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Naoya Takanashi,
Yohsuke Tomosada,
Julio Villena,
Kozue Murata,
Takuya Takahashi,
Eriko Chiba, Masanori Tohno,
Tomoyuki Shimazu,
Hisashi Aso,
Yoshihito Suda,
Shuji Ikegami,
Hiroyuki Itoh,
Yasushi Kawai,
Tadao Saito,
Susana Alvarez,
Haruki Kitazawa
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ABSTRACT: BACKGROUND: Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). RESULTS: All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1alpha and MCP-1 in BIE cells by activating both NF-kappaB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-kappaB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3). CONCLUSIONS: BIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.
BMC Microbiology 03/2013; 13(1):54. · 3.04 Impact Factor
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ABSTRACT: We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT.
Veterinary Immunology and Immunopathology 12/2011; 144(3-4):410-6. · 2.08 Impact Factor
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Tomoyuki Shimazu,
Julio Villena, Masanori Tohno,
Hitomi Fujie,
Shoichi Hosoya,
Takeshi Shimosato,
Hisashi Aso,
Yoshihito Suda,
Yasushi Kawai,
Tadao Saito,
Seiya Makino,
Shuji Ikegami,
Hiroyuki Itoh,
Haruki Kitazawa
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ABSTRACT: The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.
Infection and immunity 11/2011; 80(1):276-88. · 4.21 Impact Factor
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Shoichi Hosoya,
Julio Villena,
Tomoyuki Shimazu, Masanori Tohno,
Hitomi Fujie,
Eriko Chiba,
Takeshi Shimosato,
Hisashi Aso,
Yoshihito Suda,
Yasushi Kawai,
Tadao Saito,
Susana Alvarez,
Shuji Ikegami,
Hiroyuki Itoh,
Haruki Kitazawa
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ABSTRACT: ABSTRACT: This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.
Veterinary Research 11/2011; 42(1):111. · 4.06 Impact Factor
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Eriko Chiba,
Julio Villena,
Shoichi Hosoya,
Naoya Takanashi,
Tomoyuki Shimazu,
Hisashi Aso, Masanori Tohno,
Yoshihito Suda,
Yasushi Kawai,
Tadao Saito,
Kenji Miyazawa,
Fang He,
Haruki Kitazawa
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ABSTRACT: We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.
Research in Veterinary Science 10/2011; 93(2):688-94. · 1.65 Impact Factor
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Hitomi Fujie,
Julio Villena, Masanori Tohno,
Kyoko Morie,
Tomoyuki Shimazu,
Hisashi Aso,
Yoshihito Suda,
Takeshi Shimosato,
Noriyuki Iwabuchi,
Jin-Zhong Xiao,
Tomoko Yaeshima,
Keiji Iwatsuki,
Tadao Saito,
Muneo Numasaki,
Haruki Kitazawa
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ABSTRACT: A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyer's patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyer's patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.
FEMS Immunology & Medical Microbiology 06/2011; 63(1):129-39. · 2.44 Impact Factor
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ABSTRACT: Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.
Veterinary Immunology and Immunopathology 05/2011; 143(1-2):148-54. · 2.08 Impact Factor
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ABSTRACT: We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1alpha, IL-1beta and IL-18, are down-regulated after 24h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods.
Biochemical and Biophysical Research Communications 02/2010; 394(1):81-6. · 2.48 Impact Factor
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ABSTRACT: Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.
Animal Science Journal 10/2009; 80(5):597-604. · 0.86 Impact Factor
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ABSTRACT: Lymphotoxin-beta (LT-beta) has been suggested to be a regulator of secondary lymphoid structure development. In the present study, we isolated porcine LT-beta (poLT-beta) from adult swine spleens. The open reading frame encoded a predicted 246-amino acid polypeptide exhibiting higher similarity to the human than the mouse LT-beta protein. Expression of LT-beta mRNA in various swine tissues was analyzed by real-time PCR, and it was found to be higher in the ileal Peyer's patches (Pps) of adults than in newborns. In addition, ligand stimulation of toll-like receptors 2, 4, and 9, which are activated by bacterial components, increased LT-beta expression only in neonatal ileal Pps. These results suggest that colonization by commensal bacteria may affect the maturation of neonatal ileal Pps by the induction of LT-beta via toll-like receptors. LT-beta may therefore be useful for studying the development of the intestinal immune system at parturition in both swine and humans.
