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学一致的临床和实验室特征，具有多种激发抗原，包括病毒，化学品和药物。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，有时在血细胞计数正常化后数年，演变为其他血液学疾病，如阵发性睡眠性血红蛋白尿症（PNH），其来源于造血干细胞的克隆。在临床研究中，我们已经完成了对122例严重疾病患者的长期随访分析，这些患者接受了抗胸腺细胞球蛋白（ATG）和环孢菌素（CSA）治疗，其反应率约为70%，但复发风险较高，发生骨髓增生异常或染色体异常的风险较低。一个令人惊讶和重要的结果是，不仅存在血液学应答，而且在治疗后3或6个月时血细胞计数改善的稳健性之间存在强的、高度显著的相关性，这预测了生存和进展（但不是复发）的结果。在我们的高剂量环磷酰胺和ATG的随机试验的长期结果分析中，我们观察到复发，细胞遗传学变化，并持续异常糖基磷酸肌醇锚定蛋白缺陷细胞发生在两个武器，表明更有毒的化疗方案不能预防或减轻这些临床并发症。为了诱导这种免疫介导疾病的耐受性，我们制定了一项方案，在早期治疗中加入新型免疫抑制药物吗替麦考酚酯，并延迟环孢霉素的给药。到目前为止，已经招募了54名患者; 3个月时血液学缓解率为62%，6个月时为74%。这些值与我们用ATG加环孢素治疗重型再生障碍性贫血的经验相当。1名患者死亡，4名患者复发;研究需要成熟，以确定这种方法与标准方案相比的优势。其他临床研究已经描述了AA与阵发性睡眠性血红蛋白尿（PNH）的关系：几乎一半的AA患者同时存在这些情况，并且在疾病表现时存在扩增克隆的证据，而不是作为晚期事件。在诊断和治疗后确实会发生细胞遗传学异常，但结局差异很大，与难治性全血细胞减少症、白血病进展和7号单体死亡相比，8号三体对免疫抑制治疗具有持续的反应性或依赖性，生存率较好。我们的实验室研究主要集中在AA的免疫病理生理学、病毒抗原的鉴定和晚期克隆进化的机制上。病因学研究集中在AA肝炎后综合征中的未知肝炎病毒（见Z 01 HL 02319-14 HB）。为了更好地表征免疫应答和靶抗原，我们分离并永生化了辅助性和细胞毒性T细胞克隆，并根据TCR偏斜的模式确定了它们的T细胞受体库。发现特异性V-β TCR在患者体内和疾病患者中占主导地位，随着成功的免疫抑制而下降，并且在正常T细胞中检测不到。通过对患者样本进行免疫表型分析，我们测量到与疾病状态相关的效应细胞毒性淋巴细胞（CD 28阴性CD 8细胞）数量增加。相反，AA的特征在于调节性NK-T细胞的重要群体的显著缺乏，在许多其他自身免疫性疾病中也可以看到这种减少。其他研究已经检查了ATG的作用机制：对造血的体外刺激作用已显示为非特异性。负责晚期克隆性疾病，PNH和骨髓增生异常（MDS）的机制的实验室调查，使用敏感的流式细胞术和荧光原位杂交（FISH）检测。对于PNH，已经观察到从患者骨髓获得的正常和缺陷型CD 34细胞的显著差异：正常CD 34细胞由于与Fas表达相关的凋亡而具有较差的体外存活，这与体内损伤一致，可能来自免疫攻击。这些结果与我们发现的组织相容性抗原HLA-DR 2与骨髓衰竭综合征中存在扩展的PNH克隆之间存在强相关性的结果是互补的。其他正在进行的PNH研究包括对PNH患者中糖基磷酸肌醇连接蛋白从正常输血红细胞向缺陷红细胞和白色血细胞体内转移的临床观察。与正常细胞相比，PNH中GPI相关蛋白降解途径的研究正在进行中，包括朊病毒蛋白的正常类似物PrPc的研究。三体8，而不是单体7，也与造血相关的免疫异常有关，因为细胞遗传学异常细胞比正常细胞更可能表达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 clinical research, we have completed analyses of long-term follow-up of a large cohort of 122 patients with severe disease who were treated with antithymocyte globulin (ATG) and cyclosporine (CSA), which shows a response rate of about 70% but with a high risk of relapse and a more modest risk of development of myelodysplasia or chromosomal abnormalities. A surprising and important result is that there is a strong, highly significant correlation between not only the presence of a hematologic response but the robustness of blood count improvement at 3 or 6 months post-treatment, which is predictive of outcome, both survival and evolution (but not relapse). In analysis of long-term results from our randomized trial of high dose cyclophosphamide and ATG, we observe relapse, cytogenetic changes, and persistence of abnormal glyocsylphosphoinositol-anchored protein-deficient cells occurring in both arms, indicating that the more toxic chemotherapeutic regimen does not prevent or alleviate these clinical complications. In an effort to induce tolerance in this immune-mediated disease, we have instituted a protocol that incorporates the novel immunosuppressive drug mycophenolate mofetil in early treatment and delays adminsstration of cyclosporine. To date, 54 patients have been enrolled; at 3 months the hematologic response rate is 62%, and at 6 months the rate is 74%. These values are equivalent to our experience in