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To investigate whether the oral intake of slowly digestible α-glucan (SDG) could have a trophic (i.e., thickening) effect on their ileal mucosae, for 10 d, rats were given control (non-SDG), 10% isomaltodextrin (IMD) or 10% resistant maltodextrin (RMD) diets. In addition, experimental rat groups were further divided into two groups each and their diets either had or had not 1% sodium carboxymethylcellulose (CMC) added as a thickening agent. In the jejuna and the ilea, compared with control rats, the villus length and the mucosal thickness, but not the crypt depth, were significantly greater in the RMD-fed rats, with the trophic effect being weaker in the IMD-fed rats than in the RMD-fed rats. The colonic crypt depth was significantly greater in SDG groups than in the control group. The concentration of plasma glucagon-like peptide (GLP)-2 in the portal veins of the RMD group but not the IMD group was significantly higher than in the control group, with no effect of CMC supplementation on its concentration. The concentrations of cecal short-chain fatty acids did not significantly increase with SDG supplementation except for propionate concentration of the IMD-supplemented rats, compared with those in the control rats. We concluded that SDGs, especially RMD, thickened the mucosae of the rat distal small intestines. In particular, this effect of RMD but not IMD could have resulted from increased glucose available as a secretagogue of the trophic hormone GLP-2, in the ileum.

SCFA increase serotonin (5-hydroxytryptamine, 5-HT) synthesis and content in the colon in vitro and ex vivo, but little is known in vivo. 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 (Tph1), chromogranin A (Chga), Slc6a4 and monoamine oxidase A (Maoa) 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 Bifidobacterium pseudolongum 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 B. pseudolongum (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 B. pseudolongum could contribute to the reduction of 5-HT content in the colonic mucosa via diminishing EC cells.

Pectin enhances mucin secretion in the rat small intestine. However, what structural features of pectin to stimulate mucin secretion remain unclear. The study aimed to clarify active constituents of pectin using a human goblet cell line, HT29-MTX. Various pectins at 100 mg/L commonly stimulated MUC5AC secretion, irrespective of their differences in molecular size, plant origin and degree of methoxylation, whereas other dietary fiber materials at 100 mg/L did not show any effects, except fucoidan. Hairy region concentrate (HRC) and its further fractions (F1-F3) were prepared by polygalacturonase treatment of citrus pectin and successive anion exchange chromatography. Neutral sugars, such as galactose and arabinose were enriched in these fractions. HRC and F1-F3 at 30 mg/L significantly increased MUC5AC secretion, which were 3 times more potent compared with a starting material (citrus pectin). Further, a dose-dependent study showed that F1 significantly increased MUC5AC secretion from at 0.3 mg/L, much stronger than that of mucin-secretagogue lipopolysaccharides. Rats consumed 5% apple pectin diet showed significant increases of luminal mucin contents and Muc2 expression in the small intestine, while the luminal mucin contents in rats consumed 1.5% HRC diet were increased by 24% compared to those in rats consumed control diet, but the difference did not reach significant. Thus, HRC is supposed to be active constituents of mucin-secretory effect of pectin in vitro. At present, however, the effect of HRC has not been verified in vivo.

Although lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein mainly produced by hepatocytes, it has also been proposed to be a pro-inflammatory adipokine. Obesity and the consumption of a high-fat diet (HFD) are reportedly associated with elevated levels of LPS in plasma and free fatty acids (FFAs) in white adipose tissue (WAT). We examined whether circulating LPS or local FFAs are responsible for the HFD-induced increase of LBP in WAT. Male C57BL/6J mice were fed either a normal-fat diet (NFD) or an HFD. The mRNA levels in the liver and mesenteric WAT (mWAT), total FFA content in mWAT, and LBP and LPS concentrations in plasma were determined. The Lbp mRNA level in mWAT was higher in mice fed the HFD than in those fed the NFD for 3, 7, or 28 days or 14 weeks, whereas the hepatic Lbp mRNA level did not diller between the groups. The Lbp mRNA level in mWAT was also increased by the HFD in germ-free mice, which do not have gut microbiota, the source of LPS. The plasma LPS level did not show a significant correlation with the mWAT Lbp mRNA level. The total FFA content in mWAT was higher in mice fed the HFD than in those fed the NFD and positively correlated with the Lbp mRNA level. Supplementation with palmitic acid increased the Lbp mRNA level in 3T3-L1 adipocytes. We propose that local FFAs, but not circulating LPS, are the trigger for increased Lbp expression in mWAT of mice fed the HFD.

