = 0.013 and P < 0.001, respectively. In the correlation study, low PG I/II ratio does not associate with chronic atrophic gastritis severity score in Thais non-cancer cases. However, there is a trend, but not significant convert correlation between IL-8 mRNA expression level and low PG I/II ratio in Thai positive H. pylori infection. The high expression of IL-8 gene demonstrates a poorer prognosis by stage and histology.
CONCLUSION:Predominant gastric mucosal IL-8 mRNA expression level, H. pylori infection, and low PG I/II ratio are relative risks for Thai gastric cancer without correlation with cagA mutation.
PMCID: PMC3660819  PMID: 23704827
Gastric cancer; CagA mutation; Interleukine-8 mRNA expression; Helicobacter pylori; Pepsinogen I/II ratio
4.  A lipid peroxidation product 9-oxononanoic acid induces phospholipase A2 activity and thromboxane A2 production in human blood 
Lipid peroxidation products are known to cause toxicity by reacting with biologically significant proteins, but the inducing role of peroxidation products has been not noted to produce degenerative disease-related eicosanoids. Here, 9-oxononanoic acid (9-ONA), one of the major products of peroxidized fatty acids, was found to stimulate the activity of phospholipase A2 (PLA2), the key enzyme to initiate arachidonate cascade and eicosanoid production. An exposure of fresh human blood to the atmosphere at 37°C accumulated 9-ONA, increasing peroxide value and thiobarbituric acid reactive substances in the blood. The lipid peroxidation was accompanied by significant increases of PLA2 activity and thromboxane B2 (TxB2) production, which is a stable metabolite of thromboxane A2 (TxA2) and a potent agonist of platelet aggregation. These events were abolished by standing the blood under nitrogen. The addition of organically synthesized 9-ONA resumed the activity of PLA2 and the production of TxB2. Also, 9-ONA induced platelet aggregation dose-dependently. These results indicated that 9-ONA is the primary inducer of PLA2 activity and TxA2 production, and is probably followed by the development of diseases such as thrombus formation. This is the first report to find that a lipid peroxidation product, 9-ONA, stimulates the activity of PLA2.
PMCID: PMC3652295  PMID: 23704812
9-oxononanoic acid; lipid peroxidation; phospholipase A2; thromboxane A2; arachidonate cascade
5.  Intestinal Anti-Inflammatory Activity of Lentinan: Influence on IL-8 and TNFR1 Expression in Intestinal Epithelial Cells 
PLoS ONE  2013;8(4):e62441.
Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. It is unknown whether β-1,3;1,6-glucan can induce immune suppressive effects. Here, we study intestinal anti-inflammatory activity of Lentinula edodes-derived β-1,3;1,6-glucan, which is known as lentinan. Dextran sulfate sodium (DSS)-induced colitis mice were used to elucidate effects of lentinan in vivo. In the cellular level assessment, lentinan was added into a co-culture model consisting of intestinal epithelial Caco-2 cells and LPS-stimulated macrophage RAW264.7 cells. Ligated intestinal loop assay was performed for assessing effects of lentinan on intestinal epithelial cells (IECs) in vivo. Oral administration of lentinan (100 µg/mouse) significantly ameliorated DSS-induced colitis in body weight loss, shortening of colon lengths, histological score, and inflammatory cytokine mRNA expression in inflamed tissues. Lentinan reduced interleukin (IL)-8 mRNA expression and nuclear factor (NF)-κB activation in Caco-2 cells without decreasing of tumor necrosis factor (TNF)-α production from RAW264.7 cells. Flow cytometric analysis revealed that surface levels of TNF receptor (TNFR) 1 were decreased by lentinan treatment. A clathrin-mediated endocytosis inhibitor, monodansylcadaverine, canceled lentinan inhibition of IL-8 mRNA expression. Moreover, lentinan inhibited TNFR1 expression in Caco-2 cells in both protein and mRNA level. Lentinan also inhibited TNFR1 mRNA expression in mouse IECs. These results suggest that lentinan exhibits intestinal anti-inflammatory activity through inhibition of IL-8 mRNA expression associated with the inhibition of NF-κB activation which is triggered by TNFR1 endocytosis and lowering of their expression in IECs. Lentinan may be effective for the treatment of gut inflammation including IBD.
