Yukinori Yoshimura

Hiroshima University, Hirosima, Hiroshima, Japan

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Publications (147)180.8 Total impact

  • A. Huang · N. Isobe · T. Obitsu · Y. Yoshimura
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    ABSTRACT: The aim of this study was to determine the role of fatty acids for sperm survival in the sperm storage tubules (SSTs) of the hen oviduct. The mucosa tissues of uterovaginal junction (UVJ) of White Leghorn laying hens with or without artificial insemination using semen from Barred Plymouth Rock roosters were collected. The lipid density in the epithelium of UVJ and SST was analyzed by Sudan black B staining. The expressions of genes encoding lipid receptors and lipases were assayed by polymerase chain reaction in UVJ mucosa and SST cells isolated by laser microdissection. Fatty acid composition was analyzed by gas chromatography, and sperm were cultured with or without the identified predominant fatty acids for 24 hours to examine their effect on sperm viability. The lipid droplets were localized in the epithelium of UVJ mucosa and SSTs. The expression of genes encoding very low–density lipoprotein receptor(VLDLR), low-density lipoprotein receptor (LDLR), and fatty acid translocase (FAT/CD36) were found in SST cells. Expression of genes encoding endothelial lipase (EL), lipase H (LIPH), adipose triglyceride lipase (ATGL), and lipoprotein lipase (LPL) were found in UVJ. In contrast, only ATGL was found in SST cells, and its expression was significantly upregulated after artificial insemination. In UVJ mucosal tissues, five fatty acids, namely myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1n9), and linoleic acid (C18:2n6), were identified as predominant fatty acids. The viability of sperm cultured with 1 mM oleic acid or linoleic acid was significantly higher than the sperm in the control culture without fatty acids. These results suggest that lipids in the SST cells may be degraded by ATGL, and fatty acids including oleic acid and linoleic acid may be released into the SST lumen to support sperm survival.
    No preview · Article · Dec 2015 · Theriogenology
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    ABSTRACT: It has been reported that goat cathelicidin-2, an antimicrobial peptide, localizes in leukocytes and is present in milk. Here, we examined whether cathelicidin-2 is secreted by leukocytes. Different concentrations (10(5) -10(8) cells/mL) of blood leukocytes were cultured for 0-48 h with or without lipopolysaccharide (LPS). After culture, the concentrations of cathelicidin-2 in the conditioned media were measured. Blood was collected from male goats 0-24 h after the intravenous injection of Escherichia coli O111:B4 LPS. The plasma cathelicidin-2 concentrations were determined and the blood leukocytes immunostained with anti-cathelicidin-2 antibody to calculate the proportion of cathelicidin-2-positive cells in the total leukocytes. When higher concentrations of leukocytes were cultured, the cathelicidin-2 concentrations in the media increased significantly, whereas the addition of LPS to the media caused no further increase. The plasma cathelicidin-2 concentrations did not increase with time after LPS infusion. The proportion of cathelicidin-2-positive cells in the total leukocytes was significantly reduced 1 h after LPS injection compared with that at 0 h, but increased again at 6 h and thereafter. These results suggest that cathlicidin-2 is secreted by leukocytes even without LPS stimulation, whereas LPS may be required for cathelicidin-2-containing leukocytes to be recruited from the blood to tissues showing inflammation. © 2015 Japanese Society of Animal Science.
    Full-text · Article · Jul 2015 · Animal Science Journal
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    Yukinori Yoshimura
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    ABSTRACT: Avian β-defensins (AvBDs) attack various microorganisms and may have efficacy to protect tissues from infection by unforeseen pathogenic microbes. This article describes the mechanism by which AvBDs are expressed in the innate immune system in hen reproductive organs. Both ovary and oviduct express Toll-like receptors (TLRs), which recognize microbe-associated molecular patterns, and express and produce AvBDs mRNA and peptides, which are antimicrobial peptides. The interaction of TLRs with their ligands upregulates the expression of AvBDs and proinflammatory cytokines, and proinflammatory cytokines also induce expression of AvBDs. The synthesized AvBDs are predicted to kill the microbes. However, strategies to enhance innate immune functions have not yet been established. Breeding of birds that have a higher ability to synthesize antimicrobial peptides in response to pathogens may be one such strategy. Because the ovary and oviduct are unique organs regulated by the endocrine system, consideration of the role of gonadotropic and gonadal hormones may also be important for the enhancement of local defense function. Physiological information about the mechanism of pathogen recognition and AvBD synthesis is essential to enhance immunodefense functions in the reproductive organs. © 2015 Poultry Science Association Inc.
