Diamond-Blackfan anemia: promotion of marrow erythropoiesis in vitro by recombinant interleukin-3

To clarify the defective erythropoiesis in eight patients with Diamond- Blackfan anemia, we studied their bone marrow response in vitro to recombinant human interleukin-3 (IL-3) and recombinant granulocyte- macrophage colony-stimulating factor (GM-CSF). In an erythropoietin- containing assay system, specimens from six of the eight patients yielded low numbers of erythroid colonies compared to control values, and in five of these no erythropoietin dose-response could be elicited. Addition of IL-3, GM-CSF or both to cultures from the six patients had no effect on CFU-E-derived colonies. In contrast, IL-3 but not GM-CSF induced a marked increase in the number (183%) and size of the BFU-E- derived colonies in five of the six cases and partially corrected the impaired dose-response to erythropoietin in four. Bone marrow from the other two patients yielded numbers of CFU-E and BFU-E colonies comparable to controls and manifested similar increments in colonies with increasing concentrations of erythropoietin. When IL-3 was added to these cultures, further increments were observed in the number and size of BFU-E colonies. We conclude that IL-3 enhanced the marrow erythropoiesis in most of the patients and exerted a corrective effect on the aberrant colony formation in the presence of erythropoietin. The data raise the possibility of IL-3 as a therapeutic agent in Diamond- Blackfan anemia.     

Case report: isoimmune inhibition of erythropoiesis following ABO-incompatible bone marrow transplantation.

A 26-year-old ABO-O positive patient with aplastic anemia received a bone marrow transplant from his genotypically HLA identical, but ABO-A positive, brother. Engraftment of myeloid and megakaryocytic lineages occurred within 4 weeks but pure red cell aplasia and transfusion dependent anemia persisted for 160 days. The authors postulated that the failure of erythropoiesis was due to a high titer of anti-A isohemagglutinins. They tested this hypothesis with clonal cell cultures and flow cytometric analysis of ABO antigen expression by colony forming cells in vitro. During the period of prolonged red cell aplasia, the patient had normal numbers (85 +/- 12 per 10(6) cells) of circulating donor derived, burst forming units-erythroid (BFU-E). Immunophenotypic analysis of erythroid burst colonies derived from culture of the patient's bone marrow cells showed that 91 +/- 5% of 274 nucleated red cells were A-antigen positive, confirming full donor engraftment. Autologous plasma and complement added on day 1 of culture did not affect the colony growth (82.5 +/- 15 per 10(6) cells). However, when the addition of complement was delayed until day 7 of culture, there was 90% inhibition of BFU-E (7.5 +/- 5 per 10(6) cells) compared to controls (p less than 0.0004). Based on this, the authors propose a model for expression of ABO antigens during erythropoiesis, in which BFU-E do not express ABO antigens but their progeny do. The data support the hypothesis that the mechanism of prolonged pure red cell aplasia after ABO-incompatible bone marrow transplantation is complement mediated immune destruction of erythroid progenitors past the stage of BFU-E in differentiation.     

Fetal erythropoiesis following bone marrow transplantation

"Fetal" erythrocytes are present in older children and certain adults with hematologic disorders. To determine if regenerating bone marrow produces such cells, we examined the blood of seven allogeneic bone marrow transplant recipients. Six patients were engrafted with donor cells, while on e patients recovered autologous bone marrow after rejection of a marrow transplant. All seven patients had fetal hemoglobin levels of up to 10% by 100 days after transplant. In three patients, the Ggamma to Agamma ratio in the fetal hemoglobin was "newborn", while in one it was "adult". Gamma chain synthesis in blood and bone marrow never exceeded 20% of total non-alpha globin synthesis. The fetal hemoglobin was heterogeneously distributed in the cells. High titer i antigen also appeared. All fetal characteristics declined by 200 days. Erythropoiesis during bone marrow recovery appears to be associated with an accelerated, albeit partial, recapitulation of ontogeny.     

Bone marrow transplantation with interleukin-2-activated bone marrow followed by interleukin-2 therapy for acute myeloid leukemia in mice

We have investigated approaches to induce graft-versus-leukemia (GVL) effect in autologous bone marrow transplantation (ABMT) without graft-versus-host disease to improve survival and cure in leukemia. The present study shows that bone marrow transplantation (BMT) using syngeneic bone marrow activated with interleukin-2 (ABM) for 24 hours in vitro, followed by interleukin-2 (IL-2) therapy, was superior to BMT with fresh, syngeneic bone marrow (FBM) in terms of survival and cure in mice with acute myeloid leukemia (P less than .001) and led to normal hematopoietic reconstitution. Addition of IL-2 therapy after BMT with FBM did not improve the results over BMT with FBM alone (P = .98). These results suggest that the GVL effect of ABMT can be enhanced by using ABM for BMT followed by IL-2 therapy without compromising engraftment.     

