Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

In Vitro Production of Optically Active Octahydrocurcumin by Human Intestinal Bacterium.

Enterococcus avium, producing 5R-hexahydrocurcumin metabolized tetrahydrocurcumin to octahydrocurcumin in vitro. Based on a detailed analysis of the two secondary alcohols, the metabolite obtained from tetrahydrocurcumin via 5R-hexahydrocurcumin was identified as 3R,5R-octahydrocurcumin. The activities of 5R-hexahydrocurcumin and 3R,5R-octahydrocurcumin were compared to those of the synthetic compounds, using monocyte chemoattractant protein-1 produced via murine adipocytes in vitro. The optically active curcuminoids reduced the cytokine production similar to tetrahydrocurcumin without any difference in their stereochemistry.

Production of Optically Active Hexahydrocurcumin by Human Intestinal Bacterium in Vitro.

A hexahydrocurcumin-producing bacterium named 2a1-2b was isolated from human feces. It was observed that the bacterium had more than 99% similarity with Enterococcus avium ATCC14025T according to 16S ribosomal DNA (rDNA) sequence. The strain 2a1-2b produced optically active 5R-hexahydrocurcumin (enantiomeric excess (e.e.) > 95%) from tetrahydrocurcumin but not from curcumin. Our results showed that intestine is an important place for producing hexahydrocurcumin.

Equol Inhibits Mushroom Tyrosinase in Vitro through Tight Binding.

Equol, an intestinal metabolite of daidzein, inhibited more potently mushroom tyrosinase in vitro than other inhibitors, genistein and kojic acid. We investigated the mechanism underlying tyrosinase inhibition by equol. Treating racemic equol with tyrosinase produced 3'-hydroxyequol. Because the optical activity of the product showed <25% enantiomeric excess, the reaction was not highly stereospecific. Using enzyme-linked immunosorbent assays with an anti-equol monoclonal antibody, we observed that equol bound to pre-coated tyrosinase in a dose-dependent manner. Our results suggested the formation of a stable equol-tyrosinase complex.

Persimmon fruit tannin-rich fiber reduces cholesterol levels in humans.

Bile acid-binding agents are known to lower blood cholesterol levels and have been clinically used for the treatment of hypercholesterolemia. We previously showed that tannin-rich fiber from young persimmon (Diospyros kaki) fruits had bile acid-binding properties. In this study, we performed a randomized, double-blind, placebo-controlled trial to investigate the hypocholesterolemic effects of tannin-rich fiber in humans. The subjects (n = 40, plasma total cholesterol levels 180-259 mg/dl) were divided into 3 groups and ingested cookie bars containing 0 g (placebo group, n = 14), 3 g (low-dose group, n = 13), or 5 g (high-dose group, n = 13) of tannin-rich fiber 3 times daily before meals for 12 weeks. Plasma total cholesterol levels decreased significantly in the low-dose (12 weeks, p < 0.005) and high-dose (6 weeks, p < 0.05; 12 weeks, p < 0.001) groups. In addition, plasma low-density lipoprotein cholesterol levels decreased significantly in the high-dose group (6 weeks, p < 0.05; 12 weeks, p < 0.001). These improvements were not accompanied by changes in plasma high-density lipoprotein cholesterol or plasma triglyceride levels. Our findings indicate that tannin-rich fiber from young persimmon fruits is a useful food material for treating hypercholesterolemia.

Complete genomic sequence of the O-desmethylangolensin-producing bacterium Clostridium rRNA cluster XIVa strain SY8519, isolated from adult human intestine.

The O-desmethylangolensin-producing Clostridium rRNA cluster XIVa strain SY8519 was isolated from the intestinal flora of a healthy human as a key isoflavonoid-metabolizing bacterium. Here, we report the finished and annotated genomic sequence of this organism.

Complete genomic sequence of the equol-producing bacterium Eggerthella sp. strain YY7918, isolated from adult human intestine.

Eggerthella sp. strain YY7918 was isolated from the intestinal flora of a healthy human. It metabolizes daidzein (a soybean isoflavonoid) and produces S-equol, which has stronger estrogenic activities than daidzein. Here, we report the finished and annotated genomic sequence of this organism.

