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1.  Thioesterase Superfamily Member 2 (Them2) and Phosphatidylcholine Transfer Protein (PC-TP) Interact To Promote Fatty Acid Oxidation and Control Glucose Utilization 
Molecular and Cellular Biology  2014;34(13):2396-2408.
Thioesterase superfamily member 2 (Them2) is a mitochondrion-associated long-chain fatty acyl coenzyme A (CoA) thioesterase that is highly expressed in the liver and oxidative tissues. Them2 activity in vitro is increased when it interacts with phosphatidylcholine transfer protein (PC-TP), a cytosolic lipid binding protein. Them2−/− and Pctp−/− mice exhibit enhanced hepatic insulin sensitivity and increased adaptive thermogenesis, and Them2−/− mice are also resistant to diet-induced hepatic steatosis. Although we showed previously that a Them2–PC-TP complex suppresses insulin signaling, the enzymatic activity of Them2 suggests additional direct involvement in regulating hepatic nutrient homeostasis. Here we used cultured primary hepatocytes to elucidate biochemical and cellular mechanisms by which Them2 and PC-TP regulate lipid and glucose metabolism. Under conditions simulating fasting, Them2−/− and Pctp−/− hepatocytes each exhibited decreased rates of fatty acid oxidation and gluconeogenesis. In results indicative of Them2-dependent regulation by PC-TP, chemical inhibition of PC-TP failed to reproduce these changes in Them2−/− hepatocytes. In contrast, rates of glucose oxidation and lipogenesis in the presence of high glucose concentrations were decreased only in Them2−/− hepatocytes. These findings reveal a primary role for Them2 in promoting mitochondrial oxidation of fatty acids and glucose in the liver.
PMCID: PMC4054321  PMID: 24732803
2.  Metabolomics Evaluation of Serum Markers for Cachexia and Their Intra-Day Variation in Patients with Advanced Pancreatic Cancer 
PLoS ONE  2014;9(11):e113259.
Cancer cachexia is a multifactorial syndrome characterized by progressive loss of weight and muscle atrophy. Using metabolomics, we investigated serum markers and their intra-day variation in advanced pancreatic cancer patients with cachexia.
Patients were enrolled in two groups: those with or without cachexia. Blood samples collected at 6:30 AM, 11:30 AM, 4:30 PM, and 9:30 PM were analyzed using metabolomics, and serum levels of IL-6, TNF-α, and leptin were measured and compared between the two groups. Intra-day variation was then evaluated.
Twenty-one patients were enrolled in total. In the cachexia group (n = 9), median body weight loss rate over 6 months was greater, performance status was poorer, and anorexia was more severe than in the non-cachexia group (n = 12). Each metabolites level showed substantial intra-day variation, and some of them displayed significant differences between the two groups. Levels of paraxanthine remained markedly lower in the cohort with cachexia at all measurement points. Besides, median IL-6 and TNF-α levels appeared higher and leptin concentration appeared lower in the cachexia group, albeit without statistical significance.
Some metabolites and some serological marker levels were affected by cancer cachexia. Although paraxanthine levels were consistently lower in patients with cachexia, we identified that many metabolites indicated large intra- and inter-day variation and that it might be necessary to pay attention to intra-day variation in metabolomics research.
PMCID: PMC4239056  PMID: 25411961
3.  Metabolomics for Biomarker Discovery in Gastroenterological Cancer 
Metabolites  2014;4(3):547-571.
The study of the omics cascade, which involves comprehensive investigations based on genomics, transcriptomics, proteomics, metabolomics, etc., has developed rapidly and now plays an important role in life science research. Among such analyses, metabolome analysis, in which the concentrations of low molecular weight metabolites are comprehensively analyzed, has rapidly developed along with improvements in analytical technology, and hence, has been applied to a variety of research fields including the clinical, cell biology, and plant/food science fields. The metabolome represents the endpoint of the omics cascade and is also the closest point in the cascade to the phenotype. Moreover, it is affected by variations in not only the expression but also the enzymatic activity of several proteins. Therefore, metabolome analysis can be a useful approach for finding effective diagnostic markers and examining unknown pathological conditions. The number of studies involving metabolome analysis has recently been increasing year-on-year. Here, we describe the findings of studies that used metabolome analysis to attempt to discover biomarker candidates for gastroenterological cancer and discuss metabolome analysis-based disease diagnosis.
PMCID: PMC4192679  PMID: 25003943
metabolomics; biomarker; serum; gastroenterological cancer; mass spectrometry
4.  Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes 
Gut Pathogens  2014;6:27.
Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype.
Based on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains.
Gene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.