Heterogeneous ontogeny of erythropoiesis after bone marrow ablation and allogeneic bone marrow grafting

Thirty-two patients who underwent bone marrow transplantation for hematologic malignancies were studied for hemoglobin-F as an indicator for fetal erythropoiesis. Two different patterns of response were noted. One group of patients who had an elevated HbF level prior to marrow grafting later showed a marked reactivation of HbF synthesis, whereas the other group of patients who had normal HbF levels prior to transplantation failed to exhibit such an increase. This phenomenon occurred independently of ages of marrow graft donors or recipients, the type of underlying hematologic malignancy, or remission induction therapy prior to preparation for transplantation, pretransplant hemoglobin levels, transfusion with red blood cells, red cell volume, and production of reticulocytes.     

Hematologic and immunologic effects of the systemic administration of recombinant interleukin-2 after autologous bone marrow transplantation

T cells from allogeneic bone marrow grafts are responsible for a graft versus leukemia effect. Use of recombinant Interleukin-2 (rIL-2) after autologous bone marrow transplantation (BMT) may enhance immune function and hopefully reproduce the allogeneic reaction. We report here the hematologic and immunologic changes observed in the first 10 patients of a phase 1 trial studying the infusion of IL-2 after autologous BMT. All patients had high-risk malignancies and received 6 days of a constant infusion of IL-2 (Eurocetus, Amsterdam, The Netherlands) at dose of 3 x 10(6) Cetus Units/m2/d, 79 +/- 12 days after autologous BMT. Clinical toxicities involving cutaneous, cholestatic, gastrointestinal, and hemodynamic effects occurred during IL-2 treatment but reversed in all cases. Completion of treatment was 91% of the scheduled dose of IL-2. Hematologic toxicity was moderate and transient with no graft failure. Increases in eosinophil and lymphocyte counts were significant (P less than .05). Stimulation of the immune system was intense and prolonged, manifested by increase numbers of CD3+, CD3+DR+, CD3+ CD25+ lymphocytes, and natural killer (NK) cells (all P less than .01), and increase of Lymphokine-activated killers (LAK) and NK activities (P less than .01 and P less than .05). This study establishes the feasibility of a 6-day administration of rIL-2 after autologous BMT leading to a major immune activation 2.5 months after BMT.     

Potential use of interleukin-6 in bone marrow transplantation: effects of recombinant human interleukin-6 after syngeneic and semiallogeneic bone marrow transplantation in mice

The potential of recombinant glycosylated human interleukin-6 (rhIL-6) for enhancing immunohematopoietic reconstitution and survival after syngeneic and semiallogeneic bone marrow transplantation (BMT) in BALB/c mice subjected to total body irradiation (TBI) was investigated. rhIL-6 produced enhanced reconstitution of white blood cells as assessed on days 8 and 14 after syngeneic BMT and of platelets as assessed on day 10. Moreover, rhIL-6 treatment produced significant improvement of survival in lethally irradiated mice receiving either syngeneic or semiallogeneic BMT with limiting number of BM cells. This effect of IL-6 was not seen with large BM cell inocula producing high survival by themselves. rhIL-6 showed no toxic effects and did not affect the survival of mice that were lethally irradiated but not reconstituted by BM cells. However, the sensitivity of mice to sublethal irradiation was increased by rhIL-6 in the absence of BM cell transplantation. In experimental conditions inducing graft-versus-host disease (GVHD), in which lethally irradiated (BALB/c x C57BL/6)F1 mice received mixtures of BM and spleen cells from C57BL/6 donors, rhIL-6 was found to enhance GVHD manifestations. No consistent enhancement of T-cell in vitro proliferative responses to allogeneic spleen cells or T- and B-cell-dependent mitogens were seen in the splenocytes obtained from recipients of syngeneic or semiallogeneic BMT. Our data suggest that rhIL-6 may be useful in BMT procedures to enhance thrombopoiesis and hematologic recovery, as well as to increase overall survival rates. In addition, the potentiation of GVHD, which is considered to correlate with graft-versus-leukemia effects, may be of interest in enhancing GVHD-dependent antitumor effects in protocols combining radiochemotherapy with BMT.     

