Hideyuki Yoshida

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of <i>IL-17A</i> expression. We have characterized the <i>IL-17A</i> promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated <i>IL-17A</i> promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated <i>IL-17A</i> promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes irreversible joint damage and significant disability. However, the fundamental mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unknown. Here, we show that signal transducer and activator of transcription 3 (STAT3) is the key mediator of both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines highly expressed in RA patients, such as IL-1β, tumor necrosis factor alpha and IL-6, activated STAT3 either directly or indirectly and in turn induced expression of IL-6 family cytokines, further activating STAT3 in murine osteoblastic and fibroblastic cells. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), a cytokine essential for osteoclastogenesis, and STAT3 deficiency or pharmacological inhibition promoted significant reduction in expression of both IL-6 family cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen-induced arthritis, in vivo through significant reduction in expression of IL-6 family cytokines and RANKL, inhibiting both inflammation and joint destruction. Leukemia inhibitory factor expression and STAT3 activation by IL-1β were mainly promoted by IL-6 but still induced in IL-6-deficient cells. Thus, our data provide new insight into RA pathogenesis and provide evidence that inflammatory cytokines trigger a cytokine amplification loop via IL-6-STAT3 that promotes sustained inflammation and joint destruction.

Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical "interferon signature genes" that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.

Abstract Sexual dimorphism in the mammalian immune system is manifested as more frequent and severe infectious diseases in males and, on the other hand, higher rates of autoimmune disease in females, yet insights underlying those differences are still lacking. Here we characterize sex differences in the immune system by RNA and ATAC sequence profiling of untreated and interferon-induced immune cell types in male and female mice. We detect very few differentially expressed genes between male and female immune cells except in macrophages from three different tissues. Accordingly, very few genomic regions display differences in accessibility between sexes. Transcriptional sexual dimorphism in macrophages is mediated by genes of innate immune pathways, and increases after interferon stimulation. Thus, the stronger immune response of females may be due to more activated innate immune pathways prior to pathogen invasion.

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.

Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator for cytokines; however, the role of SOCS1 in Th17 differentiation has not been clarified. We generated T cell-specific SOCS1-deficient mice and found that these mice were extremely resistant to a Th17-dependent autoimmune disease model, experimental autoimmune encephalomyelitis. SOCS1-deficient naive CD4(+) T cells were predominantly differentiated into Th1 and poorly into Th17 in vitro. These phenotypes were canceled in IFN-gamma(-/-) background, suggesting that a large amount of IFN-gamma in SOCS1-deficient T cells suppressed Th17 differentiation. IL-6 plus TGF-beta enhanced retinoic acid receptor-related orphan receptor (ROR)-gammat expression and suppressed IFN-gamma production in wild-type T cells, whereas these effects were severely impaired in SOCS1-deficient T cells. These phenotypes can be partly explained by STAT3 suppression by enhanced SOCS3 induction through hyper-STAT1 activation in SOCS1-deficient T cells. In addition, SOCS1-deficient T cells were much less sensitive to TGF-beta. Suppression of Th1 differentiation by TGF-beta was impaired in SOCS1-deficient T cells. TGF-beta-mediated Smad transcriptional activity was severely inhibited in SOCS1-deficient cells in the presence of IFN-gamma. Such impairment of TGF-beta functions were not observed in SOCS3-overexpressed cells, indicating that suppression of Smads was independent of SOCS3. Therefore, SOCS1 is necessary for Th17 differentiation by suppressing antagonistic effect of IFN-gamma on both STAT3 and Smads. Induction of SOCS3 can partly explain IFN-gamma-mediated STAT3 suppression, while other mechanism(s) will be involved in IFN-gamma-mediated Smad suppression. SOCS1-deficient T cells will be very useful to investigate the molecular mechanism for the STAT1-mediated suppression of Th17 development.

Objectives. Human endogenous uveitis is one of the sight-threatening diseases associated with variety of systemic disorders, such as Behcet's disease and sarcoidosis. Recently, biosynthesized antibodies against inflammatory cytokines have been recognized to be useful to control the regional inflammation. In this study, we focused on the possibility of IL-6-based biological therapies for endogenous uveitis. We initially confirmed the significant increase of several inflammatory soluble factors including IL-6 in the vitreous fluids from refractory/chronic engogenous uveitis patients.

Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50–100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II–rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

Interleukin 10 (IL-10) and regulatory T cells (Tregs) maintain tolerance to intestinal microorganisms. However, Il10(-/-)Rag2(-/-) mice, which lack IL-10 and Tregs, remain healthy, suggesting the existence of other mechanisms of tolerance. Here, we identify suppressor of cytokine signalling 1 (SOCS1) as an essential mediator of immune tolerance in the intestine. Socs1(-/-)Rag2(-/-) mice develop severe colitis, which can be prevented by the reduction of microbiota and the transfer of IL-10-sufficient Tregs. Additionally, we find an essential role for prostaglandin E2 (PGE2) in the maintenance of tolerance within the intestine in the absence of Tregs. Socs1(-/-) dendritic cells are resistant to PGE2-mediated immunosuppression because of dysregulated cytokine signalling. Thus, we propose that SOCS1 and PGE2, potentially interacting together, act as an alternative intestinal tolerance mechanism distinct from IL-10 and Tregs.

Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified topoisomerase 1 as a cardinal Aire partner that colocalized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.

JAK is believed to be an essential tyrosine kinase that mediates signals from the cytokine receptor to its downstream events. JAK associates with the cytoplasmic domain of the type I cytokine receptor superfamily and upon the ligand stimulation it can be activated, resulting in the receptor phosphorylation. In signaling from gp130, a common signal transducer for the IL-6 family cytokines, STAT3, a transcription factor that contains an SH2 domain, is recruited by phosphotyrosines on gp130 and is subsequently phosphorylated by gp130-associated JAKs. In this study, we attempted to find a new target for JAK that is directly activated by JAK, independent of gp130 tyrosine phosphorylation, by using a yeast two-hybrid system. In the process we found that the JH2 domain of JAK1, JAK2 or JAK3 could specifically associate with the carboxy-terminal portion of STAT5, but not with STAT3 or STAT1. The interaction was confirmed using both a transient expression system in a cell line and a GST-fusion protein binding assay. Furthermore, we showed that the activation of STAT5 via gp130 did not need any phosphotyrosines on gp130 while that of STAT3 strictly depended on phosphotyrosines on gp130. Mutations of STAT5 that eliminated the interaction with JAK1 reduced the activation of STAT5 upon the gp130 stimulation, although such mutants could be still activated through erythropoietin receptor. These results indicate that STATs are activated through cytokine receptors by two distinct mechanisms, one dependent on receptor tyrosine phosphorylation and the other mediated by the JAK-STAT direct interaction.

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.

The amino acid sequences of two closely related peptides from Gila monster (Heloderma suspecturn) venom are reported.Helospectin I is a 38-residue peptide, His-Ser-Asp-Ala-Thr-Phe-Thr-Ala-Glu-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala-Leu-Gln-Lys-Tyr-Leu-Glu-Ser-Ile-Leu-Gly-Ser-Ser-Thr-Ser-Pro-Arg-Pro-Pro-Ser-Ser, and helospectin I1 is a 37-residue peptide identical to helospectin I except that it lacks serine 38.Helospectins are pancreatic secretagogues with structures and bioactivities similar to vasoactive intestinal peptide and other members of the glucagon superfamily.The relative significance of helospectin-I and helospectin-I1 is presently unknown.Comparison of the 28 residues of vasoactive intestinal peptide with residues 1-28 of helospectin shows that identical amino acids occur in 15 positions.Since members of the glucagon superfamily have similar structures but different biological actions, it is possible that helospectin is more closely related to a mammalian peptide awaiting discovery.We previously reported that Gila monster venom contains a peptide(s) which stimulates amylase secretion from dis- persed guinea pig pancreatic acini by a mechanism involving activation of adenylate cyclase (1).The venom increases enzyme secretion and cellular CAMP to the same extent as VIP,' secretin, or PHI.The Gila monster secretagogue(s) have a higher affinity for VIP-preferring receptors than for secretin-preferring receptors.Interestingly, the secretagogue(s) do not cross-react with VIP-specific antiserum, indicating structural differences.We now report the amino acid sequences of helospectin-I (HS-I) and helospectin-I1 (HS-11) from Gila monster venom.