    Preview · Article · Feb 2015 · Poultry Science
  • Takahiro Nii · Naoki Isobe · Yukinori Yoshimura
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    ABSTRACT: The aim of this study was to determine whether the egg-laying phase and estrogen affect the induction of cytotoxic cells in response to avian infectious bronchitis (IB) virus at early stage of infection in the oviduct. Attenuated IB virus (aIBV group) or its vehicle (control group) was introduced to the oviductal magnum lumen of White Leghorn hens in the laying and molting phase, as well as molting hens injected with estradiol benzoate (M-EB hens) or corn oil (M-oil hens). Oviductal isthmus and uterus were collected 24h after injection. The frequency of CD8(+) and TCRγδ(+) T cells expression was examined by immunohistochemistry, followed by image analysis. The expression of the genes of toll-like receptor 7 (TLR7), natural killer cell receptor (BNK), cytotoxic substances (granzyme, perforin), and cytokines (CXCL12, CX3CL1, and IFNγ) were examined by real-time polymerase chain reaction analysis. The frequency of CD8(+) and TCRγδ(+) T cells in the isthmus, and CD8(+) cells in the uterus was significantly higher in the aIBV group compared to the control group of laying and M-EB hens. The expression of all the genes examined in this study in the isthmus, and CX3CL1 and IFNγ expression in the uterus was significantly higher in the aIBV group in the laying and M-EB hens. These results suggested that infection with IB virus causes an immune response involving the influx of cytotoxic cells and upregulation of cytokines in the isthmus and uterus at early stage of infection. This response was stronger during the laying phase compared to the molting phase, probably due to the effect of estrogen. Copyright © 2015 Elsevier B.V. All rights reserved.
    No preview · Article · Jan 2015 · Veterinary Immunology and Immunopathology
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    Elsayed S. I. Mohammed · Yasumi Igarashi · Naoki Isobe · Yukinori Yoshimura
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    ABSTRACT: The aim of this study was to determine the effects of probiotics-feeding on the gene expression and protein localization of avian β-defensins (AvBDs) in the proventriculus of broiler chicks. Male broiler chicks were arranged in 3 groups: control group, probiotics group I and probiotics group II, which were fed with starter rations containing 0%, 0.2% or 0.4% probiotics, respectively, from day 0 (D0; at one day old) to D14. Proventriculi in all groups were collected at D0, D7 and D14 for analysis of AvBDs expression and AvBD12 protein localization. The expression of AvBDs genes was examined by reverse transcription-PCR and changes in the expression upon probiotics-feeding were examined by real-time PCR. The AvBD12 localization was examined by immunohistochemistry, and density of immunoreaction products was examined by image analysis under a microscope. Out of 14 AvBDs genes, seven AvBDs were detected in the proventriculus of chicks, namely, AvBD1, 2, 4, 6, 7, 10 and 12. The expression of the 7 detected genes did not show any significant differences between control and probiotics groups at D7 and D14. The immunoreactive (ir) -AvBD12 was localized in surface epithelium and cells in the connective tissues of proventricular glands. The ir-AvBD12 density in surface epithelium was significantly higher at D7 than at D0 or D14 in control group. At D7 and D14, the ir-AvBD12 density was significantly lower in probiotics groups than in control group. The ir-AvBD12 cells in proventricular gland increased in number with age; however, there were no significant differences between control and probiotics groups at D7 and D14. These results suggest that, although probiotics-feeding does not affect the gene expression of AvBDs, it may induce AvBD12 secretion from the surface epithelium of the proventriculus in broiler chicks.
