Pathogenesis And Treatment Of Aplastic Anemia

今年的年度报告既包括细胞生物科在骨髓衰竭疾病方面的工作，也包括病毒发现科的工作。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，具有多种可能的激发抗原，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与克隆性血液病相关，尤其是阵发性睡眠性血红蛋白尿(PNH)和骨髓增生异常综合征(MDS)。在临床上，研究的方向是对再障患者进行更有效的免疫抑制，并将免疫抑制方案应用于相关的骨髓衰竭综合征。在目前的临床方案中，我们的主要研究是直接比较两种商业上可获得的抗胸腺细胞球蛋白，马ATG和兔ATG；已有70多名患者入选，初步浏览数据表明，这些药物并不等同。这项研究的一个组成部分是确定小剂量环孢素是否能防止复发，这是成功的免疫抑制治疗后的一个常见问题。在其他方案中，我们正在继续研究兔ATG和抗CD52的单抗(CamPath)在单程马ATG难治性再生障碍性贫血中的作用。CamPath现在也用于复发性再生障碍性贫血的研究环境，避免使用环孢素A。回顾我们在再生障碍性贫血方面的大量经验，使我们能够根据确诊时的血细胞计数，确定对免疫抑制治疗的反应和长期生存的新预测模型。此外，我们注意到再生障碍性贫血患者的短期和长期存活率都有了显著的改善，部分原因是更好的支持性护理，以及重复免疫抑制和干细胞移植等替代疗法的使用。在实验室，我们的免疫介导的骨髓衰竭小鼠模型继续产生有趣的结果。除了证明1型细胞因子反应在这些动物中占主导地位外，我们还表明T调节细胞可以消除疾病，与在再生障碍性贫血患者中观察到的低T-regs平行，并提示这些细胞在自身免疫性疾病中的潜在用途。我们还利用与基因敲除动物的杂交来确定在我们的模型中，Fas-配体细胞死亡途径，而不是穿孔素-颗粒酶，在介导骨髓衰竭中起主导作用。在再生障碍性贫血的人类免疫系统研究中，我们发现了信号转导通路中的损伤，包括Zeta链表达减少和SAP蛋白表达减少，这与免疫反应失调是一致的。在与人类疾病相关的端粒生物学研究中，我们将我们最初对急性髓细胞白血病TERT突变的观察扩展到包括对加拿大患者中一种罕见多态性的合作研究，这再次表明AML中有更高的患病率。最初在一个门诺派大家族中观察到的TERT突变与肝硬变的关系，现在已经扩展到其他家庭，包括详细的组织病理学，揭示了炎症和纤维化的不寻常组合。人们正在探索端粒缩短和基因组不稳定之间的关系，有一些早期成功的证据，有几种策略。我们利用来自端粒修复复合体突变的疾病患者的细胞，以及来自相同家族的临床健康者的细胞。到目前为止的结果包括通过荧光原位杂交(FISH)检测到体外组织培养后非整倍体率的增加，通过传统细胞遗传学和特殊核型(SKY)确定的端到端染色体连接，以及建立了一种对来自正常和患者骨髓的有限数量的单个集落的人造血细胞进行比较基因组杂交(CGH)的方法。在我们的病毒发现小组中，我们开发了一种在悬浮培养的正常人造血细胞中繁殖B19细小病毒的有效方法。一个重要的新发现是，细小病毒感染劫持了红系细胞的分化程序。在涉及微阵列、实时聚合酶链式反应和免疫印迹的研究中，确定了多种重要的红细胞生成基因的调节，因为它们在病毒感染的早期阶段，在靶细胞凋亡之前受到影响。在基因表达模式的这种戏剧性变化中，E2F介导的控制基因表达似乎是关键。最后，我们对血清阴性肝炎患者的组织样本进行了高通量测序，并确定了可能导致这种疾病以及肝炎相关性再生障碍性贫血和儿童暴发型肝炎的潜在新的感染源。

This years annual report incorporates both the work of the Cell Biology Section in bone marrow failure diseases and also of the Virus Discovery Section. 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 clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). In the clinic, studies have been directed towards more effective immunosuppression in AA and the application of immunosuppressive regimens to related bone marrow failure syndromes. Among current clinical protocols, our major study is a direct comparison two commercially available antithymocyte globulins, horse ATG versus rabbit ATG; more than 70 patients have been enrolled and a preliminary glimpse at the data suggests that these agents are not equivalent. A component of this study is to determine if low dose cyclosporine will prevent relapse, a common problem after successful immunosuppressive therapy. In other protocols, we are continuing to examine the role of rabbit ATG and the monoclonal antibody anti-CD52 (campath), in aplastic anemia refractory to a single course of horse ATG. Campath also is now utilized in a research setting for relapsed aplastic anemia, avoiding the use of cyclosporine. Retrospective examination of our large experience with aplastic anemia has allowed us to determine a new predictive model for response to immunosuppressive therapy and long-term survival, based on blood counts at presentation. Also, we have noted a striking improvement in both short and long-term survival in patients with aplastic anemia, due in part to better supportive care and also to the employment of alternative therapies, such as repeat immunosuppression and stem cell transplantation. In the laboratory, our murine model of immune-mediated bone marrow failure continues to yield interesting results. In addition to demonstrating that a type 1 cytokine response dominates in these animals, we have also shown that T-regulatory cells can abrogate disease, parallel with the observation of low T-regs in patients with aplastic anemia and suggesting potential utility of these cells in autoimmune diseases. We also have utilized cross-breeding with knock-out animals to determine that the Fas-ligand cell death pathway, rather than perforin-granzyme, dominates in mediating bone marrow failure in our model. In studies of the human immune system in aplastic anemia, we have identified lesions in modulating signal transduction pathways, including decreased zeta chain expression and diminished sap protein expression, consistent with dysregulated immune response. In studies of telomere biology as they related to human disease, we have extended our original observations of TERT mutations in acute myeloid leukemia to include collaborative studies of an infrequent polymorphism in Canadian patients, again demonstrating a higher prevalence in AML. The relationship of TERT mutations to hepatic cirrhosis, observed initially in a large Mennonite kindred, has now been extended to other families, including detailed histopathology disclosing an unusual combination of inflammation and fibrosis. The relationship between telomere shortening and genomic instability is being pursued, with some evidence of early success, with several strategies. We utilize cells from patients with telomere repair complex mutations with disease as well as clinically healthy persons from the same families who also have mutations. Results to date include the detection of an increased rate of aneuploidy after in vitro tissue culture, as determined by fluorescent in situ hybridization (FISH), end-to-end chromosome joining by conventional cytogenetics as well as spectrakaryotyping (SKY), and the establishment of a method to perform comparative genomic hybridization (CGH) on limited numbers of human hematopoietic cells from individual colonies from normal and patient bone marrows. In our virus discovery group, we have developed an efficient method of propogating B19 parvovirus in normal human hematopoietic cells in suspension culture. An important new observation is that parvovirus infection hijacks the erythroid cells differentiation program. In studies involving microarray, real time PCR, and immunoblot, determined regulation of multiple significant erythropoietic genes as they are affected at early stages of viral infection, prior to apoptosis of target cells. E2F-mediated control gene expression appears to be critical in this dramatic alteration in the pattern of gene expression. Finally, we have subjected tissue samples from patients with seronegative hepatitis to high throughput sequencing and identified potential novel infectious agents that may be responsible for this disease as well as for hepatitis-associated aplastic anemia and fulminant hepatitis of childhood.