Type I interferons (IFN-α/β) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-α/β production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-α/β induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-κB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-β induction in this process, since normal IFN-α/β production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-β promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-β, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-β by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-κB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons. Type I interferons (IFN-α/β) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-α/β production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-α/β induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-κB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-β induction in this process, since normal IFN-α/β production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-β promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-β, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-β by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-κB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons. TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway.Journal of Biological ChemistryVol. 284Issue 52PreviewVOLUME 283 (2008) PAGES 36211–36220 Full-Text PDF Open Access The innate immune system serves as a first line defense against viral infection. Host antiviral responses are initiated thorough the recognition of viral components by PRRs, 2The abbreviations used are: PRRpattern recognition receptorTRAFTNF receptor-associated factorIFNinterferonTLRToll-like receptorRLHRIG-I-like helicasedsRNAdouble-stranded RNAJNKc-Jun N-terminal kinaseNF-κBnuclear factor-κBDCdendritic cellTRIFTIR domain containing adaptor-inducing IFN-βIKKIκB kinaseTIMTRAF-interacting motifMAP3KMAP kinase kinase kinaseMEFmouse embryonic fibroblastVSVvesicular stomatitis virussiRNAsmall interfering RNAHAhemagglutininRANTESregulated on activation normal T cell expressed and secretedcDCconventional DCMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseG3PDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionIP-10Interferon-γ inducible protein 10 including TLRs and RIG-I (retinoic acid-inducible gene I)-like helicases (RLHs) (1Beutler B. Eidenschenk C. Crozat K. Imler J.L. Takeuchi O. Hoffmann J.A. Akira S. Nat. Rev. Immunol. 2007; 7: 753-766Crossref PubMed Scopus (162) Google Scholar, 2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8918) Google Scholar, 3Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1444) Google Scholar). Upon recognition, the PRRs trigger intracellular signaling pathways that induce the production of antiviral mediators, such as type I interferons (IFN-α/β), IFN-stimulated genes, inflammatory cytokines, and chemokines, such as IP-10. The expression of type I IFNs and other antiviral proteins suppresses viral replication and facilitates the adaptive immune responses. dsRNA is one of the viral components recognized by TLR3 and RNA helicases, such as RIG-I and MDA5 (melanoma differentiation-associated protein 5). TLR3 recognizes extracellular viral dsRNA internalized into the endosomes in a certain type of cells, such as DCs, whereas RIG-I and MDA5 detect intracellular viral dsRNA in various types of cells, including fibroblasts (4Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1302) Google Scholar, 5Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3139) Google Scholar, 6Andrejeva J. Childs K.S. Young D.F. Carlos T.S. Stock N. Goodbourn S. Randall R.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17264-17269Crossref PubMed Scopus (823) Google Scholar, 7Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4965) Google Scholar). pattern recognition receptor TNF receptor-associated factor interferon Toll-like receptor RIG-I-like helicase double-stranded RNA c-Jun N-terminal kinase nuclear factor-κB dendritic cell TIR domain containing adaptor-inducing IFN-β IκB kinase TRAF-interacting motif MAP kinase kinase kinase mouse embryonic fibroblast vesicular stomatitis virus small interfering RNA hemagglutinin regulated on activation normal T cell expressed and secreted conventional DC mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase mitogen-activated protein kinase glyceraldehyde-3-phosphate dehydrogenase reverse transcription Interferon-γ inducible protein 10 The viral recognition by TLR3 and RIG-I/MDA5 results in rapid induction of type I IFNs through the activation of their intracellular signaling molecules (1Beutler B. Eidenschenk C. Crozat K. Imler J.L. Takeuchi O. Hoffmann J.A. Akira S. Nat. Rev. Immunol. 2007; 7: 753-766Crossref PubMed Scopus (162) Google Scholar, 2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8918) Google Scholar, 3Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1444) Google Scholar). For instance, TLR3 interacts with an adaptor molecule, TRIF (8Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar, 9Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Science. 2003; 301: 640-643Crossref PubMed Scopus (2526) Google Scholar), which in turn activates two IKK family proteins, TBK1 (TANK-binding kinase-1) and IKK-i (also known as IKKϵ) (10Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (590) Google Scholar). Both TBK1 and IKK-i subsequently activate a transcription factor, IRF3, resulting in the initial expression of IFN-β (11Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2100) Google Scholar, 12Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1374) Google Scholar). Another IRF (IFN-regulatory factor) family member, IRF7, which is induced by the initial IFN-β, elicits further induction of type I IFN genes, including IFN-α and IFN-β (13Honda K. Takaoka A. Taniguchi T. Immunity. 2006; 25: 349-360Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar). Stimulation with TLR3 ligand also activates other transcription factors, including NF-κB and AP-1, which is thought to synergize with IRF3 to induce type I IFN genes (14Theofilopoulos A.N. Baccala R. Beutler B. Kono D.H. Annu. Rev. Immunol. 2005; 23: 307-336Crossref PubMed Scopus (1022) Google Scholar, 15Kim T.K. Maniatis T. Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). On the other hand, RIG-I/MDA5 bind to intracellular RNA through the C-terminal helicase domain and initiate downstream signaling cascades through the N-terminal CARD domains (4Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1302) Google Scholar, 5Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3139) Google Scholar, 6Andrejeva J. Childs K.S. Young D.F. Carlos T.S. Stock N. Goodbourn S. Randall R.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17264-17269Crossref PubMed Scopus (823) Google Scholar). The CARD domains interact with another CARD containing molecule, IPS-1 (IFN-β promoter stimulator-1; also known as MAVS, VISA, and CARDIF) (16Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2036) Google Scholar, 17Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2523) Google Scholar, 18Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1521) Google Scholar, 19Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1976) Google Scholar), which activates TBK1/IKK-i via TRAF3, resulting in the activation of IRF3, IRF7, and NF-κB (20Häcker H. Redecke V. Blagoev B. Kratchmarova I. Hsu L.C. Wang G.G. Kamps M.P. Raz E. Wagner H. Häcker G. Mann M. Karin M. Nature. 2006; 439: 204-207Crossref PubMed Scopus (748) Google Scholar, 21Oganesyan G. Saha S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Nature. 2006; 439: 208-211Crossref PubMed Scopus (714) Google Scholar). Therefore, both TLR3 and RIG-I/MDA5 pathways converge at the TBK1/IKK-i kinase complex. IPS-1 contains multiple TRAF-interacting motifs (TIMs) in the proline-rich region, which can associate with the C-terminal TRAF domain of TRAF3 (22Saha S.K. Pietras E.M. He J.Q. Kang J.R. Liu S.Y. Oganesyan G. Shahangian A. Zarnegar B. Shiba T.L. Wang Y. Cheng G. EMBO J. 2006; 25: 3257-3263Crossref PubMed Scopus (348) Google Scholar). Furthermore, IPS-1 has been shown to interact with other TRAF family members, such as TRAF6 and its downstream MAP3K, TAK1 (transforming growth factor-β-activated kinase 1) (18Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1521) Google Scholar). Both TRAF6 and TAK1 have been demonstrated to play a critical role in the production of proinflammatory cytokines in macrophages and DCs triggered by MyD88 (myeloid differentiation factor 88)-mediated signals from the Toll-like/IL-1 receptor family (23Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (767) Google Scholar, 24Kobayashi T. Walsh P.T. Walsh M.C. Speirs K.M. Chiffoleau E. King C.G. Hancock W.W. Caamano J.H. Hunter C.A. Scott P. Turka L.A. Choi Y. Immunity. 2003; 19: 353-363Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). The TRAF6/TAK1 signal activates a canonical IKK complex, IKKα/β/γ, resulting in the activation of NF-κB as well as MAPK cascades leading to the activation of AP-1 (25Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar). Although TRAF6/TAK1 has been implicated in proinflammatory cytokine production induced by TLR ligands, the involvement of these molecules in the regulation of type I IFN production induced by the RLH pathway is largely unknown. In this report, we show that MEFs lacking TRAF6 exhibit a defect in the production of IFN-α/β in response to VSV infection and dsRNA, poly(I:C). On the contrary, MEFs lacking TAK1 showed normal induction of IFN-α/β. We also show that another MAP3K, MEKK1, instead of TAK1 is involved in the expression of IFN-β upon dsRNA stimulation. Both TRAF6 and MEKK1 are required for NF-κB activation but not for IRF3 activation, implicating the requirement of these molecules for the optimal induction of type I IFN in the cytosolic viral recognition systems. Cells and Reagents—Wild-type and TRAF6-deficient MEFs were prepared from day 13.5–14.5 embryos, as described previously (24Kobayashi T. Walsh P.T. Walsh M.C. Speirs K.M. Chiffoleau E. King C.G. Hancock W.W. Caamano J.H. Hunter C.A. Scott P. Turka L.A. Choi Y. Immunity. 2003; 19: 353-363Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). TAK1-deficient MEFs have been described previously (23Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (767) Google Scholar). MEFs and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fatal bovine serum (Cell Culture Technologies, Switzerland), penicillin, streptomycin, and glutamine (Nacalai Tesque, Japan). Conventional DCs were generated from the spleen of 2-week-old wild-type or TRAF6 knock-out mice, as described previously (24Kobayashi T. Walsh P.T. Walsh M.C. Speirs K.M. Chiffoleau E. King C.G. Hancock W.W. Caamano J.H. Hunter C.A. Scott P. Turka L.A. Choi Y. Immunity. 2003; 19: 353-363Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Briefly, erythrocyte-removed splenic cells were cultured in RPMI 1640 containing 10% fetal bovine serum and granulocyte-macrophage colony-stimulating factor for 7 days. Less adherent cells were collected by gentle pipetting and used as DCs. All experiments using these mice were approved by and performed according to the guidelines of the animal ethics committee of Kyushu University (Fukuoka, Japan). Poly(I:C) was purchased from Sigma. The anti-FLAG, M2 (Sigma), anti-T7 (Novagen), anti-HA, HA.11 (Covance), anti-IRF3 (Zymed Laboratories Inc.), and anti-p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies were used. The anti-phospho-IκBα, anti-phospho-p38, anti-phospho-JNK, anti-IκBα, anti-p38, and anti-JNK antibodies were purchased from Cell Signaling Technology. IKK-2 Inhibitor VI, U0126, SP60025, and SB203580 were purchased from Calbiochem. Plasmids—The expression vectors for FLAG-IPS-1, FLAG-RIG-I, and IFN-β-luciferase reporter plasmid have been described (16Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2036) Google Scholar). T7-IPS-1 and mutant RIG-I (FLAG-RIG-IN) were subcloned into T7 vectors. The T7-tagged MEKK3 expression vector T7-MEKK3, T7-Lys(391)-Met MEKK3 mutant (kn-MEKK3), and T7-Lys(432)-Met MEKK1 mutant (kn-MEKK1) were generated by PCR (26Hirano M. Osada S. Aoki T. Hirai S. Hosaka M. Inoue J. Ohno S. J. Biol. Chem. 1996; 271: 13234-13238Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The expression vectors for FLAG-TRAF, HA-TAK1, HA-TAB-1, FLAG-IRF3, and NF-κB-Luc have been described previously (16Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2036) Google Scholar, 25Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar). Western Blotting—Cells were lysed in 0.5% Triton X-100 extraction buffer containing 20 mm HEPES (pH 7.4), 150 mm NaCl, 1.5 mm MgCl2, 2 mm dithiothreitol supplemented with phosphatase and protease inhibitor mixture (Nacalai Tesque). The debris was removed by centrifugation at 14,500 rpm for 10 min at 4 °C. The cell lysates were resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The membranes were immunoblotted with various antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG (Jackson ImmunoResearch Laboratories), using Chemi-Lumi One L Western blotting detection reagents (Nacalai Tesque). For native PAGE assay, MEFs were seeded at 4 × 105 cells/well in 6-well plates. 24 h later, cells were stimulated with 10 μg/ml of poly(I:C) using FuGENE HD for 3 h and then harvested. The cells were lysed with native PAGE sample buffer (62.5 mm Tris-Cl, pH 6.8, 15% glycerol). The extracts were separated on a 7.5% native PAGE and then immunoblotted with anti-IRF3 antibody. RT-PCR and Real Time PCR—Total RNA was extracted from MEFs using the RNA-iso (Takara) according to the manufacturer's protocol, and then cDNA was synthesized from 1 μg of total RNA with the High Capacity cDNA reverse transcription kit (Applied BioSystems, CA). The cDNA was used as a template for PCR using KOD-plus DNA polymerase (TOYOBO, Japan) or real time PCR using the SYBR Green system (Applied BioSystems) according to the manufacturer's protocol. Gene-specific primer sequences are as follows: G3PDH, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′; IFN-α, 5′-ATGGCCTCGCCCTTTGCTTTACTG-3′ and 5′-TTTCTGCTCTGACAACCTCCCAGG-3′; IFN-β, 5′-TCCAAGAAAGGACGAACATTCG-3′ and 5′-TGAGGACATCTCCCACGTCCA-3′; IP-10, 5′-GTGTTGAGATCATTGCCACG-3′ and 5′-GCTTACAGTACAGAGCTA-3′; RANTES, 5′-CCCTCACCATCATCCTCACT-3′ and 5′-CCTTCGAGTGACAAACACGA-3′. Luciferase Reporter Assay—For the reporter gene assays, HEK293T cells were seeded at 1 × 105 cells/well in 12-well plates, or MEFs were seeded at 1 × 105 cells/well in 6-well plates. After 24 h, cells were transfected with a reporter plasmid and expression plasmids as indicated, using polyethyleneimine or FuGENE HD (Roche Applied Science). After 24 h, the cells were lysed, and luciferase activity was measured by using the luciferase assay system and chemiluminescent reagents (Promega). A plasmid containing the β-galactosidase gene was used to normalize for transfection efficiency. Detection of Cell Death—MEFs were seeded at 5 × 104 cells/well in a 24-well plate 1 day prior to treatment. The cells were left untreated or infected with VSV at the indicated multiplicity of infection. After 12 h, cells were trypsinized and collected with the supernatants, and the cell viability was determined by propidium iodide (Sigma) and Annexin V (Sigma) staining using a flow cytometer (FACSCalibur; BD Biosciences). Confocal Microscopy—MEFs were seeded on a glass coverslip 1 day prior to treatment. The cells were transfected with 10 μg/ml poly(I:C) for 3 h, fixed in 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 in PBS. After incubation in blocking buffer (1% bovine serum albumin in PBS) for 5 min, the cells were incubated with anti-IRF3 and anti-p65 antibodies, followed by ALEXA-488 and ALEXA-546-conjugated secondary antibodies (Molecular Probes). The cells were observed under a confocal laser microscope (LSM510 META; Carl Zeiss). Virus Preparation—VSV was propagated with vero cells. The titration of VSV was determined by plaque assay using vero cells. RNA Interference—For siRNA experiments, double-stranded RNA duplexes composed of 21-nucleotide sense and antisense oligonucleotides were synthesized. RNA oligonucleotides used for targeting mouse MEKK1 and mouse MEKK3 in this study were as follows: MEKK1#1 sense, 5′-GGUAGGAGCUGUGAUGUAUdTdT-3′; MEKK1#2 sense, 5′-GCUGCUUAUUCUCUAGAAAdTdT-3′; MEKK3 sense, 5′-GGAGGUGAGUGCUCUGGAGdTdT-3′. MEFs were seeded at 2 × 105 cells/well in 6-well plates 24 h prior to transfection. Lipofectamine RNAiMAX (Invitrogen) was used to transfect MEFs with 40 nm siRNA, according to the manufacturer's instructions. 24 h after transfection, knockdown efficiency of the transfected siRNA on MEKK1 and MEKK3 mRNA expression was confirmed by RT-PCR. The primers were as follows: MEKK3, 5′-TACCTGAGCGACAACAGCAC-3′ and 5′-TTCGGGGAAATGTTCTTCTG-3′; MEKK1, 5′-CGGAACACCATCCAGAAGTT-3′ and 5′-TCTCCTGATTCCTTCGCTGT-3′. TRAF6 Is Required for Type I IFN Production Induced by Intracellular dsRNA—TRAF6 is an essential adaptor protein for production of proinflammatory cytokines, such as IL-6 and IL-12, in macrophages and DCs in response to TLR ligands (24Kobayashi T. Walsh P.T. Walsh M.C. Speirs K.M. Chiffoleau E. King C.G. Hancock W.W. Caamano J.H. Hunter C.A. Scott P. Turka L.A. Choi Y. Immunity. 