PMCID: PMC3632531  PMID: 23630633
6.  Hepatitis B virus and host factors 
PMCID: PMC3924661  PMID: 24570927
7.  Chromosome Painting In Silico in a Bacterial Species Reveals Fine Population Structure 
Molecular Biology and Evolution  2013;30(6):1454-1464.
Identifying population structure forms an important basis for genetic and evolutionary studies. Most current methods to identify population structure have limitations in analyzing haplotypes and recombination across the genome. Recently, a method of chromosome painting in silico has been developed to overcome these shortcomings and has been applied to multiple human genome sequences. This method detects the genome-wide transfer of DNA sequence chunks through homologous recombination. Here, we apply it to the frequently recombining bacterial species Helicobacter pylori that has infected Homo sapiens since their birth in Africa and shows wide phylogeographic divergence. Multiple complete genome sequences were analyzed including sequences from Okinawa, Japan, that we recently sequenced. The newer method revealed a finer population structure than revealed by a previous method that examines only MLST housekeeping genes or a phylogenetic network analysis of the core genome. Novel subgroups were found in Europe, Amerind, and East Asia groups. Examination of genetic flux showed some singleton strains to be hybrids of subgroups and revealed evident signs of population admixture in Africa, Europe, and parts of Asia. We expect this approach to further our understanding of intraspecific bacterial evolution by revealing population structure at a finer scale.
PMCID: PMC3649679  PMID: 23505045
fineSTRUCTURE; homologous recombination; phylogenetic network; human evolution; Helicobacter pylori
8.  Reduction in Non-Protein Respiratory Quotient Is Related to Overall Survival after Hepatocellular Carcinoma Treatment 
PLoS ONE  2013;8(3):e55441.
Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that can occasionally lead to the shortening of life expectancy. We aimed to make a new and more accurate prognostic model taking into account the course of disease after TACE.
Methodology/Principal Findings
We performed a prospective cohort study involving 100 HCC patients who underwent TACE at Kobe University Hospital. Indirect calorimetry and blood biochemical examinations were performed before and 7 days after TACE. Time-dependent and time-fixed factors associated with 1-year mortality after TACE were assessed by multivariate analyses. A predictive model of 1-year mortality was established by the combination of odds ratios of these factors. Multivariate analyses showed that the ratio of non-protein respiratory quotient (npRQ) (7 days after/before TACE) and Cancer of Liver Italian Program (CLIP) score were independent factors of 1-year mortality after TACE (p = 0.014 and 0.013, respectively). Patient-specific 1-year mortality risk scores can be calculated by summarizing the individual risk scores and looking up the patient-specific risk on the graph.
The short-term reduction of npRQ was a time-dependent prognostic factor associated with overall survival in HCC patients undergoing TACE. CLIP score was a time-fixed prognostic factor associated with overall survival. Using the prediction model, which consists of the combination of time-dependent (npRQ ratio) and time-fixed (CLIP score) prognostic factors, 1-year mortality risk after TACE would be better estimated by taking into account changes during the course of disease.
PMCID: PMC3592854  PMID: 23520445
9.  Adherence to the preventive strategies for nonsteroidal anti-inflammatory drug- or low-dose aspirin-induced gastrointestinal injuries 
Journal of Gastroenterology  2013;48(5):559-573.
