Proliferating dendritic cell progenitors in human blood.

CD34+ cells in human cord blood and marrow are known to give rise to dendritic cells (DC), as well as to other myeloid lineages. CD34+ cells are rare in adult blood, however, making it difficult to use CD34+ cells to ascertain if DC progenitors are present in the circulation and if blood can be a starting point to obtain large numbers of these immunostimulatory antigen-presenting cells for clinical studies. A systematic search for DC progenitors was therefore carried out in several contexts. In each case, we looked initially for the distinctive proliferating aggregates that were described previously in mice. In cord blood, it was only necessary to deplete erythroid progenitors, and add granulocyte/macrophage colony-stimulating factor (GM-CSF) together with tumor necrosis factor (TNF), to observe many aggregates and the production of typical DC progeny. In adult blood from patients receiving CSFs after chemotherapy for malignancy, GM-CSF and TNF likewise generated characteristic DCs from HLA-DR negative precursors. However, in adult blood from healthy donors, the above approaches only generated small DC aggregates which then seemed to become monocytes. When interleukin 4 was used to suppress monocyte development (Jansen, J. H., G.-J. H. M. Wientjens, W. E. Fibbe, R. Willemze, and H. C. Kluin-Nelemans. 1989. J. Exp. Med. 170:577.), the addition of GM-CSF led to the formation of large proliferating DC aggregates and within 5-7 d, many nonproliferating progeny, about 3-8 million cells per 40 ml of blood. The progeny had a characteristic morphology and surface composition (e.g., abundant HLA-DR and accessory molecules for cell-mediated immunity) and were potent stimulators of quiescent T cells. Therefore, large numbers of DCs can be mobilized by specific cytokines from progenitors in the blood stream. These relatively large numbers of DC progeny should facilitate future studies of their Fc epsilon RI and CD4 receptors, and their use in stimulating T cell-mediated resistance to viruses and tumors.

Mixed colony formation in vitro by the heterogeneous compartment of multipotential progenitors in human bone marrow.

The physiology of the human haemopoietic primitive progenitor populations can be studied in normal and disease states by clonal in vitro cultures in which the primitive progenitor cells proliferate and differentiate to form mixed colonies. For many applications it is essential that such assays detect a high proportion of primitive progenitor cells. We describe an in vitro assay which detects a high incidence of human CD34+ multipotential progenitor cells. Bone marrow mononuclear cells (MNC) or selected CD34+ cells were plated at low cell concentrations in semisolid agar cultures with synergizing growth factor combinations. The optimum growth factor combination of conditioned medium from Mia PaCa-2 cells (Mia-CM), recombinant granulocyte-macrophage colony-stimulating factor and recombinant stem cell factor (SCF) supported the formation of macroscopic (> or = mm) colonies (97% of which were multilineage), at an average incidence of 250/10(5) MNC. The colony-forming cells (human colony-forming unit, type A) detected, showed a low cycling status (7.3%) and the macroscopic colonies had a high replating efficiency (46%), reflecting their probable primitive nature. This assay should prove invaluable, for studies on the regulation of proliferation of the multipotential compartment and in studies involving the assessment of these cells in transplantation and neoplastic disease.

The role of erythropoietin, megakaryocyte colony-stimulating factor, and T-cell-derived factors on human megakaryocyte colony formation: evidence for T-cell-mediated and T-cell-independent stem cell proliferation.

Recent studies suggest that megakaryocytopoiesis is governed by a dual-level regulatory process, with megakaryocyte colony-stimulating factor (Meg-CSF) primarily influencing proliferation of the committed precursors and thrombopoietin required for megakaryocyte ploidy amplification and for maturation. The authors have examined different sources of Meg-CSF in a microagar culture system with a view to their capacity to enhance megakaryocyte colony formation directly or via an indirect T-lymphocyte- or monocyte-mediated effect. The comparative influences of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), erythropoietin (Epo), sera of patients with severe aplastic anemia, and direct PHA addition to the culture were evaluated for their capacity to enhance megakaryocytic colony formation as well as for the maturation rate of megakaryocytes (Mk) grown in our microagar culture system. Each treatment by itself enhanced colony formation from unseparated low-density cells. Removal of T-lymphocytes and monocytes from the bone marrow sample caused a cessation of the enhancing effect of direct PHA addition to cultures stimulated with Epo, but did not influence the enhancing activities of severe aplastic anemia serum (SAA), PHA-LCM, and Epo. The results show that SAA serum, Epo, and PHA-LCM induced Mk colony formation directly and therefore may act via a common mechanism. Differences, however, were observed concerning their colony-stimulating potency and their influence on the Mk maturation rate.