To investigate whether oral intake of highly branched α-glucan isomaltodextrin (IMD) could stimulate ileal glucagon-like peptide-1 (GLP-1) secretion, we examined (1) the digestibility of IMD, (2) the digestion and absorption rates of IMD, in rat small intestine and (3) portal GLP-1 concentration in rats given IMD. In Expt 1, ileorectostomised rats were given a 3 % IMD diet for 10 d. Separately, a 16-h in vitro digestion of IMD, using porcine pancreatic α-amylase and brush-border membrane vesicles from rat small intestine, was conducted. In Expt 2, upon 24-h fasting, rats were given any of glucose, IMD and high-amylose maize starch (HAMS) (1 g/kg of body weight). In Expt 3, caecectomised rats were given 0·2 % neomycin sulphate and a 5 % IMD diet for 10 d. The in vivo and in vitro digestibility of IMD was 70–80 %. The fraction of IMD digested in vitro for the first 120 min was 67 % of that in maize starch. The AUC for 0–120 min of plasma glucose concentration was significantly lower in HAMS group and tended to be lower in IMD group than in the glucose group. Finally, we also observed that, when compared with control rats, glucose of IMD significantly stimulated and improved the concentration of portal active GLP-1 in antibiotic-administered, caecectomised rats. We concluded that IMD was slowly digested and the resulting glucose stimulated GLP-1 secretion in rat small intestine. Oral delivery of slowly released IMD glucose to the small intestine probably exerts important, yet unknown, physiological effects on the recipient.

We investigated whether non-digestible saccharide fermentation-derived hydrogen molecules (H2) in rat colon could improve the in vivo reduction–oxidation (redox) balance via regeneration of α-tocopherol, by assessing their effect on hydroxyl radicals, the α-tocopherol concentration and the redox balance. In Expt 1, a Fenton reaction with phenylalanine (0 or 1·37 mmol/l of H2) was conducted. In Expt 2, rats received intraperitoneally maize oil containing phorone (400 mg/kg) 7 d after drinking ad libitum water containing 0 or 4 % fructo-oligosaccharides (FOS) (groups CP and FP, respectively). In Expt 3, rats unable to synthesise ascorbic acid drank ad libitum for 14 d water with 240 mg ascorbic acid/l (group AC), 20 mg of ascorbic acid/l (group DC) or 20 mg of ascorbic acid/l and 4 % FOS (group DCF). In the Fenton reaction, H2 reduced tyrosine produced from phenylalanine to 72 % when platinum was added and to 92 % when platinum was excluded. In Expt 2, liver glutathione was depleted by administration of phorone to rats. However, compared with CP, no change in the m-tyrosine concentration in the liver of FP was detected. In Expt 3, net H2 excretion was higher in DCF than in the other rats after 3 d of the experiment. Furthermore, the concentrations of H2 and α-tocopherol and the redox glutathione ratio in perirenal adipose tissue of rats were significantly higher in DCF than in DC. To summarise, in rat colon, fermentation-derived H2 further shifted the redox balance towards a more reducing status in perirenal adipose tissue through increased regeneration of α-tocopherol.

SCFA increase serotonin (5-hydroxytryptamine, 5-HT) synthesis and content in the colon in vitro and ex vivo, but little is known in vivo. 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 (Tph1), chromogranin A (Chga), Slc6a4 and monoamine oxidase A (Maoa) 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 Bifidobacterium pseudolongum 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 B. pseudolongum (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 B. pseudolongum could contribute to the reduction of 5-HT content in the colonic mucosa via diminishing EC cells.

The effects of fructo-oligosaccharides (FOS) on gut-barrier function are still controversial in human and animal studies. Diet conditions would be a major factor for the controversy in animal studies. We fed rats a semi-purified (SP) or a non-purified diet (NP) with or without FOS (60 g/kg diet) for 9 (experiment 1) or 10 d (experiment 2). We assessed microbial fermentation, gut permeability, and inflammatory responses in the cecum (experiment 1), and mucus layer in the cecum, intestinal transit time and microbiota composition (experiment 2). FOS supplementation induced a very acidic fermentation due to the accumulation of lactate and succinate in SP, while short-chain fatty acids were major products in NP. Gut permeability estimated by urinary chromium-EDTA excretion, bacterial translocation into mesenteric lymph nodes, myeloperoxidase activity, and expressions of the inflammatory cytokine genes in the cecal mucosa were greater in SP+FOS than in SP, but these alterations were not observed between NP and NP+FOS (experiment 1). FOS supplementation destroyed the mucus layer on the epithelial surface in SP, but not in NP. Intestinal transit time was 3-fold longer in SP+FOS than in SP, but this was not the case between NP and NP+FOS. Lower species richness of cecal microbiota was manifest solely in SP+FOS (experiment 2). These factors suggest that impact of FOS on gut permeability and inflammatory responses in the cecal mucosa quite differs between SP and NP. Increased gut permeability in SP+FOS could be evoked by the disruption of the mucus layer due to stasis of the very acidic luminal contents.

