SUMMARY Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure 32-␣-Man- (132) (18,55), and the O3-and O5-specific polysaccharides of Klebsiella strains are identical to the O8 and O9 polysaccharides of E. coli (8,18,29). Mannans of algal origin were found to exert antitumor activity (37). Such an activity could later be attributed also to the mannan-containing LPS of E. coli and Klebsiella strains (13, 37).The genetics of LPS biosynthesis in enteric bacteria is well documented in recent reviews (33,43,48,55). Two mechanisms, block and monomeric, have been described for O-polysaccharide synthesis (49). In the block mechanism, observed for Salmonella typhimurium and related Salmonella serotypes, the oligosaccharide repeating units are assembled on undecaprenol phosphate (antigen carrier lipid [ACL]) under the direction of rfb genes. The first sugar transferred was found to be galactose-1-phosphate, and the corresponding transferase gene was termed rfbP (48). The repeating units are polymerized under the direction of the rfc gene, which may be located outside of or within the rfb gene cluster (41). The chain length is controlled by the rol gene, located between gnd and his (3, 4). The monomeric mechanism, experimentally proven only for E. coli O8 and O9 (18, 55), consists of the direct and sequential transfer of the monosaccharide residues from their nucleotideactivated precursors to the nonreducing end of the growing polysaccharide chain.The synthesis of some O polysaccharides requires the rfe gene. According to this requirement, LPS biosynthesis can also be divided into rfe-dependent and rfe-independent pathways. The rfe gene, first described by Mäkelä et al. (31), was found to be essential for the synthesis of the O polysaccharide in Salmonella strains of O groups C1 and L, and E. coli O8 and O9 (18, 33) and more recently in E. coli O4, O7, O18, O75, and O111 (1, 23a). It was reported to determine the tunicamycinsensitive transfer of N-acetylglucosamine (GlcNAc)-1-phosphate from UDP-GlcNAc to undecaprenol monophosphate

Big mitogen-activated protein (MAP) kinase (BMK1), a member of the mammalian MAP kinase family, is activated by growth factors. The activation of BMK1 is required for growth factor-induced cell proliferation and cell cycle progression. We have previously shown that BMK1 regulates c-jun gene expression through direct phosphorylation and activation of transcription factor MEF2C. MEF2C belongs to the myocyte enhancer factor 2 (MEF2) protein family, a four-membered family of transcription factors denoted MEF2A, -2B, -2C, and -2D. Here, we demonstrate that, in addition to MEF2C, BMK1 phosphorylates and activates MEF2A and MEF2D but not MEF2B. The blocking of BMK1 signaling inhibits the epidermal growth factor-dependent activation of these three MEF2 transcription factors. The sites phosphorylated by activated BMK1 were mapped to Ser-355, Thr-312, and Thr-319 of MEF2A and Ser-179 of MEF2D both in vitro and in vivo. Site-directed mutagenesis reveals that the phosphorylation of these sites in MEF2A and MEF2D are necessary for the induction of MEF2A and 2D transactivating activity by either BMK1 or by epidermal growth factor. Taken together, these data demonstrate that, upon growth factor induction, BMK1 directly phosphorylates and activates three members of the MEF2 family of transcription factors thereby inducing MEF2-dependent gene expression.

Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniue 0 3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4-deoxy-~-arabinopyranose than that of LEN-1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-~-arabinopyranose. In the sensitive-type LEN-1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-I .Keywords: Klebsiella pneumoniae ; lipopolysaccharide; lipid A ; structure ; polymyxin.In gram-negative enteric bacteria such as Escherichia coli and Salmonella typhimurium, resistance to the polycationic cyclic peptide antibiotic polymyxin B is associated with extensive cationic substitution of phosphate groups of the cell surface lipopolysaccharide (LPS) [l -61. Phosphate groups occur in LPS in the lipid part (termed lipid A) and in the proximal inner core oligosaccharide [7]. Such capping of LPS phosphates by 2-aminoethanol and 4-amino-4-deoxy-~-arabinopyranose (~-Arap4N) results in a shift of the electrostatic net charge of the LPS molecule towards cationicity, which decreases the polymyxin-binding tendency of the LPS and, as a consequence, that of the bacterial cell [I, 2, 8-10]. The degree of substitution of LPS phosphates appears to be genetically regulated. In S. typhimurium, capping of LPS phosphate takes place as a result of mutations in the prnr gene locus, and structural investigation of the wild-type and resistant-type lipopolysaccharides by phosphorus NMR has revealed that the ester-linked 4'-phosphate of lipid A becomes almost stoichiometrically esterified with ~-Arap4N, and the glycosidically linked diphosphate of lipid A becomes esterified with 2-aminoethanol in the LPS of polymyxin resistant-type pmrA 166, FIN-00300 Helsinki, FinlandA: in the LPS of polymyxin-resistant mutants, ~-Arap4N and 2-aminoethanol were shown to be present in lipid A in linkages similar to those found in S. typhimurium [6]. Capping of LPS phosphates, which is expected to affect the surface charge of bacteria, thus seems to be a general phenomenon, which may have important implications to bacterial virulence.Whereas E. coli and S. typhimurium differ very little in their lipid A and inner co...

