阵发性睡眠性血红蛋白尿克隆扩增机制的研究进展

阵发性睡眠性血红蛋白尿是一种溶血性疾病,以GPI-AP缺陷的异常造血细胞的出现为特征.临床表现是以与睡眠有关的、间歇发作性的血红蛋白尿为特征,可伴有全血细胞减少和反复血栓形成,其常见的并发症是感染、下肢静脉血栓塞,部分患者转化为骨髓增生异常综合征、急性白血病.近年来研究证实本病是造血干细胞PIG-A基因突变所致的克隆病.本文参阅近几年的文献,对阵发性睡眠性血红蛋白尿克隆扩增机制的研究现况作一综述.     

特发性造血障碍病理机制研究的新进展

本文以再生障碍性贫血、阵发性睡眠性血红蛋白尿症及骨髓增生异常综合征为中心，根据造血障碍的分子生物学研究结果，探讨这些疾病的病理分析研究的新进展，综述特发性造血障碍研究的现状。     

特发性造血障碍病理机制研究的新进展

本以再生障碍性贫血、阵发性睡眠性血红蛋白尿症及骨髓增生异常综合征为中心，根据造血障碍的分子生物学研究结果，探讨这些疾病的病理分析研究的新进展，综述特发性造血障碍研究的现状。     

多色流式细胞术测定CD55及CD59表型在阵发性睡眠性血红蛋白尿诊断中的应用

阵发性睡眠性血红蛋白尿(paroxysmal nocturnal hemoglobinuria, PNH)是一种获得性红细胞异常性溶血性疾病,以睡眠时出现阵发性血红蛋白尿为主要特征.发作期尿潜血阳性,并有其他血管内溶血表现.尿含铁血黄素试验持续阳性,蔗糖溶血试验阳性,酸溶血Ham's试验阳性等.随着科学研究的进展,现在PNH已被确认是造血干细胞基因突变的克隆性疾病,其异常血细胞膜上糖基磷脂酰肌醇(glycosylphosphatidylinositol, GPI)生成障碍,导致PNH患者血细胞膜上GPI连接蛋白如分化群(cluster of differentiation, CD)55、CD59等的表达明显减低和缺乏,因而对正常血清中的补体特别敏感而发生溶血.因此,利用免疫荧光染色标记针对CD55、CD59的单克隆抗体,采用流式细胞术检测PNH患者血细胞CD55、CD59表达的数量,对PNH的诊断有重要意义.现将结果报告如下.     

阵发性睡眠性血红蛋白尿症与血栓发生的研究进展

阵发性睡眠性血红蛋白尿症(PNH)是一种克隆性造血干细胞功能紊乱所致罕见性骨髓功能衰竭性疾病,以血管内溶血性贫血,全血细胞减少和血栓为表现。PNH虽是良性疾病,但其并发症严重影响患者生活质量和生存时间,其中最常见严重并发症是血栓。     

合成的GPI-Pr基因及其与PNH的关系

糖基磷脂酰肌醇锚蛋白及阵发性睡眠性血红蛋白尿症是近年来的研究热点之一。本文总结了近年来发现的GPI-锚合成过程中的基因——PIG-A、PIG-H、PIG-C、PIG-B、PIG-L、PIG-F,并着重介绍了GPI-Pr和PIG-A基因与PNH发病的关系及有关PNH的最新研究进展。     

阵发性睡眠性血红蛋白尿的分子基础

阵发性睡眠性血红蛋白尿发病的直接原因，是患者血细胞膜上缺乏多种糖化肌醇磷脂锚蛋白。近年研究表明，其缺乏的基础在于锚本身的改变，而后者改变的关键在于锚合成的起始步骤发生障碍。ＰＩＧ基因改变可能是上述障碍的根本原因。     

阵发性睡眠性血红蛋白尿症溶血机制的研究现状

阵发性睡眠性血红蛋白尿症(paroxysmalnocturnal hemoglobinuria,PNH)是一种后天获得的血细胞膜缺陷病。只有对血细胞膜缺陷有深入了解,才能阐明该病的溶血机制。一、PNH 病态血细胞的来源主要有克隆学说和干细胞学说二种观点。1970年Oni等发现一女性PNH患者,其葡萄     