Coronary vein infusion of multipotent stromal cells from bone marrow preserves cardiac function in swine ischemic cardiomyopathy via enhanced neovascularization.

Few reports have examined the effects of adult bone marrow multipotent stromal cells (MSCs) on large animals, and no useful method has been established for MSC implantation. In this study, we investigate the effects of MSC infusion from the coronary vein in a swine model of chronic myocardial infarction (MI). MI was induced in domestic swine by placing beads in the left coronary artery. Bone marrow cells were aspirated and then cultured to isolate the MSCs. At 4 weeks after MI, MSCs labeled with dye (n=8) or vehicle (n=5) were infused retrogradely from the anterior interventricular vein without any complications. Left ventriculography (LVG) was performed just before and at 4 weeks after cell infusion. The ejection fraction (EF) assessed by LVG significantly decreased from baseline up to a follow-up at 4 weeks in the control group (P<0.05), whereas the cardiac function was preserved in the MSC group. The difference in the EF between baseline and follow-up was significantly greater in the MSC group than in the control group (P<0.05). The MSC administration significantly promoted neovascularization in the border areas compared with the controls (P<0.0005), though it had no affect on cardiac fibrosis. A few MSCs expressed von Willebrand factor in a differentiation assay, but none of them expressed troponin T. In quantitative gene expression analysis, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) levels were significantly higher in the MSC-treated hearts than in the controls (P<0.05, respectively). Immunohistochemical staining revealed VEGF production in the engrafted MSCs. In vitro experiment demonstrated that MSCs significantly stimulated endothelial capillary network formation compared with the VEGF protein (P<0.0001). MSC infusion via the coronary vein prevented the progression of cardiac dysfunction in chronic MI. This favorable effect appeared to derive not from cell differentiation, but from enhanced neovascularization by angiogenic factors secreted from the MSCs.

Bile acid-binding ability of kaki-tannin from young fruits of persimmon (Diospyros kaki) in vitro and in vivo.

The bile acid-binding ability of a highly polymerized tannin (kaki-tannin) extracted from dried-young fruits of persimmon (Diospyros kaki) was examined. The kaki-tannin was composed mainly of epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Bile acid-binding ability of kaki-tannin was examined against cholic acid, glycocholic acid, taurocholic acid and deoxycholic acid in vitro, and its effect on fecal bile acid excretion in mice was also examined. Although the bile acid-binding ability of kaki-tannin was weaker than that of cholestyramine, kaki-tannin adsorbed all the bile acids tested and significantly promoted fecal bile acid excretion in mice when supplied at 1% (w/w) in the diet.

Effects of isoflavone-rich red clover extract on blood glucose level: A randomized, double-blind, placebo-controlled trial.

High blood glucose is associated with increased risk of various diseases. Red clover (RC; Trifolium pratense L.) is an edible legume whose sprout is rich in isoflavones such as formononetin and biochanin A. We examined the effects of RC extract on postprandial and fasting blood glucose level, using a randomized, double-blind, placebo-controlled trial with 36 participants, aged 25 to 64 years, who were randomly assigned to receive either 1.91 g of RC extract (containing 8 mg formononetin and 1.8 mg biochanin A) or placebo. Each participant ingested the assigned test food daily for 8 weeks, and at the oral maltose tolerance test (OMTT). Initially, the two groups did not significantly differ in OMTT results. However, fasting insulin levels at 8 weeks were significantly lower in the RC group (4.76 µIU/ml at Week 0 to 4.01 µIU/ml at Week 8) with a significant interaction (P = 0.046). Subgroup analysis showed that change in blood glucose level (blood glucose ΔC) tended to decrease late in the trial period during OMTT in the ≤50-year-old RC group, as did fasting blood glucose and insulin levels at 8 weeks; hemoglobin A1c was also significantly reduced in this subgroup (5.36% at Week 0 to 5.28% at Week 8) with a significant interaction (P = 0.040). These results suggest that the daily intake of RC could reduce blood glucose, particularly for those ≤50 years old. Formononetin-an α-glucosidase inhibitor-is considered to be the major functional molecule for these effects. Therefore, intake of RC that contains formononetin might help blood glucose control.

