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

血小板活化因子(PAF)是一种由多种细胞合成的磷脂，参与炎症和过敏反应，被认为是一种重要的生物介质，参与了多种临床疾病，包括哮喘、血栓形成、循环障碍和移植物排斥反应。从生产细胞释放到血液中的PAF被血浆/血清PAF乙酰水解酶(PAF-AH)迅速水解为无活性的溶解PAF。我们发现了一些日本家庭的血清PAF-AH缺乏，这似乎是通过常染色体隐性遗传传播的。哮喘患儿PAF乙酰水解酶缺乏的概率在症状较重(中、重度)时显著增高(P&lt；0.01)。因此，我们认为血清PAF乙酰水解酶缺陷可能是导致AS患儿严重呼吸道症状的遗传因素之一。此外，为了阐明PAF-AH缺陷是否是…异常所致为了了解更多的血清酶蛋白和什么样的细胞产生PAF-AH，我们的目的是建立PAF-AH酶蛋白的免疫分析方法。首先，通过磷钨酸沉淀、Tween20增溶、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柱层析，从人混合血清中纯化PAF-AH为单一条带。接下来，我们从兔体内制备了PAF-AH抗血清，并将上述纯化的PAF-AH注射到其中。该抗血清不仅与纯化的PAF-AH有交叉反应，而且在免疫电泳法上也能与不含PAF-AH活性的脂蛋白发生交叉反应。因此，为了获得高度特异性的PAF-AH抗体，我们正在用PAF-AH免疫的BALB/C小鼠的脾细胞与骨髓瘤细胞融合的杂交瘤细胞制备单抗。另一方面，我们阐明了(1)川崎病患者血清PAF-AH活性显著高于健康儿童，(2)系统性红斑狼疮(SLE)患者在病程中发生变化，(3)大鼠开始水浸应激导致胃部病变的发生，并伴随着血清PAF-AH活性的显著升高。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PAF-AH缺乏的人血清中鉴定了携带蛋白，该血清提取了人中性粒细胞产生的PAF。较少

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