驗認創新的p110δ 和Btk 基因的突變/多型性及分析其衍生蛋白質特性來闡明他們在原發性B細胞免疫缺陷的致病

此計劃的目的為尋找在未知原因的原發性B 細胞免疫缺陷病童的致病機轉是否為p110δ 基因突變所造成。但因根據外國資料, 原發 性B 細胞免疫缺陷的病童大部(約85%)為 Btk 基因突變所引起, 所以應須先行檢查Btk 基因； 不過, 因為目前台灣尚未有方便且可 靠的分子生物學方法來在原發B 細胞免疫缺 陷病童中確定診斷為XLA 疾病﹐也無XLA 病童的Btk 基因突變型本土資料。在國科會 補助下(NSC91-2314-13-002-189/NSC92- 2314-B-002-211) ﹐ 我們已建立用genomic DNA-PCR and direct sequencing 的方法來從 患有原發B 細胞免疫缺陷疾病的病童中篩檢 及確定其Btk 基因突變位置及多型式從而來 證實診斷為XLA 疾病。目前, 從所篩檢病童 中, 已成功確立4 個病童其Btk 基因突變位 置及型式以及其家族的基因篩檢。 同時, 亦 已建立用RT-PCR and direct sequencing 的方 法來篩檢在Btk 基因檢查確定無突變但仍不 知原因的原發B 細胞免疫缺陷病童的PI3 kinase p110δ基因是否有多型性及突變以證 實p110δ基因的突變可能是某些未知原因的 原發性B 細胞免疫缺陷的致病機轉。目前, 在3 位病人發現其p110δ基因有一整段 Exon14 刪除。這段被刪除的基因位於PI3 kinase 的accessory domain, 它在PI3 和 PI4- kinase 是conserved 的,其功能為受脢質的呈 現。This study is to explore the possibility that some patients with defects in B-cell immunodeficiency of unknown etiology might have mutations in PI3 kinase p110δ. Although, according to the foreign data, the majority of primary B-cell immunodeficiency (about 85%) is due to Btk mutation. We need to exclude the possibility of Btk gene mutation before we check the p110δgene. However, at present, there is still no convenient and reliable molecular method for definite diagnosis of XLA in patients with primary B-cell immunodeficiency in Taiwan. There is no Btk gene mutation and polymorphism data for Taiwanese XLA patients, either. Under this grant support of NSC (NSC91-2314-13-002- 189/NSC92-2314-B-002-211), we have established the methods to screen the patients with primary B-cell defect by genomic DNAPCR and direct sequencing analysis to identify the Btk gene mutations and then establish the diagnosis of the XLA patients. Until now, we have already successfully identified 4 patients of primary B-cell immunodeficiency with Btk gene mutation and screened their family members. We also have established the RTPCR and direct sequencing methods to screen the potential p110 δ gene polymorphism and mutation in the remaining primary B-cell immunodeficiency patients without Btk mutations. Until now, we have identified three patient with 2 different sizes of his 3rd PCR amplified fragment. After cloning and comparison with human p110δsequence, the nucleotides 2,007-2,150 were completely missed in the shorter band. These missed nucleotides coded for the entire Exon 14(inreading frame deletion). The missed Exon 14 is located in PI3K accessory domain which is conserved in all PI3 and PI4-kinase with the function for substrate presentation

