Pathogenesis And Treatment Of Aplastic Anemia

再生障碍性贫血的发病机制和治疗

• 批准号: 6683979
• 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/6683979
• 关键词: T cell receptor; aplastic anemia; autoimmune disorder; blood disorder chemotherapy; bone marrow disorder; cell population study; cyclophosphamide; cytotoxic T lymphocyte; fluorescent in situ hybridization; helper T lymphocyte; hematopoietic stem cells; hepatitis virus; histocompatibility typing; human subject; human therapy evaluation; immunohematology; immunotherapy; interferons; mycophenolate mofetil; natural killer cells; pathologic process; patient oriented research; prions; 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 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.

再生障碍性贫血(AA)和其他类型的骨髓衰竭具有符合自身免疫病理生理学的临床和实验室特征，具有多种激发抗原，包括病毒、化学物质和药物。无论其具体病因如何，大多数患者在接受免疫抑制治疗后，血液学有所改善。再生障碍性贫血的一个重要临床特征是，有时在血细胞计数正常化数年后，它会演变为其他血液疾病，如发作性睡眠性血红蛋白尿(PNH)，这些疾病源于造血干细胞克隆。在临床上，我们采用了一种方案，在标准的抗胸腺细胞球蛋白中加入霉酚酸酯，以及延迟添加环孢素，以努力诱导出现严重再生障碍性贫血患者的耐受性。随着应计几乎完成，3个月和6个月的血液学应答率可与标准治疗相媲美；复发、演变和生存分析将需要更长的评估期。在实验室中，工作集中在未知病毒(见Z01 HL 02319-14 HB)刺激再生障碍性贫血、异常免疫反应以及克隆进化为其他血液疾病的问题上。为了更好地表征免疫反应，我们利用流式细胞术和分型的方法来确定T细胞的V-βTCR家族的扩张和扩增家族中CDR3表达的偏斜，作为抗原驱动的克隆性T细胞增殖的指标。特别是在CD8细胞毒性淋巴细胞群体中，我们发现很大比例的再生障碍性贫血和PNH患者表现出寡克隆性T细胞增殖，我们已经在这些家庭中确定了单个CDR3序列。CDR3的分子测定与免疫抑制前后的疾病状态相关，但其模式因使用ATG或环磷酰胺而不同。CDR3克隆型被认为是具有相似人类白细胞抗原背景的患者免疫反应的标志。最终，CDR3感兴趣区域可能被整合到重组人TCR基因中，并转导到永生化效应细胞中，用于确定抗原特异性。我们还使用DNA芯片分析来检测CD34造血细胞，包括正常和来自骨髓衰竭综合征患者的细胞。来自再生障碍性贫血患者的有限数量的CD34细胞已经被汇集并预先扩增了cRNA。对于这种疾病，靶细胞显示与免疫系统激活、细胞凋亡和应激相关的基因表达增强，而增殖基因表达减少。虽然正常表型和PNH表型的配对细胞株在转录水平上几乎没有差异，但来自单个患者的配对原代CD34细胞在假定为PIG-A阴性的异常群体中免疫和凋亡基因显著上调；这些结果与我们之前使用类似细胞群体的细胞培养数据一致。在骨髓发育不良中，已经观察到来自骨髓发育不良患者明确的细胞遗传学异常的CD34细胞表达上调和下调的基因。8号三体，而不是7号单体，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。