Aplastic Anaemia with Marrow Defective in Formation of Fibroblastoid Cell Colonies in Vitro

The formation of fibroblastoid colonies by marrow cells in vitro has been used as a putative assay for a stem cell of haemopoietic stroma. Bone marrow from one patient with aplastic anaemia did not form any of these colonies, while its growth in diffusion chambers as an indirect measure of a haemopoietic stem cell was even better than normal. On the other hand, marrow from the other aplastic anaemia patient showed only quantitative decrease in the formation of fibroblastoid colonies and simultaneously grew very poorly in diffusion chambers. These patients were indistinguishable by the cytological examination of their marrow, however, the peripheral blood abnormalities were expressed less severely in the first patient. These results suggest the contribution of the defect in marrow cells, which form fibroblastoid colonies in vitro to the development of aplastic anaemia in these patients.     

Stem Cell Transplantation for Aplastic Anemia

Survival of patients with aplastic anemia treated with transplantation of bone marrow has improved significantly over the past several decades. Allogeneic bone marrow transplantation (BMT) for patients with HLA-identical siblings is now the first-line therapy, and long-term survival of approximately 90% can be expected with cyclophosphamide/antithymocyte globulin conditioning and postgrafting methotrexate/cyclosporine immunosuppression. The outcome of unrelated donor BMT has also improved significantly with the identification of a preparative regimen with less toxicity combined with the development of high-resolution DNA-based HLA typing to identify the optimal unrelated marrow donor. Patients with fully HLA-matched unrelated donors should be considered candidates for transplantation prior to exposure to repeat courses of immunosuppression. Future progress in hematopoietic stem cell transplantation for aplastic anemia will be directed toward further decreasing the acute toxicity and decreasing the delayed effects of the conditioning regimens while maintaining highly reliable rates of sustained engraftment with prevention of acute and chronic graft-versus-host disease.     

The Colony Forming Cell in the Myeloproliferative Disorders and Aplastic Anaemia

Bone marrow colony forming cell (CFC) concentration and the proportion of CFC in DNA synthesis were studied in myeloproliferative disorders and aplastic anaemia. Growth patterns of bone marrow cells in agar cultures were able to supplement traditional morphological and clinical criteria in the diagnosis of these haematolog-ical conditions. Bone marrow CFC concentration tended to be increased in chronic myeloid leukaemia (CML) and polycythaemia vera (PV), but decreased in myelo-fibrosis, erythroleukaemia, paroxysmal nocturnal haemoglobinuria (PNH) and the aplastic phase of aplastic anaemia. The proportion of CFC in DNA synthesis was decreased in CML, myelofibrosis and aplastic anaemia, but increased in blastic transformation, PV, PNH and during regeneration from aplastic anaemia. The proportion of CFC in DNA synthesis in bone marrow from patients with CML in blastic transformation was directly related to the percentage of myeloblasts in the bone marrow. CFC kinetics in blastic transformation have been demonstrated to be different from those in acute leukaemia.     

Lymphocyte DNA in Aplastic Anaemia

Using ultracentrifugation on alkaline sucrose density gradients the DNA of lymphocytes from 14 patients with aplastic anaemia and 23 controls was studied before and after exposure to bleomycin, an agent known to cause strand breaks in DNA. Before exposure to bleomycin the DNA from aplastic patients had more strand breaks than the DNA from the controls of similar ages. Following exposure to bleomycin an abnormal number of DNA strand breaks was produced in 10 of 14 patients and this molecular evidence of drug sensitivity correlated well with the sensitivity of the proliferating lymphocytes to bleomycin in tissue culture. Furthermore, two relatives of patients with aplastic anaemia showed similar abnormality of DNA before and after exposure to bleomycin and increased sensitivity to bleomycin in tissue culture. These results suggest that an abnormality of DNA structure and/or repair may be present in some patients with aplastic anaemia.     

Immune Complexes in Aplastic Anaemia

S ummary . Immune complexes (IC) have been detected in nine out of 15 patients presenting with idiopathic aplastic anaemia using the polymorphonuclear neutrophil immunohistochemical technique. Immunosuppressive treatment undertaken in one patient produced a gradual recovery, the bone marrow repopulation being paralleled by the disappearance of IC. The significance of IC in aplastic anaemia and the relationship with possible pathogenesis and therapy of the disease are discussed.     

Acquired Aplastic Anaemia in Adults

A follow-up study of 10 patients suffering from acquired aplastic anaemia, comprising methacrylate-embedded bone marrow biopsies and CFU cultures, is presented. The haematopoietic recovery patterns and changes in the inflammatory infiltration after permanent engraftment could be distinguished from those in non-transplanted patients. After anti-thymocyte globulin treatment followed by allogeneic bone marrow infusion, the recovery pattern resembled that in non-transplanted patients. The persistently low colony-forming capacity in some patients could be explained by the existence of lymphoid inhibitory cells, which suggests an immunologic auto-destructive mechanism.     

