Human long-term bone marrow cultures in aplastic anemia

Long-term bone marrow cultures (LTMC) were initiated with marrow from five normal subjects and eight patients with aplastic anemia (AA). Near confluent to confluent adherent layers developed in all cultures from normal subjects and AA patients. When present, the 'cobblestone' areas in LTMC from AA subjects were smaller than those observed in the LTMC from normal subjects. The decline in total and viable cell numbers in the LTMC was similar for both normal subjects and AA patients. Granulocyte-macrophage colony-forming units (CFU-gm) were present in nonadherent cells (NAC) from normal LTMC for a mean of 5.2 weeks. CFU-gm were present in the NAC of only two of the eight AA cultures for one week. The absent or small 'cobblestone' areas and the absence of CFU-gm production in AA-LTMC suggest a decrease in the reproductive potential of adherent hematopoietic stem cells, which may be the result of either an abnormal hematopoietic stem cell or an abnormal stromal microenvironment or both.     

Bone marrow transplantation for leukemia and aplastic anemia: management of ABO incompatibility.

Between February 1971 and October 1980, 34 patients with leukemia or aplastic anemia received bone marrow transplants from HLA-identical siblings whose lymphocytes did not react in a mixed leukocyte culture. The donors of 10 patients were ABO-incompatible, and for five pairs the ABO incompatibility was major. Plasma exchanges followed by a red blood cell exchange transfusion reduced the anti-A titres to 1:4 or less in these patients. The ABO incompatibility had no adverse effect on the results of marrow transplantation. Twenty-two patients, including 16 of the 20 who received their transplant after Jan. 1, 1980, are still living. Seven of the 15 patients with acute leukemia have survived 89 to 466 days, and 4 of the 6 with chronic myelogenous leukemia (CML) have survived 117 to 545 days. Of the 13 patients with aplastic anemia, 11 are alive up to 8 years after transplantation. Marrow transplantation, when possible, is the treatment of choice for young patients with acute leukemia in remission and for patients with aplastic anemia. Marrow transplantation may also prove to be effective in patients with CML.     

The value of cell cultures in leukemias, aplastic anemias and bone marrow transplantations

In a survey different methods of culture are represented for the purpose of identifying and quantifying haemopoietic stem cells (CFU-GEMM, CFU-GM, CFU-E/BFU-E) in human bone-marrow or peripheral blood respectively. On the basis of findings from international medical literature their validity is explained in the diagnostics and prognosis of some haematological diseases, such as acute and chronic myeloic leukemia, aplastic anemia, preleukemia. Special attention is given to their significance within bone-marrow transplantation. Their importance in evaluating transplantations after their preceding in-vitro manipulation as to the separation of rest tumour and T-cells is particularly referred to.     

Analysis of cytotoxic effector cell function in patients with leukemia or aplastic anemia before and after marrow transplantation

Cytotoxic effector cells mediating natural killing (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC) were studied in patients with leukemia or aplastic anemia before and/or after marrow transplantation. Before transplantation, about one-third to one-half of the patients were deficient in cytotoxic activity. In patients with leukemia, this was most likely due to large numbers of circulating blast cells diluting or replacing the effector cells. In patients with aplastic anemia there was an apparent absence of the effector cells in a proportion of the patients. After marrow transplantation, cytotoxic activity in all three systems returned to normal rapidly, by 30 days, and remained so through 100 days. However, about 20% of patients studied beyond 1 year were deficient in these functions. There were no significant associations between cytotoxic activity and important clinical parameters including infections, graft-vs-host disease, and recurrence of leukemia. Our findings do not support an immunosurveillance role for NK against leukemia after marrow transplantation. Furthermore, they point out the need for new in vitro approaches for meaningful monitoring of marrow transplant patients. Finally, our results showed a significant discordance between NK, ADCC, and LDCC activities in these immunologically perturbed individuals, indicating that either different cell populations or different cellular mechanisms are involved in these cytotoxic functions.     

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

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.     

Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia

Aplastic anemia or bone marrow failure often develops as an effect of chemotherapeutic drug application for the treatment of various pathophysiological conditions including cancer. The long-term bone marrow injury affects the basic hematopoietic population including hematopoietic stem/progenitor cells (HSPCs). The present study aimed in unearthing the underlying mechanisms of chemotherapeutics mediated bone marrow aplasia with special focus on altered redox status and associated effects on hematopoietic microenvironment and epigenetic status of hematopoietic cells. The study involves the development of busulfan and cyclophosphamide mediated mouse model for aplastic anemia, characterization of the disease with blood and marrow analysis, cytochemical examinations of bone marrow, flowcytometric analysis of hematopoietic population and microenvironmental components, determination of ROS generation, apoptosis profiling, expressional studies of Notch-1 signaling cascade molecules, investigation of epigenetic modifications including global CpG methylation of DNA, phosphorylation of histone-3 with their effects on bone marrow kinetics and expressional analysis of the anti-oxidative molecules viz; SOD-2 and Sdf-1. Severe hematopoietic catastrophic condition was observed during aplastic anemia which involved peripheral blood pancytopenia, marrow hypocellularity and decreased hematopoietic stem/progenitor population. Generation of ROS was found to play a central role in the cellular devastation in aplastic marrow which on one hand can be correlated with the destruction of hematopoiesis supportive niche components and alteration of vital Notch-1 signaling and on other hand was found to be associated with the epigenetic chromatin modifications viz; global DNA CpG hypo-methylation, histone-3 phosphorylation promoting cellular apoptosis. Decline of anti-oxidant components viz; Sdf-1 and SOD-2 hinted towards the irreversible nature of the oxidative damage during marrow aplasia. Collectively, the findings hinted towards the mechanistic correlation among ROS generation, microenvironmental impairment and epigenetic alterations that led to hematopoietic catastrophe under aplastic stress. The findings may potentiate successful therapeutic strategy development for the dreadful condition concerned.     