To investigate whether oral intake of highly branched α-glucan isomaltodextrin (IMD) could stimulate ileal glucagon-like peptide-1 (GLP-1) secretion, we examined (1) the digestibility of IMD, (2) the digestion and absorption rates of IMD, in rat small intestine and (3) portal GLP-1 concentration in rats given IMD. In Expt 1, ileorectostomised rats were given a 3 % IMD diet for 10 d. Separately, a 16-h in vitro digestion of IMD, using porcine pancreatic α-amylase and brush-border membrane vesicles from rat small intestine, was conducted. In Expt 2, upon 24-h fasting, rats were given any of glucose, IMD and high-amylose maize starch (HAMS) (1 g/kg of body weight). In Expt 3, caecectomised rats were given 0·2 % neomycin sulphate and a 5 % IMD diet for 10 d. The in vivo and in vitro digestibility of IMD was 70-80 %. The fraction of IMD digested in vitro for the first 120 min was 67 % of that in maize starch. The AUC for 0-120 min of plasma glucose concentration was significantly lower in HAMS group and tended to be lower in IMD group than in the glucose group. Finally, we also observed that, when compared with control rats, glucose of IMD significantly stimulated and improved the concentration of portal active GLP-1 in antibiotic-administered, caecectomised rats. We concluded that IMD was slowly digested and the resulting glucose stimulated GLP-1 secretion in rat small intestine. Oral delivery of slowly released IMD glucose to the small intestine probably exerts important, yet unknown, physiological effects on the recipient.

We investigated whether non-digestible saccharide fermentation-derived hydrogen molecules (H2) in rat colon could improve the in vivo reduction-oxidation (redox) balance via regeneration of α-tocopherol, by assessing their effect on hydroxyl radicals, the α-tocopherol concentration and the redox balance. In Expt 1, a Fenton reaction with phenylalanine (0 or 1·37 mmol/l of H2) was conducted. In Expt 2, rats received intraperitoneally maize oil containing phorone (400 mg/kg) 7 d after drinking ad libitum water containing 0 or 4 % fructo-oligosaccharides (FOS) (groups CP and FP, respectively). In Expt 3, rats unable to synthesise ascorbic acid drank ad libitum for 14 d water with 240 mg ascorbic acid/l (group AC), 20 mg of ascorbic acid/l (group DC) or 20 mg of ascorbic acid/l and 4 % FOS (group DCF). In the Fenton reaction, H2 reduced tyrosine produced from phenylalanine to 72 % when platinum was added and to 92 % when platinum was excluded. In Expt 2, liver glutathione was depleted by administration of phorone to rats. However, compared with CP, no change in the m-tyrosine concentration in the liver of FP was detected. In Expt 3, net H2 excretion was higher in DCF than in the other rats after 3 d of the experiment. Furthermore, the concentrations of H2 and α-tocopherol and the redox glutathione ratio in perirenal adipose tissue of rats were significantly higher in DCF than in DC. To summarise, in rat colon, fermentation-derived H2 further shifted the redox balance towards a more reducing status in perirenal adipose tissue through increased regeneration of α-tocopherol.

The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases (IBDs); however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin not only establishes a first-line barrier against pathogens, but also serves as a niche for microbial growth. We hypothesized that dysbiosis may cause abnormal mucin utilization and microbial metabolic dysfunction. To test this hypothesis, we analyzed short-chain fatty acids (SCFAs) and mucin components in the stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn’s disease (CD) patients from Japan. The levels of n-butyrate were significantly lower in the stools of both the CD and UC patients than in those of the healthy subjects. Correlation analysis identified 7 bacterial species positively correlated with n-butyrate levels, among which the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans act as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in the colonic lamina propria. Importantly, the availability of mucin-associated O-glycans to the microbiota was significantly reduced in n-butyrate-deficient UC patients. Taken together, our findings highlight the biological significance of the symbiosynthesis pathway in the production of n-butyrate, which maintains gut immune homeostasis.

Using barley cultivars differing widely in β-glucan content, we aimed to determine their effects on small intestinal macronutrient digestion in 24 ileorectostomised rats. The rats were fed 1 of 4 experimental diets, each containing a different barley variety, for 11 d. The diets had a content of 0, 2.1, 2.6 and 4.3 g of β-glucan/100 g. Feed intake and faecal excretion of fat, protein, starch, and non-starch polysaccharides were determined in the final 5 d of the study and apparent macronutrient digestibility calculated. Higher dietary levels of β-glucan (2.6% and 4.3%) lowered feed intake (by 15 and 19%, respectively) but final body weight was only lowered by the 4.3% β-glucan diet relative to rats fed the 0% β-glucan diet (all ps

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.