K-Cell Activity in Aplastic Anaemia

K-cell activity was measured by specific cytotoxicity and cytotoxic capacity in 29 patients with aplastic anaemia. 9 patients had a reduction of the specific cytotoxicity and 12 had a decreased cytotoxic capacity. The decreased cytotoxic capacity correlates with the severity of aplastic anaemia. Some of the possible causes of the reduction of this activity were investigated.     

Severe Aplastic Anaemia: Correlation of in Vitro Tests with Clinical Response to Immunosuppression in 20 Patients

S ummary . Colony formation in agar (CFU-c) was studied in 20 patients with severe aplastic anaemia by three different assays: (1) cultures of light density untreated marrow cells; (2) cultures of marrow cells manipulated in order to enhance colony formation (pretreatment with antilymphocytic globulin, ALG, or 6-methylprednisolone, 6-MPr, T cell depletion, adherent cell (AC) depletion, depletion of both AC and T cells), and (3) co-culture of putative suppressor T cells with autologous T-depleted marrow cells.
By the first assay, all patients showed poor colony formation (1 1.5 colonies/10⁵ cells; normal controls 46.18 colonies/10⁵ cells). By the second assay, ALG and 6-MPr had no significant effect on colony formation. Removal of adherent cells proved equally without effect on colony growth. On the contrary, removal of T cells enhanced significantly ( P < 0.001) colony formation in 10 out of 20 patients. By the third assay, colony formation of marrow cells (deprived of T lymphocytes) was inhibited by the addition of autologous T cells in six patients studied.
All patients were given high dose bolus 6-MPr as first treatment on admission: only patients who had detectable suppressor T cells in their marrow achieved a complete autologous haematologic reconstitution after 6-MPr or after 6-MPr and ALG.
The results of this study indicate that detection of CFU-c/suppressor T cells correlates with responses to immunosuppressive regimens, and may thus help to identify patients with immune mediated aplastic anaemia.     

Granulocytic Progenitor Cells in Aplastic Anaemia

The characteristics and the concentration of granulopoietic colony forming cells (CFC) were examined in 22 patients with aplastic anaemia at different stages of their disease. Additionally the ability of the patients' peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (CSA) was studied. The ability of the patients' cells to generate CSA was shown to be unaffected. However, the incidence of CFC within the marrow and peripheral blood suspensions was significanlty reduced in all patients. The results suggest a reduced compartment size of CFC even in those patients who have recovered from aplastic anaemia. This may indicate that the disturbances in the preceding compartments of the haemopoietic cell renewal system still persist after recovery from the acute bone marrow failure.     

Erythroid Colony Forming Cells in Aplastic Anaemia

The concentration and erythropoietin dependence of erythropoietic progenitor cells (CFU-E) were examined in 13 patients with aplastic anaemia at different stages of their disease. The CFU-E incidence was shown to be quantitatively diminished in aplastic anaemia but tended to recover to normal values if the disease recovered. In addition the CFU-E showed a qualitatively different response to stimulation by erythropoietin, being resistant to low concentrations but responsive to concentrations greater than 0.2 U/ml whereas there was a linear response in the controls up to 0.5 U/ml.     

111Indium-Chloride Bone Marrow Scintigraphy in Aplastic Anaemia

Bone marrow scintigraphy, using ¹¹¹Indium-chloride, was performed in 24 patients with acquired aplastic anaemia to investigate: (1) a possible relationship between bone marrow scintigraphy and peripheral blood cell values, (2) a possible relationship between scintigraphy and histology of the bone marrow and (3) the ability to distinguish various aplastic anaemia subtypes with bone marrow scintigraphy. For this purpose a semi-quantitative scoring of scintigraphic results was used. Only a weak correlation was found between the radionuclide studies and blood counts. It appeared that an abnormal ¹¹¹In-scintigraphic activity in the pelvis was related to an abnormal quality and quantity of haematopoietic tissue. To study a correlation with histological subtype grading, the patients were grouped in 4 categories based on clinical-histological results. Thus it could be demonstrated that the presence of ¹¹¹In-activity in long bones (‘scintigraphic extension’) is an important parameter in distinguishing patients who are believed to suffer from a primary stem-cell defect, from patients who may suffer from an auto-aggressive disorder.     

Impairment of Immunological Function in Aplastic Anaemia*

Summary: Since the bone marrow is now believed to be the source of many of the cells participating in immune reactions immunological function was tested in eleven patients with aplastic anaemia. Nine were lymphocytopenic and seven were monocytopenic. Various abnormalities were detected, the most common being failure to show intradermal hypersensitivity in six patients and failure to produce antibody to tetanus toxoid in six. Immunological impairment may be a factor in some of the clinical manifestations of aplastic anaemia.     

Inherited Aplastic Anaemia with Increased Endoreduplications: a New Syndrome or Fanconi's Anaemia Variant?

Two sisters with aplastic anaemia without other congenital anomalies are described. Peripheral blood cytogenetic studies revealed an increase in endoreduplications in the absence of other unstable chromosome anomalies. Increased expression of T-antigen following SV40 virus infection in vitro was demonstrated in both sisters, as well as other normal family members. We feel that these patients represent a variant of Fanconi's anaemia. The importance of performing chromosome studies in idiopathic aplastic anaemia is emphasized.     