PMCID: PMC4079918  PMID: 24995043
Whole-genome sequencing; Helicobacter pylori; Clarithromycin; Multidrug efflux; TolC homolog
5.  Role of Fc Receptors as a Therapeutic Target 
It has been forty years since the discovery of Fc Receptors and their function. Fc Receptors include the IgG receptors (FcγR), high-affinity IgE receptor (FcεRI), IgA and IgA/IgM receptors, and neonatal Fc receptor for IgG (FcRn). In particular, the FcγRs have been well known to play an important role in many biologic processes including those associated with the response to infection and cancer as well as in the pathogenesis of immune-mediated diseases. Both positive and negative regulatory function has ascribed to Fc receptors and FcγRs in particular which serve to establish a threshold for immune cell activation. In other cases, Fc receptors such as FcRn possess a novel structure and function by playing a major role in the transport of IgG across polarized epithelial barriers at mucosal surfaces and in the regulation of IgG half-life. These diverse functions highlight the potential effectiveness of targeting Fc receptors for therapeutic purposes. This review summarizes new information available in the therapeutic applications of this biology.
PMCID: PMC3990358  PMID: 19275696
Fc receptors; FcRn; IgG; FcγR
6.  The transfer of maternal antigen-specific IgG regulates the development of allergic airway inflammation early in life in an FcRn-dependent manner 
Asthma is a chronic inflammatory airway disease characterized by airway hyperreactivity, increased mucus production, and reversible airway contraction. Asthma is a complex genetic trait caused by environmental factors in genetically predisposed individuals. The transportation of maternal antigen-specific IgG via amniotic fluid, placenta and breast milk plays an important role in passive immunity. First, to examine whether maternal passive immunity by the transportation of antigen-specific IgG via FcRn regulates allergic airway inflammation, ovalbumin-immunized FcRn+/− female mice were bred with FcRn−/− male mice to evaluate the degree of ovalbumin-induced allergic airway inflammation of FcRn−/− offspring. Maternal passive immunity regulated allergic airway inflammation in an FcRn-dependent manner. Second, to examine the role of maternal antigen-specific IgG1 injection into mothers, we intravenously injected ovalbumin-specific IgG1 into wild-type or FcRn+/− mice immediately after they gave birth. The offspring were sensitized and challenged with ovalbumin. Antigen-specific IgG1 administered to lactating mice reduced allergic airway inflammation in their offspring in an FcRn-dependent manner. Last, to exclude the factor of maternal passive immunity other than ovalbumin-specific IgG1, we administered ovalbumin-specific IgG1 orally to offspring after birth. Oral administration of ovalbu-min-specific IgG1 to offspring during the lactating period prevented the development of allergic airway inflammation in an FcRn-dependent manner. These data show that the transfer of maternal antigen-specific IgG regulates the development of allergic airway inflammation early in life in an FcRn-dependent manner.
PMCID: PMC3990410  PMID: 20362552
Allergic airway inflammation; Asthma; IgG; FcRn; Breast milk
7.  Immune and non-immune functions of the (not so) neonatal Fc receptor, FcRn 
Seminars in immunopathology  2009;31(2):223-236.
Careful regulation of the body’s immunoglobulin-G (IgG) and albumin concentrations is necessitated by the importance of their respective functions. As such, the neonatal Fc receptor (FcRn) which, as a single receptor, is capable of regulating both of these molecules, has become an important focus of investigation. In addition to these essential protection functions, FcRn possesses a host of other functions that are equally as critical. During the very first stages of life, FcRn mediates the passive transfer of IgG from mother to offspring both before and after birth. In the adult, FcRn regulates the persistence of both IgG and albumin in the serum as well as the movement of IgG, and any bound cargo, between different compartments of the body. This shuttling allows for the movement not only of monomeric ligand but also of antigen/antibody complexes from one cell type to another in such a way as to facilitate the efficient initiation of immune responses towards opsonized pathogens. As such, FcRn continues to play the role of an immunological sensor throughout adult life, particularly in regions such as the gut which are exposed to a large number of infectious antigens. Increasing appreciation for the contributions of FcRn to both homeostatic and pathological states is generating an intense interest in the potential for therapeutic modulation of FcRn binding. A greater understanding of FcRn’s pleiotropic roles is thus imperative for a variety of therapeutic purposes.
PMCID: PMC3898171  PMID: 19495758
Neonatal Fc receptor; Immunoglobulin G; Albumin; Immune complexes; Gastrointestinal tract
8.  The Immunologic Functions of the Neonatal Fc Receptor for IgG 
Journal of clinical immunology  2012;33(Suppl 1):9-17.