As the aging of the population advances, the use of nonsteroidal anti-inflammatory drugs (NSAIDs) and/or low-dose aspirin (LDA) is increasing. Their use is accompanied by a risk of serious complications, such as hemorrhage or perforation of the gastrointestinal tract. Therefore, gastroprotective strategies upon the prescription of NSAIDs/LDA are outlined in several guidelines or recommendations. Because all NSAIDs including cyclooxygenase (COX)-2 inhibitors have cardiovascular (CV) toxicity, recent guidelines are based on not only GI risks but also CV risks of NSAID users. Assessment of the adherence to evidence-based guidelines or recommendations for the safe prescription of NSAIDs/LDA in clinical practice is an important issue. Here, we summarize randomized controlled trials (RCTs) on the preventive effects of antisecretory drugs for NSAID- or LDA-induced peptic ulcers. Then, we describe preventive strategies upon the prescription of NSAIDs/LDA outlined in several guidelines or recommendations, and describe studies on adherence and outcomes of adherence to these preventive strategies. Finally, we discuss strategies to increase the adherence rate, and changing pattern of GI events associated with NSAIDs/LDA. In Japan, the preventive strategies upon the prescription of NSAIDs/LDA are expected to spread rapidly because the use of proton pump inhibitors for the prevention of recurrence of NSAID- or LDA-induced peptic ulcers and the use of COX-2 for the palliation of acute pain were recently approved under the national health insurance system. Further studies on adherence to the preventive strategies and the outcomes of adherence, which include both GI events and CV events, in the Japanese population are required.
PMCID: PMC3654181  PMID: 23460386
Adherence; Preventive strategy; Nonsteroidal anti-inflammatory drug; Low-dose aspirin; Gastrointestinal injury
10.  TNF-α -857C>T Genotype is Predictive of Clinical Response after Treatment with Definitive 5-Fluorouracil/cisplatin-based Chemoradiotherapy in Japanese Patients with Esophageal Squamous Cell Carcinoma 
Background: Genotypes of tumor necrosis factor alpha (TNF-α) and its surface receptors, TNFRSF1A and TNFRSF1B, have been examined in terms of the progression, metastasis, clinical efficacy, and prognosis of various cancers; however, little is known about their effects on clinical outcome in patients with esophageal squamous cell carcinoma (ESCC). In this study, TNF-α and TNFRSF1A genotypes were retrospectively evaluated in terms of predicting clinical response, long-term survival, and severe acute toxicities in 46 male Japanese ESCC patients treated with definitive 5-fluorouracil (5-FU)/cisplatin (CDDP)-based chemoradiotherapy (CRT).
Methods: A course consisted of the continuous infusion of 5-FU at 400 mg/m2/day for days 1-5 and 8-12, the infusion of CDDP at 40 mg/m2/day on days 1 and 8, and radiation at 2 Gy/day on days 1-5, 8-12, and 15-19, with a second course being repeated after a 2-week interval. The TNF-α -1031T>C (rs1799964), -863C>A (rs1800630), -857C>T (rs1799724), -308G>A (rs1800629), -238G>A (rs361525), TNFRSF1A -609G>T (rs4149570), and 36A>G (rs767455) genotypes were evaluated.
Results: The TNF-α -857C>T genotype was found to be predictive of clinical response, i.e., complete response or not (P = 0.010, Fisher's exact test), but had no effect on long-term survival (CC-857 vs. CT-857 + TT-857, P = 0.072, Fisher's exact test, P = 0.070, Log-rank test).
Conclusions: The TNF-α -857C>T genotype was found to be predictive of clinical response and was more likely to predict long-term survival in Japanese ESCC patients receiving definitive 5-FU/CDDP-based CRT. Further clinical investigations with a larger number of patients or experiments in vitro should be performed to assess the predictive value of this genotype following CRT.
PMCID: PMC3804799  PMID: 24151445
esophageal squamous cell carcinoma; chemoradiotherapy; clinical response; prognosis; tumor necrosis factor.
11.  Alteration of non-protein respiratory quotient after hepatocellular carcinoma treatment can be related to des-γ-carboxy prothrombin before treatment 
SpringerPlus  2012;1:55.
Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that would occasionally lead to energy malnutrition through therapeutic hypoxic stress. We aimed to clarify the correlation between the energy malnutrition after TACE and low tolerability for hypoxia of non-tumoral liver before TACE.
