Production of trout offspring from triploid salmon parents

Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.     

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.     

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.     

Intraocular vaccination with an inactivated highly pathogenic avian influenza virus induces protective antibody responses in chickens

Because it is expected to induce cross-reactive serum and mucosal antibody responses, mucosal vaccination against highly pathogenic avian influenza (HPAI) is potentially superior to conventional parenteral vaccination. Here, we tested whether intraocular vaccination with an inactivated AI virus induced protective antibody responses in chickens. Chickens were inoculated intraocularly twice with 10⁴ hemagglutination units of an inactivated H5N1 HPAI virus. Four weeks after the second vaccination, the chickens were challenged with a lethal dose of the homologous H5N1 HPAI virus. Results showed that most of the vaccinated chickens mounted positive antibody responses. The median serum hemagglutination inhibition titer was 1:80. Addition of CpG oligodeoxynucleotide 2006 or cholera toxin to the vaccine did not enhance serum antibody titers. Cross-reactive anti-hemagglutinin IgG, but not IgA, was detected in oropharyngeal secretions. In accordance with these antibody results, most vaccinated chickens survived a lethal challenge with the H5N1 HPAI virus and did not shed the challenge virus in respiratory or digestive tract secretions. Our results show that intraocular vaccination with an inactivated AI virus induces not only systemic but also mucosal antibody responses and confers protection against HPAI in chickens.     

Pathological characteristics of thyroid glands from Japanese Black Cattle living in the restricted area of the Fukushima Daiichi Nuclear Power Plant accident

To study the effect of ionizing radiation on thyroid glands, 66 Japanese Black cattle residing in the restricted area of the Fukushima Daiichi Nuclear Power Plant in 2013–2017 were pathologically examined. There were no clinical symptoms of thyroid disease in these cattle. Three cases of goiter and seven of atrophy were found in two among the four farms examined. Cases of goiter exhibited normal morphological structure without mass or nodule formation in thyroid glands. Cellular atypia or capsular invasion of the follicular epithelium was absent. The estimated integrated dose of external radiation in goiter cases ranged from maximum 797 mSv to minimum 24 mSv. All lobules in the seven atrophic thyroid glands were affected, but pathological findings, such as inflammatory cell infiltration or stromal fibrosis, were not observed. The estimated integrated dose of external radiation in atrophic thyroids ranged from maximum 589 mSv to minimum 8 mSv. Immunohistochemical analysis of anti‐nitroguanosine and the TUNEL method in goiter and atrophic thyroid glands did not reveal any positive findings. The present study indicates that there was no significant relationship between a radiation effect and pathological findings in any thyroid glands.     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

Preparation of Photocrosslinked Fish Elastin Polypeptide/Microfibrillated Cellulose Composite Gels with Elastic Properties for Biomaterial Applications

Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.     

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.