OBJECTIVES: Intestinal mucins escape digestion and enter the large bowel where they are degraded by microbiome. To what extent and how mucins impact large-bowel physiology remain unclear. This study examined the large-bowel fermentation characteristics of mucins and mucin-derived O-glycan sugars and whether mucins and N-acetylglucosamine (GlcNAc) affect gut immunity. METHODS: Mucin secretion from the terminal ileum was determined from feces of ileorectostomized male Wistar rats (age 6 weeks) fed AIN76-based control diet (CD) for 15 d (Expt. 1). Normal male Wistar rats (age 6 weeks; 4 weeks for Expt. 4) were fed CD ± porcine stomach mucin (PM) at 6 or 12g/kg diet, equivalent to 1.5 and 3 times daily mucin secretion, for 14 d (Expt. 2); CD ± GlcNAc, fucose, or N-acetylneuraminic acid at 10g/kg diet for 14 d (Expt. 3); or CD ± PM (15 g/kg diet) or GlcNAc (10 g/kg diet) for 29 d (Expt. 4). Short-chain fatty acids (SCFAs), microbial composition, and cecal O-glycan content were assessed. IgA(+) plasma and regulatory T cells and inflammatory cytokine expression in the cecum were evaluated (Expt. 4). RESULTS: Daily mucin secretion corresponded to 43.2 mmol of O-glycans. PM was efficiently fermented in the cecum, as evidenced by comparable amounts of cecal O-glycans between groups. PM-fed rats harbored more mucin-degrading bacteria. Cecal SCFA concentrations, particularly n-butyrate, were higher in 12g/kg PM diet-fed rats versus CD (P

Background: The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases (IBDs); however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin not only establishes a first-line barrier against pathogens, but also serves as a niche for microbial growth. Methods: To elucidate relationships between dysbiosis, abnormal mucin utilization, and microbial metabolic dysfunction, we analyzed short-chain fatty acids (SCFAs) and mucin components in the stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn's disease (CD) patients from Japan. Findings: The levels of n-butyrate were significantly lower in the stools of both the CD and UC patients than in those of the healthy subjects. Correlation analysis identified 7 bacterial species positively correlated withn-butyrate levels, among which the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans act as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in the colonic lamina propria. Importantly, the availability of mucin-associated O-glycans to the microbiota was significantly reduced inn-butyrate-deficient UC patients. Interpretation: Our findings demonstrated that mucin O-glycans facilitate symbiosynthesis of n-butyrate by gut microbiota. Abnormality in the mucin utilization may lead to decreased n-butyrate production in UC patients. Funding Statement: This study was supported by grants from the Japan Society for the Promotion of Science (#16H01369, 17KT0055, and 18H04680 to KH), Health Labour Sciences Research Grant (KH and JK), AMED-Crest (#16gm0000000h0101, 17gm1010004h0102, and 18gm1010004h0103 to KH and JK), AMED (#18ek0109303h0001 to KH and JK), Yakult Foundation (KH), Keio Gijuku Academic Development Funds (KH), The Aashi Grass Foundation, and The Canon Foundation (JK). Declaration of Interests:The authors have declared that no competing interests exist. Ethics Approval Statement: The protocol was approved by the ethics committees of Osaka University (#13165-2), The University of Tokyo (#25-42-1122), Keio University (#150421-1), Shizuoka University (#14-11), and NIBIOHN (#72) prior to subject inclusion. All animal experiments were performed according to protocols approved by the Animal Use Committees of Keio University and Shizuoka University. The rats and mice were maintained in accordance with the guidelines for the care of laboratory animals of Shizuoka University and Keio University, respectively.