We performed a prospective cohort study involving 100 HCC patients who underwent TACE at Kobe University Hospital. Indirect calorimetry was performed before and 7 days after TACE, and non-protein respiratory quotient (npRQ) as an indicator of the energy malnutrition was measured. Blood biochemical examinations were also performed before TACE. As an indicator of hypoxic marker, des-γ-carboxy prothrombin (DCP) was measured before TACE. The correlation between npRQ ratio (7 days after/before TACE) and DCP (before TACE) was statistically examined. Spearman’s correlation coefficient test showed that npRQ ratio (Day 7/Day 0) was significantly related to DCP (Day 0) (p=0.0481, r=-0.2033). On the other hand, npRQ ratio (Day 7/Day 0) was not related to alpha fetoprotein (Day 0) (p=0.6254, r=-0.0494).
The npRQ reduction after TACE was related to a high value of DCP before TACE. The energy malnutrition after TACE would originate from low tolerability for hypoxia of non-tumoral liver. The HCC patients with a high value of DCP before TACE would clinically have a high risk of the energy malnutrition after TACE.
PMCID: PMC3725920  PMID: 23961374
Des-γ-carboxy prothrombin; Energy malnutrition; Hepatocellular carcinoma; Hypoxic stress; Indirect calorimetry; Non-protein respiratory quotient; Transcatheter arterial chemoembolization
12.  Principles of Quality Controlled Endoscopic Submucosal Dissection with Appropriate Dissection Level and High Quality Resected Specimen 
Clinical Endoscopy  2012;45(4):362-374.
Endoscopic submucosal dissection (ESD) has enabled en bloc resection of early stage gastrointestinal tumors with negligible risk of lymph node metastasis, regardless of tumor size, location, and shape. However, ESD is a relatively difficult technique compared with conventional endoscopic mucosal resection, requiring a longer procedure time and potentially causing more complications. For safe and reproducible procedure of ESD, the appropriate dissection of the ramified vascular network in the level of middle submucosal layer is required to reach the avascular stratum just above the muscle layer. The horizontal approach to maintain the appropriate depth for dissection beneath the vascular network enables treatment of difficult cases with large vessels and severe fibrosis. The most important aspect of ESD is the precise evaluation of curability. This approach can also secure the quality of the resected specimen with enough depth of the submucosal layer.
PMCID: PMC3521937  PMID: 23251883
Endoscopic submucosal dissection; Gastro intestinal neoplasms; Quality control; Dissection level; Vessel network
13.  1,635 Endoscopic submucosal dissection cases in the esophagus, stomach, and colorectum: complication rates and long-term outcomes 
Surgical Endoscopy  2012;27(3):1000-1008.
Endoscopic submucosal dissection (ESD) enables en bloc resection of early gastrointestinal neoplasms; however, most ESD articles report small series, with short-term outcomes performed by multiple operators on single organ. We assessed short- and long-term treatment outcomes following ESD for early neoplasms throughout the gastrointestinal tract.
We performed a longitudinal cohort study in single tertiary care referral center. A total of 1,635 early gastrointestinal neoplasms (stomach 1,136; esophagus 138; colorectum 361) were treated by ESD by single operator. Outcomes were complication rates, en bloc R0 resection rates, and long-term overall and disease-specific survival rates at 3 and 5 years for both guideline and expanded criteria for ESD.
En bloc R0 resection rates were: stomach: 97.1 %; esophagus: 95.7 %; colorectum: 98.3 %. Postoperative bleeding and perforation rates respectively were: stomach: 3.6 and 1.8 %; esophagus: 0 and 0 %; colorectum: 1.7 and 1.9 %. Intra criteria resection rates were: stomach: 84.9 %; esophagus: 81.2 %; colorectum: 88.6 %. Three-year survival rates for lesions meeting Japanese ESD guideline/expanded criteria were for all organ-combined: 93.4/92.7 %. Five-year rates were: stomach: 88.1/84.6 %; esophagus: 81.6/57.3 %; colorectum: 94.3/100 %. Median follow-up period was 53.4 (range, 0.07–98.6) months. Follow-up rate was 94 % (1,020/1,085). There was no recurrence or disease-related death.
