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Ohtani, M.,Hikima, J.i.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.5
Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.
Hikima, J.i.,Ohtani, M.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science 2011 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.35 No.3
Both PU.1 and C/EBPα transcription factors play important roles in myeloid development and inflammatory response. These transcripts were cloned from the Japanese flounder (Paralichthys olivaceus) and were highly conserved with those of other vertebrates. PU.1 mRNA was mainly expressed in lymphoid tissues while C/EBPα mRNA was widely expressed in all tissues examined. Higher levels of PU.1 mRNA were expressed in the IgM<SUP>+</SUP> cells of both PBL and KL, while C/EBPα expression was higher only in the IgM<SUP>-</SUP> cells of KL. The expression of C/EBPα mRNA was induced only in KL stimulated with LPS. Interestingly, PU.1 mRNA expression was induced by Edwardsiella tarda, whereas the expression of C/EBPα mRNA was induced by Streptococcus iniae infection. Both PU.1 and C/EBPα drove transcription from the LPS-responsive region of the Japanese flounder TNFα gene, suggesting that both PU.1 and C/EBPα induced by bacterial infection are involved in inflammation mediated through TNFα expression.
Immunoglobulin genes and their transcriptional control in teleosts
Hikima, J.i.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.35 No.9
Immunoglobulin (Ig), which exists only in jawed vertebrates, is one of the most important molecules in adaptive immunity. In the last two decades, many teleost Ig genes have been identified by in silico data mining from the enormous gene and EST databases of many fish species. In this review, the organization of Ig gene segments, the expressed Ig isotypes and their transcriptional controls are discussed. The Ig heavy chain (IgH) locus in teleosts encodes the variable (V), the diversity (D), the joining (J) segments and three different isotypic constant (C) regions including Cμ, Cδ, and Cζ/τ genes, and is organized as a ''translocon'' type like the IgH loci of higher vertebrates. In contrast, the Ig light (L) chain locus is arranged in a ''multicluster'' or repeating set of VL, JL, and CL segments. The IgL chains have four isotypes; two κ L1/G and L3/F), σ (L2) and λ. The transcription of IgH genes in teleosts is regulated by a VH promoter and the Eμ3' enhancer, which both function in a B cell-specific manner. The location of the IgH locus, structure and transcriptional function of the Eμ3' enhancer are important to our understanding of the evolutional changes that have occurred in the IgH gene locus.
Cha, I.S.,Kwon, J.,Mun, J.Y.,Park, S.B.,Jang, H.B.,Nho, S.W.,del Castillo, C.S.,Hikima, J.i.,Aoki, T.,Jung, T.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 Developmental and comparative immunology Vol.38 No.4
Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.
Jang, H B,Kim, Y R,Cha, I S,Noh, S W,Park, S B,Ohtani, M,Hikima, J,Aoki, T,Jung, T S Blackwell Publishing Ltd 2011 Journal of fish diseases Vol.34 No.7
<P><B>Abstract</B></P><P>Although the major capsid proteins (MCPs) of lymphocystis disease virus (LCDV) have been characterized, little is known about the host‐derived immune response to MCPs and other LCDV antigenic proteins. To identify antigenic proteins of LCDV that could be used as vaccine candidates in olive flounder, <I>Paralichthys olivaceus</I>, we analysed the viral proteins responsible for its virulence by applying immuno‐proteomics. LCDV proteins were separated by one‐dimensional gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with homogeneous <I>P.?olivaceus</I> antisera elicited by LCDV natural infection and vaccination with formalin‐killed LCDV. Four immune‐reactive proteins were obtained at 68‐, 51‐, 41‐ and 21 kDa using antisera collected from natural infection while two proteins at 51‐ and 21 kDa exhibited response to antisera from vaccinated fish, indicating that the latter two proteins have vaccine potential. Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and nanoelectrospray MS/MS, the 51 and 21 kDa proteins were identified as MCP and an unknown protein, respectively.</P>
Cha, I.S.,Castillo, C.S.d.,Nho, S.W.,Hikima, J.i.,Aoki, T.,Jung, T.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.8
Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.
Ohtani, M.,Hikima, J.i.,Jung, T.S.,Kondo, H.,Hirono, I.,Takeyama, H.,Aoki, T. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.34 No.2
Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 x His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.
Quynh, N.T.,Hikima, J.i.,Kim, Y.r.,Fagutao, F.F.,Kim, M.S.,Aoki, T.,Jung, T.S. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.44 No.2
DDX41, a receptor belonging to the DExD family, functions as a DNA sensor in the mammalian cytoplasm and mediates the antiviral response in host cells. Here, the olive flounder DDX41 was found to have 2267-bp long and encodes a putative protein of 614 amino acid residues. The olive flounder DDX41 mRNA was presented in all tested tissues, and was distinctly expressed in fish naturally infected with LCDV. High expression levels were observed in the heart, liver, kidney and stomach. Furthermore, the olive flounder DDX41 mRNA expression increased significantly in adherent (monocyte-like) cells following stimulation with a DNA virus. Reporter assays showed that the transcriptional activity of the IFN-I promoter was enhanced in DDX41-overexpressing HINAE cells treated with C-di-GMP (dinucleotides). Overexpression of DDX41 also induced the antiviral and inflammatory cytokine gene expression through cytoplasmic C-di-GMP treatment. These results suggest that DDX41 functions as a cytosolic DNA sensor that is capable of inducing antiviral activity and inflammatory responses in the olive flounder.
Jung, C.Y.,Hikima, J.i.,Ohtani, M.,Jang, H.B.,del Castillo, C.S.,Nho, S.W.,Cha, I.S.,Park, S.B.,Aoki, T.,Jung, T.S. Academic Press 2012 FISH AND SHELLFISH IMMUNOLOGY Vol.33 No.2
Interferon gamma (IFN-γ) is a cytokine that plays a very important role in defining Th1 immune response in all vertebrates. In this study, recombinant IFN-γ (rIFN-γ) from the olive flounder (Paralichthys olivaceus) was produced in an Escherichia coli system using a pET expression vector. Stimulation of whole kidney leukocytes (immune-related cells) in vitro with the resulting rIFN-γ significantly induced the gene expression of interleukin-1β (IL-1β), signal transducer and activator of transcription 1 (STAT1), CXCL13-like chemokine (CXCL13), and IFN-γ. rIFN-γ also weakly induced the expression of IL-1β, tumor necrosis factor-α (TNF-α), CXCL13, and IFN-γ in olive flounder-derived HINAE (non-immune) cells. The effects of rIFN-γ against Edwardsiella tarda infection in vivo were assessed by intraperitoneally injecting a mixture of rIFN-γ (100 ng) and E. tarda (1 x 10<SUP>5</SUP> CFU/ml) into the olive flounder. The survival rate in the rIFN-γ-injected group was 60% compared to 0% in the group treated with E. tarda only, demonstrating that olive flounder IFN-γ is effective in reinforcing immune responses and preventing against edwardsiellosis.