Hiroki Korenaga

Miyazaki University, Миядзаки, Miyazaki, Japan

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Publications (25)39.75 Total impact

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    Dataset: 237
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    ABSTRACT: Vaccines (subunit and DNA) targeting major envelope proteins VP19 and/or VP28 of white spot syndrome virus (WSSV) in penaeid shrimp were developed and elicited good protection against white spot disease (WSD). However, the immune responses in shrimp after administration of these vaccines are not well understood. In this study, we developed a DNA vaccine encoding the VP28 envelope protein in kuruma shrimp (Marsupenaeus japonicus) and confirmed the potentiality of protection against WSSV infection. The efficacy of the DNA vaccine against WSSV infection was confirmed by WSSV artificial challenge at 7 days post vaccination in kuruma shrimp. However, the efficacy of the vaccine did not last 30 days post vaccination. The transcript of VP28 gene derived from expression vector in tissues of vaccinated shrimp was analyzed by RT-PCR. The transcript of VP28 gene was detected in various tissues including muscle, hemolymph, gill, intestine, stomach, heart, hepatopancreas and lymphoid organ tested at 1, 3 and 7 days post vaccination. Subsequently, the expression of innate immune-related genes in intestine and lymphoid organ was analyzed at 1, 3 and 7 days post vaccination. The expression of innate immune-related genes such as Rab7, penaeidin, lysozyme, and crustin was up-regulated upon DNA vaccination. These results suggest that DNA vaccination induces significant protection against WSSV by stimulating innate immune responses in kuruma shrimp.
    11/2013;
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    Dataset: 236
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    Dataset: 212
    Hiroki Korenaga, Tomoya Kono, Masahiro Sakai
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    ABSTRACT: Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (10(5) cfu g(-1)) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P < 0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.
    Marine Biotechnology 09/2013; · 2.74 Impact Factor
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    ABSTRACT: Monocytes are antigen-presenting cells (APCs) in mammals. Antigen presentation by monocytes via costimulatory molecules was recently confirmed in the Japanese pufferfish Fugu rubripes (also known as Takifugu rubripes) (Sugamata et al., 2009[1]). However, no detailed investigations examining how cytokine gene expression regulates the activation/differentiation of these cells have been conducted to date. Therefore, in this study, the expression of cytokine genes in Fugu monocytes stimulated with TLR agonists (LPS, polyI:C, and IMQ) was profiled. First, the morphological changes and phagocytic activity of stimulated monocytes were examined. At 5days post-stimulation, the ratio of activated to inactivated monocytes increased (as determined by FCM analysis), and the phagocytic activity of the cells was higher (5-15%) than that of nonactivated cells. Analysis of cytokine gene expression in stimulated monocytes revealed that the genes encoding CSF-1b and IFNγrel are important cytokines in activation/differentiation of monocytes/macrophages, and that genes encoding inflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α are upregulated by stimulation. The present study revealed the types of cytokines expressed in Fugu monocytes stimulated with TLR agonists.
    International immunopharmacology 07/2013; · 2.21 Impact Factor
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    ABSTRACT: The important role played by cytokines in host innate immunity and the interaction of subsets of immune and inflammatory cells through cytokines offer avenues for immune interventions. We investigated 16 cytokine gene responses in the Japanese pufferfish, Takifugu rubripes orally treated with a heat-killed lactic acid bacterium (LAB), Lactobacillus paracasei spp. paracasei (strain 06TCa22) (Lpp) isolated from a Mongolian dairy product at 1mgg(-1)body weightd(-1) for 3days. Additionally, we assessed superoxide anion production (SAP) and phagocytic activity (PA) of head kidney cells and resistance to Vibrio harveyi infection in treated fish. Significant up-regulation of pro-inflammatory (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immunity inducing (IL-12p35, IL-12p40 and IL-18), antiviral/intra-cellular pathogen killing (I-IFN-1 and IFN-γ), anti-inflammatory (IL-10) and peripheral T cell expansion and survival controlling (IL-2, IL-7, IL-15, IL-21 and TGF-β1) cytokines was observed in the treated fish. Furthermore, significantly increased SAP, PA and pathogen resistance were observed in the treated fish compared to untreated fish. Our results indicate the enhancement of cytokine mediated immunity in T. rubripes by the use of the heat-killed Lpp as a potential immunostimulant and would be of great use in immunomodulation trials with the possibility to monitor positive immune response.