In this large series by single operator, ESD was associated with high curative resection rates and low complication rates across the gastrointestinal tract. Disease-specific and overall long-term prognosis for patients with lesions within intra criteria after curative resection appeared to be excellent.
PMCID: PMC3572381  PMID: 23052530
Endoscopic submucosal dissection; Complication; Postoperative; Prognosis; Neoplasms
14.  Metastatic Renal Cell Carcinoma Change Vascularity 
Case Reports in Urology  2012;2012:654617.
Several molecular targeted agents have been approved for clinical use for metastatic renal cell carcinoma (mRCC). A case of a 32-year-old woman with mRCC is presented. These tumors could change vascularity by administration of molecular agents. We could select a drug timely based on findings of computed tomography. To our knowledge, this is the first report that tumor's character change induced by molecular targeted agents can be detected and the efficacy of molecular targeted agents can be predicted.
PMCID: PMC3437274  PMID: 22970402
15.  Clinical utility of protein induced by vitamin K absence in patients with chronic hepatitis B virus infection 
Biomedical Reports  2012;1(1):122-128.
Hepatitis B virus (HBV) is a leading cause of hepatocellular carcinoma (HCC). α-fetoprotein (AFP) is a common tumor marker for the diagnosis of HCC, although not for protein induced by the absence of vitamin K or antagonist-II (PIVKA-II). The present study aimed to evaluate the role of PIVKA-II in the diagnosis of HCC in HBV-infected Vietnamese patients. A total of 166 consecutive HBV-infected Vietnamese patients were enrolled, including 41 HCC, 43 liver cirrhosis (LC), 26 chronic hepatitis (CH) and 56 asymptomatic carriers (ASC). AFP was examined using ELISA, while PIVKA-II was analyzed using Eitest PIVKA-II. The cut-off level of AFP and PIVKA-II was 20 ng/ml and 40 mAU/ml, respectively. Although the markers, AFP (344±356 ng/ml) and PIVKA-II (16,200±25,386 mAU/ml), were the highest in the HCC groups, only PIVKA-II in HCC was significantly higher compared to the other groups (P<0.001). The univariate analysis demonstrated that age over 50, male, genotype C, AFP and PIVKA-II were risk factors of LC and HCC. Results of the receiver operating characteristics (ROC) analysis showed that PIVKA-II was more sensitive to HCC compared to AFP. Moreover, PIVKA-II was strongly correlated with the portal venous thrombosis in HCC, as opposed to AFP. Results of the multivariate analysis demonstrated that PIVKA-II was the strongest independent risk factor of LC and HCC. In conclusion, PIVKA-II is likely to be a better marker for the diagnosis of HCC in chronic HBV-infected Vietnamese patients.
PMCID: PMC3956828  PMID: 24648907
hepatitis B virus; hepatocellular carcinoma; protein induced by vitamin K absence or antagonist-II; Vietnam
16.  Molecular Characterization of Helicobacter pylori VacA Induction of IL-8 in U937 Cells Reveals a Prominent Role for p38MAPK in Activating Transcription Factor-2, cAMP Response Element Binding Protein, and NF-κB Activation1 
Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IκBα ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca2+ entry (SKF96365), and intracellular Ca2+ channels (dantrolene) blocked VacA-induced IL-8 production. Furthermore, an intracellular Ca2+ chelator (BAPTA-AM), which inhibited VacA-activated p38 MAPK, caused a dose-dependent reduction in VacA-induced IL-8 secretion by U937 cells, implying a role for intracellular Ca2+ in mediating activation of MAPK and the canonical NF-κB pathway. VacA stimulated translocation of NF-κBp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which is a p38MAPK substrate and binds to the AP-1 site of the IL-8 promoter, inhibited VacA-induced IL-8 production. VacA activated an IL-8 promoter containing an NF-IL-6 site, but not a mutated AP-1 or NF-κB site, suggesting direct involvement of the ATF-2/CREB binding region or NF-κB-binding regions in VacA-induced IL-8 promoter activation. Thus, in U937 cells, VacA directly increases IL-8 production by activation of the p38 MAPK via intracellular Ca2+ release, leading to activation of the transcription factors, ATF-2, CREB, and NF-κB.
