Pathogenesis And Treatment Of Aplastic Anemia

再生障碍性贫血的发病机制和治疗

• 批准号: 6966935
• 负责人: NEAL S YOUNG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966935
• 关键词: T cell receptor; aplastic anemia; autoimmune disorder; blood disorder chemotherapy; bone marrow disorder; cell population study; cyclophosphamide; cyclosporines; cytotoxic T lymphocyte; fluorescent in situ hybridization; helper T lymphocyte; hematopoietic stem cells; hepatitis virus; histocompatibility typing; human subject; human therapy evaluation; immunohematology; immunosuppression; immunotherapy; interferons; natural killer cells; pathologic process; patient oriented research; relapse /recurrence; tissue /cell culture

Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with stem cell clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). Accrual has been terminated for a research study of 100 patients with severe AA on presentation that added mycophenolate mofetil, as well as delayed addition of cyclosporine, to standard ATG, in an effort to induce tolerance. Hematologic response rates at 3 and 6 months were comparable to those achieved by standard treatment; relapse, evolution, and survival analyses are underway. Current protocols randomize previously untreated severe AA patients to either ATG plus cyclosporine for two years (long course immunosuppression) or to ATG, cyclosporine and rapamycin for six months (more intensive early treatment). For refractory disease, patients are randomized to treatment with either rabbit ATG or CAMPATH-1, a monoclonal antibody to T cells. Daclizumab, a monoclonal antibody that binds to the interleukin-2 receptor and has relative specificity for activated T cells, has been successful in about 30% of patients with moderate AA, and is now being applied to early relapse of severe AA (without profound neutropenia) and in single lineage marrow failure sates, pure red cell aplasia and amegakaryocytic thrombocytopenic purpura. In the laboratory, efforts have concentrated on the incitement of AA by an unknown virus (see Z01 HL 02319-14 HB), the aberrant immune response, and the problem of clonal evolution. In efforts to more specifically characterize the immune response, we have utilized the methods of flow cytometry and spectratyping to determine expansion of V-beta TCR families of T cells and skewing of CDR3 expression within expanded families, as indicators of antigen-driven clonal T cell proliferation. We are exploiting CD8 cytotoxic lymphocyte oligoclonal expansions detection of individual CDR3 sequences, evidence of an antigen-driven immune response, to monitor patients during and after theapy, especially as predictors of hematologic relapse. We have employed DNA chip analysis to examine CD34 hematopoietic cells, both normal and from patients with marrow failure syndromes, first for AA and subsequently for cytogetically defined myelodysplastic syndromes that commonly evolve from immune marrow failure (trisomy 8 and monosomy 7) and the clonal disease paroxysmal nocturnal hemoglobinuria (PNH), closely associated with AA. In PNH, paired primary CD34 cells from individual patients show marked upregulation of immune and apoptosis genes in the abnormal putative PIG-A-negative population; the PNH clone pattern was close to normal, and additionally there was little difference in the transciptome of CD34 cells obtained from patients with predominantly hemolytic compared to marrow failure clinical variants. In MDS, specifically up- and down-regulated genes have been observed for