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.

再生障碍性贫血（AA）和其他类型的骨髓衰竭具有与自身免疫病理生理学一致的临床和实验室特征，具有多种假定的刺激抗原，包括病毒、化学物质、药物和肿瘤抗原。无论其具体病因是什么，大多数患者在免疫抑制治疗后都会出现血液学改善的情况。 AA 的一项重要临床特征是其与干细胞克隆性血液疾病的关联，尤其是阵发性睡眠性血红蛋白尿 (PNH) 和骨髓增生异常综合征 (MDS)。一项针对 100 名严重 AA 患者的研究已终止，该研究在标准 ATG 中添加吗替麦考酚酯以及延迟添加环孢菌素，以诱导耐受。 3 个月和 6 个月时的血液学缓解率与标准治疗达到的效果相当；复发、进化和生存分析正在进行中。目前的方案将以前未经治疗的严重 AA 患者随机分配到 ATG 加环孢素两年（长期免疫抑制）或 ATG、环孢素和雷帕霉素六个月（更强化的早期治疗）。对于难治性疾病，患者被随机分配接受兔 ATG 或 CAMPATH-1（一种 T 细胞单克隆抗体）治疗。 Daclizumab 是一种单克隆抗体，与白细胞介素 2 受体结合，对活化的 T 细胞具有相对特异性，已成功治疗约 30% 的中度 AA 患者，目前正应用于重度 AA 的早期复发（无严重中性粒细胞减少症）以及单系骨髓衰竭、纯红细胞再生障碍性贫血和无巨核细胞血小板减少性紫癜。在实验室中，工作重点集中在未知病毒引发的 AA（参见 Z01 HL 02319-14 HB）、异常的免疫反应和克隆进化问题上。为了更具体地表征免疫反应，我们利用流式细胞术和谱分型方法来确定 T 细胞 V-β TCR 家族的扩展和扩展家族内 CDR3 表达的偏差，作为抗原驱动的克隆 T 细胞增殖的指标。我们正在利用 CD8 细胞毒性淋巴细胞寡克隆扩增检测单个 CDR3 序列（抗原驱动的免疫反应的证据）来监测治疗期间和治疗后的患者，特别是作为血液学复发的预测因子。我们采用 DNA 芯片分析来检查正常和骨髓衰竭综合征患者的 CD34 造血细胞，首先检查 AA，然后检查细胞遗传学定义的骨髓增生异常综合征，这些综合征通常由免疫性骨髓衰竭（8 三体性和 7 单体性）和克隆性疾病阵发性睡眠性血红蛋白尿症 (PNH) 演变而来，与与骨髓衰竭密切相关 AA。在 PNH 中，来自个体患者的配对原代 CD34+ 细胞在异常的假定 PIG-A 阴性群体中显示出免疫和凋亡基因的显着上调； PNH 克隆模式接近正常，此外，与骨髓衰竭临床变异相比，从主要溶血患者获得的 CD34+ 细胞转录组几乎没有差异。在 MDS 中，已观察到来自患有明确的骨髓增生异常细胞遗传学异常的患者的 CD34+ 细胞的特定上调和下调基因。 8 三体（而非 7 单体）也与造血相关的免疫异常有关，因为细胞遗传学异常的细胞比正常细胞更有可能表达 Fas 并发生凋亡。 8 三体中的非整倍体细胞具有凋亡性，因为它们表达 Fas 和膜联蛋白。 8 三体中 T 细胞的使用模式类似于 AA。分离的 CD8 T 细胞克隆显示出针对细胞遗传学异常细胞的优先活性，表明它们对部分转化的造血细胞克隆有反应。 7 号单体是一种综合征，通常与难治性全血细胞减少症或急性白血病导致的致命病程相关，染色体异常细胞在体外对 G-CSF 异常敏感，高浓度的这种细胞因子似乎会选择先前存在的少数 7 号单体细胞，只能通过灵敏的荧光原位杂交方法检测到。实验室已探索了 8 三体和 7 单体的分子机制。对于 8 三体性，寡克隆 T 细胞反应似乎针对非整倍体克隆，但细胞因细胞凋亡受阻而免于完全死亡；微阵列研究表明，一种假定的抗原是 WT1 的基因产物，在这种形式的 MDS 中过度表达，并且可能针对异常克隆。在与长期中性粒细胞减少症和粒细胞集落刺激因子 (G-CSF) 治疗相关的单体 7 中，G-CSF 受体的截短形式存在于非整倍体细胞中，通过非分化发出增殖信号，并且可能在高内源性 G-CSF 或施用细胞因子的条件下被选择。在基于将亲代淋巴结细胞输注到 F1 受体中的 AA 小鼠模型中，共移植实验证明了一种显着的造血干细胞杀伤“无辜旁观者”机制，这可能解释了有限数量的活化 T 细胞在人类骨髓衰竭疾病中的一些非凡效力。我们发现端粒酶修复复合物的基因与晚发型 AA 和 MDS 中的哺乳动物细胞有丝分裂时的端粒长度有关。 TERC（RNA 成分）和 DKC1 这两个基因会导致称为先天性角化不良的体质性 AA 疾病。在对先证者的亲属进行的研究中，先证者表现出明显的获得性 AA，没有身体耻辱，但其他家庭成员表现出轻度血液学异常，我们在所有受影响的成员中发现了 TERC 的两种新突变。尽管仅有轻度贫血、红细胞巨细胞增多或血小板减少症，但骨髓细胞明显减少，并且 CD34+ 细胞和功能性集落形成祖细胞含量较低。一名家庭成员在高龄时被误诊为骨髓增生异常综合征，他和先证者都对雄激素的使用表现出良好的治疗反应（通常对遗传性骨髓衰竭综合征有效）。在中年出现骨髓衰竭的患者中，端粒酶本身的 TERT 基因中存在多种不同的突变，这些突变以前与人类疾病无关。在另一名 AA 患者中，TERC 缺失，且骨髓疾病家族史显着阳性，端粒长度正常，但端粒单链突出端明显被侵蚀；端粒突出缩短可能先于端粒缩短。组织相容性抗原和细胞因子启动子多态性已被认为是 AA 的免疫系统危险因素；我们现在提出，TERC 和 TERT 异常通过加速端粒缩短，导致干细胞区室大大减少，从而成为造血系统骨髓衰竭的危险因素。体外实验还表明，端粒酶活性可能受到雄激素的调节，这为在体质性骨髓衰竭状态下使用雄性激素提供了一种机制。最后，对正常个体和 MDS 中 CD34+ 细胞线粒体 DNA 序列的研究已扩展到 CD34+ 细胞、T 细胞、B 细胞和粒细胞的单细胞测定；这种方法可能有助于追踪干细胞、测量哺乳动物突变率以及检测微小残留恶性疾病。