Abnormal marrow fibroblasts in aplastic anemia

Human bone marrow fibroblasts (BMF) were grown in vitro from normal (N) subjects and patients with aplastic anemia (AA). Growth studies in vitro revealed that both the N-BMF and AA-BMF had a logarithmic growth phase of eight days. Population doubling time for six of the 12 N-BMF and seven of the 12 AA-BMF was greater than 50 h. Five of the 12 AA-BMF studied in contrast to only two of the 12 N-BMF had a population doubling time of less than 50 h. The remaining four N-BMF had a population doubling time of greater than 100 h. During a similar duration in the logarithmic phase of growth, the AA-BMF underwent an average of 2.499 population doublings in comparison to 1.586 doublings by N-BMF (P = less than 0.01). The AA-BMF grew in multiple layers compared with the N-BMF, which usually grew as a monolayer. At the end of the logarithmic phase of growth, the AA-BMF also had a significantly higher number of cells per dish than the N-BMF (P = 0.02 on analysis of variance and covariance). These data suggest that a subgroup of AA-BMF grows faster than N-BMF and that the AA-BMF lack cell-to-cell inhibition. Testosterone, 3 alpha-etiocholanolone, and dexamethasone at 1 X 10(-8)M concentration, a physiological concentration, stimulated the growth of N-BMF as evidenced by increase in cell numbers and radioactive thymidine (3H-TdR) uptake. While dexamethasone had a stimulating effect on growth of N-BMF, it suppressed the growth of AA-BMF. Specific binding of radioactive dexamethasone (3H-dexa) was determined both for the N-BMF and AA-BMF. Specific binding sites for dexamethasone (Bmax) present on the N-BMF ranged from 460 to 770 fmol/mg protein). Bmax for AA-BMF was low (27-215 fmol/mg protein). In addition, the dissociation constant (Kd) was ten times lower for AA-BMF (1.0 X 10(-7) M) than for N-BMF (1.1 X 10(-8) M). The observations on the growth studies, the paradoxical response to dexamethasone, and the difference in the number of binding sites for dexamethasone indicate that the marrow fibroblasts from patients with aplastic anemia are abnormal.     

Epstein-Barr virus in the bone marrow of patients with aplastic anemia

STUDY OBJECTIVE: To determine whether Epstein-Barr virus is present in the bone marrow of patients with aplastic anemia. DESIGN: Assay of fresh and fixed bone marrow specimens for Epstein-Barr virus using immunofluorescence for nuclear antigen, Southern analysis with an Epstein-Barr virus specific probe, and in-situ hybridization. SETTING: Governmental medical referral center. PATIENTS: Five patients were studied prospectively: three who previously had infectious mononucleosis, one with a recent viral pneumonitis, and one who was asymptomatic. Stored DNA samples from other patients with aplastic anemia were also screened. MEASUREMENTS AND MAIN RESULTS: Epstein-Barr virus DNA and protein were detected in the bone marrow of 5 patients studied prospectively and in 1 of 40 patients studied retrospectively. As estimated by in-situ hybridization, about 3% to 5% of marrow cells were infected with virus in those patients who had not received acyclovir. In contrast, Epstein-Barr virus DNA was not detected in peripheral blood DNA of these patients, nor were Epstein-Barr virus proteins or DNA found in the bone marrow of normal donors, patients with other hematologic diseases, or in 1 patient with acute infectious mononucleosis. Analysis of DNA fragments by hybridization with Epstein-Barr virus probes showed a pattern dissimilar to the type of Epstein-Barr virus usually associated with infectious mononucleosis. CONCLUSIONS: Aplastic anemia may be associated with Epstein-Barr virus more commonly than suspected by history. Localization of the virus in the bone marrow supports a causative role for Epstein-Barr virus in bone marrow failure.     

Methimazole-induced aplastic anemia caused by hypocellular bone marrow with plasmacytosis.

Aplastic anemia is a rare but severe complication of methimazole (MMI) treatment for Graves' disease. We present a case of a 53-year-old Japanese female who had been treated with 30 mg/d of MMI for 30 days for Graves' disease and was subsequently admitted to the Japan Self Defense Forces (JSDF) Central Hospital with a mild sore throat and high-grade fever that began the previous day. The patient had a reduced white blood cell count (WBC) count of 0.9 x 10(3) per microliter with severe granulocytopenia and increased lymphocytes, a platelet count of 49 x 10(3) per microliter, and hemoglobin of 10.6 g/dL. Bone marrow (BM) aspirates showed hypocellular bone marrow with plasmacytosis. Because of poor recovery of her peripheral blood values after withdrawal of MMI, she was given transfusions of platelets and erythrocytes thereafter. This is the second report of plasmacytosis in bone marrow of MMI-induced aplastic anemia, and suggests that immunogenic mechanisms may cause this rare complication.     

