BH3 Inhibitor Sensitivity and Bcl-2 Dependence in Primary Acute Lymphoblastic Leukemia Cells.

BH3 mimetic drugs may be useful to treat acute lymphoblastic leukemia (ALL) but the sensitivity of primary tumor cells has not been fully evaluated. Here, B-lineage ALL cell cultures derived from a set of primary tumors were studied with respect to sensitivity to the BH3 mimetics ABT-263 and ABT-199 and to Bcl-2 dependence and function. These ALL cells each expressed high levels of Bcl-2 and exhibited great sensitivity to ABT-263 and ABT-199, which induced rapid apoptotic cell death.

Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors.

Objective: B-lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease.

Cytokine-dependent proliferation of human CD34+ progenitor cells in the absence of serum is suppressed by their progeny's production of serine proteinases.

In this study, we demonstrate that the synthesis and release of serine proteinases by hematopoietic cells affects the in vitro proliferation of hematopoietic progenitor cells (HPCs) in response to proteins, including hematopoietic growth factors (HGFs), transferrin, insulin, and albumin in serum-free cultures. In serum-free cultures, bone marrow mononuclear cells or the CD34- progeny of the CD34+ cells were shown to release the serine proteinases human neutrophil elastase (HNE), cathepsin G (Cath G), and proteinase 3 (Pr3). In the absence of serum, we showed that HNE, Cath G, and Pr3 rapidly and dose-dependently degraded HGF and other proteins present in the medium, resulting in decreased proliferation of HPCs.

Colony growth of human hematopoietic progenitor cells in the absence of serum is supported by a proteinase inhibitor identified as antileukoproteinase.

Serum contains many growth factors and nutrients that stimulate colony formation of hematopoietic progenitor cells (HPC) in semisolid cultures. In the absence of serum, no proliferation of HPCs could be obtained in semisolid medium cultures of partially purified bone marrow cells in the presence of multiple hematopoietic growth factors, insulin, cholesterol, and purified clinical-grade human albumin. This appeared to be due to a suppressive activity induced by monocyte- and T lymphocyte-depleted accessory cells on CD34+ HPCs.

Recognition of minor histocompatibility antigens on lymphocytic and myeloid leukemic cells by cytotoxic T-cell clones.

Clinical studies indicated an enhanced antileukemic effect of allogeneic bone marrow transplantation (BMT), as compared with autologous BMT. After allogeneic HLA-identical BMT, donor-derived cytotoxic T lymphocytes (CTLs) directed at minor histocompatibility (mH) antigens on the recipients, tissues can be shown. To evaluate the antileukemic reactivity of mH antigen-specific CTLs, we analyzed the expression of mH antigens on circulating lymphocytic and myeloid leukemic cells.

Effect of mast cell growth factor (c-kit ligand) on clonogenic leukemic precursor cells.

Mast cell growth factor (MGF), the ligand for the c-kit receptor, has been shown to be a hematopoietic growth factor that preferentially stimulates the proliferation of immature hematopoietic progenitor cells (HPC). We studied the effect of MGF on the in vitro growth of clonogenic leukemic precursor cells in the presence or absence of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or erythropoietin (EPO). Leukemic blood and bone marrow cells from patients with various types of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) in chronic phase, as well as bone marrow samples from patients with myelodysplastic syndromes (MDS) were studied.

Growth inhibition of clonogenic leukemic precursor cells by minor histocompatibility antigen-specific cytotoxic T lymphocytes.

Minor histocompatibility (mH) antigens appear to play a major role in bone marrow transplantation (BMT) using HLA-identical donors. Previously, we reported the isolation of major histocompatibility complex (MHC)-restricted mH antigen-specific cytotoxic T lymphocytes (CTL) from patients with graft-vs.-host disease or rejection after HLA-identical BMT.

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta.

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks.

Interleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony-stimulating factor.

Pure interleukin 1 (IL 1) was found to stimulate established human bone marrow stromal layers in long-term culture to produce colony-stimulating activity (CSA). Maximal concentrations in the culture medium were reached 24 hours after a single IL 1 pulse. The effect could be neutralized by a specific rabbit anti-IL 1 antiserum.

Human hematopoietic progenitor cells in long-term cultures express HLA-DR antigens and lack HLA-DQ antigens.

The expression of HLA-DR antigenic determinants on human hematopoietic progenitor cells (HPC) capable of differentiating into mature blood cells, as determined in semisolid cultures, has been demonstrated previously (3-7). Here, we investigated the expression of class II determinants on HPC responsible for the sustained proliferation of colony-forming units of granulocyte/macrophage (CFU-GM), of multilineage HPC (CFU-GEMM, granulocyte/erythrocyte/macrophage/megakaryocyte), and burst-forming units of erythroid cells (BFU-E) in liquid long-term cultures. Using both fluorescence-activated cell sorting and complement-dependent cytotoxicity assays, HLA-DR determinants could be identified on virtually all these HPC capable of proliferating in long-term cultures.

