Publishing type：Research paper (scientific journal)  

Six sets of fecal, milk, and uterine samples from dairy cows and two sets of airborne dust and water samples from the cowshed were collected from two farms in Ho Chi Minh City at 20–40 days postpartum. The microbiota was characterized using Illumina sequencing of the V4 hypervariable region of the 16S rRNA genes. The predominant species in the fecal microbiota was not as predominant in the milk microbiota, but occasionally appeared in the uterine microbiota. The microbiota from the airborne dust and water samples did not show a relation with either the milk or the uterine microbiota, whereas Moraxellaceae, the most abundant family in the airborne dust and water microbiota, was detected at low proportions in the milk microbiota. The farm-to-farm difference was more apparent for the milk microbiota than for the fecal and uterine microbiota. It is noteworthy that Dermacoccaceae, Bacillaceae, and Methylobacteriaceae were the prevalent families in the microbiota of several milk and uterine samples; however, these findings did not help understand the role of the microbiota in fertility and subsequent conception. Despite the small number of dairy cows examined in this study, this report is the first to characterize the fecal, milk, uterine, airborne dust, and water microbiota in dairy farms in southern Vietnam.

DOI： 10.17582/journal.aavs/2022/10.7.1525.1531

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Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

DOI： 10.12938/bmfh.2021-061

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.nutres.2021.06.009

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Publishing type：Research paper (scientific journal)   Publisher：Cambridge University Press (CUP)  

<title>Abstract</title>
SCFA increase serotonin (5-hydroxytryptamine, 5-HT) synthesis and content in the colon <italic>in vitro</italic> and <italic>ex vivo</italic>, but little is known <italic>in vivo</italic>. We tested whether dietary indigestible saccharides, utilised as a substrate to produce SCFA by gut microbiota, would increase colonic 5-HT content in mice. Male C57BL/6J mice were fed a purified diet and water supplemented with 4 % (w/v) 1-kestose (KES) for 2 weeks. Colonic 5-HT content and enterochromaffin (EC) cell numbers were lower in mice supplemented with KES than those without supplementation, while monoamine oxidase A activity and mRNA levels of tryptophan hydroxylase 1 (<italic>Tph1</italic>), chromogranin A (<italic>Chga</italic>), <italic>Slc6a4</italic> and monoamine oxidase A (<italic>Maoa</italic>) genes in the colonic mucosa, serum 5-HT concentration and total 5-HT content in the colonic contents did not differ between groups. Caecal acetate concentration and <italic>Bifidobacterium pseudolongum</italic> population were higher in KES-supplemented mice. Similar trends were observed in mice supplemented with other indigestible saccharides, that is, fructo-oligosaccharides, inulin and raffinose. Intragastric administration of live <italic>B. pseudolongum</italic> (10⁸ colony-forming units/d) for 2 weeks reduced colonic 5-HT content and EC cell numbers. These results suggest that changes in synthesis, reuptake, catabolism and overflow of 5-HT in the colonic mucosa are not involved in the reduction of colonic 5-HT content by dietary indigestible saccharides in mice. We propose that gut microbes including <italic>B. pseudolongum</italic> could contribute to the reduction of 5-HT content in the colonic mucosa via diminishing EC cells.

DOI： 10.1017/s0007114521001306

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Language：English   Publishing type：Research paper (scientific journal)  

Diet is considered the most influential factor in modulating the gut microbiota but how dietary protein sources differ in their modulatory effects is not well understood. In this study, soy, meat (mixture of beef and pork), and fish proteins (experiment 1) and soy, milk (casein), and egg proteins (experiment 2) were fed to rats with cellulose (CEL) and raffinose (RAF); the microbiota composition and short-chain fatty acid concentration in the cecum were determined. Egg protein feeding decreased the concentration of acetic acid and the richness and diversity of the cecum microbiota. RAF feeding increased the concentrations of acetic and propionic acids and decreased the richness and diversity of the cecum microbiota. When fed with CEL, the abundance of Ruminococcaceae and Christensenellaceae, Akkermansiaceae and Tannerellaceae, and Erysipelotrichaceae enhanced with soy protein, meat and fish proteins, and egg protein, respectively. The effects of dietary proteins diminished with RAF feeding and the abundance of Bifidobacteriaceae, Erysipelotrichaceae, and Lachnospiraceae increased and that of Ruminococcaceae and Christensenellaceae decreased regardless of the protein source. These results indicate that, although the effect of prebiotics is more robust and distinctive, dietary protein sources may influence the composition and metabolic activities of the gut microbiota. The stimulatory effects of soy, meat, and egg proteins on Christensenellaceae, Akkermansiaceae, and Erysipelotrichaceae deserve further examination to better elucidate the dietary manipulation of the gut microbiota.

