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。在临床研究中，我们完成了122例重症患者的长期随访分析，这些患者接受抗胸腺细胞球蛋白（ATG）和环孢素（CSA）治疗，有效率约为70%，但复发风险高，发生骨髓发育不良或染色体异常的风险较低。一个令人惊讶和重要的结果是，不仅血液学反应的存在，而且在治疗后3或6个月的血细胞计数改善的稳健性之间存在很强的、高度显著的相关性，这是预后的预测，包括生存和进化（但不是复发）。在我们的高剂量环磷酰胺和ATG随机试验的长期结果分析中，我们观察到在两组患者中都出现了复发、细胞遗传学改变和异常糖基磷酸肌醇锚定蛋白缺陷细胞的持续存在，这表明毒性更强的化疗方案并不能预防或减轻这些临床并发症。为了在这种免疫介导的疾病中诱导耐受性，我们制定了一项方案，将新型免疫抑制药物霉酚酸酯纳入早期治疗和延迟给予环孢素。迄今为止，已有54名患者入组；3个月时血液反应率为62%，6个月时为74%。这些数值相当于我们用ATG加环孢素治疗严重再生障碍性贫血的经验。1名患者死亡，4名复发；这项研究需要成熟，以确定这种方法与标准方案相比的优势。其他临床研究已经证实了AA与阵发性夜间血红蛋白尿（PNH）的关系：这两种关系在几乎一半的AA患者中同时存在，并且扩大克隆的证据在疾病出现时就存在，而不是作为晚期事件出现。在诊断和治疗后确实会发生细胞遗传学异常，但其结果有很大的不同，与难治性全血细胞减少症、白血病演变和单体7死亡相比，三体8患者的持续反应性或对免疫抑制治疗的依赖性和良好的生存率。我们的实验室研究主要集中在AA的免疫病理生理，病毒抗原的鉴定，以及晚期克隆进化的机制。病因学研究集中于一种未知的肝炎病毒在AA型肝炎后综合征（见Z01 HL 02319-14 HB）。为了更好地表征免疫反应和靶抗原，我们分离和永生化了辅助性和细胞毒性T细胞克隆，并根据TCR倾斜的模式确定了它们的T细胞受体库。特异性v - β TCR在患者和疾病患者中占主导地位，随着免疫抑制的成功而下降，并且在正常T细胞中未被检测到。通过对患者样本进行免疫分型，我们测量了与疾病状态相关的效应细胞毒性淋巴细胞（cd28阴性CD8细胞）数量的增加。相反，AA的特点是显著缺乏重要的调节性NK-T细胞群，在许多其他自身免疫性疾病中也可见到这种减少。其他研究已经检验了ATG的作用机制：体外对造血的刺激作用已被证明是非特异性的。实验室研究导致晚期克隆性疾病的机制，包括PNH和骨髓异常增生（MDS），使用了敏感的流式细胞术和荧光原位杂交（FISH）测定。对于PNH，从患者骨髓中获得的正常CD34细胞和缺陷CD34细胞存在显著差异：正常CD34细胞由于Fas表达相关的凋亡而体外存活较差，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-DR2与骨髓衰竭综合征中扩增PNH克隆的存在之间的强烈关联是互补的。其他正在进行的PNH研究包括PNH患者体内糖基磷酸肌醇连接蛋白从正常输注的红细胞转移到缺陷红细胞和白细胞的临床观察。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PrPc的研究，PrPc是朊病毒蛋白的正常类似物。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 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.