    International immunopharmacology 07/2013; · 2.21 Impact Factor
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    ABSTRACT: With the aim of evaluating the effect of a Mongolian dairy product derived Lactobacillus paracasei spp. paracasei (strain 06TCa22) (Lpp) on the cytokine-mediated immune responses to Vibrio harveyi infection, we examined 16 cytokine expressions in the Japanese pufferfish, Takifugu rubripes. Fish were orally treated with the heat-killed Lpp at 1 mg g(-1) body weight d(-1) for 3 days. At 24 h post treatment, fish were infected by an intramuscular injection of 0.1 mL V. harveyi bacterial suspension (10(8) cfu mL(-1)). Additionally, superoxide anion production (SAP) and phagocytic activity (PA) of head kidney cells were assessed during 120 h post infection period. Significant up-regulation of pro-inflammatory (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immune inducing (IL-12p35, IL-12p40 and IL-18), antiviral/ intra-cellular pathogen killing (I-IFN-1 and IFN-γ), anti-inflammatory (IL-10) and lymphocyte agonistic (IL-2, IL-7, IL-15, IL-21 and TGF-β1) cytokines was observed in the treated fish compared to control ones during the pathogen infection. Furthermore, significantly increased SAP and PA (P<0.01; 0.05) were recorded in the treated fish compared to untreated fish. These results suggest the beneficial role of Lpp in enhancement of cytokine-mediated immunity in the Japanese pufferfish against V. harveyi infection and application of this product as a potential fish immunostimulant.
    Fish &amp Shellfish Immunology 06/2013; · 2.96 Impact Factor
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    ABSTRACT: Cytokine responses in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells to heat-killed lactic acid bacteria probiotics isolated from the Mongolian dairy products were investigated by transcriptomic examination. The HK cells were incubated with two heat-killed bacteria, namely Lactobacillus paracasei spp. paracasei (strain 06TCa22) and L. plantarum (strain 06CC2) and the responses of 16 cytokine genes at 0 (control), 1, 4, 8, 12, 24 and 48h post-stimulation were assayed by multiplex RT-PCR analysis (GenomeLab Genetic Analysis System, GeXPS; Beckman Coulter, Inc.). The 16 genes included in the assay were pro-inflammatory cytokines (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immune regulators (IL-12p35, IL-12p40 and IL-18), antiviral (I-IFN-1 and IFN-γ) and other regulatory (IL-2, IL-7, IL-15, IL-21, IL-10 and TGF-β1) cytokines. Despite the differences in the transcriptional profiles, expression of all the cytokines tested here was significantly elevated by both the probiotic bacterial stimulants compared with the unstimulated control. Therefore, this in vitro study has demonstrated the modulation of cytokine defense mechanisms in the HK cells by the two heat-killed probiotics indicating their potentiality as novel immunostimulants to fish. However, strain-dependent varied expression of important cytokines (cell-mediated immune regulators, antiviral and anti-inflammatory cytokines) suggests better efficacy of L. paracasei spp. paracasei strain as fish immunostimulant. Further in vivo studies to elucidate the cytokine regulation networks will validate our present observations. A careful evaluation of ant-inflammatory properties may be undertaken using single strain to affirm the immunostimulatory capability. Moreover, application timings and frequency to assess the longevity of immunostimulant effects and to make the application cost-effective need to be evaluated before any practical use in aquaculture.
    Fish &amp Shellfish Immunology 02/2013; · 2.96 Impact Factor
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    Tomoya Kono, Hiroki Korenaga
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    ABSTRACT: CD4(+) T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4(+) T cells from the blood of Japanese pufferfish, Fugu rubripes, and to characterize their cytokine expression profile. We produced a specific antibody against Fugu CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted Fugu CD4(+) cells were characterized by morphology and expression analysis of cell marker genes. Fugu CD4(+) cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated Fugu CD4(+) cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.