DOI： 10.3934/microbiol.2021001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Serotonin (5-hydroxytryptamine [5-HT]) synthesized and released in enterochromaffin (EC) cells participates in various functions in the gastrointestinal tract by acting on a diverse range of 5-HT receptors (HTRs) expressed on smooth muscle, enteric neurons, and epithelial cells. We previously observed that genes encoding HTR2A, HTR2B, and HTR4 are expressed in murine intestinal organoids, suggesting the expression of these HTRs in intestinal epithelial cells. The present study investigated the localization of these HTRs in the murine intestine by immunofluorescence staining. HTR2A, HTR2B, and HTR4 localized in individual solitary cells in the epithelium, while HTR2C was observed in the lamina propria. In the epithelium, HTR2A, HTR2B, and HTR4 colocalized with 5-HT, and HTR4 colocalized with glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Murine intestinal organoids show a colocalization pattern that is similar to in vivo HTR2A and HTR4 with 5-HT, GLP-1, and PYY. Intraperitoneal and intragastric administration of tegaserod, an HTR4 agonist, failed to alter plasma GLP-1 levels in fasted mice. However, intragastric but not intraperitoneal administration of tegaserod reduced dietary lipid-induced increases of plasma GLP-1 levels. This action of tegaserod was inhibited by co-administration of RS39604, an HTR4 antagonist. These results suggest that murine ileal GLP-1/PYY-producing enteroendocrine (EE) cells express HTR4, while 5-HT-producing EC cells express HTR2A, HTR2B, and HTR4. In addition, the observations regarding in vivo GLP-1 secretion suggest that HTR4 signaling in ileal EE cells suppresses dietary lipid-induced GLP-1 secretion. We thus propose that EC and EE cells may interact with each other through paracrine signaling mechanisms.

DOI： 10.1007/s00424-020-02453-7

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Language：English   Publishing type：Research paper (scientific journal)  

Microbiota of individual cow milk, bulk tank milk, and feces of Jersey cows were examined. Samples were collected from two farms (F1 and F2) in cool (November, Nov) and hot (July, Jul) seasons. Milk yield and milk composition were similar between the two farms and between the two seasons. Prevalent taxa of the fecal microbiota, i.e. Ruminococcaceae, Bacteroidaceae, Lachnospiraceae, Rikenellaceae, and Clostridiaceae, were unaffected by the farm and season. Relative abundance of milk microbiota for Pseudomonadaceae, Enterobacteriaceae, and Streptococcaceae (F1 > F2) and Lactobacillaceae, Bifidobacteriaceae, and Cellulomonadaceae (F1 < F2) were different between the two farms, and those for Staphylococcaceae, Bacillaceae, Ruminococcaceae, and Veillonellaceae (Nov < Jul) and Methylobacteriaceae and Moraxellaceae (Nov > Jul) were different between the two seasons. The microbiota of bulk tank milk was numerically different from that of individual cow milk. Principal coordinate analysis indicated that the milk microbiota was unrelated to the fecal microbiota. The finding that relative abundance of Pseudomonadaceae and Moraxellaceae appeared greater than those reported for Holstein milk suggested that higher protein and fat content may result in a greater abundance of proteolytic and lipolytic taxa in Jersey cow milk.

DOI： 10.1111/asj.13441

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Language：English   Publishing type：Research paper (scientific journal)  

