R Kirisawa

Rakuno Gakuen University, Ebetsu, Hokkaido, Japan

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Publications (75)89.23 Total impact

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    ABSTRACT: This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1β in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 µg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.
    Journal of Veterinary Medical Science 06/2011; 73(11):1399-404. · 0.88 Impact Factor
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    ABSTRACT: The present study examined localization of cholecystokinin receptor (CCK-R) mRNA in the muscle layer of the ovine omasum and role of CCK-R type 1 (CCK-1R) in the regulation of muscle contraction of the omasum. We demonstrated that not only CCK-R type 2 (CCK-2R) mRNA but also CCK-1R mRNA is highly expressed in the muscle layer of the ovine omasum. Application of CCK-8 to muscle strips of the greater curvature of the ovine omasum at 1-100 nM induced tonic contraction in a concentration-dependent manner, and the contractile effect of CCK-8 was inhibited by both CCK-1R antagonist lorglumide (IC(50) 2.7 and 7.9 microM in the longitudinal and circular muscle, respectively) and CCK-2R antagonist PD135,158 (IC(50) 51.4 microM in the longitudinal muscle), indicating that not only CCK-2R but also CCK-1R is functionally expressed in the plasma membrane of smooth muscles in the omasum and mediates action of exogenous CCK. Contractile effect of intravenous infusion of CCK-8 (1-30 pmol/kg/min) on omasal contraction was also confirmed in the in vivo experiments using conscious sheep in the absence and presence of atropine infusion (14.4 nmol/kg/min), and showed that circulating CCK increases omasal electromyographic (EMG) activity at lower plasma concentration than that it inhibits ruminal contractions. Taking account of our previous results in the in vivo study using other CCK-1R antagonist, it is suggested that circulating CCK, even at normal range of plasma concentration, plays a physiological role as a regulator of omasal contractions in sheep and CCK-1R mediates the action of CCK.
    Domestic Animal Endocrinology 08/2008; 35(2):231-44. · 2.38 Impact Factor
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    ABSTRACT: The aim of this study was to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples and to determine whether a relationship exists between BoTV and diarrhea in Japan. Ninety-nine diarrheic and 114 normal fecal samples from calves in Hokkaido Prefecture and 38 diarrheic fecal samples from calves in 10 other prefectures were examined by reverse transcription (RT)-PCR with primers designed in the spike (S) gene for the presence of BoTV. The specimens were also examined for the presence of other enteric pathogens, bovine rotavirus, coronavirus and Cryptosporidium spp. BoTV RNA was detected in 15 (15.2%) of the 99 diarrheic samples from Hokkaido and in 9 (23.7%) of the 38 diarrheic samples from the other prefectures. The incidence of BoTV in control specimens was 7.0%. In 11 of the 15 BoTV-positive specimens from Hokkaido, BoTV was the only pathogen detected among those examined, and 11 BoTV-positive specimens were obtained from calves less than 2 weeks of age. Rotavirus was confirmed to be associated with calf diarrhea, but coronavirus and Cryptosporidium spp. were not. Nucleotide sequences of 17 different BoTV RT-PCR products were determined. Phylogenetic analysis based on the sequences revealed that Japanese BoTVs could be classified into at least two groups. This study showed that BoTV is a common virus in fecal specimens of calves with diarrhea in Japan and may be an important pathogen of cattle, principally in young calves less than 2 weeks of age.
    Journal of Veterinary Medical Science 06/2007; 69(5):471-6. · 0.88 Impact Factor
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    ABSTRACT: The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In the cells exposed to heat (37 to 45 degrees C, 0 to 60 min), the survival rate decreased sharply in a temperature- and time-dependent manner, especially at 42 and 45 degrees C. Cells exposed at 40 degrees C, however, showed little change in survival rate and morphology. Gelatin zymograms revealed that proMMP-2 and -9 were the only two MMPs remaining in the supernatant of the cultured tendinocytes, including that of untreated cells. Addition of TNFalpha and IL-1beta to the culture medium of tendinocytes accelerated proMMP-9 synthesis considerably. Heating the tendinocytes (40 degrees C) led to a three-fold increase in proMMP-9 synthesis in a short time. Only TNFalpha was detected in tendinocytes after heat exposure for 30 and 60 min. In contrast, IL-1beta was under the detectable level in ELISA. Cooling of heat-exposed cells from 40 degrees C to 37 degrees C considerably down-regulated cellular proMMP-9 synthesis. Furthermore, proMMP-9 level was greatly reduced in cells treated at lower temperatures, 20 degrees C and 5 degrees C. These findings support our hypothesis that hyperthermia in the horse tendon induces tendinocytes to synthesize pro-inflammatory cytokines and that the synthesis of these cytokines results in the up-regulation of gelatinases.