Olfactory receptor neurons (ORNs) were infected upon intranasal inoculation with the R404BP strain of neurovirulent influenza A virus. Virus-infected neurons and a small fraction of neighbouring uninfected neurons displayed apoptotic neurodegeneration substantiated by the immunohistochemistry for activated caspase-3 molecules and the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling method. However, virus infection was restricted within the peripheral neuroepithelium and all mice survived the infection. Virus-infected ORNs revealed upregulated expression of the Fas ligand molecules, activating the c-Jun N-terminal kinase signal transduction pathway. In addition, Iba1-expressing activated microglia/macrophages appeared to partake in phagocytic activities, eventually clearing apoptotic bodies. These results raise the possibility that induction of apoptosis in olfactory receptor neurons at an early stage of infection may provide protective effects against invasion of the neurovirulent virus from the peripheral to the CNS.

The apoptotic cell death induced in D-galactosamine-sensitized mice by administration of lipopolysaccharide was characterized. Administration of lipopolysaccharide caused apoptotic cell death in livers of D-galactosamine-sensitized mice. Apoptotic cells were also detected in the kidney, thymus, spleen, and lymph node. Severe hepatic apoptosis in D-galactosamine-sensitized mice was reproduced by transfer of the sera from mice injected with D-galactosamine and lipopolysaccharide. The hepatocyte apoptosis induced by lipopolysaccharide was completely prevented by an anti-tumor necrosis factor alpha antibody but not by an anti-gamma interferon antibody. Administration of recombinant tumor necrosis factor alpha into D-galactosamine-sensitized mice also caused hepatocyte apoptosis. Lipopolysaccharide-induced hepatocyte apoptosis in D-galactosamine-sensitized mice did not seem to be mediated by Fas antigen. It was suggested that lipopolysaccharide-induced hepatic injury and failure in D-galactosamine-sensitized mice was due to the apoptotic cell death of hepatocytes caused by tumor necrosis factor alpha released in the circulation.

The effect of quercetin on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied. Quercetin pretreatment significantly inhibited NO production in an LPS-stimulated RAW 264.7 murine macrophage cell line. Post-treatment with quercetin partially inhibited NO production. The inhibitory action of quercetin was due to neither the cytotoxic action nor altered LPS binding. The expression of inducible-type NO synthase (iNOS) was markedly down-regulated by quercetin. Quercetin suppressed the release of free nuclear factor (NF)-kappaB by preventing degradation of IkappaB-alpha and IkappaB-beta. Moreover, quercetin blocked the phosphorylation of extracellular signal regulated kinase 1/2 (Erk 1/2), p38, and c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and, further, the activity of tyrosine kinases in LPS-stimulated RAW cells. Quercetin also inhibited interferon (IFN)-gamma-induced NO production. Taken together, these results indicate that the inhibitory action of quercetin on NO production in LPS- and/or IFN-gamma-stimulated macrophages might be due to abrogation of iNOS protein induction by impairment of a series of intracellular signal pathways.

The expression of heat shock proteins (HSPs) as stress-induced proteins was studied in mice injected with D-galactosamine (D-GalN) and lipopolysaccharide (LPS) as an experimental endotoxic shock model. The expression of constitutive type heat shock protein 70 (HSC70) was significantly reduced in livers of mice injected with D-galactosamine and lipopolysaccharide, while its expression was unaffected in livers of mice injected with D-galactosamine or lipopolysaccharide alone. The expression of other constitutive type heat shock proteins, namely HSP60, HSP32 and HSP25 was also reduced in mice injected with D-galactosamine and lipopolysaccharide. On the other hand, inducible type HSP70 was detected in livers from mice injected with D-galactosamine and lipopolysaccharide, but not in livers from mice injected with D-galactosamine or lipopolysaccharide alone. Simultaneous injection of anti-tumor necrosis factor (TNF)-alpha antibody prevented the liver from reduced expression of constitutive type HSC70, and lead to marked expression of inducible type HSP70 in the liver. Reduced expression of constitutive type HSC70 was also found when D-galactosamine and recombinant TNF-alpha was injected. Therefore, TNF-alpha was suggested to play a critical role on altered expression of constitutive HSC70 and inducible type HSP70 in response of D-galactosamine-sensitized mice to lipopolysaccharide.