Aplastic anemia. Immunosuppressive therapy in a multi center trial in Japan

We evaluated the biologic characteristics and effectiveness of immunosuppressive agents in patients with a severe form of aplastic anemia in a multicenter study. Treatment with a bolus of methylprednisolone (mPSL) was associated with a good response and a partial response in 19.4% and 1.4% of the patients, respectively. In contrast, two kinds of antilymphocyte globulin (ALG) were effective for 9.4% and 15.6% of the severe form of aplastic anemia, and two kinds of antithymocytoglobulin (ATG) were effective in 30.8% and 37.2% of the patients, although the difference between ALG and ATG was not statistically significant. We recommend that patients diagnosed as having severe aplastic anemia should be referred as soon as possible for treatment with immunosuppressive agents or bone marrow transplantation, the latter depending on disease severity, age, and potential availability of an HLA-identical sibling donor.     

The thymus in patients with allogeneic bone marrow transplants.

The thymus glands from 11 patients with aplastic anemia or acute leukemia who received allogeneic bone marrow transplants were studied at autopsy. All showed marked cortical involution. In the short-term survivors the medulla and perivascular spaces were lymphocyte-depleted and the epithelial cells formed pseudorosettes. In those surviving over 2 months, increasing numbers of small lymphocytes were present, presumably reconstituted with donor lymphocytes. Phagocytosis of cellular debris was frequent, especially in patients with graft-versus-host reaction (GVHR) or treated with anithymocyte globulin (ATG). Plasma cells were numerous in perilobular tissue and were occasionally found within the medulla. The findings are compatible with the concept that the thymus plays an important role in the immune deficiency experienced after allogeneic bone marrow transplantation and in the subsequent lymphoid reconstitution.     

Treatment of aplastic anemia with cyclosporin A,methylprednisolone, and antithymocyte globulin

Summary Twenty-three patients with aplastic anemia (18/23 with severe aplastic anemia) were treated with an immunosuppressive regimen consisting of cyclosporin A (CsA) and methylprednisolone (MP) (n=7) or CsA, MP, and antithymocyte globulin (ATG;n=16). Nineteen patients are alive with a follow-up of 4 to 25 months; three patients died of infections and one of a gastrointestinal hemorrhage. Within 3 months, improvement of hematopoiesis was seen in 14 patients (61%). First signs of a response after 23 to 88 days were followed by complete remission in eight patients, partial remission in three patients, and minimal improvement in three patients. Two of the patients with only minimal improvement were treated with a second course of immunosuppression and reached a complete remission and partial remission. Interestingly, remission proved to be dependent on the continued administration of CsA in four of five patients with partial or complete remission who could be evaluated up to now. Thus, CsA must have been effective in the induction and/or maintenance of remission in three patients. This observation is a very strong argument for the role of T cells in the pathogenesis of at least some cases of aplastic anemia and warrants further evaluation of the role of CsA in the treatment of aplastic anemia.Abbreviations ALG Antilymphocyte globulin - ATG Antithymocyte globulin - CR Complete remission - CsA Cyclosporin A - MI Minimal Improvement - MP Methylprednisolone - NR No response - PR Partial remission - SAA Severe aplastic anemia - SGPT Serum alanine aminotransferase     

Evaluation of angiogenesis and vascular endothelial growth factor expression in the bone marrow of patients with aplastic anemia

It is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 +/- 0.7 events per field, versus controls, 5.9 +/- 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 +/- 35 pg/ml versus controls, 444 +/- 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression.     

Foamy cell syndrome associated with repeated platelet transfusions

Variable numbers of foamy cells (macrophages with foamy cytoplasm) were noted in generalized organs from four patients who received repeated were noted in generalized organs from four patients who received repeated platelet transfusions. The underlying disease in three cases was aplastic anemia, and the remaining case was chronic myelocytic leukemia. In two patients (aplastic anemia and chronic myelocytic leukemia) bone marrow transplantation (BMT) was done. Opportunistic infection was noted in three out of four cases. The foamy cells were stained black with Sudan black B. Variable amounts of materials immunoreactive with antihuman platelet antibody were demonstrated in most of the foamy cells. Ultrastructurally, the foamy cells contained myelin-like materials. The foamy cells described here resembled those demonstrable in the spleen from patients with idiopathic thrombocytopenic purpura. We suggest that the foamy appearance of the macrophage results from incomplete intracellular degradation of phagocytosed platelets.     