阵发性睡眠性血红蛋白尿症补体不敏感红细胞囊泡化现象的观察

经蛇毒因子溶血法纯化阵发性睡眠性血红蛋白尿症补体不敏感红细胞，用过氧化氢、中性多形核白细胞和卵黄磷脂酰胆碱人工脂质体分别与之孵育，通过检测上清乙酰胆碱脂酶活性了解红细胞囊泡化释放情况。结果显示阵发性睡眠性血红蛋尿症红细胞用过氧化氢或中性多形核白细胞诱导后上清中乙酰胆碱脂酶活性与正常对照无明显差别（Ｐ＞０．０５）；而在卵黄磷脂酰胆碱人工脂质体诱导下，随孵育时间延长，可见明显囊泡释放，第４小时乙酰胆碱     

尿激酶受体的研究进展

尿激酶受体 (u PAR)是由体内多种细胞表达的单链膜糖蛋白 ,介导尿激酶催化的纤溶酶原活化与纤溶过程 ,并参与基质的水解、信号传递和细胞的粘附 ,在肿瘤侵润和转移、炎症与血栓形成中具有重要作用。u PAR的检测可作为肿瘤预后及炎症、阵发性睡眠性血红蛋白尿等多种疾病状态的判断指标 ,调节u PAR的表达与功能可能成为肿瘤、炎症以及动脉粥样硬化治疗的一个新的途径。     

Hematopoietic progenitor cells in the blood and bone marrow in various hematologic disorders

Hematopoietic progenitor cells are present in the blood and the bone marrow. Changes in the numbers of hematopoietic progenitor cells reflect alteration of pluripotent stem cells. We discuss such changes in common hematologic diseases including aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and thalassemia. In aplastic anemia, the numbers of burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) are much decreased; the decrease still exists after recovery from therapy. In PNH, the numbers of progenitor cells are low, even in the presence of marrow hypercellularity. In thalassemia, the numbers of progenitor cells are much increased; more pronounced in splenectomized patients.     

Paroxysmal nocturnal hemoglobinuria and the age of therapeutic complement inhibition

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease of hematopoietic stem cells due to a mutation in the PIG-A gene leading to a deficiency of GPI-anchored proteins. Lack of two specific GPI-anchored proteins, CD55 and CD59, leads to uncontrolled complement activation that result in both intravascular and extravascular hemolysis. Free hemoglobin leads to nitric oxide depletion that mediates the pathophysiology of some of the common clinical signs of PNH. Clinical symptoms of PNH include evidence of hemolytic anemia, bone marrow failure, smooth muscle dystonias and thromboses. Treatment options for patients with PNH include bone marrow transplantation, a therapy associated with high morbidity and mortality, or treatment with the complement inhibitor eculizumab. Eculizumab is a first-in-class anti-complement drug that in PNH has been shown to block complement-mediated hemolysis, reduce transfusion dependency, reduce thromboembolic complications and improve the quality of life (QoL) of patients.     

Investigation on apoptosis in paroxysmal nocturnal hemoglobinuria (PNH) granulocytes]

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disordr characterized by increased susceptibility of erythrocytes to complement-mediated hemolysis. Recently, PNH is a stem cell disorder with all hematopoietic lineages affected, which have deficiencies of glycosylphosphtidylinositol-anchored membrane proteins due to the phosphatidylinositol glycan-class A (PIG-A) gene abnormalities. However, it is unknown how PNH clones with PIG-A gene abnormalities increase in bone marrow. The possibility has been suggested that resistance of PNH cells to apoptosis causes the increase. We studied two-color or single-color flow cytometric analysis using Annexin V and propidium iodide or 7-amino actinomycin D for evaluation of spontaneous apoptosis in peripheral blood granulocytes from PNH patient (n = 5) and healthy volunteers (n = 5), respectively. Apoptotic granulocytes were evaluated before and after 6-, 12-, 18- and 24-hour cultures without serum. Flow cytometric analyses showed that there were no significant differences of the number of proportion of apoptotic cells between them. This fact reveals that the sensitivity of PNH cells to apoptosis is similar to that of normal cells, suggesting that PNH clones should not be increased according to resistance to apoptosis as an intrinsic characteristic.     

