Publisher：Journal of Marine Science and Engineering  

In the evolution of the complement system, a major humoral innate immune factor, the existence of multiple isotypes of the complement components is considered as a key strategy to enhance innate immune defense. Complement C4 is also diversified in a wide range of vertebrate species including teleost fish, possibly supporting the robust activation mechanism of the complement. To better understand the functional diversity of C4 isotypes in the teleost complement system, two C4 isotypes, C4-1 and C4-2, sharing only 32% amino acid sequence identity, were examined for binding specificities towards model target molecules representing microbe antigens and towards Gram-positive and -negative bacteria. The results suggest that C4-1 and C4-2 behave similarly in binding to the tested targets, despite the predicted difference in binding specificity based on the thioester catalytic site. The participation of C4-1 in the classical and lectin pathways of complement activation was also explored using pathway-specific activating enzyme complexes, C1r/s and MBL-MASP2. As a result, C4-1 can be activated in both the classical and the lectin pathways, at higher efficiency in the classical pathway. Taken together, the present results imply that both C4-1 and C4-2 isotypes are fully functional in the complement activation cascades, probably playing comparable roles in innate immunity.

DOI： 10.3390/jmse11040707

Web of Science

Scopus

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-022-13408-6

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-022-13408-6

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.dci.2020.103886

Language：English   Publishing type：Research paper (scientific journal)  

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8
⁺
cytotoxic T cells (CTLs) in teleost fish are generated by vaccination with exogenous antigens. In the present study, we investigated whether antigen-specific CD8
⁺
CTLs in ginbuna crucian carp can be elicited by intestinal immunization with an exogenous antigen without any adjuvant. The IFNγ-1 and T-bet mRNA expressions were up-regulated in intestinal leukocytes following the administration of formalin-inactivated crucian hematopoietic necrosis virus (FI-CHNV), whereas the down-regulation of these genes was observed in kidney leukocytes. Furthermore, an increase in the percentage of proliferating CD8
⁺
cells was detected in the posterior portion of the hindgut, suggesting that the virus-specific CTLs are locally generated in this site. In addition, cell-mediated cytotoxicity against CHNV-infected syngeneic cells and the in vivo inhibition of viral replication were induced by immunization with FI-CHNV. Unexpectedly, intraperitoneal immunization with FI-CHNV induced a type I helper T cell (Th1)-response in the intestine, but not in the kidney; however, its effect was slightly lower than that reported after intestinal immunization. These findings suggest that the posterior portion of the intestine is an important site for generating virus-specific CTLs by vaccination with the inactivated vaccine.

DOI： 10.1016/j.dci.2018.12.009

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Infectious diseases have been found to be a major cause of mortality in fish hatcheries. Probiotics have been introduced to replace antibiotics commonly used for treatment of bacterial infection in aquaculture. This study was conducted to isolate, screen, and evaluate the probiotic Bacillus spp. for potential use as a feed supplement to enhance fish growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti. Bacillus aerius strain B81e was selectively isolated from the intestine of healthy catfish and chosen based on its probiotic properties both in vitro and in vivo. This bacterium produced a bacteriocin-like substance and exhibited a broad-spectrum antibacterial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens Aeromonas hydrophila and Streptococcus agalactiae. The susceptibility to all 8 antibiotics tested implies that it is unlikely to be an antibiotic-resistant bacterium. B. aerius strain B81e possessed interesting adhesion properties as shown by its high percentages of hydrophobicity, auto-aggregation, co-aggregation with fish pathogens A. hydrophila FW52 and S. agalactiae F3S and mucin binding. The strain B81e survived simulated gastrointestinal conditions, producing protease and lipase but not β-haemolysin. The study also evaluated the effects of dietary supplementation with strain B81e on growth performance, innate immunity, and the disease resistance of P. bocourti against A. hydrophila infection. Fish with a mean body weight of 69 g were fed strain B81e at 0 (control) and 10⁷ CFU g⁻¹ feed (test) for 60 days. Various growth and immune parameters were examined at 30 and 60 days post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 14 days post-infection. Results showed that the administration of strain B81e for 60 days had significant effects (p < 0.05) on weight gain, specific growth rate and feed utilization efficiency of P. bocourti. Dietary administration of strain B81e increased the serum lysozyme and bactericidal activities of P. bocourti significantly throughout the experimental period whereas the alternative complement, phagocytic and respiratory burst activities were significantly (p < 0.05) higher in the test fish compared to the control fish after 60 days of feeding. In addition, the fish fed a strain B81e supplemented diet had a significantly higher (p < 0.05) post-challenge survival rate than the control fish. The results in this study indicate that B. aerius B81e has beneficial effects on growth performance, innate immunity and disease resistance of P. bocourti. This is the first report on the probiotic roles of B. aerius in aquaculture.

DOI： 10.1016/j.fsi.2017.11.032

Language：English   Publishing type：Research paper (scientific journal)  

To develop an accurate diagnosis of “new ulcer disease” in koi carp, we produced four monoclonal antibodies (mAbs) against a strain of atypical Aeromonas salmonicida isolated from koi carp. These mAbs did not cross-react with an isolate of atypical A. salmonicida from Japanese flounder and other pathogenic bacteria. Re-isolation from artificially infected koi carp was achieved by selecting the blue colonies on agar medium containing Coomassie brilliant blue, and some of the colonies were detected by immunofluorescent staining using the mAbs. These results suggested that the mAbs can distinguish atypical A. salmonicida from koi carp from resident aeromo-nads.