TEL-JAK2 致癌基因在血癌生成機轉的訊息傳遞路徑

JAK/STAT 在血液細胞的訊息傳遞路徑中扮演著非 常重要的角色。藉由JAK/STAT 使cytokine 的訊 息能藉由刺激細胞膜上的receptor 一步步地傳達 到細胞核內以啟動基因的表達。此路徑傳遞血液 細胞絕大部分的生長和分化訊息。目前文獻顯示 所有須與cytokine receptor superfamily 結合 的cytokine 都能活化JAK tyrosine kinase family。 因為JAK/STAT 路徑在血液細胞訊息傳遞路徑中扮 演著非常重要的角色，所以推論JAK/STAT 路徑亦 應在血癌的生成機轉中佔重要的角色。愈來愈多 的證據支持此學說，最近TEL-JAK2 致癌基因在三 個血癌病人細胞中被發現。此致癌基因能使細胞 變性轉型的能力是來自於藉由TEL 的PNT domain 使JAK2 的kinase domains 彼此結合 (oligomerization)而持續保有JAK2 kinase 的活 性。 TEL-JAK2 的致癌生成機轉, 主要來自於其kinase domain 上的酪氨酸基被持續磷酸化。尋找TELJAK2 的下游受脢質蛋白, 對了解癌症的生成機轉 之謎, 是很重要的. 我們已clone hTEL-mJAK2 , 且證明TEL-JAK2 的持續保有酪氨酸激脢的特性, 能使 Ba/F3 細胞株由cytokine-dependent 轉型 成cytokine-independent. 而且我們利用TELJAK2 的持續保有酪氨酸激脢的特性, 發現一個持 續被酪氨酸磷酸化的受脢質蛋白, 只存在於已轉 型的Ba/F3/ TEL-JAK2 細胞, 但不出現在未轉型 的Ba/F3/ TEL-JAK2 細胞. 目前正進而研究它的 生物特性, 以了解血癌的生成機轉.The JAK/STAT (Janus protein tyrosine kinase/Signal Transducers and Activators of Transcription) pathway has emerged as a major signal transduction mechanism in hematopoietic system, linking cell surface receptors on the membrane to transcriptional events in the nucleus. This pathway plays critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. It was demonstrated that all the cytokines that utilize receptors of the cytokine receptor superfamily were capable of activating members of the JAK tyrosine kinase family. The importance of the JAK/STAT pathway in cytokine signaling for the hematopoietic cells suggests a potential role in the pathogenesis of hematological malignancies. Some evidences have supported this hypothesis. The TEL-Jak2 fusion oncogene has recently been found in 3 leukemia patients. The oligomerization by the PNT domain of TEL leads to constitutive activation ( phosphorylation) of the Jak2 tyrosine kinase domain, which is necessary for cellular transformation. The constitutive tyrosine phosphorylation of the tyrosine kinase domain of TEL-Jak2 plays the critical role for the leukemogenesis. To try to find the downstream substrate of the TEL-Jak2 is very important for the understanding the signal transduction pathway for the leukemogenesis. We have constructed the hTELmJAK2 oncogene including the human TEL-specific oligomerization domain and the catalytic domain of murine JAK2. And our data also showed the TELinduced oligomerization of TEL-JAK2 resulted in the constitutive activation of its tyrosine kinase activity and conferred cytokine (IL-3)-independent proliferation to the IL-3-dependent Ba/F3 hematopoietic cell line. We have also identified one strongly constitutively tyrosine-phosphorylated protein in the Ba/F3/TEL-JAK2 cells which is not found in the Ba/F3 parent cells by using the character of the constitutively activated catalytic activity of TEL-JAK2. We are studying the biological character of this down-stream substrate of TEL-JAK2 oncogene to elucidate the signal transduction pathway for leukemogenesis in the hematopoietic system

闡明創新的 p110delta 和Btk 基因在原發性B細胞免疫缺陷的致病機轉角色

此計劃的目的為尋找在未知原因的原發性B 細胞免疫缺陷病童的致病機轉是否為p110δ 基因突變所造成。但因根據外國資料, 原發 性B 細胞免疫缺陷的病童大部(約85%)為 Btk 基因突變所引起, 所以應須先行檢查Btk 基因； 不過, 因為目前台灣尚未有方便且可 靠的分子生物學方法來在原發B 細胞免疫缺 陷病童中確定診斷為XLA 疾病﹐也無XLA 病童的Btk 基因突變型本土資料。在國科會 補助下(NSC91-2314-13-002-189)﹐我們已建 立用genomic DNA-PCR and direct sequencing 的方法來從患有原發B 細胞免疫缺陷疾病的 病童中篩檢及確定其Btk 基因突變位置及多 型式從而來證實診斷為XLA 疾病。目前, 從 所篩檢病童中, 已成功確立4 個病童其Btk 基因突變位置及型式以及其家族的基因篩 檢。 同時, 亦已建立用RT-PCR and direct sequencing 的方法來篩檢在Btk 基因檢查確 定無突變但仍不知原因的原發B 細胞免疫缺 陷病童的PI3 kinase p110δ基因是否有多型 性及突變以證實p110δ基因的突變可能是某 些未知原因的原發性B 細胞免疫缺陷的致病 機轉。目前, 在1 位病人發現其p110δ基因 有一整段Exon14 刪除。這段被刪除的基因 位於PI3 kinase 的accessory domain, 它在 PI3 和 PI4-kinase 是conserved 的,其功能為受 脢質的呈現。This study is to explore the possibility that some patients with defects in B-cell immunodeficiency of unknown etiology might have mutations in PI3 kinase p110δ. Although, according to the foreign data, the majority of primary B-cell immunodeficiency (about 85%) is due to Btk mutation. We need to exclude the possibility of Btk gene mutation before we check the p110δgene. However, at present, there is still no convenient and reliable molecular method for definite diagnosis of XLA in patients with primary B-cell immunodeficiency in Taiwan. There is no Btk gene mutation and polymorphism data for Taiwanese XLA patients, either. Under this grant support of NSC (NSC91-2314-13-002- 189), we have established the methods to screen the patients with primary B-cell defect by genomic DNA-PCR and direct sequencing analysis to identify the Btk gene mutations and then establish the diagnosis of the XLA patients. Until now, we have already successfully identified 4 patients of primary B-cell immunodeficiency with Btk gene mutation and screened their family members. We also have established the RT-PCR and direct sequencing methods to screen the potential p110 δ gene polymorphism and mutation in the remaining primary B-cell immunodeficiency patients without Btk mutations. Until now, we have identified one patient with 2 different sizes of his 3rd PCR amplified fragment. After cloning and comparison with human p110δsequence, the nucleotides 2,007-2,150 were completely missed in the shorter band. These missed nucleotides coded for the entire Exon 14(inreading frame deletion). The missed Exon 14 is located in PI3K accessory domain which is conserved in all PI3 and PI4-kinase with the function for substrate presentation

