Gnotobiology symposium abstracts

Symposium 2-1 Nitrogen as an eco-regulator in the gastrointestinal tract of mammals Tore Midtvedt Microbiology & Tumor Biology Center, Karolinska Institute, Stockholm, Sweden Nitrogen is an essential compound for all living organisms. It constitute the major part of our atmosphere. Some microrganisms are the only living organisms capable of transforming atmospheric nitrogen into many compounds of biological interest. In mammals, mechanisms have been established for reducing the faecal and urinary loss of nitrogen-containing molecules. These nitrogen-saving mechanisms are most important in herbivores, followed by omnivores and carnivores. Two major mechanisms will be focus upon  a. Nitrate cycle  b. Urea cycle Regarding both substrates, comparative studies in germfree and conventional animals have shown that microorganisms in the oro-intestinal tract play a compartmentalized, pivotal role in the endo-exo-circulation of these compounds; to the benefit for themselves as well as for the host. 2-2 Role of gut microflora in metabolism of phytoestrogens I Rowland Northern Ireland Centre for Food and Health University of Ulster, Coleraine, BT52 1SA, UK Fax: [phone] The isoflavone phytoestrogens genistein and daidzein are found in soy beans and soy products, where they are present mainly in the form of their glycosidic conjugates, genistin and daidzin. When ingested, the latter are hydrolysed and further metabolized. Genistein can be converted to the hormonally inert p-ethyl phenol, whilst daidzein can be reduced to the isoflavan equol and o-desmethylangolensin (O-Dma). The biological consequences of this metabolism are not fully elucidated. The lignan phytoestrogens secoisolariciresinol and matairesinol glycosides, derived from cereal brans and flaxseed, undergo analogous reactions and are converted to enterolactone and enterodiol. There is marked interindividual variation in phytoestrogen metabolism in human subjects. For example, in a recent study in our laboratory, 8/23 subjects (35%) excreted large amounts of equol, mean 10905±2147 nmol/24 h, (approximately 200 times that of poor equol producers). Subjects who were good equol producers also had high levels of O-Dma in plasma indicating that equol and O-Dma do not represent alternative pathways of daidzein metabolism. There is increasing evidence that the metabolism of both isoflavones and lignans is bacterially mediated. In order to provide unequivocal evidence for the role of the gut microflora in the absorption and metabolism of these phytoestrogens, we have investigated their metabolism in germ free (GF) rats and rats associated with human faecal bacteria (human flora associated [HFA] rats). Furthermore, we have investigated whether certain metabolic characteristics (high equol-producing and low equol-producing status) of human intestinal floras can be transferred to GF rats. Germ-free rats fed a soy-isoflavone containing diet excreted large quantities of daidzein and genistein in urine indicating that the gut microflora is not required for the absorption of isoflavones. The isoflavone metabolites equol, O-desmethylangolensin and the lignan enterolactone were not detectable in urine from the GF rats, but were present in HFA rat urine, indicating that they were products of gut microflora activity. Colonization of GF rats with a faecal flora from a human subject with the capacity to convert daidzein to equol, resulted in the rats excreting substantial amounts of the metabolite. In contrast, equol was undetectable in urine of HFA rats associated with a faecal flora from a low equol-producing subject. The results therefore show that the inability of some subjects to produce equol is a consequence of the lack of specific components of the gut microflora. There is evidence from in vitro experiments and epidemiological studies that metabolism is important in the biological activity of isoflavonoid and lignan phytoestrogens. We are currently involved in comparative studies of the activity of parent compounds and bacterial metabolites in assays for anti-cancer activity We thank the EC (FAIR CT-95-0894; QLK1-2000-00266) for financial support. 2-3 Recent advances in physiological functions of menaquinone-4 (MK-4), a unique vitamin K₂ not derived from intestinal microflora. Michio Komai, Hitoshi Shirakawa, Yusuke Ohsaki Although it is known that several vitamins, including menaquinones (MK-n = vitamin K₂), biotin, vitamin B12, and pantothenic acid, are microbiologically synthesized in the distal gastrointestinal tract of mammals, the contribution of these vitamins to human nutrition remains unclear. During our studies, we have gradually recognized that MK-4 accumulates in various tissues of germfree animals fed a MK-4-free diet (Kimura, S., Komai, M., Sato, H., 1991). Accordingly, we have focused on clarifying the mechanism of MK-4 formation in several tissues, using both in vitro tissue homogenates and in vivo experiments with rats, mice, and bovine kidney. Our results indicate that MK-4 is produced without enzymatic participation of microflora in diverse tissues other than gastrointestinal mucosa (stomach, small intestine, and colon) from ingested or added vitamin K analogues, including vitamin K₁ and MK-n (MK-6, MK-7, and MK-10), by conversion of side-chain replacement to geranylgeranyl group at 3-position of naphthoquinone ring. Even though experimental results indicate that MK-4 has unique functions involved in cell differentiation, induction of apoptosis, and stimulation of transcription of a ligand for a nuclear receptor, these cannot completely explain the physiological significance of MK-4 synthesis in various tissues. To elucidate the biological role of MK-4 production outside the gastrointestinal system, we used germfree rats to eliminate MK-n synthesized by intestinal flora and employed DNA microarray techniques to identify patterns of gene expression. We summarize our findings as follows: 1) MK-4, instead of K₁, may suppress inflammation through down-regulation of IL-6 expression; 2) MK-4 is a key factor involved in testicular steroidogenesis. 2-4 Regulation of xenobiotic metabolism in intestine by nuclear receptor/dioxin receptor-cytochrome P450 pathway. Shigeyuki Uno and Makoto Makishima Department of Biochemistry, Nihon University School of Medicine, Tokyo, Japan Fax: [phone] E-mail: [email protected] Nuclear receptors are ligand-inducible transcription factors that have been shown to play important roles in lipid and xenobiotic metabolisms. The vitamin D receptor (VDR) is a nuclear receptor known to mediate the biological actions of the active form of vitamin D. Recently we found that VDR responds to secondary bile acids produced by intestinal microflora and regulates their detoxification though induction of cytochrome P450 (CYP) 3A genes. Since CYP3A family is involved in metabolism of xenobiotics containing drugs in intestine and liver, VDR may be a regulator of xenobiotics metabolism. CYP1 family (Cyp1a1, 1a2, 1b1) is another enzyme involved in xenobiotic metabolism. It has a broad tissue distribution, is expressed very early in gestation, and is transcriptionally regulated by the Ah receptor (AHR). Although AHR does not belong to the nuclear receptor superfamily, it acts as a receptor for xenobiotics such as the smoking-associated contaminants benzo[a]pyrene (BaP). Toxicity of BaP is known to be due to AHR-mediated metabolic activation of this compound. We investigated the role of CYP1A1 in metabolic activation of BaP, comparing Cyp1a1(-/-) knockout with Cyp1a1(+/ + ) wild-type mice––following oral BaP. Cyp1a1(-/-) mice died within 30 days whereas Cyp1a1(+/ + ) mice appeared healthy 1 year later. The rate of BaP clearance was 6-fold greater in wild-type than knockout mice. The cause of death in Cyp1a1(-/-) mice receiving oral BaP was hypoglobulia, including toxic chemical depression of the bone marrow. DNA post-labeling studies demonstrated statistically significantly higher BaP-DNA adduct levels in Cyp1a1(-/-) than Cyp1a1(+/ + ) in liver, small intestine, spleen and marrow. The same results were obtained also in AhR(-/-). We conclude that, despite previous studies in vitro and in cell culture to the contrary, the present data indicate that––in the intact animal––inducible CYP1A1 regulated by AHR is much more important in detoxification and protection against oral BaP toxicity than in mice having this enzyme genetically absent. Whether the protection comes primarily from the liver, the GI tract, or something in the context of the immune system––remains to be determined. These studies will greatly enhance our understanding of lead to the elucidation of molecular mechanism of bile acid-related diseases such as colon cancer and the design of drugs to shield against such toxicity of CYP1A1 substrates and AHR ligands, in the diet and especially for cigarette smokers. 3-1 Fibroblasts rescue butyric acid-induced T-cell apoptosis at the inflammatory sites of anaerobic-bacterial infection in mucous membrane. K. Ochiai ¹⁾ and T. Kurita-Ochiai ^{2) 1)} Department of Microbiology, Nihon University School of Dentistry, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310 and ²⁾ Department of Microbiology, Nihon University School of Dentistry at Matsudo, Matsudo-shi, Chiba-ken, 271-8587, Japan, Recently, it was reported that periodontpathic bacteria cause local chronic disease, could be not only a source of focus infection, but also holding resources of inflamed materials. It is recognized that periodontal diseases are infectious and that periodontal tissue breakdown results from the interaction of specific anaerobic bacteria and host immune mechanisms. Short-chain fatty acids (SCFA) produced by periodontpathic bacteria, easily penetrate into oral mucosa and greatly inhibit T- and B-cell proliferation and cytokine production. BA is also known released by colonic bacteria and exerts immunomodulatory properties. BA induces apoptosis in murine and human T cells. We reported that BA-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. We demonstrated that human gingival fibroblasts (HGF) rescue BA-induced T-cell apoptosis via the proinflammatory cytokines, such as IL-6 and IL-11, which were produced in fibroblasts stimulated with BA. We assessed whether the T cell adhesion to HGF regulates the susceptibility of T cells to BA-induced apoptosis. The number of Jurkat cells adhered to HGF was significantly increased by addition of BA. All Jurkat cells adhered to Gin-1 cells were live cells in contrast to non-adhered cells drop into apoptosis. BA-induced T-cell apoptosis is down-regulated by the adhesion to HGF through the interaction with the adhesion molecule such as CD44, VLA-2 and VLA-5, expressed on T cells stimulated with BA. BA is a causative agent in gingival inflammation and may exert immunomodulation through T- and B-cell apoptosis in gingival tissue. However, HGF rescue BA-induced T-cell apoptosis at the inflammatory sites of anaerobic-bacterial infection in mucous membrane. 3-2 Bacterial interactions in oral biofilms H. Kuramitsu, A. Ikegami, M. Yamada, and B.-Y. Wang State University of New York, Buffalo, NY, USA FAX: [phone] Oral biofilms (dental plaque) have been demonstrated to be responsible for two of the most common human infectious diseases, dental caries and periodontal diseases. Although specific pathogens have been associated with each disease, it is likely that the virulence properties of these organisms are influenced by the presence of other plaque bacteria. In order to investigate the effects of such interactions we have initiated studies involving two model systems, Streptococcus gordonii-S. mutans and Porphyromonas gingivalis-Treponema denticola. We have demonstrated that S. gordonii can antagonize several virulence properties of S. mutans, ie., bacteriocin production, transformation, and colonization by both protease and protease-independent mechanisms. Besides antagonistic interactions, plaque organisms can also have beneficial effects on other members of this oral community. P. gingivalis can enhance the colonization of the oral spirochete T. denticola in vitro. Confocal microscopic examination of such synergistic biofilms indicated that P. gingivalis initially colonizes inert surfaces followed by interactions with T. denticola. Such interactions require the motility of the spirochete as well as the RgpB protease and major fimbrial protein, FimA, of P. gingivalis. These results suggest that the composition of dental plaque can have a major impact on the virulence of oral biofilms depending on the relative proportions of individual organisms. Such a demonstration suggests that such information could be used as basis for maintaining the oral health of individuals. 3-3 Clinical analysis of anti neutrophil cytoplasmic antibody against bactericidal/permeability increasing protein (BPI-ANCA) in biofilm formed Pseudomonas aeruginosa lung infections Osamu Kobayashi Infectious disease department, Kyorin university school of medicine [Objection] It is well known that persistently colonization of biofilm formed Pseudomonas aeruginosa on lung in patients with diffuse panbronchiolitis (DPB), bronchiectasis (BE) and Cystic fibrosis (CF) induce immunological modification to the host side. Previous basic studies have indicated that the phagocytic activity of neutrophil was significantly inhibited by anti neutrophil cytoplasmic antibody against bactericidal/permeability increasing protein (BPI-ANCA) in a dose dependent manner in vitro (P < 0.01), and in clinical studies, high titers of serum BPI-ANCA played an important role in the clinical course of disease such in patients with DPB, BE and CF. However, the influence of BPI-ANCA on the clinical picture and prognosis of in patients of these disease has not been clarified in detail. In this time, I am going to make discussion with the clinical role of such autoantibody in chronic biofilm formed P. aeruginosa lung infections. [Materials and Methods] Sixteen patients of DPB and 34 patients with BE are sampled. Relationship between serum BPI-ANCA titers and clinical symptoms, respiratory function, chest X-ray findings, and bacteria detected were evaluated. Serum BPI-ANCA was tested with ELISA testing kit (BPI IgG kit, GENESIS diagnosis, Cambridge, UK). [Results] Serum BPI-ANCA titer was 1) correlated with the severity of clinical symptoms, in patients with biofilm formed chronic P. aeruginosa lung infections (P < 0.01), 2) decreased with the improvement of the clinical picture (P < 0.05), 3) significantly higher in patients with in patients with far-advanced lesions on chest X-rays than with milder lesions (P < 0.05) and in patients with reduced pulmonary function (P < 0.05), 4) significantly higher in the patients with poor prognosis (P < 0.05). [Conclusion]The above results suggests that BPI-ANCA, an autoimmune factor, appears during the course of chronic biofilm formed P. aeruginosa lung infections, and that this autoimmune factor may make chronic lung infections more intractable, by inhibiting the phagocytic activity of neutrophil for P. aeruginosa. 