    PLoS ONE 01/2013; 8(6):e66364. · 3.73 Impact Factor
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    ABSTRACT: In fish, T cell lineage commitment has not been studied, although there are reports related to CD4 and CD8 positive cells. This study describes the cloning and analysis of a master regulator involved in this process, the Th-POK gene in Japanese pufferfish, Takifugu rubripes. The fugu Th-POK cDNA was composed of 1901bp, with a 75bp 5'-UTR, a 131bp 3'-UTR, and a 1692bp open reading frame which translates into a peptide of 564 amino acid residues. The deduced fugu Th-POK protein contained a BTB / POZ domain, krüppel motif (H/C linker) and krüppel-like zinc finger DNA binding domain with C2H2 structure. Homology analysis of fugu Th-POK (ZBTB7B) with other known ZBTB7 members (ZBTB7A, 7C) showed low identity, and the phylogenetic tree analysis showed the fugu Th-POK clustered with the mammalian Th-POK, away from other ZBTB7 members. The analysis of transcriptional control region of Th-POK gene suggested that the 5'-flanking region and intron 1 includes numerous canonical binding motifs for transcription factors regulating T cells development. The genomic organization of the fugu Th-POK gene was composed of three exons and two introns, and its structure was identical to that of its human counterpart. Comparison of the fugu and human genomes showed that high levels of conserved synteny existed around the Th-POK gene. The high expression of the fugu Th-POK gene in unstimulated tissues was seen in head kidney, muscle, skin and gills. Moreover, the expression of the fugu Th-POK gene in thymic cell s was increased by LPS, polyI:C and PHA stimulation.
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 11/2012; · 1.61 Impact Factor
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    ABSTRACT: To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.
    Veterinary Immunology and Immunopathology 11/2012; · 1.88 Impact Factor
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    ABSTRACT: Immune stimulant effects of a nucleotide-rich baker's yeast extract (Vertex IG20) were investigated in the kuruma shrimp, Marsupenaeus japonicus by examining expression of anti-microbial peptides/proteins (AMPs) such as penaeidin (MjPen), crustin (MjCrus) and lysozyme (MjLyz) genes. Furthermore, to confirm that the baker's yeast extract-induced AMPs were functional, we also assessed the effect of its oral administration on resistance to Vibrio nigripulchritudo infection in the kuruma shrimp. Our results demonstrate that baker's yeast extract-injected and fed shrimps displayed a significant up-regulation of MjPen, MjCrus and MjLyz gene expression in the lymphoid organ. Moreover, significantly increased (P<0.01) resistance to the bacterial pathogen in term of better post infection survival (66.6%) was observed in the shrimp fed with the yeast extract-incorporated diet compared with the control diet fed group (8.3%). The present study indicates the immunostimulatory effects of the nucleotide-rich baker's yeast extract on the kuruma shrimp immune system and supports its potential use in aquaculture.
    Aquaculture 11/2012; 366–367:40-45. · 2.01 Impact Factor
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    ABSTRACT: The mononuclear phagocyte system is composed of monocytes, macrophages and dendritic cells and has crucial roles in inflammation, autoimmunity, infection, cancer, organ transplantation and in maintaining organismal homeostasis. Interleukin-34 (IL-34) and macrophage colony stimulating factor (MCSF), both signalling through the MCSF receptor, regulate the mononuclear phagocyte system. A single IL-34 and MCSF gene are present in tetrapods. Two types of MCSF exist in teleost fish which is resulted from teleost-wide whole genome duplication. In this report, we first identified and sequence analysed six IL-34 genes in five teleost fish, rainbow trout, fugu, Atlantic salmon, catfish and zebrafish. The fish IL-34 molecules had a higher identity within fish group but low identities to IL-34s from birds (27.2-33.8%) and mammals (22.2-31.4%). However, they grouped with tetrapod IL-34 molecules in phylogenetic tree analysis, had a similar 7 exon/6 intron gene organisation, and genes in the IL-34 loci were syntenically conserved. In addition, the regions of the four main helices, along with a critical N-glycosylation site were well conserved. Taken together these data suggest that the teleost IL-34 genes described in this report are orthologues of tetrapod IL-34. Comparative expression study of the three trout MCSFR ligands revealed that IL-34, MCSF1 and MCSF2 are differentially expressed in tissues and cell lines. The expression of MCSF1 and MCSF2 showed great variance in different tissues and cell lines, suggesting a role in the differentiation and maintenance of specific macrophage lineages in specific locations. The relatively high levels of IL-34 expression across different tissues suggests a homeostatic role of IL-34 for the macrophage lineage in fish. One striking observation in the present study was the lack of induction of MCSF1 and MCSF2 expression but the quick induction of IL-34 expression by PAMPs and inflammatory cytokines in cell lines and primary head kidney macrophages in rainbow trout. In a parasitic proliferative kidney disease (PKD) model, the expression of IL-34 but not the dominant MCSF2 was affected by PKD, suggesting an involvement of macrophage function in this disease model. Thus IL-34 expression is sensitive to inflammatory stimuli and may regulate macrophage biology once up-regulated.
