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，具有多种刺激性抗原，包括病毒、化学物质和药物。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演变，有时在血球计数正常化数年后，演变为其他血液学疾病，如阵发性夜间血红蛋白尿（PNH），这源于造血干细胞的克隆。在临床上，我们采用了一种方案，将霉酚酸酯和环孢素延迟添加到标准抗胸腺细胞球蛋白中，以努力诱导严重再生障碍性贫血患者的耐受性。随着累积几乎完全，3个月和6个月的血液学缓解率与标准治疗的效果相当；复发、进化和生存分析将需要更长的评估周期。在实验室，研究工作集中在一种未知病毒（见Z01 HL 02319-14 HB）引起再生障碍性贫血、异常免疫反应以及克隆进化到其他血液病的问题上。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。特别是在CD8细胞毒性淋巴细胞群中，我们发现大部分再生障碍性贫血和PNH患者表现出低克隆T细胞扩增，并且我们已经确定了这些家族中的单个CDR3序列。CDR3的分子测量与免疫抑制前和免疫抑制后的疾病状态相关，但模式不同取决于是否使用ATG或环磷酰胺。在HLA背景相似的患者中寻找CDR3克隆型作为免疫反应的标记。最终，CDR3感兴趣的区域可能被整合到重组人TCR基因中，然后转导到永生化效应细胞中，以确定抗原特异性。我们还使用DNA芯片分析来检测正常和骨髓衰竭综合征患者的CD34造血细胞。来自再生障碍性贫血病例的有限数量的CD34细胞已被汇集并预先扩增了cRNA。对于这种疾病，靶细胞显示出与免疫系统激活、凋亡和应激相关的基因表达增强的遗传程序，而增殖基因表达降低。虽然正常和PNH表型配对的细胞系在转录组上的总体差异不大，但来自个体患者的配对原代CD34细胞在异常推定的猪- a阴性群体中显示出免疫和凋亡基因的显著上调；这些结果与我们以前使用类似细胞群的细胞培养数据一致。在骨髓发育不良中，在骨髓发育不良中有明确的细胞遗传学异常的患者的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。

Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of inciting antigens, including viruses, chemicals, and drugs. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its evolution, sometimes years after normalization of blood counts, to other hematologic diseases such as paroxysmal nocturnal hemoglobinuria (PNH), which derive from clones of hematopoietic stem cells. In the clinic, we have employed a regimen that incorporates mycophenolate mofetil, as well as delayed addition of cyclosporine, to standard antithymocyte globulin, in an effort to induce tolerance in patients with severe aplastic anemia on presentation. With accrual almost complete, hematologic response rates at 3 and 6 months are comparable to those achieved by standard treatment; relapse, evolution, and survival analyses will require longer periods of evaluation. In the laboratory, efforts have concentrated on the incitement of aplastic anemia by an unknown virus (see Z01 HL 02319-14 HB), the aberrant immune response, and the problem of clonal evolution to other hematologic diseases. 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. Expecially within the CD8 cytotoxic lymphocyte population, we found that a large proportion of aplastic anemia and PNH patients show oligoclonal T cell expansion, and we have identified individual CDR3 sequences within these families. Molecular measurment of CDR3 correlates with disease status pre- and post-immunosuppression, but the patterns differ depending on whether ATG or cyclosphosphamide is used. CDR3 clonotypes are sought as markers of the immune response in patients of similar HLA background. Ultimately, CDR3 regions of interest may be incorporated into recombinant human TCR genes and in turn transduced into immortalized effector cells, for determiantion of antigen specificity. We also have employed DNA chip analysis to examine CD34 hematopoietic cells, both normal and from patients with marrow failure syndromes. Limited numbers of CD34 cells from aplastic anemia cases have been pooled and cRNA pre-amplified. For this disease, target cells show evidence an enhanced genetic program of expression of genes related to immune system activation, apoptosis, and stress, and decreased expression of proliferation genes. While paired cell lines of normal and PNH phenotype show little overall difference in transcriptomes, paired primary CD34 cells from individual patients show marked upregulation of immune and apoptosis genes in the abnormal putative PIG-A-negative population; these results are consistent with our previous cell culture data using similar cell populations. In myelodysplasia, 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.