4-1 Probiotics in infectious diseases – an overview Torkel Wadström Department of Laboratory Medicine, Section Bacteriology Lund University Hospital SE 223 62 Lund, Sweden Fax [phone] A number of probiotic based concepts were studied to modulate the gut microflora and found to inhibit various gastrointestinal (GI) pathogens, including rotavirus in in vitro organ culture and animal models. However, the great costs and poor “political will” in the food and dairy companies to evaluate the most promising candidates from those studies in clinical trials has delayed the development of probiotics to treat such GI infections including travellers and other forms of diarrhoea. Most promising results to prevent neonatal and weanling diarrhoea in animal medicine accumulated more rapidly based on new findings on how probiotic bacteria associate with the gut mucus layer, stabilize gut epithelial junctions, prevent pathogen adherence, and inhibit growth of GI pathogens. Interestingly, immunomodulatory properties of lactic acid bacteria (LAB) and other gram positive microbes (Bacillus, Clostridium butyricum, Propionebacteria etc.) indicate that a gut flora modulation can stabilize gut barrier functions and suppress development of inflammatory bowel disease (IBD)–like syndromes as well as pediatric atoptic diseases. Use of probiotics to prevent urinary tract infections, vaginosis and related microbial disorders are other examples of promising fields to develop probiotic prevention and treatment strategies to diminish antibiotic overuse and selection of antibiotic resistant pathogens as for many GI infections. Some “turbo- mixtures” of probiotic microbes at a high dose and often in combination with specific prebiotic fibres show promising results to prevent translation of gut microbes to the liver and other extraintestinal organs after surgery and in intensive care units. The effect of probiotic bacterium Clostridium butyricum on Clostridium difficile infection Motomichi Takahashi^1,2, Haruhiko Taguchi², Takako Osaki² and Shigeru Kamiya^{2 1}Miyarisan Pharmaceutical Co., Ltd., ²Department of Infectious Diseases, Kyorin University School of Medicine. Clostridium butyricum MIYAIRI 588 strain is a probiotic agent used for protection against infection with enteropathogenic microorganisms such as enterohemorrhagic Escherichia coli O157: H7 (EHEC) and/or Salmonella enterica serovar Enteritidis. The Japanese Ministry of Health, Labor and Welfare approves this probiotics, for treatment of several gastrointestinal diseases. In this symposium, I present the antagonistic interaction between C. butyricum and Clostridium difficile. We examined an antagonistic interaction between C. butyricum and C. difficile in vitro and in vivo using germ-free mice. Six out of seven gnotobiotic mice mono-associated with C. difficile died within 2 days. Haemorrhagic enterocolitis was observed from distal ileum to the caecum in dead mice. In contrast, eight out of 10 gnotobiotic mice, which were treated with C.butyricum survived without any diarrhea after infection with C. difficile. Result of the microbiological examination shows that effects of C. butyricum on the prevention of lethally infection of C. difficile caused by not only growth inhibition but also inhibition of toxin production by C. difficile. In addition, Kuroiwa et al. reported that C.butyricum using prescription drug prevent the detection of C. difficile and/or its toxin in the patients who receiving several antibiotics. These results indicated that C. butyricum MIYAIRI 588 strain has a considerable beneficial effect on the C. difficile infections. 4-3 The suppression of the Helicobacter pylori-induced interleukin-8 production in vitro and within the gastric mucosa by a live lactobacillus strain Yasuhiro Koga, M.D. Laboratory for Infectious Diseases, Tokai University School of Medicine, Isehara 259-1193, Japan Accumulating evidence indicates that interleukin-8 plays a major role in the mucosal inflammation caused by Helicobacter pylori infection. In this study, we attempted to elucidate whether Lactobacillus gasseri OLL2716 (LG21) can inhibit the H.pylori-induced production of IL-8. A coculture system including MKN45 cells, H.pylori and LG21 was set up for that analysis. As a result, LG21 with the number one-hundredth less than that of H.pylori significantly suppressed the production of IL-8. However this number of LG21 could not suppress the IL-8 production by the cells stimulated with tumor necrosis factor-a. Moreover, UV- or heat-treated LG21 could not suppress the IL-8 production, thus ruling out the possibility that such suppression is due to a nonspecific hindrance of the contact between H.pylori and the host cell as a result of the overcrowding by LG21. In this coculture system, LG21 also inhibited the tyrosine phosphorylation of CagA, a H.pylori-derived molecule delivered to the host cell through a type IV secretion system and then it was phosphorylated at the tyrosine residues, whereas LG21 could not inhibit the adhesion of H.pylori to the cell. It was therefore suggested that LG21 suppresses H.pylori-induced IL-8 production by inhibiting the type IV secretion system. The IL-8 level in the gastric biopsy specimens from H. pylori-infected humans also revealed a significant reduction after LG21 intake. 4-4 Differential effects of two probiotic strains, L. Casei Shirota and B. Breve Yakult, on the relief of the inflammatory bowel disease Yoshinori Umesaki Yakult Central Institute for Microbiological Research, Yaho 1796, Kunitachi, Tokyo, 186-8650, Japan, E-mail:[email protected] It is well known that the intestinal microorganisms are playing a key role in the development of the inflammatory bowel disease (IBD), Crohn disease (CD) or ulcerative colitis (UC). Clinical trials for the prevention of IBD using probiotics are now focused on the two aspects, immunoregulations and improvement of intestinal environments. In this paper, we would like to introduce the attempts to improve the IBD using our two probiotic strains, L. casei Shirota (LcS) and B. breve Yakult (BbrY). Firstly we conducted randomized controlled studies using fermented milk with BbrY, a member of the indigenous colonic flora, on UC patients, where abnormal microbial balance such as a reduction of Bifidobacterium was reported. By daily administration of the BbrY-fermented milk to the patients with active colitis, clinical and endoscopic scores were significantly improved, in accordance with the improvement of the intestinal flora and the increase in butyric acid concentration. Moreover, it was shown that daily ingestion of the BbrY-fermented milk was effective on the maintenance of remission, at least within a year. In vitro studies on HT-29 cell line showed that the culture supernatant of bifidobacterial strains bore the inhibitory activity toward TNF-alpha stimulated IL-8 secretion. Secondly, we tried to repress IBD including Crohn disease by LcS. In Crohn disease model mouse, SAMP1/Yit, oral administration of LcS improved the histological score of the ileitis. In this case, a clear inhibition of IL-6/Stat 3 pathway by LcS was observed in the lamina propria cells. Interestingly, the down-regulation of IL-6 by LcS preparation was also observed in the peripheral blood mononuclear cells obtained from IBD patients including Crohn disease. Conclusion: The two probiotic strains, LcS and BbrY are hopeful candidates for probiotic therapy for IBD through immunoregulation and improvement of intestinal environments, respectively. Genetically Modified Lactococcus: Novel Tools For Medicine Steidler L.¹, Neirynck, S.¹, Vandenbroucke, K.², Remaut, E.², Rottiers, P.^{2 1}Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland, Fax: [phone]Department for Molecular Biomedical Research, Ghent University, Ghent, Belgium. Fax: [phone] Lactococcus lactis can be genetically modified (GM) to secrete fully functional cytokines and various other regulatory proteins derived from eukaryotes. By simple topical application, these GM L. lactis can actively deliver such cytokines to the mucosa. The mechanism by which this occurs involves in situ synthesis of the recombinant proteins. By use of this principle we developed novel therapeutic approaches for the treatment of inflammatory bowel disease (IBD) i.e. Crohn's disease and ulcerative colitis. IBD is prominent in public healthcare and affects over 2 per 1000 individuals in Europe and the US. Because IBD requires long-term, anti-inflammatory medication, patients are often subject to a spectrum of disagreeable side effects. Interleukin-10 (IL-10) is a cytokine that acts to suppress inflammation. When administered by injection, IL-10 suffers from similar problems as conventional immune suppressants: high IL-10 levels are required to be effective and concomitantly lead to adverse reactions. Administration of L. lactis that secrete murine IL-10 both cure and prevent IBD in mice [1]. Targeted delivery by use of this GM L. lactis allows dramatic reduction of the effective dose and may therefore lead to a more effective therapy. Trefoil factors (TFF) are compact, non-mitogenic peptides that are important in the protection and repair of the intestinal epithelium. Accordingly, they are promising tools for treatment of acute colitis. GM lactococci that express TFF cure and prevent acute colitis [2]. This is remarkable because purified, luminal administered TFF are sequestered by the mucus. For this reason, TFF delivery by GM L. lactis requires less than 2500 fold lower amounts of TFF. The deliberate release of live GM bacteria, as would occur following medical application, raises legitimate concerns. We established adequate means for inheritable growth control of engineered L. lactis by genetically exchanging the chromosomal thymidylate synthase gene for the IL-10 gene [3]. 5-1 Peptide-mediated cell-cell communication in Gram-positive bacteria Jiro Nakayama (Dept. of Bioscience and Biotechnology, Kyushu Univ., Japan) e-mail: [email protected], Tel/Fax: [phone] Bacterial cells often communicate with each other by using chemical signal. Gram-positive bacteria often use peptide molecule as a communication signal within same species, while Gram-negative bacteria often use N-acylhomoserine lactone for this purpose. First example of the peptide-mediated cell-cell communication in Gram-positive bacteria was a peptide pheromone-induced conjugal transfer of a plasmid in enterococci. The peptide pheromones were found to be a seven- or eight-residue oligopeptides. These peptide pheromones induce the conjugal transfer of a corresponding plasmid specifically at picomolar concentrations. In the 1990s, a term “quorum sensing (QS)” was introduced to explain cell-density dependent regulatory system frequently found in bacteria. QS of Gram-positive bacteria is also usually controlled by peptide-mediated QS. Some lactic acid bacteria control bacteriocin production by the peptide-mediated QS. Staphylococci control expression of some virulence factors by QS mediated by cyclic autoinducing peptide, the so-called agr system. Recent genome sequence data has revealed the peptide-mediated QSs in some Gram-positive species. For example, Enterococcus faecalis carries fsr system that is homologous to agr system of staphylococci. We found that a peptide lactone termed GBAP functions as an autoinducing peptide in the fsr system, which induces virulent-related extracellular proteases. Lactobacillus plantarum carries lam system that is also homologous to agr system and we found a peptide thiolactone termed LamD558 as an autoinducing peptide which may be involved in control of biofilm formation. Furthermore, gene clusters which are likely to encode those cyclic peptide-mediated QS systems were found from genome data of some species of clostridia and listeria. Especially, a family of proteins (AgrB family) commonly found in these QS gene clusters are suspected to function as biosynthetic enzymes of cyclic autoinducing peptides. Amino acid sequence alignmnent of the AgrB family proteins showed two conserved histidine and cysteine residues which are well-known catalytic residues of cysteine protease family, suggesting that AgrB family proteins may process and cyclize the prepeptide to form the mature cyclic autoinducing peptide. Now we are screening inhibitors targeting this biosynthetic enzyme, which may offer a novel means of treating virulent and/or antibiotic resistant infections and quorum sensing researches in vivo or in vitro. 