    Biomedical Research 11/2006; 27(5):233-41. · 1.26 Impact Factor
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    ABSTRACT: cDNA generated from lipopolysaccharide-stimulated equine peripheral blood mononuclear cells was used to amplify and clone type I and type II equine interleukin-1 receptors (IL-1RI and IL-1RII) using primers derived from semi-conserved regions between human and mouse IL-1RI and IL-1RII sequences, respectively. 5' and 3' terminal sequences of equine IL-1RI and IL-1RII were amplified by 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of equine IL-1RI demonstrated 77, 64 and 63% similarity with human, mouse and rat sequences, respectively. The predicted amino acid sequence of equine IL-1RII demonstrated 70, 60 and 58% similarity with human, mouse and rat sequences, respectively. Recombinant equine soluble IL-1RI and IL-1RII produced in insect cells bound recombinant equine IL-1alpha and IL-1beta. Furthermore, both receptors suppressed the growth inhibitory activities of equine IL-1alpha and IL-1beta toward A375 cells in a dose-dependent manner, indicating that the present equine IL-1RI and IL-1RII cDNA encodes biologically active proteins.
    Veterinary Immunology and Immunopathology 03/2006; 109(3-4):219-31. · 1.88 Impact Factor
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    ABSTRACT: We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.
    Archives of Virology 01/2006; 150(12):2549-65. · 2.03 Impact Factor
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    ABSTRACT: The IL1R is composed of two kinds of molecule, type I (IL1R I) and type II (IL1R2). IL1R1 contributes to IL-1 signaling, whereas the IL1R2 has no signaling property and acts as a decoy for IL-1. In this study, we developed a bovine IL1R2-specific sandwich ELISA to examine the sIL1R2 concentration in serum and milk from dairy cows. The concentration of colostral IL-1beta was examined to estimate the correlation to sIL1R2. The results showed that the sIL1R2 concentration in sera and milk changes with the stages of lactation. The serum sIL1R2 concentrations were 5.56+/-0.69 ng/ml (colostrum), 3.14+/-0.72 ng/ml (the early stage of lactation) and 5.76+/-1.25 ng/ml (the late stage of lactation). The milk sIL1R2 concentrations were 1.83+/-0.47 ng/ml (colostrum), 0.73+/-0.22 ng/ml (the early stage of lactation) and 2.92+/-0.56 ng/ml (the late stage of lactation). The concentrations of IL1R2 in sera and milk were significantly higher at the late stage of lactation and colostrum than that of the early stage of lactation. The reduction rates of sIL1R2 levels from the colostrum to the early stage of lactation were 43.6% (serum) and 61% (whey). IL-1beta was detected in all the colostrum (995.9+/-346.6 ng/ml). Significant correlation was observed between the levels of colostral IL-1beta and IL1R2 (r=0.75).
    Cytokine 12/2005; 32(3-4):132-6. · 2.52 Impact Factor
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    ABSTRACT: Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R1 in tendinocytes. In this study, we focused on four signaling factors, TNFalpha, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and some samples were also subjected to reverse transcription-polymerase chain reaction (RT-PCR), PCR-Southern blot analysis and in situ hybridization to detect the expression of TNFalpha mRNA. Distribution of the four factors differed depending on the tendon condition, normal, inflamed or scar-formed. In the normal tendon, large amounts of TRAF2 were found in tendinocytes, but the amounts of TNF-R1 were small. TNFalpha mRNA was expressed most highly in the inflamed tendon. TNF-R1, which was only faintly detected in the normal tendon, was detected at a high level in the inflamed tendon, and the amounts of TRAF2 and caspase-3 also increased. Activated caspase-3 was only detected in the inflamed tendon. TNFalpha mRNA was also expressed in the scar-formed tendon, though it showed weak signals, and the expression levels of TNF-R1, TRAF2 and caspase-3 proteins were very low. Two distinct intracellular signaling pathways of TNFalpha, which lead to cell survival and apoptosis, might be present in tendinocytes mediated through TNF-R1. These results, which reflect the dynamism of TNFalpha, provide important clues for means to prevent tendinopathy.