Production of interleukin-1 by bone marrow myeloma cells

Plasma cells isolated from bone marrow (BM) aspirates of 12 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed for production of cytokines with bone-resorbing activity, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lymphotoxin (LT). Culture supernatants of plasma cells from MM, but not from MGUS or normal donor, invariably contained high amounts of IL-1-beta and lower amounts of IL-1-alpha. With a single exception, TNF/LT biologic activity was not detected in the same supernatants. IL-6 was present in two of five supernatants tested. Normal B lymphocytes released both IL-1 and TNF/LT activities for four days after activation in vitro; however, production of these cytokines ceased at the final stage of plasma cell. Unexpectedly, the mRNA extracted from MM plasma cell hybridized with TNF- and LT- specific, as well as IL-1-specific probes, although the culture supernatants did not contain detectable TNF/LT biologic activity. When tested in the fetal rat long bone assay, MM plasma cell supernatants displayed a strong osteoclast-activating factor (OAF) activity, which was greatly reduced but not completely abolished by neutralizing anti- IL-1 antibodies. Anti-TNF or anti-LT antibodies were ineffective in the same test. We conclude that the IL-1 released in vivo by malignant plasma cells has a major role in pathogenesis of lytic bone lesions of human MM.     

c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.     

Abnormalities in the regulation of erythropoiesis by bone marrow fibroblasts in aplastic anemia

The influence of bone marrow fibroblasts in healthy subjects and patients with aplastic anemia on normal erythroid colony formation was studied using the methylcellulose method. These fibroblasts were treated with methylprednisolone as well. Bone marrow fibroblasts of healthy subjects and patients with aplastic anemia, and the supernatant of their conditioned medium significantly inhibited normal erythroid colony formation. A significantly marked inhibition of normal erythroid colony formation was observed of bone marrow fibroblasts (or the conditioned medium) of the aplastic anemia, when compared with that of the bone marrow of healthy subject fibroblasts (or the conditioned medium). By treating both groups of the bone marrow fibroblasts with methylprednisolone the inhibition was slightly improved. From the above experimental results, it was suggested that the bone marrow fibroblasts and the conditioned medium inhibited normal erythroid colony formation through humoral factors secreted by the fibroblasts, and through contact between the fibroblasts and erythroid colony formation cells.     

Aplastic anemia associated with in vitro inhibition of erythropoiesis by bone marrow-adherent cells

A patient with aplastic anemia that evolved following pure red cell aplasia is described. Cultures of the patient's marrow cells revealed greatly reduced numbers of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors, but normal numbers of multipotential (CFU-GEMM) precursors. The BFU-E/CFU-GEMM and CFU-E/BFU-E ratios in the patient's marrow cell cultures were also reduced. T cell- or antibody-mediated inhibition of in vitro erythropoiesis could not be demonstrated in this patient. However, the patient's marrow-adherent cells suppressed the growth of autologous and allogeneic BFU-E and CFU-E, without influencing the growth of CFU-GEMM. Medium conditioned by the patient's adherent cells failed to inhibit the growth of normal erythroid precursors. Our findings suggest a role for marrow-adherent cells in the pathogenesis of aplastic anemia in this patient.     

Synergistic effect of recombinant interleukin-2 on the inhibition of growth of adenocarcinoma 755 by mitomycin

The increase in life-span (ILS) of tumor-bearing mice caused by recombinant interleukin-2 (RIL-2) was studied in the solid tumor adenocarcinoma 755 system. With long-term treatment (Days 5-12), RIL-2 (10(5) U/mouse) showed a weak effect (24% ILS), but short-term RIL-2 treatment (Days 5-8 or 9-12) had hardly any effect. Mitomycin, at 2 mg/kg (maximum nontoxic dose), caused 35% ILS with Days 5-8 treatment and only 12% ILS with Days 9-12 treatment. Sequential treatment with mitomycin (Days 5-8) and RIL-2 (Days 9-12) markedly enhanced antitumor activity (88% ILS). This value is significantly greater than that of mitomycin alone or RIL-2 alone (P less than 0.01). Furthermore, mitomycin followed by RIL-2 markedly augmented killing activity of spleen cells that are cytotoxic in vitro. These results indicate that RIL-2 markedly affects the inhibition of growth of a tumor treated with an antitumor agent.     