CD34 cells from patients with well-defined cytogenetic abnormalities in myelodysplasia . Trisomy 8, but not monosomy 7, also is associated with immune abnormalities related to hematopoiesis, as cytogenetically abnormal cells are more likely than normal cells to express Fas and to be apoptotic. Aneuploid cells in trisomy 8 are apototic, as they express Fas and annexin. The pattern of T cell usage in trisomy 8 resembles AA. Isolated CD8 T cell clones show preferential activity against cytogenetically abnormal cells, suggesting that they are reactive to a partially transformed clone of hematopoietic cells. Monosomy 7, a syndrome usually associated with aa fatal course due to refractory pancytopenia or acute leukemia, chromosomally aberrrant cells are abnormally sensitive to G-CSF in vitro, and high concentrations of this cytokine appear to select for pre-existing minor populations of monosomy 7 cells only detectable by the sensitive fluorescent in situ hybridization methodology. Molecular mechanisms for both trisomy 8 and monosomy 7 have been explored in the laboratory. For trisomy 8, oligoclonal T cell responses appear to be directed to the aneuploid clone, but cells are salvaged from fully undergoing cell death as result of blocked apoptosis; a putative antigen, suggested by the micoarray studies, the gene product of WT1, is overexpressed in this form of MDS and may target the abnormal clone. In monosomy 7, which has been associated with prolonged neutropenia and treatment with granulocyte colony stimulating factor (G-CSF), a truncated version of the G-CSF receptor, which signals proliferation by not differentiation, is present in the aneuploid cells and may be selected under conditions of high endogenous G-CSF or adminstered cytokine. In a mouse model of AA based on infusion of parental lymph node cells into F1 recipients, a prominent "innocent bystander" mechanism of hematopoietic stem cell killing has been demonstrated in co-transplantation experiments, likely explaining some of the extraordinary potency of limited numbers of activated T cells in human marrow failure diseases. We have implicated genes of the telomerase repair complex, which acts to preserve telomere length at mitosis in mammalian cells, in late onset AA and MDS. Two genese, TERC (for the RNA component) and DKC1, cause the constitutional AA disease called dyskeratosis congenita. In studies of kindreds with probands presenting with apparently acquired AA, without physical stigmata but with other family members showing mild hematologic abnormalities, we identified two novel mutations in TERC in all affected members. Despite only mild anemia, erythrocyte macrocytosis, or thromboycotopenia, marrows were strikingly hypocellular and showed low content of CD34 cells and functional colony-forming progenitor cells. One family member had been misdiagnosed with MDS at an advanced age, and both he and the proband showed good therapeutic response to instition of androgens (frequently effective in inherited marrow failure syndromes). Multiple different mutations in the TERT gene for the telomerase enzyme itself, not previously associated with disease in humans, in patients with onset of marrow failure in middle-age. In an AA additional patient with a deletion in TERC, and a strikingly positive family history of marrow disease, telomere length was normal but telomere single strand overhang was markedly eroded; telomere overhang shortening may precede telomere shortening. Histocompatability antigens and cytokine promoter