PMCID: PMC3404739  PMID: 18354227
17.  Enhanced expression of CCL20 in human Helicobacter pylori-associated gastritis 
Clinical Immunology (Orlando, Fla.)  2008;130(3):290-297.
CC chemokine ligand 20 (CCL20) attracts CC chemokine receptor 6 (CCR6)-expressing cells. Using endoscopic biopsies taken from the gastric antrum of 42 subjects infected with H. pylori and 42 uninfected subjects, mucosal CCL20 mRNA and protein levels were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. CCL19 mRNA and protein levels, as well as CCL21 mRNA levels, were also measured. The CCL20 mRNA and protein levels were significantly elevated in H. pylori-positive patients and substantially decreased after successful eradication. CCL19 and CCL21 expression levels were comparable in the H. pylori-infected and the uninfected groups. The CCL20 concentrations correlated with the degree of chronic gastritis. Immunohistochemistry and the in vitro infection assay showed that CCL20 was principally produced by the gastric epithelium. CCR6-expressing cells, including CD45RO+ memory T lymphocytes and fascin+-CD1a+ immature dendritic cells, infiltrated close to the CCL20-expressing epithelial cells. The CCL20/CCR6 interaction may be involved in the development of H. pylori-associated gastritis.
PMCID: PMC3404125  PMID: 19006683
CC chemokine ligand 20; CC chemokine receptor 6; H. pylori; Dendritic cells; cag pathogenicity island
18.  A Novel Serum Metabolomics-Based Diagnostic Approach for Colorectal Cancer 
PLoS ONE  2012;7(7):e40459.
To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer.
Methodology/Principal Findings
We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0–2 colorectal cancer (82.8%).
Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.
PMCID: PMC3394708  PMID: 22792336
19.  Genome-Wide Survey of Mutual Homologous Recombination in a Highly Sexual Bacterial Species 
Genome Biology and Evolution  2012;4(5):628-640.
The nature of a species remains a fundamental and controversial question. The era of genome/metagenome sequencing has intensified the debate in prokaryotes because of extensive horizontal gene transfer. In this study, we conducted a genome-wide survey of outcrossing homologous recombination in the highly sexual bacterial species Helicobacter pylori. We conducted multiple genome alignment and analyzed the entire data set of one-to-one orthologous genes for its global strains. We detected mosaic structures due to repeated recombination events and discordant phylogenies throughout the genomes of this species. Most of these genes including the “core” set of genes and horizontally transferred genes showed at least one recombination event. Taking into account the relationship between the nucleotide diversity and the minimum number of recombination events per nucleotide, we evaluated the recombination rate in every gene. The rate appears constant across the genome, but genes with a particularly high or low recombination rate were detected. Interestingly, genes with high recombination included those for DNA transformation and for basic cellular functions, such as biosynthesis and metabolism. Several highly divergent genes with a high recombination rate included those for host interaction, such as outer membrane proteins and lipopolysaccharide synthesis. These results provide a global picture of genome-wide distribution of outcrossing homologous recombination in a bacterial species for the first time, to our knowledge, and illustrate how a species can be shaped by mutual homologous recombination.
PMCID: PMC3381677  PMID: 22534164
homologous recombination; horizontal transfer; population genomics; species; Helicobacter pylori
20.  Primary Mucinous Adenocarcinoma of the Testis 
Case Reports in Medicine  2012;2012:685946.
Ovarian-type surface epithelial neoplasms of the testis and paratestis are uncommon, and the mucinous subtype is particularly rare. These tumors represent a counterpart to ovarian cancer. Malignant tumors have the potential for metastatic spread and are often fatal. The case of a 59-year-old man with testicular mucinous adenocarcinoma is presented. Computed tomography indicated involvement of the paraaortic and pelvic lymph nodes, so chemotherapy was initiated. To the best of our knowledge, this is the second paper regarding responsiveness to chemotherapies used in ovarian cancer.