2003; 19: 353-363Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). However, the role of TRAF6 in antiviral immunity and type I IFN production has not been clarified. To address this issue, we first infected TRAF6-deficient MEFs with the IFN-sensitive prototypic rhabdovirus, VSV. TRAF6-deficient MEFs were more vulnerable to virus infection than wild-type MEFs. Virus-induced cytolysis and Annexin V-positive fraction, including early apoptotic cells and dead cells, were markedly increased after 12-h infection in TRAF6-deficient MEFs compared with wild-type cells (Fig. 1A). This was probably due to reduced IFN production in TRAF6-deficient MEFs, because the enhanced cell death of the mutant MEFs was rescued by adding exogenous IFN-β (Fig. 1A). To investigate the role of TRAF6 in type I IFN induction, MEFs as well as conventional DCs (cDCs) from wild-type and TRAF6-deficient mice were infected with VSV. Induction of IFN-α, IFN-β, and IP-10 was almost completely diminished in TRAF6-deficient cDCs (Fig. 1B) and remarkably reduced in TRAF6-deficient MEFs (Fig. 1C, left). To mimic intracellular dsRNA generated during viral replication, we also transfected synthetic dsRNA, poly(I:C), into MEFs, using a lipid-based transfection reagent, FuGENE HD. Up-regulation of IFN-α/β mRNA, but not IP-10 mRNA, was also severely impaired in TRAF6-deficient MEFs in response to poly(I:C) (Fig. 1C, right). These data suggest that TRAF6 is required for type I IFN production induced by intracellular dsRNA. TRAF6 Is Involved in the RLH/IPS-1 Pathway to Activate the IFN-β Promoter—To understand the molecular mechanism by which TRAF6 induces type I IFNs in the context of intracellular dsRNA recognition, we first transfected wild-type MEFs with poly(I:C) using FuGENE HD and then examined the effect of inhibitors of various signaling molecules downstream of TRAF6 on IFN-β production (Fig. 2A). The up-regulation of mRNA expression for IFN-β and IP-10 induced by poly(I:C) was strongly suppressed by inhibitors of IKK (IKK-2 inhibitor VI), JNK (SP60025), and p38 (SB203580) but not by an inhibitor of MEK (upstream of ERK) (U0126), suggesting that the activation of IKK, p38 and JNK pathways is required for IFN-β expression. Next we transfected HEK293T cells with IRF3, TRAF6, or both constructs along with an IFN-β promoter-luciferase reporter gene (Fig. 2B). Although overexpression of TRAF6 can activate both IKK and MAPK pathways required for IFN-β expression, the IFN-β promoter was only marginally activated by TRAF6 overexpression. However, cotransfection of TRAF6 and IRF3 exhibited a strong activation of the promoter, indicating that TRAF6 and IRF3 cooperatively activate the IFN-β promoter. Taken together, these results support the notion that the optimal activation of IFN-β promoter by intracellular dsRNA requires the coordinate activation of transcription factors IRF3, NF-κB, and ATF-2/c-Jun (14Theofilopoulos A.N. Baccala R. Beutler B. Kono D.H. Annu. Rev. Immunol. 2005; 23: 307-336Crossref PubMed Scopus (1022) Google Scholar, 15Kim T.K. Maniatis T. Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Next we investigated whether TRAF6 functions at the downstream of the RIG-I/IPS-1 pathway. Overexpression of either RIG-IN (constitutively active form of RIG-I) or IPS-1 activated IFN-β promoter in HEK293T cells, whereas this was strongly suppressed by forced expression of the dominant negative form of TRAF6 (DN-TRAF6) in a dose-dependent manner (Fig. 2C, left). Similarly, DN-TRAF6 suppressed NF-κB activation induced by RIG-IN or IPS-1 (Fig. 2C, right). To ensure the role of TRAF6 in the RLH-mediated pathway, we further examined the IFN-β reporter assay in TRAF6-deficient MEFs. As shown in Fig. 2D, the activation of IFN-β promoter as well as NF-κB induced by forced expression of IPS-1 or transfection with poly(I:C) was severely impaired in TRAF6-deficient cells. These results suggest that RLH/IPS-1 pathway activates NF-κB via TRAF6, which is essential for the full activation of the IFN-β promoter. To further dissect the signaling pathways downstream of TRAF6, we assessed the activation and nuclear translocation of IRF3. As shown in Fig. 2E, the homodimerization of IRF3, reflecting the activation of the molecule, was induced by poly(I:C) in MEFs even in the absence of TRAF6. To observe the nuclear translocation of IRF3 as well as NF-κB subunit, p65, we stimulated wild-type and TRAF6-deficent MEFs with poly(I:C) along with FuGENE HD and then stained the cells with anti-IRF3 and anti-p65 antibodies (Fig. 2F). Three hours after stimulation, ∼30% of knock-out MEFs exhibited nuclear translocation of IRF3, which was comparable with that of wild-type MEFs. These results indicate that the activation and nuclear translocation of IRF3 are independent of TRAF6. However, nuclear translocation of p65 was impaired in TRAF6-dficient MEFs. Although all of the wild-type MEFs exhibited co-localization of p65 and IRF3 in the nucleus, more than 50% of TRAF6-deficient MEFs showed cytoplasmic retention of p65 but nuclear translocation of IRF3 (Fig. 2F). These data are consistent with previous results showing a defective NF-κB activation in the absence of functional TRAF6 in the reporter assay (Fig. 2, C and D). Moreover, poly(I:C)-induced phosphorylation of JNK and p38 was severely impaired in TRAF6-deficieint MEFs (Fig. 2G). Taken together, TRAF6 appears to contribute to the activation of NF-κB and MAPK pathways rather than IRF3 activation in the RLH/IPS-1 signaling pathway in MEFs. TAK1 Is Not Essential for RLH/IPS-1-mediated IFN-β Induction—TAK1 is essential for the TRAF6-dependent activation of NF-κB and MAPKs in Toll-like/IL-1 receptor signaling (23Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (767) Google Scholar). Hence, we next examined whether TAK1 is involved in the RLH/IPS-1 signaling pathway. First we analyzed the ability of wild-type and TAK1-deficient MEFs to induce type I IFNs in response to VSV infection and poly(I:C) transfection. As shown in Fig. 3A, the induction of IFN-α/β in TAK1-deficient MEFs was comparable with that in wild-type MEFs. IFN-β promoter reporter activity enhanced by IPS-1 or poly(I:C) was not impaired in TAK1-deficient MEFs (Fig. 3B, left). NF-κB activity was also normally elevated by forced expression of IPS-1 in TAK1-deficient MEFs (Fig. 3B, middle), whereas lipopolysaccharide stimulation failed to up-regulate the NF-κB activity in the knock-out cells (Fig. 3B, right). The dimerization of IRF3 induced by poly(I:C) was also not affected by TAK1 deficiency (Fig. 3C). When we examined the nuclear translocation of p65 and IRF3, co-localization of both transcription factors in the nuclei was observed in both wild-type and TAK1-deficient MEFs at the same ratio (Fig. 3D). These results suggest that TAK1 is not required for type I IFN induction in the RLH/IPS-1-mediated signaling pathway. MEKK1 Is a Candidate MAP3K to Induce IFN-β in the RLH/IPS-1 Pathway—Since TAK1 was not necessary for type I IFN induction in the RLH/IPS-1 pathway, we sought a responsible MAP3K involved in the IPS-1/TRAF6-mediated signal. MEKK1 (MEK kinase-1) and MEKK3 have been implicated in the activation of IKK and MAPKK, leading to the activation of NF-κB and JNK (27Symons A. Beinke S. Ley S.C. Trends Immunol. 2006; 27: 40-48Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To examine whether MEKK1 or MEKK3 is involved in the RIG-I/IPS-1 pathway, we performed an IFN-β and NF-κB reporter assay using kinase-inactive mutants of MEKK1 (knMEKK1) and MEKK3 (knMEKK3). As shown in Fig. 4A, IPS-1- and RIG-IN-induced IFN-β promoter and NF-κB activities were inhibited efficiently by knMEKK1 and to a lesser extent by knMEKK3. Next we examined the effect of overexpression of MEKK1 and MEKK3. The forced expression of IRF3, MEKK1, or MEKK3 alone was not sufficient for the activation of the IFN-β promoter; however, cotransfection of IRF3 and MEKK1, but not MEKK3 synergistically activated the promoter activity (Fig. 4B). In order to confirm the significance of endogenous MEKK1 and MEKK3, we performed siRNA-mediated knockdown experiments. Wild-type MEFs were transfected with scrambled control, MEKK1, or MEKK3 siRNA construct, and then endogenous gene expression of MEKK1 as well as MEKK3 was sufficiently suppressed (Fig. 4C). The knockdown of MEKK1, but not MEKK3, resulted in a severe reduction of IFN-β mRNA expression induced by poly(I:C) (Fig. 4C). Induction of mRNA for IP-10 was slightly suppressed in the MEKK1 knockdown MEFs. Significant reduction of IFN-α and IFN-β levels by knockdown of MEKK1 with different siRNA oligonucleotides was also confirmed by real time PCR (Fig. 4D). Furthermore, the suppression of type I IFNs by siRNA for MEKK1 was reverted by forced expression of rat MEKK1 (Fig. 4E). These results indicate that endogenous MEKK1 but not MEKK3 is required for type I IFN production induced by cytoplasmic dsRNA. Recent studies have shown that the RLH family of intracellular receptors detect viral nucleic acid and signal through IPS-1 (also known as MAVS, CARDIF, and VISA) during viral infection (1Beutler B. Eidenschenk C. Crozat K. Imler J.L. Takeuchi O. Hoffmann J.A. Akira S. Nat. Rev. Immunol. 2007; 7: 753-766Crossref PubMed Scopus (162) Google Scholar, 2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8918) Google Scholar, 3Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1444) Google Scholar). IPS-1 recruits TRAF3, which in turn activates TBK1 and IKK-i, leading to the activation of IRF3 (22Saha S.K. Pietras E.M. He J.Q. Kang J.R. Liu S.Y. Oganesyan G. Shahangian A. Zarnegar B. Shiba T.L. Wang Y. Cheng G. EMBO J. 2006; 25: 3257-3263Crossref PubMed Scopus (348) Google Scholar). IPS-1 also activates NF-κB via the FADD/RIP1 pathway mediated by caspase-8 and -10 (16Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2036) Google Scholar, 28Takahashi K. Kawai T. Kumar H. Sato S. Yonehara S. Akira S. J. Immunol. 2006; 176: 4520-4524Crossref PubMed Scopus (156) Google Scholar). In this study, we have found that TRAF6 and MEKK1 were involved in the downstream pathway of IPS-1, which facilitates NF-κB and MAPKs to enhance the type I IFN promoter. It has been shown that there are two distinct types of TIM; one is the PXEXX(acidic or aromatic residue) motif for TRAF6, and the other is the PXQX(T/S) motif for other TRAFs, such as TRAF2 and TRAF3 (29Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (543) Google Scholar). IPS-1 contains multiple TIMs, and TRAF1, -2, -3, -5, and -6 can interact with IPS-1 (data not shown) (see Refs. 18Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1521) Google Scholar and 22Saha S.K. Pietras E.M. He J.Q. Kang J.R. Liu S.Y. Oganesyan G. Shahangian A. Zarnegar B. Shiba T.L. Wang Y. Cheng G. EMBO J. 2006; 25: 3257-3263Crossref PubMed Scopus (348) Google Scholar). Furthermore, TRAF3 is required for the activation of IRF3 but not NF-κB (21Oganesyan G. Saha S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Nature. 2006; 439: 208-211Crossref PubMed Scopus (714) Google Scholar), whereas TRAF6 is required for the activation of NF-κB and MAPK but not IRF3. Thus, the IPS-1·TRAF complex is a divergent point of the RLH/IPS-1-mediated signal (Fig. 5). A similar case of the signal divergence regulated by TRAF6 and TRAF3 in the context of MyD88-dependent signaling pathway has been reported (20Häcker H. Redecke V. Blagoev B. Kratchmarova I. Hsu L.C. Wang G.G. Kamps M.P. Raz E. Wagner H. Häcker G. Mann M. Karin M. Nature. 2006; 439: 204-207Crossref PubMed Scopus (748) Google Scholar). IKK and MAPK activities result from the activation of upstream MAP3Ks, such as TAK1, MEKK1, MEKK3, and NIK. In viral infection or dsRNA stimulation, TAK1-deficient cells normally induced type I IFN, and we could not detect the activation of TAK1 in wild-type cells in response to dsRNA (data not shown). Our findings suggest that TAK1 is not very necessary for virus-induced type I IFN production. The TAK1-independent mechanism should be controlled by TRAF6 in the RLH/IPS-1 signal pathway. Thus, we have made an attempt to identify the responsible MAP3K. TRAF6-interacting protein, ECSIT, has been shown to mediate the activation of NF-κB and JNK in TLR pathway (30Kopp E. Medzhitov R. Carothers J. Xiao C. Douglas I. Janeway C.A. Ghosh S. Genes Dev. 1999; 13: 2059-2071Crossref PubMed Scopus (276) Google Scholar). ECSIT binds to and activates MEKK1, which in turn activates IKK. MEKK3 has also been implicated in the activation of IKK and MAPKK, leading to the activation of NF-κB and JNK. For instance, the activation of NF-κB and JNK by TLR8 is TAK1-independent but MEKK3-dependent (31Qin J. Yao J. Cui G. Xiao H. Kim T.W. Fraczek J. Wightman P. Sato S. Akira S. Puel A. Casanova J.L. Su B. Li X. J. Biol. Chem. 2006; 281: 21013-21021Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Thus, it might be possible that MEKK1 and/or MEKK3 is involved in IPS-1/TRAF6-mediated IKK and MAPK activation. Our results indicate that MEKK1 is involved in the activation of the IFN-β promoter in the RLH/IPS-1-mediated anti-viral signal pathway. Forced expression of MEKK1 and IRF3 cooperatively activated the IFN-β promoter, whereas knockdown of MEKK1 resulted in the reduction of IFN-β production. The knMEKK1 suppressed the NF-κB activation. Therefore, although the precise mechanism currently remains unclear, MEKK1 seems to be necessary for the activation of IPS-1/TRAF6 pathway. Further analysis is necessary to understand how MEKK1, but not TAK1, is activated during viral infection. IPS-1 localizes in the outer mitochondrial membrane; thus, one possibility is that the localization of IPS-1 may induce the downstream kinase differently from that in TLR pathway. IPS-1-deficient mice were found to be defective in the production of type I IFNs in response to all RNA viruses recognized by either RIG-I or MDA5, suggesting a central role in the production of type I IFNs upon viral infection (32Kumar H. Kawai T. Kato H. Sato S. Takahashi K. Coban C. Yamamoto M. Uematsu S. Ishii K.J. Takeuchi O. Akira S. J. Exp. Med. 2006; 203: 1795-1803Crossref PubMed Scopus (409) Google Scholar, 33Sun Q. Sun L. Liu H.H. Chen X. Seth R.B. Forman J. Chen Z.J. Immunity. 2006; 24: 633-642Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). IRF3 and IRF7 were also shown to be indispensable for type I IFN production (34Honda K. Yanai H. Negishi H. Asagiri M. Sato M. Mizutani T. Shimada N. Ohba Y. Takaoka A. Yoshida N. Taniguchi T. Nature. 2005; 434: 772-777Crossref PubMed Scopus (1748) Google Scholar, 35Sato M. Suemori H. Hata N. Asagiri M. Ogasawara K. Nakao K. Nakaya T. Katsuki M. Noguchi S. Tanaka N. Taniguchi T. Immunity. 2000; 13: 539-548Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). On the other hand, we found that TRAF6-deficient MEFs did not completely lose the ability to induce type I IFNs, probably due to partial NF-κB activation by other mechanisms. It has been reported that IPS-1 interacts with both FADD and RIP1 (16Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2036) Google Scholar). Both FADD and RIP1 have been reported to be critical for the induction of type I IFNs in response to poly(I:C) (36Balachandran S. Thomas E. Barber G.N. Nature. 2004; 432: 401-405Crossref PubMed Scopus (246) Google Scholar). Thus, we speculate that the absence of TRAF6 in the RLH/IPS-1 pathway is compensated for by the signal mediated by FADD and RIP1 to induce type I IFNs. Further investigation is necessary to clarify this point. dsRNA is also recognized by TLR3 in certain cell types, and TRAF6 has been shown to be involved in the pathway depending on TRIF. However, the function of TRAF6 in TLR3 signaling remains controversial. The activation of NF-κB by poly(I:C) is abrogated in TRAF6-deficient MEFs, whereas NF-κB activation occurs normally in TRAF6-deficient macrophages in response to dsRNA (37Gohda J. Matsumura T. Inoue J. J. Immunol. 2004; 173: 2913-2917Crossref PubMed Scopus (238) Google Scholar, 38Jiang Z. Mak T.W. Sen G. Li X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3533-3538Crossref PubMed Scopus (309) Google Scholar). Cell type-specific involvement of TRAF6 was suggested in the TRIF-mediated NF-κB activation. Nevertheless, in our data, TRAF6-deficient cDCs infected with VSV showed a severe defect in the production of type I IFNs, indicating that TRAF6 plays an essential role in cDC for type I IFN production in response to RNA viral infection. In the present study, we examined the role of TRAF6, TAK1, MEKK1, and MEKK3 in the RLH/IPS-1-mediated IFN-β induction. Based on data published recently and presented here, we propose a model for a RLH-IPS-1-mediated signaling pathway via TRAF6 (Fig. 5). Upon RNA viral infection, RIG-I or MAD5 recognizes the viral dsRNA and then recruits IPS-1 through the CRAD domain. IPS-1 associates with TRAF3, TRAF6, FADD, and RIP1. TRAF3 activates TBK1/IKK-i, leading to the activation of IRF3, whereas TRAF6 may interact with MEKK1, which in turn contributes to the activation of IKK and MAPKK, leading to the activation of NF-κB and AP-1. These transcription factors cooperatively activate the IFN-β expression. We thank Dr. T. Fujita (Kyoto University) for RIG-I cDNA, M. Ohtsu and K. Fukidome for technical assistance, Y. Nishi for manuscript preparation, and H. Shiraishi for helpful discussion and support.