Severe aplastic anemia induced by ticlopidine: report of two cases.

Aplastic anemia is a rare side-effect associated with ticlopidine therapy. We report two cases of severe aplastic anemia developed after the use of ticlopidine. A 51-year-old woman took ticlopidine at 500 mg/day for 49 days to prevent a secondary stroke. She developed fever and dizziness within 49 days of initiating ticlopidine therapy. A 70-year-old woman was started on ticlopidine after coronary stent insertion. Fifty days after starting ticlopidine, she developed fever and dizziness. Both patients showed pancytopenia and were diagnosed as aplastic anemia which were confirmed by bone marrow examination. Both patients were hospitalized and received antibiotics, blood products and hematopoietic growth factors. Four and seven weeks after the withdrawal of ticlopidine, the hematologic parameters of each patient improved. A complete blood count should be monitored during ticlopidine therapy to check for cytopenia.     

Granulocyte-macrophage colony-stimulating factor in the sera of patients with aplastic anemia

Summary To clarify the role of growth factors in the pathophysiology of aplastic anemia we measured serum granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in 33 aplastic anemia patients by a specific and sensitive enzyme-linked immunosorbent assay. GM-CSF serum levels of patients with aplastic anemia were significantly higher than in healthy volunteers. GM-CSF levels were correlated with the severity of aplastic anemia but not with the absolute neutrophil count. Since T lymphocytes are one of the main sources of GM-CSF, our data provide further evidence for in vivo T lymphocyte activation in aplastic anemia. GM-CSF serum levels are higher in patients responding to immunosuppressive treatment than in nonresponders. Elevated serum GM-CSF might be predictive of a good response to immuno-suppressive therapy. GM-CSF serum levels are lower immediately after treatment with antilymphocyte globulin/antithymocyte globulin (ALG/ATG) than corresponding pretreatment values. Thus we cannot confirm the hypothesis that ALG/ATG effects in vivo are mediated by stimulating the release of growth factors. We conclude that in aplastic anemia the primary defect is a failure in GM-CSF response rather than in GM-CSF supply.Abbreviations AA aplastic anemia - ALG antilymphocyte globulin - ATG antithymocyte globulin - CyA cyclosporine A - ELISA enzyme-linked immunosorbent assay - Epo erythropoietin - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN- interferon- - IL interleukin - MP methylprednisolone - nSAA nonsevere aplastic anemia - rh recombinant human - SAA severe aplastic anemia - TNF- tumor necrosis factor- Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

Osteonecrosis of the hip in patients with aplastic anemia

The incidence and clinical and magnetic resonance imaging features of osteonecrosis of the hip were evaluated in patients with aplastic anemia. Two hundred and forty-one patients with aplastic anemia were examined using MR imaging of bone marrow during the five years from 1994 to 1998. Osteonecrosis of the hip was observed on MR imaging in nineteen (15 males and 4 females, mean age 35 yr) of the 241 patients. It was present in both hips in 14 patients, and there were five cases with unilateral occurrence, with a total of 33 involved hips. All except for five hips with associated bone marrow edema revealed increased fatty marrow conversion in the proximal femoral metaphysis. In nine patients, osteonecrosis was detected without any pain. Five patients already had osteonecrosis before any medication was administered. Twelve patients received antilymphocyte globulin, and seven patients received a low dose of steroids before the MR diagnosis of osteonecrosis. Osteonecrosis of the hip frequently develops in patients with aplastic anemia (7.9%), associated with fatty marrow conversion of the proximal femoral metaphysis.     

