Immunological study on human serum PAF(platelet-activating factor) acetylhydrolase deficiency

血小板活化因子（PAF）是一种由多种参与炎症和过敏反应的细胞合成的磷脂，被认为是一种重要的生物介质，参与许多不同的临床疾病，包括哮喘，血栓形成，循环系统疾病和移植排斥反应。PAF从生产细胞释放到血液中，经血浆/血清PAF乙酰水解酶（PAF- ah）迅速水解为无活性的溶血opaf。我们发现一些日本家庭存在血清PAF-AH缺乏症，其表现为常染色体隐性遗传。哮喘患儿出现PAF乙酰水解酶缺乏的概率在症状严重组（中度和重度）中显著增高（P<0.01）。根据这些观察结果，我们提出血清PAF乙酰水解酶缺乏可能是导致哮喘儿童出现严重呼吸道症状的一个遗传因素。此外，为了阐明PAF-AH缺乏症是否由血清酶蛋白异常引起，以及哪种细胞产生血清PAF-AH，我们旨在建立PAF-AH酶蛋白的免疫测定方法。首先，通过磷钨酸沉淀、Tween 20溶解、DEAE Cellulofine AM离子交换柱、Q- sepharose柱、TSK-Gel HW-60高效液相色谱柱、羟基磷灰石高效液相色谱柱、Asahipak GS-510高效液相色谱柱和Mono Q FPLC柱等层析方法纯化人血清PAF-AH，使其呈单带电泳。接下来，我们从兔子身上制备了PAF-AH抗血清，并将上述纯化的PAF-AH注射到兔体内。该抗血清不仅与纯化的PAF-AH具有交叉反应性，而且在免疫电泳上与不含PAF-AH活性的脂蛋白也具有交叉反应性。因此，为了获得对PAF-AH具有高度特异性的抗体，我们正在利用经PAF-AH免疫的BALB/C小鼠脾细胞与骨髓瘤细胞融合形成的杂交瘤制备单克隆抗体。另一方面，我们阐明了(1)川萨基病患者血清PAF-AH活性显著高于健康儿童，(2)系统性红斑狼疮（SLE）患者血清PAF-AH活性在疾病过程中发生变化，(3)大鼠水浸应激的发生引起胃病变的发生，血清PAF-AH活性显著升高。此外，尽管有报道称中性粒细胞明显保留了它们合成的PAF，但我们在人PAF- ah缺陷血清中发现了一种载体蛋白，它可以提取人中性粒细胞产生的PAF。少

Platelet-activating factor(PAF) is a phospholipid synthesized from many cell types involved in inflammatory and allergic reactions, and is thought to be a significant biological mediator involved in many diverse clinical conditions including asthma, thrombosis, circulatory disorders and graft rejection. PAF, which is released from producing cells into blood, is hydrolyzed rapidly by plasma/serum PAF acetylhydrolase PAF-AH) to be inactive lysoPAF. We found some Japanese families with serum PAF-AH deficiency, which appeared to be transmitted by autosomal recessive heredity. The probability of PAF acetylhydrolase deficiency among asthmatic children was significantly higher in groups with severe symptoms (moderate and severe)(P<0.01). From these observations, we proposed that deficiency of serum PAF acetylhydrolase might be one hereditary factor leading to severe respiratory symptoms in as thmatic children.Furthermore, in order to elucidate whether PAF-AH deficiency results from abnormalit … More y of serum enzyme protein and what kind of cells produces serum PAF-AH, we aimed to develop the immunoassay for PAF-AH enzyme protein. First, we purified PAF-AH from human pooled serum to be a single band on electrophoresis through Phosphotungstic acid precipitation, solubilization with Tween 20, chromatographies using DEAE Cellulofine AM ion exchange column, Q-Sepharose column, TSK-Gel HW-60 HPLC column, Hydroxyapatite HPLC column, Asahipak GS-510 HPLC column and Mono Q FPLC column. Next, we prepared PAF-AH antiserum from rabbits into which above described purified PAF-AH was injected. This PAF-AH antiserum had cross-reactivity with not only purified PAF-AH but also lipoprotein containing no PAF-AH activity on immunoelectrophoresis. Hence, in order to obtain antibody highly specific for PAF-AH, we are in progress to prepare monoclonal antibody using the resulting hybridomas that spleen cells from BALB/C mouse immunized with PAF-AH were fused with myeloma cells.On the other hand, we elucidated that (1) serum PAF-AH activity in subjects with Kawasakis disease was significantly higher than that in healthy children, (2) that in subjects with systemic lupus erythematosus(SLE ) changed during the course of the sickness, and (3) the onset of water-immersion stress toward rat caused the development of gastric lesions associated with a significant increase in serum PAF-AH activity. Furthermore, although it has been reported that neutrophils apparently retain the PAF they synthesize, we identified the carrier protein in human PAF-AH deficient serum which drew out PAF produced in human neutrophils. Less