Latent TGF-β-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics.

The present paper provides the principles for chemiluminescence of luminol-type compounds and their wide and powerful application to the detection system in liquid chromatography and capillary electrophoresis as derivatization reagents. The reagents can be classified into two types, chemiluminescence labeling and chemiluminogenic reagents. The former reagents are highly chemiluminescent themselves and used for tagging their intense chemiluminophores to analytes, whereas the latter are weakly chemiluminescent but generate intense chemiluminescence by reaction with analytes. The liquid chromatographic methods utilizing chemiluminescence derivatizing reactions with luminol-type reagents allow the analytes to be detected at pmol-sub-fmol levels. Furthermore, the chemiluminogenic reactions show high selectivity owing to their selective reaction against the analytes permitting facile and reproducible detection.

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Abstract Glial cell line‐derived neurotrophic factor (GDNF) has been shown to possess potent neurotrophic effects on dopaminergic (DA) neurons. We attempted the transplantation of encapsulated GDNF‐producing cells to generate a stable supply of GDNF in the brain to promote neuroprotective and restorative effects for DA neurons. We established baby hamster kidney (BHK) cells and introduced GDNF cDNA to produce human GDNF (BHK‐GDNF). These BHK‐GDNF cells, or nontransfected BHK cells (BHK‐Control), were encapsulated into hollow fibers, and the polymer encapsulated cells were unilaterally implanted into the striatum of adult rats, either before or after the administration of 6‐hydroxydopamine into the same striatum. The encapsulated BHK‐GDNF cells produced GDNF continuously in the striatum for up to 6 months. The rats that received a BHK‐GDNF capsule showed a significant decrease in rotational behaviour compared to those that received a BHK‐control capsule. Preservation of the nigrostriatal pathway was significantly greater in those that received a BHK‐GDNF capsule than in those that received a BHK‐control capsule. This indicates that encapsulated GDNF‐producing cells can supply GDNF in a stable fashion and have protective and restorative effects on host DA neurons. Our results support a role for this grafting technique in the treatment of Parkinson's disease. © 2002 Wiley‐Liss, Inc.

To improve the fluorescence characteristics, especially emission wavelength, of coumarins, various 3-substituted-6-methoxycoumarin derivatives were synthesized, and then benzocoumarin derivatives were also synthesized in expectation of the shift to the longer wavelength region by the extension of the conjugated system. Their fluorescence properties were investigated spectrophotometrically in acetonitrile and evaluated from the viewpoint of the intramolecular charge transfer (ICT) between push- and pull-substituents in the ground and the excited states. Among them, benzocoumarin derivatives especially fluoresced in the longer wavelength around 540 nm with remarkably large Stokes shifts beyond 10000 cm−1. Using such fluorophores, some novel fluorescence derivatization reagents for carboxylic acids, alcohols, phenols, and amines were preliminarily prepared as an example, and their derivatized products were also found to fluoresce in the longer wavelength region with large Stokes shifts.

Recently, T cell cytokines such as IL-17 and IFN-γ have been shown to play important roles in the progression of brain injury induced by ischemia. We have shown that IL-23 from infiltrated macrophages activates γδT cells, thereby inducing IL-17 from these cells. However, deletion of the IL-23 gene in mice showed a more dramatic protective effect against brain ischemia reperfusion (I/R) model than γδT cell depletion did, suggesting that IL-23 plays some other pivotal role in brain injury in addition to its role in IL-17 induction. To develop therapeutic methods based on these findings, we examined the effect of the JAK kinase inhibitor CP-690550 and an anti-IL12/23 monoclonal antibody on an I/R model. CP-690550 efficiently inhibited IL-17 production from memory T cells in vitro and partly suppressed infarct volume increase after I/R. Anti-p40 antibody, which blocks both IL-12 and IL-23, efficiently suppressed I/R injury and improved recovery of neurological deficits. The number of IL-17-producing cells was decreased by anti-p40 antibody treatment. Thus the JAK inhibitor and anti-p40 antibody, both of which have already been under trial for the treatment of several human inflammatory diseases, appear to be promising therapeutic agents for the amelioration of stroke.

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

A liquid chromatographic (LC) derivatization method for simple and selective determination of catecholamines and indoleamines in human urine has been developed. This method uses "fluorous interaction" in which perfluoroalkyl compounds show affinity with each other. The amino groups of native fluorescent analytes are precolumn derivatized with a non-fluorescent fluorous isocyanate, 2-(perfluorooctyl)ethyl isocyanate, and the fluorous-labeled analytes are retained in the fluorous LC column, whereas underivatized substances are not. Only the retained fluorous-fluorescent analytes are detected fluorometrically at appropriate retention times, and retained amines without fluorophores are not detected. In this study, 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, and metanephrine were used as the representative of catecholamines. Tryptophan, 5-hydroxytryptophan, and 5-hydroxytryptamine were used as the representative indoleamines. This method was applied to determine eight biogenic amines in urine from healthy humans. The fluorous-labeled amines could be separated by fluorous LC column under conditions of isocratic elution within 35 min and simultaneously determined without interference from contaminants in biological samples. The detection limits for eight biogenic amines were 31-640 fmol on column. Calibration curves of them were linear over the range of at least 10-100 nmol/mL urine (r² > 0.9989) with good repeatability.

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

While group-2 innate lymphoid cells (ILC2s) are highly proliferative in allergic inflammation, the removal of overactivated ILC2s in allergic diseases has not been investigated. We previously showed that chronic airway allergy induces "exhausted-like" dysfunctional ILC2s expressing T cell immunoreceptor with Ig and ITIM domains (TIGIT). However, the physiological relevance of these cells in chronic allergy remains elusive. To precisely identify and monitor TIGIT+ ILC2s, we generated TIGIT lineage tracer mice. Chronic allergy stably induced TIGIT+ ILC2s, which were highly activated, apoptotic, and were quickly removed from sites of chronic allergy. Transcripts from coding genes were globally suppressed in the cells, possibly due to reduced chromatin accessibility. Cell death in TIGIT+ ILC2s was enhanced by interactions with CD155 expressed on macrophages, whereas genetic ablation of Tigit or blockade by anti-TIGIT antagonistic antibodies promoted ILC2 survival, thereby deteriorating chronic allergic inflammation. Our work demonstrates that TIGIT shifts the fate of ILC2s toward activation-induced cell death, which could present a new therapeutic target for chronic allergies.

The thymus is a highly specialized organ that plays an indispensable role in the establishment of self-tolerance, a process characterized by the "education" of developing T-cells. To provide competent T-cells tolerant to self-antigens, medullary thymic epithelial cells (mTECs) orchestrate negative selection by ectopically expressing a wide range of genes, including various tissue-restricted antigens (TRAs). Notably, recent advancements in the high-throughput single-cell analysis have revealed remarkable heterogeneity in mTECs, giving us important clues for dissecting the mechanisms underlying TRA expression. We overview how recent single-cell studies have furthered our understanding of mTECs, with a focus on the role of Aire in inducing mTEC heterogeneity to encompass TRAs.

Background/AimsInfection after major surgery, such as massive hepatectomy, induces liver dysfunction, occasionally leading to multiple organ failure and death. We demonstrated the anti-inflammatory effects and functional mechanisms of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a newly synthesized free radical scavenger, on an experimental model of endotoxemia after partial hepatectomy in rats.MethodsRats were treated with lipopolysaccharide (LPS) 48 h after 70% hepatectomy. Edaravone was administered intravenously before LPS-treatment.ResultsEdaravone markedly improved the survival rate of LPS-treated rats after hepatectomy and inhibited increases in serum levels of AST and LDH. Histopathological analysis demonstrated that edaravone prevented inflammatory changes in the liver, kidney and spleen. Edaravone inhibited the formation of one of the markers of oxidative damage, malondialdehyde. Increases in inflammatory cytokines and cytokine-induced neutrophil chemoattractant (CINC) in serum and liver tissue were inhibited in the edaravone-treated group. An electrophoretic mobility shift assay revealed that edaravone inhibited the activation of the transcription factor, nuclear factor-kappa B (NF-κB). Edaravone also reduced the induction of inducible nitric oxide synthase (iNOS).ConclusionsEdaravone prevents endotoxin-induced liver injury after partial hepatectomy not only by attenuating oxidative damage, but also by reducing the production of inflammatory cytokines, CINC and iNOS, in part through the inhibition of NF-κB activation. Infection after major surgery, such as massive hepatectomy, induces liver dysfunction, occasionally leading to multiple organ failure and death. We demonstrated the anti-inflammatory effects and functional mechanisms of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a newly synthesized free radical scavenger, on an experimental model of endotoxemia after partial hepatectomy in rats. Rats were treated with lipopolysaccharide (LPS) 48 h after 70% hepatectomy. Edaravone was administered intravenously before LPS-treatment. Edaravone markedly improved the survival rate of LPS-treated rats after hepatectomy and inhibited increases in serum levels of AST and LDH. Histopathological analysis demonstrated that edaravone prevented inflammatory changes in the liver, kidney and spleen. Edaravone inhibited the formation of one of the markers of oxidative damage, malondialdehyde. Increases in inflammatory cytokines and cytokine-induced neutrophil chemoattractant (CINC) in serum and liver tissue were inhibited in the edaravone-treated group. An electrophoretic mobility shift assay revealed that edaravone inhibited the activation of the transcription factor, nuclear factor-kappa B (NF-κB). Edaravone also reduced the induction of inducible nitric oxide synthase (iNOS). Edaravone prevents endotoxin-induced liver injury after partial hepatectomy not only by attenuating oxidative damage, but also by reducing the production of inflammatory cytokines, CINC and iNOS, in part through the inhibition of NF-κB activation.

Inflammation has been shown to contribute to both tumor development and antitumor immunity. However, conditions determining these opposing effects are not well understood. Suppressor of cytokine signaling 1 (SOCS1) has been shown to play an important role in regulating inflammation and tumor development. It has been reported that silencing of SOCS1 gene in dendritic cells potentiates antitumor immunity, while SOCS1 ‐deficiency in whole organs except for T and B cells enhances inflammation‐mediated colon tumor development. To determine which types of cells are important for the suppression of tumor development by SOCS1 ‐deficiency, we employed the conditional knockout strategy. SOCS1 gene was deleted in macrophages and neutrophils by crossing SOCS1‐flox/flox mice with LysM‐cre mice. Resulting conditional knockout (cKO) mice showed enhanced sensitivity to endotoxin shock. SOCS1‐ cKO mice survived much longer than wild‐type mice after B16 melanoma transplantation. Colon carcinogenesis induced by 1,2‐dimethylhydrazine (DMH) plus dextran sulfate sodium (DSS) was also reduced in SOCS1 ‐cKO mice. SOCS1 ‐deficiency in monocytic cells enhanced tumor‐killing activity of macrophages and tumor‐specific cytotoxic T cell activity. These results suggest that inflammation induced by SOCS1 ‐deficiency in monocytes potentiates antitumor immune responses rather than tumor‐promoting inflammation. ( Cancer Sci 2009; 100: 730–736)

Th17 cells, which have been implicated in autoimmune diseases, require STAT3 signaling activated by IL-6 or IL-23 for their development. Other Th1 and Th2 cytokines such as IL-2, IFN-γ and IL-4 strongly suppress Th17 development. Recently, CP-690,550 (tofacitinib), originally developed as a JAK3 inhibitor, has been shown to be effective in phase III clinical trials of rheumatoid arthritis and collagen-induced arthritis (CIA) models, but the precise mechanism of the effect, especially with respect to Th17 cells, is poorly understood. To our surprise, a low dose CP-690,550 was found to accelerate the onset of experimental autoimmune encephalomyelitis (EAE) at a concentration that suppressed CIA. At an early stage after immunization, more IL-17 production was observed in 15 mg/kg body weight CP-690,550-treated mice than in untreated mice. In vitro, CP-690,550 inhibited both Th1 and Th2 development, while promoting Th17 differentiation at 10–50 nM concentrations. Enhancement of Th17 by CP-690,550 is probably due to suppression of IL-2 signaling, because anti-IL-2 antibodies cancel the Th17-promoting effect of CP-690,550. CP-690,550 selectively inhibited IFN--induced STAT1, IL-4-induced STAT6 and IL-2-induced STAT5 at 3–30 nM, while suppression of IL-6-induced STAT3 phosphorylation required a concentration greater than 100 nM. In HEK293T cells, CP-690,550 less effectively suppressed JAK1-mediated STAT3 phosphorylation compared with JAK3. These results suggest that CP-690,550 has a different effects among JAKs and STATs, thereby affecting helper T cell differentiation, and murine autoimmune disease models.

Microfibrils are exracellular matrix components necessary for elastic fiber assembly and for suspending lenses. We previously reported that latent TGF-β binding protein 2 (LTBP-2), a microfibril-associated protein, is required for forming stable microfibril bundles in ciliary zonules. However, it was not understood why Ltbp2 null mice only showed an eye-specific phenotype, whereas LTBP-2 is abundantly expressed in other tissues containing microfibrils in wild type mice. Here, we show that LTBP-4, another microfibril-associated protein, compensates for the loss of LTBP-2 in microfibril formation. Ltbp2/4S double knockout (DKO) mice showed increased lethality due to emphysema, which was much more severe than that found in Ltbp4S null mice. Elastic fibers in the lungs of Ltbp2/4S DKO mice were severely disorganized and fragmented. Cultured mouse embryonic fibroblasts (MEFs) from Ltbp2/4S DKO embryos developed reduced microfibril meshwork in serum-free conditions, whereas the microfibril formation was restored by the addition of either recombinant LTBP-2 or -4. Finally, ectopic expression of LTBP-4 in the whole body restored ciliary zonule microfibril bundles in the eyes of Ltbp2 null mice. These data suggest that LTBP-2 and -4 have critical overlapping functions in forming the robust structure of microfibrils in vitro and in vivo.

To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system.