5-1 Quorum Sensing in Helicobacter pylori Shigeru Kamiya and Takako Osaki Department of Infectious Diseases, Kyorin University School of Medicine, Mitaka, Tokyo, 181-8611 Japan [email protected] < sp = 1 > < sp = 1 > Quorum sensing (QS) in Helicobacter pylori with production of autoinducer-2(AI-2) regulated by luxS gene has been reported, but its association with pathogenesis in H. pylori infection has not been clarified. We have determined the effects of a defined luxS mutation on the virulence of H. pylori. The luxS mutant displayed reduced colonization rate compared to its wild-type parent strain, TK1402 in a Mongolian gerbil model. The luxS mutant (HpKY08) was generated by natural transformation of PCR-amplified luxS::cat gene from TN2 DNA fragment (kind gift from Drs Ogura & Berg, Washington Univ.) into H. pylori TK1402 strain. The production of AI-2 by luxS mutant was nearly one-hundredth of that by TK1402 parent strain. At 1 month after infection, H. pylori was not detected by culture in luxS mutant strain infected gerbils. In contrast, H. pylori was detected by culture and RT-PCR in all the TK1402-infected gerbils (n = 5). No significant difference in the number of H. pylori adhered to gastric mucosa at 1 hr after inoculation was detected between luxS mutant and TK1402-inoculated gerbils. At 3 month after infection, serum antibody titers of TK1402-infected gerbils were significantly higher than that of luxS mutant infected gerbils and non-infected gerbils. Gastric inflammation in gastric mucosa and increasing of antibody to H. pylori was observed in TK1402-infected gerbil. Motility of luxS mutant was significantly weaker than that of TK1402, but there was no significant difference in adhesion activity and resistance to acidic conditions (pH = 3) in the presence of urea between luxS mutant and TK1402 strains. According to DNA microarray data, 58 genes including flaA, flaB and flaG were overexpressed in the luxS mutant, but 13 genes including omp31 and omp32 were underexpressed compared to gene expression in the wild type. These results indicate that QS in H. pylori is important for colonization of H. pylori in gastric mucosa of Mongolian gerbil. The decrease of infectivity by luxS mutant was implied to be associated with the decreased motility. 5-3 Evaluation of the role of luxS-mediated quorum sensing in the pathogenicity of Escherichia coli O157:H7 using germfree mice Byeonghwa Jeon, Kazuhiro Hirayama, Kikuji Itoh Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan The effect of a luxS mutation on the pathogenicity of Escherichia coli O157:H7 was investigated. A luxS mutation in E. coli O157:H7 did not cause any difference in Shiga toxin (Stx) production or the lethality in germfree BALB/c mice. However, Stx production by the luxS mutant was lower than that of the wild-type in vitro. Interestingly, Stx production was significantly lower under microaerobic conditions, probably due to the lower expression level of recA under microaerobic compared with aerobic conditions. In order to investigate bacterial interactions between non-pathogenic E. coli and E. coli O157:H7 via luxS-mediated quorum sensing, a model system was constructed using germfree mice and a luxS mutant of non-pathogenic E. coli from a murine isolate. Two groups of gnotobiotic mice were pre-colonized with either non-pathogenic E. coli or its isogenic luxS mutant and challenged with a luxS mutant of E. coli O157:H7. However, administration of the luxS mutant of E. coli O157:H7 to E. coli-monoassociated mice did not cause any differences in bacterial colonization or lethality in the mice. 5-4 Chemical Control of Quorum Sensing in Gram-Negative BacteriaTsukasa Ikeda¹, Tomohiro Morohoshi¹, Norihiro Kato¹, Takenori Ishida², Junichi Kato², Hisao Ohtake^{3 1}Department of Applied Chemistry, Utsunomiya University, Utsunomiya, Japan, Fax: [phone]Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Japan ³Department of Biotechnology, Osaka University, Suita, Japan Quorum sensing is one of the bacterial cell-to-cell communication mechanisms depending on cell population density. Several kinds of N-acyl-L-homoserine lactones (AHLs) have been identified as one of the signal compounds involved in this mechanism among a lot of gram-negative bacteria and called autoinducers. Quorum sensing controls production of several virulence factors and biofilm formation in gram-negative bacteria. For instance, Pseudomonas aeruginosa possesses two quorum sensing systems, las- and rhl-quorum sensing systems. The las system has been shown to activate the expression of several virulence factors, such as LasB elastase, LasA protease, alkaline protease, exotoxin A, and pyoverdin. The rhl system controls the expression of rhlI and rhlAB, which codes for a rhamnosyltransfarase required for rhamnolipid production. Therefore, the quorum sensing systems are an excellent target to reduce the pathogenicity of gram-negative bacteria. In this work, several kinds of chemically control methods of quorum sensing will be presented using several kinds of synthetic AHL analogues, AHL host compounds and AHL trap materials against not only P. aeruginosa, but also other kinds of gram-negative bacteria. One of the chemically synthesized AHL analogues, N-acyl-cyclopentylamide showed strong inhibitory effects on quorum sensing activities in P. aeruginosa and Serratia marcescens. Several kinds of cyclodextrins (CDs) and their derivatives showed complex formation with AHLs in the bacterial medium and also showed inhibitory effects on quorum sensing activities. Moreover, CD immobilized hydrogel sheets were prepared to also depress the quorum sensing activities. 6-2 Incidence and risk factors of early severe infections after unrelated cord blood transplantation. An Eurocord-Netcord registry analysis. Vanderson Rocha, Sylvie Chevret, Irina Ionescu, Federico Garnier, Eliane Gluckman on behalf of Eurocord-Netcord. Hopital Saint Louis, Paris, France. Fax [phone] We have analyzed retrospectively 510 UCBT performed from 1994 to 2002 in 55 centers. The median age was 6.9 years, the median follow-up for survivors was 36.6 months; 77% patients had hematologic maligancies, 13% inborn errors and 10% bone marrow failure. Conditioning was TBI based in 50% and associated to ATG/ALG in 86%. CsA and corticoides for GVHD prophylaxis was used in most of the patients. The donor was more frequently 5/6 or 4/6 HLA disparate. The median number of nucleated cells and CD34+ cells infused was 3.8x10⁷/kg and 1.6 x10⁵/kg. In 95% of the cases patients were transplanted in a HEPA room. Prophylaxis of infections varied among centers. In all patients, the definitons of infections during 100 days after UCBT proposed by the IWP were followed. Results: cumulative incidence (CI) of neutrophil recovery, acute GVHD (II-IV), and 100-days mortality were 75%, 38% and 32% respectively. CI at day-100 of first overall, bacterial, viral and fungal infections were respectively 69%, 49%, 32% and 10%. During the first 100 days, 686 severe infections episodes were diagnosed in 352 patients. A total of 404 episodes were from bacteria origin: 276 gram +; 124 gram- and 4 others, 189 severe viral infections or diseases episodes (142 CMV, 21 adenovirus, 12 EBV and 12 Herpes simplex or HHV6 and 2 para-myxo-virus), 54 episodes of severe fungal infections (26 candidemia, 20 aspergillus and 8 other), 5 episodes were of toxoplasmosis and 34 episodes were not classified. In a multivariate analysis the following factors decreased the cumulative hazard of 1) bacterial infections: shorter time to engraftment (p < 0.001); 2) viral infections: negative CMV serology (p < 0.001); less than 3 out of 6 HLA disparate graft (p = 0.03) and shorter time to engraftment (p = 0.002); and finaly for 3) fungal infections: recipient's age <16 years (p = 0.004), malignant disease (p = 0.03), shorter time to engraftment (p = 0.02); and absence of acute GVHD III-IV (p = 0.03). Considering all types of infections, period of UCBT performed (after 1998) was also associated with a decreased hazard of severe infections at day 100. In this retrospective analysis, bacterial infections appeared early, followed by viral and fungal infections during the first 100-days after UCBT. Delayed engraftment was frequently associated with increased risk of all types of infections. Aproaches that will improve time to engraftment after UCBT might decrease the incidence and severity of early infections after UCBT. 6-3 Viral infection in cord blood transplantation Satoshi Takahashi, M.D. The Institute of Medical Science, The University of Tokyo, Japan Viral infection remains a significant cause of mortality and morbidity following cord blood transplantation (CBT). Reactivated herpes viruses infections such as cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella-zoster virus (VZV) are common after allogeneic stem cell transplantation. We here report a single institutional observation addressing viral infectious complications after CBT. CMV infection after CBT was compared with that after bone marrow transplantation from HLA (human leucocyte antigen)-matched related (R-BMT) and unrelated (U-BMT) donors. Positive CMV antigenaemia was seen in 79% of CMV-seropositive patients at a median of 42 d after CBT, but in zero of four CMV-seronegative patients. This did not differ significantly from values of positive antigenemia in seropositive patients observed after R-BMT and U-BMT (66%, P = 0.22, and 60%, P = 0.15 respectively). Twenty five percent of HSV-seropositive patients developed HSV disease at a median of 92 days and the cumulative incidence of HSV disease was 27% at 12 months after CBT. None of nine HSV-seronegative patients developed HSV disease. The cumulative incidence of VZV reactivation after CBT was 80% at 30 months and median time of reactivation was 5 months after CBT (range 1.7-26 months). All the cord blood recipients who reactivated with herpes viruses responded well to antiviral therapy, except one has developed fatal CMV encephalitis. These results suggest that recovery of virus-specific immune responses is delayed, but might be established within certain period after CBT. 6-4 Fungal infection in cord blood transplantation Shigesaburo Miyakoshi Toranomon Hospital, Japan Fungal infection is one of the most fetal complications after stem cell transplantation (SCT). When fluconazole has been available since 1990, and used for prophylaxis against fungal infection after SCT, incidence of candida infections have been reduced, on the other hand, other pathogens are hi-lighted, especially aspergillus infection. Invasive Aspergillosis mortality is higher in SCT than in chemotherapy and epidemiology of invasive Aspergillosis in SCT consists of 3 phases, neutropenic periods, 70 to 90 days when acute GVHD occurs, and late period occurring chronic GVHD, incidence of fungal infections may be depend on the patients’ immune function after SCT. In late years, nonmyeloabrative and reduced intensity SCT for elderly patients have been developing, and transplanting from many kinds of stem cell sources such as bone marrow and peripheral blood stem cells of sibling donor, unrelated donor, and HLA mismatched donor as well as umbilical cord blood, but some investigators reported that intensity of conditioning regimens were not influenced to incidence of aspergillus infection among myeloabrative regimens and the others. Generally speaking, the risks of fungal infection after SCT are GVHD, use of steroid for GVHD, and prolonged period to engraftment in any setting of SCT. The incidence of fungal infections under the situation of cord blood transplantation is unclear and not understood. Today, I am going to present about the incidence of fungal infection after umbilical cord blood transplantation, especially using reduced intensity regimens. Free paper 1-1 Effect of clarithromycin on Mycoplasma pneumoniae infection in gnotobiotic mice Haruhiko Taguchi, Satoshi Kurata, Taki Manzoku, Takako Osaki, and Shigeru Kamiya Department of Infectious Diseases,Kyorin University School of Medicine Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan Tel: [phone] Ext: 3463 Fax: [phone]. E-mail: [email protected] We evaluated the antimicrobial activity of clarithromycin to Mycoplasma pneumoniae in gnotobiotic mice and attempted to show its anti-inflammatory activity in mouse pneumonia induced by sonicated crude antigen of M. pneumoniae. Treatment with clarithromycin reduced the burden of M. pneumoniae in the lung of this model, demonstrating histologically that clarithromycin has beneficial effect on M. pneumoniae infection. These results suggest that the changes in inflammation with clarithromycin treatment in M. pneumoniae infected mice were likely a consequence of the antimicrobial activity of clarithromycin. However, beneficial effects with a resultant decrease in inflammation by clarithromycin treatment were histologically observed in mouse pneumonia induced by M. pneumoniae sonicated antigen. These results indicate that clarithromycin has microbiologically and histologically beneficial effects on M. pneumoniae infection, and that its anti-inflammatory activity is independent of its antimicrobial activity. 1-2 EPEC infection in gnotobiotic piglets Splichal I.¹, Oswald E.², Trebichavsky I.¹, Hojna H.¹, Splichalova A.^{1 1}Institute of Microbiology, ASCR, Novy Hradek, Czech Republic, Fax: +(420) 491 478 264 ²Ecole Nationale Veterinaire, Toulouse, France, Fax: +(33) 561 193 975 Enteropathogenic E.coli (EPEC) are considered an important cause of diarrhea. Disease occurs after an intimate contact of bacteria with the host enterocyte and destruction of adjacent microvilli (adherence/effacement lesion). In this study, 1-week-old germfree pigs were intragastrically infected in isolators with 5x10⁶ CFU of human EPEC strain B171 (serogroup O111) or porcine EPEC strain1390 (serogroup O45). Clinical signs of enteritis (fever, diarrhea, vomitting and anorexia) were observed in the first day. Experimental animals were sacrificed seven days after the infection. Bacteria were cultivated from all samples of the gut, mesenteric lymph nodes and lungs but they were mostly absent in blood, spleen and liver. The levels of IL-8 but not IL-4, IL10 and IFN-γ were detected by ELISA in intestinal lavage of infected piglets on contrary to germ-free controls. No cytokine levels were detected in plasma of all groups. This work was supported by grant of the Czech Scientific Foundation 523/05/0249 and the Research Concept AV OZ 50 200 510 of the Institute of Microbiology of the Academy of the Czech Republic. 