the treatment of severe aplastic anemia with ATG plus cyclosporine. One patient has died and 4 have relapsed; the study will need to mature in order to determine the advantage of this approach compared to standard regimens. Other clinical studies have characterized the relationship of AA to paroxysmal nocturnal hemoglobinuria (PNH): these are concurrent in almost half of AA patients,and evidence of an expanded clone is present at disease presentation, not as a late event. Cytogenetic abnormalities do occur after diagnosis and treatment but have widely different outcomes, with continued responsiveness or dependence on immunosuppressive therapies and good survival in trisomy 8 compared to refractory pancytopenia, leukemic evolution, and death in monosomy 7. Our laboratory studies have focused on the immune pathophysiology of AA, identification of a viral antigen, and the mechanism of late clonal evolution. Studies of etiology focus on an unknown hepatitis virus in the post-hepatitis AA syndrome (see Z01 HL 02319-14 HB). In efforts to better characterize the immune response and target antigens, we have isolated and immortalized helper and cytotoxic T cell clones and determined their T cell receptor repertoire based on the pattern of TCR skewing. Specific V-beta TCR were found to dominate within a patient and among patients with disease, declined with successful immunosuppression, and were not detectable in normal T cells. By immunophenotyping of patient samples, we have measured increased numbers of effector cytotoxic lymphocytes (CD28-negative CD8 cells) which correlate with disease status. Conversely, AA is characterized by a marked deficiency of an important population of regulatory NK-T cells, a decrease seen also in many other autoimmune disease. Other studies have examined the mechanism of action of ATG: in vitro stimulatory effects on hematopoiesis have been shown to nonspecific. Laboratory investigations of the mechanisms responsible for late clonal disease, both PNH and myelodysplasia (MDS), have used sensitive flow cytometric and fluorescent in situ hybridization (FISH) assays. For PNH, a marked difference in normal and deficient CD34 cells obtained from patient bone marrow has been observed: the normal CD34 cells have poor in vitro survival due to apoptosis associated with Fas expression, consistent with in vivo damage, probably from immune attack. These results are complementary to our finding of a strong association between a histocompatability antigen, HLA-DR2, and the presence of an expanded PNH clone in bone marrow failure syndromes. Additional ongoing studies of PNH include clinical observations of in vivo transfer of glycosylphosphoinositol-linked proteins from normal transfused erythrocytes to deficient red and white blood cells in PNH patients. Examination of pathways of GPI-linked protein degradation in PNH compared to normal cells are ongoing, including studies of PrPc, the normal analogue of the prion protein. 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.