Characterization of an O-desmethylangolensin-producing bacterium isolated from human feces.

A bacterium that converted daidzein to O-desmethylangolensin was isolated from the feces of healthy humans. It was an obligately anaerobic, nonsporeforming, nonmotile and Gram-positive rod. The isolate used glucose, sucrose, raffinose, maltose, and fructose as carbon sources. It did not hydrolyze gelatin, esculin, or starch. The strain was urease, acid phosphatase, and arginine dihydrolase positive. It was catalase, oxidase, H(2)S, and indole negative. The major products of glucose fermentation were butyrate and lactate. Its mol% G+C was 51.2. The major cellular fatty acids were C(16:0) DMA, C(16:0), and C(16:0) aldehyde. The structural type of cell wall peptidoglycan was suggested to be A1gamma. The isolate was susceptible to beta-lactam, cefem, and macrolide antibiotics and resistant to aminoglycoside and quinolone antibiotics. The bacterium was related to Eubacterium ramulus ATCC29099(T), Eubacterium rectale ATCC33656(T), and species of the genus Roseburia, but the highest 16S rRNA gene similarity to these described species was only 94.4%, consistent with its being classified as a novel genus. Based on the above, the isolate, named strain SY8519, was identified as belonging to a novel genus in the Clostridium rRNA cluster XIVa.

Bile acid-binding activity of young persimmon (Diospyros kaki) fruit and its hypolipidemic effect in mice.

The hypolipidemic effects and bile acid-binding properties of young persimmon (Diospyros kaki) fruit were examined. In an animal experiment, male C57BL/6.Cr mice (n = 5) were fed an AIN-76-modified high fat diet supplemented with 2% or 5% (w/w) dried young persimmon fruit (YP) for 10 weeks. The intake of YP significantly enhanced fecal bile acid excretion and lowered the concentration of hepatic lipids and plasma cholesterol. Analysis of gene expression in liver tissue showed that 2% or 5% YP up-regulated the expression of the sterol regulatory element-binding protein-2 gene. In the 5% group, there were increased expressions of the genes for cholesterol 7alpha-hydroxylase and the low-density lipoprotein receptor. Next, the bile acid-binding ability of YP was analysed in vitro using cholic acid (CA). In 100-2000 microM CA solutions, 1% (w/v) YP adsorbed approximately 60% of CA, while dried mature persimmon fruit adsorbed approximately 20% of CA. The positive control, cholestyramine, adsorbed approximately 80% of CA in the 100-2000 microM CA solutions. A crude tannin extract from YP, which contained 54.7% condensed tannins, adsorbed approximately 78% of CA in the 2000 microM CA solutions. These results suggest that the ability of YP to bind bile acid contributes to its hypolipidemic effect in mice.

Benzyl Isothiocyanate Produced by Garden Cress (Lepidium sativum) Prevents Accumulation of Hepatic Lipids.

We determined the physiological effects of glucotropaeolin-rich lyophilized garden cress sprout powder (GC) administered to fasting and nonfasting mice. High-performance liquid chromatography analysis revealed glucotropaeolin (57.4±1.1 mg/g dry weight) as a major phytochemical constituent of GC. Decreasing tendency in body weight and feeding efficiency ratio were detected in the group of mice fed 0.05% (w/w) GC (GC0.05). Nonfasting mice exhibited significantly lower liver weights that were unchanged after fasting. Decreased total lipid (TL) and triglyceride (TG) levels in the liver were detected in the nonfasted GC0.01 and GC0.05 groups, but only in TLs of the fasted groups. The levels of plasma TGs and nonesterified fatty acids of the GC0.05 group, which remained unchanged during nonfasting, decreased after fasting. To determine its effects on the accumulation of lipids in the liver, the glucotropaeolin aglycone, benzyl isothiocyanate (BITC), was added to the liver-derived HepG2 human cell line cultured in a medium containing a high concentration of D-glucose (4,500 mg/L D-glucose) (HG group) or 1 mM oleic acid (SO group). Toxicity was not detected when cells were treated with as much as 5 µM BITC; however, lipid accumulation was inhibited by BITC in a concentration-dependent manner in the HG groups. The same effect was observed when 2 µM BITC was added to the diet of the SO groups. These results suggest that moderate levels of GC or BITC are useful for reducing liver and plasma TGs.

