Biomedical subjects
Publications and source records attributed to Tadao Saito.
Twenty lactobacilli isolated from human feces were studied for binding to the human blood type B-antigen [Galalpha1-3 (Fucalpha1-2) Gal-] and H-antigen (Fucalpha1-2Gal-] expressed sugar chains in human intestinal mucosa. We found two strains, L. gasseri OLL2755 and L. gasseri OLL2877 that firmly adhere to human B-antigen, and we found L. gasseri OLL2827 bound to the H-antigen.
Samples of milk from a Bryde's whale and a Sei whale contained 2.7 g/100 mL and 1.7 g/100 mL of hexose, respectively. Both contained lactose as the dominant saccharide along with small amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc (3'-N-acetylneuraminyllactose), Neu5Ac(alpha2-6)Gal(beta1-4)Glc (6'-N-acetylneuraminyllactose) and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c). The dominance of lactose in the carbohydrate of these milks is similar to that of Minke whale milk and bottlenose dolphin colostrum, but the oligosaccharide patterns are different from those of these two species, illustrating the heterogeneity of milk oligosaccharides among the Cetacea.
To clarify the crucial role of Toll-like receptor (TLR) 2 and TLR9 in immature gut-associated lymphoid tissues (GALT), we focused on the expression of TLR2 and TLR9 and the immune responses induced by their ligands in the GALT of presuckling newborn swine. Quantitative real-time PCR revealed that TLR2 and TLR9 mRNA were expressed at detectable levels in all tested tissues (heart, thymus, lung, spleen, liver, kidney, skeletal muscle, duodenum, jejunum, ileum, ileal Peyer patches (Pps), and mesenteric lymph nodes (MLN)). In particular, in immature intestinal tissues and GALT, TLR2 and TLR9 mRNA were expressed at higher levels in ileal Pps and MLN than in the duodenum, jejunum, and ileum. We confirmed that the TLR2 and TLR9 proteins were also highly expressed and that their ligands were preferentially recognized by TLR2- or TLR9-expressing cells in the MLN and ileal Pps. Zymosan, CpG2006, and lactic acid bacteria could promote mitogenesis and production of multiple cytokines by the MLN and ileal Pps. In addition, double immunostaining for cytokeratin 18 and either TLR2 or TLR9 revealed that both TLR2 and TLR9 are strongly expressed in the columnar membranous (M) cells. Interestingly, while the apical membrane of the columnar M cells strongly expressed TLR2 protein and preferentially recognized zymosan, both "TLR2 expression on the apical membrane" and "TLR2-mediated zymosan binding" were negligible in neighboring enterocytes. These results indicate that TLR2 and TLR9 allow MLN and ileal Pps to respond to a variety of bacterial components immediately after birth, thereby providing newborns with a host defense system.
Milk of an Asian elephant (Elephas maximus), collected at 11 days post partum, contained 91 g/L of hexose and 3 g/L of sialic acid. The dominant saccharide in this milk sample was lactose, but it also contained isoglobotriose (Glc(alpha1-3)Gal(beta1-4)Glc) as well as a variety of sialyl oligosaccharides. The sialyl oligosaccharides were separated from neutral saccharides by anion exchange chromatography on DEAE-Sephadex A-50 and successive gel chromatography on Bio Gel P-2. They were purified by high performance liquid chromatography (HPLC) using an Amide-80 column and characterized by 1H-NMR spectroscopy. Their structures were determined to be those of 3'-sialyllactose, 6'-sialyllactose, monofucosyl monosialyl lactose (Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc), sialyl lacto-N-neotetraose c (LST c), galactosyl monosialyl lacto-N-neohexaose, galactosyl monofucosyl monosialyl lacto-N-neohexaose and three novel oligosaccharides as follows: Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, and Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. The higher oligosaccharides contained only the type II chain (Gal(beta1-4)GlcNAc); this finding differed from previously published data on Asian elephant milk oligosaccharides.