Cyclic nigerosylnigerose (CNN) is a cyclic oligosaccharide. Oral administration of CNN promotes immunoglobulin A (IgA) secretion in the gut. IgA is a major antibody secreted into the gut and plays a crucial role in suppressing gut inflammation due to commensal gut microbiota. To investigate the effect of administration of CNN to promote IgA secretion on gut inflammation, experimental colitis was induced with dextran sulfate sodium (DSS) in Balb/c mice after 6 weeks of CNN pre-feeding. The severity of colitis was evaluated based on a disease activity index (DAI), the gene expression of inflammatory cytokines, and a histological examination. The CNN-treated mice with DSS-induced colitis (CNN-DSS group) showed significantly lower DAI scores and mRNA levels of interleukin-1 compared with the CNN-untreated mice with DSS-induced colitis (DSS group). Histological examination of the colon revealed that the pathological score was significantly lower in the CNN-DSS group compared with the DSS group due to the reduced infiltration of immune cells. The number of goblet cells was significantly higher in the CNN-DSS group compared with the DSS group. The IgA concentration and the ratio of microbiota coated with IgA were evaluated in the cecal content. Although there was no difference in the IgA concentration among groups, a higher proportion of cecal microbiota were coated with IgA in the CNN-DSS group compared with that in the DSS group. These results suggest that CNN might preserve goblet cells in the colon and promote IgA coating of gut microbiota, which synergistically ameliorate gut inflammation in mice with DSS-induced colitis.

DOI： 10.12938/bmfh.2020-012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Taylor and Francis Ltd  

Soy, meat (mixture of pork and beef), and fish proteins were fed to rats with and without prebiotic raffinose (RAF), and the composition and fermentation of gut microbiota were examined. Bifidobacterium spp. populations were higher, and propionic acid concentration was lower in soy protein-fed than meat protein-fed rats. Likewise, Enterobacteriaceae populations were higher in fish protein-fed rats than other rats. RAF feeding increased Bifidobacterium spp. and decreased Faecalibacterium prausnitzii populations regardless of the dietary protein source. Interactions between dietary proteins and RAF were shown for Lactobacillus spp. and Clostridium perfringens group
the increase of Lactobacillus spp. populations by RAF was seen only for soy protein-fed rats, whereas the reduction of C. perfringens group by RAF was evident in fish and meat protein-fed rats. It is concluded that dietary proteins may differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, with differences observed between plant and animal proteins.

DOI： 10.1080/09637486.2017.1382454

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s12079-017-0403-9

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Other Link： http://link.springer.com/content/pdf/10.1007/s12079-017-0403-9.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The microbiota of whole crop corn silage and feces of silage-fed dairy cows were examined. A total of 18 dairy cow feces were collected from six farms in Japan and China, and high-throughput Illumina sequencing of the V4 hypervariable region of 16S rRNA genes was performed. Lactobacillaceae were dominant in all silages, followed by Acetobacteraceae, Bacillaceae, and Enterobacteriaceae. In feces, the predominant families were Ruminococcaceae, Bacteroidaceae, Clostridiaceae, Lachnospiraceae, Rikenellaceae, and Paraprevotellaceae. Therefore, Lactobacillaceae of corn silage appeared to be eliminated in the gastrointestinal tract. Although fecal microbiota composition was similar in most samples, relative abundances of several families, such as Ruminococcaceae, Christensenellaceae, Turicibacteraceae, and Succinivibrionaceae, varied between farms and countries. In addition to the geographical location, differences in feeding management between total mixed ration feeding and separate feeding appeared to be involved in the variations. Moreover, a cow-to-cow variation for concentrateassociated families was demonstrated at the same farm; two cows showed high abundance of Succinivibrionaceae and Prevotellaceae, whereas another had a high abundance of Porphyromonadaceae. There was a negative correlation between forage-associated Ruminococcaceae and concentrate-associated Succinivibrionaceae and Prevotellaceae in 18 feces samples. Succinivibrionaceae, Prevotellaceae, p-253418B5, and Spirochaetaceae were regarded as highly variable taxa in this study. These findings help to improve our understanding of variation and similarity of the fecal microbiota of dairy cows with regard to individuals, farms, and countries. Microbiota of naturally fermented corn silage had no influence on the fecal microbiota of dairy cows.

