Immune Pathophysiology of Aplastic Anemia and Immunosuppressive Treatments

再生障碍性贫血的免疫病理生理学和免疫抑制治疗

• 批准号: 9157323
• 负责人: NEAL S YOUNG
• 金额: $ 295.83万
• 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/9157323
• 关键词: Activities of Daily Living; Acute Myelocytic Leukemia; Affect; Agreement; Anemia; Animal Experiments; Animal Model; Animals; Antithymoglobulin; Aplastic Anemia; Apoptosis; Basic Science; Benzene; Blood Platelets; Bone Marrow; Bone Marrow Transplantation; Candidate Disease Gene; Categories; Cell Count; Cell Line; Cells; Cessation of life; Chemical Exposure; Clinical; Clinical Protocols; Clinical Research Protocols; Clonal Evolution; Collaborations; Colony-Stimulating Factors; Complete Blood Count; Complication; Cyclosporine; Data; Disease; Dose; Drug usage; Dysmyelopoietic Syndromes; Effector Cell; Elderly; Equus caballus; Erythrocytes; Erythropoietin; Event; Evolution; Fatty acid glycerol esters; Flow Cytometry; Functional disorder; Genomics; Growth Factor; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Cell Growth Factors; Hematopoietic stem cells; Hemorrhage; Hepatitis; Human; Immune; Immune system; Immunologics; Immunosuppression; Immunosuppressive Agents; In complete remission; Induction of Apoptosis; Infection; Inflammatory; Interferon Type II; Intervention; Investigation; Laboratories; Lead; Link; Lymphocyte; Marrow; Mediating; Mediation; Medical; Megakaryocytes; Molecular; Monitor; Monosomy 7; Mutate; Mutation; Myelogenous; Myelopoiesis; National Heart, Lung, and Blood Institute; Pancytopenia; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Platelet Count measurement; Population; Pregnancy; Protocols documentation; Pure Red-Cell Aplasia; Recovery; Refractory Disease; Regimen; Regulatory T-Lymphocyte; Relapse; Risk; Role; Scientist; Sickle Cell Anemia; Signal Transduction; Sirolimus; Stem cell transplant; Stem cells; T-Lymphocyte; Testing; Therapeutic immunosuppression; Thrombopoietin; Time; Transplantation; Tumor Necrosis Factor Ligand Superfamily Member 6; Up-Regulation; White Blood Cell Count procedure; Work; animal data; bone marrow failure syndrome; cohort; conditioning; cytokine; cytopenia; cytotoxic; experience; falls; high risk; interest; mimetics; mouse model; peripheral blood; prevent; research and development; research study; response

In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene; it is an idiosyncratic complication of some medical drug use; it occurs as a rare event in pregnancy and following seronegative hepatitis; and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. Clinical protocols in the Branch have centered on utilization of the synthetic thrombopoietin mimetic eltrombopag in bone marrow failure. First, we have continued accruing patients to our extension trial of eltrombopag as a single agent in patients with refractory disease; the response rate of 40-50% has been maintained and the rate of clonal evolution to myelodysplastic syndrome and acute myeloid leukemia has not increased. Genomics are being employed in order to determine whether mutations in candidate genes are responsible for untoward late hematologic effects. Eltrombopag is also being employed as a single agent in patients with low-risk myelodysplastic syndrome and in moderate aplastic anemia or single lineage bone marrow failure, such as pure red cell aplasia. Responses have been seen in all categories, in some cases approaching 50% hematologic recovery. However, as yet, there is not clear evidence that MDS in its usual presentation in the