Development of Rapid Rvaluation Methods for Immunopotentiators of Fish.

为了建立鱼类免疫增强剂的快速评价方法，以真菌来源的β-1，3-葡聚糖和褐藻制备的海藻酸钠为免疫增强剂的模型材料，对鲤鱼的免疫增强剂的作用机理进行了分析。β-1，3-葡聚糖激活鲤鱼补体，腹腔注射β-1，3-葡聚糖可提高哺乳动物头肾吞噬细胞CR3样补体受体的表达水平。因此，补体受体表达增加所赋予的吞噬细胞吞噬活性的增强可能在提高宿主对细菌感染的抵抗力中起重要作用。在这些发现的基础上，我们分离了鲤鱼C3a片段，这将是一个很好的补体激活指示剂，并建立了鲤鱼C3a的检测方法以及受体的玫瑰花环试验。另一方面，腹腔注射海藻酸钠后，既没有观察到补体的激活，也没有观察到吞噬细胞活性的增强。相反，海藻酸钠诱导了头肾白细胞向腹膜腔的大量迁移，表明海藻酸钠刺激了驻留在腔内的白细胞释放趋化细胞因子。这些结果表明，这些多糖类免疫增强剂对鱼类宿主防御系统的增强作用有不同的机制。细胞因子的功能和免疫化学检测不适用于FISH，因为关于FISH细胞因子的数据积累很少。然而，基因表达的分析将有助于克服这些困难，因为基因表达是由免疫增强剂特异性刺激的。

To develop rapid evaluation methods of immunopotentiators in fish, the mechanism of the immunopotentiation in carp was analyzed using a fungi-derived beta-1,3-glucan and sodium alginate prepared from a brown algae as model materials of immunopotentiators. The beta-1,3-glucan activated the complement of carp and intraperitoneal administration of the beta-1,3-glucan elevated the expression level of mammalian CR3-like complement receptors on head kidney phagocytes. Therefore, the enhancement of phagocytic activity of the phagocytes which is endowed by the increase in complement receptors expression probably plays an important role in elevation of host defense against bacterial infection. On the basis of these findings, we have isolated carp C3a fragment, which would be a good indicator for complement activation, and developed the a detection method of carp C3a as well as a rosette assay of the receptors. On the other hand, neither activation of complement nor enhancement of phagocyte activity was observed after intraperitoneally injected sodium alginate. Rather, sodium alginate induced a great migration of head kidney leukocytes to the peritoneal cavity, indicating that sodium alginate stimulate the release of chemotactic cytokines from resident leukocytes in the cavity. These results show that there are distinct mechanisms underlying the enhancing effects of those polysaccharide immunopotentiators on the fish host defense system. Functional and immunochemical assays of cytokines are not applicable to fish, because few data has been accumulated on fish cytokines. However, analysis of gene-expression, which is specifically stimulated by the administration of immunopotentiator, will help to overcome the difficulties.