<h2>Summary</h2> Impaired production of thymic regulatory T cells (Tregs) is implicated in the development of Aire-dependent autoimmunity. Because Tregs require agonistic T cell receptor stimuli by self-antigens to develop, reduced expression of self-antigens from medullary thymic epithelial cells (mTECs) has been considered to play a major role in the reduced Treg production in Aire deficiency. Here, we show that mTECs abnormally express co-inhibitory receptor CTLA-4 if Aire is non-functional. Upon binding with CD80/CD86 ligands expressed on thymic dendritic cells (DCs), the ectopically expressed CTLA-4 from Aire-deficient mTECs removes the CD80/CD86 ligands from the DCs. This attenuates the ability of DCs to provide co-stimulatory signals and to present self-antigens transferred from mTECs, both of which are required for Treg production. Accordingly, impaired production of Tregs and organ-specific autoimmunity in Aire-deficient mice are rescued by the depletion of CTLA-4 expression from mTECs. Our studies illuminate the significance of mTEC-DC interaction coordinated by Aire for the establishment of thymic tolerance.

Abstract The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire’s primary targets while distinguishing them from the secondary targets.

A highly sensitive and selective fluorometric determination method for ornithine and lysine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase liquid chromatography (LC). The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PSE. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled derivatives of monoamines. The structures of the derivatives and the emission of excimer fluorescence were confirmed by LC with mass spectrometry and with three-dimensional fluorescence detection system, respectively. The PSE derivatives of ornithine and lysine could be separated by reversed-phase LC on ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-microl injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.

A simple, selective and sensitive determination method of tricyclic antidepressants (imipramine, desipramine, amitriptyline, nortriptyline and clomipramine) has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence detection. The method is based on the detection of aliphatic secondary or tertiary amino moieties in antidepressants with post-column tris(2,2′-bipyridyl)ruthenium(III) chemiluminescence. Five antidepressants were separated on a trimethylsilyl (TMS) column using a mixture of 50 mM phosphate buffer (pH 7.0) and acetonitrile (55:45, v/v) as a mobile phase. The eluate was on-line mixed with acidic tris(2,2′-bipyridyl)ruthenium(III) solution and the generated chemiluminescence was detected. The detection limits (signal-to-noise ratio=3) for antidepressants examined were 3.2–33.1 pg (12–105 fmol), for an injection volume of 20 μl. This HPLC system could sensitively detect other tri- or tetracyclic antidepressants and was successfully applied to the determination of imipramine and amitriptyline in human plasma after purification by two-step liquid–liquid extraction.

Pirfenidone has antiinflammatory effects in animals with endotoxemia. We reported that pirfenidone inhibits the enhancement of inflammatory cytokines and inducible nitric oxide synthase (iNOS) in liver of endotoxin-treated rats, leading to the prevention of hepatic injury. However, the mechanisms involved in suppression of these gene inductions are obscure. Studies were performed to investigate whether pirfenidone directly influences iNOS induction in hepatocytes.Cultured hepatocytes were treated with interleukin-1beta (IL-1beta) in the presence and absence of pirfenidone, and iNOS induction and its signal including NF-kappaB were analyzed.Pirfenidone inhibited the induction of iNOS mRNA and protein, resulting in the decrease of nitric oxide production. Gel shift assay demonstrated that pirfenidone inhibited the activation of NF-kappaB. Consistent with this observation, transfection experiments revealed that pirfenidone decreased transcriptional activation of iNOS gene promoter. In contrast, pirfenidone had no effect on the degradation of IkappaB, and could not prevent nuclear translocation of p50/p65. Finally, pirfenidone inhibited the activation of Akt and up-regulation of IL-1 receptor stimulated by IL-1beta.Results indicate that pirfenidone inhibits the induction of iNOS gene expression at a step of NF-kappaB DNA binding, but not its nuclear translocation, partly through the inhibition of IL-1 receptor induction in hepatocytes.

A method for the determination of tetrabromobisphenol A (TBBPA) in human serum utilizing solid-phase extractions (SPEs) and liquid chromatography (LC) with electrospray ionization tandem MS (MS/MS) has been developed. After purification and concentration of TBBPA using consecutive SPEs on reversed-phase and normal-phase cartridges, the serum sample was subjected to LC. TBBPA was separated on a C18 reversed-phase column by gradient elution with a mixture of water, methanol, and acetonitrile as the mobile phase, and then detected with electrospray ionization MS/MS in negative ion mode. 13C12-TBBPA was suitable as an internal standard for the reproducible determination of TBBPA in human serum samples (5 g). The method has been validated in TBBPA concentration range of 5-100 pg per g serum, and the recoveries in the concentration range were higher than 83.3%. The repeatabilities of the proposed method of non-spiked control serum (6.3 pg per g serum) and spiked serum (added 5-100 pg per g serum) were within 10.0% as relative standard deviations. The limit of quantification (LOQ) for TBBPA was 4.1 pg per g serum, which was corresponded to 0.63 fmol on column.

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)–induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient livers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody–induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-α-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-α. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced liver injury. Conclusion: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis. (HEPATOLOGY 2008.)

Abstract A method combining hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry (MS/MS) was developed for the determination of polar organophosphorus pesticides (OPPs; acephate, methamidophos, monocrotophos, omethoate, oxydemeton‐methyl, and vamidothion) in water samples. To extract the polar OPPs and minimize matrix effects from the water sample, an activated carbon cartridge was used for pretreatment. After pretreatment of the water sample, the eluate from the activated carbon cartridge was directly injected into the HILIC/MS/MS system. The OPPs were separated on an Atlantis HILIC silica column by isocratic elution with a mixture of acetonitrile, isopropanol, and ammonium formate buffer as a mobile phase, and they were detected by positive electrospray ionization MS/MS in the selected reaction monitoring mode. The method was validated at 0.05, 0.5, and 5 µg/L levels in water samples, and the recoveries of polar OPPs were between 76.4 and 98.6%. The limits of detection were between 0.13 and 1.0 pg on‐column, and the limits of quantification were between 0.43 and 3.4 pg on‐column. The method can be applied to the determination of trace amounts of OPPs in environmental water samples. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Atopic dermatitis (AD) is a common pruritic inflammatory disease triggered by a defective skin barrier and immunodysregulation. AD has been considered a typical example of a Th2 response associated with allergic disease. In the early phases of the disease, symptoms include IgE hyperproduction, eosinophil accumulation, and mast cell activation; in the chronic phase, a Th1-dominant immune response is also observed at the sites of AD skin lesions. The role of IL-17-producing Th (Th17) cells in AD has not been established. In the current study, we found that pyridone 6 (P6), a pan-JAK inhibitor, delayed the onset and reduced the magnitude of skin disease in an AD-like skin-disease model of NC/Nga mice. P6 reduced IFN-γ and IL-13, whereas it enhanced IL-17 and IL-22 expression. In vitro, P6 also inhibited both Th1 and Th2 development, whereas it promoted Th17 differentiation from naive T cells when present within a certain range of concentrations. This was probably because P6 strongly inhibited STAT1, STAT5, and STAT6 phosphorylation, whereas STAT3 phosphorylation was less efficiently suppressed by P6 at the same concentration. Furthermore, IL-22 protects keratinocytes from apoptosis induced by IFN-γ, and administration of IL-17 and IL-22 partially ameliorated skin diseases in NC/Nga mice. These results suggested that the JAK inhibitor P6 is therapeutic for AD by modulating the balance of Th2 and Th17.

Th2 cytokines and their downstream Janus kinase (JAK)-signal transducer and activation of transcription (STAT) pathways play a critical role in allergic asthma. We studied the effects of a pan-JAK inhibitor, pyridone 6 (P6), on asthmatic responses in a mouse model and investigated the mechanism for its biological effects. Mice were sensitized and challenged by ovalbumin (OVA). P6 treatment during the challenge phase suppressed eosinophilia in bronchoalveolar lavage (BAL) fluids but did not affect airway hyperresponsiveness (AHR). To improve the efficacy of the JAK inhibitor, P6 was encapsulated in polylactic-coglycolic acid nanoparticles (P6-PLGA). P6-PLGA treatment just before OVA challenge suppressed both airway eosinophilia and AHR. Although the IL-13 levels in BAL fluids and the OVA-specific IgE levels in serum after the challenge phase treatment with P6-PLGA were similar to those after a sham treatment, the eotaxin levels in BAL fluids and lung mCLCA3/Gob-5 expression were decreased in P6-PLGA-treated mice. Interestingly, the local IL-13 levels and serum OVA-specific IgE were decreased, while IL-17-producing T cells were increased by P6-PLGA treatment during the sensitization plus challenge phases. In vitro, P6 strongly suppressed the differentiation of Th2 from naive CD4 T cells, but it partly enhanced Th17 differentiation. P6 potently suppressed IL-13-mediated STAT6 activation and mCLCA3/Gob-5 expression in mouse tracheal epithelial cells. These findings suggest that the JAK inhibitor P6 suppresses asthmatic responses by inhibiting Th2 inflammation and that application of PLGA nanoparticles improves the therapeutic potency of P6.

We have developed a novel method for the determination of biogenic amines (dopamine, norepinephrine, 3-methoxytyramine, normetanephrine, serotonin, tyramine, tryptamine, 5-methoxytryptamine, and histamine) utilizing liquid chromatography with electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) combined with a separation-oriented derivatization technique. Using this approach, primary amino groups in the target amines were selectively dialkylated with a perfluorinated aldehyde reagent (2H,2H,3H,3H-perfluoroundecan-1-al) through reductive amination. The derivatives were directly injected onto an LC column containing perfluoroalkyl-modified stationary phase and were separated via gradient elution using a water/methanol/trifluoroacetic acid mixture and trifluoroethanol with formic acid as mobile phases. Matrix-induced signal suppression effects were eliminated because the binary fluorous-labeled amines were strongly retained on the fluorous-phase LC column, whereas the nonfluorous derivatives, including matrix components and monofluorous-labeled compounds such as the derivatization reagent, were poorly retained under the separation conditions. The linear dynamic ranges of the target amines were established over a concentration range of 0.01–1 nM (r > 0.9978), and the limits of detection were found to be 7.8–26 amol on column. The feasibility of this method was further evaluated by applying it to human plasma samples.

Ossification of the posterior longitudinal ligament (OPLL) is a pathological ossification in the spinal ligament, with formation of ectopic bone mainly through endochondral ossification. Bone morphogenetic proteins (BMPs) and activins are multifunctional proteins that belong to the transforming growth factor-beta superfamily and that have been implicated in the formation of new bone and cartilage. BMPs and activins signal via type I and type II receptors for BMPs (BMPRs) and activins (ActRs), respectively. OP-1/BMP-7 binds to BMPR-II and ActR-II and forms complexes with BMPR-IA and -IB and ActR-I. We studied the expression of BMPR-IA, -IB, and -II, ActR-I, ActR-II, and OP-1/BMP-7 by immunohistochemistry in ossified ligament tissues of patients with OPLL and control ligament tissues from patients with cervical disc herniation. The expression of BMPRs and ActRs was elevated in OPLL compared with controls. Expressions of BMPR-IA, -IB, and -II were observed not only in chondrocytes at the fibrocartilage tissue around the calcified zone but also in fibroblast-like spindle cells at the nonossified ligament. ActR-I and -II were found co-localized in the hypertrophic chondrocytes near the calcified zone and in the ossified tissue. OP-1/BMP-7 was expressed in chondrocytes near the calcified zone. In the control cases, the BMPRs and ActRs were only weakly expressed in the fibrocartilage tissue at the site of ligament attachments to bone and OP-1/BMP-7 was not detected. Enhanced expression of BMPRs at the nonossified ligament in OPLL patients suggests that these cells have a greater potential to differentiate into osteogenic cells than ligament cells from non-OPLL patients. The high expression of BMPRs and ActRs in the ectopic ossified ligament suggests that BMPs and activin may be tightly involved in the pathological ossification process of OPLL.

Edaravone has an anti-inflammatory effect in experimental models of various organ injuries. We reported that edaravone reduces the induction of inducible nitric oxide synthase (iNOS) as well as pro-inflammatory cytokines in endotoxin-treated rats with partial hepatectomy, leading to the prevention of liver injury. Studies were performed to investigate the mechanisms involved in the inhibition of iNOS expression by edaravone in hepatocytes. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of edaravone, and iNOS and its signal were analyzed. Edaravone decreased the expression of iNOS mRNA and its protein stimulated by IL-1β, resulting in the reduction of NO production. Edaravone inhibited the activation of transcription factor NF-κB through IκB degradation and the up-regulation of type I IL-1 receptor through PI3K/Akt activation, which are essential signals for iNOS induction. Further transfection experiments with iNOS promoter-luciferase construct having iNOS 3′-UTR revealed that edaravone decreased the stability of iNOS mRNA. In support of this observation, edaravone decreased the expression of iNOS antisense-transcript, which stabilizes iNOS mRNA by interacting with its 3′-UTR and RNA-binding protein. Edaravone may inhibit the induction of iNOS gene expression at steps of promoter transactivation and mRNA stabilization in cytokine-stimulated hepatocytes.

We have developed a novel method for selective and sensitive analysis of sialic acids (N-acetylneuraminic, N-glycolylneuraminic, and 2-keto-3-deoxy-D-glycero-D-galactonononic acid) utilizing liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with a fluorous derivatization technique. In this method, the carboxylic groups in the sialic acids are derivatized via amidation with heptadecafluoroundecylamine, a commercially available perfluoroalkylamine reagent. This reaction proceeds rapidly and readily at room temperature in the presence of a condensation reagent. Subsequently, the derivatives are retained specifically on an LC column with a perfluoroalkyl stationary phase by means of a fluorophilic or 'fluorous' interaction, and detected by positive electrospray ionization MS/MS. The detection limits of the examined sialic acids are in the range of 60-750 amol on column. We show that the proposed method can be used to analyze trace amounts of sialic acids in biological samples.