Lack of cross-resistance with gemcitabine and cytarabine in cladribine-resistant HL60 cells with elevated 5'-nucleotidase activity.

Cross-resistance patterns between chemotherapeutic agents have implications for the treatment of hematologic and other diseases. Previous in vitro models have shown cross-resistance between the purine analog 2-chlorodeoxyadenosine (cladribine) and the pyrimidine analogs 2',2'-difluorodeoxycytidine (gemcitabine) and 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside, cytarabine) with reduced deoxycytidine kinase (dCK) activity as the underlying determinant of resistance. In this study, we continuously exposed the human promyelocytic leukemia cell line HL60 to as much as 1024 nM cladribine. After limiting dilution, the cladribine concentrations that caused 50% growth inhibition (IC50) of the two clones R13 and R23 were 33.3- and 18.7-fold, respectively, higher than the IC50 of the parental HL60 cells (8.7+/-1.3 nM). These cladribine-resistant clones, however, showed no cross-resistance to gemcitabine and only 3.3- and 2.7-fold resistance to cytarabine, respectively. Characterization of both clones revealed stably elevated levels of purine-specific "high-Michaelis constant (Km)" 5'-nucleotidase (5'-NT) messenger RNA expression and specific activity, whereas pyrimidine-specific "low-Km" 5'-NT activity was undetectable, and dCK activity was only marginally decreased in R13. Thus, the ratio of dCK (specific for cladribine) to high-Km 5'-NT activity in R13 and R23 was reduced to 65.3% and 63.7%, respectively. These results show that changes of high-Km 5'-NT activity can induce cladribine resistance, without cross-resistance to gemcitabine.

Proliferation and differentiation of human erythropoiesis in vitro: effect of different human lipoprotein species.

In order to permit erythroid proliferation in agar, a serum-free system was developed based on McCoy's 5A medium at pH 8.0, containing deionized and delipidated bovine serum albumin, iron-saturated transferrin, and crude erythropoietin (step III). Addition of the entire complement of serum lipoproteins, termed lipoprotein fraction I (density, less than 1.21 g/ml), increased the number of erythroid colony-forming units and erythroid burst-forming units to numbers indistinguishable from those observed with media containing serum. Moreover, terminal differentiation occurred to fully hemoglobinized normoblasts and even mature erythrocytes. Under these conditions, the erythropoietin dose-response curve obtained when not using serum was comparable to that with serum. Colony formation also displayed a linear relationship to seeding densities down to limiting dilutions. For differential analysis of the main density lipoprotein fractions, sequential ultracentrifugation was performed to isolate very low-density lipoproteins, low-density lipoproteins (LDL), high-density lipoproteins2 (HDL2), and HDL3, which were then added individually to serum-free cultures. Of the main lipoprotein classes, LDL exhibited the most proliferative capacity, followed by HDL2 and HDL3. Our findings indicate that when serum is substituted by well defined compounds including highly purified lipoprotein fractions, a serum-like proliferation and differentiation of human erythropoietic progenitor cells in agar can be obtained.

Erythropoietin controls heme metabolic enzymes in normal human bone marrow culture.

Erythropoietin (Epo) was found to act as a concentration-dependent inducer of aminolevulinic acid (ALA) synthase and porphobilinogen (PBG) deaminase in normal human bone marrow in culture. Epo increased enzymatic activities in individual plated nucleated cells. At a low concentration of Epo, heme oxygenase activity did not change in human bone marrow erythroid progenitor cells. However, Epo at a concentration of 2 U/ml increased heme oxygenase as demonstrated by an increase in both the enzyme protein and its mRNA. In experiments with an inhibitor of heme synthesis, succinylacetone (SA), Epo failed to stimulate erythroid colony-forming unit (CFU-E) growth, but this CFU-E inhibition by SA was completely overcome by the addition of hemin. Epo nevertheless potentiated induction of ALA synthase in the presence of SA. Hemin exerted its regulatory role by negative feedback on ALA synthase in the presence of SA and Epo. Heme potentiated Epo action and resulted in the increase of human marrow erythroid progenitor cell proliferation and differentiation and a concomitant stimulation of ALA synthase and PBG deaminase. The potentiating effects of hemin on CFU-E growth were observed in human bone marrow cells cultured in media supplemented with fetal calf serum or serum-free media with interleukin 3 (IL-3). These results indicate that Epo is a potent inducer of ALA synthase and PBG deaminase in normal human bone marrow. In addition, our results may explain the mechanisms by which heme potentiates Epo or IL-3 enhancement of erythropoiesis. 1) Heme may stimulate the translation of several globin and nonglobin mRNAs, including those of ALA synthase and PBG deaminase; 2) as endogenous cellular heme synthesis reaches optimal levels, heme exerts its regulatory role on ALA synthase by negative feedback inhibition. Additionally, an increase in cellular heme may lead to an increase in its own degradation by induction of heme oxygenase.