DOI： 10.1007/s00253-017-8348-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Orally administered Lactobacillus strains, including L. plantarum No.14 and L rhamnosus GG, reportedly reduce inflammatory cytokine production in mice. The present study tested our idea that circulating exosomes mediate the action of Lactobacillus strains. The lipopolysaccharide-induced production of TNF-alpha and IL-6 in vitro was attenuated in peritoneal exudate cells (PECs) isolated from C57BL/6N mice that had been fed L. plantarum No.14. When PECs were cultured for 24 h with exosomes isolated from the serum of mice fed L plantarum No.14 or L. rhamnosus GG, accumulation of both TNF-alpha and of the corresponding mRNA was lowered. Growth in the presence of these exosomes also decreased the production of TNF-alpha and IL-6 by the murine macrophage cell line RAW264.7. In contrast, supplementation with exosome-depleted serum of mice fed L plantarum No.14 or L. rhamnosus GG failed to affect the production of TNF-alpha and IL-6 by RAW264.7 cells. When PECs and RAW264.7 cells were cultured for 24 h with PKH67-labeled exosomes isolated from murine serum, fluorescent signal was observed inside the cells, suggesting that these cells incorporate serum exosomes. We propose that the anti-inflammatory activity of orally administered L plantarum No.14 and L rhamnosus GG is mediated, at least in part, by circulating exosomes. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.05.152

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The microbiota of pre-ensiled crop and silage were examined using denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS). Wilted Italian ryegrass (IR), whole crop corn (WC), and wilted alfalfa (AL) silages stored for 2 months were examined. All silages contained lactic acid as a predominant fermentation product. Across the three crop species, DGGE detected 36 and 28 bands, and NGS identified 253 and 259 genera in the pre-ensiled crops and silages, respectively. The NGS demonstrated that, although lactic acid bacteria (LAB) became prevalent in all silages after 2 months of storage, the major groups were different between crops: Leuconostoc spp. and Pediococcus spp. for IR silage, Lactobacillus spp. for WC silage, and Enterococcus spp. for AL silage. The predominant silage LAB genera were also detected by DGGE, but the presence of diverse non-LAB species in pre-ensiled crops was far better detected by NGS. Likewise, good survival of Agrobacterium spp., Methylobacterium spp., and Sphingomonas spp. in IR and AL silages was demonstrated by NGS. The diversity of the microbiota described by principal coordinate analysis was similar between DGGE and NGS. Our finding that analysis of pre-ensiled crop microbiota did not help predict silage microbiota was true for both DGGE and NGS.

DOI： 10.1007/s00253-016-7900-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Although diet has an important influence on the composition of gut microbiota, the impact of dietary protein sources has only been studied to a minor extent. In this study, we examined the influence of different dietary protein sources regarding the effects of prebiotic oligosaccharides on the composition and metabolic activity of gut microbiota. Thirty female rats were fed casein and soy protein isolate with cellulose, raffinose (RAF), and fructooligosaccharides (FOS). Microbiota composition was examined by real-time qPCR and denaturing gradient gel electrophoresis. Dietary protein source affected cecum microbiota; acetic acid concentration and Lactobacillus spp. populations were greater with soy protein than with casein. Prebiotic oligosaccharides had distinctive effects on gut microbiota; RAF increased the acetic acid concentration and Bifidobacterium spp. populations, and FOS increased the butyric acid concentration regardless of the dietary protein. Likewise, Bifidobacterium sp., Collinsella sp., and Lactobacillus sp. were detected in microbiota of the rats fed RAF, and Bacteroides sp., Roseburia sp., and Blautia sp. were seen in microbiota of the rats fed FOS. Interactions between dietary proteins and prebiotic oligosaccharides were observed with Clostridium perfringens group populations and cecum IgA concentration. RAF and FOS decreased C. perfringens group populations in casein-fed rats, and the combination of soy protein and RAF substantially increased cecum IgA concentration. These results indicate that dietary proteins can differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, depending on the type of carbohydrate polymers involved.

DOI： 10.1111/1750-3841.13391

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Intestinal organoids were recently established as an ex vivo model of the intestinal epithelium. The present study investigated the serotonin (5-hydroxytryptamine, 5-HT) system using organoids. Organoids from murine small intestinal and colonic crypts were successfully cultured. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that small intestinal and colonic organoids express mRNAs encoding tryptophan hydroxylase-1 (TPH1) (the rate-limiting enzyme of 5-HT synthesis), serotonin reuptake transporter (SERT), 5-HT receptor (HTR)2A, HTR2B, and HTR4. SERT mRNA levels were significantly higher in the small intestine than in the colon in both the mucosal tissues and organoids, as estimated by quantitative real-time RT-PCR. Although the 5-HT concentration and levels of chromogranin A (CgA) (an enteroendocrine cell marker), TPH1, and HTR4 mRNAs were significantly higher in the colonic mucosa than the small intestinal mucosa, they were the same in small intestinal and colonic organoids. There were no significant differences in HTR2A and HTR2B mRNA levels between the small intestine and colon in either the mucosal tissues or organoids. Immunofluorescence staining showed that the number of CgA-positive cells in the colonic organoids appeared to increase upon culturing with acetate. Acetate supplementation significantly increased CgA, TPH1, and HTR4 mRNA levels in the colonic organoids. We propose that organoids are useful for investigating the 5-HT system in the intestinal epithelium, even though colonic organoids may require gut microbiota derived factors such as short-chain fatty acids. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.03.165