Background: The mechanism underlying transient increases in immunoglobulin (Ig) A concentrations in the cecal contents of rats fed fructo-oligosaccharide (FOS) is unclear.Objective: This study was designed to test whether increased IgA concentrations represent one aspect of the inflammatory response to increased permeability induced by FOS in the cecum.Methods: Seven-week-old male Wistar rats were fed a fiber-free semipurified diet (FFP) with or without supplemental FOS (60 g/kg diet) for 9 or 58 d [experiment (expt.) 1], 7 d (expt. 2), or 7 or 56 d (expt. 3). In addition to measuring IgA concentrations in cecal content, we assessed gut permeability, inflammatory responses (expt. 1), the number of IgA plasma cells in the cecal lamina propria, polymeric Ig receptor (pIgR) expression in the cecal mucosa (expt. 2), and the condition of the cecal mucus layer (expt. 3).Results: The cecal IgA concentration in the FOS-fed rats was 15-fold higher than that of the rats fed FFP for 9 d (P < 0.05). Gut permeability estimated by urinary chromium-EDTA excretion, bacterial translocation to mesenteric lymph nodes, myeloperoxidase activity, and expression of inflammatory cytokine genes in the cecal mucosa was greater in the FOS-fed rats than in the rats fed FFP for 9 d. These effects were not observed in the rats fed FOS for 58 d (expt. 1). Accompanying the higher cecal IgA concentration, pIgR protein and the number of IgA plasma cells in the cecal mucosa were higher in the FOS-fed rats than in the rats fed FFP for 7 d (expt. 2). Destruction of the mucus layer on the epithelial surface, as evidenced by Alcian blue staining in the cecal sections, was evident in the rats fed FOS for 7 d, but the mucus layer appeared normal in the rats fed FOS for 56 d (expt. 3).Conclusions: These findings suggest that transient increases in cecal IgA concentrations induced by FOS in rats are associated with mucosal inflammation in response to increased gut permeability; these are presumably evoked by disruption of the cecal mucus barrier. The observed responses could contribute to the maturation of the gut immune system.

We examined the effects of fructo-oligosaccharides (FOS) on IgA and mucin secretion in the rat cecum after different ingestion periods. Rats were fed a control diet or a diet containing FOS for 1, 2, 4, and 8 wk. FOS ingestion greatly increased IgA and mucin concentrations at 1 and 2 wk, but the effects were disappeared or attenuated at 4 and 8 wk. After 1 wk, FOS induced higher lactobacilli and lactate concentrations and lower cecal pH in the cecum, but the alterations were moderated with the prolonged ingestion accompanying with increasing short-chain fatty acid concentrations. At 1 and 2 wk, FOS increased IgA plasma cells and polymeric immunoglobulin receptor expression in the cecal mucosa and strongly depressed fecal mucinase activities related to the lower cecal pH. These findings may explain the FOS-induced early elevation of IgA and mucin. Clearly, FOS effects on IgA and mucin secretion considerably differ depending on the ingestion period.

The study aimed to examine the effects of insoluble and soluble fibers on mucin sialylation and sulfation in the small intestine. First, diets containing soluble [konjac mannan (KM), psyllium, or guar gum; 50 g/kg) or insoluble (polystyrene foam, wheat bran, or cornhusk; 80 g/kg) fiber were fed to rats for 13 d. The fiber-fed groups had more goblet cells in the ileum than the fiber-free control group. High-iron diamine/alcian blue staining showed more sialylated mucin-producing cells in the fiber-fed groups than in the control, whereas sulfated mucin-producing cells were fewer (insoluble fibers) or unchanged (soluble fibers). Second, feeding KM (50 g/kg) and beet fiber (BF) (80 g/kg) diets for 7 d yielded a higher ileum Siat4C expression than the control, but Gal3ST2 and Gal3ST4 expression was comparable. Luminal mucin content correlated with sialic acid (r = 0.96; P < 0.001) or sulfate (r = 0.62; P < 0.01), but the slope of the sialic acid-derived equation was greater than that of the sulfate-derived equation, indicating a preferred increase in sialylated mucins. Third, rats were fed the control diet for 10 d while receiving antibiotic treatment. Analysis of the luminal mucin showed that sialylated mucins were more vulnerable to bacterial degradation than sulfated mucins. Finally, a study of bromo-deoxyuridine incorporation in rats fed a BF diet indicated that goblet cell proliferation accompanied by increased sialylated mucin appeared to be related to accelerated ileal epithelial cell migration. We conclude that intestinal goblet cell responses to insoluble and soluble fibers are characterized by increases in sialylated mucin production.