Careful regulation of the body’s immunoglobulin G (IgG) and albumin concentrations is necessitated by the importance of their respective functions. As such, the neonatal Fc receptor (FcRn), as a single receptor, is capable of regulating both of these molecules and has become an important focus of investigation. In addition to these essential protection functions, FcRn possesses a number of other functions that are equally as critical and are increasingly coming to attention. During the very first stages of life, FcRn mediates the passive transfer of IgG from mother to offspring both before and after birth. In the adult, FcRn regulates the persistence of both IgG and albumin in the serum as well as the movement of IgG, and any bound cargo, between different compartments of the body via transcytosis across polarized cells. FcRn is also expressed by hematopoietic cells; consistent with this, FcRn regulates MHC class II presentation and MHC class I cross-presentation by dendritic cells. As such, FcRn plays an important role in immune surveillance throughout adult life. The increasing appreciation for FcRn in both homeostatic and pathological conditions is generating an intense interest in the potential for therapeutic modulation of FcRn binding to IgG and albumin.
PMCID: PMC3548031  PMID: 22948741
Neonatal Fc receptor; FcRn; maternal IgG; transcytosis
9.  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
10.  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
11.  Metabolic Profiling of Oxidized Lipid-Derived Volatiles in Blood by Gas Chromatography/Mass Spectrometry with In-Tube Extraction 
Mass Spectrometry  2013;2(1):A0018.
Once lipids are oxidized, various volatiles are produced by cleavage of the fatty acid side chain. Considering the variety of lipids present in the body, a large number of possible volatiles might originate from oxidized lipids. However, only specific volatiles such as aldehydes are exclusively examined in current studies, and there is no reported method for the exhaustive analysis of all volatiles. We developed a sensitive analytical method for the detection of all possible volatiles for multimarker profiling, applying a new extraction method called in-tube extraction. Oxidized phosphatidyl choline standards were prepared in vitro and analyzed in order to determine the potential variety of volatiles. Over 40 compounds, including alcohols, ketones, and furanones, were identified in addition to the aldehydes reported previously. Based on this result, we applied our analytical method to mouse plasma and identified 12 volatiles, including 1-octen-3-ol, which is correlated to disease states. To determine the volatile profile after oxidation, we oxidized plasma in vitro under various conditions and identified 27 volatiles, including 1-octen-3-ol and benzaldehyde. The generation capacity of each volatile was different. This method allows sensitive and exhaustive analysis of various volatiles in addition to aldehydes.
PMCID: PMC3967009  PMID: 24860708
in tube extraction; volatile; lipids; oxidation; metabolic profiling
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells 
Respiratory Research  2011;12(1):42.
There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.
In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).
In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.
Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.
PMCID: PMC3079623  PMID: 21477339
21.  Resolvin E1, an endogenous lipid mediator derived from eicosapentaenoic acid, prevents dextran sulfate sodium induced colitis 
Inflammatory bowel diseases  2010;16(1):87-95.
Resolvin E1 (RvE1), an endogenous lipid mediator derived from eicosapentaenoic acid (EPA), has been identified in local inflammation during the healing stage. RvE1 reduces inflammation in several types of animal models including peritonitis and retinopathy, and blocks human neutrophil transendothelial cell migration. The RvE1 receptor ChemR23 is expressed on myeloid cells such as macrophages and dendritic cells. The aim of this study was to determine whether RvE1 regulates colonic inflammation when the innate immune response of macrophages plays a key role in the pathogenesis and tissue damage.
RvE1 receptor, ChemR23, was expressed in mouse peritoneal macrophages as defined by flow cytometry. Peritoneal macrophages were pretreated with RvE1, followed by lipopolysaccharide (LPS) stimulation whereupon of the transcriptional levels of proinflammatory cytokines were analyzed. RvE1 treatment led to the inhibition of proinflammatory cytokines including TNF-α and IL-12p40. In HEK293 cells, pretreatment with RvE1 inhibited TNF-α-induced nuclear translocation of NF-κB in a ChemR23 dependent manner. These results suggested that RvE1 could regulate pro-inflammatory responses of macrophages expressing ChemR23. Therefore, we investigated the beneficial effects of RvE1 in dextran sulfate sodium (DSS) induced colitis. RvE1 treatment led to amelioration of colonic inflammation.
These results indicate that RvE1 suppresses pro-inflammatory responses of macrophages. RvE1 and its receptor may therefore be useful as therapeutic targets in the treatment of human inflammatory bowel disease (IBD) and other inflammatory disorders.
PMCID: PMC3070396  PMID: 19572372
Resolvin E1; macrophage; NF-κB; DSS-induced colitis
22.  Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity 
Previously, we reported that vitamin K3 (VK3), but not VK1 or VK2 (=MK-4), inhibits the activity of human DNA polymerase γ (pol γ). In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.