Effect of deoxycytidine on 2-chloro-deoxyadenosine-mediated growth inhibition of normal human erythroid and myeloid progenitor cells.

The cytotoxic effect of chlorodeoxyadenosine (CdA) on lymphocytes and monocytes requires phosphorylation by the enzyme deoxycytidine kinase and can be antagonized by coadministration of deoxycytidine (dCyt), a competitive substrate of deoxycytidine kinase. It has also been shown for lymphocytes that coadministration of 3-aminobenzamide (3-ABA), an inhibitor of the enzyme poly-(ADP ribose) synthetase, is activated by CdA-mediated DNA strand breaks, consumes intracellular nicotinamide-dinucleotide (NAD) and can antagonize the lethal effect of CdA. Recent in vitro studies have shown that not only growth of lymphocytes and monocytes, but also colony formation by erythroid and myeloid progenitors derived from normal human bone marrow, is inhibited by CdA in a dose-dependent manner. In this study we examined the effect of various doses of dCyt (10(-6) to 10(-3) M) on CdA-mediated growth inhibition of erythroid and myeloid progenitor cells in vitro. Our results show that colony formation by human bone marrow-derived progenitor cells--CFU-E (colony-forming unit erythroid), BFU-E (burst-forming unit erythroid) and CFU-GM (colony-forming unit granulocyte/macrophage)-in semisolid medium is protected by a high, but clinically achievable and non-toxic, concentration of dCyt (> 10(-4) M) against the inhibitory effects of coadministered high concentrations of CdA. The protective effect of dCyt was markedly different on the various subclasses of progenitor cells, however. Thus, with coadministration of 10(-4) M dCyt, the CFU-E colony formation could be restored to almost 100% despite the presence of high concentrations of CdA (160 nM) compared to control cultures, whereas the colony formation of BFU-E and CFU-GM was restored to only 50%. At a concentration of 10(-3) M dCyt, colony formation of BFU-E and CFU-GM was raised to 80% of control cultures even in the presence of high concentrations of CdA (160 nM). Further experiments in which 3-ABA was coadministered to CdA-treated cultures showed that in all concentrations tested (0.3 to 5 mM) 3-ABA was not able to prevent CdA-mediated cytotoxicity on bone marrow progenitors. Based on these studies, we suggest that the CdA toxicity on CFU-E is mainly mediated by phosphorylation by deoxycytidine kinase, whereas additional mechanisms may be operative in BFU-E and CFU-GM. Considerable biochemical differences seem to exist between hematopoietic stem cells on the one hand and lymphocytes and monocytes from peripheral blood on the other.

A miniaturized agar culture system for cloning human erythropoietic progenitor cells.

The micro-agar-culture technique for cloning early and late erythropoietic progenitor cells (BFU-E and CFU-E) was further modified and miniaturized in order to study the optimal growth conditions with a minimal consumption of erythropoietin (EP). Using microtiter plates, the total incubation volume was lowered from 0.5 to 0.1 ml and thus reduced the necessary amount of EP and cells by a factor of 5. The method consists of a 50 microliter agar layer, in which the mononuclear cells are suspended, and a 50 microliter liquid overlayer containing bovine serum albumin (BSA), transferrin (TF), and EP. After a seven- or 14-day incubation, the whole agar layers were fixed, transferred to microscopic slides, dried, stained using the Pappenheim method, and permanently preserved. The influences of FCS, BSA, and TF in the presence of EP were studied on the proliferation of human bone marrow CFU-E and BFU-E. The variation of FCS concentration showed an optimum at 10%. The addition of BSA in the presence of optimal concentrations of EP markedly increased the number of BFU-E, but not CFU-E. Furthermore, the threshold concentration of EP required for the initial burst formation could be reduced by half in the presence of BSA. By the addition of TF, a further increase in the number of BFU-E was obtained.

