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，也包括在细小病毒方面的病毒发现科的工作，因为后者由于凯文·布朗博士的离职而被并入前者。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。多项研究正在进行中。虽然我们观察到第一次出现的再生障碍性贫血对单抗CamPath的一些反应，但当很明显抗CD52不太可能像标准治疗那样有效时，我们的三臂随机方案的这一组很早就停止了。在这个患者群体中，马和兔ATG的比较仍在继续。在对标准马ATG和环孢素耐药的AA中，兔ATG和CamPath均显示出约30%的抢救活性。我们还在测试CamPath在治疗复发性再障和MDS方面的作用。我们参与的eculuzimab的多中心试验已经完成；除了导致FDA批准这种现已上市的Soliris药物用于治疗PNH中血管内溶血引起的症状和/或严重贫血的适应症外，对终点的进一步分析表明，单抗在降低PNH中主要死亡原因-血栓形成方面也显著有效。在实验室中，对造血细胞的免疫破坏的研究在几个方向上继续进行。我们已经研究了ATGs的作用机制，特别是在马和兔产品在治疗再生障碍性贫血方面似乎不一样有效的情况下。在细胞培养和分子研究中，我们已经证实兔ATG在体外可以诱导CD25-FoxP3-CD4细胞产生T调节细胞，而马ATG缺乏这一活性。为了深入研究T细胞，现在对同意这一程序的患者在治疗前和完成六个月的疗程后进行淋巴细胞分离，以进行细胞因子基因表达的分子研究。初步数据表明，在完全恢复造血能力的患者中持续存在的T细胞克隆缺乏伽马干扰素和其他1型细胞因子的产生，因此可能是耐受的。在以端粒修复为重点的获得性再生障碍性贫血的遗传学研究中，我们从一个携带新的TERT突变的门诺族大家族中扩展了我们的发现，揭示了先证者父亲的急性髓系白血病与先证者的姑姑和两个更远的女性亲戚的严重肝病的关系，他们都携带着这种突变。在两项独立的Cohert研究中，一项是在100名巴西AML患者中，另一项是在MD Anderson癌症中心收集的92名患者中，我们发现这种白血病的TERT突变率约为6%。体外研究表明，这些是功能丧失改变；通过研究非骨髓组织和家庭成员的生殖系遗传。同样，在收集的肝移植患者样本中的初步数据也显示，有几个患者存在TERT突变。这些结果表明端粒修复与白血病的发生有关，也与严重的肝病有关，在这一点上，其特点是纤维化和炎症的结合。在我们对雄性激素在血液病中作用机制的研究中，我们在体外证明了合成的和天然的雄激素以及雌二醇增加了TERT基因的转录和端粒酶的活性。他莫昔芬以及抗雌激素和抗雄激素受体药物的抑制活性表明，雄激素芳构化为雌二醇以及雌激素和雄激素受体都参与了对造血细胞的这种作用。我们正在继续我们的假设，即由于染色体融合和基因组不稳定，端粒缩短会导致非整倍体，就像在小鼠模型中所证明的那样。在病毒发现小组与细小病毒相关的研究中，已经有了一些信号方面的进展。首先，重组B19细小病毒疫苗目前正在人类志愿者中进行第二阶段第一阶段试验，以确定诱导中和抗体所需的适当剂量的空衣壳，然后计划在镰状细胞疾病儿童中进行第三阶段试验，目的是防止一过性再生障碍性危机。其次，我们开发了一个实用的B19细小病毒的感染性克隆，它有助于通过分子研究和共聚焦显微镜来确定特定病毒基因在人类细胞感染中的作用。最后，我们利用我们大量获得CD34细胞并诱导红系分化的能力，开发了一种高效的B19细小病毒细胞培养系统。CD34细胞来源的CD36细胞概括了正常的红细胞生成，包括Glogoside受体的表达，它们也是一种更敏感的方法来检测中和抗体，用于我们的疫苗试验，以及病毒与其已知致病靶细胞相互作用的分子和细胞生物学研究。

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 in parvoviruses, as the latter has been incorporated into the former due to the departure of Dr. Kevin Brown. 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. Multiple studies are in progress. While we observed some responses in first presentation aplastic anemia to the monoclonal antibody CAMPATH, this arm of our three-arm randomized protocol was discontinued early when it became clear that anti-CD52 was unlikely to be as effective as standard therapy. A comparison of horse and rabbitt ATG continues in this patient population. In AA refractory to standard horse ATG plus cyclosporine, both rabbit ATG and CAMPATH show activity about 30% salvage rate. We are also testing CAMPATH in the treatment of relapsed AA and in MDS. Our participation in a mulit-center trial of eculuzimab was completed; in addition to leading to FDA approval for this agent, now marketed as Soliris, for the indications of symptoms and/or severe anemia due to intravassular hemolysis in PNH, further analysis of end points has shown that the monoclonal antibody is also strikingly effective in reducing the rate of thrombosis, the major cause of death in PNH. In the laboratory, studies of immune destruction of hematopoietic cells has continued in several directions. We have examined the mechanism of action of the ATGs, especially as horse and rabbit products do not appear to be equally effective in the treatment of aplastic anemia. In cell culture and molecular studies, we have confirmed that rabbit ATG in vitro induces T-regulatory cells from CD25-FoxP3-CD4 cells while horse ATG lacks this activity. In order to study T-cells intensively, lymphocytopheresis