Fluorous derivatization followed by fluorous-phase liquid chromatographic (LC) separation exploits the affinity between perfluoroalkyl compounds for highly selective and quantitative isolation of various analytes. However, the applicability of this technique as a simple pretreatment for fluorometric determination in clinical settings has not been fully explored. Here we show the applicability of this technique to the clinical determination of non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma. Naproxen and felbinac, widely used native-fluorescent NSAIDs with a carboxyl group, can have toxic effects at acute doses, and were therefore chosen as representative NSAIDs. Samples were precolumn derivatized with a non-fluorescent fluorous amine, which allowed highly selective retention of only derivatized substances in the fluorous LC column. Thus, subsequently, only the retained fluorous-labeled and fluorescent analytes were detected fluorometrically at appropriate retention times. The detection limits for these two drugs were less than 11 fmol on column. Correlation curves were liner over the range of 0.04–10 and 5–250 nmol/mL plasma for both two drugs (r > 0.999) with good repeatability. Thus, this method offers a simple, sensitive, and selective solution for determination of NSAIDs in clinical settings.

RATIONALE A separation‐oriented derivatization method using a specific fluorous affinity between perfluoroalkyl‐containing compounds was applied to selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of sialyl oligosaccharides. The perfluoroalkyl‐labeled sialyl oligosaccharides could be selectively retained on an LC column with the perfluoroalkyl‐modified stationary phase and effectively distinguished from non‐derivatized species. METHODS Sialyl oligosaccharides (3′‐sialyllactose, 6′‐sialyllactose, sialyllacto‐ N ‐tetraose a, sialyllacto‐ N ‐tetraose b, sialyllacto‐ N ‐tetraose c, and disialyllacto‐ N ‐tetraose) were derivatized with 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11‐heptadecafluoroundecylamine via amidation in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (condensation reagent). The obtained derivatives were directly injected onto the fluorous LC column without any pretreatments and then detected by positive electrospray ionization MS/MS. RESULTS The method enabled accurate determination of the sialyl oligosaccharides in biological samples such as human urine and human milk, because there was no interference with matrix‐induced effects during LC/MS/MS analysis. The limits of detection of the examined sialyl oligosaccharides, defined as signal‐to‐noise ( S / N ) = 3, were in the range 0.033–0.13 nM. Accuracy in the range 95.6–108% was achieved, and the precision (relative standard deviation) was within 9.4%. CONCLUSIONS This method enabled highly selective and sensitive analysis of sialyl oligosaccharides, enabling accurate measurement of even their trace amounts in biological matrices. The proposed method may prove to be a powerful tool for the analysis of various sialyl oligosaccharides. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Perfluoroalkyl-containing compounds have a unique ‘fluorous’ property that refers to the remarkably specific affinity they share. Fluorous compounds can be easily isolated from non-fluorous species on the perfluoroalkyl-functionalized stationary phases used in fluorous solid-phase extraction and fluorous liquid chromatography by means of fluorous–fluorous interactions (fluorophilicity). Recently, this unique specificity has been applied to the highly selective enrichment and analysis of different classes of biogenic and related compounds in complex samples. Because the biogenic compounds are generally not ‘fluorous’, they must be derivatized with appropriate perfluoroalkyl group-containing reagent in order to utilize fluorous interaction. In this review, we introduce the application of fluorous affinity techniques including derivatization methods to biogenic sample analysis.

The existence of D-amino acids in mammals has been increasingly reported, and clarified that D and L enantiomers have different biological functions, distributions and metabolic pathways. Some D-amino acids were also reported to have a correlation with diseases, and the screening of biomarkers based on quantitative enantioselective metabolomics is highly expected. However, the amounts of most D-amino acids, especially the metabolic-related chiral amino acids, are usually at trace levels in biological samples. Therefore, highly selective and sensitive analytical methods are essential for their accurate determination. In this focusing review, multi-dimensional high-performance liquid chromatographic (HPLC) systems for the precise determination of metabolic-related chiral amino acids are introduced with their application to biological samples. For the highly-sensitive determination, target amino acids were precolumn-derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The multi-dimensional HPLC systems were composed of a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension. By using these systems, various proteinogenic amino acids were clearly observed in the tissues and physiological fluids of rodents. The existence of metabolic-related chiral amino acids in various biological samples were also elucidated.

RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear.To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis.The corpus tissue of RUNX3R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate.RUNX3R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer.

Abstract Aire, the defect of which is responsible for the development of autoimmunity, is predominantly expressed in medullary thymic epithelial cells, and it controls a wide variety of genes, including those of tissue-restricted Ags, for establishing thymic tolerance. Aire is also expressed from APCs in the periphery, called extrathymic Aire-expressing cells (eTACs), and their complementing role to thymic tolerance has been suggested. eTACs are composed of two distinct classes of APCs, conventional dendritic cell (cDC)–type and group 3 innate lymphoid cell (ILC3)-like–type expressing retinoic acid receptor–related orphan receptor γt (RORγt). Although the essential role of Aire in the latter in the Th17-mediated immune response against Candida albicans has been reported, the role of Aire in the cDC-type eTACs for this action has not been examined. Furthermore, the significance of Aire in the production of the transcriptome of the cDC-type eTACs remains unknown. We have approached these issues using a high-fidelity Aire-reporter mouse strain. We found that although the cDC-type eTACs dominated ILC3-like–type eTACs in number and they served as efficient APCs for the immune response against an exogenous Ag as well as for the C. albicans–specific Th17 immune response, loss of Aire in cDC-type eTACs showed no clear effect on these functions. Furthermore, loss of Aire showed no major impact on the transcriptome from cDC-type eTACs. These results suggested that Aire in cDC-type eTACs may not have a cell-intrinsic role in the immune response in contrast to the role of Aire in ILC3-like–type eTACs.

The transplantation of encapsulated dopamine-secreting cells into the striatum represents one potential means of treating Parkinson's disease. The present study investigated the ability of encapsulated PC12 cells, which are derived from rat pheochromocytoma, to supply L-dopa and dopamine into the primate brain in the long term and to effect functional improvement in the animals. Following polymer encapsulation, PC12 cells were transplanted into the striatum of hemiparkinsonian monkeys. The secretion of L-dopa and dopamine from the encapsulated cells, the morphology of these cells, the histology of the host striatum surrounding the capsule, and functional changes in the host animals were examined 1, 6, and 12 months after transplantation. Analysis of retrieved capsules revealed that the PC12 cells survived and continued to release L-dopa and dopamine even 12 months after transplantation. The histological response of the host brain surrounding the capsules was minimal and there were no signs of immunological rejection or tumor formation. The physical condition of the host animals was good for 12 months, and hematologic and cerebrospinal fluid analysis revealed that no animals suffered from infection or immunological reaction. These PC12 cell-grafted monkeys showed improvements in hand movements after transplantation, effects that lasted for at least 12 months. These results further support the potential use of this approach for the treatment of Parkinson's disease.

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.

A highly selective and sensitive fluorometric method for the determination of bisphenols has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyryl chloride (PBC), followed by reversed-phase liquid chromatography (LC). The bisphenols, containing two phenolic hydroxyl groups in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PBC. The derivatives afforded intramolecular excimer fluorescence (440–520 nm) which can clearly be discriminated from normal fluorescence (360–420 nm) emitted from PBC and monopyrene-labeled derivatives of monophenols. The PBC derivatives of bisphenols could be separated by reversed-phase LC on an octyl column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for bisphenols were 3.0–5.0 fmol, for a 20 μl injection. The method was successfully applied to the determination of bisphenol A in hot water in contact with commercially available baby bottle samples after solid-phase extraction.

In order to clarify the possible involvement ofN-methyl-d-aspartate (NMDA) receptors in mediating striatal Fos protein induction and behavioral sensitization after methamphetamine administration, we examined the effects of non-competitive NMDA receptor antagonist MK-801 on these phenomena in rats. A single administration of 1.0 and 5.0 mg/kg methamphetamine resulted in a dose-dependent increase in Fos-immunoreactive cells in the medial striatum. Prior exposure to 5.0 mg/kg methamphetamine enhanced ipsilateral rotational behavior in response to subsequent methamphetamine administration in unilateral nigral-lesioned rats (sensitization). Pretreatment with 1.0 mg/kg MK-801 completely prevented both the expression of striatal Fos protein and the development of acute behavioral sensitization following a single injection of 5.0 mg/kg methamphetamine. These results suggest that NMDA receptor-mediated mechanisms contribute to the expression of striatal Fos protein associated with behavioral sensitization that follows exposure to methamphetamine.

In order to deliver glial cell line-derived neurotrophic factor (GDNF) into the brain, we have established a cell line that produces GDNF in a continuous fashion by genetic engineering. These cells were encapsulated and grafted into parkinsonian model rats that had received unilateral intrastriatal injection of 6-hydroxydopamine 2 weeks earlier. Neurochemical analysis showed that GDNF has been produced from the capsule for 6 months after grafting and histological analysis revealed good survival of GDNF-producing cells in the capsule 6 months after grafting. The density of nigrostriatal dopaminergic fibers in the striatum as well as the number of dopaminergic cell bodies in the substantia nigra recovered significantly after GDNF-producing cell grafting. These results suggest the possible application of GDNF-producing cell grafting for the treatment of Parkinson's disease.

A fluorescence derivatization LC method is a powerful tool for the analysis with high sensitivity and selectivity of biological compounds. In this review, we introduce new types of fluorescence derivatization LC analysis methods. These are (1) detection-selective derivatization methods based on fluorescence interactions generated from fluorescently labeled analytes: excimer fluorescence derivatization and fluorescence resonance energy transfer (FRET) derivatization; (2) separation-selective derivatization methods using the fluorous separation technique: fluorous derivatization, F-trap fluorescence derivatization, and fluorous scavenging derivatization (FSD).

Novel luminol-related compounds containing a 2-arylbenzothiazole moiety were synthesized by the reaction of a series of aromatic aldehydes with 7-amino-6-sulfanylphthalazine-1,4(2H,3H)-dione (ASPH). The chemiluminescent properties of the ASPH derivatives were examined and compared with luminol and ASPH. All compounds produced chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in sodium hydroxide solution. The chemiluminescence intensities of the chemiluminophores were influenced by the concentrations of hydrogen peroxide, potassium hexacyanoferrate(III) and sodium hydroxide. The ASPH derivatives of the aromatic aldehydes examined were found to produce chemiluminescence 1.1–40 times more intensely than luminol.

A highly sensitive and selective fluorimetric determination method for dicarboxylic acids (C5-C12) has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid hydrazide (PBH), followed by reversed-phase liquid chromatography (LC). The carboxylic acids were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acids. The structures of the derivatives and the emission of excimer fluorescence were studied by LC with mass spectrometry and with spectrofluorimetry, respectively. The PBH derivatives of the carboxylic acids could be separated by reversed-phase LC on an ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) were 1.3 fmol to undetectable for a 20-microl injection.

In this paper, we introduce a novel approach for the highly selective and sensitive analysis of native fluorescent bioamines (indoleamines and catecholamines). This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the bioamines' amino groups. In this detection process, we monitored the FRET from the native fluorescent moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 15 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated the FRET most effectively. The OPA derivatives of the native fluorescent bioamines emitted OPA fluorescence (445 nm) through an intermolecular FRET process when they were excited at the excitation maximum wavelengths of the native fluorescent bioamines (280 nm). The generation of FRET was confirmed through comparison with the analysis of a nonfluorescent amine (isoleucine) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the indoleamines and catecholamines when performing LC on an ODS column. The detection limits (signal-to-noise ratio, 3) for the indoleamines and catecholamines, at a 20-μL injection volume, were 17−120 and 28−200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than those of systems that take advantage of both native fluorescence detection (i.e., without derivatization) and the conventional detection of OPA derivatives. Furthermore, this method provides enough selectivity and sensitivity for the determination of the indoleamines present in the urine of healthy humans.

During hepatic ischemia/reperfusion (I/R), proinflammatory cytokines such as tumor necrosis factor α and interleukin (IL) 1β stimulate the induction of inducible nitric oxide synthase (iNOS) in hepatocytes, followed by massive production of nitric oxide. We hypothesized that I/R upregulated the susceptibility of hepatocytes to confer the induction of iNOS gene expression. This study was designed to investigate whether cell susceptibility occurs in response to I/R and to delineate the mechanisms underlying the susceptibility. Hepatocytes were isolated from rats with hepatic I/R or sham, cultured, and treated with IL-1β. The iNOS induction and its signal including inhibitor κB (IκB) kinase/nuclear factor κB (NF-κB) and Akt/type 1 interleukin 1 receptor (IL-1R1) were analyzed. Hepatocytes isolated from rats with I/R markedly increased the production of nitric oxide when stimulated by IL-1β as compared with sham control. Ischemia/R also increased the levels of iNOS protein and its messenger RNA. Furthermore, I/R enhanced the activation of transcription factor NF-κB and the transactivation of iNOS promoter. However, I/R had no effects on the degradation of IκB and the nuclear translocation of p65 subunit of NF-κB. In contrast, I/R increased the phosphorylation of Akt and the upregulation of IL-1R1 induction, which is essential signal for the transcriptional activation of iNOS in addition to IκB kinase/NF-κB. These results demonstrate that I/R may augment hepatocyte susceptibility for the induction of iNOS gene expression through the enhancement of IL-1R1. ABBREVIATIONS-NO, nitric oxide; I/R, ischemia/reperfusion; IL-1β, interleukin 1β; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor κB; IL-1R1, type 1 interleukin 1 receptor

In animal models of liver injury, proinflammatory cytokines are implicated in inducing iNOS, which is followed by the production of NO in hepatocytes. Previously we have reported that the up-regulation of type I IL-1 receptor (IL-1RI) is required for the transcriptional activation of iNOS gene, in concert with the activation of transcription factor NF-kappaB. In this study, we found three alternatively spliced isoforms of IL-1RI in primary cultured rat hepatocytes: two (long and short) membrane-bound and one soluble IL-1RI. Interleukin (IL)-1beta markedly augmented the mRNA levels of long and short IL-1RI with time, but was less effective for soluble IL-1RI. Two membrane-bound IL-1RI were localized in the intracellular fraction, whereas soluble IL-1RI was released into the culture medium. Cotransfection experiments with iNOS promoter-luciferase constructs revealed that the overexpression of long and short IL-1RI, but not soluble IL-1RI, significantly increased the transactivation of iNOS promoter and the stabilization of its mRNA. In contrast, the addition of conditioned medium containing soluble IL-1RI reduced the induction of iNOS and NO production stimulated by IL-1beta. These results further suggest that the enhancement of IL-1RI isoforms may contribute to the regulation of iNOS induction in hepatocytes.

