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: 8149485
• 负责人: NEAL S YOUNG
• 金额: $ 228.44万
• 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/8149485
• 关键词: Aplastic Anemia; Functional disorder; Immune; Immunosuppressive Agents; Aplastic Anemia; Functional disorder; Immune; Immunosuppressive Agents

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. In the last year, we completed enrollment to our main clinical protocol for treatment of recent onset severe aplastic anemia. The study was initiated as a triple randomization among horse ATG (standard therapy in the United States for many years); rabbit ATG (newly introduced into the American market and used interchangeably with hATG); and Campath, a monoclonal antibody directed against CD52 with broad specificity to lymphocytes. The third arm was discontinued due to a low response rate after the treatment of about a dozen patients (see below). Entry into the hATG versus rATG protocol continued to completion, with 60 patients in each arm. The results of this study will be made public at the American Society of Hematology Meetings in December, when data analysis is complete, but they are likely to show marked and unexpected differences in hematologic response rates and possibly survival for two agents that have been employed indiscriminately for this disease - - therefore likely to change practice world-wide. Second, Campath, a highly immunosuppressive monoclonal antibody, has been employed in a variety of settings. The drug has been administered to 27 aplastic anemia patients refractory to hATG therapy; Campath appeared to be equivalent to rATG in inducing hematologic responses, in about 30% of refractory patients and > 50% of relapsed patients. However, only 3 of 16 patients with therapy-nave recent onset severe aplastic anemia responded, and Campath was judged to be inferior to standard hATG and therefore abandoned in this setting. The Branch has also pioneered another monoclonal antibody, daclizumab, which is directed to the interleukin 2 receptor of T cells. Long-term outcomes in patients with moderate aplastic anemia and pure red cell aplasia were reported: 7/28 moderate aplastic anemia patients achieved long-term hematologic recovery, and 38% of patients with pure red cell aplasia showed long-term transfusion independence. The toxicity profile for dacluzimab is excellent; unfortunately, the monoclonal antibody is no longer produced for the American market. In other clinical studies, retrospective examination of mortality in severe aplastic anemia has disclosed a marked decrease in deaths overall, especially in patients who failed to respond to an initial course of hATG. Multi-parameter analysis suggested that this improvement was mainly due to the introduction of superior anti-fungal therapies, their greater effectiveness and earlier implementation in severely neutropenic patients. Additionally, patients have been retreated with immunosuppression and referred earlier and more often to stem cell transplant due to decreased risks from this procedure. Also reported from the Clinical Center was the effectiveness of granulocyte transfusions in the setting of severe aplastic anemia; this product has been life-saving in patients who were severely neutropenic but not yet responded to ATG. Laboratory investigation of aplastic anemia has exploited several different modalities. We have created a mouse model of immune-mediated bone marrow failure, based on runt disease: lymph node cells from a parental strain are infused into F1 hybrid animals, resulting in rapid destruction of the host bone marrow, pancytopenia, and death from infection and bleeding. This model is dependent on lymphocytes, type 1 cytokines, and a potent innocent bystander effect. In recent work, we have demonstrated that T-bet, an important transcription factor that polarizes T-cells to type 1 cytokine production, and dysregulation of which has been implicated in human aplastic anemia, also plays a role in this animal model, as demonstrated in genetically defective animals. Lymph node cells from T-bet knock-out animals failed to induce bone marrow failure, although other cytokines were increased and associated with mild blood count abnormalities. In other murine experiments, the perforin/granzyme pathway was shown to be less important than the Fas/FasL pathway in inducing bone marrow cell destruction. Aplastic anemia in men and mouse was also addressed in imaging studies. We developed a novel methodology employing confocal microscopy to examine intact bone marrow to unprecendented depths, approximately 200 microns, in combination with multicolor antibody or flourochrome staining. This method allows digital reconstruction of the three dimensional architecture of a tissue. Confocal imaging reveals marked expansion in the number and size of fat cells in both experimental immune mediated bone marrow failure in the mouse and in humans with aplastic anemia, as well as rapid invasion of the murine bone marrow by CD8 effector T-cells. Imaging also allows visualization of the boney and vascular architecture of the marrow and of engraftment over time after marrow transplantation in animals. Aplastic anemia has also been investigated utilizing conventional and advanced flow cytometric methods. We have characterized the role of T-helper type 17 (Th17) lymphocytes, which have been associated with other autoimmune diseases. These cells are increased in patients with aplastic anemia at presentation and their presence correlated with disease activity; furthermore, Th17 and CD4 regulatory T-cells were inversely correlated. Th17 cell numbers also rise in the lymph node infusion model of bone marrow failure, although in the mouse model they appear to be of secondary importance to type 1 cytokine lymphocytes. We have also utilized multiplex cytokine analysis to examine bone marrow failure. When many dozens of cytokines levels are determined in serum or plasma, distinct signatures appear for aplastic anemia and a diagnostically confusing entity, hypocellular myelodysplastic syndrome. Multiplex cytokine analysis has also been applied to serum sickness, and certain cytokines correlate both with the concentrations of human antibody directed against xenoprotein and with the occurrence of clinical serum sickness. Certain cytokines are only produced in the presence of adequate platelet and megakaryocyte numbers, and in vitro, these cells can be shown to be the source of the proteins.