Red Clover (Trifolium pratense L.) Sprout Prevents Metabolic Syndrome.

We examined the prevention effect of red clover (Trifolium pratense L.) sprout on metabolic syndrome using a high-carbohydrate and high-fat diet (Western diet; WD)-induced male C57BL/6J obese model mouse. Red clover sprout-lyophilized powder (RC) contained 3.5 mg/g dry-weight of formononetin as a major phenolic compound, as analyzed by high performance liquid chromatography. Supplementation of 0.3% (w/w) RC in a WD (WD+RC) showed an anti-obesity effect and ameliorated lipid metabolism in the obese model mice. Additionally, fasting plasma glucose levels were significantly reduced in the WD+RC group. Administration of 0.1 mg/kg formononetin reduced the postprandial blood glucose level, as assessed using the oral maltose tolerance test. However, no significant formononetin intake effect was observed on the plasma insulin level. These results suggest that the formononetin contained in red clover sprout inhibits α-glucosidase and thereby contributes to reducing the postprandial blood glucose response in mice.

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats.

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Isolation and characterization of a novel equol-producing bacterium from human feces.

An equol-producing bacterium was newly isolated from the feces of healthy humans and its morphological and biochemical properties were characterized. The cells were obligate anaerobes. They were non-sporulating, non-motile, gram-positive bacilliform bacteria with a pleomorphic morphology. The strain was catalase-positive, and oxidase-, urease-, and indole-negative. The only other sugar utilized by the strain was glycerin. The strain also degraded gelatin, but not esculin. It was most closely related to Eggerthella hongkongensis HKU10, with 93.3% 16S rDNA nucleotide sequence homology. Based on these features, the isolate was identified as a novel species of the genus Eggerthella. It was named Eggerthella sp. YY7918. Strain YY7918 converted substrates daidzein and dihydrodaidzein into S-equol, but did not convert daidzin, glysitein, genistein, or formononetin into it. An antimicrobial susceptibility assay indicated that strain YY7918 was susceptible to aminoglycoside-, tetracycline-, and new quinolone-antibiotics.

Hypolipidemic effect of young persimmon fruit in C57BL/6.KOR-ApoEshl mice.

We investigated the hypolipidemic effects of young persimmon fruit (YP) on apolipoprotein E-deficient C57BL/6.KOR-ApoEshl mice. These mice exhibited higher plasma cholesterols, except for high-density lipoprotein (HDL), and lower plasma HDL cholesterol than C57BL/6.Cr mice that had the same genetic background as the C57BL/6.KOR-ApoEshl mice. Male C57BL/6.KOR-ApoEshl mice (n=5) were fed a diet supplemented with dry YP, Hachiya-kaki, at a concentration of 5% (w/w) for 10 weeks. YP treatment significantly lowered plasma chylomicron, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) cholesterols, and triglyceride, and this response was accompanied by an elevation of fecal bile acid excretion. In the liver, sterol regulatory element binding protein-2 gene expression was significantly higher in mice fed YP, while the mRNA and protein levels of the LDL receptor did not change. These results indicate that acceleration of fecal bile acid excretion is a major mechanism of the hypolipidemic effect induced by YP in C57BL/6.KOR-ApoEshl mice.

Daidzein reductase of Eggerthella sp. YY7918, its octameric subunit structure containing FMN/FAD/4Fe-4S, and its enantioselective production of R-dihydroisoflavones.