    Journal of Veterinary Medical Science 11/2005; 67(10):985-91. · 0.88 Impact Factor
  • The Veterinary record 08/2005; 157(4):118-20. · 1.80 Impact Factor
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    ABSTRACT: The spleen plays an important role in cellular immunity leading to parasite elimination, especially in hemo-protozoan parasite infection. In the present study, we quantified the serum IFN-gamma in splenectomized (SP) and non-splenectomized (NSP) cows infected with Theirelia sergenti (TS) to investigate whether the spleen plays a role in the Th1-type cytokine responses in cows following parasite infection. A transient increase in IFN-gamma was observed in the early stages of infection in the NSP cows, and the cows did not develop parasitemia. In contrast, the SP cows showed no IFN-gamma response at the early stage of infection, and the cows developed parasitemia following anemia. The NSP cows produced IgG1 and IgG2 antibodies to TS-specific, whereas the SP cows showed increases only in IgG1. Increased PBMC proliferation by cocanavalin A stimulation was observed concomitant with the IFN-gamma response. Pretreatment with IFN-gamma suppressed the propagation of TS in the SP cattle. These results suggest that the spleen plays an important role in the resistance to TS infection, leading to a Th1-type immune response. IFN-gamma might contribute to the activation of immunocytes and play an important role in the immune response to resist TS proliferation.
    Veterinary Parasitology 02/2005; 127(2):105-10. · 2.38 Impact Factor
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    ABSTRACT: A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(-) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.
    Nucleic Acids Research 02/2005; 33(6):e65. · 8.81 Impact Factor
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    ABSTRACT: Scars formed after tendonitis result in altered tissue mechanical properties after injury. The interaction of collagen molecules with decorin affects collagen fibrogenesis, and scar tissue is fragile as a consequence of a large amount of decorin in the scar. We hypothesized that scar formation could be prevented by controlling decorin expression in tendinocytes. As a preliminary experiment, we treated tendinocytes with decorin antisense oligodeoxynucleotides (ODNs). Tendinocytes were isolated from Achilles tendons of New Zealand white rabbits and treated with ODN. When tendinocytes were transfected with decorin sense ODN, there was no alteration, whereas decorin antisense ODN-treated tendinocytes showed suppression of transforming growth factor (TGF)-beta1 production. Decorin and TGF-beta1-production of tendinocytes is regulated by decorin gene suppression. The results showed that the antisense approach is an attractive therapeutic strategy not only for preventing decorin deposition in scar tissue, which decreases collagen fibril diameter, but also for controlling TGF-beta1 production, which leads to organ fibrosis.
    Connective Tissue Research 02/2005; 46(1):18-26. · 1.79 Impact Factor
  • Journal of Veterinary Medical Science - J VET MED SCI. 01/2005; 67(10):985-991.
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    ABSTRACT: Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were treated with 10 ng/ml equine TNFalpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF -R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFalpha-treated cells (inflamed condition). Intense TRAF2 and NF-kappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in vitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.
    The Japanese journal of veterinary research 12/2004; 52(3):135-44. · 0.65 Impact Factor
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    ABSTRACT: Reproductive diseases after parturition are a serious problem in dairy cattle. It is important to predict postpartum reproductive diseases early and to develop prophylaxis. The objectives of this study were to demonstrate changes in the peripheral blood concentration of interleukin-6 (IL-6) before parturition, which was mainly produced by T helper 2 type (Th2) cells, and to investigate a correlation between the IL-6 concentration and the occurrence of the postpartum retained placenta, endometritis and/or follicular cyst in dairy cattle. Twenty-seven Holstein-Friesian cows were used for this study. Thirteen had no clinical disease, 8 had retained placenta, 4 were diagnosed with endometritis by vaginal inspection, and 2 were diagnosed with follicular cyst by rectal palpation at 1 and 2 months after parturition. Blood samples were collected 60 days pre- and post-partum. They used for IL-6, progesterone (P(4)) and estradiol-17beta (E(2)) concentration determination. This study showed that the IL-6 concentration prepartum was higher than postpartum. Low levels of IL-6 and P(4) in peripheral blood prepartum tended to affect retained placenta and a high level of IL-6 prepartum tended to affect endometritis. These results indicate that measurement of change in the IL-6 concentration during pregnancy is one useful tool for predicting crisis in postpartum reproductive diseases in dairy cattle.