elderly with cytopenias has a high response rate to eltrombopag; MDS following aplastic anemia has responded to this therapy. Our major clinical research protocol is the combination of conventional immunosuppression (horse antithymocyte globulin and cyclosporine), with eltrombopag in treatment-nave severe aplastic anemia. This protocol has now accrued about 75 patients in three cohorts. In the first cohort of 31 patients, eltrombopag was initiated two weeks after the first dose of ATG and continued for six months. In the second cohort, eltrombopag exposure was limited to the first three months; in the third and current cohort, eltrombopag is administered with ATG treatment and continued for six months. Overall and complete response rates are similar in the three cohorts, with 75-90% of patients responding by three or six months, and with a complete response rate approximately 30%. Both rates are much higher than observed historically in patients who received ATG and cyclosporine (approximately 65% and 10%, respectively). The rate of clonal evolution appears to be lower than in patients with refractory disease, although some cases of evolution to monosomy 7 requiring stem cell transplant intervention have occurred. Long-term survival data will be accrued, as will genomic analysis of patients at landmark time points to monitor for somatically mutated clones, especially in patients who evolved to MDS/AML. Negotiations are underway for continuation of further trials of eltrombopag in hematologic disease at the Clinical Center, under a Collaborative Research and Development Agreement, now with Novartis. In the basic research laboratory, we are interested in the mechanism of action of eltrombopag in aplastic anemia, as patients have high endogenous thrombopoietin levels; erythropoietin and myeloid colony stimulating factors are ineffective in patients with bone marrow failure, presumably because patients are already producing large quantities of hematopoietic growth factors in physiologic response to cytopenias. One curious observation is that thrombopoietin levels remain elevated in patients post-immunosuppressive therapy for long periods of time, even with complete blood count recovery. We hypthothesize that thrombopoietin is regulated by at least in part by megakaryocyte mass, which may not return to normal in aplastic anemia even when platelet counts are adequate. In collaboration with Andre LaRochelle, a tenure-track scientist, and with Cynthia Dunbar we also participate in mechanistic experiments utilizing primary cells and hemapoietic cell lines. Following on work of others, we are investigating whether eltrombopag can circumvent a block in hemapoietic growth factor signal transduction in the presence of inflammatory cytokines such as gamma-interferon. In animal models, with which our laboratory has had extensive experience, we have completed investigations of the potentially dual biologic roles of gamma interferon. Others have claimed that gamma interferon increases hematopoietic stem cell number and drives myelopoiesis. However, in our studies, gamma interferon increases phenotypically defined hematopoietic stem cells, but these cells have poor functional ability; as in transplant experiments. Likely, Sca-1 upregulation on stem cells is responsible for this discrepancy. Conversely, gamma interferon induces Fas expression and apoptosis, especially in the presence of Fas ligand or activated cytotoxic effector cells. Our recent data are therefore consistent with historical