S-Equol is a metabolite of daidzein, a type of soy isoflavone, and three reductases are involved in the conversion of daidzein by specific intestinal bacteria. S-Equol is thought to prevent hormone-dependent diseases. We previously identified the equol producing gene cluster (eqlABC) of Eggerthella sp. YY7918. Daidzein reductase (DZNR), encoded by eqlA, catalyzes the reduction of daidzein to dihydrodaidzein (the first step of equol synthesis), which was confirmed using a recombinant enzyme produced in Escherichia coli. Here, we purified recombinant DZNR to homogeneity and analyzed its enzymological properties. DZNR contained FMN, FAD, and one 4Fe-4S cluster per 70-kDa subunit as enzymatic cofactors. DZNR reduced the CC bond between the C-2 and C-3 positions of daidzein, genistein, glycitein, and formononetin in the presence of NADPH. R-Dihydrodaidzein and R-dihydrogenistein were highly stereo-selectively produced from daidzein and genistein. The Km and kcat for daidzein were 11.9 µM and 6.7 s-1, and these values for genistein were 74.1 µM and 28.3 s-1, respectively. This enzyme showed similar kinetic parameters and wide substrate specificity for isoflavone molecules. Thus, this enzyme appears to be an isoflavone reductase. Gel filtration chromatography and chemical cross-linking analysis of the active form of DZNR suggested that the enzyme consists of an octameric subunit structure. We confirmed this by small-angle X-ray scattering and transmission electron microscopy at a magnification of ×200,000. DZNR formed a globular four-petal cloverleaf structure with a central vertical hole. The maximum particle size was 173 Å.

Rapid and convenient detection of urinary equol by thin-layer chromatography.

Equol, a metabolite of daidzein for some intestinal microflora, is known to retain highly estrogenic activity and is of wide interest in relation to human health. However, not all humans can produce equol. In this study, detection of urinary equol using thin-layer chromatography (TLC) was performed to distinguish between equol producers and non-producers. After 36 h of soy food intake, urine, collected from 7 volunteers, was hydrolyzed, purified by reverse phase silica gel column and applied to normal phase TLC. Consequently, equol was clearly separated from the urine samples and discriminated the equol producers in this system. The detection limit of equol was at least 20 ng.

Effects of G-CSF on cardiac remodeling and arterial hyperplasia in rats.

Although granulocyte colony-stimulating factor (G-CSF) has been shown to prevent cardiac remodeling after acute myocardial infarction, the mechanism and safety of G-CSF treatment acute myocardial infarction remain controversial. The purpose of the present study was to investigate in a rat model the mechanisms underlying the beneficial effect of G-CSF in acute myocardial infarction and to determine whether G-CSF treatment aggravates vascular remodeling of injured artery after acute myocardial infarction. Sprague-Dawley rats received transplanted bone marrow cells from green fluorescent protein (GFP) transgenic rats. Acute myocardial infarction was induced by ligation of the left coronary artery. After 24 h, the right carotid artery was injured with a balloon catheter. G-CSF (100 microg/kg/day) or saline was injected subcutaneously for 5 consecutive days after induction of acute myocardial infarction. G-CSF treatment significantly improved left ventricle function and reduced infarct size in rats with acute myocardial infarction. Expression of mRNA for the angiogenic cytokines was significantly higher in the infarction border area in the G-CSF group than in the control group. The surviving cardiomyocytes in infarction area were more in the G-CSF group. GFP-positive cells were gathered in the infarction border area in both groups; G-CSF did not increase cardiac homing of GFP-positive bone marrow cells in contrast to control group. Most GFP-positive cells were CD68-positive (macrophages). It was difficult to find bone marrow-derived cardiomyocytes in the infarcted area. G-CSF treatment inhibited neointima formation and increased reendothelialization of the injured artery. GFP-positive cells were identified most in the adventitia of the injured artery. A few cells in the neointima and reendothelialization were GFP positive. In conclusion, administration of G-CSF appears to be effective for treatment of left ventricular remodeling after acute myocardial infarction and does not aggravate vascular remodeling. The effect of G-CSF on cardiac and vascular remodeling may occur mainly through a direct action on the heart and arteries.