Haruki Kitazawa

Phosphate group requirement for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus

The mitogenic activity of extracellular polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 and NCFB2483 was examined in murine lymphocytes. The extracellular polysaccharide from Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 was fractionated into neutral and acidic polysaccharides by anion-exchange chromatography, while that of Lactobacillus delbrueckii ssp. bulgaricus NCFB2483 were all fractionated into neutral polysaccharide(s). The acidic polysaccharide stimulated mitogenic responses of murine splenocytes and Peyers patches but not of thymocytes. The optimal concentration of the acidic polysaccharide at the highest stimulation was 160 microg/ml. A significant increase of mitogenic activity was initiated at 24 h, and the highest response was obtained after stimulation for 48 h. The acidic polysaccharide purified by high performance liquid chromatography also had substantial mitogenic activity, and the molecular weight was estimated to be 1.2 x 10(6). The acidic polysaccharide was a phosphopolysaccharide consisting of glucose, galactose and phosphorus. Dephosphorylation by hydrofluoric acid degradation reduced the mitogenic activity in lymphocytes. The phosphopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus 1073R-1 is a potent B-cell-dependent mitogen in which the phosphate group acts as a trigger of the mitogenic induction.

Cell surface Lactobacillus plantarum LA 318 glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) adheres to human colonic mucin

Aims:  To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM).

Strong immunostimulation in murine immune cells by Lactobacillus rhamnosus GG DNA containing novel oligodeoxynucleotide pattern.

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‐γ) and tumour necrosis factor alpha (TNF‐α) mRNA expression and IFN‐γ/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.

Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli

ABSTRACT Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of α-helices, the spectra of the bacteriocins did not coincide. The results of d- and l-amino acid composition analysis showed that two residues and one residue of d-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different d-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in d-amino acid contents in the molecules.

Immunobiotic Lactobacillus jensenii Elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the toll-like receptor signaling pathway

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.

Cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Lactobacillus plantarum LA 318 recognizes human A and B blood group antigens

Lactobacillus plantarum LA 318 is a potential probiotic strain isolated from normal human intestinal tissue that shows high adhesion to human colonic mucin mediated by the bacterial cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report the adhesion mechanism of the lactobacilli is in part due to GAPDH binding to human ABO-type blood group antigens expressed on human colonic mucin (HCM). After periodate oxidation of HCM, adhesion of L. plantarum LA 318 bacterial cells significantly decreased compared to normal HCM. A BIACORE binding assay of GAPDH to blood group antigens was then performed. High binding was observed to A and B group antigens, while binding to H group antigen was lower (P<0.01). No interaction was observed between GAPDH and various monosaccharides. Furthermore, GAPDH binding to the B-trisaccharide biotinyl polymer (BP)-probe [Galalpha1-3 (Fucalpha1-2) Gal-] was significantly higher as compared to B-disaccharide, Lewis D-trisaccharide, 3-fucosyl-N-acetylglucosamine and alpha-N-acetylneuraminic acid BP-probes. The data suggests the trisaccharide structure is important in binding to the blood group antigens. The binding of GAPDH to HCM significantly decreased after incubation with NAD+. This suggests that the NAD binding domain on GAPDH may be related to binding to HCM.

Modulation of Intestinal TLR4-Inflammatory Signaling Pathways by Probiotic Microorganisms: Lessons Learned from Lactobacillus jensenii TL2937

The intestinal mucosa plays a critical role in the host’s interactions with innocuous commensal microbiota and invading pathogenic microorganisms. Intestinal epithelial cells (IECs) and gut associated immune cells recognize the bacterial components via pattern-recognition receptors (PRRs) and are responsible for maintaining tolerance to the large communities of resident luminal bacteria while being also able to mount inflammatory responses against pathogens. Toll-like receptors (TLRs) are a major class of PRRs that are present on IECs and immune cells which are involved in the induction of both tolerance and inflammation. A growing body of experimental and clinical evidence supports the therapeutic and preventive application of probiotics for several gastrointestinal inflammatory disorders in which TLRs exert a significant role. This review aims to summarize the current knowledge of the beneficial effects of probiotic microorganisms with the capacity to modulate the immune system (immunobiotics) in the regulation of intestinal inflammation in pigs, which are very important as both livestock and human model. Especially we discuss the role of TLRs, their signaling pathways, and their negative regulators in both the inflammatory intestinal injury and the beneficial effects of immunobiotics in general, and Lactobacillus jensenii TL2937 in particular. This review article emphasizes the cellular and molecular interactions of immunobiotics with IECs and immune cells through TLRs and their application for improving animal and human health.