    Full-text · Article · Jan 2015 · The Journal of Poultry Science
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    Ahmad M Abdel-Mageed · Naoki Isobe · Yukinori Yoshimura
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    ABSTRACT: The immune response in the lower part of the hen oviduct is of crucial importance to protect the oviductal tissues from infection by microorganisms colonizing the cloaca. The aim of this study was to examine whether different TLRs can recognize their ligands to induce expression of proinflammatory cytokines and avian β-defensins (AvBDs) in the uterus and vagina of laying hens. The mucosal tissues of the uterus and vagina were collected, cultured in TCM-199 medium and stimulated with or without different ligands of TLRs, namely Pam3CSK4 (TLR2), poly I:C (TLR3), flagellin (TLR5), R848 (TLR7), and CpG-ODN (TLR21) and incubated for 3h. The expression of IL1B in the uterus and vagina was upregulated by all TLR ligands tested. The expression of IL6 in the uterus and vagina was upregulated by poly I:C and CpG-ODN, and it was also upregulated by Pam3CSK4 in the uterus and by R848 in the vagina. The expression of AvBD10 was upregulated by poly I:C in the uterus and by flagellin in the vagina. On the other hand, the AvBD10 expression was downregulated by CpG-ODN in the uterus and by R848 in the vagina, whereas its expression was not affected by Pam3CSK4 in both tissues. The expression of AvBD12 in the uterus and vagina was not affected by any TLR ligands except for CpG-ODN, which downregulated its expression in the vagina. These results suggest that TLR2, 3, 5, 7, and 21 in the uterine and vaginal tissues are functionally active in inducing proinflammatory cytokines in response to their specific ligands, although the effect on the expression of AvBDs is limited. Proinflammatory cytokines induced by interaction of TLRs with their ligands may play roles in the defense against infectious microorganisms. Copyright © 2014 Elsevier B.V. All rights reserved.
    Full-text · Article · Dec 2014 · Veterinary Immunology and Immunopathology
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    G. W. Zhang · S. J. Lai · Y. Yoshimura · N. Isobe
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    ABSTRACT: Psoriasin (S100A7) is a member of the S100 protein family of calcium-binding proteins and plays a crucial role in local host defences. The present study aimed to identify the expression of S100A7 in the goat mammary gland and in milk. The goat S100A7 coding DNA sequence was identified using direct sequencing. An S100A7 antibody was raised in rabbits by immunization with a synthetic S100A7 peptide consisting of 13 amino acids. Messenger RNA expression and protein localization in different regions of a healthy mammary gland were detected by reverse transcription-polymerase chain reaction and immunohistochemistry. Changes in the concentration of S100A7 in the milk after an intramammary infusion of Escherichia coli lipopolysaccharide (LPS) were examined by an enzyme immunoassay. The goat S100A7 peptide had 98% and 86% sequence similarity to that of sheep and bovines, respectively. The S100A7 mRNA expression was higher in the teat and udder skin than in the cistern and parenchyma of the mammary gland. Immunoreactive S100A7 was localized in the epithelial cells of the alveolus and gland cistern, and stratified squamous epithelium of the teat. Psoriasin as a secreted protein was detectable in healthy milk, and an intramammary LPS infusion increased the concentration of S100A7 in the milk. The results suggest that S100A7 is produced in the epithelial cells of the mammary gland and is secreted into the milk.