Adherent lactic acid bacteria (LAB) in the human intestine were investigated using the surface plasmon resonance technique with the biosensor BIACORE-1000. Ninety-three LAB strains were isolated from human feces and evaluated for binding to human blood type-A antigen [GalNAcalpha1-3 (Fucalpha1-2) Gal-: A-trisaccharide] expressed in the intestinal mucosa. Eleven strains showed strong adherence to an A-trisaccharide biotinyl polymer (BP) probe, and slightly or no adherence to a B-trisaccharide BP probe. Four strains with high adherence (high A/B ratio) were selected and their surface layer proteins (SLPs) were evaluated for A-antigen ligand binding using BIACORE. The SLP from L. brevis strain OLL2772 showed a single band at ca. 48 kDa by SDS-PAGE analysis and it had a very strong adherence to the human A-antigen, as shown using an anti-A lectin blocking technique. A partial N-terminal sequence of the band showed strong homology to an S-layer protein of L. brevis ATCC8287T. The probiotic LAB binds to human blood type-A antigen expressed in the intestinal mucosa which may aid in colonization of the gut.
Lactobacillus gasseri OLL2716 has recently been discovered as a probiotic that suppresses the growth of Helicobacter pylori and reduces gastric mucosal inflammation in humans. This has resulted in the development of a new type of probiotic yoghurt 'LG21' in Japan. In our previous study, we found an immunostimulatory AT5ACL oligodeoxynucleotide (AT-ODN) containing a unique core sequence (5'-ATTTTTAC-3') in L. gasseri JCM1131(T). Interestingly, although the AT-ODN does not contain any CpG sequences, it exerts mitogenic activity in B cells and augments Th-1-type immune responses via Toll-like receptor 9. These findings prompted us to identify strong immunostimulatory non-CpG AT-ODNs that contain the 5'-ATTTTTAC-3' motif in the genomic sequence of L. gasseri OLL2716. We identified 280 kinds of AT-ODNs in the L. gasseri OLL2716 genome. Mitogenicity and NF-kappaB gene reporting assays showed that 13 of the 280 AT-ODNs were strongly immunostimulatory when in the TLR9 transfectant. Of these, AT-ODNs LGAT-145 and LGAT-243 were the most potent. With respect to the induction of Th-1-type cytokines, LGAT-243 had the greatest activity and was more potent than the swine prototype, ODN D25. We further found that a six-base secondary loop structure containing a self-stabilized 5'-C...G-3' stem sequence is important for potent immunostimulatory activity. These results show for the first time that AT-ODNs with a specific loop and stem structure are important factors for immunostimulatory activity. Finally, we found that novel strong immunostimulatory non-CpG AT-ODNs exist in the genome of probiotic lactic acid bacteria.
The immunostimulatory oligodeoxynucleotide (ODN) BL07 (5'-GCGTCGGTTTCGGTGCTCAC-3') was identified from the genomic DNA of the probiotic strain Bifidobacterium longum BB536. ODN BL07 stimulated B-lymphocyte proliferation and induced interleukin-12 (IL-12) production in macrophage-like J774.1 cells. ODNs BL07 and BL07S (modified with phosphorothioate backbone) significantly inhibited immunoglobulin E (IgE) production and stimulated interferon-gamma (IFN-gamma) and IL-12 production, but did not affect IL-4 secretion in murine splenic cells of ovalbumin-primed BALB/c mice. These ODNs also significantly inhibited production of IgE in purified murine B cells in the presence of IL-4 and anti-CD40. The results suggest the potential of ODNs BL07 and BL07S in preventing IgE-related immune responses and the possible involvement of ODN BL07 in the antiallergic efficacy of B. longum BB536.
We have reported the antiallergic activities of the immunostimulatory oligodeoxynucleotide (ODN) BL07S, identified from genomic DNA of Bifidobacterium longum BB536 from in vitro and in vivo studies. The present study evaluated the efficiency of ODN BL07S in preventing allergic responses by oral administration. Oral administration of BL07S suppressed serum ovalbumin (OVA)-specific immunoglobulin (Ig) E levels and improved the OVA-specific IgG2a/IgG1 ratio. ODN BL07S increased Th1 cytokine and decreased Th2 cytokine production in splenocytes. These results suggest that immunostimulatory ODNs are potentially associated with the antiallergic effects of probiotics.