Recovery of hematopoiesis after blood-group-incompatible bone marrow transplantation with red-blood-cell-depleted grafts.

Severe hemolytic transfusion reactions may complicate major blood-group-incompatible bone marrow transplantations (BMT). Probably the most appropriate way to avoid this complication is removal of the incompatible red blood cells (RBC) from the bone marrow graft. In this report we describe a method to eliminate incompatible RBCs that is based on repeated dilution of the graft with donor-and-recipient--compatible third-party erythrocytes.

Complement-dependent cytotoxicity in the analysis of antigenic determinants on human hematopoietic progenitor cells with HLA-DR as a model.

Complement-dependent cytotoxicity (CDC) assays using anti-Ia antisera have resulted in controversial conclusions about the expression of HLA-DR determinants on human hematopoietic progenitor cells (HPC). The expression of these antigens on CFU-E in particular could often not be demonstrated. Since this is contradictory to our own results, we decided to study the influence of both the antibody and complement concentrations in CDC assays using murine monoclonal and polyclonal anti-Ia (HLA-DR "backbone") antibodies and human anti-HLA-DR typing sera.

The in vitro growth pattern of human bone marrow in methylcellulose stimulated by different concentrations of conditioned medium.

The proliferation of human bone marrow in methylcellulose stimulated by various concentrations of conditioned medium (CM) and observed at various intervals was studied. The growth kinetics of granulocytic aggregates was found to differ from monocytic clusters and colonies. Granulocytic aggregates showed a consistent and reproducible dose-response relationship; at day 7, the maximum number of granulocytic aggregates was found at 4% CM.

Polymorphic and monomorphic HLA-DR determinants on human hematopoietic progenitor cells.

The expression of monomorphic Ia-like antigens and polymorphic (allotypic) HLA-DR determinants on CFU-GM, BFU-E, CFU-E, and CFU-GEMM was studied in bone marrow and peripheral blood cells from normal healthy individuals. Using various polyclonal and monoclonal anti-Ia-like antibodies, the presence of HLA-DR backbone antigens was shown on all hematopoietic progenitor cells (HPC) studied, both in complement-dependent cytotoxicity assays and in fluorescence-activated cell sorting (FACS). The expression of allotypic determinants was demonstrated on all HPCs, using the HLA-DR typing sera anti-HLA-DR1, 2, 3, 4, 5, and 7.

The impact of the composition of the bone marrow graft on engraftment and graft-versus-host disease.

To study the impact of the composition of the bone-marrow graft on engraftment and graft-versus-host disease (GvHD), we analyzed the data on 29 patients with acute leukemia in remission and 11 patients with aplastic anemia. All of them received bone-marrow grafts from HLA matched, MLC nonreactive, sibling donors, were nursed in laminar down-flow isolators with selective gut decontamination, and received GvHD prophylaxis with methotrexate. The number of nucleated cells in the marrow graft/kg body weight of the recipient had no relation with the rapidity of engraftment or with the occurrence and severity of GvHD.

Serum inhibitors in aplastic anaemia.

To study the frequency and clinical importance of serum factors inhibiting CFU-c in patients with aplastic anaemia, sera from 21 patients were analysed. The sera of eight patients showed inhibition of at least one normal bone marrow, but strong and consistent inhibition of two or more normal bone-marrow samples was found in only two cases. In both of these two patients the inhibition disappeared after successful therapy.

Anti-thymocyte globulin effective in the treatment of aplastic anemia does not stimulate granulocyte precursor cells.

To find out whether the preparations of anti-lymphocyte or anti-thymocyte globulin (ATG), successfully used in our center for the treatment of patients with aplastic anemia, were stimulatory for hematopoietic precursor cells, we studied the effect on CFUC in 30 normal bone marrow samples. Although in 2 out of 30 cases stimulation was observed, the overall result for both the horse anti-lymphocyte globulin and the rabbit anti-thymocyte globulin was dose-dependent and complement-dependent inhibition. Neither in the absence, nor in the presence of complement was there any indication of consistent stimulation of CFUC.

Acquired aplastic anemia in adults. IV. Histological and CFU studies in transplanted and non-transplanted patients.

A follow-up study of 10 patients suffering from acquired aplastic anaemia, comprising methocrylate-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.

Freezing of canine lymphocytes for use in mixed leukocyte culture and cell-mediated lympholysis tests.

A satisfactory and reproducible technique of cryopreservation of canine lymphocytes for use in mixed leukocyte culture (MLC) and cell-mediated lympholysis tests has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to -50 C at a controlled rate (-1 C/min) and stored at -169 C.

One-way nonstimulation of mixed leukocyte culture in dog families.

Fifteen dog families were studied in mixed leukocyte culture. In eight families of seven different breeds, one-way nonstimulation in mixed leukocyte culture was observed. This could be explained by lymphocyte-defined (LD) homozygosity in most instances.