Swine Toll-like receptor 9 recognizes CpG motifs of human cell stimulant

Complementary DNA (cDNA) encoding swine Toll-like receptor 9 (sTLR9) was isolated from Peyers patches (Pps) of gut-associated lymphoid tissue (GALT). The complete open reading frame (ORF) of sTLR9 contains 3093 bp coding deduced 1030 amino acid residues. The amino acid sequence of sTLR9 was characterized by a signal peptide followed by multiple leucine-rich repeats, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (TIR). The sTLR9 showed a higher amino acid identity with humans (81.8%) and felis catus (86.7%) than mice (74.9%). The HEK293T cells transfected with pCXN2.1-FLAG DNA containing the sTLR9 cDNA were expressed sTLR9 as a membrane-bound molecules, which were reactive with anti-sTLR9 rabbit polyclonal antibody. Moreover, the transfectant was responsible for the CpG oligo DNA. sTLR9 was preferentially expressed in Pps and mesenteric lymph nodes (MLNs), and its degree was approximately three times higher than a spleen but weak in the other tissues by the real-time quantitative PCR analyses. The strong expression of sTLR9 in Pps and MLNs and its recognizing CpG DNA for human cell stimulant are shown first in this study, which may help in understanding the intestinal immune system mediated by a bacterial DNA through TLR9.

Immunostimulatory oligodeoxynucleotide from Bifidobacterium longum suppresses Th2 immune responses in a murine model

We have reported previously that novel immunostimulatory sequence (ISS) oligodeoxynucleotide (ODN) BL07S from a probiotic strain of Bifidobacterium longum inhibited immunoglobulin (Ig) E production in vitro. However, whether ISS‐ODNs from probiotics regulate T helper type 2 (Th2)‐polarized immune reactions in vivo remains unclear. To evaluate the inhibitory effects of ODN BL07S on type I allergic response, BALB/c mice were injected with or without ODN BL07S in the presence of ovalbumin (OVA) on days 0 and 14. Serum Ig levels (IgE, IgG1 and IgG2a) and cytokine levels (interferon (IFN)‐γ, interleukin (IL)‐12, IL‐4, IL‐5, IL‐10 and IL‐13) were investigated in splenocyte cultures from days 14–28. Production of OVA‐specific and total IgE were significantly suppressed by administration of ODN BL07S, but not by ODN BL06S, a non‐ISS‐ODN. Compared to controls, ODN BL07S induced significantly lower levels of Th2 cytokines (IL‐4 and IL‐5) in splenocyte cultures, and significantly higher levels of serum OVA‐specific IgG2a. These effects of ODN BL07S on modulation of Th2 immune response were dose‐dependent. The present results demonstrate that ODN BL07S from genomic DNA of B. longum BB536 prevents antigen‐induced Th2 immune responses in vivo, suggesting that ISS‐ODNs from probiotics might be useful in preventing allergic disease.

Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection

BackgroundSome studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. However, the capacity of immunobiotics to improve protection against respiratory syncytial virus (RSV) infection was not investigated before.ObjectiveThe aims of this study were: a) to evaluate whether the nasal administration of Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by TLR3/RIG-I activation; b) to investigate whether viability of Lr05 or Lr06 is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of Lr05 and Lr06 to improve the resistance of infant mice against RSV infection.ResultsNasally administered Lr05 and Lr06 differentially modulated the TLR3/RIG-I-triggered antiviral respiratory immune response. Lr06 administration significantly modulated the production of IFN-α, IFN-β and IL-6 in the response to poly(I:C) challenge, while nasal priming with Lr05 was more effective to improve levels of IFN-γ and IL-10. Both viable Lr05 and Lr06 strains increased the resistance of infant mice to RSV infection while only heat-killed Lr05 showed a protective effect similar to those observed with viable strains.ConclusionsThe present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by TLR3/RIG-I activation in the respiratory tract and to increase the resistance of mice to the challenge with RSV. Comparative studies using two Lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(I:C) or RSV challenges, conferring different degree of protection and using distinct immune mechanisms. We also demonstrated in this work that it is possible to beneficially modulate the respiratory defenses against RSV by using heat-killed immunobiotics.