No abstract

Optimal functioning of the immune system is crucial to human health, and nutrition is one of the major exogenous factors modulating different aspects of immune function. Currently, no single marker is available to predict the effect of a dietary intervention on different aspects of immune function. To provide further guidance on the assessment and interpretation of the modulation of immune functions due to nutrition in the general population, International Life Sciences Institute Europe commissioned a group of experts from academia, government and the food industry to prepare a guidance document. A draft of this paper was refined at a workshop involving additional experts. First, the expert group defined criteria to evaluate the usefulness of immune function markers. Over seventy-five markers were scored within the context of three distinct immune system functions: defence against pathogens; avoidance or mitigation of allergy; control of low-grade (metabolic) inflammation. The most useful markers were subsequently classified depending on whether they by themselves signify clinical relevance and/or involvement of immune function. Next, five theoretical scenarios were drafted describing potential changes in the values of markers compared with a relevant reference range. Finally, all elements were combined, providing a framework to aid the design and interpretation of studies assessing the effects of nutrition on immune function. This stepwise approach offers a clear rationale for selecting markers for future trials and provides a framework for the interpretation of outcomes. A similar stepwise approach may also be useful to rationalise the selection and interpretation of markers for other physiological processes critical to the maintenance of health and well-being.

Although many Lactobacillus strains used as probiotics are believed to modulate host immune responses, the molecular natures of the components of such probiotic microorganisms directly involved in immune modulation process are largely unknown. We aimed to assess the function of polysaccharide moiety of the cell wall of Lactobacillus casei strain Shirota as a possible immune modulator which regulates cytokine production by macrophages. A gene survey of the genome sequence of L. casei Shirota hunted down a unique cluster of 10 genes, most of whose predicted amino acid sequences had similarities to various extents to known proteins involved in biosynthesis of extracellular or capsular polysaccharides from other lactic acid bacteria. Gene knockout mutants of eight genes from this cluster resulted in the loss of reactivity to L. casei Shirota-specific monoclonal antibody and extreme reduction of high-molecular-mass polysaccharides in the cell wall fraction, indicating that at least these genes are involved in biosynthesis of high-molecular-mass cell wall polysaccharides. By adding heat-killed mutant cells to mouse macrophage cell lines or to mouse spleen cells, the production of tumor necrosis factor alpha, interleukin-12 (IL-12), IL-10, and IL-6 was more stimulated than by wild-type cells. In addition, these mutants additively enhanced lipopolysaccharide-induced IL-6 production by RAW 264.7 mouse macrophage-like cells, while wild-type cells significantly suppressed the IL-6 production of RAW 264.7. Collectively, these results indicate that this cluster of genes of L. casei Shirota, which have been named cps1A, cps1B, cps1C, cps1D, cps1E, cps1F, cps1G, and cps1J, determine the synthesis of the highmolecular-mass polysaccharide moiety of the L. casei Shirota cell wall and that this polysaccharide moiety is the relevant immune modulator which may function to reduce excessive immune reactions during the activation of macrophages by L. casei Shirota.

Protein D has previously been demonstrated to be associated with Escherichia coli ribosomes by the radicalfree and highly reducing method of two-dimensional polyacrylamide gel electrophoresis. In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on the E. coli genetic map, between ompC and sfcA. The gene consists of 45 codons, coding for a protein of 5,096 Da. The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase. For these reasons, protein D was named SRA (stationaryphase-induced ribosome-associated) protein and its gene was named sra. The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S). In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription of sra in the stationary phase.The ribosomal proteins of Escherichia coli were systematically characterized in the beginning of the 1970s, and a unified nomenclature, S1 through S21 for the 30S subunit and L1 through L34 for the 50S subunit, was proposed on the basis of their behavior in two-dimensional gel electrophoresis (17,44,45,46). Using a modified and improved method, termed radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis (RFHR 2-D PAGE), we discovered four additional proteins, proteins A, B, C, and D, that were associated with E. coli ribosomes (38,39,40).The gene for protein A (40) was located between infC (initiation factor 3) and rplT (ribosomal protein L20) (26,27,31) at 38 min, suggesting that the three constitute a new ribosomal protein operon in E. coli. The gene for protein B (40) was found to be identical to the protein X gene of the spc operon (8). Proteins A and B were consequently named L35 and L36 and their genes were named rpmI and rpmJ, respectively, in accordance with the standard nomenclature for E. coli ribosomal proteins and their genes. Protein C (41) is likely to be the full-length product of the rpmE gene, coding for L31, because the N-terminal amino acid sequence completely matches that of L31 and the molecular mass of protein C is larger than that of L31 by about 1,000 Da. More recently, L31 was proved to arise through cleavage of protein C by protease VII, an outer membrane enzyme (A. Wada, unpublished data).Protein D is a small basic protein that can be isolated from the 30S subunit after dissociation of the 70S ribosome. It migrates much faster than S21 and to a point below the L32 spot upon RFHR 2-D PAGE of purified 70S ribosomal proteins. Thus, it is the smallest protein component of the 30S subunit of E. coli. When the 70S ribosome was dissociated into the core particle and split ...

SummaryInterleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signals play key roles in the pathogenesis of inflammatory bowel disease (IBD). We previously described that both intact cells and a cell wall-derived polysaccharide-peptidoglycan complex (PSPG) in a strain of lactobacillus [Lactobacillus casei Shirota (LcS)] inhibited IL-6 production in lipopolysaccharide (LPS)-stimulated lamina propria mononuclear cells (LPMCs) isolated from murine IBD. Diets with LcS improve murine IBD by suppression of IL-6 synthesis in LPMCs. Moreover, LcS supplementation with fermented milk ameliorates disease activity in patients with active ulcerative colitis. Here, we focused on the specific roles of PSPG in LcS concerning their anti-inflammatory actions. PSPG derived from LcS, and no other strain of lactobacilli, inhibited IL-6 production in LPS-stimulated murine IBD LPMCs. Purified PSPG-I from LcS inhibited IL-6 synthesis in LPS-stimulated murine IBD LPMCs through the inhibition of nuclear factor-jB. The anti-IL-6 action of LcS PSPG was abrogated by masking with monoclonal anti-PSPG-I. Furthermore, PSPG-I-negative L. casei strains (PSPG-I-negative mutant LcS: LC DPSPG-I , L. casei ATCC 334) did not inhibit IL-6 production. Finally, we confirmed the effects of PSPG-I on LcS in the models of both IBD and colitis-associated cancer (CAC). In the IBD model, ingestion of LcS improved ileitis and inhibited activation of IL-6/STAT3 signaling, while ingestion of the LC DPSPG-I strain did not. In the CAC model, treatment with LcS, but not the LC DPSPG-Istrain, showed tumour-suppressive effects with an inhibition of IL-6 production in the colonic mucosa. These results suggested that a specific polysaccharide component in an L. casei strain plays a crucial role in its anti-inflammatory actions in chronic intestinal inflammatory disorders.Keywords: colitis-associated cancer; inflammatory bowel disease; interleukin-6; Lactobacillus; polysaccharide-peptidoglycan complexPlease cite this article in press as: Matsumoto S. et al. A component of polysaccharide peptidoglycan complex on Lactobacillus induced an improvement of murine model of inflammatory bowel disease and colitis-associated cancer, Immunology (2009)