    Full-text · Article · Oct 2014 · The Veterinary Journal
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    Naoki Isobe · Chihiro Iwamoto · Hirokazu Kubota · Yukinori Yoshimura
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    ABSTRACT: The aim of the present study was to examine the effect of the somatic cell count (SCC) in milk on reproductive performance, such as pregnancy status in the prepartum period and ovarian function in the postpartum period, in dairy cows. Blood samples were collected every week from one month prepartum to parturition in order to measure the concentrations of 13,14-dihydro-15-keto-PGF2α (PGFM), estrone sulfate (E1S) and progesterone. Milk samples were collected three times per week in both the prepartum (for one month before the dry period) and postpartum periods (for 3 months immediately after parturition) to measure the SCC. Progesterone was also determined in the whole milk of postpartum cows to define the day of the first ovulation. In the prepartum period, the maximum SCC negatively correlated with the pregnancy period (r = –0.77), but not the calf birth weight. Positive and negative correlations were observed between the average SCC and PGFM or progesterone concentrations in plasma, respectively (r = 0.84 or –0.92, respectively), at 39 weeks of pregnancy. In the postpartum period, a correlation was observed between the day of the first ovulation and both the average and maximum SCC (r = –0.74 and –0.75, respectively), whereas days open was not related to the SCC. These results suggest that a high SCC in the prepartum period may advance parturition by increasing PGF2α and decreasing progesterone and that the first ovulation in the postpartum period was affected by a high SCC.
    Preview · Article · Sep 2014 · Journal of Reproduction and Development
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    ABSTRACT: The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day −6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days −6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 (<5 × 106 cell/mL) were significantly higher and lower than those in cows with a higher SCC, respectively. These results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factor in milk of cows treated for clinical mastitis.
    No preview · Article · Sep 2014 · Animal Science Journal
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    Takahiro Nii · Naoki Isobe · Yukinori Yoshimura
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    ABSTRACT: The aim of this study was to determine the mechanism by which the avian infectious bronchitis virus (IBV) affects eggshell formation. Attenuated IBV (aIBV group) or vehicle (control group) was injected into the oviductal magnum lumen of White Leghorn laying hens. The changes in the expression of genes related to eggshell formation (collagen types I and V, and CaBP-D28 K), densities of cytotoxic cells (CD8+ cells and TCR-γδ+ T cells) as well as gene expression of molecules related to cytotoxic immunoreaction (B-NK, perforin, granzyme and IL-2) and proinflammatory cytokines (IL-1β, IL-6 and IFN-γ) were examined by quantitative RT-PCR or immunohistochemistry in the isthmus and uterus. Gene expression of IL-1β and IL-6 receptors in the tubular gland cells of the isthmus and uterus was analyzed by RT-PCR. Gene expression of collagen type I, but not collagen type V, in the isthmus and CaBP-D28 K in the uterus was decreased in the aIBV group compared with that in the control. The frequencies of CD8+ cells and TCR-γδ+ T cells in the isthmus and uterus were significantly higher in the aIBV group than in the control group. The expression of cytotoxic molecular and proinflammatory cytokines was also higher in the aIBV group than in the control. The expression of IL-6 receptor, but not IL-1β receptor, was identified in the tubular gland cells in the isthmus and uterus. These results suggest that IBV infection causes disorder of eggshell formation by disturbing gene expression of collagen type I in the isthmus and CaBP-D28 K in the uterus, probably via the effects of substances from cytotoxic cells and proinflammatory cytokines.
    Full-text · Article · May 2014 · Theriogenology
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    R.X. Lan · F. Liu · Z.B. He · C. Chen · S.J. Liu · Y. Shi · Y.L. Liu · Y. Yoshimura · M. Zhang
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    ABSTRACT: The aim of this study was to investigate the presence and localisation of gonadotropin releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR) and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR and PGRMCI, mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles, and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all of the receptors were primarily localised in the granulosa and theca cells. In addition, LHR was also localised in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors except GnRHRI in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR and PGRMCI decreased from the preantral follicles (primordial, primary and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, while FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.