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Blackwell Publishing Ltd  

Intestinal epithelial cells sense short-chain fatty acids (SCFAs) to secrete non-neuronal acetylcholine (ACh). However, the roles of luminal SCFAs and epithelial ACh under normal and pathological conditions remain unknown. We examined ileal contractile responses to SCFAs at different ages and their mucosal cholinergic alterations under inflammatory conditions. Ileal contractile responses to SCFAs in 1-day-old pups to 7-week-old mice were compared using an isotonic transducer, and responses to an intraperitoneal injection of lipopolysaccharide (LPS) were analyzed in 7-week-old mice. The mRNA expression levels of a SCFA activate free fatty acid receptor, acetylcholinesterase (AChE), choline acetyltransferase (Chat), and choline transporter-like protein 4 (CTL4) were measured using real-time quantitative RT-PCR. AChE was analyzed by histochemical and optical enzymatic assays. Atropine-sensitive ileal contractile responses to SCFAs occurred in all 1-day-old pups, but were frequently desensitized after the weaning period. These contractile responses were not inhibited by tetrodotoxin and did not appear when the mucosal layer had been scraped off. Contractile desensitization in 7-week-old mice was abolished in the presence of the AChE inhibitor, eserine, which was consistent with increased AChE activity after weaning. Ileal contractions to SCFAs in adult mice were restored by LPS, which significantly increased the epithelial mRNA expression of Chat and CTL4. Atropine-sensitive ileal contractile responses to SCFAs constitutively occur in the newborn period, and are desensitized during developmental stages following the up-regulated expression of AChE in the villous mucosa, but are restored under inflammatory conditions possibly via the release of epithelial ACh.

DOI： 10.14814/phy2.12759

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Influenza is a major cause of respiratory tract infection. Although most cases do not require further hospitalization, influenza periodically causes epidemics in humans that can potentially infect and kill millions of people. To countermeasure this threat, new vaccines need to be developed annually to match emerging influenza viral strains with increased resistance to existing vaccines. Thus, there is a need for finding and developing new anti-influenza viral agents as alternatives to current treatments. Here, we tested the antiviral effects of an extract from the stems and roots of Salacia reticulata (SSRE), a plant rich in phytochemicals, such as salacinol, kotalanol, and catechins, on H1N1 influenza virus-infected mice. Following oral administration of 0.6 mg/day of SSRE, the incidence of coughing decreased in 80% of mice, and only one case of severe pulmonary inflammation was detected. Moreover, when compared with mice given Lactobacillus casei JCM1134, a strain previously shown to help increase in vitro natural killer (NK) cell activity, SSRE-administered mice showed greater and equal NK cell activity in splenocytes and pulmonary cells, respectively, at high effector cell: target cell ratios. Next, to test whether or not SSRE would exert protective effects against influenza in the absence of gut microbiota, mice were given antibiotics before being inoculated influenza virus and subsequently administered SSRE. SSRE administration induced an increase in NK cell activity in splenocytes and pulmonary cells at levels similar to those detected in mice not treated with antibiotics. Based on our results, it can be concluded that phytochemicals in the SSRE exerted protective effects against influenza infection putatively via modulation of the immune response, including enhancement of NK cell activity, although some protective effects were not necessarily through modulation of gut microbiota. Further investigation is necessary to elucidate the molecular mechanisms underlying the protective effects of SSRE against influenza infection.