Five Lactobacillus strains (2 L. gasseri, 2 L. plantarum and 1 L. reuteri) were cultured in modified MRS medium containing fatty acids (FAs) instead of Tween 80 for 24 h at 37 degrees C, to learn the effect of saturated and unsaturated FAs on the Lactobacillus growth. Free FAs included palmitic (16:0), palmitoleic (c9-16:1), stearic (18:0), oleic (c9-18:1), elaidic (t9-18:1), cis-vaccenic (c11-18:1), vaccenic (t11-18:1), linoleic (c9, c12-18:2), conjugated linoleic (c9, t11- and t10, c12-18:2), alpha-linolenic (c9, c12, c15-18:3), alpha-eleostearic (c9, t11, t13-18:3), eicosapentaenoic (20:5), and docosahexaenoic (22:6) acids. Among free FAs, oleic acid stimulated the growth of all Lactobacillus strains, whereas palmitoleic acid had almost no affect on the Lactobacillus growth. Saturated FAs such as stearic and palmitic acids inhibited or did not affect the Lactobacillus growth. Polyunsaturated FAs such as alpha-linolenic, eicosapentaenoic and docosahexaenoic acids strongly inhibited the Lactobacillus growth at 7.6 x 10(-4) m. Octadecenoic acids such as oleic, elaidic, cis-vaccenic and vaccenic acids remarkably promoted the growth of L. gasseri, regardless of the different double bond positions and configurations. When oleic or cis-vaccenic acid was incubated with L. gasseri, the FAs was transformed to cyclopropane FAs (methyleneoctadecanoic acids) after incorporation into the cells. On the other hand, trans FAs such as elaidic and vaccenic acids incorporated into the cells were not converted to another FAs. Conjugated linoleic and alpha-eleostearic acids having a trans double bond promoted the Lactobacillus growth. The growth of L. gasseri was also stimulated by trans-rich free FAs from hydrogenated canola and fish oils. These results showed that octadecenoic acid and trans FAs had strong promotion activities for the Lactobacillus growth due to their incorporation into membrane lipids.
Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.
Toll-like receptor 2 (TLR2) has been shown to mediate cell signaling in response to microbial cell wall components, such as peptidoglycan, lipoteichoic acid, microbial lipoprotein, and zymosan. In this study, we cloned the swine TLR2 and used it to transfect Chinese hamster ovary K-1 cells. We demonstrated that the swine TLR2-expressing transfectant can bind not only zymosan from yeast cell wall components but also intact lactic acid bacteria, resulting in the activation of nuclear factor-kappaB. These findings suggest that the swine TLR2-expressing transfectant can be very useful for the primary screening of immunobiotic microorganisms.
The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.
Toll-like receptor 9, which is expressed on the surface of antigen presenting cells and which was recently identified in the cytoplasmic follicle, recognizes bacterial CpG oligodeoxynucleotides (ODNs), resulting in the induction of a potent immune response. However, in our previous study, we found that TLR9 potentially recognizes not only CpG ODN but also non-CpG ODN such as AT ODN. Therefore, in the present study, to investigate this possibility, we elucidated the effects of AT ODN on T(H)-1, T(H)-2 type cytokine induction via TLR9 by real-time quantitative PCR analysis and ELISA of the swine TLR9 transfectant. The results demonstrated that the T(H)-1 type cytokines such as interleukin (IL)-12p70 and interferon (IFN)-gamma were strongly induced by AT ODN compared to the unexposed controls, while T(H)-2 type cytokines were not induced. These results indicate that the AT ODN can augment the T(H)-1 immune response, which plays an important role in prevention of allergic responses. Moreover, the swine TLR9 transfectant demonstrated its usefulness for evaluation of immunostimulation by bacterial DNA through the detection of T(H)-1, T(H)-2 type cytokine induction via TLR9 signaling.
Whole cells, cell wall components and some soluble factors from Lactobacillus rhamnosus GG (LGG) are known to invoke immune responses as they interact with animal and human immune cells. In the present study, we found that chromosomal DNA from LGG is a potent inducer of splenic B cell proliferation, CD86/CD69 expression and cytokine production in mice. In the genomic DNA of LGG we discovered TTTCGTTT oligodeoxynucleotide (ODN) ID35, which has a potent activity in a number of immunostimulatory assays. Phosphorothioate backbone is not required for the activity of ID35. The ODN ID35 showed levels of activity comparable with those induced by the murine prototype ODN 1826 in B cell proliferation, CD86/CD69 expression, interleukin (IL)-6, IL-12, IL-18, interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) mRNA expression and IFN-gamma/IL-12p70 protein production assays. Additionally, ID35 appeared to be equally active in both murine and human immune cells. These stimulatory effects are due to TTTCGTTT motif located in the 5' end of ID35. In this study we demonstrate for a first time that, DNA from LGG is a factor of immunobiotic activity. Furthermore, ODN ID35 is the first ODN, with such a strong immunostimulatory activity to be found in immunobiotic bacterial DNA.
