| Co-Investigator(Kenkyū-buntansha) |
INANAMI Osamu HOOKAIDO UNIVERSITY, GRADUATE SCHOOL OF VETERINARY MEDICINE ASSOC., PROFESSOR, 大学院・獣医学研究科, 助教授 (10193559)
KIRISAWA Rikio RAKUNO GAKUEN UNIVERSITY, SCHOOL OF VETERINARY MEDICINE, ASSOC., PROFESSOR, 獣医学部, 助教授 (70153252)
HAYASHI Masanobu RAKUNO GAKUEN UNIVERSITY, SCHOOL OF VETERINARY MEDICINE, PROFESSOR, 獣医学部, 教授 (10130337)
KUWABARA Mikinori HOOKAIDO UNIVERSITY, GRADUATE SCHOOL OF VETERINARY MEDICINE, PROFESSOR, 大学院・獣医学研究科, 教授 (10002081)
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| Budget Amount *help |
¥9,200,000 (Direct Cost: ¥9,200,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
The above-entitled study was conducted to characterize the leukocyte functions of dairy cows during peripartum period, and to investigate the modulation of the host defence capability. Major results of this study were summarized as follows:
Significantly decreased luminal-dependent chemiluminescent response was found in neutrophils from cows around parturition, compared to those of control cows. Similar changes were found in neutrophils stimulated with aggregated bovine IgG. These results were in consistent with previous findings that functions of neutrophils are impaired in peripartum period.
Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium( [Ca2+}i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA by neutrophil lysates from normal cattle and 3 cattle with leukocyte adhesion deficiency were determined to evaluate the apoptosiss of normal and CD 18 deficient neutrophils. The rate of
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apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation-time, whereas no apparent increase in apoptosis was found in CD 18-deficient neutrophils. Aggregated bovine IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. A delay in apoptosis was demonstrated in CD 18-deficient bovine neutrophils and this appeared to be closely associated with lowered signaling via [Ca2+]I, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates. These results suggest that decreased functional activity of neutrophils from peripartum cows may be associated with changes in the rate of apoptosis.
In vitro kinase assay revealed that Akt was unable to catalyze p47 phox phosphorylation. The activation of cPKC ( classical isotypes of PKC) and PKC-delta after fMLP stimulation was dependent on P13K (phosphoinositide 3-kinase). P13K inhibitors reduced the activation of phospholipase C gamma 2 without affecting tyrosine phosphorylation on it. P13K regulates the -phosphorylation of NADPH oxidase component p47phox by controlling diacylglycerol-dependen' t PKCs but not Akt.
Decreased tyrosine phosphorylation in neutrophils, detected by p38 and MAPK, also found in cows around parturition. This result indicate that the role of p38 and MAPK in bovine neutrophils is related to this signal transduction and related mechanisms. The costimulative effects of complement receptor type 3 (CR3) with IgG-Fc receptors on neutrophils were demonstrated in peripartum dairy cows, however the functional responses detected by tyrosine products were diminished, compared to that of control cows.
Micronutrients such as selenium (Se), biotin, zinc, copper (Cu) play a significant role in host defence capability in dairy cows. The concentrations of alpha-tocopherol and Se in serum from perinatal dairy cows were determined. Serum alpha-tocopherol concentrations in serum from cows at 1-2 weeks before parturition were significantly decreased, compared to those of cows with 3-4 weeks before parturition. No significant changes in Se concentration was found in serum from cows before and after parturition. Six hours after administration of alpha-tocopherol to the cows, alpha-tocopherol concentration started to increase and peak concentration (607.2 ug/dl) was found at 48 hours after administration. The concentration of Se increased greatly (88.3+8.3 ppb), and then decreased rapidly to 24 hours, thereafter Se levels were gradually decreased until 3 weeks after Se administration. The concentration of Se in leukocytes was significantly increased at 48 hours after Se administration, and decreased significantly until 2 to 3 weeks after administration. The changes in the concentration of Se in leukocytes were monitored. It appears that the changes found in this study are associated with the times that shows the increased functions of leukocytes after administration of Se.
Biotin acts as a cofactor for enzymes and play a key role in glycogenesis, fatty acid synthesis, and amino acid catabolism. Effects of dietary biotin supplementation on serum biotin levels and physical properties of sole horn of dairy cows were evaluated. The serum biotin level in biotin- supplemented cows after 10 months of supplementation was significantly higher than that in control cows. Significantly harder quality of sole horn was observed in dairy cows after biotin- supplementation.
Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks (SSB) were investigated in LEC rats.When LEC rats were treated with trientine, the observed number of cells with DNA damage decreased. This result show the importance of the role of Cu on host defense in relation to the scavenges of the superoxides produced by neutrophils.
Lactoferrin (LF) is an iron-binding antimicrobial protein present in milk and mucosal surfaces, and it is thought to play a role in preimmune host defense. The concentrations of LF in milk from normal cows and cows with mastitis were measured to determine whether the LF concentration in milk is influenced by the age of the cows, the stage of lactation, number of milk somatic cells and the presence of pathogens. Significantly higher LF concentrations in milk were found in cows with subclinical mastitis, in latter stage of lactation, pathogens with Staphylococcus aureus and with environmental streptocci, and in milk with increasing number of SCC.
The effect of administering lactoferrin hydrolysate (LFH) into the mammary glands of dairy cows with subclinical mastitis was evaluated to know whether lactoferrin and LFH have an immune modulating activity or not. Seven ml of a preparation of LFH was infused into 35 quarters of 25 cows with subclinical mastitis. The mean somatic cell counts (SCC) peaked one day after administration of LFH and the counts were significantly decreased on days 7, 14 and 21 compared to those before the administration of LFH. The mean lactoferrin concentration levels in the milk peaked on days 2 or 3 and then gradually decreased to day 14m returning to the level before the administration of LFH. It appears that administration of LFH may have a therapeutic effect when infused into the quarters of cows with subclinical mastitis.
In summary, a series of this study revealed that the functional characteristics of neutrophils in dairy cows around parturition. The various biological functions of alpha- tocopherol, Se, biotin and zinc on dairy cows and on experimental conditions were determined in seeking the biological modifiers for peripartum dairy cows. alpha- tocopherol and Se were administered to dairy cows, and their changes in leukocytes were monitored. Infusion of LF and LFH into mammary glands of dairy cows was examined for studying the host responses in the dairy cows. Less
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