DOI： 10.3389/fimmu.2016.00115

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

Non-neuronal and atropine-sensitive ileal contractile responses to short chain fatty acids (SCFAs) are detected in the neonatal stage, and change with age or inflammatory conditions. However, the roles of luminal SCFAs in developmental changes have not yet been elucidated. We examined ileal contractile responses to SCFAs in mice colonized with different SCFA-producing intestinal microbiota under normal and inflammatory conditions. Using conventional (Conv), germ-free (GF), and gnotobiotic mice infected with Bifidobacterium (GB-bif), Propionibacterium (GB-prop), or Lactobacillus (GB-lact), ileal contractions were measured in 1-day-old neonates and 7-week-old mice using an isotonic transducer. Contractions occurred in all 1-day-old neonates, and were significantly desensitized in the adult stage in the Conv, GB-bif, and GB-prop groups, but not in the GF and GB-lact groups. An injection of lipopolysaccharide frequently restored desensitized contractions; however, the contraction rate did not change in the GF and GB-lact groups. The relative mRNA expression of a SCFA receptor (GPR43) or nicotinic acetylcholine receptor alpha 7 was weaker in the GF group (0.3-fold or 0.4-fold expression level, respectively) than in the Conv group. In conclusion, the luminal inhabitation of SCFA-producing bacteria may potentiate the regulation of non-neuronal and atropine-sensitive ileal contractile responses to SCFAs under healthy and inflammatory conditions.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Although leptin and adiponectin are the predominant adipokines, how their circulating levels are regulated is incompletely understood. The present study tested whether intestinal epithelial cells influence the expression and secretion of these adipokines by adipocytes. Leptin gene expression and secretion by cultured human primary adipocytes and Simpson-Golabi-Behmel Syndrome adipocytes increased upon coculture with human enterocytic Caco-2 cells or incubation in conditioned medium of Caco-2 cells. Although adiponectin secretion increased, its mRNA levels decreased. Tissue homogenate of the ileum (but not the jejunum, colon, or liver) of nonobese C57BL/6J mice also stimulated leptin and adiponectin secretion by cultured murine 3T3-L1 adipocytes. However, ileal homogenate of obese KK-Ay mice had no effect on leptin and adiponectin secretion. We propose that as yet unidentified humoral factors released from intestinal epithelial cells are involved in regulating circulating leptin and adiponectin levels. Decreased production of such factors may contribute to hyperphagia in KK-Ay mice. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2015.01.118

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Small intestinal epithelium is a self-renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt-villus axis. We examined whether dual labeling with different thymidine analogues, 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the click reaction, respectively. We successfully observed BrdU- and EdU-positive cells on the epithelium with no cross-reaction. In addition, we observed an almost complete overlapping of BrdU- and EdU-positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU- and EdU-positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5m/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.

DOI： 10.1111/dgd.12191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.04.114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Among domestic animals, teat order is only observed in the pig. In order to achieve the healthy growth and weaning of piglets, it is important to elucidate if volume of colostrum secretion and immunoglobulin A (IgA) and IgG concentrations differ among the teats of a sow. Nine sows were used to evaluate the difference in colostrum secretion volume (CSV) and four of these sows were assessed for IgA and IgG concentrations from each teat. Samples were collected five times during 21h following parturition. Teats were assigned anatomical locations of teat (1 to 7) from anterior to posterior. The CSV of anterior (locations 1 and 2) and middle teats (locations 3-5) was significantly higher than those of posterior teats (locations 6 and 7) throughout the experiment except for 18h post-parturition (P<0.05). The CSV of the teats at location 1 was significantly higher at most collection times than those at locations 6 and 7. A positive correlation of CSV was observed with IgA and IgG concentrations from 12h and 6h post-parturition, respectively (P<0.05). The results suggest that anterior teats secrete greater volumes of colostrum and that these tend to contain higher IgA and IgG than posteriors teats.

DOI： 10.1111/asj.12211

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Fluorescein isothiocyanate (FITC)-labeled EC-12 was cocultured with peritoneal macrophage and the amount of EC-12 phagocytosed by peritoneal macrophages was measured using a microplate fluorometer. Peritoneal macrophages from toll-like receptor (TLR) 2-, TLR7-, and MyD88- deficient knockout (KO) mice exhibited similar levels of EC-12 phagocytosis to those from wild-type mice. Similarly, dectin-1 neutralization of peritoneal macrophages had no effect on EC-12 phagocytosis. However, blockade of the mannose receptor (MR) significantly decreased the amount of EC-12 phagocytosed by peritoneal macrophages; the same effect was observed in bone marrow-derived macrophages and dendritic cells. Our findings suggest that MR plays a major role in EC-12 phagocytosis by the APCs.

DOI： 10.1002/mbo3.99

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). Post-weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial-resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilisDB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21-day-old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.