再生障碍性贫血全身骨髓显像与预后的分析

目的 :利用99Tcm 硫胶体骨髓显像对不同类型再生障碍性贫血 (再障 )显影骨髓进行半定量分析 ,并探讨与预后的关系。方法 :观察 2 7例重型再障 (SAA)及 2 5例慢型再障 (CAA) 99Tcm 硫胶体全身骨髓显像分布类型 ,按骨髓分布及活性分级计算显影骨髓量值 (M ) ,一年内重复显像并随访 3年。结果 :SAA全身骨髓分布不均匀及灶状增生现象少见 ,骨髓不显影及总量减少者占 73 .1 % ,SAA Ⅰ型及SAA Ⅱ型骨髓M值分别为 1 .39±1 .68;1 .81± 2 .0 2 ,均明显低于CAA(3 .91± 1 .39;t =5 .2 5及 4 .2 0 ,P <0 .0 0 1 ) ,1年内骨髓分布类型及显影骨髓量无明显改变。当M值 >3时 ,患者 3年治疗有效率为 80 .0 % ;当M值≤ 3时 ,患者 3年治疗有效率为 1 4 .8% ;M值≤ 1时 ,患者死亡率占 93 .3 %。结论 :结合M测定及显像骨髓分布特点有助于再障分型的进一步确定 ,并对估计患者的预后有指导意义     

Pregnancy after bone marrow transplantation for severe aplastic anemia

Two women suffering from severe aplastic anemia were treated by bone marrow transplantation (BMT). They became pregnant 17 and 40 months after the procedure. During the first 20 weeks of pregnancy, one of them continued to receive cyclosporin A (CSA) to prevent graft-versus-host disease. Her CSA serum levels during these first 4 months ranged from 35 to 280 ng/ml. The course of pregnancy was uneventful and a healthy boy was delivered at term by Cesarean section. The other patient was shown to have rejected the transplanted marrow 4 months after BMT; autologous regeneration had occurred. During the last 2 months of her pregnancy, the hemoglobin and platelet numbers decreased again. Nevertheless, a healthy girl was delivered at term. Three months after delivery this patient's hematologic parameters are almost back to normal.     

Current status of allogeneic bone marrow transplantation in acquired aplastic anemia

Bone marrow transplantation is an effective therapy for aplastic anemia. Infusion of allogeneic hematopoietic stem cells after high-dose immune suppression restores normal hematopoiesis in most patients and long-term follow-up has confirmed the durability of donor hematopoiesis. However, success of this approach is limited by transplant-related complications, such as graft failure, graft-versus-host disease, and various organ toxicities. Long-term survival rates range from less than 40% to more than 90% in reported series. These rates have improved over the past 20 years due to significant reductions in graft-versus-host disease, interstitial pneumonitis, and early transplant-related mortality. Most long-term survivors have excellent performance status. Late effects such as cataracts, thyroid disorders, joint problems, and therapy-related cancers are observed, especially in patients who received radiation for pretransplant conditioning. Results are best in young patients transplanted with bone marrow from a human leukocyte antigen (HLA)-identical sibling; early transplantation is appropriate in this group. For older patients or those without an HLA-identical related donor, transplants are better reserved for those who fail to respond to immunosuppressive therapy.     

Selection of patients for bone marrow transplantation in severe aplastic anemia

Despite androgens and intensive supportive care, satisfactory survival in severe aplastic anemia remains at 20% or less. Histocompatible bone marrow transplantation can restore normal hematopoiesis in approximately 40% of similarly severe individuals. Delay of transplantation for 3 wk after diagnosis allows time for proper evaluation and for many spontaneous recoveries. Further delay increases risks of fatal complications and decreases chances for successful transplantation while the incidence of spontaneous remission declines. When available, early histocompatible bone marrow transplantation may be the treatment of choice for severe aplastic anemia.     