    Journal of Veterinary Medical Science 12/2004; 66(11):1403-8. · 0.88 Impact Factor
  • Journal of Veterinary Medical Science - J VET MED SCI. 01/2004; 66(11):1403-1408.
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    ABSTRACT: The relationship of passage-induced mutant genes 1 and 71 of an attenuated equine herpesvirus 1 (EHV-1) with virulence was analysed by constructing nine recombinant EHV-1 viruses by homologous recombination. Gene 1 or/and gene 71 of a virulent EHV-1 strain, HH1, was replaced by a mutant gene 1 or/and 71 of an attenuated HH1 strain, BK343, respectively. The beta-galactosidase gene of Escherichia coli was inserted within the gene 1 or 71 coding sequence of HH1 to inactivate the genes. Virus replications of these recombinant viruses in cell cultures were similar, but release of the gene 71-inactivated virus from infected cells was delayed compared to that of the other viruses. Plaque sizes of the recombinant viruses were similar to those of HH1, but those of BK343 were significantly smaller, indicating an effect of some unknown factor(s) on viral cell-to-cell spread. The growth abilities of the recombinant viruses with a mutant gene 1 or/and 71 in lungs of mice were similar to those of HH1, but those of gene 71-inactivated viruses were reduced to the level of BK343 and the titers were about 100-times lower than those of the other recombinant viruses. These results indicate that the mutant genes 1 and 71 of BK343 might not confer an attenuated nature to EHV-1.
    Veterinary Microbiology 10/2003; 95(3):159-74. · 3.13 Impact Factor
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    ABSTRACT: We demonstrated the experimental vertical transmission of Borna disease virus (BDV) in pregnant BALB/c mice. Giessen strain He/80 of BDV was used in the present study. Six six-week-old mice were inoculated intraperitoneally with 10(5) 50% tissue culture infective doses (TCID50), and were bred immediately. Four pregnant mice were sacrificed under anaesthesia on the 10th and 14th days after vaginal plug formation. Nine newborns from two maternal mice were sacrificed under anaesthesia on the 7th day after birth. Positive signals with RT-nested PCR techniques for BDV p24-RNAs were seen in the fetuses, placentas and brains of all newborn mice. No immunopositivities for BDV p40 were found in the fetuses or placentas at 10 days' gestation. BDV p40 immunopositivities were found in neurons of the fetal brains and in decidual cells of the placentas at 14 days' gestation. They were also found in neurons of the brains of newborn mice. At 10 days' gestation, no positive signals for BDV p40 sense or antisense riboprobes were seen in the fetal brains or placentas. Positive signals were found in neurons of the fetal brains and decidual cells of the placentas at 14 days' gestation. Positive signals for BDV p40 sense and antisense riboprobes were found in almost all neurons throughout the brains of nine newborn mice. These results suggest that persistent infection with BDV in newborn mice may be induced by vertical transmission during gestation.
    Archives of Virology 09/2003; 148(8):1557-68. · 2.03 Impact Factor
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    ABSTRACT: To obtain basic information on the state of proinflammatory cytokines in newborn calves, we determined the kinetics of 5 cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist) in sera of newborns during the first 4 weeks of life. At birth, none of the 5 cytokines were detected in almost all serum samples, but the cytokines became detectable within 12 hr after being fed colostram. The mean concentrations of the cytokines reached peak levels by 24 hr and then gradually decreased and became undetectable by 4 weeks after birth. Cytokine mRNA expressions in peripheral blood mononuclear cells of newborns were observed without reference to the cytokine concentrations in sera. Serum cytokines detected in newborn calves are probably colostral origin.
    Journal of Veterinary Medical Science 08/2003; 65(7):813-6. · 0.88 Impact Factor
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    ABSTRACT: Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist, IL-1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL-2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL-1beta, TNF-alpha or IFN-gamma significantly enhanced the ConA response, whereas IL-1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL-2 mRNA expression in ConA-stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA-stimulated controls, the expression levels became comparable after pretreatment with IL-1beta, TNF-alpha or IFN-gamma. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL-1beta, TNF-beta and IFN-gamma. These results suggest that pretreatment of neonatal PBMC with IL-1beta, TNF-alpha or IFN-gamma promotes mitogenic response to ConA through up-regulating the production of IL-2 and the expression of the mature IL-2 receptor.
    Microbiology and Immunology 02/2003; 47(6):461-8. · 1.55 Impact Factor