Biochemical Genetics 02/2009; 47(1-2):126-36. · 0.86 Impact Factor
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ABSTRACT: In this study, we isolated a complementary DNA encoding nucleotide-binding oligomerization domain-1 (NOD1) from Peyer's patches (Pps) of swine gut-associated lymphoid tissues (GALT). The complete open reading frame of porcine NOD1 contains 2862 bp, encoding a 953-amino acid polypeptide. The porcine NOD1 amino acid sequence is more closely related to the human sequence (83.8% identity) than the mouse counterpart (79.2% identity). To examine the subcellular expression and function of porcine NOD1, we overexpressed it in human embryonic kidney 293 cells. Immunostaining with an anti-porcine NOD1 polyclonal antibody revealed that the protein was expressed in transfectants as an intracellular membrane-bound molecule. In the transfected cells, both gamma-d-glutamyl-meso-diaminopimelic acid, and meso-diaminopimelic acid and meso-lanthionine activated nuclear factor-kappa B. Quantitative real-time PCR detected NOD1 mRNA in multiple tissues isolated from adult and newborn swine, including the esophagus, duodenum, jejunum, ileum, ileal Pps, colon, spleen, and mesenteric lymph nodes. In the newborn and adults, NOD1 was highly expressed in the esophagus and GALT, such in the ileal Pps and mesenteric lymph nodes. Furthermore, Toll-like receptor and NOD1 ligands as well as immunobiotic lactic acid bacteria enhanced the expression of NOD1 in GALT of adult and newborn swine. Our results should help clarify how the intestinal immune system is modulated by low-molecular weight peptidoglycan fragments through NOD1.
Molecular Immunology 04/2008; 45(6):1807-17. · 2.90 Impact Factor
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ABSTRACT: To study the immune responses of porcine intestinal epithelial cells to gram-negative bacteria via toll-like receptors (TLRs), originally established porcine intestinal epitheliocyte (PIE) cells were treated with lipopolysaccharide (LPS) or swine-specific enterotoxigenic Escherichia coli (ETEC). Real-time quantitative PCR revealed that PIE cells expressed TLR1-9 and MD-2 mRNAs, preferentially expressed TLR4/MD-2. Immunostaining of PIE cells revealed that TLR4 was precisely expressed in PIE cells at the protein level. PIE cells treated with LPS had up-regulated expression of several TLRs (TLR2, 3, 4, 5 and 8), type 1 helper T (Th1) cytokines (interleukin (IL)-1alpha, IL-1beta, IL-6, IL-15, 18, leukemia inhibitory factor (LIF), and interferon (IFN)-beta), and chemokines (monocyte chemoattractant protein (MCP)-1 and IL-8). ETEC enhanced the expression of TLR2, Th1 type cytokines (IL-1alpha, IL-12p35 and IL-6) and chemokines (MCP-1 and IL-8). These results indicate that PIE induces inflammatory responses by up-regulating Th1 cytokines and chemokines in response to LPS or ETEC, suggesting that PIE is a useful cell line for studying inflammatory responses via TLR4/MD-2 in intestinal epithelial cells.