A study of bone marrow failure syndrome in children

Background: Bone marrow failure syndrome (BMFS), or aplastic anemia, includes peripheral blood single cytopenias, as well as pancytopenia due to inability of the marrow to effectively produce blood cells. Aim: To study the clinico-hematological profile and etiological factors of bone marrow failure syndrome in children. Setting and Design: This prospective study was carried out in the Department of Pediatrics of a university teaching hospital over 36 months. Materials and Methods: Children with pancytopenia (Hb 9 /L, platelet count < 100 x 10 9 /L) and bone marrow cellularity < 25% were included in the study. History of exposure to drugs, socioeconomic status, ethnicity and occupation of father were noted. Bone marrow aspiration; trephine biopsy; Ham test; viral studies for hepatitis A, B and C; and cytogenetic investigations were carried out. Statistical Analysis: Relative risk was estimated by odds ratio (OR) with 95% confidence interval (CI) in matched cases and controls. Results: Of the 53 children studied, 6 (11.3%) were diagnosed as Fanconi anemia. Two cases had features of myelodysplastic syndrome. Forty-five children were labeled as acquired aplastic anemia, of whom one had evidence of hepatitis B infection and two patients (5.8%) had paroxysmal nocturnal hemoglobinuria. Aplastic anemia was more common in children from family with lower socioeconomic status; in Muslims; and where the father's occupation was weaving, dyeing and painting. However, the number was small to make statistically significant conclusions. No correlation could be established with exposure to drugs. Conclusion: Fanconi anemia was responsible for approximately one-tenth of the cases of bone marrow failure syndrome. Majority of the patients had acquired aplastic anemia. Hepatitis B infection was an uncommon cause of acquired aplastic anemia.     

骨髓间充质干细胞对再生障碍性贫血患者T细胞的免疫抑制

背景：骨髓间充质干细胞对再生障碍性贫血患者T细胞增殖的影响国内报道较少,而骨髓间充质干细胞是否通过抑制T细胞增殖实现对再生障碍性贫血患者的免疫调节目前尚无定论。 目的：观察人骨髓间充质干细胞对再生障碍性贫血患者T细胞的免疫调节作用。 方法：体外分离培养、扩增人骨髓间充质干细胞并通过形态学特征以及流式细胞术进行表面标志鉴定,将骨髓间充质干细胞分别与正常人和再生障碍性贫血患者外周血提取的T淋巴细胞共培养7 d。应用ELISA法检测各培养上清液中T淋巴细胞分泌的白细胞介素2、γ-干扰素、白细胞介素4及白细胞介素10水平。 结果与结论：再生障碍性贫血组患者培养上清液中T淋巴细胞分泌的白细胞介素2、γ-干扰素水平明显高于正常人(P < 0.05),白细胞介素4、白细胞介素10低于正常人(P < 0.05)。骨髓间充质干细胞可下调白细胞介素2、γ-干扰素表达,同时上调白细胞介素4、白细胞介素10表达,从而调节再生障碍性贫血患者的免疫紊乱。     

Effect of interleukin 10 on the hematopoietic progenitor cells from patients with aplastic anemia.

The overproduction of cytokines with inhibitory effects on hematopoiesis is considered to play a role in the pathogenesis of aplastic anemia. While interleukin 10 (IL-10) is a cytokine production inhibitory factor, the possibility of immunosuppressive therapy using IL-10 for aplastic anemia has not been explored. In this study, therefore, we examined the effect of IL-10 on progenitor cells obtained from seven patients with severe aplastic anemia. Our study indicated that IL-10 dramatically enhanced the erythroid colony formation in a dose-dependent manner in two of the seven cases examined. When we examined the concentration of cytokines in the culture supernatants of unstimulated bone marrow cells, the spontaneous production of interferon-gamma (IFN-gamma) was observed in one of these two cases, and this production was completely inhibited by addition of IL-10. These findings suggested that IL-10 enhanced the erythroid colony formation by inhibiting the pathological production of IFN-gamma in this case. This study provides an experimental support for the clinical application of IL-10 in some patients with aplastic anemia.     