1-3 Effect of the indigenous microbiota on the resistance to infection L. Q. Vieira, R. Duarte, A.M. Silva, H. P. Silva, M. R. Oliveira, J. R. Nicoli Departamento de Bioquímica Imunologia and Departamento de Microbiologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG, Brazil. Our group has been interested on the effect of the indigenous microbiota on the immune response of hosts to infection. To address this issue, we have been utilizing germfree, mono-associated and conventional mice. Several infection models have been used. One of them was infection with the intracellular parasite Leishmania major. Studies using mice infected with L. major allowed the establishment of the dichotomy of the immune response to infections. Hence, susceptible mice present a type 2 response, while resistant mice present a type 1 response. The use of germfree mice allowed us to determine that the type response is not enough for the resistance to this parasite, since germfree mice are able to mount a type 1 response that is similar to that of conventional animals but are susceptible to infection. This susceptibility seems to be caused by a failure of germfree mice macrophages to kill the parasite. Therefore, germfree mice do not control parasite growth. Similarly, germfree mice are more susceptible to infection by another intracellular parasite, Trypanosoma cruzi. In this model, we have shown a higher mortality in germfree animals. Conventional mice present higher levels of IFN-γ, TNF-α and NO. In addition, the concentration of the IgG2a isotype waw shiehr in sera from germfree mice. When these mide were monoassotiated with components of the dominant and sub-dominant human intestinal microbiota, namely Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp, there was a direct correlation between higher survival in monoassotiated mice and a more potent type 1 response. Finally, we have shown that treatment with the probiotic Lactobacillus delbruckeii augments resistance of conventional mice to infection with the bacterium Listeria monocytogenes, This higher resistance was translated in a lower mortality of treated mice. The mechanism of this higher resistance is still obscure, since the number of bacteria in the viscera was similar in treated and non-treated mice. Besides, we could not detect higher levels of IFN-γ in sera of resistant mice. We conclude that the microbiota may prime the immune system to resistance to infections by intracellular parasites. 1-4 DNA stability and transformation studies of Acinetobacter baylyi in the gastrointestinal tract of gnotobiotic mice Lise Nordgård¹, Tore Midtvedt², Terje Traavik¹, and Kaare M. Nielsen^{1,3 1}Norwegian Institute of Gene Ecology, Science Park, N9294 Tromsø, Norway, ²Laboratory of Medical Microbial Ecology, Karolinska Institute, Stockholm, Sweden, ³Department of Pharmacy, Faculty of Medicine, University of Tromsø, N9037 Tromsø, Norway Background: Considerably amounts of foreign DNA are introduced into the gastrointestinal (GI) tract with food ingestion. Studies have shown that minor proportions of this DNA survive the passage through the GI tract and that it can be absorbed to reach white blood cells, the spleen and the liver, or be present in feces (Schubbert et al 1994). Furthermore, several bacterial species that are present in the GI tract are known to be competent for natural transformation (Lorenz and Wackernagel, 1994), suggesting that some bacteria can take up food-ingested DNA through natural transformation. The use of antibiotic resistance genes as marker genes in genetically modified organisms has put forward the question whether such antibiotic resistance genes can transform bacteria present in the GI tract in vivo. Aim: To quantify and characterize the stability of food-ingested DNA (chromosomal DNA and DNA present in bacterial lysates) in the GI tract of mice. To determine if natural transformation of the naturally competent bacterium Acinetobacter baylyi strain BD413 (AcBD413) occurs in gnotobiotic mice during the bacterial colonization process. Methods: For DNA stability studies, gnotobiotic mice were feed either purified bacterial chromosomal DNA or bacterial cell lysates containing chromosomal DNA. The mice were sacrificed at various time points and the gastrointestinal tract content sampled, the DNA content obtained through a bead-beating based Fast Prep protocol, and the quantity and quality of DNA determined through Real Time PCRs using primer set amplifying DNA fragments of increasing length. For the horizontal gene transfer studies, bacterial suspensions of BD413 cells was selected for the in vivo studies of natural transformation. Chromosomal DNA or bacterial lysates (both harbouring a selectable kanamycin-resistance gene) were given one day prior to the addition of the bacterial suspension. Of the 4 mice given DNA, only two were colonized with BD4103 cells and the reminder used as control of sterility. Samples of the GI tract contents from the stomach, the small intestine, the cecum and the large intestine were suspended in 4 ml 0,85% NaCl. Determination of the bacterial colonization level was done by plating 100 ul of ten-fold dilutions of the sample contents on Luria-Bertoni (LB) medium with selective antibiotics. CFU counts were determined after incubation of the plates at 32°C for 72h. Identification of transformants was done by plating 100 ul of ten-fold dilutions of the contents from the different parts of the GI tract on LB medium with transformant-selective antibiotics. The number transformants were determined after incubation of the plates at 32°C for 72h Results and conclusions: Our newly developed real-time PCR protocol allow a precise quantification of the presence of food-ingested DNA in different parts of the GI tract; enabling determination of the relative contribution of the normal microflora of mice in degrading food-ingested DNA. We observe that A. baylyi strain BD413 is capable of maintaining high numbers of CFU in the lower part of the GI of mice, although transformants remains to be detected. We are currently characterizing in detail the bacterial colonization process in order to describe the bacterial growth dynamics that may be necessary to observe the hypothesised occurrence of in vivo transformation of A. baylyi strain BD413. 1-5 The epidemiology of febrile neutropenia and the clinical outcome of antibiotic cycling Yoichi Tatsumi, Junichi Miyatake, Tetsuaki Sano, Mitsuhiro Matsuda, Takashi Ashida, Yasuhiro Maeda, and Akihisa Kanamaru1-5 The Hematology Department, Kink University School of Medicine, Japan1-5 Fax: [phone] E-mail; [email protected] In spite of the recent progress of chemotherapy and stem cell transplantation against hematological malignancies, the bacterial and fungal infection is still the most life threatening complication during treatment of hematological malignancies. In addition, recent increase of Methicillin resistant Staphylococcus aureus (MRSA) due to the abuse of certain antibiotics such as carbapenem, became a serious social problem. We analyzed microorganisms that caused bacteremia/fungicemia in our department during in the last 18 years. The results of isolated microorganisms between the recent period from 1997 to 2002 and the past period from 1985 to 1996 revealed the decrease of gram-negative bacteria and increase of gram-positive bacteria. Among the isolated microorganisms, staphylococcal species were predominant and MRSA has been increasing in the recent period. Moreover, pseudomonas aeruginosa was the next predominant but decreased in the recent period. When the microorganisms from the blood cultures of febrile neutropenia (FN) patients and from any samples of whole hospitalized patients were compared, the gram-negative bacteria were still dominantly isolated than gram-positive bacteria in the whole hospitalized patients, however, inversed ratio was recognized in the hematological patients and the whole patients. Then, we employed antibiotic cycling of four kinds of antibiotics for the primary treatment of FN, whether the resistant bacteria might decrease or not. The antibiotics were changed periodically in every three-months. For example, we used carbapenem for the first three months, penicilline for the second, fluoroquinolones for the third, and the fourth generation Cephalosporins for the last three months. The clinical efficacy of each antibiotic was evaluated at the end of final period. There was dramatic decrease of MRSA and MRSE during the penicillin-period, and drug resistant pseudomonas were also decreased during the same period. Our results indicated that antibiotic cycling might be useful for febrile neutropenia. To confirm the results, we are now performing second cycle of antibiotic cycling. 2-1 Injection of antigen-pulsed dendritic cells induces specific T cell proliferation and results in therapeutic vaccine effect against long-term Helicobacter pylori infection in mice Gotoh K, Otsu S, Yamagata J and Nishizono A Department of Infectious Diseases, Faculty of Medicine, Oita University, Oita, Japan Fax: [phone] e-mail: [email protected] Helicobacter pylori causes persistent infection of the stomach and results in gastroduodenal diseases. The current study tested the immunotherapeutic effects of intravenous injection of H. pylori specific antigen-pulsed dendritic cells (DCs) can reduce the colonized bacteria and the possible role of DCs for elimination of H. pylori. Mouse bone marrow-derived DC cell line, Jaws II cells, were incubated with live-H. pylori, formalin killed-H. pylori, and whole cell sonicated (WCS) bacterial lysate for 48 hr. H. pylori WCS pulsed-Jaws II cells showed significant secretion of TNF-α in the culture supernatant and expression of MHC class I, class II and co-stimulatory molecules on their surface, which resulted in induction of their maturation and acquisition of antigen presentation. Naïve T cells co-cultured with WCS-pulsed Jaws II cells showed potentiation of IFN-γ and IL-10 secretion of proliferation of T cell population in vitro. WCS pulsed-Jaws II cells were injected into mice with persistent H. pylori-infection and the curative rate was determined by reduction of bacterial load and evaluated by systemic and localized immune responses. Injection of WCS pulsed Jaws II cells reduced bacterial burden to less than 1/100 and upregulated IFN-γ and IL-10 production in the gastric tissue and correlated with the in vitro findings of cytokine response. The strong activation of CD4- and/or CD8-positive T cells induced by H. pylori antigen, especially WCS, suggests that pulsed-DCs can induce eradicative immune responses against H. pylori. The results indicate that specific antigen pulsed-DCs can induce Th1 and Th2 cellular immune response capable of bacterial eradication. 2-2 IL-18 is a critical factor for VEGF-enhanced migration in human gastric cancer cell lines Daeho Cho Department of Life Science, Sookmyung Women's University, Seoul, Korea, Fax: [phone] Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, which is a critical step in metastasis of tumor cells. Because cell migration is also important in metastasis, we examined human gastric cancer cell line, SNU-601, to investigate the relationship between VEGF and migration, and mechanism involved in VEGF-controlled migration. We confirmed that VEGF receptor-2 was strongly expressed on SNU-601 cells, and migration of SNU-601 cells was significantly increased by rhVEGF-165. Because interleukin (IL)-18 is associated with malignant progression of tumors, the effect of IL-18 binding protein (IL-18BP) was tested to identify the factor involved in the increased migration. The increased migration of SNU-601 cells was markedly reduced by IL-18BP. Next, to investigate whether rhVEGF-165 affect IL-18 production directly, RT-PCR and ELISA were used. IL-18 production was significantly enhanced by rhVEGF-165 at mRNA and protein level. Reactive oxygen intermediates (ROIs) level and ERK1/2 phosphorylation were then tested to determine signal transduction pathway. VEGF-enhanced IL-18 production was blocked by using antioxidant N-acetyl-L-cysteine and ERK1/2 specific inhibitor. Accordingly, rhVEGF-165 increased ROIs level and activated ERK1/2. Taken together, these results indicate that rhVEGF-165 enhances IL-18 production via ROIs generation and ERK1/2 phosphorylation, resulting in increased migration in gastric cancer cells. 2-3 Influence of intestinal non-culturable bacteria on colitis developing in SCID mice reconstituted with CD4 + CD45RB^high T cells and structural changes of tight junctions (TJs) Stepankova R.¹, Powrie F.³, Kofronova O.², Kozakova H.¹, Hudcovic T. ¹, Hrncir T.¹, Tlaskalova-Hogenova H.^{1 1}Department of Immunology and Gnotobiology and ²Laboratory of Electron Microscopy, Institute of Microbiology, Prague, Czech Republic, ³Sir William Dunn School of Pathology, University of Oxford, UK Background and aims: The transfer of the CD4 + CD45RB^high subset of T cells isolated from spleen of immunocompetent BALB/c mice into conventional SCID mice (mice with severe combined immunodeficiency) leads to the development of chronic intestinal inflammation. The aim of the study was to analyze the influence of intestinal bacteria on the development of colitis and the structure of tight junctions. Methods: CD4 + CD45RB^high subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and 3,5x10⁵ cells were transferred. i.p. into immunodeficient SCID mice. Recipient SCID mice were colonized by a defined cocktail of microflora (Bacillus sp., Lactobacillus lactis, delbruckii sp. bulgarcius, fermentum, catenaforme, Bacteroides distasonis, thetaiotaomicron, Peptococcus micros, ascharolyticus, Acinetobacter sp., Staphylococcus sciuri), and segmented filamentous bacteria (SFB). Other recipient SCID mice were monoassociated only with SFB bacteria, control SCID mice were either germfree or colonized with conventional microflora. 