Paroxysmal nocturnal hemoglobinuria: Correction of abnormal phenotype by somatic cell hybridization

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disorder thought to result from a somatic mutation in a hemopoietic stem cell. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in proteins attached to the cell membrane via a glycosylphosphatidylinositol structure, called the GPI anchor, and the primary lesion in PNH is thought to be a defect in the biosynthesis of the GPI anchor. We have recently established permanent lymphoblastoid cell lines that have the PNH phenotype and we report now the isolation of human-human somatic cell hybrid clones obtained by fusing them with normal lymphoblastoid cells. In all of 21 hybrid clones, obtained from five different patients, the expression of three different GPI-linked proteins on the hybrid cells was normal. These findings indicate that the PNH mutant gene is recessive with respect to the normal allele and that a recessive mutation can cause a clonal preneoplastic disorder.     

Post-Allogeneic Hematopoietic Stem Cell Transplantation Eculizumab as Prophylaxis Against Hemolysis and Thrombosis for Patients with Hematologic Disorders Associated with Paroxysmal Nocturnal Hemoglobinuria Clones

Paroxysmal nocturnal hemoglobinuria (PNH) is frequently seen in the context of other aplastic anemia and myelodysplastic syndromes and is associated with hemolysis and increased thromboembolic events. Allogeneic hematopoietic stem cell transplantation (alloHCT) is the sole curative treatment but is associated with significant morbidity. The terminal complement inhibitor eculizumab reduces hemolysis and thromboembolic events and is the sole Food and Drug Administration-approved therapy for PNH. Prophylactic administration of this agent in the early post-transplantation setting to prevent hemolysis and thrombosis has not been described in the literature. We describe our institutional experience of 8 patients with PNH who underwent alloHCT and who received at least 1 dose of eculizumab within 30 days of alloHCT for prevention of thrombosis and hemolysis. One patient with underlying aplastic anemia who received bone marrow stem cells failed to engraft. Another patient experienced steroid-refractory grade IV acute graft-versus-host disease and died of a fungal infection. The other patients engrafted well; no hemolysis, thrombotic events, or infections associated with encapsulated bacteria occurred in any of the 8 patients.     

Glycosylphosphatidylinositol-linked proteins are required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development

Surface proteins tethered to the membrane through a glycosylphosphatidylinositol (GPI) anchor are deficient in the blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) as result of a somatic mutation, in a hematopoietic stem cell, of the X-linked phosphatidylinositolglycan complementation group A (PIG-A) gene. In PNH patients, compared to the large numbers of GPI-deficient myeloid cells, the proportion of GPI-deficient lymphocytes tends to be low, and therefore the impact of GPI deficiency on immune function has been unclear. We have obtained complementation by Pig-a(-) embryonic stem (ES) cells of Rag(-/-) blastocysts, and we show that Pig-a(-) ES cells are able to reconstitute the T cell and B cell compartments of Rag(-/-) mice. Although these mice were immunologically competent, by comparison with appropriate controls we detected several abnormalities: (1) increased levels of IgG; (2) high frequency/titers of anti-nuclear antibodies; (3) markedly reduced delayed hypersensitivity; and (4) impaired activation-induced lymphocyte death in vitro. In some cases, aging Pig-a(-)/Rag(-/-) chimeric mice developed lymphadenopathy and polyclonal T cell and B cell expansion. Thus, GPI-linked proteins are not required for lymphocyte development but they are required for normal lymphocyte function and for maintaining normal peripheral lymphoid homeostasis.     