病毒性肝炎合并再生障碍性贫血的临床研究

目的 研究病毒性肝炎合并再生障碍性贫血的临床特征,探讨这类疾病的临床转归及治疗方法。方法 对2004年4月至2009年9月在地坛医院住院诊断为病毒性肝炎并且合并再生障碍性贫血的25例患者进行回顾性分析,本组病例中再生障碍性贫血诊断均通过骨穿确诊,收集患者的临床资料,包括肝病病史、药物过敏史、住院期间用药史、生化检查等等,对其进行描述性分析。结果 25例病毒性肝炎患者在经组织病理学诊断同时合并再生障碍性贫血,其中17例为男性,8例为女性;12例为慢性乙型肝炎患者,4例为慢性丙型肝炎患者,l例为急性戊型肝炎患者,l例为巨细胞病毒感染所致肝炎,7例为未分型病毒性肝炎患者,7例诊断为重型肝炎;仅有3例患者在入院前有干扰素短期使用史,其余患者未使用影响血象药物;治疗过程中使用集落细胞细胞刺激因子者6例,使用丙种球蛋白者9例,使用糖皮质激素者3例,使用促红素者1例,仅用口服药物升血细胞治疗者2例,输红细胞者6例,输血小板者2例;好转出院者20例,自动出院者3例,因重型肝炎其他合并症死亡者2例。结论 病毒性肝炎合并再生障碍性贫血的治疗以治疗原发性肝病及核苷类似物抗病毒治疗为主,血液病方面对症支持治疗,随肝病好转血液病逐渐同时恢复,预后较好。     

FOAMY CELL SYNDROME ASSOCIATED WITH REPEATED PLATELET TRANSFUSIONS

Variable numbers of foamy cells (macrophages with foamy cytoplasm) were noted in generalized organs from four patients who received repeated platelet transfusions. The underlying disease in three cases was aplastic anemia, and the remaining case was chronic myelocytic leukemia. In two patients (aplastic anemia and chronic myelocytic leukemia) bone marrow transplantation (BMT) was done. Opportunistic infection was noted in three out of four cases. The foamy cells were stained black with Sudan black B. Variable amounts of materials immunoreactive with antihuman platelet antibody were demonstrated in most of the foamy cells. Ultrastructurally, the foamy cells contained myelin-like materials. The foamy cells described here resembled those demonstrable in the spleen from patients with idiopathic thrombocytopenic purpura. We suggest that the foamy appearance of the macrophage results from incomplete intracellular degradation of phagocytosed platelets.     

Spontaneous clinical and cytogenetic remission of aplastic anemia in a patient with del(13q)

We describe the case of a 64-year-old Japanese man with pancytopenia. Bone marrow biopsy findings were consistent with aplastic anemia. The patient was treated by transfusions without immunosuppressive therapy. Chromosome analysis of bone marrow cells at 6 months after onset showed a 46,XY,del(13) (q14q22) karyotype. The pancytopenia resolved gradually over the next 5 years; chromosome analysis of bone marrow cells at that time yielded normal findings. To our knowledge, this is the first report of spontaneous hematologic and cytogenetic remission of aplastic anemia. These findings suggest that the abnormal clone with deletion of the long arm of chromosome 13 was not sufficient for clonal evolution in aplastic anemia in this case.     

The treatment of severe aplastic anemia: outcomes of bone marrow transplantation and immunosuppressive therapy in a single institution of Korea

The present study represents an analysis of 96 patients with severe aplastic anemia (SAA) treated in Seoul National University Hospital, Seoul, Korea between 1990 and 1999. Twenty-two patients were treated by allogeneic bone marrow transplantation (BMT) from HLA identical sibling donors and 74 by immunosuppressive therapy (IS) with antithymocyte globulin (ATG) or antilymphocyte globulin (ALG). There was no statistical difference between the two treatment groups in age, sex, disease duration, and previous transfusion amount. In the BMT group, grade II-IV acute graft versus host disease (GVHD) developed in 10% and chronic GVHD occurred in 33% of patient. Only one patient died from complication of transplantation (veno-occlusive disease). Of 74 patients who received IS treatment, 45% achieved a complete or partial response. Twenty patients died among IS treatment group. Major causes of death were hemorrhage (40%) and infection (55%). In the BMT group, the 5-yr overall survival (OS) was 95% after a median follow-up of 42 months. In the IS group, the 5-yr OS was 70% after a median follow-up of 49 months (p=0.04). In conclusion, the long-term survival rates of SAA in Koreans receiving BMT or IS were excellent compared with the Western data. Further evaluation on the prognosis of aplastic anemia in Asians should be done.     