Biochimica et Biophysica Acta 03/2008; 1780(2):134-44. · 4.66 Impact Factor
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Masanori Tohno,
Wataru Ueda,
Yuko Azuma,
Tomoyuki Shimazu,
Shinichiro Katoh,
Ji Ming Wang,
Hisashi Aso,
Haruhiko Takada,
Yasushi Kawai,
Tadao Saito,
Haruki Kitazawa
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ABSTRACT: The nucleotide-oligomerization domain (NOD) 2 is an important molecule involved in host defense. In this study, we report the cloning and characterization of porcine NOD2 (poNOD2) cDNA. The open reading frame of poNOD2 contains 3042 bp which encode 1013 amino acid residues. The putative poNOD2 protein shares higher level of homology with human counterpart (81.6% amino acid identity) than the mouse protein (76.6% amino acid identity). In order to determine the function of poNOD2, we established human embryonic kidney (HEK) 293 cells transfected to express poNOD2 cDNA. We found that poNOD2 was expressed not only in the cytoplasm but also in the inner side of the plasma membrane of HEK293 cells. HEK293 cells expressing poNOD2 responded to muramyl dipeptide (MDP) by activation of the nuclear factor kappa B (NF-kappaB). Quantitative real-time PCR revealed that poNOD2 mRNA was expressed by a number of tissues isolated from adult and newborn swine such as esophagus, duodenum, jejunum, ileum, ileal Peyer's patches (Pps), colon, spleen, and mesenteric lymph nodes (MLNs). In the newborn swine, the expression of poNOD2 mRNA was detected at higher levels in MLNs and spleen as compared to other tissues. In the adult swine, the highest expression was observed in ileal Pps. Furthermore, Toll-like receptor (TLR) and NOD2 ligands as well as immunobiotic lactic acid bacteria (LAB) enhanced the expression of NOD2 in gut-associated lymphoid tissues (GALT) in adult and newborn swine. Our results implicate NOD2 as an important immunoregulator in the swine intestinal immunity.
Molecular Immunology 02/2008; 45(1):194-203. · 2.90 Impact Factor
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ABSTRACT: We isolated cDNA encoding porcine MyD88 (poMyD88) from Peyer's patches (Pps) of GALT. The complete open reading frame (ORF) of poMyD88 contains 879 bp encoding a deduced 293 aa residues. The amino acid sequence of poMyD88 was characterized by N-terminal death, intermediate and C-terminal Toll/IL-1 receptor (TIR) domains. The putative poMyD88 protein shares a higher level of homology with its human (87.2% amino acid identity) than with its mouse (77.4% amino acid identity) counterpart. Overexpression of poMyD88 participated in the further enhanced activation of NF-kappaB in human embryonic kidney (HEK) 293 cells expressing porcine TLR2 and porcine TLR4/MD-2, but not porcine RP105/MD-1 after stimulation with the corresponding ligands. The expression levels of MyD88 were highest in the spleen and mesenteric lymph nodes (MLNs), and lower in digestive tissues of newborn swine. In adult swine, the expression levels in the digestive tissues were lower than those in MLNs and the spleen. These results suggest that an MyD88-dependent signaling pathway is present in newborn as well as in adult swine and that it is involved in the innate immune system of these animals.
Cellular & molecular immunology 11/2007; 4(5):369-76. · 2.99 Impact Factor
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ABSTRACT: Recent interest has focused on the importance of intestinal immunity for the host defense, but to date, not much is known about the underlying mechanisms. The toll-like receptor (TLR) family plays an important role in host defense through recognizing bacterial pathogen-associated molecular patterns. Our recent research on the physiological function of food products has investigated the immunoregulatory effects of probiotic lactic acid bacteria (LAB) via TLR. Studies of swine, which often substitute for a human model, have demonstrated intestinal immunoregulation by the probiotic LAB mediated by TLR in the gut. On the basis of our study, efforts have also been made to develop a molecular immunoassay system for probiotic LAB and find novel immunostimulatory DNA sequences from probiotics and high potential immunobiotic LAB strains via TLR signaling. These findings may provide important clues at the molecular level on TLR signal transduction pathways and recognition mechanisms for the ligands. They also provide impetus to further delineate the activation mechanism of the innate immune response. In addition to identifying immunoregulatory factor immunogenics from LAB, a better understanding of intestinal immune regulation through cytokine networks holds out promise for basic food immunology research and the development of immunobiotic foods to prevent specific diseases.