PMCID: PMC3328900  PMID: 22550498
21.  Magnifying endoscopy in upper gastroenterology for assessing lesions before completing endoscopic removal 
Any prognosis of gastrointestinal (GI) cancer is closely related to the stage of the disease at diagnosis. Endoscopic submucosal dissection (ESD) and en bloc endoscopic mucosal resection (EMR) have been performed as curative treatments for many early-stage GI lesions in recent years. The technologies have been widely accepted in many Asian countries because they are minimally invasive and supply thorough histopathologic evaluation of the specimens. However, before engaging in endoscopic therapy, an accurate diagnosis is a precondition to effecting the complete cure of the underlying malignancy or carcinoma in situ. For the past few years, many new types of endoscopic techniques, including magnifying endoscopy with narrow-band imaging (ME-NBI), have emerged in many countries because these methods provide a strong indication of early lesions and are very useful in determining treatment options before ESD or EMR. However, to date, there is no comparable classification equivalent to “Kudo’s Pit Pattern Classification in the colon”, for the upper GI, there is still no clear internationally accepted classification system of magnifying endoscopy. Therefore, in order to help unify some viewpoints, here we will review the defining optical imaging characteristics and the current representative classifications of microvascular and microsurface patterns in the upper GI tract under ME-NBI, describe the accurate relationship between them and the pathological diagnosis, and their clinical applications prior to ESD or en bloc EMR. We will also discuss assessing the differentiation and depth of invasion, defying the lateral spread of involvement and targeting biopsy in real time.
PMCID: PMC3319956  PMID: 22493543
Magnifying endoscopy with narrow-band imaging; Upper gastroenterology; Assessment; Endoscopic submucosal dissection; Endoscopic mucosal resection
22.  Sequence Heterogeneity in NS5A of Hepatitis C Virus Genotypes 2a and 2b and Clinical Outcome of Pegylated-Interferon/Ribavirin Therapy 
PLoS ONE  2012;7(2):e30513.
Pegylated-interferon plus ribavirin (PEG-IFN/RBV) therapy is a current standard treatment for chronic hepatitis C. We previously reported that the viral sequence heterogeneity of part of NS5A, referred to as the IFN/RBV resistance-determining region (IRRDR), and a mutation at position 70 of the core protein of hepatitis C virus genotype 1b (HCV-1b) are significantly correlated with the outcome of PEG-IFN/RBV treatment. Here, we aimed to investigate the impact of viral genetic variations within the NS5A and core regions of other genotypes, HCV-2a and HCV-2b, on PEG-IFN/RBV treatment outcome. Pretreatment sequences of NS5A and core regions were analyzed in 112 patients infected with HCV-2a or HCV-2b, who were treated with PEG-IFN/RBV for 24 weeks and followed up for another 24 weeks. The results demonstrated that HCV-2a isolates with 4 or more mutations in IRRDR (IRRDR[2a]≥4) was significantly associated with rapid virological response at week 4 (RVR) and sustained virological response (SVR). Also, another region of NS5A that corresponds to part of the IFN sensitivity-determining region (ISDR) plus its carboxy-flanking region, which we referred to as ISDR/+C[2a], was significantly associated with SVR in patients infected with HCV-2a. Multivariate analysis revealed that IRRDR[2a]≥4 was the only independent predictive factor for SVR. As for HCV-2b infection, an N-terminal half of IRRDR having two or more mutations (IRRDR[2b]/N≥2) was significantly associated with RVR, but not with SVR. No significant correlation was observed between core protein polymorphism and PEG-IFN/RBV treatment outcome in HCV-2a or HCV-2b infection. Conclusion: The present results suggest that sequence heterogeneity of NS5A of HCV-2a (IRRDR[2a]≥4 and ISDR/+C[2a]), and that of HCV-2b (IRRDR[2b]/N≥2) to a lesser extent, is involved in determining the viral sensitivity to PEG-IFN/RBV therapy.