以樹突細胞為主的免疫療法治療在小鼠的殘餘血癌疾病模式(1/2)

我們的初步資料顯示﹐腫瘤抗原-pulsed 的樹狀細胞能夠引起專一性及保護性的抗血 癌效果﹐並且能夠明顯的延長有植入小鼠RL male 1 血癌細胞株的BALB/c 小鼠的存活 時間。根據以上研究資料﹐我們提出此研究計劃來測試以樹狀細胞為主的免疫療法﹐ 用來作為臨床化學療法後的殘餘腫瘤的輔助治療﹐以期避免腫瘤的再復發。 在今年﹐我們已建立小鼠血癌疾病模式（圖一）﹐並藉由化學治療來治療已建立起 明顯的血癌疾病﹐使其達到緩解（圖二）﹐並觀察其會復發與否（圖三）﹐以研究由 骨髓細胞所衍生的樹狀細胞所引發的腫瘤專一性免疫反應。我們將藉由檢定各種體外 的免疫反應和體內的抗殘餘血癌效果﹐以評估各種的pulsed(TA 或TA/adeno-IL12)及非 pulsed 的樹狀細胞的效用﹐及最佳的疫苗施打方案。Our preliminary results have demonstrated that vaccination with tumor antigen(TA)-pulsed DCs could induce a specific protective anti-leukemia effect and significantly prolonged survival of BALB/c mice that were challenged with murine RL male 1 leukemia. Based on our preliminary data, we propose this study to test that the capacity of DC-based immunization may have clinical implications as the adjuvant treatment of residual leukemia after the traditional induction chemotherapy to prevent the recurrence of the disease. In this year, we have established the murine leukemia model (Figure 1) and by initial chemotherapy for the established frank leukemia status to induce remission (Figure 2), monitor relapse or not(Figure 3) and investigate the effect of BM-derived DCs in inducing tumor-specific immune response. We will evaluate the efficiency of various pulsed (TA or TA/adeno-IL12) or unpulsed-DCs by both in vitro immune response assays and in vivo antiresidual leukemia effect. We will also elucidate the novel optimal immunization schedule for inducing the competent anti-residual tumor immune response

Essential, Nonredundant Role for the Phosphoinositide 3-Kinase P110 Delta in Signaling by the B-Cell Receptor Complex

Many receptor and nonreceptor tyrosine kinases activate phosphoinositide 3-kinases (P13Ks). To assess the role of the delta isoform of the p110 catalytic subunit of P13Ks, we derived enzyme-deficient mice. The mice are viable but have decreased numbers of mature B cells, a block in pro-B- cell differentiation, and a B1 B-cell deficiency. Both immunoglobulin M receptor-induced Ca2+ flux and proliferation in response to B-cell mitogens are attenuated. Immunoglobulin levels are decreased substantially . The ability to respond to T-cell-independent antigens is markedly reduced, and the ability to respond to T-cell- dependent antigens is completely eliminated. Germinal center formation in the spleen in response to antigen stimulation is disrupted. These results define a nonredundant signaling pathway(s) utilizing the delta isoform of p110 P13K for the development and function of B cells