Clonic granulomaous disease (CGD) and its deficients in bactricidal activity-Basic research for gene therapy for CGD-

克隆性肉芽肿病（CGD）及其杀菌活性缺陷-CGD基因治疗的基础研究-

• 批准号: 09660311
• 负责人: INANAMI Osamu
• 金额: $ 2.11万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660311/
• 关键词: clonic granulomaous disease (CGD); neutrophil; superoxide; NADPH oxidase; gene therapy; phagocytosis; signal transduction; kinases; CGD; シグナル伝達; NADPHオキシターゼ; リン酸化酵素; 生体防御; NADPHオキシダーゼ; 電子スピン共鳴法; ケミルミネッセンス; p47phox; p67phox

This project was performed to clarify the signal transduction mechanisms for NADPH oxidase activation and phagocytotis by using normal neutrophil, human clonic granulomaous disease (CGD) and bovine leukocyte adhesion deficiency (BLAD) which were genetic deficient in p47phox and beta2-integrin CR3 corresponding to the receptor of complement iC3b, respectively The various reagents to inhibit NADPH oxidase-related signal transduction were used for this purpose. We found that inhibitors for protein kinase C (PKC), phosphatidyl inositol 3 kinase (P1 3-kinase) and p38 mitogen-activated protein kinase (p38 MAPK) were dose-dependently inhibited superoxide generation from serum-opsonized zymosan (s-OZ)-stimulated neutrophils from BLAD and normal calves, although the stimulation of BLAD neutrophils with s-OZ brought about lower generation of superoxide than that of normal neutrophil. These results indicated that the lack of beta2-integrin CR3 did not influence signal transduction pathways of NAD … More PH oxidase but reduced superoxide production from NADPH oxidase. This reduced NADPH oxidase activity in BLAD was partially recovered by transfusion of CD 18-positive granulocytes to disease animal. Furthermore, P1 3-kinase and p38 MAPK but not PKC are shown to be required for phagocytotic activity in normal neutrophil. Concerning the intracellular mechanisms of NADPH oxidase activation, the p47phox, one component of NADPH oxidase. is known to be phosphorylated extensively on serines that are located among its C-terminal amino acids and this phosphorylation is a trigger for the activation of NADPH oxidase. By using site-directed mutagenesis of p47phox and p47phox-deficien B cells from human clonic granulomaous disease (COD), it was showed that the phosphorylation of serines 3031304, 359(370 and possibly serine 379 must take place in order to activate the oxidase. These results seem to be important in not only understanding the signal transduction mechanism of NADPH oxidase activity but also development of therapy for BLAD and p47phox-deficien COD such as granulocyte transfusion and gene therapy. Less

本课题利用正常中性粒细胞、人克隆性肉芽肿病（CGD）和牛白细胞粘附缺陷症（BLAD）细胞，研究NADPH氧化酶激活和吞噬细胞炎的信号转导机制，这些细胞均为补体iC 3b受体p47 phox和β 2-整合素CR 3基因缺陷型，抑制NADPH氧化酶相关信号转导的各种试剂用于此目的。我们发现蛋白激酶C（PKC）、磷脂酰肌醇3激酶（P13-kinase）和p38丝裂原活化蛋白激酶（p38 MAPK）的抑制剂可剂量依赖性地抑制血清调理酵母多糖（s-OZ）刺激的BLAD和正常小牛中性粒细胞产生超氧化物，尽管s-OZ刺激BLAD中性粒细胞产生的超氧化物低于正常中性粒细胞。这些结果表明，β 2-整合素CR 3的缺失并不影响NAD的信号转导途径 ...更多信息 PH氧化酶，但减少NADPH氧化酶的超氧化物产生。通过将CD 18阳性粒细胞输注给患病动物，BLAD中这种降低的NADPH氧化酶活性部分恢复。此外，P13-激酶和p38 MAPK而不是PKC被证明是正常中性粒细胞吞噬活性所需的。关于NADPH氧化酶激活的细胞内机制，p47 phox是NADPH氧化酶的一种组分。已知在位于其C-末端氨基酸之间的丝氨酸上广泛磷酸化，并且这种磷酸化是NADPH氧化酶活化的触发剂。通过对人克隆性肉芽肿病（COD）p47 phox和p47 phox缺陷型B细胞进行定点突变，表明丝氨酸303、304、359、370和可能的丝氨酸379的磷酸化必须发生以激活氧化酶。这些结果不仅对了解NADPH氧化酶活性的信号转导机制，而且对BLAD和p47 phox缺陷型COD的治疗如粒细胞输注和基因治疗的发展具有重要意义。少

项目成果

Osamu Inanami: "H_2O_2-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells" Antioxidant and Redox Signaling. in press. (1999)

Osamu Inanami：“中国仓鼠 V79 细胞中磷脂酰肌醇 3-激酶调节的 H_2O_2 诱导的 SAPK/JNK 激活”抗氧化和氧化还原信号传导。

Inanami,O.et al.: "Lipid peroxides and antioxidants in serum of neonatal calves." Am J Vet Res.(in press). (1999)

Inanami,O.等人：“新生犊牛血清中的脂质过氧化物和抗氧化剂。”

Osamu Inanami: "Oral administration of (-) catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil. Free Radical Research" Free Radical Research. 29・4. 359-365 (1998)

Osamu Inanami：“口服（-）儿茶素可以防止沙鼠缺血再灌注引起的神经元死亡。自由基研究”29・4（1998）。

Hajime Nagahata: "Survival of transfused CD18-positive granulocytes and their chemiluminescent response in a heifer with leukocyte adhesion deficiency" Journal of Veterinary Medical Science. 60・2. 261-262 (1998)

Hajime Nagahata：“白细胞粘附缺陷的小母牛中输注的 CD18 阳性粒细胞的存活及其化学发光反应”《兽医医学杂志》60・2（1998 年）。

Osamu Inanami: "Attenuation of caspase 3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells. International Journal of Radiation Biology, 75 (2) , 155-163" International Journal of Radiation Biology. in press. (1999)

Osamu Inanami：“Trolox 对 X 射线照射的 MOLT-4 细胞进行后处理可减弱 caspase 3 依赖性细胞凋亡。国际放射生物学杂志，75 (2) , 155-163”国际放射生物学杂志。