DOI： 10.1111/asj.12003

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Language：English   Publishing type：Research paper (scientific journal)  

Weaning piglets are continuously exposed to various viruses. The antiviral effects of lactic acid bacteria (LAB) have been confirmed mainly in humans and mice, while few studies have been conducted in livestock. In this study, we evaluated the effect of oral administration of Enterococcus faecalis strain EC-12 (EC-12) on the gene expressions of antiviral cytokines in weaning piglets. Piglets were allocated to the EC-12-administered group (E group) and the no-treatment control group (C group). The small intestinal tissue, the mesenteric lymph node (MLN) cells and the splenocytes were collected from the piglets. The tissue and cells were co-cultured with a live vaccine of porcine reproductive respiratory syndrome virus, porcine epidemic diarrhea virus or EC-12. After the incubation, the gene expressions of IFN-γ and TNF-α in the tissue and cells were evaluated. The gene expressions of IFN-γ in the MLN cells and TNF-α in the splenocytes were significantly higher in the E group than in the C group. However, the increase in the gene expression of antiviral cytokines was observed independently of the antigen treatments. The results of the present study suggest that oral administration of EC-12 did not increase the response of immune cells to specific viral antigens but increased the baseline gene expression of antiviral cytokines.

DOI： 10.12938/bmfh.32.123

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

A large amount of secretory immunoglobulin A (S-IgA) is secreted in the alimentary tract of mammals. It has been reported that S-IgA coats a portion of commensal intestinal bacteria in human and mouse. However, S-IgA-coated bacteria have not been studied in pigs and calves. In this study, we evaluated the distribution of S-IgA-coated commensal intestinal bacteria in each portion of the gastrointestinal tracts of pigs and calves. Immunoglobulin G (IgG)-coated bacteria were also analyzed because a considerable amount of IgG is secreted in the gastrointestinal tracts of pigs, and in particular, calves. S-IgA- or IgG-coated bacteria were detected in all the segments of the gastrointestinal tracts of pigs and calves. The proportion of S-IgA-coated bacteria to total bacteria (i.e. S-IgA coating ratio) varied in the segments of the gastrointestinal tract in pigs, whereas those of calves were nearly the same throughout the gastrointestinal tract. The S-IgA and IgG coating ratios were higher in pigs than in calves for all segments of the gastrointestinal tract.

DOI： 10.1111/j.1740-0929.2012.01026.x

Scopus

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Aim: Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon.
Methods: Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I-sc) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt.
Results: Luminal addition of methyl-beta-cyclodextrin (10 mm) and mastoparan (30 mu m), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I-sc, but did not affect increases in I-sc induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 mu m), a G alpha(q/11)-selective inhibitor, markedly inhibited propionate-induced increase in I-sc, but did not affect I-sc responses to ACh and EFS. Both methyl-beta-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and G alpha(q/11), but not GPR43. Uncoupling of G alpha(q/11) from flotillin-1 in lipid rafts occurred under desensitization of the I-sc response to propionate.
Conclusions: These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via G alpha(q/11).

DOI： 10.1111/j.1748-1716.2011.02331.x

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Some commensal intestinal bacteria in humans and mice are coated with secretory immunoglobulin A (S-IgA). It has been suggested that the S-IgA coating of commensal bacteria does not occur at random and thus identification of S-IgA-coated bacterial genera/species should help in elucidating the interaction between S-IgA and commensal intestinal bacteria, but no method of identifying the genera/species of S-IgA-coated bacteria has been established. To identify S-IgA-coated bacterial composition, we developed a method combining immunohistochemical detection of S-IgA and subsequent 16S rRNA targeted fluorescence in situ hybridization (FISH) analysis. Human and mouse fecal S-IgA coated bacterial composition was evaluated by this newly developed method with 10 frequently-used FISH probes. Fecal S-IgA-coated bacterial composition was successfully analyzed by this method, and this suggests that Enterobacteriaceae is preferably coated with S-IgA, whereas Bacteroides/Prevotella and Lactobacillus/Enterococcus groups appear to be poorly coated with S-IgA.