PMCID: PMC3083694  PMID: 21541047
vitamin K; MK-2; DNA polymerase λ; enzyme inhibitor; anti-inflammation
23.  Gamma Interferon Produced by Antigen-Specific CD4+ T Cells Regulates the Mucosal Immune Responses to Citrobacter rodentium Infection▿  
Infection and Immunity  2010;78(6):2653-2666.
Citrobacter rodentium, a murine model pathogen for enteropathogenic Escherichia coli, colonizes the surface of intestinal epithelial cells and causes mucosal inflammation. This bacterium is an ideal model for investigating pathogen-host immune interactions in the gut. It is well known that gene transcripts for Th1 cytokines are highly induced in colonic tissue from mice infected with C. rodentium. However, it remains to be seen whether the Th1 or Th2 cytokines produced by antigen-specific CD4+ T cells provide effective regulation of the host immune defense against C. rodentium infection. To investigate the antigen-specific immune responses, C. rodentium expressing ovalbumin (OVA-C. rodentium), a model antigen, was generated and used to define antigen-specific responses under gamma interferon (IFN-γ)-deficient or interleukin-4 (IL-4)-deficient conditions in vivo. The activation of antigen-specific CD4+ T cells and macrophage phagocytosis were evaluated in the presence of IFN-γ or IL-4 in vitro. IFN-γ-deficient mice exhibited a loss of body weight and a higher bacterial concentration in feces during OVA-C. rodentium infection than C57BL/6 (wild type) or IL-4-deficient mice. This occurred through the decreased efficiency of macrophage phagocytosis and the activation of antigen-specific CD4+ T cells. Furthermore, a deficiency in antigen-specific CD4+ T-cell-expressed IFN-γ led to a higher susceptibility to mucosal and gut-derived systemic OVA-C. rodentium infection. These results show that the IFN-γ produced by antigen-specific CD4+ T cells plays an important role in the defense against C. rodentium.
PMCID: PMC2876554  PMID: 20351140
24.  An FcRn-dependent role for anti-flagellin in pathogenesis of immunoglobulin G-mediated colitis in mice 
Gastroenterology  2009;137(5):1746-56.e1.
Background & Aims
The neonatal receptor for immunoglobulin (Ig)G (FcRn) protects monomeric IgG from catabolism in parenchymal and hematopoietic cells. In dendritic cells, FcRn also promotes presentation of antigens in association with IgG. Since IgGs with anti-bacterial specificity are a hallmark of inflammatory bowel disease, we sought to determine their significance and relationship to FcRn expression in antigen presenting cells, focusing on IgGs specific for flagellin.
Levels of circulating anti-flagellin IgG were induced in wild-type and FcRn−/− mice, followed by induction of colitis with dextran sodium sulfate (DSS). Bone marrow chimera models were used to localize the site of FcRn action.
Wild-type mice that received anti-flagellin IgG exhibited more severe colitis following administration of DSS, compared to mice that received control IgG. Wild-type mice immunized with flagellin exhibited significantly more severe colitis in response to DSS administration than that observed in similarly treated FcRn−/− mice. In chimera studies, FcRn−/− mice given wild-type bone marrow and immunized with flagellin exhibited significantly more colitis than wild-type mice given FcRn−/− bone marrow and immunized with flagellin. Serum anti-flagellin IgG levels were similar in both sets of chimeric mice, consistent with the equal participation of hematopoietic and non-hematopoeitic cells in FcRn-mediated IgG protection.
Anti-bacterial IgG antibodies are involved in the pathogenesis of colitis; this pathway requires FcRn in antigen presenting cells, the major subset of hematopoietic cells that express FcRn.
PMCID: PMC2787451  PMID: 19664634
25.  Neonatal Fc Receptor: From Immunity to Therapeutics 
Journal of Clinical Immunology  2010;30(6):777-789.
The neonatal Fc receptor (FcRn), also known as the Brambell receptor and encoded by Fcgrt, is a MHC class I like molecule that functions to protect IgG and albumin from catabolism, mediates transport of IgG across epithelial cells, and is involved in antigen presentation by professional antigen presenting cells. Its function is evident in early life in the transport of IgG from mother to fetus and neonate for passive immunity and later in the development of adaptive immunity and other functions throughout life. The unique ability of this receptor to prolong the half-life of IgG and albumin has guided engineering of novel therapeutics. Here, we aim to summarize the basic understanding of FcRn biology, its functions in various organs, and the therapeutic design of antibody- and albumin-based therapeutics in light of their interactions with FcRn.
PMCID: PMC2970823  PMID: 20886282
FcRn; Fcgrt; Brambell; IgG; Fc; albumin; transcytosis; transport; recycling

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