    Full-text · Article · May 2014 · Theriogenology
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    S. Srisaikham · Y. Yoshimura · N. Isobe

    Full-text · Conference Paper · Mar 2014
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    B Ariyadi · N Isobe · Y Yoshimura
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    ABSTRACT: We previously reported that bacterial lipopolysaccharide (LPS), a ligand of Toll-like receptor 4 (TLR4), induced mucin mRNA to enhance the mucosal barrier in the hen vagina. The aim of this study was to determine the intracellular signaling molecules for that mucin induction, and the effect of molting and estrogen on their expression. The expression of TLR4, its adaptor molecules, and transcriptional factors in the vaginal mucosa of laying and molting hens treated with or without estradiol was examined by reverse-transcription PCR. The expression of mucin in the cultured mucosal tissue stimulated by LPS together with inhibitors of transcriptional factors was analyzed by quantitative reverse-transcription PCR. The expression of TLR4, its adaptor molecule, namely, myeloid differentiation factor 88 (MyD88) or Toll-interleukin 1 receptor domain-containing adaptor-inducing IFN-β (TRIF), and transcriptional factors, namely, cFos and cJun, declined in molting hens compared with that in laying hens, and were upregulated by estradiol. In vagina of laying hens, mucin expression was upregulated by LPS, whereas it was suppressed by inhibitors of transcriptional factors, namely, ALLN (an inhibitor of IκB proteolysis), BAY-117085 (an NFκB inhibitor), U0126 [a mitogen-activated protein kinase (MAPK) inhibitor], and transhinone IIA [an activated protein 1 (AP-1) inhibitor]. These results suggest that a MyD88-dependent pathway downstream of TLR4 and transcriptional factors of NFκB and AP-1 participate in the induction of mucin expression by LPS in the vaginal mucosa. These signaling functions may decline during molting owing to the decline in the level of circulating estrogen. Such mucin expression system may play a role in the mucosal barrier against infection in the vaginal mucosa.
    Preview · Article · Mar 2014 · Poultry Science
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    Gong-Wei Zhang · Song-Jia Lai · Yukinori Yoshimura · Naoki Isobe
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    ABSTRACT: Cathelicidins are a family of antimicrobial peptides found in neutrophils and the epithelium that have broad-spectrum activity against bacteria. This study aimed to investigative the mRNA expression of cathelicidins and protein localization of cathelicidin-2 in the goat mammary gland and its secretion into milk. The mRNA expression of cathelicidins was examined in different regions of the mammary gland by reverse transcription PCR. A cathelicidin-2 antibody was raised in rabbits by immunization with a synthetic cathelicidin-2 peptide consisting of 17 amino acids. The protein localization of cathelicidin-2 was investigated in the mammary gland by immunohistochemistry. Skim milk was collected before (0h) and 2, 4, 8, 12, and 24h after the intramammary infusion of lipopolysaccharide and saline, and the concentration of cathelicidin-2 was examined by an enzyme immunoassay. The mRNAs of cathelicidin-1, 2, 3, 6, and 7 were expressed in both the teat and deep region of the mammary gland from healthy and mastitic goats. Immunoreactive cathelicidin-2 was not localized in the epithelial cells of the teat skin, teat cistern, or mammary alveoli, whereas it was localized in polymorphonuclear cells in the mammary gland and those collected from the blood and milk. Cathelicidin-2 was detected in skim milk by Western blotting. The concentration of cathelicidin-2 in milk increased 4h after the intramammary infusion of lipopolysaccharide. These results suggest that cathelicidin-2 is expressed in polymorphonuclear cells and is secreted into milk in goat.
    Full-text · Article · Feb 2014 · Veterinary Microbiology
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    ABSTRACT: The ovary and oviduct of the hen are susceptible to various pathogenic microorganisms, and infections in these organs may not only contaminate the eggs, but also disorder the egg formation. The immune function of the ovary and oviduct is essential to protect these tissues from infection as well as for the production of hygienic eggs. This paper reviews recent studies on the host defence system in the reproductive organs with reference to their innate immune functions, with emphasis on the important role of Toll-like receptors and avian β-defensins in this defence system.