DOI： 10.3147/jsfp.53.36

Language：English   Publishing type：Research paper (scientific journal)  

rag1 ^-/- zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1 ^-/- zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1 ^-/- fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1 ^-/- fish possess innate protection equivalent to that of rag1 ^-/- fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.

DOI： 10.1038/s41598-017-08000-2

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.imbio.2016.06.188

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.imbio.2016.06.187

Language：English   Publishing type：Research paper (scientific journal)  

Teleost IL-4/13B is a cytokine related to mammalian IL-4 and IL-13, of which hitherto the function had not been studied at the protein level. We identified an IL-4/13B gene in common carp (Cyprinus carpio) and expressed the recombinant protein (rcIL-4/13B). RcIL-4/13B was shown to stimulate proliferation of IgM⁺ B cells, because after four days of stimulation the IgM⁺ fraction of carp kidney and spleen leukocytes had formed many cell colonies, whereas such colonies were not found in the absence of rcIL-4/13B stimulation. After nine days of incubation with rcIL-4/13B these cells had proliferated to more than 3-to-7-fold higher numbers when compared to untreated cells. The proliferating cells contained a majority of IgM⁺ cells but also other cells, as indicated by FACS and RT-PCR analyses. The important conclusion is that in fish not only IL-4/13A has B cell stimulating properties, as a previous publication has shown, but also IL-4/13B.

DOI： 10.1016/j.fsi.2015.12.043

Language：English   Publishing type：Research paper (scientific journal)  

Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb⁺ thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb^- leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb^- leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

DOI： 10.1016/j.dci.2015.05.002

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.dci.2015.05.002

Language：English   Publishing type：Research paper (scientific journal)  

In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane- bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host-cell protection is an evolutionarily conserved mechanism in regulation of the complement system.

DOI： 10.4049/jimmunol.1303179

Language：English   Publishing type：Research paper (scientific journal)  

Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5-3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.

DOI： 10.3389/fimmu.2014.00445

Event date： 2012.6

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Country：Japan  

Event date： 2024.11

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Event date： 2023.12

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Event date： 2023.9

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Event date： 2023.9

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Event date： 2023.8 - 2023.9

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Event date： 2023.8 - 2023.9

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Event date： 2022.1

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Event date： 2021.9

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Event date： 2021.9

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Event date： 2020.3

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Event date： 2020.3

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Event date： 2020.2

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2020.1

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Country：Japan  

Event date： 2019.12

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Event date： 2019.12

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Event date： 2019.9

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Event date： 2019.9

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Event date： 2019.6

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Venue：Las palmas de Gran Canaria   Country：Spain  

Event date： 2019.3

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Event date： 2019.3

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Event date： 2018.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：Nagasaki, Japan   Country：Japan  

Event date： 2018.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Fukushima, Japan   Country：Japan  

Reproductive Management Committee for Sand Tiger Sharks (RMCS) is working with six aquariums to exchange rearing record since 2015. In RMCS, under different water temperature and photoperiod condition, male breeding behavior is observed in different season, spring (seasonal change) and autumn (constant). MARINEWORLD started to analyze changes in blood levels of steroid hormones(17-β-Estradiol, Progesteron, Teststeron) since 2015. Many investigations are still undergoing to reveal a mechanism of reproduction of C. taurus and to make a success in breeding of this shark in the future. Sand tiger sharks are distributed around Ogasawara islands in Japan, but biological and ecological data of them is very limited. We started data collection program (photo identification survey) in cooperation with local diving services to complement the lack of data.

Event date： 2018.9

Language：English   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2018.9

Language：Japanese  

Country：Japan  

Event date： 2018.9

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.9

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

In early stage of viral infection, viral-specific nucleic acids including single/double-stranded RNA (ss/dsRNA) are recognized by innate pattern recognition receptors, such as Toll-like receptors (TLRs), thereby type-I interferon (IFN) is produced. In mammal, plasmacytoid dendritic cells are known as professional type-I IFN producing cells against viral infection, which possess interferon-regulatory factor 7 (IRF7) signaling pathway to produce huge amount of the IFN. In the present study, we show that thrombocytes in common carp (Cyprinus carpio) have potent ability to produce type-I IFN in response to synthetic viral molecule mimicries. Magnetic-sorted mAb+ thrombocytes and other peripheral blood leukocytes (PBLs) were incubated with polyinosinic:polycytidylic acid (poly(I:C), analog of dsRNA), resiquimod (R-848, ligand of TLR7/8 which detect ssRNA), or CpG motif oligodeoxynucleotides (ODN2007 and GpC motif nucleotides for control). qPCR analysis showed that thrombocytes had produced large amount of type-I IFN (ccIFNαβ) compared with the PBL fraction. R-848 acutely activated the IFN production in both fractions, whereas upregulating of the IFN expression by poly(I:C) were detected only in the PBL fractions. CpG DNA never enhanced type-I IFN expression but upregulated several inflammatory cytokines such as interleukin-6, suggesting that different signal pathways were activated by those ligand recognitions. After the R-848 stimulation, expression level of IRF7 were dramatically increased in thrombocytes, suggesting that thrombocytes activated the IRF7 signaling pathway for the IFN production. Those pattern-recognition characteristics, type-I IFN producing ability, and existence of the specialized IFN-producing pathway of carp thrombocytes are analogous to those of mammalian plasmacytoid dendritic cells, indicating that thrombocytes are important components for antiviral immunity in bony fish and can be a new target for the strategy of vaccine development.

Event date： 2018.8 - 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.8

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Event date： 2018.3

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Event date： 2017.10

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Event date： 2017.9

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Venue：Shinagawa, Tokyo   Country：Japan  

Event date： 2017.9

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Event date： 2017.9

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Event date： 2011.5

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