Agranulocytosis, aplastic anemia, and leukemia: relevance to arachidonic acid metabolism

Several agents including drugs, chemicals and viruses are known to induce agranulocytosis, aplastic anemia, and leukemia. The recent identification, characterization and cloning of several peptide regulatory factors, including granulocyte-macrophage-colony stimulating peptide regulatory factor (GM-CSF), erythropoietin, and interleukins and a study of their actions, suggest that agents producing agranulocytosis, aplastic anemia, and leukemia may interfere with the action of these factors. The agents that are capable of inducing these diseases and the various peptide regulatory factors have positive and/or negative actions on the prostaglandin system. Prostaglandins are known to be involved in the maturation and differentiation of the progenitor cells of the bone marrow and in erythropoietin-mediated erythropoiesis. Since prostaglandins influence immune response, modify genetic damage induced by drugs and chemicals, modulate gene action, and have feed-back control on the actions of peptic regulatory factors, it is likely that prostaglandins are involved in the pathogenesis of agranulocytosis, aplastic anemia, and leukemia. If so, this may lead to new therapeutic strategies in these hematological conditions.     

Effect of Danggui Buxue Tang on immune-mediated aplastic anemia bone marrow proliferation mice

To investigate the pharmacological effects of Danggui Buxue Tang (DBT) on immune-mediated aplasia anemia mice. The model of immune-mediated aplasia anemia mice was induced by means of ⁶⁰Co γ-ray irradiation and mixed cells of thymus and lymphnode of DBA/2 mice infusion through tail vein, the parameters tested indices were as following: blood picture, bone marrow nucleated cell count (BMNC), murine colony-forming unit-megakaryocytes (CFU-GM) of bone marrow cells, murine colony-forming unit-erthroid (CFU-E) and burst forming unit-erythroid (BFU-E). The results showed that DBT could not only withstand significantly decreation of blood cells by immune-mediated, but also stimulate on the growth of bone marrow colony cell and increase the weight of hemopoietic progenitor of bone marrow. Therefore, DBT had an obvious treat effect on immune-mediated aplasia anemia models mice.     

Prognosis in acquired aplastic anemia. An approach in the selection of patients for allogeneic bone marrow transplantation.

In a retrospective study, factors influencing the eventual course and outcome of patients suffering from aplastic anaemia were analysed. Of the 62 patients with pancytopenia concomitant with an aplastic or hypoplastic bone marrow, 36 have died during the first 24 months following diagnosis, 27 of these 36 cases within the first six months. 26 patients survived more than two years after presentation, but a further six of these cases succumbed during the following months. Of the 62 cases, 46 had neutrophil counts under 1 X 10(9)/l and platelet counts under 20 X 10(9)/l. These cases were subdivided according to their reticulocyte levels. In the age group of patients between 10 and 35 years, all those patients having more than 10,000/mul reticulocytes survived more than two years with only one exception, whereas only one of the 11 patients having reticulocyte counts below 10,000/mul has survived. In our series, the initial neutrophil and platelet counts were not of predictive value in identifying poor prognosis patients. The initial reticulocyte counts, however, appear to be a valid parameter for selecting patients in two groups, one with a favorable, and the other with an extremely unfavorable prognosis on conservative treatment. Young individuals with a poor predicted prognosis could thus be defined as possible candidates for bone marrow transplantation.     

Icariin protects bone marrow mesenchymal stem cells in aplastic anemia by targeting MAPK pathway

Molecular Biology Reports - Icariin, the main pharmacological active flavonoid extracted from Epimedi herba, can regulate cellular processes in diverse diseases. The aim of this study was to...     

Monosomy 7 in donor cell-derived leukemia after bone marrow transplantation for severe aplastic anemia: Report of a new case and review of the literature

Monosomy 7 arises as a recurrent chromosome aberration in donor cell leukemia after hematopoietic stem cell transplantation. We report a new case of donor cell leukemia with monosomy 7 following HLA-identical allogenic bone marrow transplantation for severe aplastic anemia (SAA). The male patient received a bone marrow graft from his sister, and monosomy 7 was detected only in the XX donor cells, 34 months after transplantation. The patient’s bone marrow microenvironment may have played a role in the leukemic transformation of the donor hematopoietic cells.     

Bone marrow transplantation in a patient with drug-induced aplastic anemia

A 23-year-old woman gravely ill with Pseudomonas septicemia secondary to presumed drug-induced bone marrow aplasia received marrow transplantation from two male HL-A identical sibling donors. She had a successful engraftment with excellent but temporary clinical improvement. Subsequently she succumbed to graft-versus-host disease manifested by Pseudomonas and Candida albicans septicemia, cytomegalovirus pneumonitis, three phases of dermatitis, nausea, vomiting, dysphagia, diarrhea, fever, edema and bone pain, with gradual but complete graft suppression by the 74th day after the transplantation. A second marrow transplant on the 70th day was unsuccessful.     

Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma.

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.     

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.