8-12 weeks after the cell transfer three parts of colon and terminal ileum were evaluated by histology and scanning electron microscopy. Immunohistological examination for CD4+, IL-10R, and IL-18R expressing cells and TJ structural proteins was performed. Bacteria were detected by in situ hybridization (FISH) using 16S rRNA oligonucleotide probes. Results: Colitis was present only in conventional SCID mice and in mice colonized with the defined cocktail microflora plus SFB bacteria. Monoassociation of SCID mice with SFB bacteria or with a cocktail of defined microflora did not lead to intestinal inflammation. SFB adhering to terminal ileum enterocytes were detected in colitic mice by scanning electron microscopy. Altered tight junctions (TJs) structure was detected in colitic mice using antibodies against ZO-1 and claudin-1 proteins. Increased numbers of CD4+ T cells in lamina propria and higher expression of IL-18R on colonocytes were found. Conclusion: Structural changes of TJs are an important feature of intestinal inflammation. Supported by EU contract QLGI-1999-00050, grant 303/04/0849 (Grant Agency of the CzechRepublic). 2-4 A linezolid antibiotic significantly enhances some murine immune responses R. Culbreath, B. Riling, M. Gooch, S. Khazaeli, D.J. Kitz S. Illinois University Edwardsville, Illinois 62026 USA [phone] Zyvox is a newer linezolid-class antibiotic produced by Pfizer and targeted for treatment of gram-positive bacterial infections, especially drug resistant Staphylococcus aureus, Enterococcus and Streptococcus species. Our studies were designed to determine if Zyvox was able to alter certain murine immune responses. Using peritoneal-derived phagocytic cells elicited by thioglycolate Difco, Zyvox had no measurable effect on neutrophil killing of yeast targets, while macrophage killing of yeasts was significantly boosted over a range of drug concentrations. The drug also significantly enhanced delayed type hypersensitivity response to the contact sensitizing chemical dinitrofluorobenzene Sigma. However Zyvox's effect on organ clearance of candidal yeasts administered intravenously was less clear, although yeast CFU from spleen, liver and kidney were all reduced in drug-treated animals, only the kidneys reduction was statistically significant compared to controls. These findings support the hypothesis that Zyvox may benefit the host through non-specific enhancement of certain immune responses. This work was supported in part by the LS-AMP Scholars Program, and the Max Baer Heart Fund, Fraternal Order of Eagles (Granite City, IL). 2-5 Effect of diet and lifestyle on risk of gastrointestinal infections and allergy in early life Elisabeth Norin, PhD Microbiology & Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden There are many differences in diet and lifestyle characteristics within Europe. For example, in the UK, breastfeeding rates are low and many infants are weaned before 3 months of age. In contrast, in Sweden breast feeding rates are high and infants are weaned later. Studying these differences in diet and lifestyle in different parts of Europe would allow identification of foods and feeding practices associated with gastrointestinal infections and allergy and to evaluate biomarkers of risk. One of the main ways that diet and environment influence the infant is through their effects on the gut bacterial flora and its metabolism. It is also well established that there are major differences between the bacterial colonisation of breastfed and formula fed infants. This is related to differences in different bacterial metabolites and bacterial enzymes. A better information with regard to biomarkers associated with risk of infection and allergy would furthermore allow improved targeting of dietary, feeding and product advices. This ongoing EU project INFABIO will show the influence of diet, feeding patterns and lifestyle in allergic and healthy infants on biomarkers and risk factors for allergy, infection and illness in several European countries with characteristically different feeding behaviour, complementary feeding and adult diets and lifestyles. Two intervention studies are also involved in the project – in the UK and in Spain – where it will be evaluated whether the addition of pre- or probiotics to infant formula can influence biomarkers and risk factors for allergy and infection. This project will also support a survey of consumers’ beliefs and attitudes to infant feeding and infant functional foods and identify factors influencing food choice and purchasing across Europe. 3-1 Culture-independent analysis of bacterial colonization in ex-germfree mice with human intestinal microbiota Kibe, R.^{1, 2}, Sakamoto, M.², Yokota, H.¹, Ishikawa, H.³, Aiba, Y.³, Koga, Y.³ and Benno, Y.^{2 1}Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan ²Microbe Division/ Japan Collection of Microorganisms, RIKEN BioResource Center, Saitama, Japan ³Department of Infections Disease, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan Fax No: [phone] (Microbe Division/ Japan Collection of Microorganisms, RIKEN BioResource Center) Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of human intestinal bacteria. Although many bacteria in human intestine have not yet been cultivated, there are few reports about HFA mice using molecular techniques to determine the composition of microbiota including unidentified bacteria. 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis were used to reveal the shifts in dominant bacteria of the intestinal microbiota in HFA mice after the administration of human fecal specimens. Moreover, the vertical transmission of intestinal microbiota in their offspring was also investigated. Common or characteristic T-RFs were detected from all samples. Increased T-RFs with time were at 184 and 196 bp, in contrast decreased T-RFs with time were at 366 bp. These T-RFs were assigned by 16S rRNA gene sequences. Most of intestinal bacteria with unique shifts in the intestine of HFA mice were unidentified bacteria. To compare the whole bacteria in samples, the dendrogram analysis based on the similarity of T-RFLP patterns among samples was used. These results indicated that microbial compositions of HFA mice and their offspring reflected the inoculum although they have some host-specific modifications. These host-specific modifications would be reflected by host-bacterial interactions, bacteria-bacterial interactions, physiological condition and diet differences between the human and mice. We need clarify the details of the reason based on this research. 3-2 Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite Sobko T, Reinders C, Jansson E Å, Norin E, Midtvedt T, Lundberg JO Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle in nature. However, little is known about NO production from commensal bacteria inhabiting the human intestinal tract. Considering the profound biological significance of NO in the gut we wanted to study if also luminal bacterial can generate NO. We used a sensitive and specific chemiluminescence assay to measure NO generation over time from human faeces incubated anaerobically on agar with/without nitrate and nitrite added in gas-tight bags. Also, the NO generating capacity was studied in gut commensals and probiotics when mono-inoculated on the plates. Finally, luminal NO levels were measured in vivo in germ-free, conventional rats, and in rats mono-colonized with Lactobacilli. In an anaerobic environment human faeces generated NO both from nitrate and nitrite. Lactobacilli and Bifidobacteria generated much NO from nitrite but only a few of strains produced NO from nitrate and at much lower levels. E. coli, Bacteroides and Clostridiae did not produce any significant amounts of NO either with nitrate nor nitrite. Nitrate reduction to nitrite is most likely enzymatic while nitrite to NO is predominantly non-enzymatic and caused by bacterial generation of acid, catalysing spontaneously decomposition of nitrite to NO. NO generation in the gut was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-colonized with Lactobacilli. We conclude that NO can be generated by the anaerobic gut commensal flora in the presence of nitrate or nitrite. Effective NO generation from nitrate seems to require sequential reduction involving several different bacteria. The biological influence of this NO on the intestinal ecosystem needs to be further studied. 3-3 Correlation between faecal iso-butyric and iso-valeric acids in different species Collinder E, Cardona ME, Tjellström B, Norin E, Midtvedt T Microbiology and Tumor Biology Centre, Karolinska Institutet, Nobels väg 16, S-171 77 Stockholm, Sweden The branched iso-butyrate and iso-valerate have been found to correlate strongly in different hindgut-fermenting species, although there are most likely few situations in the nature in which two variables are totally correlated, or closely. Of the short-chain fatty acids (SCFAs) in the hindgut, acetate, propionate and butyrate are mostly derived from carbohydrates, while iso-butyrate and iso-valerate from protein sources. We have investigated the totals of faecal SCFAs and the correlation between the fatty acids iso-butyrate and iso-valerate in four different species. The species were fed on different diets and housed in different environments. High differences in the total output of SCFAs were observed within and between species. Despite these differences, a remarkable correlation between the iso-butyric and the iso-valeric acids was found. The fact that the correlation is strong, irrespectively of age, diet and living conditions, suggests an endogenous common protein source actually reaching the hindgut of the species investigated; most probably deriving from intestinal sloughed cells. 3-4 Nutritional support of gomeostasis basis regulation systems is priority direction of preventive and rehabilitation medicine Shenderov B. A. Moscow State University of Food Processing; Moscow State Research Institute of Epidemiology and Microbiology, Russia During evolution all living being has grown up basis (water, mineral, microecology, symbiosis regulation system, oxidant/antioxidant) and secondary (immune, endocrine, nervous) regulation systems for gomeostasis support. Basis systems intrinsic to all earthly living being irrespective of their evolutionary and structural organization. The secondary systems are present in eucaryotic organisms only. In higher animals including homo sapiens there is hierarchism of co-operations among separate sections of unified gomeostasis system. Dysbalance in basis gomeostasis systems usually predisposes disorders in nervous-immune-endocrine system. The restoration of basis regulation system(s) is frequently enough to prevent the transition from functional disorders to the pathological processes and does not require interference in the work of gomeostasis secondary systems. Nutrition is supposed to be the most important factor that determines the health, physical and mental development of human being. Nutritional effects carries out through basis and indirectly secondary regulatory systems for gomeostasis support on the molecular, cell, near-cell space levels as well as levels of separate organs, tissues in the whole organism. Incidentally nutritional (functional) ingredients co-operate one with other and with the cell bioactive modulators. Production of such modulators is often induced with nutritional components. Understanding what way the nutrients can intervene in the structure, metabolic and behavior reactions of separate cells and the whole organism will discover the outlook for nutrition optimization of work of different host physiological systems. So keeping human being of today with food containing necessary quantity of “good” water answering the sanitary and physiology requirements, bioavailable essential minerals, probiotic bacteria or prebiotic substances supporting the host normal microflora balance, other bioactive compounds of various origin which can optimize the work of symbiosis regulation system and oxidant/antioxidant system is the priority direction of preventive and rehabilitation medicine. 3-5 Non-bacterially synthesized menaquinone-4 (vitamin K₂) is a key factor involved in testicular steroid metabolism Shirakawa H., Minegishi Y., Ohsaki Y. and Komai M. Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan Vitamin K (K) is well known for its role in post-translational activation of a family of nascent proteins to Gla-proteins, such as blood clotting factors and bone Gla-proteins. There are two forms of naturally occurring K: K₁, plant-synthesized phylloquionone; and K₂, a family of homologues designated as menaquinone-n (MK-n), which are synthesized by microorganisms including intestinal microflora. However, reports indicate that K analogues are converted into MK-4 in various organs outside the gastrointestinal tract without involvement of bacterial enzymes. Although studies indicate that MK-4 has unique functions related to cell differentiation, induction of apoptosis, and stimulation of transcription of a ligand for the nuclear receptor, these cannot totally explain the physiological significance of MK-4 synthesis in diverse tissues. To help elucidate the biological role of MK-4 production, we used germfree rats to eliminate MK-n synthesized by intestinal flora, and identified patterns of gene expression as a function of K status. Wistar germfree rats were fed either K-deficient, Control (normal level of K₁), K₁-supplemented, or MK-4-supplemented diets. We obtained testicular gene expression profiles using DNA microarray techniques. Expression of genes involved in cholesterol synthesis and isoprenoid metabolism was down-regulated in the K-deficient group compared with K₁-supplemented animals. Quantitative RT-PCR analysis revealed that testicular MK-4 concentrations were positively correlated with expression of CYP11a, a rate-limiting enzyme involved in testicular testosterone synthesis. Moreover, plasma testosterone concentrations were reduced in K-deficient relative to Control and MK-4-supplemented animals. Together, these results suggest that testicular MK-4 could be a key factor involved in steroid hormone synthesis via regulation of gene expression related to steroidogenesis. Poster 1 Anti-inflammatory and anti-microbial activities of clarithromycin in gnotobiotic mouse monoassociated with Mycoplasma pneumoniae Satoshi Kurata, Haruhiko Taguchi, Taki Manzoku, Takako Osaki and Shigeru Kamiya Department of Infectious Diseases, Kyorin University School of Medicine Shinkawa, 6-20-2, Mitaka-si, Tokyo 181-8611, Japan Tel:[phone] ex.3469 Fax:[phone] E-mail:[email protected] Mycoplasma pneumoniae is a pathogenic agent responsible for numerous outbreaks of acute respiratory infection. However, the mechanism by which primary atypical pneumonia caused by M. pneumoniae has not been clarified. We evaluated the antimicrobial activity of clarithromycin to M. pneumoniae in gnotobiotic mice and attempted to show its anti-inflammatory activity in mouse pneumonia induced by sonicated crude antigen of M. pneumoniae In Addition, we examined the effect of clarithromycin on cytokine production by human alveolar type II-like epithelial cells A-549. As a result, clarithromycin showed antibacterial activity in the gnotobiotic mice. Pulmonary inflammation in the SPF mice caused by M. pneumoniae antigen and cytokine production by A-549 cells were reduced by the treatment with clarithromycin. From the present study, it was demonstrated that clarithromycin has anti-inflammation activity as well as anti-microbial activity against M. pneumoniae infection. 2 Antagonism of human infant intestinal flora against Escherichia coli O157:H7 infection in gnotobiotic mice related to the effects of organic acids Yoshika Momose, Kazuhiro Hirayama, Kikuji Itoh Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. The protective role of intestinal flora against enteric infection is well known. Escherichia coli O157:H7 is one of the major causes of human enteritis. The aim of this study was to elucidate the mechanisms to prevent E. coli O157:H7 infection in human infants. We inoculated human infant feces into germfree mice and produced baby-flora-associated mice (BFA-3 and BFA-4). Orally challenged E. coli O157:H7 was eliminated from BFA-3, whereas BFA-4 became a carrier of E. coli O157:H7. Intestinal bacteria of BFA-3 and BFA-4 were isolated, and the mixtures were inoculated into germfree mice to produce two groups of gnotobiotic mice (GB-3 and GB-4). GB-3 and GB-4 reflected well the original characteristics of BFA-3 and BFA-4 related to whether or not E. coli O157:H7 was eliminated. The proliferation of challenged E. coli O157:H7 was suppressed in the cecum of GB-3 compared with that of GB-4. The growth rate of E. coli O157:H7 was faster in the GB-4 cecal suspension than in that of GB-3 in vitro under anaerobic conditions. Furthermore, acetic and lactic acid were detected from GB-3, and acetic and propionic acid from GB-4. The combination of acetic and lactic acid had a slight but not significant effect on the growth of E. coli O157:H7. However, this combination suppressed the motility of E. coli O157:H7 more than the combination of acetic and propionic acid under anaerobic conditions. The organic acid profile in the cecum might affect the elimination of E. coli O157:H7. Along with organic acids, other factors, such as nutritional substances, contained in the cecum may contribute to the growth and elimination of E. coli O157:H7. 3 Effect of circular lactic acid on the survival of gnotobiotic mice infected with enterohemorrhagic E. coli or murine cytomegalovirus Noriaki Nukanobu¹, Keiko Shimizu¹, Seiki Tazume¹, Mami Tsukiji^2, Mitsugu Hirano³, Yasushi Takei¹, Mikio Watanabe⁴, Masahiro Murakami^{5 1}Tokai University School of Health Sciences, Isehara, Japan ²Kanagawa Prefectural University of Human Service, Yokosuka, Japan ³Animal Care Co. Ltd., Tokyo, Japan ⁴Tokai University School of Science, Hiratuka, Japan ⁵Amato Pharmaceutical Co. Ltd., Kyoto, Japan The aim of the present study was to examine the effect of circular lactic acid (CLA) on the pathogenicity of enterohemorrhagic E. coli (EHEC) or murine cytomegalovirus (MCMV) in germfree (GF) mice. Five- to twelve-weeks-old BALB/c GF male and female mice were used. In the case of EHEC-infected GF mice, sterilized CLA solution was administered per os using a stomach probe. EHEC was administered per os using a stomach probe on day 3 after the mice were placed on the CLA. The effect of CLA on the survival rate and EHEC adhesion to intestinal mucosal surfaces EHEC-infected GF mice were examined. The mice survival rate administered CLA was high in comparison with control group. This result indicated the administration of CLA protected GF mice from death by EHEC infection. There was no significant difference between the number of adherent bacteria of CLA injected and control group. From this result, CLA are not able to function as inhibitors of EHEC adhesion to intestinal mucosal surfaces. The verotoxin titres were high in the caecum of control group, but were lower in CLA-injected mice. This finding suggests the possible action of CLA as a toxigenicity-blocking agent with regard to EHEC. In the case of MCMV-infected GF mice, survival rate of MCMV-infected GF mice were showing no difference between the two groups. From this result, it was concluded that CLA do not exert inhibitory effects on protection of MCMV. 4 Synergy between levofloxacin and fosfomycin against Pseudomonas aeruginosa biofilms in a capillary biofilm system Kariyama R.¹, Mitsuhata R.¹, Uehara S.¹, Monden K.¹, Kato J.² and Kumon H.^{1 1}Okayama University, Okayama, Japan, (FAX) 086-231-3986 ²Hiroshima University, Higashi-Hiroshima, Japan To identify antibiofilm agents, experimental models of complicated urinary tract infections (UTI) are utilized. Kumon et al. used modified Robbins devices and reported the synergy between ofloxacin and fosfomycin against Pseudomonas aeruginosa biofilms. More recently, we began using a capillary biofilm system as an in vitro model. P. aeruginosa OP14-210 isolated from a patient with catheter-associated UTI was used. Biofilms were grown in glass capillary tubes under continuous flow conditions with artificial urine, and were observed by confocal laser scanning microscopy. To evaluate the effects of potential antibiofilm agents, levofloxacin (LVFX 10×MIC: 192 µg/ml) and fosfomycin (FOM 3×MIC: 80 µg/ml) were tested. BacLight staining was applied to assess the effects of treatment on the number of viable cells, and their distribution in biofilms. A GFP (green fluorescent protein)-producing strain was also used. The thickness of 2-day biofilms did not vary markedly after 18-h treatment with LVFX or FOM, either alone or in combination. After combined treatment with LVFX and FOM, live and dead cells were distributed throughout the vertical profile of the biofilms, while a higher proportion of dead cells was observed in the upper third of the biofilms after treatment with LVFX alone. Evenly distributed live cells dominated after no treatment or treatment with FOM alone. GFP-producing 1-day biofilms were irregular after 24-h treatment with both LVFX and FOM. Our previous findings regarding the synergy between fluoroquinolones and FOM were confirmed using the present capillary biofilm system. Potential antibiofilm agents are currently under investigation. 5 Gastric mucosal inflammation and serum antibody in the Mongolian gerbil by infection with Helicobacter pylori Takako Osaki, Shigehito Nakagawa¹⁾, Taki Manzoku, Tomoko Hanawa, Satoshi Kurata, Haruhiko Taguchi & Shigeru Kamiya Department of Infectious Diseases, Kyorin University School of Medicine, ¹⁾ Sanofi Aventis Group, Aventis Pharma. Ltd. During long-term infection on Mongolian gerbils with H. pylori, we examined how H. pylori contributes to the autoimmune response by checking the relation between the histopathological changes in the stomach and the antibody titers against the various antigens including HSP60 and Lewis antigens. Mongolian gerbils were inoculated with 1ml inoculums of 10⁹ CFU/ml of H. pylori TK1402 intragastrically for 2 consecutive days. Both microaerobic cultivation and real-time RT-PCR using primers of 16SrRNA evaluated colonization of H. pylori TK1402. Determination of serum antibody titers against various antigens, and both macroscopic and histopathological examinations of stomach were carried out at 0.5, 1, 2, 3, 4, 5 and 6 months after inoculation with H. pylori. Persistent infection with H. pylori in gastric mucosa was detected by real-time RT-PCR 6-month after infection, but no H. pylori was isolated 4 months after infection by cultivation. Infiltration with neutrophils and mononuclear cells was observed since 2 months after infection. Both intestinal metaplasia and gastric atrophy were also detected since 2 months after infection. The results by ELISA indicated that antibody titers against whole H. pylori antigens, H. pylori heat shock protein 60 (HSP60), and Escherichia coli GroEL were significantly higher in the infected gerbils than those of non-infected gerbils. After long-term infection with H. pylori for 18 months, marked atrophy of gastric mucosa and multiple cysts in the submucosa were observed in the glandular stomach of the infected gerbils. In addition, squamous cell papilloma with hyperkeratosis was observed in cardia of all the infected gerbils. These results indicate that evaluation of bacterial colonization during long-term infection can be done by real-time RT-PCR. Furthermore, it is possible that the raised antibodies to these antigens following long-term infection with H. pylori might play an important role for induction of mucosal damages in the stomach of the infected gerbils. However, the correlation between the production of these antibodies and the induction of gastric squamous cell papilloma remains to be determined. 6 Comparison of virulence-related genes by multiplex pcr among Escherichia Coli isolates from Kenya and Japan M.W. Njoroge^1,2, C.C.Bii¹, H. Taguchi², T.T Ouko¹, L.W Muita¹, N.Wamae¹ and S. Kamiya^{1 1}Center for Microbiology research, Kenya Medical Research Institute, Nairobi, Kenya. ²Division of Medical Microbiology, Department of Infectious Diseases, Kyorin University School of Medicine. Introduction: Escherichia coli is a major cause of bacterial gasteroenteritis that has been considered as an agent responsible for outbreaks in Kenya and developing countries, strains like EPEC, ETEC, and EAggEC are important pathotypes isolated from stool specimens. Methods: One hundred and twenty nine (129) E.coli isolates from Kenya (n = 82) and Japan (n = 47) from stool of children under 5 years presenting with diarrhea were investigated. Standard microbiological procedures, analytical profile index (API) 20E (Bio merieux, Marcy, France) was used for identification, and drug susceptibility was done using Disc Diffusion. Multiplex PCR was used to detect virulence-related genes; aggR, It, eaeA, vt, and astA. Results: There were 41 O-serogroups of E. coli detected (29) from Kenya and (12) from Japan. E coli strains from Kenya were most resistant to TET (70.7%), AMP (65.9%), SXT (68.3%) and CHL (22.0%) compared to Japanese strains. Multiplex PCR detected virulence-related genes in 82% of Kenyan isolates and 25% of Japan isolates. No virulence-related genes were detected in 74.5% of E. coli isolates from Japan Conclusion: Emerging multiple drug resistant has worsened management E. coli enteritis in Kenya and further studies needs to be carried out to ascertain the significant between multiple drug resistance and presence of virulence-related genes. 7 Immunoblot for detection of Helicobacter infections in laboratory mice I. Nilsson, B. Barup, I-S Quis, T. Wadström Dept Laboratory Medicine, Div Medical Microbiology, Lund University, Lund, Sweden Background: Mouse models for various human diseases are widely used in biomedical research. Some murine enteric biletolerant Helicobacter spp cause hepatobiliary and intestinal tract diseases in inbred laboratory mice strains, while other species probably belong to the gut indigenous microflora. This is a first systematic attempt to develop a specific serology to analyse the immune response in naturally colonized and infected mouse colonies. Aim: To evaluate the antibody responses to antigens of H. bilis, H. hepaticus and H. ganmani in mice sera obtained from five animal houses. Material and methods: Sera from 88 mice (BalbC, C57BL/6, C3H, IL10 deficient, ICOS -/-, CD8 -/-, IgA -/-xIL, IL18 -/-, SPF) were analysed by immunoblot using cell surface proteins of H. bilis (CCUG 38995) and H. hepaticus (CCUG 33637). For the H. ganmani (CCUG 47872) a whole cell lysate was employed. Proteins were separated in an 8-16% gradient gel and following transfer to PVDF membrane. Strips were probed with sera diluted 1/50. For interpretation, stained band patterns were compared to sera of experimentally infected mice of each species. From 33 of these mice genetic material were analysed by PCR-DGGE for comparison with results obtained by immunoblot. Results: A specific antibody reactivity to H. bilis was found in 23%, to H. hepaticus in 17% and to H. ganmani in 19% of tested mice sera and agreement between the immunoblot and PCR-DGGE was found in 82%, 91%, and 91%, respectively. Conclusions: Infection with enteric Helicobacter seems to be common in mice kept for animal experiments, independent of mouse strain. Intestinal and hepatic naturally infected laboratory mice may represent confounding factors in the interpretation of animal bioassays. A serological test with high specificity, such as immunoblot, could be of great value in health controls of laboratory mice. Sacrificing animals is not necessary using immunobased tests. 