polymorphisms have been suggested as immune system risk factors for AA; we now propose TERC and TERT abnormalities as responsible, through acclerated telomere shortening, for greatly diminished stem cell compartments and therefore hematopoietic system risk factors for marrow failure. In vitro experiments also suggest that telomerase activity may be modulated by androgens, providing a mechanism for the utility of male hormones in constitutional marrow failure states. Finally, studies of mitochondrial DNA sequence of CD34 cells in normal individuals and in MDS have been extended to single cell assays for CD34 cells, T cells, B cells and granulocytes; this methodology may prove useful in tracking stem cells, measuring the mammalian mutation rate, and to detect minimal residual malignant disease.

性障碍性贫血（AA）和其他类型的骨髓衰竭具有临床和实验室特征，与自身免疫性病理生理学一致，具有多样性的推定煽动性抗原，包括病毒，化学物质，医疗药物和肿瘤抗原。无论其具体的病因如何，大多数患者在免疫抑制疗法后会以血液学改善反应。 AA的一个重要临床特征是它与干细胞克隆血液学疾病的关联，尤其是阵发性夜间夜间血红蛋白尿（PNH）和骨髓增生异常综合征（MDS）。已终止了对100名严重AA患者的研究研究，从而增加了霉酚酸酯的莫菲蒂，并延迟添加了环孢菌素的ATG，以诱导耐受性。 3和6个月时的血液学反应率与标准治疗获得的血液反应率相当；复发，进化和生存分析正在进行中。当前的方案将先前未经治疗的严重AA患者随机进行ATG加环孢霉素（长期免疫抑制）或ATG，环孢菌素和雷帕霉素六个月（更强化的早期治疗）。对于难治性疾病，患者被随机地用兔ATG或CAMPATH-1（一种与T细胞的单克隆抗体）治疗。 Daclizumab是一种与白介素-2受体结合并具有活化T细胞具有相对特异性的单克隆抗体，已在大约30％的中度AA患者中成功，现在已适用于严重AA的早期复发（无深层中性粒细胞减少），并且在单位层次失败的术语中，单个层次失败的杂物和单一的红细胞疗法，纯净的Amegre phrors phrors prinies toth amegren thits Amegren totire totire tithity phrorcy prohrecy totire totire totire totire totire totility。在实验室中，努力集中在未知病毒（请参阅Z01 HL 02319-14 HB），异常免疫反应以及克隆进化的问题上。为了更具体地表征免疫反应，我们利用了流式细胞仪和光谱型的方法来确定T细胞的V-beta TCR家族的扩展以及在扩展家族中CDR3表达的偏斜，作为抗原驱动的克隆T细胞增殖的指标。我们正在利用CD8的细胞毒性淋巴细胞寡聚膨胀检测单个CDR3序列（抗原驱动的免疫反应的证据），以监测屈曲期间和之后的患者，尤其是作为血液学复发的预测指标。 We have employed DNA chip analysis to examine CD34 hematopoietic cells, both normal and from patients with marrow failure syndromes, first for AA and subsequently for cytogetically defined myelodysplastic syndromes that commonly evolve from immune marrow failure (trisomy 8 and monosomy 7) and the clonal disease paroxysmal nocturnal hemoglobinuria (PNH), closely associated with aa。在PNH中，来自个别患者的成对的原代CD34细胞在异常假定的猪a阴性种群中表现出明显的免疫和凋亡基因上调。 PNH克隆模式接近正常状态，此外，与骨髓衰竭临床变异相比，从主要溶血性患者获得的CD34细胞的转盘差异很小。在MDS中，已经观察到来自骨髓增生性良好的细胞遗传学异常患者的CD34细胞的特异性上调基因。三体术8而不是单肌也不是与造血相关的免疫异常有关的，因为细胞遗传学异常细胞比正常细胞更有可能表达FAS并凋亡。三体构体中的非整倍体细胞是尖体，因为它们表达FAS和膜联蛋白。第8三体中T细胞使用的模式类似于AA。分离的CD8 T细胞克隆表现出与细胞遗传异常细胞的优先活性，这表明它们对造血细胞的部分转化的克隆有反应。单肌7是一种综合征，通常与AA致命病程有关，这是由于难治性全周年症或急性白血病而导致的，染色体异常细胞对体外对G-CSF的敏感性异常，并且这种细胞因子的高浓度似乎仅用于可在较小的7个细胞中选择较小的单细胞群体，可在效率下效率地进行效率。在实验室中探索了三体和单肌第7三体性的分子机制。对于第8三体性，寡克隆T细胞反应似乎针对非倍型克隆，但由于细胞凋亡的阻滞而被完全经历细胞死亡的细胞挽救。 MicoArray研究提出的一种推定的抗原是WT1的基因产物，以这种MDS形式过表达，并可能针对异常克隆。在单肌第7中，它与延长的中性粒细胞减少症和粒细胞菌落刺激因子（G-CSF）的治疗相关，G-CSF受体的截断版本是在非倍倍型细胞中存在的，而不是通过分化来表示，并且可以在甲状腺倍倍倍型细胞中选择，并且可以在内源性G-CSF高CSF的cysinsf cytokine cytokine cytokine中选择。在基于亲本淋巴结细胞输注F1受体的AA的小鼠模型中，在共同移植实验中已经证明了造血干细胞杀死的突出的“无辜旁观者”机制，这可能解释了人类Marrow衰竭疾病中有限数量的活性T细胞的一些非凡效力。我们牵涉到端粒酶修复复合物的基因，该复合物在哺乳动物细胞中的有丝分裂时保持端粒长度，在晚期AA和MDS中。两个Genese TERC（用于RNA成分）和DKC1引起宪法AA病，称为Dyskeratosis congenita。在对具有显然获得的AA的概率的亲属研究中，没有物理污名，但其他家庭成员表现出轻度的血液学异常，我们确定了所有受影响成员的TERC中的两个新型突变。尽管仅轻度贫血，红细胞大细胞增多症或血小板性骨质减少症，但骨髓却非常低细胞，并且表现出较低的CD34细胞含量和功能性集群形成的祖细胞。一个家庭成员在高龄误诊了MDS，他和该证券对雄激素的插入表现出良好的治疗反应（通常在遗传的骨髓衰竭综合症中有效）。端粒酶本身的TERT基因中的多个不同的突变，以前与人类疾病相关，中期骨髓衰竭的患者。在TERC中删除的AA额外患者中，骨髓疾病的阳性家族病史，端粒长度正常，但端粒单链悬垂已明显侵蚀。端粒悬空可能是端粒缩短之前的。组织相容性抗原和细胞因子启动子多态性已被认为是AA的免疫系统风险因素。现在，我们建议通过加速端粒缩短，将TERC和TERT异常作为负责，以大大减少干细胞室，从而导致骨髓衰竭的造血系统危险因素。体外实验还表明，端粒酶活性可能由雄激素调节，为雄性激素在宪法骨髓衰竭状态下的实用性提供了一种机制。最后，对正常个体和MDS中CD34细胞的线粒体DNA序列的研究已扩展到CD34细胞，T细胞，B细胞和颗粒细胞的单细胞分析。该方法可能证明可用于跟踪干细胞，测量哺乳动物突变率并检测最小残留恶性疾病。