DOI： 10.1271/bbb.90801

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

We evaluated the effects of long-term daily cows&apos; milk (CM) administration on insulin resistance induced by a high-sucrose diet. F344 rats, aged 3 weeks, were divided into two groups according to diet (dextrin-fed v. sucrose-fed). These groups were further divided into two groups receiving either CM or artificial milk (AM; isoenergetic emulsion of egg white protein, maltose, lard and minerals). Rats were fed a sucrose- or dextrin-based diet for 7 weeks and orally administered CM or AM at 25 ml/kg following an 8 h fast on a daily basis. Insulin sensitivity was evaluated via postprandial changes in serum glucose and insulin, oral glucose tolerance tests, and fasting serum insulin and fructosamine concentrations. The sucrose-fed rats showed an overall decrease in insulin sensitivity, but postprandial insulin levels were lower in the CM-treated subgroup than in the AM-treated subgroup. Peak serum glucose and insulin concentrations were highest in the sucrose-fed rats, but CM administration reduced peak glucose and insulin values in comparison with AM administration. By area under the curve analysis, insulin levels after feeding and glucose loads were significantly lower in the CM-treated groups than in the AM-treated groups. The CM-treated groups also demonstrated lower fasting insulin and fructosamine levels than the AM-treated groups. Improved insulin sensitivity due to CM administration seemed to be associated with reduced duodenal GLUT2 mRNA levels and increased propionate production within the caecum.

DOI： 10.1017/S0007114509990365

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

It is reported that some, but not all, bacteria in human faeces are coated with secretory immunoglobulin A (S-IgA). We evaluated the proportion of S-IgA-coated bacteria to total intestinal bacteria (S-IgA coating ratio) in the gastrointestinal tract of two different strains of mice supplied by two different suppliers. The S-IgA coating ratio was significantly different in each gastrointestinal segment and between mouse suppliers. The amount of non-bacteria-bound IgA (free IgA) in each gastrointestinal segment indicated that this difference in the S-IgA coating ratio might not be due to the amount of secreted IgA. Furthermore, immunoblotting analysis revealed that only a small amount of IgA (&lt; 5% to free-IgA) was used for the coating. This indicates that, although sufficient S-IgA was secreted to coat the entire intestinal population of bacteria, only some part of the bacteria were coated with S-IgA. This study suggests that the amount of luminal S-IgA may not determine the S-IgA coating ratio, and that the amount of IgA coating intestinal commensal bacteria is very small.

DOI： 10.1111/j.1574-695X.2009.00568.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

The immune system in juvenile calves is immature, so calves are susceptible to several diarrheal and respiratory diseases. Oral administration of lactic acid bacteria (LAB) is known to improve the growth performance and prevent diarrheal and respiratory diseases by stimulating the immune system in juvenile calves. Most of the immunostimulation by LAB is achieved by their cell wall components, and therefore we evaluated the immunostimulation of the cell preparation of Enterococcus faecalis strain EC-12 (EC-12) in juvenile calves in a clinical field. Twenty-nine 1-week old calves were used. Fourteen calves were administered 0.2% (w/w) of an EC-12 preparation that supplemented a milk replacer, and other calves were not supplemented. Feces and serum was collected at day 0, 7 and 49 after the administration to measure the IgA and IgG concentration. The fecal IgA concentration was increased by EC-12 administration at day 49, and the serum IgA concentration was also increased at day 7. These results suggested that oral administration of EC-12 in juvenile calves might have an immunostimulatory effect and provide earlier recovery of IgA levels in mucosal immunity.

DOI： 10.1111/j.1740-0929.2008.00621.x

Web of Science

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Language：English   Publisher：KARGER  

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Although there have been many fascinating studies on cryptdins, the information for each cryptdin isoform was not completely provided. In this study, the postnatal changes in the gene expression of cryptdin 1-6 were evaluated, and the patterns of change were compared between conventional and germ-free mice. Two patterns of postnatal change were observed: gene expression of cryptdins 1, 3 and 6 increased gradually, and that of cryptdins 2 and 5 increased rapidly. Gene expression of cryptdin 4 increased gradually in the ileum but rapidly in the jejunum. Conventional mice showed significantly higher gene expression for all isoforms than germ-free mice. Interestingly, the difference in the gene expression for cryptdin 2, 4 and 5 between the jejunum and ileum seemed to be increased by the presence of the luminal bacteria. The results indicate that cryptdin isoforms develop differently depending on the isoform type, and that the gene expression of all cryptdin isoforms was affected by the presence of the luminal bacteria.

DOI： 10.1111/j.1574-695X.2008.00390.x

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