Few common carbohydrate epitopes consisting of terminal beta-(1,2)-xylose and/or alpha-(1,3)-fucose residues are shared by a variety of glycoproteins from plants, insects and parasitic worms, termed cross-reactive carbohydrate determinant (CCD), and frequently recognized by IgE antibodies of patients with food and/or respiratory allergy, though clinical relevancy of such CCD-specific IgE is still controversial. Attention has also been focused on CCDs from the undesired post-translational modification of recombinant therapeutic proteins produced by transgenic plants and insects. In the present study, to clarify immunogenic potentials of CCD-bearing glycoproteins, the antibody response to a model plant glycoprotein, horseradish peroxidase (HRP) was investigated in a mouse model. C3H/He mice were immunized with HRP plus Al(OH)(3) or Freund's adjuvant, and IgG and IgE responses to CCDs in addition to HRP were analyzed by ELISA using some distinct glycoproteins with known N-glycan structures. IgE response to HRP was induced remarkably, whereas that to CCD was weaker and delayed. Moreover, apparent ratio of the CCD-specific antibodies to HRP-specific ones tended to be higher in IgG2a and IgG2b isotypes than IgG1, IgG3 and IgE. In contrast to rabbit antibodies, the CCD-specific antibodies from the mice gave poor reactivity with bromelain and honeybee phospholipase A2, suggesting the critical role of both beta-(1,2)-xylose and alpha-(1,3)-mannose in the CCD-recognition by the mouse antibodies. Moreover, the mouse antibodies showed weaker cross-reactivity to pollen- and insect-derived glycoproteins than the rabbit ones. Thus, in this mouse model, not only IgE but also IgG2 antibody responses to CCDs were induced by immunizing with a CCD-bearing glycoprotein, suggesting that CCDs affected not only Th2-type but also Th1-type antibody response at least in C3H/He mice.

Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.

Summary Background NC/Nga mice are known to show a spontaneous outbreak of atopic-like dermatitis accompanied by a marked elevation in serum IgE levels when reared in a conventional environment. The specific effects of such a strong serum IgE response on the development of the dermatitis and specific antigens recognized by the IgE antibodies are still uncertain. Objective and methods To characterize the IgE of NC/Nga mice, we established IgE-secreting hybridoma clones from spleen cells of NC/Nga mice spontaneously developing dermatitis and identified variable-region genes and specific antigens of the IgE monoclonal antibodies (mAbs). Serum polyclonal IgE, as well as IgG1 and IgG2a, specific for the identified antigen were also analysed. Results Four IgE-producing hybridoma clones were established. Variable-region nucleotide sequences of the IgE mAbs showed that these clones did not necessarily share common germline gene segments (V, D or J) for each variable region, and several somatic mutations had occured in the V gene segments. Through antigen screening, histone H3 was identified to be an auto-antigen recognized by three of the four IgE mAbs. Serum IgE as well as IgG1 specific for histone H3 were almost undetectable in 6-week-old mice, but rapidly increased by 10–12 weeks of age. This age-dependent increase in the serum anti-histone H3 IgE was roughly in parallel with the onset of dermatitis, and slightly preceding total IgE elevation. The serum-specific IgE level correlated well with a dermatitis-severity score of each mouse at 12–16 weeks of age, and weakly with the severity of ear erosion of each mouse over 28 weeks of age. Furthermore, immunologically detectable histone-H3 antigens were observed in skin tissue sections from the dermatitis sites. Conclusion In NC/Nga mice, anti-histone H3 auto-antibodies may contribute, at least in part, to the considerably elevated serum IgE and might play some roles in the development and exacerbation of dermatitis.

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII(250-270) peptide (residues 250-270) (GluA-4XCII(250-270)) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII ( 250-270 ), but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII(250-270) fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII(250-270) peptide was immunochemically estimated to be about 1 microg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 microg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.

We histologically examined the effects of elemental diet (ED) on the goblet cell profile in the rat small intestine. The sulfomucin goblet cells were predominant throughout the small intestine in the control group, while sialomucin goblet cells were manifest in the duodenum and jejunum in the ED group. Next, we investigated the possible relevance of luminal osmolality to the goblet cell profile. Gastric osmolality in the ED group was within the physiological range. Meanwhile, ingestion of high glucose diet elevated gastric osmolality and increased the number of sialomucin goblet cells in the duodenum and jejunum. Further, it turned out that the lower sulfur contents in ED was not related to the unique goblet cell profile by ED ingestion. It is inductively suggested that the influx of high concentrations of low molecular nutrients into the small intestine could be associated with the goblet cell alteration, but the alteration was not necessarily due to the changes in the gastric osmolality by ED ingestion.

A new aspect of protein immunogenic and allergenic properties has become important recently, when there is a higher chance that our immune system will be exposed to novel protein antigens and/or familiar protein antigens with an unprecedented high frequency and large amount. These proteins are innocuous, nontoxic, and noninvasive by themselves, and include various natural proteins from the environment and recombinant proteins from industry. The technical term allergenic has been used for such proteins and their abilities to induce specific IgE production and to cross-link IgE/Fc epsilonRI on the surface of mast cells and basophiles have been recognized. As for the environmental proteins, some physicochemical properties (solubility, stability, and permeability across a mucosal epithelium) of the proteins indirectly play important roles in their allergenic potential because they do not originate from invasive pathogens as vehicles. Indeed, several lines of experimental evidences have been accumulated indicating that all proteins are absorbed across mucosal epithelia by transcellular transport and/or through interstitial spaces among the epithelial cells but not at equal levels. Some animal models have been established for natural sensitization to some allergenic proteins by feeding or intragastric administration without an adjuvant and, in a few cases, some symptoms resembling human allergy and even anaphylaxis have been induced by oral challenge with the proteins. Sometimes, even to self-proteins, the immunogenic or allergenic potential is given by post-translational modifications and possibly by unknown structural/conformational alterations, when they are exogenous self-proteins, such as recombinant human proteins for drug use. Despite the accumulation of knowledge and the progress in analytical technology on protein allergenicity, it is still crucial to predict the allergenic potential of novel and unused proteins. However, some animal models are applicable for assessing the relative allergenic potential of processed proteins in comparison with that of native proteins in preclinical studies.