Histological differences between sternal and iliac bone marrow in various hematological diseases

Even in normal state, hematopoietic activity is different in terms of the site of bone marrow as well as age. The activity may also differ with bone marrow site in various hematological diseases. We chose 32 cases with various hematological diseases and examined their bone marrow histologically at two different sites (sternum and posterior iliac crest) at almost the same time. We studied the histological differences in 5 cases of malignant lymphoma, 15 cases of aplastic anemia, 8 cases of myelodysplastic syndrome, 2 cases of myeloproliferative syndrome and 2 cases of acute leukemia. In malignant lymphoma, the iliac marrow was slightly hypoplastic compared with sternal marrow but there were no differences in the components of hematopoietic cells, that were thought to reflect normal hematopoietic activity for their respective ages. In 5 among 15 cases of aplastic anemia, nodular hyperplastic areas were observed in sternal marrow even though iliac marrow was severely hypoplastic. The presence of a nodular hyperplastic area is useful for differential diagnosis between aplastic anemia and refractory anemia, because such a histological change was not observed in the cases of refractory anemia. In myeloproliferative syndrome and acute leukemia, there were no differences between sternal and iliac marrow. They were hyperplastic and had almost the same hematopoietic components.     

Complete hematopoietic recovery after continuous iron chelation therapy in a patient with severe aplastic anemia with secondary hemochromatosis

A 16-yr-old male patient with hemochromatosis due to multiple packed red blood cell transfusions was referred to our emergency center for the treatment of severe aplastic anemia and dyspnea. He was diagnosed with aplastic anemia at 11-yr of age. He had received continuous transfusions because an HLA-matched marrow donor was unavailable. Following a continuous, approximately 5-yr transfusion, he was noted to develop hemochromatosis. He had a dilated cardiomyopathy and required diuretics and digitalis, multiple endocrine and liver dysfunction, generalized bleeding, and skin pigmentation. A total volume of red blood cell transfusion before deferoxamine therapy was about 96,000 mL. He received a regular iron chelation therapy (continuous intravenous infusion of deferoxamine, 50 mg/kg/day for 5 days q 3-4 weeks) for approximately seven years after the onset of multiple organ failures. His cytopenia and organ dysfunctions began to be gradually recovered since about 2002, following a 4-yr deferoxamine treatment. He showed completely normal ranges of peripheral blood cell counts, heart size, and liver function two years ago. He has not received any transfusions for the last four years. This finding suggests that a continuous deferoxamine infusion may play a role in the immune regulation in addition to iron chelation effect.     