in now being performed in patients who consent to this procedure prior to therapy and at the completion of a six month course in order to perform molecular studies of cytokine gene expression. Preliminary data suggests that T-cell clones that persist in patients who have fully recovered hematopoietic capacity lack production of gamma-interferon and other type-1 cytokines and therefore may be tolerant. In studies of the genetics of acquired AA with a focus of telomere repair, we have extended our findings from a large mennonite kindred bearing a novel TERT mutation, which disclosed a relationship to both acute myeloid leukemia in the proband's father and to severe hepatic disease in the proband's aunt and two more distant female relatives all of whom bear the mutation. In two separate cohert studies, one of 100 Brazilian patients with AML and the other of 92 patients collected at MD Anderson Cancer Center, we found a rate of approximately 6% of TERT mutations in this leukemia. In vitro studies have suggested that these are loss of function alterations; germline inheritance by study of non-bone marrow tissues and of family members. Similarly, preliminary data among samples collected of patients who have undergone liver transplant have also show several with TERT mutations. These results implicate telomere repair in the initiation of leukemia and also in severe liver disease, characterized at this point as a combinnation of fibrosis and inflammation. In our studies of the mechanism of action of male hormones in hematologic disease, we have demonstrated in vitro that synthetic and natural androgens and also estrodiol increase transcription of the TERT gene and of telomerase activity. The inhibitory activity of tamoxofin and of both anti-estrogen and anti-androgen receptor agents indicates that both aromatization of androgens to estrodiol and the estrogen and androgen receptors are involved in this effect on hematopoietic cells. We are pursuing our hypothesis that telomere shortening results in aneuploidy due to chromosome fusion and genomic instability, as has been demonstrated in murine models. In studies from the Virus Discovery Group related to parvoviruses, there have been a number of signal advances. First, a recombinant B19 parvovirus vaccine is now in its second phase 1 trial among human volunteers to determine the appropriate dose of empty capsids needed to elicit neutralizing antibodies, prior to a planned phase III trial in children with sickle cell disease with the goal of preventing transient aplastic crisis. Second, we have developed a practical infectious clone of B19 parvovirus, which has been useful to define by molecular studies and confocal microscopy the role of specific viral genes in infection of human cells. Finally, we have utilized our ability to obtain CD34 cells in bulk and to induce erythroid differentiation to develop a highly productive cell culture system for B19 parvovirus. CD34 cell-derived CD36 cells recapitulate normal erythropoeisis, including expression of the glogoside receptor, and they are useful also as a much more sensitive method to measure neutralizing antibodies, of use in our vaccine trials, and for molecular and cell biology studies of virus interaction with its known pathogenic target cell.