    Molecular Immunology 10/2012; 53(4):398-409. · 2.65 Impact Factor
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    H. Korenaga, T. Kono, M. Sakai
    Cytokine; 09/2012
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    ABSTRACT: Immune stimulant effects of a nucleotide-rich baker's yeast extract (Vertex IG20) were investigated in the kuruma shrimp, Marsupenaeus japonicus by examining expression of anti-microbial peptides/proteins (AMPs) such as penaeidin (MjPen), crustin (MjCrus) and lysozyme (MjLyz) genes. Furthermore, to confirm that the baker's yeast extract-induced AMPs were functional, we also assessed the effect of its oral administration on resistance to Vibrio nigripulchritudo infection in the kuruma shrimp. Our results demonstrate that baker's yeast extract-injected and fed shrimps displayed a significant up-regulation of MjPen, MjCrus and MjLyz gene expression in the lymphoid organ. Moreover, significantly increased (P<0.01) resistance to the bacterial pathogen in term of better post infection survival (66.6%) was observed in the shrimp fed with the yeast extract-incorporated diet compared with the control diet fed group (8.3%). The present study indicates the immunostimulatory effects of the nucleotide-rich baker's yeast extract on the kuruma shrimp immune system and supports its potential use in aquaculture.
    Aquaculture 09/2012; 366-367:40-45. · 2.01 Impact Factor
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    ABSTRACT: Cytokines are important regulators of the immune system and investigation of their functions may prove useful for the development of vaccines and immunostimulants for aquaculture. We therefore investigated the cytokine [interleukin (IL)-1β, IL-10, IL-12 p35 and p40, tumor necrosis factor (TNF)-α, CXC-chemokine and interferon (IFN)-α and γ] responses of the common carp, Cyprinus carpio L., upon treatment with a commercial baker's yeast extract (CW-I) that contained nucleotides and β-glucan. Additionally, to confirm that the CW-I-induced cytokines were functional, we also assessed the effect of CW-I administration on superoxide anion production and phagocytic activities of head kidney leucocytes and resistance to Aeromonas hydrophila infection in the common carp. Our results demonstrate that baker's yeast extract-treated fish displayed a significant up-regulation of pro-inflammatory cytokine (IL-1β, IL-12 p35 and p40, TNF-α, CXC-chemokine, IFN-γ2) gene expression and a down-regulation of anti-inflammatory cytokine (IL-10) gene expression. Furthermore, significantly increased phagocytic activity and superoxide anion production in kidney cells, and resistance to a bacterial pathogen, were observed in the yeast extract-treated fish compared to nontreated fish. The current study indicates the immunostimulatory effects of a baker's yeast extract rich in nucleotides and β-glucan on the carp immune system and supports its potential use in aquaculture.