We examined the effect or not of an antibiotic treatment on dietary fiber-induced goblet cell proliferation in the rat ileum. The number of goblet cells in the ileum increased when rats consumed dietary fiber. However, this effect was maintained with a concurrent treatment of antibiotics, suggesting that the fiber effect on goblet cell response would remain irrespective of a bacterial component such as endotoxin.

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

Pollen proteins from several grass species have been identified and characterized as causative allergens in grass pollinosis. In contrast, allergenic potential of pollen proteins from rice, which belongs to the same Poaceae family, has not well been investigated, despite that a few clinical cases have been reported on rice-pollen allergy. In this study, to characterize expression and allergenic potential of pollen proteins from rice (Oryza sativa, ssp. japonica), rice putative proteins for β-expansin (EXP), a Ca(2+)-binding protein (CBP)/polcalcin, extensin (EXT), profilin (PRF) and polygalacturonase (PGA) retrieved from a rice complete cDNA database were prepared as recombinant proteins, and the antibodies to these recombinant proteins were obtained. Immuno-blotting and immuno-histological analyses showed that rice putative EXP, EXT and PGA were expressed abundantly in anther tissue and pollen granules and immuno-cross reactive with pollen proteins from timothy grass. ELISA and immuno-dot blotting analyses using serum specimens from allergic patients showed that majority of the specimens was positive in the IgE-binding to EXP and EXT, but weakly to PGA and almost negative to PRF. EXP and EXT were suggested to be potentially allergenic in the rice-pollen allergy as well as the grass pollinosis.

This study aimed to examine the mechanism for differential effects of low- (LPPS) and high-phosphorus (HPPS) potato starches and high-amylose cornstarch (HACS) on rat cecal fermentation, the n-butyrate proportion in particular. In ileorectostomized rats, the in vivo resistant starch (RS) contents were determined to be 66% (LPPS), 66% (HPPS) and 36% (HACS), but the carbohydrate/nitrogen (C/N) ratios of the ileal digesta were comparable among the respective starch diets. In intact rats fed diets including similar amounts of RS, the cecal n-butyrate proportions in the LPPS- and HPPS-fed rats were equally higher than in the HACS-fed rats. The cecal starch contents were fivefold greater in the LPPS- and HPPS-fed rats than in the HACS-fed rats. The results suggest that potato starches and HACS are not equivalent n-butyrate producers in the rat cecum and that the slower fermentation rate of potato starches relative to HACS might be responsible for the higher n-butyrate proportion.

Background: Digestion-resistant dextrin derivatives (DRDDs), including resistant maltodextrin (RM), polydextrose, and resistant glucan (RG), have been developed as low-energy foods. However, data on the resistance of DRDDs to small-intestinal digestion are scarce.Objective: We sought to determine the site and extent of DRDD breakdown in the rat intestine and to predict its energy contributions.Methods: In vitro small-intestinal resistance of DRDDs was evaluated by the AOAC method for dietary fiber measurement and by artificial digestion with the use of pancreatic α-amylase and brush-boarder membrane vesicles. In vivo small-intestinal resistance of DRDDs was determined from the feces of male ileorectostomized Sprague-Dawley rats fed a control diet or a diet containing one of the DRDDs at 50 g/kg for 9 d (period 1) and then for 10 d (period 2), during which they received 1 g neomycin/L in their drinking water. Separately, male Sprague-Dawley rats were fed the same diets for 4 wk, and the whole-gut recoveries of DRDDs were determined from feces at days 8-10.Results: Small-intestinal resistances determined in vitro by artificial digestion (RM: 70%; polydextrose: 67%; RG: 69%) were lower than those measured by the AOAC method (RM: 92%; polydextrose: 80%; RG: 82%). In the ileorectostomized rats, fecal dry-matter excretions were consistently greater in the DRDDs than in the control. The small-intestinal resistances of the DRDDs were 68%, 58%, and 62% in period 1 and 66%, 61%, and 67% during period 2 for RM, polydextrose, and RG, respectively. The resistances did not differ among the DRDDs at either time. In the normal rats, food intakes and body weight gains did not differ among the groups. The whole-gut recovery of RM (13%) was lower than that of polydextrose (33%) and RG (29%), which did not differ.Conclusions: DRDDs were more digestible in the rat small intestine than the AOAC method. The energy contribution from small-intestine digestibility, not just large-bowel fermentability, must be considered in determining the energy contribution of DRDDs. Whether humans respond similarly needs to be tested.