Long-term outcome after bone marrow transplantation for severe aplastic anemia

From January 1978 to December 2001, 133 patients with severe aplastic anemia (SAA) underwent non-T cell-depleted allogeneic bone marrow transplantation from an HLA-identical sibling donor, at the Hospital Saint Louis using either the combination of cyclophosphamide (Cy) and thoracoabdominal irradiation (TAI; n=100) or Cy and antithymocyte globulin (ATG; n=33), as a conditioning regimen. With 13.6 years of follow-up, the 10-year survival estimate was 64%. Four factors were associated with lower survival: older age, use of Cy-TAI, any form of treatment prior to transplantation (either androgens or immunosuppressive therapy, [IST]), and grade II to IV acute graft-versus-host disease (GvHD). TAI was the sole factor associated with the occurrence of acute GvHD. The risk of cancers (15-year cumulative incidence, 10.9%) was associated with older age and with the use of cyclosporine as IST before transplantation. Cumulative incidences and risk factors of nonmalignant late effect including avascular osteonecrosis and late bacterial, viral, and fungal infection were also analyzed. Improved results using Cy-ATG as conditioning can lead to more than 90% chance of cure in patients with SAA. Even if, in our experience, the role of Cy-ATG versus that of Cy-TAI remained inextricably related to the year of transplantation, the major detrimental role of the GvHD disease in the long-term outcome and its relation to TAI supports avoidance of irradiation in the conditioning regimen. Furthermore, avoidance of any IST before transplantation in patients with a sibling donor is a prerequisite for attaining such excellent results.     

再生障碍性贫血的骨髓和细胞病理

目的：研究再生障碍性贫血（AA）患者骨髓组织病理及有核细胞的超微结构变化。方法：光镜观察20例AA患者骨髓活检病理,透射电镜分析骨髓有核细胞超微结构。结果：光镜下所有患者骨髓脂肪组织增多,造血面积减小,大部分存在血浆渗出、出血和纤维细胞局灶性增生,血窦血管结构紊乱。电镜观察显示原始和早幼红细胞代偿性不典型增生,中晚幼红细胞核损伤。粒细胞呈活化和损伤状态;巨核细胞显著减少,胞浆灶性坏死;单核细胞异常增生、活化、吞噬和坏死。淋巴细胞大部分结构正常。结论：AA患者骨髓造血细胞减少和炎症反应同时存在,单核巨噬细胞活化和吞噬反应与造血细胞损伤相关。     

Disparate regulation of human fetal erythropoiesis by the microenvironments of the liver and bone marrow

The liver and the bone marrow (BM) are the major organs that support hematopoiesis in the human fetus. Although both tissues contain the spectrum of hematopoietic cells, erythropoiesis dominates the liver. Previous studies suggested that a unique responsiveness of fetal burst-forming units erythroid (BFU-E) to erythropoietin (EPO) obviates the need for cytokines with burst-promoting activity (BPA) in fetal erythropoiesis. This potential regulatory mechanism whereby fetal erythropoiesis is enhanced was further investigated. Fluorescence-activated cell sorting was used to isolate liver and BM progenitors based on their levels of CD34 and CD38 expression. The most mature population of CD34+ lineage (Lin-) cells was also the most prevalent of the three subpopulations and contained BFU-E responsive to EPO alone under serum-deprived conditions. Kit ligand (KL) also strongly synergized with EPO in stimulating the growth of these BFU-E. An intermediate subset of CD34++CD38+Lin- cells contained erythroid progenitors responsive to EPO alone, but also displayed synergism between EPO and KL, granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-3, demonstrating that erythroid progenitors that respond to cytokines with BPA do exist in fetal tissues as in the adult BM. Candidate stem cells (CD34++CD38-Lin- cells) did not respond to EPO. Synergisms among KL, GM-CSF, and IL-3, and to a lesser extent granulocyte colony-stimulating factor (G-CSF) and FLK-2/FLT-3 ligand (FL), supported the growth of primitive multipotent progenitors that became responsive to EPO. These data define the limits of EPO activity in fetal erythropoiesis to cells that express CD38 and demonstrate the potential for various cytokine interactions to be involved in regulating fetal erythropoiesis. Furthermore, a comparison of the responses of liver and BM erythroid progenitors revealed similarity in their responses to cytokines but a difference in the frequency of BFU-E among the three subpopulations examined. A higher frequency of BFU-E among the intermediate and late progenitor subsets in the liver indicates that regulatory factors acting on stem cells and their immediate progeny are partially responsible for the high content of erythropoiesis in the liver. These data implicate a critical role for the microenvironments of the liver and BM in regulating the disparate levels of erythropoiesis in these tissues.     