results in which gamma interferon has been shown to negatively regulate hematopoiesis, compatible with its role in immune-mediated bone marrow failure in humans. Finally, because relapse remains a serious problem in patients with aplastic anemia who have been treated with immunosuppressive therapy, we have readdressed the role of a specific immunomodulatory drug, rapamycin or sirolimus, in our mouse model of immune-mediated bone marrow failure. These experiments are also inspired by the successful utilization of sirolimus in the setting of high risk stem cell transplants in sickle cell disease, as performed by our colleague John Tisdale in NHLBI. In animal experiments, rapamycin is very effective in preventing stem cell destruction in the immune-mediated aplastic anemia mouse model. We have determined optimal dose and regimen. Further, there are marked differences in lymphocytes as determined by flow cytometry phenotyping of the bone marrow of affected animals, and in transcriptional profiling. Current experiments investigate the role of T-regulatory cells and direct induction of apoptosis of effector cells in this animal model. These animal data, as well as theoretical considerations, should lead to a protocol in which rapamycin is substituted for cyclosporine in an effort to induce tolerance in aplastic anemia patients after hematologic recovery.

在再生障碍性贫血中，骨髓被脂肪取代，外周血计数（白细胞、红细胞和血小板）降至极低水平，导致因贫血、出血或感染而死亡。再生障碍性贫血是年轻人的一种疾病，严重时如果不治疗几乎总是致命的。从历史上看，再生障碍性贫血与化学物质接触有关，特别是苯；它是某些药物使用的特殊并发症；它在怀孕期间和血清阴性肝炎后发生为罕见事件；以及与某些免疫状况相关的疾病。一些患者在骨髓移植后恢复了自己的骨髓功能，这一偶然观察导致了这样的推断：免疫抑制调理方案可能治疗了潜在的免疫介导的病理生理学。有目的地施用抗胸腺细胞球蛋白（ATG）已使大多数接受治疗的患者的血液学恢复。实验室数据还揭示了免疫系统的异常：通过Fas介导的途径诱导造血靶细胞凋亡的淋巴细胞群，以及表达1型细胞因子（尤其是γ-干扰素）的效应T细胞的寡克隆。血液学分支一直是再生障碍性贫血病理生理学和治疗的科学研究和医学研究的领导者。 该部门的临床方案集中于合成血小板生成素模拟物艾曲波帕在骨髓衰竭中的应用。 首先，我们继续招募患者参加艾曲波帕作为单药治疗难治性疾病患者的扩展试验；维持40-50%的缓解率，骨髓增生异常综合征和急性髓性白血病的克隆进化率没有增加。 基因组学正在被用来确定候选基因的突变是否会导致不良的晚期血液学影响。 艾曲波帕还被用作单药治疗低危骨髓增生异常综合征和中度再生障碍性贫血或单系骨髓衰竭（例如纯红细胞再生障碍性贫血）的患者。 所有类别均出现缓解，在某些情况下血液学恢复率接近 50%。 然而，迄今为止，尚无明确证据表明老年血细胞减少症的常见MDS对艾曲波帕有较高的缓解率；再生障碍性贫血后的 MDS 对这种疗法有反应。 我们的主要临床研究方案是将传统免疫抑制（马抗胸腺细胞球蛋白和环孢素）与艾曲波帕联合治疗初治的严重再生障碍性贫血。 该方案目前已在三个队列中招募了约 75 名患者。 在第一组 31 名患者中，艾曲波帕在首次服用 ATG 两周后开始使用，并持续六个月。 在第二组中，艾曲波帕暴露仅限于前三个月；在第三个队列（即当前队列）中，艾曲波帕与 ATG 治疗一起给药，并持续六个月。 这三个队列的总体缓解率和完全缓解率相似，75-90% 的患者在三到六个月内出现缓解，完全缓解率约为 30%。 这两个比率均远高于历史上接受 ATG 和环孢素治疗的患者中观察到的发生率（分别约为 65% 和 10%）。 尽管发生了一些需要干细胞移植干预的进化为 7 号单体的病例，但克隆进化的速度似乎低于难治性疾病患者。 将积累长期生存数据，并在具有里程碑意义的时间点对患者进行基因组分析，以监测体细胞突变克隆，尤其是进化为 MDS/AML 的患者。 目前正在与诺华公司就根据合作研究和开发协议在临床中心继续进行艾曲波帕治疗血液疾病的进一步试验进行谈判。 在基础研究实验室，我们对艾曲波帕治疗再生障碍性贫血的作用机制感兴趣，因为患者内源性血小板生成素水平较高；促红细胞生成素和骨髓集落刺激因子对骨髓衰竭患者无效，可能是因为患者在对血细胞减少的生理反应中已经产生大量造血生长因子。 一项奇怪的观察结果是，即使全血细胞计数恢复，患者在免疫抑制治疗后很长一段时间内血小板生成素水平仍保持升高。 我们假设血小板生成素至少部分受到巨核细胞质量的调节，即使血小板计数充足，再生障碍性贫血中的巨核细胞质量也可能无法恢复正常。 我们还与终身教授科学家安德烈·拉罗谢尔 (Andre LaRochelle) 和辛西娅·邓巴 (Cynthia Dunbar) 合作，参与利用原代细胞和造血细胞系的机械实验。 继其他人的工作之后，我们正在研究艾曲波帕是否可以在存在炎症细胞因子（例如γ-干扰素）的情况下绕过造血生长因子信号转导的阻断。 我们的实验室在动物模型方面拥有丰富的经验，我们已经完成了对γ干扰素潜在双重生物学作用的研究。 其他人声称γ干扰素可以增加造血干细胞数量并促进骨髓生成。 然而，在我们的研究中，γ干扰素增加了表型明确的造血干细胞，但这些细胞的功能能力较差；就像移植实验一样。 干细胞上 Sca-1 的上调很可能是造成这种差异的原因。 相反，γ 干扰素诱导 Fas 表达和细胞凋亡，尤其是在存在 Fas 配体或激活的细胞毒性效应细胞的情况下。 因此，我们最近的数据与历史结果一致，其中γ干扰素已被证明对造血作用具有负调节作用，与其在人类免疫介导的骨髓衰竭中的作用相一致。 最后，由于复发仍然是接受免疫抑制治疗的再生障碍性贫血患者的一个严重问题，因此我们重新研究了特定免疫调节药物雷帕霉素或西罗莫司在免疫介导的骨髓衰竭小鼠模型中的作用。 这些实验的灵感还源于我们在 NHLBI 的同事 John Tisdale 所进行的镰状细胞病高风险干细胞移植中成功利用西罗莫司的实验。 在动物实验中，雷帕霉素对于预防免疫介导的再生障碍性贫血小鼠模型中的干细胞破坏非常有效。 我们已经确定了最佳剂量和方案。 此外，通过受影响动物骨髓的流式细胞术表型分析和转录谱测定，淋巴细胞存在显着差异。 目前的实验研究了该动物模型中 T 调节细胞的作用以及效应细胞凋亡的直接诱导。 这些动物数据以及理论考虑应该导致制定一项方案，其中雷帕霉素取代环孢菌素，以努力在血液学恢复后诱导再生障碍性贫血患者的耐受性。