8 Oxytetracycline effects on murine immune function K. Walley, J. Hard, J. Knolhoff, R. Culbreath, N. Farley, S. Khazaeli, D.J. Kitz S. Illinois University Edwardsville, Illinois 62026 USA [phone] Antibacterial antibiotics have found applications for use in animal feed where they can not only influence composition of the normal flora, but also can play a role as growth factors, increasing the rate of weight gain in domestic animals. Oxytetracycline Pfizer is commonly incorporated into animal feed supplements, and our laboratory was interested in the possibility of effects on murine immune response. Using thioglycolate Difco elicited phagocytes from mice we found little drug effect on either neutrophils or macrophages. Oxytetracycline's effect on delayed type hypersensitivity response to dinitrofluorobenzene Sigma was determined, as was the drug effect on organ clearance of candidal yeasts administered intravenously. Oxytetracycline enhanced yeast clearance from the spleen and liver, but not from the kidneys. It is possible that the increased growth rates seen in domestic animals fed oxytetracycline are influenced by effects on the host immune system as well as effects on the host normal flora. This work was supported in part by the LS-AMP Scholar's Program. 9 Colonization of germ-free piglets with probiotic E. coli is associated with stimulated production of TNF-alpha and acceleration of brush border enzyme and glycoconjugate development Kozakova H.¹, Kolinska J.², Zakostelecka M.², Lojda Z.^{3† 1}Institute of Microbiology, ²Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Novy Hradek, ³First Faculty of Medicine, Charles University, Prague, Czech Republic, Fax: [phone] The effect of monocolonization with E. coli strains O83 and O86 on the production of TNF-alpha and on the development of digestive enzymes and glycoconjugates in originally germ-free (GF) colostrum-deprivated 22-day-old piglets was studied and compared with that observed in age-matched GF and conventional (CV) controls. We tested enzyme activities biochemically in enterocyte brush-border membranes and histochemically in cryosections. Compared to untreated GF piglets we found a reduced activity of lactase, ?-glutamyltransferase and alkaline phosphatase and elevated activities of maturation markers sucrase in jejunum and glucoamylase in duodenum and jejunum Colonization with E. coli O83 significantly increases production of TNF-alpha and its release into the serum compared with GF piglets. This result is in accordance with the concept that TNF-alpha is involved in cellular cross-talk between epithelial and immune cells challenged with bacteria. ELISA tests using specific lectins revealed alterations in the expression of sialylated and fucosylated glycoconjugates of the brush borders. E. coli strains contribute to precocious maturation of specific glycosylation – appearance of fucosylated glycoforms, both alpha-1,6-fucosylated and alpha-1,2-fucosylated moieties, the highest effect being seen in the duodenum. On the other hand alpha- 2,6-sialylated glycoforms were found partially reduced, predominantly in the ileum. Grants A5020101 (Acad.Sci. CR), 303/05/2249 of the Czech Science Foundation of the Czech Republic. 10 Intestinal microflora does not affect tolerance induction to birch pollen allergen in mice Kozakova H.¹ and Repa A.^2,3, Stepankova R.¹, Hrncir T.¹, Hudcovic T.¹, Tlaskalova-Hogenova H.¹, Wiedermann U.^{2 1}Institute of Microbiology of the Academy of Sciences of the Czech Republic, Novy Hradek, Czech Republic, ²Institute of Pathophysiology, University of Vienna, Austria, ³Department of Neonatology and Intensive Care, University Children's Hospital Vienna, Austria The aim of our work was to study the development of mucosal tolerance to major allergic component of birch pollen-Bet v1 in relationship with presence of intestinal microflora. Germ-free (GF) or conventionally reared (CV) BALB/c mice were intragastrically or intranasally pre-treated with Bet v 1 prior to sensitization performed by subcutaneous injections of Bet v 1 with aluminum hydroxide as adjuvans. Allergen-specific serum antibodies were determined by ELISA and allergen-specific IgE secretion was evaluated by rat basophil degranulation assay in vitro. Cellular immune response was demonstrated by spleen cell proliferation and IL-5 production was examined using ELISA kit. Allergen-specific antibody level (IgE, IgG1, IgG2a) was comparable in Bet v 1 sensitized GF and CV BALB/c mice. Oral as well as intranasal tolerance induction led to a significant reduction of allergen-specific antibody levels and IgE mediated basophil degranulation, as well as cytokine production in vitro in both GF and CV mice. Our data indicate that the absence of the microflora has no effect on the development of allergic responses or the ability to induce tolerance using a clinical relevant allergen. Supported by QLK3-CT-2000-000340 and 303/05/2249 of the Czech Science Foundation of the Czech Republic. 11 Influence Of Normal Microbiota On Some Aspects Of The Immune Response During Experimental Infection With Trypanosoma Cruzi In Mice Duarte R.¹, Silva A.M.¹, Vieira L.Q.², Crocco-Afonso L.C.³ and Nicoli J.R.^{1 1}Departamento de Microbiologia and ²Departamento de Bioquímica-Imunologia, ICB – Universidade Federal de Minas Gerais, Belo Horizonte; ³Departamento de Ciências Biológicas – ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil. To study the influence of normal associated microbiota on systemic immunological responses during experimental Chagas’ disease, germ-free and conventional NIH Swiss mice were infected with Y strain of Trypanosoma cruzi. Conventional mice showed a slightly higher survival than the germ-free ones as well as, seven days after the infection, a tendency to a lower parasitemia. Additionally, higher IFN-γ, TNF-α and NO productions (P < 0.05) by spleen cell cultures and higher blood levels of specific immunoglobulins of the IgG2a isotype (P < 0.05) were observed in conventional animals when compared to their germ-free counterparts. On the other hand, germ-free mice showed higher production of IL-10 by spleen cell cultures (P < 0.05). In conclusion, the presence of the normal microbiota induces a more efficient Th1 immune response during an experimental infection with T. cruzi in mice. 12 Serotypes and Azoles resistance in Cryptococcus neoformans MAT a from Clinical sources in Nairobi, Kenya Bii C.C.^1,2, Makimura K.³, Abe S, Taguchi H.², Mugasia OM.¹, Revathi G.⁴, Wamae CN.¹ Kamiya S.^{2 1}Center for Microbiology Research, Kenya Medical Research Institute. ²Kyorin University School of Medicine, Japan. ³Teikyo Institute of Medical Mycology, Japan. ⁴Kenyatta National Hospital, Nairobi, Kenya. Strains/methods: The serotype and mating types of eighty clinical isolates of Cryptococcus neoformans from Kenya were determined and subjected to broth microdilution susceptibility testing to amphotericin B, flucytosin, fluconazole, itraconazole and miconazole. Results: C. neoformans variety grubii (serotypes A) predominated C. neoformans variety neoformans [75/80 (93.7%) versus 3/80 (3.8%)]. Two of eighty isolates (2.5%) were identified as Cryptococcus neoformans variety gattii (serotype B). Mating experiment confirmed all the isolates to be a mating type. The 28S rDNA sequence showed that majority of the isolates were homologous with F. neoformans var. grubii (serotype A) accession no AF335984. Seventy eight (97.5%) of the isolates were susceptible [minimum inhibitory concentration (MIC) of = 0.5 µg/ml] to amphotericin B. The MIC in which 90% of the isolates were inhibited (MIC₉₀) by flucytosin and fluconazole was 64 µg/ml each while for itraconazole and miconazole were 0.5 and 2 µg/ml respectively. Fluconazole resistance (MIC =64 µg/ml) was detected in 9/80 (11.2%) of the isolates. Conclusion: The study is the first report of serotypes and mating types in C. neoformans from clinical sources in Kenya highlighting the existence of azoles resistance in a significant proportion. It further shade light to the epidemiology and virulence associated with cryptococcal infection and the need for antifungal drug resistance surveillance as prophylactic use of fluconazole increases due to human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in sub-Sahara Africa. 13 Effects of butyrate on the production of inflammatory and anti-inflammatory mediators and COX-2 expression in peritoneal macrophages from germfree mice and conventional mice Hideo Ohira, Yukiko Toyama, Chikae Katagiri, Sanae Takahashi, and Masamichi Ikeda Laboratory of Nutritional Pathology, Faculty of Nutrition, Kobegakuin University, Kobe Short-chain fatty acids (SCFAs) which are produced from dietary fiber and indigestible oligosaccharides by the fermentation of intestinal bacteria have been found to act as potent antiproliferative and differentiating agents in various cancer cell lines besides having a role as the main energy source in the colonic mucosa. However, the effect of SCFA on inflammatory responses and immune functions are unclear. We investigated the effects of butyrate on the production of inflammatory (IL-1β, TNF-α and PGE2) and anti-inflammatory (IL-10) mediators in macrophages from germfree (GF) and conventional (CV) mice. Thioglycolate-elicited peritoneal macrophages from IQI/Jic GF and CV mice were seeded into 24-well plates in culture medium. Cells were stimulated with Lipopolysaccharide (LPS), and treated with sodium butyrate. Cell-free culture supernatants were collected, and used for the assay of cytokines and PGE2. LPS induced a substantial production of the IL-1β, IL-10 and TNF-α?, and PGE2 in macrophages from GF and CV mice. Addition of butyrate to these cultures inhibited dose-dependently the production of IL-10 and TNF-α. Butyrate markedly enhanced LPS-induced release of the IL-1β and PGE2 in macrophages from CV mice, but did not in macrophages from GF mice. It also increased expression of COX-2 mRNA in both GF and CV mice. These suggest that butyrate may play a role in sitmulation of inflammatory reaction. 14 Pivotal role for PPARg in the postembryonic maturation of the alimentary tract? Sven Pettersson, Department of Tumorbiology and Microbiology, MTC, The IRIS centre, Karolinska Institutet, Stockholm, Sweden Postembryonic gut homeostasis in mammals is influenced by epigenetic factors among them microbes. The epithelial lining of the intestine is one of the most important routes of entry for microbes into our body. Hence, the host must have developed mechanisms to attenuate activation of inflammatory signalling pathways to ensure gut homeostasis along the epithelial lining. In addition, there is a need to have these signalling pathways under a stringent integrated control by the host. Unwanted perturbation of gut homeostasis will elicit activation of the innate and the adaptive immune defence machinery. This is the situation in patients with Inflammatory Bowel Disease (IBD). Here the symbiosis between the residential intestinal flora and the epithelial cells is disrupted and these patients display a chronic inflammation that involves activation of NF-kB and its subsequent downstream arming of downstream targets. One type of regulator within the colonic epithelial cells appears to be the nuclear receptor PPARg. Through the use of in vivo experiments in animal models and ex vivo experiments in cell lines, we have identified a possible molecular mechanism whereby PPARg attenuate NF-kB activity. Data will be presented showing that 1) Bacteria can regulate PPARg expression through the Toll-Like-Receptor-4 signalling pathway 2) Bacteria can regulate NF-kB activity via PPARg independent of IkB. In conclusion, PPARg may be a gatekeeper of host innate immune responses in colonic epithelial cells and a possible second messenger integrating communication between prokaryotes and eukaryotes. 