    Aquaculture 08/2012; 356–357:169–175. · 2.01 Impact Factor
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    ABSTRACT: A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay can detect expression of 30 genes simultaneously within a reaction tube, which is advantageous to the conventional analytical methods where only one gene per single reaction is detected. Therefore, to economize the cost, labor, time, sample number and experiment frequency, a novel platform for quantitative multiplexed analysis was developed for the expression study of cytokine genes in Japanese pufferfish, Takifugu rubripes. Using a custom designed multiplex RT-PCR assay and the GenomeLab Genetic Analysis System (GeXPS), expression profiles were analyzed for 19 cytokine genes including pro-inflammatory [Interleukin (IL)-1β, IL-6, IL-17A/F3, IL-18, Tumor Necrosis Factor (TNF)-α, and TNF-N], anti-inflammatory (IL-4/13A, IL-4/13B, and IL-10), T-cell proliferation/differentiation [IL-2, IL-12p35, IL-12p40, IL-15, IL-21, and Transforming Growth Factor (TGF)-β1], activation/differentiation of B-cells (IL-7, IL-6, IL-4/13A, and IL-4/13B), induction of anti-viral activity [type I-Interferon (IFN)-1 and IFN-γ], and proliferation of monocytes/macrophage progenitor cells [M-Colony Stimulating Factor (CSF)-1β] in tissues under immune stimulatory conditions. We observed that immune stimulants such as LPS and polyI:C altered cytokine gene expression. The expression profiles were different in the un-stimulated control and each immune-stimulated fish. The expression profile following the pathogenic bacterial injection was similar to that for LPS stimulation. Notably, the type I-IFN-1 and IFN-γ genes showed significant up-regulation by polyI:C stimulation but not by stimulation with LPS or bacteria. Based on the results, this technique allows rapid and sensitive analysis of cytokine genes transcribed at different conditions to evaluate the health status of fish. The constructed multiplex RT-PCR assay scopes further understanding of cytokine network complex in immune regulation in fish.
    the 12th Congress of the International Society of Developmental and Comparative Immunology, Fukuoka, Japan; 07/2012
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    ABSTRACT: In the current study, we cloned and characterized the neuromedin U (NMU) gene from the common carp Cyprinus carpio L., and identified its participation in immune responses in the teleost. Five isoforms of the preproNMU genes were generated by alternative splicing and isolated from carp. The longest form of the carp preproNMU1 (isoform 1) cDNA was composed of 803 bp, and contained an 18 bp 5'-UTR, a 212 bp 3'-UTR and a 573 bp open reading frame, which translates into a peptide comprising 190 amino acid (aa) residues. The remaining carp preproNMU isoforms were composed of 175 (preproNMU2), 158 (preproNMU3), 150 (preproNMU4) and 133 (preproNMU5) aa residues. Isoforms 1-3 contained four processing signals (KR or RR), while isoforms 4 and 5 contained only two processing signals. High homology was demonstrated among fish and other vertebral NMU at the biologically active C-terminal region (aa position 175-182). Carp preproNMU transcript variants were identified in various tissues, and the expression pattern has been shown to change depending on feeding status. Moreover, it was shown that the expression of preproNMU3 and preproNMU5 was increased following treatment with bacterial or viral mimics. Finally, we investigated the functional aspect of carp NMU using a synthetic NMU peptide. The peptide was found to increase the expression of inflammation-related cytokine genes in intestinal cells within 1 h of treatment. In addition, the activation of phagocytic cells was also stimulated by the NMU peptide. The discovery of NMU in carp allows for a further understanding of immune regulation by biologically active substances.
    Fish &amp Shellfish Immunology 11/2011; 32(1):151-60. · 2.96 Impact Factor
  • Tomoya Kono, Hiroki Korenaga, Masahiro Sakai
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    ABSTRACT: Interleukin-17 (IL-17) is a cytokine family composed of six ligands (A-F). Especially, the IL-17A and IL-17F are best characterized cytokines of IL-17 family cytokine. These are produced by Th17 cells and induce the expression of many mediators of inflammation properties. In addition, the five member of IL-17 receptor family (RA-RE) have been identified in mammals. Although the research on fish IL-17 is a little to date, this review discusses some of the recent advances in research on IL-17 ligand and receptor genes in fish. IL-17 family member was chosen from the fish genome database, and its structure and phylogeny is analyzed in detail. Moreover, invertebrate IL-17 genes are also discussed, and the isolation and current status of fish IL-17 receptor genes are summarized. Comparative genomic analysis of the IL-17 family among mammals, teleost and invertebrates provided new insights. Novel IL-17 ligand (IL-17N) was identified from teleost, moreover it was suggested that IL-17N may be a teleost specific ligand by synteny and phylogenetic analysis. On the other hand, IL-17 receptors are well conserved between mammal and teleost, the five member of IL-17 receptor family: IL-17RA-RE were found on the teleost genome. In addition, the IL-17RA gene was duplicated in tandem on the stickleback and medaka genome. Knowledge about the IL-17 ligand/receptor in fish is very limited. Therefore this review will hopefully encourage future studies of IL-17 in fish.
    Fish &amp Shellfish Immunology 12/2010; 31(5):635-43. · 2.96 Impact Factor