在性贫血中，骨髓被脂肪所取代，白细胞，红细胞和血小板的外周血计数 - - 跌至极低的水平，导致贫血，流血或感染导致死亡。 性贫血是一种年轻人的疾病，其严重形式几乎总是致命治疗。 从历史上看，性贫血与化学暴露有关，特别是苯；这是某种医学药物使用的特殊并发症。这是怀孕和血清肝炎的罕见事件。以及与某些免疫条件相关的疾病。 偶然的观察结果是，一些患者在骨髓移植后恢复了自己的骨髓功能，从而推断了免疫抑制条件方案可能已经治疗了一种潜在的免疫介导的病理生理学。 有目的的抗杀菌细胞球蛋白（ATG）导致大多数治疗患者的血液恢复。 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型细胞因子的效应T细胞的寡球，尤其是Gamma-Interterferon。 血液学分支一直是性贫血病理生理学和治疗的科学和医学研究的领导者。 在去年，我们完成了主要临床方案的入学率，以治疗最近发作严重的性贫血。 该研究是在马ATG（美国的标准疗法多年）中作为三重随机化的；兔子ATG（新引入美国市场，并与Hatg互换使用）；和Campath，一种针对CD52的单克隆抗体，对淋巴细胞具有广泛的特异性。 由于治疗约十二名患者后，第三臂因较低的反应率而停产（见下文）。 进入HATG与Ratg方案继续完成，每个手臂中有60名患者。 这项研究的结果将在12月的美国血液学会议上公开，当时数据分析完成后，它们可能显示出明显的血液学反应率明显且意外的差异，并且可能对两种因这种疾病不分青睐的药物而生存 - 因此 - 因此 - 因此可能在全球范围内改变实践。 其次，Campath是一种高度免疫抑制的单克隆抗体，已在各种环境中使用。 该药物已针对27名性贫血患者对HATG治疗难治性。 Campath在诱导血液学反应中似乎等同于Ratg，大约30％的难治性患者和> 50％的复发患者。 但是，在16例治疗nave患者中只有3例在最近发作的严重性贫血作出反应，而Campath被认为不如标准HATG，因此在这种情况下被放弃。 该分支还开创了另一种单克隆抗体Daclizumab，该抗体针对T细胞的白介素2受体。 报道了中度性障碍性贫血和纯红色细胞性肿瘤患者的长期结局：7/28个中度性障碍性贫血患者长期血液学康复，纯红色细胞性肿瘤患者中有38％的患者长期输血独立性。 Dacluzimab的毒性特征非常好；不幸的是，单克隆抗体不再为美国市场产生。 在其他临床研究中，对严重性障碍性贫血的死亡率的回顾性检查已经揭示了总体上死亡的明显减少，尤其是在对最初的HATG疗程未反应的患者中。 多参数分析表明，这种改善主要是由于引入了上等的抗真菌疗法，其更大的有效性以及对严重中性粒细胞减少症患者的更早实施。 此外，由于此手术的风险降低，患者已被免疫抑制后退，并更早地引用了干细胞移植。 临床中心还报道了粒细胞输血在严重的性贫血的情况下的有效性。该产品一直在严重中性粒细胞减少但尚未反应ATG的患者中挽救生命。 实验性障碍性贫血的实验室调查已经利用了几种不同的方式。 我们创建了一种基于Runt疾病的免疫介导的骨髓衰竭的小鼠模型：来自亲本菌株的淋巴结细胞被注入F1杂种动物中，从而迅速破坏了宿主骨髓，全细胞质，以及感染和出血和流血。 该模型取决于淋巴细胞，1型细胞因子和有效的无辜旁观者效应。 在最近的工作中，我们证明了T-BET是将T细胞偏振与1型细胞因子产生的重要转录因子，其失调与人类性贫血有关，在该动物模型中也起着作用，如遗传性动物中所证明的那样。 来自T-Bet敲除动物的淋巴结细胞未能诱导骨髓衰竭，尽管其他细胞因子增加并与轻度血细胞计数异常相关。 在其他鼠实验中，在诱导骨髓细胞破坏时，表现出培养素/颗粒酶途径不如FAS/FASL途径重要。 在成像研究中还解决了男性和小鼠的性贫血。 我们开发了一种使用共聚焦显微镜检查的新方法，以检查完整的骨髓至未固定的深度，约200微米，并结合多色抗体或粉状染色。 该方法允许数字重建组织的三维结构。 共聚焦成像揭示了在实验性免疫介导的小鼠和患有性贫血的人类中脂肪细胞的数量和大小的显着膨胀，以及CD8效应T细胞对鼠骨髓的快速侵袭。 成像还允许可视化动物骨髓移植后的骨髓和植入的骨和血管结构。 还使用常规流式细胞术方法研究了性贫血。 我们已经表征了T型17型（Th17）淋巴细胞的作用，这些淋巴细胞与其他自身免疫性疾病有关。 这些细胞在表现时患有性贫血的患者增加，并且其存在与疾病活性相关。此外，TH17和CD4调节T细胞成反比。 Th17细胞数在骨髓衰竭的淋巴结输注模型中也增加，尽管在小鼠模型中，它们似乎对1型细胞因子淋巴细胞具有次要重要性。 我们还利用多重细胞因子分析来检查骨髓衰竭。 当在血清或血浆中确定了数十个细胞因子水平时，会出现异质性贫血和诊断出令人困惑的实体，骨髓发育异常综合征的不同特征。 多路复用细胞因子分析也已应用于血清疾病，某些细胞因子与针对异种蛋白的人类抗体的浓度以及临床血清疾病的发生相关。 某些细胞因子仅在有足够的血小板和巨核细胞的存在下产生，并且在体外，这些细胞可以证明是蛋白质的来源。