The precise distribution and expression of Toll-like receptor (TLR) 9 in gut-associated lymphoid tissues (GALTs) has not been elucidated. In this study, we investigated the expression pattern of TLR9 in adult and neonatal swine GALTs by real-time quantitative PCR, western blot, confocal laser microscopy and flow cytometric analysis. The swine TLR9 gene was preferentially expressed in adult Peyer's patches (Pps) and mesenteric lymph nodes (MLNs), which contained approximately three times higher TLR9 than the spleen. Other tissues exhibited only weak expression of TLR9. In neonatal swine, elevated expression of TLR9 was detected only in MLNs. We firstly showed that highly expressive (TLR9(+)) cells were formed in Pps and MLNs. In addition, TLR9(+) cells were present not only in immune cells such as dendritic cells and B cells but also in follicle-associated epithelia (FAE) including membranous cells (M cells) in Pps. These results suggest that Pps and MLNs provide the host defense with the ability to respond to a variety of bioactive oligonucleotides (ODNs) from bacteria at a conductive site of initial immune responses.
Carbohydrates were extracted from milk of a bearded seal, Erignathus barbatus (Family Phocidae). Free neutral oligosaccharides were separated by gel filtration, anion-exchange chromatography and preparative thin layer chromatography, while free acidic oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows; lactose, 2'-fucosyllactose, lacto-N-fucopentaose IV, difucosyl lacto-N-neohexaose and difucosyl decasaccharide which contained a lacto-N-neohexaose unit as well as an additional Gal(beta1-4)GlcNAc(beta1-3) unit and two residues of non-reducing Fuc(alpha1-2). The acidic oligosaccharides were thought to contain an Neu5Ac(alpha2-6) residue linked to GlcNAc or a sulfate linked to Gal at OH-3. The sialyl oligosaccharides and sulfated oligosaccharides had a lacto-N-neohexaose unit and two non-reducing Fuc(alpha1-2) residues and some of them had in addition one or two Gal(beta1-4)GlcNAc(beta1-3) units. The milk oligosaccharides of the bearded seal were compared to those of the harbour seal, which had been studied previously.
Sialyl oligosaccharides were separated from two samples of Japanese black bear milk by extraction with chloroform/methanol, gel filtration on Bio Gel P-2, ion exchange chromatography on DEAE-Sephadex A-50 and high-performance liquid chromatography (HPLC) on a TSK gel Amido-80 column. They were characterized by (1)H-NMR spectroscopy. The structures of four sialyl oligosaccharides separated from the milk were the following: Neu5Ac(alpha2-3)Gal(beta1-4)Glc. Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]Gal(beta1-4)Glc. Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]Gal(beta1-4)Glc. Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-6)Gal(beta1-4)Glc.
Interleukin-17 (IL-17), initially reported as CTLA-8, is a proinflammatory cytokine produced mainly by activated T cells. In the present study, the cDNA of a swine IL-17 (PoIL-17) gene was cloned from activated neonatal thymocytes, and the recombinant PoIL-17 (rPoIL-17) was biologically characterized. The complete open reading frame (ORF) of PoIL-17 contains 462-bp coding deduced 153 amino acid residues, with a calculated molecular weight of 17.3 kDa. The amino acid sequence showed 72.9%, 64.9%, 64.7%, 60.1%, and 47.4% similarities with that of human, rat, mouse, Herpesvirus saimiri ORF 13, and chicken, respectively. The six cysteine residues conserved over species including the virus were observed in PoIL-17. We successfully prepared the recombinant mature form of PoIL-17 and analyzed its biologic activities for swine splenocytes. RT-PCR analysis revealed a marked upregulation of expression of IL-1beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), and monocyte chemotactic protein-1 (MCP-1) mRNA expression in splenocytes treated with 100 ng/ml rPoIL-17 for 3 h. Furthermore, a swine chemokine, alveolar macrophage-derived neutrophil chemotactic factor II (AMCF-II), which was classified into the CXC subfamily was also augmented in mRNA level. This evidence indicates that recombinat PoIL-17 expressed in Escherichia coli was biologically active and exerted similar effects to those of a human (HuIL-17) and murine IL-17 (MuIL-17). The PoIL-17 mRNA is strongly expressed in the adult heart, skin, and, interestingly, intestinal tissues, including mesenteric lymph nodes but is restricted in neonatal tissues by using real-time quantitative RT-PCR. The gene sequence and biologically active recombinat protein for PoIL-17 will be useful for elucidation of the role of IL-17 in the regulation of intestinal immune responses.