Enrichment of interleukin-2-responsive natural killer progenitors in human bone marrow

Natural killer (NK) cells can be cultured in interleukin-2 (IL-2)- containing medium from selected human bone marrow (BM) cells obtained after the elimination of mature T and NK cells. To isolate and characterize IL-2-responsive NK progenitors in the selected BM cells, we investigated the expression of IL-2 receptors (IL-2R) on these cells. Neither CD25 (IL-2R alpha) nor IL-2R beta antigen was observed on the selected BM cells before culture. However, CD25+ cells without detectable levels of IL-2R beta antigen appeared 24 hours after culture in IL-2-containing medium. Cells were sorted from each fraction of the selected BM cells 24 hours after culture after staining with anti-CD33, anti-CD34, and anti-CD25 monoclonal antibodies. The generation of NK cells (CD3- CD56+ cells) and NK activity were observed only from the CD33-/CD34-/CD25+ cell fraction after culture in IL-2-containing medium. The frequency of IL-2-responsive NK progenitors relative to the fraction was 1/231 (95% confidence range, 1/156 to 1/289), which corresponded to the frequency relative to the total number of selected BM cells when the frequency relative to the CD33-/CD34-/CD25+ cell- fraction was converted according to the percentage of these cells in the total number of selected BM cells. These results indicated that IL- 2-responsive NK progenitors were enriched in the CD33-/CD34-/CD25+ cell fraction.     

Evaluation of erythropoiesis after bone marrow transplantation: quantitative reticulocyte counting

Erythroid regeneration is an important and separate element in the engraftment process in allogeneic and autologous bone marrow transplantation (alloBMT, autoBMT). Qualitative visual reticulocyte counting has proved inadequate in the evaluation of erythropoiesis after BMT but automated flow cytometry now allows the reliable quantitation of reticulocytes even to very low levels. Reticulocyte counts and highly fluorescent reticulocyte (HFR) counts (very early reticulocytes) were estimated daily in recipients of 22 autoBMT and 14 alloBMT using a Sysmex R-1000 automated reticulocyte counter. Marrow ablation caused an immediate and rapid fall in both the reticulocyte count and the HFR. Measurable numbers of reticulocytes persisted throughout the hypoplastic period, but HFR fell to zero in the majority of both the autoBMT and alloBMT. HFR rose significantly after a median time of 14 d post-autoBMT, and 12 d post-alloBMT. Attainment of 15 x 10(9)/l reticulocytes and 0.5 x 10(9)/l HFR at day 21 post-transplant was associated with ultimate engraftment in 100% cases. Inadequate engraftment was seen in the majority of patients whose responses fell below these levels. Graft-versus-host disease was associated with a transient slight reduction in reticulocyte count. Neither episodes of infection nor blood transfusions had any significant impact on trends of reticulocytes or HFR. Automated flow cytometric reticulocyte counting has been shown to provide an accessible measure of erythroid activity which may be of predictive value in the management of patients following bone marrow transplantation.     

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.     

Stimulation of bone marrow erythropoiesis by T lymphocytes of anaemic patients with rheumatoid arthritis.

An inappropriate response of the bone marrow is implicated in the aetiology of the anaemia of chronic disease complicating rheumatoid arthritis. T lymphocyte subsets have been shown to inhibit early erythroid development in vitro in association with some cases of bone marrow failure, and an expanded peripheral blood pool of these cells is reported in rheumatoid arthritis. We have studied the role of peripheral blood T lymphocytes in erythroid bone marrow culture from seven normal volunteers and nine anaemic patients with rheumatoid arthritis and found comparable stimulation of growth in both groups.     

激活骨髓自体移植治疗白血病和淋巴瘤的疗效分析

目的 探讨激活骨髓自体移植治疗恶性血液病的疗效及影响因素。方法 ４４例为白血病和非霍奇金淋巴瘤（ＮＨＬ）患者进行激活骨髓自体移植,观察移植后造血与免疫功能重建,移植后完全缓解（ＣＲ）,移植后相关死亡、肿瘤复发率和长期无病生存率及移植物抗宿主产产表现及毒副作用。选用Ｋａｐｌａｎ－Ｍｅｉｅｒ生存曲线评估一３、５年无病生存率、ＣＯＸ回归模型分析性别、年龄、疾病种类、移植前状态（ＣＲ与未ＣＲ）,预处理方案