Characterization of host defence capability of dairy cows during peripartum period and modulation of the immunological functions.

奶牛围产期宿主防御能力的表征和免疫功能的调节。

• 批准号: 14360192
• 负责人: NAGAHATA Hajime
• 金额: $ 5.89万
• 依托单位: RAKUNO GAKUEN UNIVERSITY, SCHOOL OF VETERINARY MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360192/
• 关键词: BOVINE NEUTROPHILS; DAIRY COW; FUNCTIONAL CHARACTERISTICS; HOST DEFENCE; MICRONUTRIENTS; MODULATION; PERIPARTUM; 炎症性サイトカイン; CR3-FcγR; 細胞内情報伝達; ラクトフェリン; サイトカイン

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 … More 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

本研究旨在探讨围产期奶牛白细胞功能的变化及其对宿主防御能力的影响。本研究的主要结果如下：与对照奶牛相比，奶牛分娩前后中性粒细胞的光依赖性化学发光反应显著降低。在聚集的牛IgG刺激的中性粒细胞中发现了类似的变化。这些结果与先前的研究结果一致，中性粒细胞的功能在围产期受损。通过测定正常牛和3头白细胞黏附缺陷牛的中性粒细胞裂解物对荧光肽底物Ac-DEVD-MCA的蛋白水解，以评估正常和cd18缺陷中性粒细胞的凋亡情况。OPZ刺激后，对照中性粒细胞的凋亡率随培养时间的增加而显著增加，而cd18缺失中性粒细胞的凋亡率未见明显增加。聚集牛igg诱导的对照中性粒细胞的凋亡与cd18缺陷中性粒细胞的凋亡无显著差异。在cd18缺陷的牛中性粒细胞中发现了细胞凋亡的延迟，这似乎与[Ca2+]I信号传导降低、细胞表面膜联蛋白V表达减少以及裂解物中caspase 3活性降低密切相关。这些结果提示围产期奶牛中性粒细胞功能活性的降低可能与细胞凋亡率的变化有关。体外激酶实验显示Akt不能催化p47磷酸化。fMLP刺激后cPKC （PKC的经典同型）和PKC- δ的激活依赖于P13K（磷酸肌苷3-激酶）。P13K抑制剂在不影响酪氨酸磷酸化的情况下降低了磷脂酶C γ 2的活化。P13K通过控制二酰基甘油依赖的PKCs调控NADPH氧化酶组分p47phox的-磷酸化，而不是Akt。通过p38和MAPK检测到中性粒细胞酪氨酸磷酸化降低，在奶牛分娩前后也发现。这一结果提示p38和MAPK在牛中性粒细胞中的作用与其信号转导及其机制有关。补体受体3型（CR3）与IgG-Fc受体对中性粒细胞的共刺激作用在围产期奶牛中得到证实，但酪氨酸产物检测到的功能反应与对照奶牛相比有所减弱。微量元素硒（Se）、生物素、锌、铜（Cu）对奶牛宿主防御能力有重要影响。测定了围产期奶牛血清中α -生育酚和硒的浓度。奶牛产前1 ~ 2周血清α -生育酚浓度显著低于产前3 ~ 4周血清α -生育酚浓度。奶牛分娩前后血清硒含量无明显变化。在给牛α -生育酚6 h后，α -生育酚浓度开始升高，在给药后48 h达到峰值（607.2 ug/dl）。硒浓度急剧升高（88.3+8.3 ppb）， 24 h后迅速下降，至给硒3周后逐渐下降。给药后48 h，大鼠白细胞中硒浓度显著升高，至给药后2 ~ 3周显著降低。监测白细胞中硒浓度的变化。本研究中发现的变化似乎与给药后白细胞功能增加的时间有关。生物素是酶的辅助因子，在糖生成、脂肪酸合成和氨基酸分解代谢中起关键作用。研究了饲粮中添加生物素对奶牛血清生物素水平和足底角物理性能的影响。添加生物素10个月后，奶牛血清生物素水平显著高于对照组。添加生物素后，奶牛的鞋底质量明显变硬。研究了特色化铜螯合剂曲安汀对LEC大鼠体内铜积累和DNA链断裂的影响。经曲恩汀处理的LEC大鼠DNA损伤细胞数量明显减少。这一结果表明，铜在宿主防御中的重要作用与中性粒细胞产生的超氧化物的清除有关。乳铁蛋白（LF）是一种存在于牛奶和粘膜表面的铁结合抗菌蛋白，被认为在免疫前宿主防御中发挥作用。对正常奶牛和乳腺炎奶牛的乳中LF浓度进行测定，以确定乳中LF浓度是否受到奶牛年龄、泌乳阶段、乳体细胞数量和病原体存在的影响。亚临床乳腺炎、哺乳后期、病原菌为金黄色葡萄球菌和环境链球菌的奶牛乳中LF浓度显著升高，SCC数量增加的奶牛乳中LF浓度显著升高。研究了乳铁蛋白水解物（LFH）对亚临床乳腺炎奶牛乳腺的影响，以了解乳铁蛋白和LFH是否具有免疫调节作用。对25头亚临床乳腺炎奶牛中35 / 4的奶牛注射7 ml LFH制剂。平均体细胞计数（SCC）在给药后第1天达到峰值，在第7、14和21天与给药前比较，SCC计数显著下降。乳汁中乳铁蛋白的平均浓度在第2天和第3天达到峰值，然后逐渐下降到第14m天，恢复到给药前的水平。结果表明，将LFH注入亚临床乳腺炎的奶牛体内可能具有治疗效果。综上所述，本研究揭示了奶牛分娩前后中性粒细胞的功能特征。通过测定α -生育酚、硒、生物素和锌对奶牛的各种生物学功能，并在实验条件下寻找围产期奶牛的生物修饰剂。饲喂α -生育酚和硒，监测奶牛白细胞的变化。研究了乳牛乳腺输注LF和LFH对宿主的影响。少

项目成果

Physiological changes in the concentration of biotin in the serum and milk and in the physical properties of the claw horn in Holstein cows.

荷斯坦奶牛血清和牛奶中生物素浓度以及爪角物理特性的生理变化。

• 发表时间: 2003
• 期刊: Vet.Res.Commun. 27
• 作者: Higuchi;H.;Maeda;T.;Kawai;K.;Kasamatsu;M.;Nagahata;H.
• 通讯作者: H.

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