PMCID: PMC3271109  PMID: 22319571
23.  THRB Genetic Polymorphisms Can Predict Severe Myelotoxicity after Definitive Chemoradiotherapy in Patients with Esophageal Squamous Cell Carcinoma 
Objective: Chemotherapy-related toxicities are difficult to predict before treatment. In this study, we investigated whether thyroid hormone receptor beta (THRB) genetic polymorphisms can serve as a potential biomarker in patients with esophageal squamous cell carcinoma (ESCC).
Methods: Forty-nine Japanese patients with ESCC who received a definitive chemoradiotherapy (CRT) with 5-fluorouracil and cisplatin in conjunction with concurrent irradiation were retrospectively analyzed. Severe acute toxicities, including leukopenia, stomatitis, and cheilitis, were evaluated according to 6 single nucleotide polymorphisms (SNPs) in the gene; the intronic SNPs of rs7635707 G/T, rs6787255 A/C, rs9812034 G/T, and rs9310738 C/T and the SNPs in the 3′-untranslated region (3′-UTR) of rs844107 C/T and rs1349265 G/A.
Results: Distribution of the 4 intronic SNPs, but not the 2 SNPs in the 3′-UTR, showed a significant difference between patients with and without severe acute leukopenia. Stomatitis and cheilitis were not associated with any of the 6 analyzed SNPs. Frequency of haplotype of the 4 intronic SNPs reached approximately 97% with the 2 major haplotypes G-A-G-C (73.4%) and T-C-T-T (23.5%).
Conclusions: THRB intronic SNPs can provide useful information on CRT-related severe myelotoxicity in patients with ESCC. Future studies will be needed to confirm these findings.
PMCID: PMC3491433  PMID: 23136537
esophageal squamous cell carcinoma; thyroid hormone receptor beta; chemoradiotherapy; severe acute toxicity; prognosis.
24.  Inhibitory Effects of Glycyrrhetinic Acid on DNA Polymerase and Inflammatory Activities 
We investigated the inhibitory effect of three glycyrrhizin derivatives, such as Glycyrrhizin (compound 1), dipotassium glycyrrhizate (compound 2) and glycyrrhetinic acid (compound 3), on the activity of mammalian pols. Among these derivatives, compound 3 was the strongest inhibitor of mammalian pols α, β, κ, and λ, which belong to the B, A, Y, and X families of pols, respectively, whereas compounds 1 and 2 showed moderate inhibition. Among the these derivatives tested, compound 3 displayed strongest suppression of the production of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in a cell-culture system using mouse macrophages RAW264.7 and peritoneal macrophages derived from mice. Moreover, compound 3 was found to inhibit the action of nuclear factor-κB (NF-κB) in engineered human embryonic kidney (HEK) 293 cells. In addition, compound 3 caused greater reduction of 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced acute inflammation in mouse ear than compounds 1 and 2. In conclusion, this study has identified compound 3, which is the aglycone of compounds 1 and 2, as a promising anti-inflammatory candidate based on mammalian pol inhibition.
PMCID: PMC3138047  PMID: 21785649
25.  Evolution in an oncogenic bacterial species with extreme genome plasticity: Helicobacter pylori East Asian genomes 
BMC Microbiology  2011;11:104.
The genome of Helicobacter pylori, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian H. pylori genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains.
A phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including oipA, hopMN, babABC, sabAB and vacA-2) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (tipα), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (miaA, tilS), a DNA recombinase/exonuclease that recognizes genome identity (addA), and DNA/RNA hybrid nucleases (rnhAB). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of cagA, vacA, homC (outer membrane protein), sotB (sugar transport), and a translation fidelity factor (miaA). Large divergence was seen in genes related to antibiotics: frxA (metronidazole resistance), def (peptide deformylase, drug target), and ftsA (actin-like, drug target).
These results demonstrate dramatic genome evolution within a species, especially in likely host interaction genes. The East Asian strains appear to differ greatly from the European strains in electron transfer and redox reactions. These findings also suggest a model of adaptive evolution through proteome diversification and selection through modulation of translational fidelity. The results define H. pylori East Asian lineages and provide essential information for understanding their pathogenesis and designing drugs and therapies that target them.
PMCID: PMC3120642  PMID: 21575176

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