噬血症候群(hemophagocytic syndrome)是病毒感染如 EB 病毒，禽流感，登革熱病毒，和 SARS 冠狀病毒感染的主要死亡原因之一。噬血症候群的病人通常會伴隨出現發燒、黃疸、肝脾腫大、全血球減少(pancytopenia)，和血液凝固病變(coagulopathy)。在實驗室的診斷上，病人有溶血、膽色素增加、貧血、三酸甘油脂增加、纖維蛋白原(fibrinogen)下降等。基於我們實驗室和其他研究人員的研究，噬血症候群的致病機轉是病毒感染後，造成毒殺 T 淋巴球(cytotoxic T lymphocyte)的過度免疫反應，使得 Th1 細胞激素(cytokines)的分泌增加，進而造成巨噬細胞(macrophage)活化，而導致噬血症候群。巨噬細胞可能經由一個非任意的或專一性的過程活化，而去吞噬紅血球。在 EB 病毒感染相關的疾病已經有許多的動物模式被建立，但是 EB 病毒感染所引發的噬血症候群，卻一直沒有一個很好的動物模式來幫助解答問題。2001 年，Hayashi 等人在兔子身上以 herpesvirus papio (HVP)感染，建立了 EB 病毒相關的噬血症候群動物模式，我們實驗室過去利用此模式發現紅血球被吞噬以及造成快速死亡與病人或動物血清中出現紅血球的自體抗體(auto-antibody)有關。抗體標定的紅血球經由 Fc 受體辨識而造成血球被吞噬，因此紅血球自體抗體的產生可能是造成血球吞噬的重要原因。所以在本研究當中，主要想要去鑑定紅血球膜上的此一專一性自體抗原。我們建立了病毒噬血症候群的動物模式後，利用免疫沉澱法/二維(2D)蛋白質分析以及噬菌體呈現技術(phage display)兩種方法來鑑定特殊抗原。在蛋白質體學研究當中，我們首先從六隻感染 HVP 兔子體內分離紅血球膜蛋白，然後再使用未感染前的血清進行去除背景，接著使用已經感染後的血清進行免疫沉澱。結果顯示，發現感染後的紅血球膜蛋白和感染前正常紅血球膜蛋白的表現結果不一樣，但是不管正常紅血球膜蛋白或是感染病毒的紅血球膜蛋白經由免疫沉澱法，所表現的均為兔子紅血球表面的特殊抗原，因此更進一步將所獲得的產物再以二維電泳做蛋白質鑑定。在二維電泳的結果中，發現使用感染後的紅血球膜蛋白和未發病前的紅血球膜蛋白的膠體上有不同的點，將這些點取出進行蛋白質鑑定。我們同時也使用噬菌體呈現技術(phage display)去鑑定自體抗體所辨識的專一性胜肽序列。初步蛋白質鑑定結果顯示取出進行的點為纖維蛋白原(fibrinogen)、免疫球蛋白kappa鏈、白蛋白(albumin)、免疫球蛋白重鏈，但真正的特殊抗原為何仍待進一步研究來澄清。

　　Hemophagocytic syndrome (HPS) is a virus-associated clinical disease and may occur in several virus infections such as avian influenza, dengue fever, SARS-CoV infection, and EBV infection. HPS is characterized by fever, hepatosplenomegaly, cytopenia and coagulopathy. Serum chemistry findings may suggest hemolysis, with hyperbilirubinemia and elevation of lactate dehydrogenase. Serum fibrinogen is typically low, and there may be disseminated intravascular coagulation. Histopathologically, hemophagocytosis is seen in bone marrow, spleen, and lymph nodes. Clinical and biological manifestations result from the secretion of huge amounts of cytokines by activated T cells and macrophages. Activated macrophages may engulf erythrocytes, leukocytes, and platelets, their precursors, and cellular fragments. The production of autoantibodies might be important for hemophagocytosis. In our preliminary studies, we have shown that activated macrophages were able to specifically engulf antibody-coated RBC but not uncoated RBC. In a rabbit model of EBV-associated HS, the presence of autoantibodies against RBC is an intial event for subsequent phagocytosis of RBC and rapid mortality. In this study, we aim to identify the specific autoantigens on the RBC membrane. Two different approaches were used, i.e., co-immunoprecipitation/2D proteomic analysis and phage display. For the proteomic approach, RBC membrane proteins from six HVP-infected rabbits were firstly applied in the Western blotting by using either pre-immune and post-infected serum. Our results revealed that the presence of autoantigens increased with HVP-infection. The same protein lysates will then be used for co-immunprecipitation with post-infected serum, and the resulted products will be resolved by 2-D electrophoresis and protein identification. Our results revealed that autoantigens may be the fibrinogen, Ig kappa chain C region, albumin and Ig heavy chain region. For the identification of specific peptide sequences recognized by the autoantibodies, we performed the phage display experiments with the same post-infected antiserum. Phages carrying specific peptide sequences for the autoantibody will be selected and identified after repeated biopanning. The identified autoantigen would be very useful in the understanding of the pathogenesis of EBV-associated hemophagocytic syndrome.

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蕭冠中. Mechanism of Hemophagocytosis in Epstein-Barr virus Associated Hemophagocytic Syndrome.成功大學微生物及免疫學研究所碩士論文 2004.