多种来源造血干细胞移植治疗重型再生障碍性贫血

背景：造血干细胞移植是年轻重型再生障碍性贫血患者首选方法,但在中国多数重型再生障碍性贫血患者无合适的供者,单倍体相合或非血缘造血干细胞移植国内外目前还处于探索阶段,联合间充质干细胞移植报道少见。 目的：观察不同干细胞来源造血干细胞移植治疗重型再生障碍性贫血的疗效。 方法：10例(3~52岁)重型再生障碍性贫血患者,分别接受了亲缘HLA相合(2例),单倍体相合(5例),非血缘(3例)的外周血和/或骨髓造血干细胞移植,其中5例患者同时联合了间充质干细胞共移植。预处理方案主要为环磷酰胺、氟达拉滨和抗人胸腺球蛋白,以霉酚酸酯、环孢素A加短疗程的甲氨蝶呤预防移植物抗宿主病,单倍体相合移植的患者在此基础上加马利兰和CD25单克隆抗体;同基因的例5患者预处理方案为抗人胸腺球蛋白+甲基泼尼龙。输注间充质干细胞的量为(0.27~1.85)×10⁶/kg。接受和未接受间充质干细胞组的患者回输的造血干细胞有核细胞分别为(7.4~17.38)×10⁸/kg和(6.09~13.68)×10⁸/kg。 结果与结论：除1例单倍体相合患者移植未成功,+36 d死于并发症外,余患者移植后染色体及DNA指纹检测等说明造血干细胞移植完全供者植入。移植后中性粒细胞达到0.5×10⁹ L^-1,血小板计数≥20×10⁹ L^-1中位时间分别为12 d和13 d;其中造血功能恢复快慢的趋势是同基因移植>外周血或/和骨髓+间充质干细胞移植>单纯外周血或/和骨髓干细胞移植,而亲缘HLA全相合的52岁患者造血恢复最慢。非血缘移植例1、6患者发生了Ⅰ度急性移植物抗宿主病,单倍体相合移植的例2和例10患者发生了Ⅱ度急性移植物抗宿主病后出现了局限性的慢性移植物抗宿主病,余下患者移植后生活质量良好,无慢性移植物抗宿主病;除未接受间充质干细胞的例3患者移植后出现严重感染外,其余患者移植后再未出现严重的感染和出血。结果提示造血干细胞是安全,高效治疗重型再生障碍性贫血的方法,联合应用间充质造血干细胞者患者造血恢复快,移植并发症少。     

Antilymphocyte globulin (ALG) or antithymocyte globulin (ATG) with methylprednisone and oxymethalone in aplastic anaemia

From 1986 to 1994 we treated 26 patients of aplastic anaemia between 6 to 61 years age group with ATG/ALG, Methylprednisone and Oxymethalone. Five had very severe aplastic anaemia, 16 had severe and 5 nonsevere disease. Disease was associated with hepatitis in 5 patients and with pregnancy and drug use in 2 patients each. In others no cause could be ascertained. A total of 31 courses of treatment were given (range 1-3 courses per patient). Nine patients had complete response (34.62%) and 3 had partial response (11.54%) with an overall response rate of 46.16%. Four patients died within 2 months of starting the treatment. The median follow up was 24 months (range 6-102 months) with an overall survival probality of 45% at 2 yr. At the time of evaluation 12 patients have died, 9 are alive disease-free and 5 are alive with disease. The side effects associated with therapy were tolerable and did not require cessation of therapy in any patient. We conclude that ATG/ALG with Methylprednisone and Oxymethalone is beneficial to significant number of patients with aplastic anaemia.     

Immune-Mediated Hematopoietic Failure after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT) and often requires stem cell infusion from the same donor when the patient fails to respond to conventional therapies. We screened the peripheral blood (PB) of 14 patients who developed donor-type LGF at 2 to 132 months after allo-SCT for the presence of the markers for immune-mediated bone marrow (BM) failure. Increased glycosylphosphatidyl inositol-anchored protein-deficient (GPI-AP^-) leukocytes, which accounted for .009% to 0.147% of the total granulocytes, were detected in 5 patients (severe aplastic anemia, n?=?2; follicular lymphoma, n?=?1; acute lymphoblastic leukemia, n?=?1; myelodysplastic syndromes; n?=?1) and 4.7% to 81.2% HLA-allele–lacking leukocytes (HLA-LLs) were detected in 2 patients (acute myelogenous leukemia, n?=?1; and myelodysplastic syndromes, n?=?1). Three of the 5 patients with increased GPI-AP^- leukocytes were treated with antithymocyte globulin (ATG), and 2 patients achieved transfusion independence. These results suggest that immune mechanisms that are similar to acquired aplastic anemia underlie condition of approximately one-half of the patients with donor-type LGF, and that in patients with increased GPI-AP^- cells, donor-derived hematopoiesis may be restored by ATG therapy alone without donor stem cell infusion.