Paroxysmal nocturnal hemoglobinuria and other complement-mediated hematological disorders

The recent availability of eculizumab as the first complement inhibitor renewed the interest for complement-mediated damage in several human diseases. Paroxysmal nocturnal hemoglobinuria (PNH) may be considered the paradigm a disease caused by complement dysregulation specifically on erythrocytes; in fact, PNH is a clonal, non-malignant, hematological disorder characterized by the expansion of hematopoietic stem cells and progeny mature blood cells which are deficient in some surface proteins, including the two complement regulators CD55 and CD59. As a result, PNH erythrocytes are incapable to modulate on their surface physiologic complement activation, which eventually enables the terminal lytic complement leading to complement-mediated intravascular anemia - the typical clinical hallmark of PNH. In the last decade the anti-C5 monoclonal antibody has been proven effective for the treatment of PNH, resulting in a sustained control of complement-mediated intravascular hemolysis, with a remarkable clinical benefit. Since then, different diseases with a proved or suspected complement-mediated pathophysiology have been considered as candidate for a clinical complement inhibition. At the same time, the growing information on biological changes during eculizumab treatment in PNH have improved our understanding of different steps of the complement system in human diseases, as well as their modulation by current anti-complement treatment. As a result, investigators are currently working on novel strategy of complement inhibition, looking at the second generation of anti-complement agents which hopefully will be able to modulate distinct steps of the complement cascade. Here we review PNH as a disease model, focusing on the observation that led to the development of novel complement modulators; the discussion will be extended to other hemolytic disorders potentially candidate for clinical complement inhibition.     

Advances in the laboratory diagnosis of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare form of acquired hemolytic anemia characterized by intravascular hemolysis, bone marrow failure and a thrombotic tendency. Diagnosis and management of PNH can be challenging as patients show a wide spectrum of clinical presentation and disease course. Over the past 10–15 years, significant advances have been made in understanding the pathophysiology of this rare condition, made possible by detailed clinical, molecular and flow cytometric studies. The disease is undoubtably complex, and its origins lie in somatic mutation of the X-linked pig-a gene within a hematopoietic stem cell. Selective clonal expansion of this population occurs under conditions of marrow failure, which is almost always aplastic anemia, and probably immune mediated. The resulting expansion of cells, which are deficient in all GPI-linked antigens, accounts for much of the clinical phenotype of the condition. In particular, the absence of two GPI-linked complement regulatory molecules, CD55 (DAF) and CD59 (MIRL), from erythrocytes is responsible for the increased sensitivity of patient red cells to activated complement and the ensuing bouts of intravascular hemolysis characteristic of the condition. Reliable and reproducible diagnosis of PNH is currently best achieved by flow cytometric analysis of GPI-linked membrane antigens. Absent or reduced expression of GPI-linked antigens, in the appropriate clinical setting, is diagnostic for PNH. As a clinical condition PNH can be difficult to manage, as thrombosis, severe cytopenias and acute hemolytic episodes are frequent but unpredictable occurrences. PNH is a rare condition and patients frequently remain undiagnosed for many months. However, the availability of a diagnostic flow cytometry assay means that in patients with suspected PNH, a definitive diagnosis can be rapidly established. This consequently has a considerable impact on patient management and prognosis.     

The patterns of MHC association in aplastic and non-aplastic paroxysmal nocturnal hemoglobinuria

The deficiency of glycosyl-phosphatidylinositol (GPI)-anchored proteins in plasma membranes of PIG-A gene mutated hematopoietic stem cells (HSCs) is so far insufficient to explain the domination of paroxysmal nocturnal hemoglobinuria (PNH) clone over the normal HSC. We attempted to elucidate possible link between MHC and initial severe aplastic anemia (ISAA/PNH) type and non-aplastic (n/PNH) outcome of PNH. In 50 PNH patients assigned as ISAA/PNH (n?=?13), n/PNH (n?=?33) or nonassigned (n?=?4) and 200 ethnically matched controls we analyzed MHC associations. Our data confirmed strong associations of DRB1*15:01 (RR?=?3.51, p?=?0.0011) and DQB1*06:02 (RR?=?7.09, p?=?0.000026) alleles, especially with n/PNH subtype. B*18:01 allele was associated with increased risk of ISAA/PNH subtype (RR?=?5.25, p?=?0.0028). We conclude that both class II and class I MHC alleles are associated with different subsets of PNH. Clonal selection of PIG-A mutated cells with cognate metabolic block is associated with MHC class II alleles DRB1*15:01 and DQB1*06:02 independent from initial severe AA clone selection. MHC class I molecule B*18:01 can additionally influence the domination of PNH clone in PNH subjects with initial severe aplastic anemia.