We used recombinant rat dectin-1 proteins to newly establish sandwich ELISA for determining barley β-glucan (BβG). The ELISA method had a working range of 15–4,000 µg/L. Plasma BβG was detectable up to 24 h after an intravenous administration of BβG (1 mg/kg of body weight) to rats. This method may be an effective tool for investigating the immune modulatory effects of BβG.

Generally, soluble fibers increase small intestinal mucin secretion by increasing the number of goblet cells in a viscosity-dependent manner. The present study aimed to examine the mechanism by which low-methoxyl pectin (LPC) affects mucin secretion in the small intestine. First, diets containing 50 g/kg of low-viscosity fiber (LPC, gum arabic, guar gum, low-molecular konjac mannan, arabinogalactan, sodium alginate) or high-molecular konjac mannan (KMH) were fed to Wistar rats for 10 d. Luminal mucin was greater in the LPC and KMH groups than in the fiber-free control group, but only the KMH group had more goblet cells in the ileum compared with the other groups. Next, Sprague-Dawley rats were fed LPC, KMH, or high-methoxyl pectin (HPC) diets (50 g/kg) for 10 d. The KMH and LPC groups, but not the HPC group, had greater luminal mucin than the control group, whereas jejunum Muc2 expression was higher only in the LPC group. Sprague-Dawley rats fed the LPC diet for 1 or 3 d had greater luminal mucin and jejunum Muc2 expression than those fed the control diet. In vitro studies using HT-29MTX cells showed that, of the various fibers studied, only LPC and HPC affected mucin secretion. Finally, Wistar rats were fed the LPC diet with or without neomycin in drinking water for 10 d; neomycin treatment did not compromise the effect of LPC on mucin secretion. We conclude that LPC does not affect the number of goblet cells but can interact directly with the epithelium and stimulate small intestinal mucin secretion.

Phagocytes engulf pathogenic microbes, kill them and degrade their cellular macromolecules by hydrolytic enzymes in phagolysosomes. However, such enzymes are unable to degrade some microbial polysaccharides, and fate of such indigestible polysaccharides in phagocytes remains uncertain. Using the extracellular domain of Dectin-1 as β-glucan-specific probes, we succeeded in detection of soluble and Dectin-1-reactive β-glucan discharged from mouse RAW 264.7 and human THP-1 macrophage cell lines as well as mouse peritoneal macrophages, which had phagocytized insoluble β-glucan particles. The RAW 264.7 cell culture-supernatant containing the discharged β-glucan stimulated naïve RAW 264.7 cells, resulting in the induction of cytokine expression. Such discharge of Dectin-1-reactive β-glucan from macrophage cells was inhibited by either NADPH oxidase inhibitors (apocynin and diphenylene iodonium) or radical scavengers (N-acetyl cysteine and MCI-186). Moreover, reactive oxygen species (ROS) produced by a Cu(2+)/ascorbic acid system solubilized insoluble β-glucan particles in vitro, and a part of the solubilized β-glucan was Dectin-1 reactive and biologically active in macrophage activation. The soluble and biologically active β-glucan was degraded further during prolonged exposure to ROS. These results suggest that degraded but Dectin-1-reactive β-glucan is discharged from macrophage cells phagocytizing insoluble β-glucan particles and stimulates not only themselves again but also the other naïve phagocytes, leading to the effective elimination of infecting microbes and the ultimate breakdown and inactivation of metabolically resistant β-glucan.

We evaluated the ameliorating effects of short-chain inulin-like fructans (SIF) with different degrees of polymerization (DP) on the healing stage of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The rats were assigned to 3 groups 10 d after the colitis induction, and fed for 24 d on a control diet or diet including 60 g of DP4 or DP8/kg. The fecal myeloperoxidase (MPO) activity and IgA concentration were monitored every 7 d. The colonic MPO activities and cecal concentrations of organic acids, lactobacilli, bifidobacteria, mucin and IgA were measured at the end of the study. DP4, but not DP8, significantly reduced the colonic inflammation accompanied by higher cecal concentrations of short-chain fatty acids, propionate in particular, and lactic acid-producing bacteria. DP4 therefore accelerated the healing process of TNBS-induced colitis, even when the treatment was initiated after inducing colitis.