15 Transfer of resistance genes between Escherichia coli isolates in a gnotobiotic animal model A.M. Grønvold*, E. Norin**, H. Sørum*, T.M. L′Abée-Lund*, T. Midtvedt** *Norwegian School of Veterinary Science, Department of Pharmacology, Microbiology and Food Hygiene, P.O.Box 8146 Dep, N-0033 Oslo, Norway **Karolinska Institutet, Microbiology & Tumor Biology Center, S-171 77 Stockholm, Sweden Introduction: Genetic alteration is of outmost importance for bacteria when it comes to adaptation to changing environments. The spread of antibiotic resistance genes in a particular environment is driven by the selective pressure provided by antimicrobial agents. Today there are numerous examples of horizontal gene transfer by different mechanisms in various environments (Eede et al, 2004). The purpose of this project was to study transfer of antibiotic resistance genes between strains of Escherichia coli in an in vivo model. In addition, we wanted to study and compare the donor capability of a normal flora E. coli strain from a free-living animal with a clinical bovine pathogen E.coli strain in the course of antibiotic treatment. Materials and methods: Streptomycin resistant E. coli W9608 isolated from fecal material of an anaesthetized non-captive healthy brown bear was used as donor in the first experiment. Donor strain in the second experiment was multiresistant E. coli NVH 1067/03 isolated from a farmed newborn calf with severe diarrhoea. Recipient strain in both experiments was nalidixic acid resistant E. coli DH5a. A total of six adult male germfree NMRI/KI mice (Karolinska Institutet, Stockholm) were included in the study. The mice were caged one by one and maintained in two lightweight stainless steel isolators (Gustafsson BE, 1959). The mice were inoculated with 0.2 ml bacterial suspension by gastric intubation. At day 7 of the experiment antibiotic treatment was initiated. Four of the six mice were treated with 0.6 µg of dihydrostreptomycin-sulphate pr ml in the drinking water for 7 days. Fresh fecal samples were regularly obtained from the mice and plated on appropriate selective agar plates for counting. Transconjugant bacteria were verified by plasmid profiling and hybridization. Results: Donor and recipient strains in both experiments readily colonized the guts of the germfree mice. The numbers of transconjugants were stable the first period of both experiments. After five days with antibiotic treatment the numbers of transconjugants suddenly became significantly higher in the treated mice compared to the negative control in both experiments. The mechanism for this increase in the number of transconjugants is further investigated. Conclusion: This study indicates that selective pressure provided by antimicrobial agents result in an abrupt increase in the number of transconjugant bacteria after several days. 16 Biotherapy A new treatment in laboratory animal welfare Johannes Bergstedt, Anna-Karin Persson, Tore Midtvedt & Elisabeth Norin Microbiology & Tumor Biology Center, Karolinska Institute, Stockholm, Sweden Newly, a research group at Lund University, Sweden, found that their Sprague Dawley breeding unit were infected with a β-haemolysing group G streptococci in the vagina. This was found in an ordinary laboratory animal health control, performed at Statens Veterinärmedicinska Anstalt (SVA), Sweden – a microbe that is on FELASA′s “black list”. The animals originated from the BK/Scanbur Company in Stockholm, Sweden.There were three alternatives for treatment; • Kill all the animals and start a new breeding colony – time consuming and very expensive • Give all animals antibiotics – alterations of the flora composition, not nice to use antibiotics • Give all the animals a probiotic strain – but which? We choosed to administer all the females during the impedance measuring with Lactobacillus reuteri, and the males were inoculated on all genital areas with the same microbes, and furthermore, the same microbes were given to the other animals at the breeder with the drinking water. The administration was repeated after four weeks.Thereafter, samples from the animals were investigated in a blind fashion, and after two treatment periods, all animals were without the β-haemolysing group G streptococci. In order to avoid further transfer of infections agents between animals in the breeding colonies, new routines were applied for the mating control, as we found that the inpedens instrument was part of the infectious carrier. After this occasion, no more infections have occurred. 17 Probiotic Lactobacillus and Bifidumbacterium lectins: isolation, physico-chemical characteristics and biological activity Lakhtin V., Lakhtin M., Pospelova V.B, Shenderov B.A. Moscow State Research Institute of Epidemiology and Microbiology, Russia. Lectins (natural or artificial nonimmunoglobulin origin proteins/glycoproteins) are able to connect noncovalently with carbohydrate targets. Lectins and lectin-like compounds of probiotic lactobacilli (L.acidophilus K3-III-24; 100AIII; NK1; L. plantarum 8RA3) and bifidumbacteria (B.adolescentis MC42) have been investigated. Bacteria have been grown in semi-anaerobic condition using casein-yeast broth. Cultural media were ultracentrifugated for isolation of 30kD compounds which were then divided with isoelectrofocusing (pH 2-6) in polyacrylamide gel in the presence of urea and sucrose. Compounds isolated were electrotransferred to immobillon P. Blots received were then treated with a set of biotylated artificial linear homopolysaccharides. Determination of polymers formed was carried out using conjugate of streptividin peroxidase. Chemiluminescence registration of peroxidase bound to blots was performed using BioChemi camera. Two fields and individual compounds with mannan-alpha (phosphated or not)-and galactan-beta- connecting lectin characteristics were determined in lactobacilli and bifidumbacteria strains investigated. Lactobacilli produced spectrum of lectins or lectin-like compounds with much more affinities to polysaccharides tested than bifidumbacteria. Interaction of isolated lectins with T- and B- lymphocytes, macrophages and thrombocytes will be demonstrated. The importance of lectin-like compounds for probiotic lactobacilli and bifidumbacteria intestinal colonization, in recognition of different immune cells, in immune reaction modification, cell mitoses, blood coagulation, the other physiological and metabolic reactions will be discussed. 18 Influence of predominant bacteria from indigenous digestive microbiota on experimental infection with Trypanosoma cruzi in mice R. Duarte,¹ A.M. Silva,¹ L.Q. Vieira,² L.C.C. Afonso,³ and J.R. Nicoli^{1* 1}Departamento de Microbiologia and ²Departamento de Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte; ³Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil In order to verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with Y strain of Trypanosoma cruzi. All the mono-associations induced a predominantly Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival (P < 0.05) of E. faecalis-, B. vulgatus- and Peptostreptococcus-associated mice and increased IFN-γ and TNF-α. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, lower IL-10 production was found in mono-associated mice after infection (P < 0.05) when compared with germ-free animals, except for E. faecalis-associated animals. Interestingly, spleen cell cultures from non-infected germfree and mono-associated mice spontaneously produced higher levels (P < 0.05) of IL-10 than cultures from mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component. 19 Germfree silk protein is an excellent food additive and a resource for silk cosmetics, and germfree silkworm pupa is an ideal medium of coldyceps militaris for producing an indispensable health food Motoyuki Sumida¹, Fujiyoshi Matsubara², Daizaburo Yasunaga³, Takako Komuratani², and Keiko Gohda^{2 1}Kyoto Institute of Technology, Department of Applied Biology, Kyoto, Japan, Fax:[phone], ²Matsubara Research Institute of Germfree, All the Year-round Sericulture System, Osaka, Japan, Fax: [phone], ³With Love, Inc., Osaka, Japan, Fax:[phone] A germfree sericulture system we established produces germfree silkworm larvae, germfree silk cocoons and germfree silkworm pupae which are inside the cocoons. Germfree silkworm larvae are resources for a health food after freeze-drying. Germfree silk cocoons are resources for producing germfree silk proteins. Germfree silk proteins are resources of food additives for making special pudding, bread, tofu, noodle, coocked rice and instant bouillon. The addition gives the food 1) an additional nutritious value, 2) a good taste, and 3) functionality. 1) An additional nutritious value is due to the fact that silk protein is similar in an amino acid composition to human beings. 2) A good taste is due to a taste of silk proteins themselves and their texture. 3) Functionality is due to several effects of silk proteins functionality such as anticancer function of sericin, antioxidant function, enhancement of alcohol metabolism, stimulation of insulin secretion, suppression of increase in blood cholesterol concentration, an agent to cure Parkinson's disease and suppression of melanine formation. Germfree silk proteins are resources for producing silk cosmetics such as silk soap, silk lotion and silk cream. Health foods made from Coldyceps militaris using germfree silkworm pupae as a medium helps people maintain and enhance a healthy life. C. militaris suppresses growth of cancer cells and even cures cancer patients. We encourage people to establish many plants of germfree sericulture system all over the world and produce germfree products and start helping people who suffer from diseases and various stresses recover and live a healthy life. 20 Malate Excretion of Arabidopsis thaliana under Germfree Culture and Quantitative Trait Locus Controlling Aluminum Tolerance Kobayashi Y, Ohno T and Koyama H Laboratory of Plant Cell Technology, Faculty of Applide Biological Science, Gifu University, Gifu Japan, [phone] Aluminum in acid soil reduces yield of crop plants, which is mainly caused by the inhibition of root growth. QTL (quantitative trait loci) analysis could be one of other useful approach to clarify the complex nature of Al tolerance. QTL analysis of Aluminum (Al) tolerance was performed with Ler/Cvi recombinant inbred (RI) lines of Arabidopsis thaliana. Relative root length (plus 4 µM Al/no Al at pH 5.0) on day 5 was used as Al tolerant index for the QTL analysis. Two single factor QTLs were detected on the top of chromosome 1 and bottom of chromosome 3. These QTLs can explain 40% and 16% with positive effect of Cvi allele, respectively. Malate excretion form roots is one of Al-tolerance mechanism in Arabidopsis thaliana. Then, we measured malate excretion under Germfree Culture of ten tolerant and sensitive RI lines. Malate excretions from the RI lines having Cvi allele on the QTL of chromosome 1 were greater than those from sensitive ones (Ler allele) under the only Al stress. On the other hand, no significant difference was observed between alleles of QTL on chromosome 3. It is possible that the genomic region, top of chromosome 1, involves a key gene controlling malate excretion of Arabidopsis. In the future work, isolation and identification of this key gene would be major targets of QTL studies. 21 Clarification of novel functions of vitamin K using genome-wide expression analysis in liver of germfree rats Ohsaki Y., Shirakawa H. and Komai M. Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan Vitamin K (K) is well known for its role in the post-translational activation of a family of nascent proteins to Gla-proteins, such as blood clotting factors and bone Gla-proteins. There are two forms of naturally occurring K: K₁, plant-synthesized phylloquionone; and K₂, a family of homologues designated as menaquinone-n (MK-n), which are synthesized by microorganisms including intestinal microflora. Although it has been reported that K analogues are converted into MK-4 in organs outside the gastrointestinal system without involvement of bacterial enzymes, the physiological significance of converted MK-4 has not yet been clarified. Using germfree rats to eliminate MK-n synthesized by intestinal flora, we attempted to identify novel functions of MK-4. Wistar germfree and conventional rats were fed either K-deficient, Control (normal level of K₁) or high K₁-supplemented diet. Hepatic gene expression profiles were obtained using DNA microarray methods. Expression of acute phase proteins was up-regulated in K-deficient compared with K-supplemented animals. Northern analysis revealed that a₁-acid glycoprotein, fibrinogen ? polypeptide and metallothionein-2 were up-regulated in the K-deficient group. These results suggest that dietary K converted to MK-4 acts to suppress inflammatory response. Next, Wistar conventional rats were fed either Control or K₁-supplemented diet for 10 days. Following administration of lipopolysaccharide (LPS) intraperitoneally, plasma alanine aminotransferase and aspartate aminotransferase activities were significantly reduced in K₁-supplemented rats compared with Control animals. To investigate the mechanism of K₁-related inflammatory suppression, we measured expression of IL-6 mRNA in spleen cultured cells from mice after treatment with LPS and K. IL-6 expression was significantly suppressed by addition of MK-4, while there was no significant effect of K₁. Together, these results suggest that MK-4 converted from other K analogues could suppress inflammation through down-regulation of IL-6 expression. 22 Effects of different types of breakfast cereal on intestinal flora and skin conditions K. Hirayama¹, R. Arai¹, K. Itoh¹, R. Ide^{2, 3}, S. Mutoh^{3 1}The University of Tokyo, Tokyo, ²Kellogg (Japan) K. K., Tokyo, and ³Kagawa Nutrition University, Saitama, Japan It has been reported that intake of breakfast cereals can improve skin conditions. Improvement of the overall nutritional condition may play a role. Prebiotic effects of breakfast cereals may also contribute, because an increase of production of harmful substances in the intestine might be responsible for skin troubles. In the present study, the effects of breakfast cereals on the intestinal flora and skin conditions were investigated. Materials & Methods: Healthy young female volunteers were administered two different types of breakfast cereals, wheat bran cereal that is particularly rich in dietary fiber or brown rice cereal fortified with vitamins and minerals, for two weeks. Composition of fecal flora and intestinal environment were analyzed. Facial skin conditions were also examined. Results: After two weeks of wheat bran cereal administration, the populations of Enterobacteriaceae and Streptococcaceae significantly decreased. Fecal moisture decreased significantly and the properties of feces and defecation frequency were improved. Activity of -glucosidase increased significantly and the concentrations of putrefactive products decreased slightly. The effects of brown rice cereal on the intestinal environment were not obvious. On the other hand, brown rice cereal improved skin conditions, while the effects of wheat bran cereal were limited. Discussion: The present study suggests that dietary fiber that are particularly rich in the wheat bran cereal had beneficial effects on the colonic flora. On the other hand, well-balanced contents of vitamins, minerals and dietary fiber in the brown rice cereal might be important for skin health. The effects of long-term administration and combinations of different types of breakfast cereals should be investigated further.