Blockade of B7-H1 suppresses the development of chronic intestinal inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4(+)CD45RB(high) T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4(+)CD45RB(high) T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-gamma, IL-2, and TNF-alpha, but not IL-4 or IL-10, by lamina propria CD4(+) T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.     

Glutamate Fermentation By‐product Activates Plant Defence Responses and Confers Resistance Against Pathogen Infection

In the food industry, glutamate fermentation by‐product (GFB) is generated by purifying glutamate products from microbial fermentation. The potential applications of GFB for upgrading agricultural soil, for foliar fertility, and as plant plankton for shrimp have been studied. We examined the efficacy of GFB foliar application and determined that GFB treatment increased the resistance of Arabidopsis leaves to infection by bacterial pathogens. Microarray gene expression analysis of Arabidopsis leaves after treatment with GFB indicated that the expression of plant defence‐related genes increased. In Corynebacterium fermentation, the active substances for induction of the defence response were extracted or solubilized after treatment with heating under acidic conditions. This extract was also effective in strawberry and grape leaves for the induction of hydrogen peroxide production. These findings suggest that foliar application of GFB that contains elicitor molecules derived from fermentation bacteria is useful for plant protection in agricultural fields.     

Non-enzymatic, template-directed ligation of 2'-5' oligoribonucleotides. Joining of a template and a ligator strand.

Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.     

Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles

Mice genetically deficient in growth and differentiation factor 8 (GDF8/myostatin) had markedly increased muscle fiber numbers and fiber hypertrophy. In the regenerating muscle of mice possessing FGF6 mutation, fiber remodeling was delayed. Although myostatin and FGF6 may be important for the maintenance, regeneration and/or hypertrophy of muscle, little work has been done on the possible role of these proteins in adult muscle in vivo. Using Western blot and immunohistochemical analysis, we investigated, in rats, the distribution of myostatin, FGF6 and LIF proteins between slow- and fast-type muscles, and the adaptive response of these proteins in mechanically overloaded muscles, in regenerating muscles following bupivacaine injection and in denervated muscles after section of the sciatic nerve. The amounts of myostatin and LIF protein were markedly greater in normal slow-type muscles. In the soleus muscle, myostatin and LIF proteins were detected at the site of the myonucleus in both slow-twitch and fast-twitch fibers. In contrast, FGF6 protein was selectively expressed in normal fast-type muscles. Mechanical overloading rapidly enhanced the myostatin and LIF but not FGF6 protein level. In the regenerating muscles, marked diminution of myostatin and FGF6 was observed besides enhancement of LIF. Denervation of fast-type muscles rapidly increased the LIF, but decreased the FGF6 expression. Therefore, the increased expressions of myostatin and LIF play an important role in muscle hypertrophy following mechanical overloading. The marked reduction of FGF6 in the hypertrophied and regenerating muscle would imply that FGF6 regulates muscle differentiation but not proliferation of satellite cells and/or myoblasts.     

The development of colitogenic CD4(+) T cells is regulated by IL-7 in collaboration with NK cell function in a murine model of colitis

We previously reported that IL-7(-/-)RAG(-/-) mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4(+) T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4(+) T cells. To further investigate these roles of NK cells, RAG(-/-) and IL-7(-/-)RAG(-/-) mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T(EM)) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44(+)CD62L(-) T(EM) and unique CD44(-)CD62L(-) T cell subsets were observed in the T cell-reconstituted RAG(-/-) recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44(+)CD62L(-) T(EM) subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG(-/-) and IL-7(-/-)RAG(-/-) recipient mice through targeting of colitogenic CD4(+)CD44(+)CD62L(-) T(EM) and, possibly, of the newly observed CD4(+)CD44(-)CD62L(-) subset present at the early stage of T cell development.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.     

Nucleotides enhance the secretion of interleukin 7 from primary-cultured murine intestinal epithelial cells

Our previous studies showed that dietary nucleotides fed to mice enhanced the secretion of interleukin 7 (IL-7) and transforming growth factor β (TGF-β) from intestinal epithelial cells (IECs). To explore whether nucleotides influence IECs directly to enhance the secretion of the cytokines or not, the effects of nucleotides added in vitro on the cytokine secretion from primary-cultured murine IECs were examined. When the mixture of nucleotide 5′-monophosphates (CMP, GMP, IMP, and UMP) or individual nucleotide 5′-monophosphates were added to the primary culture of IECs derived from BALB/c mice, the secretion of IL-7, but not that of TGF-β, was increased significantly. Addition of nucleotides to the culture did not alter the number of the IECs. Secretion of IL-6 and granulocyte-macrophage colony-stimulating factor, which are known to be secreted from IECs, was not enhanced by the addition of nucleotides. These results demonstrate that nucleotides can affect IECs directly to enhance the secretion of IL-7, and suggest that the increased secretion of TGF-β from IECs by dietary nucleotides was due to indirect effects of the nucleotides, which may affect intestinal microflora or cells other than IECs that in turn influence the cytokine secretion of IECs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8(+) T cells, and gammadelta T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells is characterized by significant increase of CD4(+)NKG2D(+) T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c(+) dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4(+)CD45RB(high) T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-gamma by lamina propria CD4(+) T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4(+) T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases.     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.