Pathogenesis And Treatment Of Aplastic Anemia

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一致的临床和实验室特征，具有多种推定的刺激性抗原，包括病毒、化学物质、药物和肿瘤抗原。无论其具体病因如何，大多数患者在免疫抑制治疗后血液学改善。AA的一个重要临床特征是其与干细胞克隆血液病，特别是阵发性夜间血红蛋白尿（PNH）和骨髓增生异常综合征（MDS）的相关性。一项针对100名重度AA患者的研究已经终止，该患者在标准ATG中添加霉酚酸酯，并延迟添加环孢素，以诱导耐受性。3个月和6个月的血液学缓解率与标准治疗相当；复发、进化和生存分析正在进行中。目前的方案将先前未治疗的严重AA患者随机分配到ATG +环孢素2年（长疗程免疫抑制）或ATG +环孢素+雷帕霉素6个月（更强化的早期治疗）。对于难治性疾病，患者随机接受兔ATG或campaign -1（一种针对T细胞的单克隆抗体）治疗。Daclizumab是一种与白细胞介素-2受体结合的单克隆抗体，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（无深度中性粒细胞减少症）的早期复发和单系骨髓衰竭状态、纯红细胞发育不全和单核细胞血小板减少性紫癜。在实验室，研究工作集中在未知病毒（见Z01 HL 02319-14 HB）对AA的激发、异常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的方法来确定T细胞v - β TCR家族的扩增和扩增家族中CDR3表达的偏态，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，这是抗原驱动免疫反应的证据，用于监测治疗期间和治疗后的患者，特别是作为血液病复发的预测指标。我们采用DNA芯片分析检测了正常和骨髓衰竭综合征患者的CD34造血细胞，首先用于AA，随后用于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（8三体和7单体）和克隆性疾病阵发性夜间血红蛋白尿（PNH）演变而来，与AA密切相关。在PNH中，来自个体患者的配对原代CD34细胞在假定的异常猪- a阴性人群中显示出免疫和凋亡基因的显著上调；PNH克隆模式接近正常，此外，从以溶血为主的患者中获得的CD34细胞的转录组与骨髓衰竭临床变体相比差异不大。在MDS中，在骨髓增生异常患者的CD34细胞中观察到特异性的上调和下调基因。8三体，而不是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容易表达Fas和凋亡。8号三体的非整倍体细胞凋亡，因为它们表达Fas和膜联蛋白。8号三体的T细胞使用模式类似于AA。分离的CD8 T细胞克隆对细胞遗传学异常细胞表现出优先活性，表明它们对部分转化的造血细胞克隆有反应。7号单体是一种通常与难治性全血细胞减少症或急性白血病导致的致命过程相关的综合征，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选择了仅通过敏感的荧光原位杂交方法检测到的已有的少量7号单体细胞群。8号三体和7号单体的分子机制已经在实验室进行了探索。对于8三体，寡克隆T细胞反应似乎针对非整倍体克隆，但由于细胞凋亡受阻，细胞从完全经历细胞死亡中被挽救出来；微阵列研究表明，一种假定的抗原，即WT1的基因产物，在这种形式的MDS中过度表达，并可能靶向异常克隆。在单核细胞7中，它与中性粒细胞减少和粒细胞集落刺激因子（G-CSF）治疗有关，G-CSF受体的截断版本，通过不分化信号增殖，存在于非整倍体细胞中，可能在高内源性G-CSF或给药细胞因子的条件下被选择。在将亲代淋巴结细胞输注到F1受体的小鼠AA模型中，一种突出的“无辜旁观者”造血干细胞杀伤机制已在共移植实验中得到证实，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功效。我们发现，端粒酶修复复合体的基因在哺乳动物细胞有丝分裂时起着保持端粒长度的作用，与晚发型AA和MDS有关。两个基因，TERC （RNA成分）和DKC1，导致被称为先天性角化不良的构造性AA疾病。在先证表现为明显获得性AA的家族的研究中，没有身体上的耻辱，但其他家族成员表现出轻微的血液学异常，我们在所有受影响的成员中发现了两个新的TERC突变。尽管只有轻度贫血、红细胞巨噬症或血栓减少症，骨髓细胞明显减少，CD34细胞和功能集落形成祖细胞含量低。一位家庭成员在高龄时被误诊为MDS，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）。在中年骨髓衰竭患者中，端粒酶本身的TERT基因中存在多种不同的突变，以前与人类疾病无关。在另一名TERC缺失且骨髓疾病家族史显著阳性的AA患者中，端粒长度正常，但端粒单链悬垂明显受损；端粒悬垂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异常是负责任的，通过加速端粒缩短，大大减少了干细胞区室，因此造血系统的危险因素骨髓衰竭。体外实验还表明，端粒酶活性可能受到雄激素的调节，这为雄性激素在体质骨髓衰竭状态下的效用提供了一种机制。最后，对正常人和MDS患者CD34细胞线粒体DNA序列的研究已经扩展到CD34细胞、T细胞、B细胞和粒细胞的单细胞检测；这种方法在追踪干细胞、测量哺乳动物突变率和检测微小残留恶性疾病方面可能是有用的。

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.