    Full-text · Article · Feb 2014 · Avian biology research
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    B Ariyadi · N Isobe · Y Yoshimura
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    ABSTRACT: Mucins play an essential role as mucosal barrier to prevent invasion of pathogens in the oviductal tissue of hens. The aim of this study was to determine the effect of estradiol and lipopolysaccharide (LPS) on the mucin expression in the lower oviductal segments (vagina and uterus) of hens. The mucosal tissues of the vagina and uterus were collected from White Leghorn laying and molting hens, and molting hens with or without intramuscular injection with 1 mg of estradiol-benzoate (EB) daily for 7 d. Part of these tissues was cultured in TCM-199 culture medium with or without LPS (10, 100, or 1,000 ng/mL) for 1.5 or 3 h. Mucin expression in the mucosa of laying, molting, and EB-treated molting hens (EB-group) and in those tissues cultured with or without LPS was analyzed by quantitative reverse-transcription PCR. Cultured tissues were also processed for paraffin sections and stained with Alcian blue (AB). In both the vagina and uterus, mucin expression and density of AB-positive mucopolysaccharide were reduced in molting hens compared with laying hens, and upregulated by EB. Mucin expression in the cultured vagina and uterus tissues of laying and molting hens was upregulated by LPS in a dose- and time-dependent manner. However, there was no response to LPS for induction of mucin in the tissues of EB-group hens. The mucin expression level in the vagina and uterus tissues stimulated by LPS was lower in the EB-group hens than in laying and molting hens, and that in the uterus was lower in the molting hens than in laying hens. These results suggest that mucin expression is stimulated by LPS in the vagina and uterus of laying and molting hens. Estrogen may upregulate mucin expression in those tissues in association with epithelial development, whereas it may suppress the response to LPS for mucin induction. The mucin expression caused by LPS may enhance mucosal barrier function and play a role in preventing infections by bacteria in the vagina and uterus.
    Preview · Article · Dec 2013 · Poultry Science
  • Naoki Isobe · Ayumi Shibata · Hirokazu Kubota · Yukinori Yoshimura
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    ABSTRACT: The present study was undertaken to examine whether potential levels of innate immune factors (lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO)) in colostrum are associated with subsequent milk somatic cell count (SCC) in dairy cows. Quarter milk samples were collected daily for 1 week postpartum to measure LAP and LF concentrations and LPO activity. SCC in milk was determined weekly for 2 months postpartum and its correlations to concentrations of LAP and LF and LPO activity were examined. Only small variations of all immune factors were found among four udders in each individual cow, whereas there were great differences in these factors among cows. Negative correlation was detected only between LPO activity and mean and maximum SCC, whereas its relationship was not significant. LAP and LF concentrations were significantly correlated positively to mean, maximum and minimum SCC. These results suggest that the great difference in innate immune factors among animals and high LAP and LF concentrations in colostrum may be associated with subsequent high incidence of SCC increase.
    No preview · Article · Sep 2013 · Animal Science Journal
  • Yaqiong Huang · Naoki Isobe · Yukinori Yoshimura · Kenji Hosoda
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    ABSTRACT: Mastitis is most critical disease in dairy cows and causes huge cost in the dairy industry. To prevent and treat it, it is important to understand the mechanisms of immune function in the mammary gland. Innate immunity is non-specific acute-response immune function. Some components of innate immunity in the mammary gland are found, e.g. Lingual Antimicrobial Peptide (LAP), Lactoferrin (LF). These components are found to be localized in the alveolar epithelium of mammary gland. LAP belongs to the beta-defensin family, and plays a crucial role in killing a large variety of microorganisms. LF belongs to an iron-binding glycoprotein and has antibacterial activity. It is reported that LF has been localized immunohistochemically in mammary epithelial cells of lactating cows. Our previous study revealed that secretion of LAP into milk proceeded to that of LF after lipopolysaccharide (LPS) injection into the mammary gland. From this result, it is hypothesized that immunohistochemistry probably shows positive to either LF or LAP but not both in the alveolus vs epithelium in the mammary gland. Therefore, the aim of the present study is to investigate the immunolocalization of LAP and LF in the same bovine mammary tissue. Bovine mammary tissues were collected in the slaughterhouse and were fixed with neutralized formalin immediately. Paraffin sections (2-um thickness) were processed with antigen retrieval treatment followed by blocking with casein milk. Sections were cultured with LF antibody or LAP antibody. Immunoreaction products were visualized by incubation with a DAB. LAP and LF were localized in the cytoplasm of epithelial cell of alveolus. In some cases, LAP and LF were seen clearly in the same alveoli of section. In other cases, some epithelial cells were stained only LAP, but not LF, and other epithelial cells of alveolus were stained only LF, but not LAP. These results suggest the possibility that LAP and LF are differentially synthesized in the alveolar epithelium and may support our previous findings that their secretion occurs at the different time course.