Severe autoimmune thrombocytopenia after allogeneic bone marrow transplantation for aplastic anemia

Autoimmune thrombocytopenia (AITP) after bone marrow transplantation (BMT) was suggested to occur by immune dysregulation mainly in association with graft-versus-host disease (GVHD). Here we present a patient who developed severe AITP after BMT. A 40-year-old woman with severe aplastic anemia received a BMT from a partially HLA-matched brother. Despite myeloid and erythroid engraftments, platelet recovery was delayed. All bone marrow cells were 46,XY and were derived from the donor. Grade I acute GVHD involving skin developed from day 34 posttransplantation, but promptly responded to prednisolone in addition to a prophylactic dose of tacrolimus. With the tapering of prednisolone, thrombocytopenia progressed without substantial changes in the white blood cell count, hemoglobin concentration, or reticulocyte count. On day 188, the patient developed chronic GVHD involving skin and liver, which promptly responded to the readministration of prednisolone and increased tacrolimus. However, the patient's platelet count decreased to 9 x 10(9) cells/L on day 222. The platelet-associated immunoglobulin G (PAIgG) values were elevated. Bone marrow examination showed hypercellularity with plentiful megakaryocytes. The number of colony-forming units-megakaryocyte was within the normal range. The elevated PAIgG values and a correlation between thrombocytopenia and the intensity of the immunosuppressive agents strongly suggested a causative role of the autoimmune mechanisms for thrombocytopenia in this patient.     

Fulminant hepatic failure following bone marrow transplantation for hepatitis-associated aplastic anemia

A case of aplastic anemia associated with non-A, non-B hepatitis was initially successfully treated by bone marrow transplantation. The patient subsequently developed fulminant hepatic failure. Fulminant hepatic failure is rare in bone marrow transplantation and only occurs in association with aplastic anemia associated with viral hepatitis. This case helps to highlight the relationships between the immune system, hepatitis, and bone marrow failure.     

T-lymphocyte subpopulations in the peripheral blood and bone marrow of patients with aplastic anemia

Summary A decrease in the absolute number of total lymphocytes, OKT3⁺ and OKT4⁺ lymphocytes, and a normal number of OKT8⁺ lymphocytes were found in the peripheral blood of patients with aplastic anemia. The OKT4: OKT8 ratio was decreased in patients due to a reduction in the percentage of OKT4⁺ cells and 3 out of 18 patients had a ratio less than 1. The values of the OKT4:OKT8 ratio were not associated either with the severity of the disease or with treatment with androgens. There was no correlation between the OKT4:OKT8 ratio and the number of transfusions received by patients. On the other hand, studies performed with bone marrow lymphocytes showed that the OKT4:OKT8 ratio for both patients and controls was lower than that of the peripheral blood. Since the ratio of OKT4:OKT8 cells in aplastic and control bone marrow was similar no direct pathogenic role can be assigned to the marrow for the imbalance detected in the peripheral blood.     

Myelopoiesis and erythropoiesis of bone marrow cells cultured in vitro in patients recovered from aplastic anaemia

Methylcellulose culture assay was used to detect committed haemopoietic stem cells, CFU-C and CFU-E, in aplastic anaemia patients with autologous haemopoietic reconstitution. Severe diminution of CFU-C was found in all the patients studied and the absence of a dose-response to colony stimulating factor (CSF) was demonstrated. A reduced number of CFU-E and lower erythropoietin (Ep) sensitivity of those progenitors was detected as well. Autologous serum added to the bone marrow cultures of these patients enhanced the growth of CFU-C but inhibited CFU-E growth. According to the results presented, some residual damage at the stem cell level is suggested.     

Characterization of T-cell repertoire of the bone marrow in immune-mediated aplastic anemia: evidence for the involvement of antigen-driven T-cell response in cyclosporine-dependent aplastic anemia

To determine whether the antigen-driven T-cell response is involved in the pathogenesis of aplastic anemia (AA), we examined the complementarity-determining region 3 (CDR3) size distribution of T-cell receptor (TCR) beta-chain (BV) subfamilies in the bone marrow (BM) of untreated AA patients. AA patients who did not respond to immunosuppressive therapy and those who obtained unmaintained remission early after cyclosporine (CyA) or antithymocyte globulin (ATG) therapy exhibited essentially a normal CDR3 size pattern. In contrast, five patients who needed continuous administration of CyA to maintain remission exhibited a skewed CDR3 size pattern in a number (>40%) of BV subfamilies suggestive of clonal predominance. The skewing of CDR3 size distribution became less pronounced in one of the CyA-dependent patients when the patient achieved unmaintained remission after a 4-year therapy with CyA, whereas it persisted longer than 7 years in the other patient requiring maintenance therapy. Sequencing of BV15 cDNA for which the CDR3 size pattern exhibited apparent clonal predominance in all CyA-dependent patients showed high homology of the amino acid sequence of the CDR3 between two different patients. These findings indicate that antigen-driven expansion of T cells is involved in the pathogenesis of AA characterized by CyA-dependent recovery of hematopoiesis.