Local free fatty acids trigger the expression of lipopolysaccharide-binding protein in murine white adipose tissue.

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 differ 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.

Hairy Region Concentrate of Pectin Strongly Stimulates Mucin Secretion in HT29-MTX Cells, but to a Lessor Degree in Rat Small Intestine.

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.

The Impact of Fructo-Oligosaccharides on Gut Permeability and Inflammatory Responses in the Cecal Mucosa Quite Differs between Rats Fed Semi-Purified and Non-Purified Diets.

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.

Fructo-oligosaccharide-Induced Transient Increases in Cecal Immunoglobulin A Concentrations in Rats Are Associated with Mucosal Inflammation in Response to Increased Gut Permeability.

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.

Allergenic potential of rice-pollen proteins: expression, immuno-cross reactivity and IgE-binding.

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 ß-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.

Low-methoxyl pectin stimulates small intestinal mucin secretion irrespective of goblet cell proliferation and is characterized by jejunum Muc2 upregulation in rats.

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.

IgG2 dominancy and carbohydrate recognition specificity of C3H/He mouse antibodies directed to cross-reactive carbohydrate determinants (CCDs) bearing beta-(1,2)-xylose and alpha-(1,3)-fucose.

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.