    No preview · Conference Paper · Sep 2013
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    G W Zhang · W X Zhang · S Y Chen · Y Yoshimura · N Isobe · S J Lai
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    ABSTRACT: Dectin-1 plays a critical role in the pathogenesis of intestinal inflammation by recognizing the pathogenic agents and mediating cytokine responses. The objective of this study was to establish the association between dectin-1 polymorphisms and susceptibility to non-specific digestive disorders (NSDD) and cytokine expression in rabbits. A total of seven coding SNPs were detected in dectin-1 gene. The genetic association between SNP (ss707197675A>G) and susceptibility to NSDD was evaluated using a case-control study (178 cases and 174 controls). The results revealed that the A allele was associated with an increased risk of developing NSDD in rabbits. The AA genotype significantly increased the genetic susceptibility to NSDD with odds ratio of 4.76 (95% confidence interval, 1.92-12.50, P = 0.0002) compared with GG and GA genotypes. We also experimentally induced NSDD in another independent growing rabbit population by feeding a low-fiber diet and subsequently investigated the cytokine mRNA expression. Among the four studied cytokines, the expression levels of IFN-γ, IL-17F, and IL-22 were increased 2.8 to 6.0 fold in AA genotype compared with GG genotype (P < 0.01). The greater IL-17F and IL-22 mRNA expressions indicated a positive correlation with severe intestinal inflammation (P < 0.05). The decreased expression of IL-10 was associated with severe intestinal inflammation (P = 0.006), but IL-10 expression was not influenced by dectin-1 genotype. In conclusion, polymorphism ss707197675 of dectin-1 is related with susceptibility to NSDD and increased expression of proinflammatory cytokines, and dectin-1 could be an important candidate gene associated with NSDD in rabbits.
    Full-text · Article · Jul 2013 · Journal of Animal Science
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    ABSTRACT: The aim of this study was to determine the effects of probiotics on T cell subsets induction in the intestine of broiler chicks. Day-old male broiler chicks were fed with or without probiotics consisting of Streptococcus faecalis, Clostridium buthricum, Bacillus mesentericus (probiotics group and control group, respectively). Cryostat sections of their ileum, cecum and rectum at day 0, 7 and 14 of feeding were immunostained for CD4, CD8 and TCRγδ, and the frequencies of positive cells in the mucosal tissue were analyzed. The CD4+, CD8+, TCRγδ+ T cells were localized in the lamina propria of intestinal mucosa in all birds. At day 7 and 14, CD8+ T cells were localized also in the mucosal epithelium of all segments in the probiotics group and of cecum in control group, and TCRγδ+ T cells were observed in the mucosal epithelium of all birds. The frequencies of CD4+, CD8+, TCRγδ+ T cells were increased with age from day 0 to day 14 in both control and probiotics groups. The frequency of CD8+ T cells was significantly greater in probiotics group than control group in the ileum and rectum (P<0.05) and the cecum (P<0.01) at day 7. There were no significant differences in the frequency of CD4+ and TCRγδ+ T cells between control and probiotics groups in all intestinal segments at day 7 and 14. The ratio of CD8+/CD4+ T cells was greater than 1.0 in all tissues. The ratio in the ileum at day 7 was significantly greater in the probiotics group than control group (P<0.05). These results further suggest that probiotics cause an influx of the CD8+ T cells into the intestinal mucosa, which may enhance the intestinal immunity by CD8+ T cells in young chicks.
    No preview · Article · Jul 2013 · The Journal of Poultry Science

Publication Stats

1k Citations
180.80 Total Impact Points

Institutions

  • 1991-2015
    • Hiroshima University
      • • Graduate School of Biosphere Sciences
      • • Faculty of Applied Biological Science
      • • Division of Biological Science
      • • Graduate School for International Development and Cooperation
      Hirosima, Hiroshima, Japan