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines' amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-kappaB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-kappaB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

We developed an LC method for the sensitive and selective fluorometric determination of polythiols. This method employs pre-column intramolecular excimer-forming fluorescence derivatization with N-(1-pyrene)iodoacetamide followed by LC separation. Polythiols were converted to the corresponding dipyrene-labeled derivatives, and the derivatives afforded intramolecular excimer fluorescence (440–540 nm). After the optimization using dithiothreitol and dimercaprol as model polythiols, α-lipoic acid (LA) and α-lipoamide were determined with high sensitivity and selectivity. The detection limits for polythiols were 0.6–3.5 fmol on column. Furthermore, this method could be successfully applied to the determination of LA in commercial dietary supplements and in human urine.

We have developed a new and simple method based on “fluorous derivatization” for LC of native fluorescent compounds. This method involves the use of a column with a fluorous stationary phase. Native fluorescent analytes with target functional groups are precolumn derivatized with a nonfluorescent fluorous tag, and the fluorous-labeled analytes are retained in the column, whereas underivatized substances are not. Only the retained fluorescent analytes are detected fluorometrically at appropriate retention times, and retained substrates without fluorophores are not detected. In this study, biologically important carboxylic acids (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindoleacetic acid) and drugs (naproxen, felbinac, flurbiprofen, and etodolac) were used as model native fluorescent compounds. Experimental results indicate that the fluorous-phase column can selectively retain fluorous compounds including fluorous-labeled analytes on the basis of fluorous separation. We believe that separation-oriented derivatization presented here is the first step toward the introduction of fluorous derivatization in quantitative LC analysis.

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. Several Smad-independent pathways including the JNK and the RhoA-ROCK pathways have been implicated in the induction of RORγt, the master regulator of Th17, however, molecular mechanisms underlying Smad-independent pathway remain largely unknown. To identify novel pathways involved in Th17 differentiation, we screened 285 chemical inhibitors for known signaling pathways. Among them, we found that Kenpaullone, a GSK3-β and CDK inhibitor, efficiently suppressed TGF-β-mediated RORγt induction and enhanced Foxp3 induction in primary T cells. Another CDK inhibitor, Roscovitine, but not other GSK3-β inhibitors, suppressed Th17 differentiation and enhanced iTreg development. Kenpaullone and Roscovitine suppressed experimental autoimmune encephalomyelitis (EAE), a typical Th17-mediated autoimmune disease model. These two compounds enhanced STAT5 phosphorylation and restored IL-2 production in the presence of TGF-β. These data suggest that CDK inhibitors modulate TGF-β-signaling pathways, which restore TGF-β-mediated suppression of IL-2 production, thereby modifying the Th17/iTreg balance.

Okadaic acid (OA) group are diarrheal shellfish poison that accumulates in the midgut glands of shellfish. It is difficult to remove these poisons by normal cooking because they are thermally stable and hydrophobicity. Therefore, in order to prevent foodborne disease due to shellfish poison, analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) before shipment is necessary. Herein the selective analytical method for OA group in shellfish sample using fluorous derivatization coupled with LC-MS/MS was developed. OA group were derivatized with the fluorous alkylamine reagent by condensing agent, and the obtained derivatives were separated with fluorous LC column (Fluofix-II 120E, 250 × 2.0 mm i.d., 5 μm, Fujifilm Wako Pure Chemical). The derivatized OA group were selective retained by fluorous LC column and accurate analysis was enabled. The present method was applied to the analysis of OA and dinophysistoxin-1 (DTX-1) in scallop midgut gland which is the certified reference material provided by national metrology institute of Japan. As a result of analysis using the present method with DTX-2 as the internal standard, the quantitative value were in agreement with the certified value.

The immune system plays a dual role in human health, functioning both as a protector against pathogens and, at times, as a contributor to disease. This feature emphasizes the importance to uncover the underlying causes of its malfunctions, necessitating an in-depth analysis in both pathological and physiological conditions to better understand the immune system and immune disorders. Recent advances in scientific technology have enabled extensive investigations into gene regulation, a crucial mechanism governing cellular functionality. Studying gene regulatory mechanisms within the immune system is a promising avenue for enhancing our understanding of immune cells and the immune system as a whole. The gene regulatory mechanisms, revealed through various methodologies, and their implications in the field of immunology are discussed in this chapter.

Cyclic ADP-ribose (cADPR), a well-known stimulator of Ca2+release from the intracellular Ca2+pool, has recently emerged as a potential regulator of insulin secretion in pancreatic β cells. As recently described, BST-1 is a glycosyl-phosphatidylinositol (GPI)-anchored surface molecule that exhibits homology with CD38 andAplysiaADP-ribosyl cyclase. Like CD38, BST-1 has both ADP-ribosyl cyclase and cADPR hydrolase activities. As a step toward elucidating the physiological role of cADPR in insulin secretion, we examined whether BST-1 is expressed in pancreatic islet cells. Sensitive reverse transcription-polymerase chain reaction detected almost as abundant expression of BST-1 mRNA in pancreatic islets as CD38 mRNA. Immunohistochemical analyses utilizing mirror image sections revealed that BST-1 protein is expressed in a majority of the cells in pancreatic islets and that at least β cells and, to an even greater extent, α cells express BST-1. These observations suggest the involvement of multiple enzymes in the regulation of cADPR concentrations in pancreatic islet cells.

Pseudomonas pseudoalcaligenes KF707 grows on biphenyl and salicylate as sole sources of carbon. The biphenyl-catabolic (bph) genes are organized as bphR1A1A2(orf3)A3A4BCX0X1X2X3D, encoding the enzymes for conversion of biphenyl to acetyl coenzyme A. In this study, the salicylate-catabolic (sal) gene cluster encoding the enzymes for conversion of salicylate to acetyl coenzyme A were identified 6.6-kb downstream of the bph gene cluster along with a second regulatory gene, bphR2. Both the bph and sal genes were cross-regulated positively and/or negatively by the two regulatory proteins, BphR1 and BphR2, in the presence or absence of the effectors. The BphR2 binding sequence exhibits homology with the NahR binding sequences in various naphthalene-degrading bacteria. Based on previous studies and the present study we propose a new regulatory model for biphenyl and salicylate catabolism in strain KF707.

A simple and sensitive analytical method for the determination of polycarbamate in water samples was developed. In this method, polycarbamate was cleaved under alkaline conditions and derivatized with dimethyl sulfate to methyl dimethyldithiocarbamate (DMDC-methyl) and dimethyl ethylenebisdithiocarbamate (EBDC-dimethyl). After the solid-phase extraction of the resulting methyl derivatives, they were measured by liquid chromatography with tandem mass spectrometry (LC–MS/MS), based on reversed-phase separation and MS/MS detection with positive atmospheric pressure chemical ionization. The absolute recoveries (mean ± SD) all through the procedure from polycarbamate to DMDC-methyl and EBDC-dimethyl were 62.6 ± 4.3 and 73.5 ± 5.9%, respectively. The limits of detection and quantification of polycarbamate in the water samples were 0.061 and 0.20 μg/L in the form of DMDC-methyl, and 0.032 and 0.11 μg/L in the form of EBDC-dimethyl, respectively. The method was validated at levels of 0.25, 1.0, and 5.0 μg/L in the tap water and river water samples, and accuracy was achieved in the range of 94–109%. The proposed method can be applied to the determination of trace amounts of polycarbamate in environmental water samples.

Edaravone, a free radical scavenger, plays crucial roles in the prevention of injuries to the brain, heart, and liver. Our in vivo study indicated that edaravone prevented endotoxin-induced liver injury through inhibition of NO production in addition to reductions in oxidative products and proinflammatory cytokine induction. Studies were performed to determine whether edaravone directly influences the induction of iNOS in murine RAW264 macrophages as a substitute for Kupffer cells (resident macrophages) in the liver. RAW264 cells were treated with LPS (1 μg/mL) in the presence or absence of edaravone. NO production, iNOS induction, and its related signaling were analyzed. Edaravone (0.5 - 5 mM) decreased the production of NO stimulated by LPS in time- and dose-dependent manners, and these concentrations of edaravone had no cytotoxic effects. Edaravone decreased the levels of iNOS protein and mRNA. Transfection experiments with iNOS promoter-luciferase constructs revealed that edaravone inhibited the activities of both iNOS promoter transactivation and iNOS mRNA stabilization. However, edaravone did not have any effects on IκBα degradation or nuclear factor-κB activation. In contrast, edaravone markedly suppressed the LPS-stimulated expression of iNOS antisense-transcript, which stabilizes iNOS mRNA by interacting with its 3′-untranslated region and RNA-binding proteins. Edaravone may inhibit the induction of iNOS gene expression at the steps of its promoter transactivation in a nuclear factor-κB-independent manner and mRNA stabilization in RAW264 cells. ABBREVIATIONS-NF-κB-nuclear factor-κB; 3′-UTR-3′-untranslated region; AS-transcript-antisense-transcript

Purpose: The anti-aquaporin-4 (AQP4) antibody was recently reported to be associated with neuromyelitis optica (NMO). Optic nerve involvements in many NMO cases are bilateral and the prognosis is poor. However, it has been suggested that plasma exchange is effective for those patients when steroid pulse therapy is ineffective. Herein, we report successful treatment of a patient with NMO using double-filtration plasmapheresis (DFPP). Case: A 22-year-old woman consulted a neurologist for neck pain in March 2008. High-intensity lesions were shown in the cervical spinal cord by T2-weighted magnetic resonance imaging. On July 15, the patient was referred to our department for a headache and pain and blurred vision in the left eye. The best-corrected visual acuity was 20/50 and 20/500 in the right and left eyes, respectively, with visual field defects observed in both. After 3 courses of steroid pulse therapy, anti-AQP4 antibodies were positive. In November, the patient again noticed visual acuity loss in the left eye and was treated by additional steroid pulse therapy, which was not effective. Next, she underwent plasma exchange therapy, though it was stopped due to hypotension and dyspnea. The next day, the patient underwent DFPP treatment and visual function gradually recovered. Conclusion: It is important to consider NMO when steroid pulse therapy is not effective. We successfully and safely treated NMO in a young adult patient using DFPP.

To identify potent EP2/EP4 dual agonists with excellent subtype selectivity, a series of γ-lactam prostaglandin E analogs bearing a 16-phenyl ω-chain were synthesized and evaluated. Structural hybridization of 1 and 2, followed by more detailed chemical modification of the benzoic acid moiety, led us to the discovery of a 2-mercaptothiazole-4-carboxylic acid analog 3 as the optimal compound in the series. An isomer of this compound, the 2-mercaptothiazole-5-carboxylic acid analog 13, showed 34-fold and 13-fold less potent EP2 and EP4 receptor affinities, respectively. Structure activity relationship data from an in vitro mouse receptor binding assay are presented. Continued evaluation in an in vivo rat model of another 2-mercaptothiazole-4-carboxylic acid analog 17, optimized for sustained compound release from PLGA microspheres, demonstrated its effectiveness in a rat bone fracture-healing model following topical administration.

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).

Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine-transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization.A knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively.Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased.Addition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.

A highly selective and simple fluorimetric liquid chromatographic method for the determination of triethylenetetramine (TETA), a therapeutic drug for Wilson's disease, in human and rabbit sera is described. This method is based on intramolecular excimer-forming fluorescence derivatization, which allows spectrofluorometric discrimination of polyamino compounds from monoamino species, followed by liquid chromatography. TETA and 1,6-hexanediamine (internal standard) were converted to the corresponding excimer-forming derivatives with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester. The derivatives were separated within 20 min on a reversed-phase column using isocratic elution and detected spectofluorometrically at 480 nm with excitation at 345 nm. This method was successfully applied to the monitoring of TETA in human and rabbit sera with a simple pretreatment. The detection limit for TETA in serum was 18 ng/ml (0.13 nmol/ml) corresponding to 0.2 pmol on column at a signal-to-noise ratio of 3.

Object. This study was conducted to evaluate the effects of grafting encapsulated basic fibroblast growth factor (bFGF)—secreting cells in rat brains subjected to ischemic injury. Methods. Two cell lines were used for encapsulated grafting in this experiment, namely, a bFGF-secreting cell line established by genetic manipulation of baby hamster kidney (BHK) cells, and a naive BHK cell line. Forty-seven Sprague—Dawley rats were used in this experiment. The animals were divided into the following three groups: those receiving grafts of encapsulated bFGF-secreting cells (BHK-bFGF group); those with grafts of encapsulated naive BHK cells (naive BHK group); and those with no grafts (control group). The authors implanted encapsulated cells into the right striatum of host rats in the BHK-bFGF and naive BHK groups. Six days after grafting, the host and control animals underwent permanent right middle cerebral artery occlusion (MCAO) with an intraluminal suture procedure. The infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride staining and computerized image analysis 24 hours after MCAO. Fragmentations of DNA in the host brains were analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling 12 hours after MCAO. The authors found that the infarct volume in the BHK-bFGF group was reduced by approximately 30% compared with that in the naive BHK and control groups. In the ischemic penumbral area, the number of apoptotic cells in the BHK-bFGF group was significantly decreased compared with that in the other groups. Conclusions. The grafting of encapsulated BHK bFGF-secreting cells protected the brain from ischemic injury. Encapsulation and grafting of genetically engineered cells such as bFGF-secreting cells is thus thought to be a useful method for protection against cerebral ischemia.

The PC12 cells are well known for their ability to secrete dopamine and levodopa. In multiple animal mode encapsulated PC12 cells have been shown to ameliorate parkinsonian symptoms when transplanted into the striatum; technique is expected to be effective clinically as well. The present study was performed using nonhuman primates to ensure that the transplantation of encapsulated PC12 cells is likely to be both safe and effective in human clinical trials.Unencapsulated or encapsulated PC12 cells were implanted into the brains of Japanese monkeys (Macaca fuscata). Histological and immunocytochemical analyses were performed 1, 2, 4, and 8 weeks posttransplantation on the unencapsulated cells and 2, 4, and 8 weeks after transplantation on the encapsulated cells. The survival of the PC12 cells inside the capsule was determined by measuring the amounts of dopamine and levodopa released from the capsules a removal from the striatum. Magnetic resonance imaging was performed in both unencapsulated and encapsulated PC12 cell-grafted groups. Due to the immunological reaction of the host brain no unencapsulated PC12 cells remained in the grafted area 8 weeks after transplantation. On the contrary, encapsulated PC12 cells retrieved from the host brain continued to release dopamine and levodopa even 8 weeks after implantation. The host's reaction to the PC12-loaded capsule was much weaker than that to the unencapsulated PC12 cells.These results suggest that the transplantation of encapsulated PC12 cells could be a safe and effective treatment modality for Parkinson disease in human patients.