Circulating Inhibitors of Granulopoiesis in Patients with Aplastic Anaemia

An inhibitor of granulopoiesis has been detected in the serum of aplastic patients by its ability to reduce colony formation by normal human marrow cells in culture. The inhibitory activity in the serum was abolished when a patient was treated with azathioprine and exposure of aplastic marrow to antithymocyte globulin (ATG) in vitro released the colony forming cells from suppression. The clinical findings in three of the patients studied support the idea that the inhibitory activity detected in vitro is associated with normal marrow suppression in vivo.     

Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro

Stem cell factor (SCF) and erythropoietin (Epo) effectively supporterythroid cell development in vivo and in vitro. We have studied herean SCF/Epo-dependent erythroid progenitor cell from cord blood that canbe efficiently amplified in liquid culture to large cell numbers in thepresence of SCF, Epo, insulin-like growth factor-1(IGF-1), dexamethasone, and estrogen. Additionally, bychanging the culture conditions and by administration of Epo plusinsulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34⁺ peripheralblood stem cells and human bone marrow and can be isolated, amplified,and differentiated in vitro under the same conditions. Thus, highlyhomogenous populations of SCF/Epo-dependent erythroid progenitors canbe obtained in large cell numbers that are most suitable for furtherbiochemical and molecular studies. We demonstrate that such cellsexpress the recently identified adapter protein p62^dok thatis involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21^cip1 and p27^kip1 that are highlyinduced when cells differentiate. Thus, the in vitro system describedallows the study of molecules and signaling pathways involved inproliferation or differentiation of human erythroid cells.     

Suppression of in Vitro Granulocyte-Macrophage Colony Formation by the Peripheral Mononuclear Phagocytic Cells of Patients with Idiopathic Aplastic Anaemia

S ummary . To investigate whether phagocytic cells play a role in the pathogenesis of aplastic anaemia, we studied the effect of the mononuclear phagocytic cells in the peripheral blood from patients with idiopathic aplastic anaemia on granulocyte—macrophage colony (GM-colony) formation. There was no apparent infection in the studied patients. In nine out of 20 cases the mononuclear cells suppressed the formation of GM-colonies by normal bone marrow. Removal of the phagocytic cells by carbonyl iron powder caused a loss of this suppressive effect. These results suggest the requirement for phagocytic cells in this suppressive effect and the possible involvement of phagocyte-mediated immunological mechanism in the pathogenesis of some cases of idiopathic aplastic anaemia.     

In Vitro And in Vivo Expansion of Stem Cell Populations

Expansion of hemopoietic stem cells occurs in vivo following transplantation of limited numbers of bone marrow cells or of highly purified stem cells. Stem cell expansion can in principle be achieved in vitro and also be promoted in vivo by growth factor treatment, notably with thrombopoietin. Advances in identification of stromal elements, growth factors and culture conditions that stimulate immature hemopoietic stem cell proliferation may result in effective stem cell expansion protocols and contribute to efficient retrovirally mediated gene transfer. In vivo expansion of immature cells by growth factor treatment may both be a valid alternative and an adjuvant to in vitro expansion.     

Effect of Antilymphocyte Globulin on Granulocyte Precursors in Aplastic Anaemia

Granylocyte colony forming units (CFU-C) were studied in 22 patients with severe aplastic anaemia before and after treatment with antilymphocyte globulin (ALG). Nine patients showed a clinical response to ALG characterized by a rise in the granulocyte count to over I X 10(9)/1 within 30 d. These patients were distinguished in vitro from non-responders by an increase in CFU-C numbers after incubation of bone marrow cells with ALG, and by the presence of inhibitors of normal CFU-C in the serum in six out of seven patients tested. In responding patients bone marrow CFU-C rose while most non-responding patients showed no change or a fall in CFU-C after treatment. In addition in three out of four responding patients examined serum inhibitors disappeared after treatment. The horse ALG used in this study also stimulated normal CFU-C in vitro. This evidence is contrary to the hypothesis that ALG stimulates CFU-C in aplasia by inactivating an abnormal suppressor lymphocyte population. The nature of the stimulation by ALG remains unclear. But in practice the effect of ALG on bone marrow cells and study of CFU-C inhibitors in serum could be used to select patients likely to respond to ALG treatment.     

Indium Chloride Scintigraphy: An Index of Severity in Patients with Aplastic Anaemia

S ummary . [¹¹¹In]Indium chloride scans of the bone marrow were performed in 22 patients with idiopathic aplastic anaemia before therapeutic intervention. In 20 patients there was a marked reduction in the uptake of indium chloride by the marrow, and in two patients the uptake of indium chloride was normal. Nineteen of the 20 patients with reduced marrow uptake of indium chloride, who underwent bone marrow transplantation, died or failed to improve. One of the two patients with normal scans improved and the other has remained stable. Four patients studied both pre and post successful bone marrow transplantation had minimal marrow uptake before transplantation and normal scans after transplantation. Failure of indium-111 chloride marrow uptake correlates with poor prognosis in idiopathic aplastic anaemia.