Animal Science Journal 10/2007; 79(1):11 - 21. · 0.86 Impact Factor
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ABSTRACT: Proteins in the milk release biologically active peptides upon enzymatic digestion. In the present study, we report the identification of novel monocyte/macrophage chemotactic peptides derived from enzymatically digested bovine beta-casein, a casein family member that is a major constituent of milk. Beta-casein fragments generated by actinase E showed potent chemotactic activity for human and mouse monocytes/macrophages, but not neutrophils, T lymphocytes or dendritic cells. The fragment-induced migration of human monocytes was inhibited by pertussis toxin and was not desensitized by a variety of known chemoattractants, suggesting that the digests activate a unique G protein-coupled receptor(s). The digests were further fractionated and purified to yield 3 small peptides. One peptide Q1 designated as "beta-casochemotide-1" with the amino acid sequence of YPVEP (f114-118 of beta-casein) induced high levels of macrophage chemotaxis. It also promoted calcium mobilization in macrophages, another indication of cell activation. Our study suggests that biologically active peptides released by actinase-digested milk beta-casein may promote innate host immune responses by inducing macrophage migration and activation.
International Immunopharmacology 10/2007; 7(9):1150-9. · 2.38 Impact Factor
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ABSTRACT: In this study, we cloned the cDNAs encoding porcine RP105 (poRP105) and porcine MD-1 (poMD-1) from Peyer's patches of adult swine. The complete open reading frames of poRP105 and poMD-1 contain 1986 and 480bp and encode 661 and 159 amino acid residues, respectively. These two proteins were more similar to the human (77.6% and 76.5% amino acid identity) than the mouse counterparts (70.0% and 71.1% amino acid identity). The results of several experiments in cells cotransfected with poRP105 and poMD-1 indicated both lipopolysaccharide and extracellular phosphopolysaccharide from Lactococcus lactis subsp. cremoris (Lc.cremoris) strongly activate nuclear factor-kappaB and induce the expression of various cytokines via RP105. These effects were mediated by phosphatidylinositol 3-kinase and Bruton's tyrosine kinase. Thus, we identified extracellular polysaccharide from Lc.cremoris as an active substance that can induce immune activation via RP105 and MD-1.
Molecular Immunology 05/2007; 44(10):2566-77. · 2.90 Impact Factor
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ABSTRACT: Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial components, and it also mediates activation signals in the cell relating to the innate immune system. In order to evaluate the precise molecular immunoregulation by various strains of lactic acid bacteria (LAB) via TLR2, the swine TLR2 (sTLR2)-expressing transfectant was constructed using human embryonic kidney (HEK) 293 cells. It is demonstrated that intact immunobiotic LAB can induce immune responses through TLR2, and that different nuclear factor-κB (NF-κB) activities of various strains can be accurately detected by sTLR2-expressing HEK293 cells. Furthermore, cellular activation of NF-κB via TLR2 is reflected in enhanced binding and uptake of LAB. The sTLR2-expressing HEK293 cells were also useful for characterizing the expression pattern of type I helper T (Th1) and type II helper T (Th2) cytokines by the stimulation of immunobiotic LAB. These results suggest that sTLR2-expressing HEK293 cells may be useful in certain molecular immunoassay systems for producing new physiologically functional foods with intestinal immunomodulatory abilities, such as the maintenance of Th1/Th2 polarization.
Animal Science Journal 03/2007; 78(2):195 - 205. · 0.86 Impact Factor
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ABSTRACT: 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.
Cellular Microbiology 04/2006; 8(3):485-95. · 5.46 Impact Factor
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ABSTRACT: 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.
FEMS Immunology & Medical Microbiology 07/2005; 44(3):283-8. · 2.44 Impact Factor