A highly sensitive and selective fluorometric method for the determination of histamine and histidine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent followed by reversed-phase liquid chromatography. The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by derivatization. The derivatives afforded intramolecular excimer fluorescence (440-540 nm), which can clearly be discriminated from the normal fluorescence (360-420 nm) emitted from reagent blanks. The detection limits (signal-to-noise ratio = 3) were femto mole levels.

Abstract A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer‐forming fluorescence derivatization with a pyrene reagent, 4‐(1‐pyrene)butanoyl chloride (PBC), followed by reversed‐phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4‐(1‐pyrene)butyric acid N ‐hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 m HCl. The detection limits (signal‐to‐noise ratio = 3) for polyamines in urine were 1.1–3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean‐up procedures. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Background Recent evidence indicates that CXC chemokines such as human interleukine-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant (CINC) have mitogenic and anti-apoptotic functions in addition to their chemotactic activity toward neutrophils. In the liver, hepatocyte growth factor (HGF) is implicated as a mitogen in the regeneration of liver. However, little is known the interaction between HGF and CXC chemokines during liver injury, repair and regeneration. We hypothesized that HGF may stimulate the expression of such chemokines, which contributes to mitogenic action in liver regeneration. Materials and methods Primary cultured rat hepatocytes were treated with recombinant human HGF (rhHGF), in the absence and presence of calpain inhibitor-1 (CI-1), genistein or anti-CINC-1 antibody. Levels of CINC-1 and its mRNA, tyrosine phosphorylation of HGF receptor (c-Met), the activation of transcription factor, nuclear factor-κB (NF-κB), and activities of DNA synthesis were measured. Results rhHGF enhanced the production of CINC-1 time- and dose-dependently, which followed the increased levels of CINC-1 mRNA. Under the same conditions, rhHGF increased the tyrosine phosphorylation of c-Met. The electrophoretic mobility shift assay revealed that rhHGF activated the nuclear translocation of NF-κB and its DNA binding. Proteasome inhib-itor (CI-1) blocked the NF-κB activation and the CINC-1 production. The tyrosine kinase inhibitor (genistein) inhibited the activities of CINC-1 production and the DNA synthesis stimulated by rhHGF. However, the treatment of anti-CINC-1 antibody had no effect on the DNA synthesis. Conclusions These results demonstrate that rhHGF can stimulate the induction of CINC-1 gene expression through the activation of NF-κB via its receptor c-Met in hepatocytes. Although CINC-1 seems to be not associated with the enhancement of DNA synthesis by rhHGF, it cannot negate the possibility that CINC-1 may contribute to liver repair and regeneration. Recent evidence indicates that CXC chemokines such as human interleukine-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant (CINC) have mitogenic and anti-apoptotic functions in addition to their chemotactic activity toward neutrophils. In the liver, hepatocyte growth factor (HGF) is implicated as a mitogen in the regeneration of liver. However, little is known the interaction between HGF and CXC chemokines during liver injury, repair and regeneration. We hypothesized that HGF may stimulate the expression of such chemokines, which contributes to mitogenic action in liver regeneration. Primary cultured rat hepatocytes were treated with recombinant human HGF (rhHGF), in the absence and presence of calpain inhibitor-1 (CI-1), genistein or anti-CINC-1 antibody. Levels of CINC-1 and its mRNA, tyrosine phosphorylation of HGF receptor (c-Met), the activation of transcription factor, nuclear factor-κB (NF-κB), and activities of DNA synthesis were measured. rhHGF enhanced the production of CINC-1 time- and dose-dependently, which followed the increased levels of CINC-1 mRNA. Under the same conditions, rhHGF increased the tyrosine phosphorylation of c-Met. The electrophoretic mobility shift assay revealed that rhHGF activated the nuclear translocation of NF-κB and its DNA binding. Proteasome inhib-itor (CI-1) blocked the NF-κB activation and the CINC-1 production. The tyrosine kinase inhibitor (genistein) inhibited the activities of CINC-1 production and the DNA synthesis stimulated by rhHGF. However, the treatment of anti-CINC-1 antibody had no effect on the DNA synthesis. These results demonstrate that rhHGF can stimulate the induction of CINC-1 gene expression through the activation of NF-κB via its receptor c-Met in hepatocytes. Although CINC-1 seems to be not associated with the enhancement of DNA synthesis by rhHGF, it cannot negate the possibility that CINC-1 may contribute to liver repair and regeneration.

A method to measure the concentrations of microcystins (MCs) in water samples has been developed by incorporating pre-column fluorescence derivatization and liquid chromatography (LC). A solid-phase extraction for pretreatment was used to extract the MCs in water samples. The MCs were derivatized with excimer-forming 4-(1-pyrene)butanoic acid hydrazide (PBH). The MCs could then be detected by fluorescence after separation with a pentafluorophenyl (PFP)-modified superficially porous (core shell) particle LC column. The derivatization reactions of MCs with PBH proceeded easily in the presence of 4,6-dimethoxy-1,3,5-triazin-2-yl-4-methylmorpholinium (DMT-MM) as a condensation reagent, and the resulting derivatives could be easily separated on the PFP column. The derivatives were selectively detected at excimer fluorescence wavelengths (440-540 nm). The instrument detection limit and the instrument quantification limit of the MCs standards were 0.4-1.2 μg L(-1) and 1.4-3.9 μg L(-1), respectively. The method was validated at 0.1 and 1.0 μg L(-1) levels in tap and pond water samples, and the recovery of MCs was between 67 and 101% with a relative standard deviation of 11%. The proposed method can be used to quantify trace amounts of MCs in water samples.

The mushroom known as Reishi (Ganoderma lucidum) has been used as an herbal medicine for tumor treatment and immune system activation. Because its effects on the differentiation of effector T helper cells have not yet been fully understood, we investigated the effects of Reishi and those of its principal ingredient, β-glucan, on the activation of dendritic cells and the differentiation of Th17 cells. Reishi extracts as well as purified β-glucan (Curdran) activated DCs and caused them to produce large amounts of IL-23. β-glucan also enhanced and sustained the transcription of IL-23p19. The MEK-ERK signaling pathway positively regulates IL-23p19 transcription in β-glucan-stimulated DCs. In a mixed leukocyte reaction, Reishi-stimulated DCs preferentially induced Th17 cells. Furthermore, orally-administrated Reishi increased the percentages of Th17 cells and the transcription levels of antimicrobial peptides. Our results show that Reishi and β-glucan activate DCs to produce large amounts of IL-23, which induces Th17 differentiation both in vitro and in vivo.

We performed a comprehensive quantification of 20 amino acids in RPMI 1640 medium-cultured human colorectal adenocarcinoma cells to evaluate the efficacy of 5-fluorouracil treatment under hypoxic and hypoglycemic conditions, which mimic the tumor microenvironment. In this study, we developed a simple and comprehensive analytical method by using LC-MS/MS connected to the Intrada amino acid column, which eluted amino acids within 9 min. The present method covered a linearity range of 3.6 - 1818 μM, except for Gly (227 - 1818 μM), Ala, Asp, His (7.1 - 1818 μM each), and Trp (3.6 - 909 μM). The limits of detection were in the range of 0.02 - 38.0 pmol per injection in a standard solution. Amino acid concentration data were analyzed using principal-component analysis to represent samples on two-dimensional graphs. Linear discriminant analysis was used to classify samples on the score plots. Using this approach, the effect of 5-fluorouracil treatment could be successfully discriminated at high discrimination rates. Moreover, several amino acids were extracted from corresponding loading plots as candidate markers for distinguishing the effects of the 5-fluorouracil treatment or tumor microenvironmental conditions. These results suggest that our proposed method might be a useful tool for evaluating the efficacy of anticancer drugs in the tumor microenvironment.

Gene-knock-out studies implicate roles of lymphotoxin (LT) α β and LTβR in the initial phase of Peyers patch (PP) organogenesis. We recently identified the requirement of IL-7Rα/γc/Jak3 signal in LTα β production of IL-7Rα+ cells. These observations lead us to a hypothetical model for PP organogenesis with three cellular components. The first is the producer of the ligand for IL-7Rα, which then stimulate the IL-7Rα+ cells to produce LTα β, activating the LTβR+ cells to form an organizing center for PP organogenesis. This model is similar to that of inflammation, suggesting that PP organogenesis is a programmed version of inflammation.

4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin-2-yl)benzoic acid N-hydroxysuccinimide ester was synthesized as a highly sensitive and selective chemiluminescence derivatization reagent for primary and secondary amines in liquid chromatography. Methyl-n-octylamine, n-nonylamine and n-decylamine were used as model compounds to optimize the derivatization, separation and chemiluminescence reaction conditions. This reagent reacts selectively with amines in the presence of triethylamine to give the highly chemiluminescent derivatives, which produce chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The chemiluminescent derivatives of the three amines can be separated within 20 min by reversed-phase liquid chromatography with isocratic elution, followed by chemiluminescence detection. The detection limits (signal-to-noise ratio=3) for primary and secondary amines are at sub-fmol levels for a 20-microl injection. Furthermore, this method was applicable to the determination of amantadine in human plasma.

A practical method of synthesizing a highly selective EP4-receptor agonist 1 using Corey lactone 2 as a key intermediate was developed. Selective methanesulfonylation of the primary alcohol of the diol 8 under the newly devised conditions followed by the protection of the remaining secondary alcohol are key reactions in this new method. Further biological evaluation of 1a-b is also reported.

A liquid chromatographic (LC) method for sensitive and selective fluorometric determination of p-hydroxyphenylethylamino group containing compounds is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and a phenolic hydroxyl moiety in a molecule, were converted to the corresponding dipyrene-labeled derivatives by one-step derivatization. The dipyrene-labeled derivatives afforded intramolecular excimer fluorescence (440-540 nm), which can clearly be discriminated from the normal fluorescence (360-420 nm) emitted from reagent blanks. The derivatives of tyrosine and tyramine could be separated by reversed-phase LC on ODS column under conditions of isocratic elution. The detection limits (signal-to-noise ratio = 3) for tyrosine and tyramine were 4.5 and 2.6 fmol per 20 microL injection, which corresponded to analyte concentrations of 0.9 and 0.5 nM, respectively.

In this study, we combined a column-switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak-free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess of the unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column-switching system placed in front of an analytical reversed-phase column. The signal of the fluorous-tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type-II model mice and their control.

Metabolomic studies conducted for evaluating cancer pathogenesis and progression by monitoring the amino acids metabolic balance hold great promise for assessing current and future anticancer treatments. We performed a comprehensive quantification of 21 amino acids concentrations in cultured human colorectal adenocarcinoma cells treated with the anticancer drugs 5-fluorouracil, irinotecan, and cisplatin. A precolumn fluorescence derivatization-HPLC method involving 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was used. Amino acid concentration data were analyzed by principal-component analysis and partial least-squares multivariate statistical methods to represent samples on two-dimensional graphs. The hierarchical cluster analysis and linear discriminant analysis were used to classify the samples on the score plots. Unlike the cluster analysis approach, the linear discrimination analysis classification successfully distinguished anticancer drug-treated samples from the untreated controls. Moreover, three candidate amino acids (serine, aspartic acid, and methionine) were identified from the loading plots as potential biomarkers. Our proposed method might be able to evaluate the effectiveness of anticancer therapy even in small laboratories or medical institutions.

The design, synthesis, and biological evaluation of novel 3-aryl-indazole derivatives as peripherally selective pan-Trk inhibitors are described. Three strategies were used to obtain a potent compound exhibiting low central nervous system (CNS) penetration and high plasma exposure: 1) a structure-based drug design (SBDD) approach was used to improve potency; 2) a substrate for an efflux transporter for lowering brain penetration was explored; and 3) the most basic pKa (pKa-MB) value was used as an indicator to identify compounds with good membrane permeability. This enabled the identification of the peripherally targeted 17c with the potency, kinase-selectivity, and plasma exposure required to demonstrate in vivo efficacy in a Complete Freund's adjuvant (CFA)-induced thermal hypersensitivity model.

A liquid chromatographic (LC) method with improved selectivity for the simultaneous determination of 5-hydroxyindoles (5-HIs; 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, and 5-hydroxytryptophol) is described. This method involves precolumn derivatization with 4-(3',3',4',4',5',5',6',6',7',7',8',8',9',9',10',10',10'-heptadecafluorodecyl)benzylamine (HFBA) and separation of the derivatives using a fluorous LC column. In this study, stable benzoxazole derivatives of 5-HIs with HFBA have been obtained by a simple derivatization procedure; their fluorescent properties enabled highly sensitive detection. In addition, only the HFBA derivatives of 5-HIs has been selectively retained on the fluorous LC column via fluorous interaction whereby perfluoroalkyl compounds show affinities with each other, while the non-fluorous compounds did not. The HFBA derivatives were separated within 30 min and the detection limits for 5-HIs in a 20-μL injection volume were 1.2-14 fmol (S/N=3). Furthermore, this method was applied to the analysis of 5-HIs in the human plasma from healthy subjects.

In this study, we developed a novel direct tandem mass spectrometric method for rapid and accurate analysis of amino acids utilizing a fluorous derivatization and purification technique. Amino acids were perfluoroalkylated with 2H,2H,3H,3H-perfluoroundecan-1-al in the presence of 2-picoline borane via reductive amination. The derivatives were purified by perfluoroalkyl-modified silica-based monolithic solid-phase extraction (monolithic F-SPE), and directly analyzed by tandem mass spectrometry using electrospray ionization without liquid chromatographic separation. The perfluoroalkyl derivatives could be sufficiently distinguished from non-fluorous compounds, i.e. the biological matrix, due to their fluorous interaction. Thus, rapid and accurate determination of amino acids was accomplished. The method was validated with human plasma samples and applied to the analysis of amino acids in the plasma of mice with maple syrup urine disease or phenylketonuria.