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。

基本信息

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

项目摘要

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

进行了上述研究的研究，以表征幼头奶牛的白细胞功能，并研究宿主防御能力的调节。这项研究的主要结果总结如下：与对照母牛相比，在分娩周围的奶牛的中性粒细胞中发现了明显改善的腔依赖性化学发光反应。在用聚集的牛IgG刺激的中性粒细胞中发现了类似的变化。这些结果与以前的发现是一致的，即嗜中性粒细胞的功能在定期期间受损。刺激性诱导的中性粒细胞的生存力，核碎裂，增加细胞内钙（[Ca2+} I）的增加（[Ca2+} I），床上膜V的表达，中性粒细胞对嗜中性粒细胞和荧光蛋白质的蛋白质的蛋白质和蛋白质的蛋白质的蛋白质和蛋白质的蛋白质酸度均为蛋白质的正常，并未达到AC-DEV。 3确定了白细胞粘附缺乏症的牛，以评估正常和CD 18缺陷性嗜中性粒细胞的凋亡。 IPZ刺激的对照中性粒细胞的凋亡率显着增加，这取决于孵育时间，而在CD 18缺陷型嗜中性粒细胞中没有明显增加凋亡。在CD 18缺陷牛中性粒细胞中证明了细胞凋亡的延迟，这似乎与通过[Ca2+] I的信号传导密切相关，在细胞表面上的膜联蛋白V表达降低，裂解物中的caspase 3活性增加。这些结果表明，外周母牛中性粒细胞的功能活性降低可能与凋亡率的变化有关。在体外激酶测定法表明AKT无法催化P47 Phox磷酸化。 FMLP刺激后的CPKC（PKC经典同种型）和PKC-DELTA的激活取决于P13K（磷酸肌醇3-激酶）。 P13K抑制剂降低了磷脂酶C伽马2的激活，而不会影响其上的酪氨酸磷酸化。 P13K通过控制二酰基甘油依赖性的t PKC，而不是Akt.中性粒细胞中抑制酪氨酸磷酸化的NADPH氧化物成分P47phox的磷酸化，并在P38和MAPK中检测到，也发现了在p38和MAPK中，也发现了在Parturition周围的奶牛中。该结果表明p38和MAPK在牛中性粒细胞中的作用与该信号转导和相关机制有关。 The costimulative effects of complement receptor type 3 (CR3) with IgG-Fc receptors on neutrophils were demonstrated in peripheral 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 defense capability in dairy cows.确定了来自围产期奶牛的血清中α-生育酚和SE的浓度。与分娩前3-4周的母牛相比，分娩前1-2周的血清中血清α-生育酚浓度显着改善。分娩前后的奶牛的血清中未发现SE浓度的显着变化。在给牛α-生育酚施用后六个小时，在给药后48小时后发现α-生育酚浓度开始增加，峰值浓度（607.2 ug/dl）。 SE的浓度大大增加（88.3+8.3 ppb），然后迅速增加到24小时，此后SE水平逐渐提高到SE给药后3周。在SE给药后48小时，白细胞中SE的浓度显着增加，直到给药后2至3周都显着增加。监测白细胞中SE浓度的变化。看来，这项研究中发现的变化与施用Se.biotin后白细胞功能增加的时代有关。Biotin充当酶的辅助因子，并在糖生成，脂肪酸合成和氨基酸分解代谢中起关键作用。评估了饮食生物素补充剂对奶牛唯一角质角的血清生物素水平和物理特性的影响。补充10个月后，生物素补充母牛中的血清生物素水平明显高于对照母牛。在补充生物素后，在奶牛中观察到了唯一的角质质量的明显困难。对铜的积累和DNA链断裂的诱导（SSB）的治疗效果在LEC大鼠中研究了DNA大鼠的铜的积累（SSB）。与Trientine一起处理了Trientine大鼠，与Trientine损坏了DNA损坏的细胞。该结果表明，Cu对宿主防御的作用在中性粒细胞产生的超氧化物的清除方面的重要性是乳糖蛋白（LF）是牛奶和粘膜表面中存在的铁结合抗菌蛋白，并且认为它在Premmune宿主防御中起着作用。测量正常母牛和奶牛的牛奶中LF浓度，以确定牛奶中的LF浓度是否受母牛年龄的影响，泌乳阶段，牛奶体细胞数量和病原体的存在。 Significantly higher LF concentrations in milk were found in cows with subclinical mastitis, in later 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乳铁蛋白和LFH是否具有免疫调节活性。将7毫升的LFH制剂注入了25头乳腺炎的25个牛中的35个四分之一。与LFH给药之前相比，在LFH给药和计数后一天，平均体细胞计数（SCC）在第7、14和21天达到峰值。牛奶中的平均乳铁蛋白浓度在第2或3天达到峰值，然后逐渐降低到14m的LFH给药之前的水平。看来，当将LFH注入具有亚临床乳腺炎的奶牛的四分之一时，LFH的给药可能具有治疗作用。总而言之，这项研究表明，在分娩周围的奶牛中，嗜中性粒细胞的功能特征。在寻找外周乳制奶牛的生物学修饰剂时，确定了奶牛上α-生育酚，SE，生物素和锌的各种生物学功能。将α-生育酚和SE施用到奶牛，并监测其白细胞的变化。检查了LF和LFH对乳制奶牛的乳腺的输注，以研究奶牛中的宿主反应。较少的