Studies on differentiation of committed hemopoietic progenitor cells with monoclonal antibodies directed against myeloid differentiation antigens.

In the present study we evaluated the reactivity of monoclonal cytotoxic antibodies directed against myeloid differentiation antigens with hemopoietic precursor cells. VIM-D5 and VIM-2 inhibit the proliferation of clusters and colony formation after seven days of incubation. Day-14 CFU-GM are not affected by these antibodies. After complement-mediated cytolysis with VIM-2, the number of BFU-e was significantly reduced; however, this effect was largely abrogated by addition of leukocyte-conditioned medium to the cultures as an exogenous source of burst-promoting activity. Furthermore, the maturation of myeloid progenitor cells has been examined by delayed treatment with VIM-D5 and complement during the in vitro culture period. In these experiments a different maturation behavior of day-7 and day-14 CFU-GM was demonstrated. To study whether a cryptic carbohydrate structure is present on more immature CFU-GM, the effect of neuraminidase treatment of myeloid progenitor cells on reactivity with VIM-D5 was tested.

A micro agar culture system for cloning human erythropoietic progenitors in vitro.

A simple and reproducible micro agar culture method for cloning erythropoietic progenitor cells from human bone marrow is described. Mononuclear cells (MNC) were immobilized in an agar layer and stimulated by erythropoietin (Ep), which was added to a liquid overlayer. The cultures were routinely incubated, fixed, transferred to microscopic slides, dried, and stained, and erythroid colonies were morphologically examined. The dynamics of growth observed from days 2 to 26 of incubation in the presence of 2.4 U Ep/ml showed basically three kinds of aggregates, which reached maximum growth on different days of incubation. A close Ep dose-response relationship was found for CFUE and BFUE at a concentration of 7.5 x 10(4) seeded cells. By varying the plated cell concentration between 2.5 and 10 x 10(4) cells a linear increase in the aggregates formed was found. On the basis of their growth dynamics and morphologic composition, the existence of three populations of erythropoietic progenitor cells in human bone marrow is tentatively proposed.

A new approach to the morphological and cytochemical evaluation of human bone marrow CFUC in agar cultures.

A simple procedure is described for the routine morphological examination of different types of colonies and clusters in agar cultures. The following cytochemical methods are used for the identification of cells: naphthol-ASD-chloroacetate esterase, naphthol-ASD-acetate esterase with and without addition of sodium fluoride, Biebrich scarlet and acid phosphatase. Based on their cytochemical staining properties neutrophils, eosinophils, monocytes and macrophages can be readily identified in their different stages of maturation and an assessment made of the cellular composition of colonies and clusters.

Human erythropoiesis in vitro and the source of burst-promoting activity in a serum-free system.

Bovine serum albumin (BSA) can markedly increase the number of size of erythropoietic bursts produced by mononuclear cells from human bone marrow and peripheral blood, and reduce the threshold amount of erythropoietin (Epo) required for initial burst formation. The purpose of this study was to determine a possible burst-promoting activity (BPA) of BSA. The experiments were performed in a miniaturized agar system, in which the addition of sheep Epo to cultures with or without BSA was delayed for five days. The results obtained have shown that, with or without BSA, Epo deprivation of up to five days (an epoprival state) did not markedly decrease the number of bursts produced by unfractionated peripheral mononuclear cells compared to the number produced in the presence of Epo from the beginning of culture. Similar results were found whether the fetal calf serum (FCS) concentration was 15% or 2%. The preservation of potential BFU-e formation during the epoprival state has therefore been attributed to the ability of T-lymphocytes and/or monocytes to supply BPA. In order to reduce the endogenous amount of BPA, a nonadherent, E-rosette-negative cell fraction was cultured in the presence of Epo, with or without BSA, in serum-free medium containing transferrin (TF). Under these conditions, an equal number of bursts was obtained in FCS and in serum-free medium containing Epo, BSA and TF, whereas no BFU-e growth was found in the presence of Epo and TF, but without BSA. If Epo was withheld for up to five days, the capacity to form erythroid colonies was still retained by the monocyte- and T-lymphocyte-depleted cell fraction in the continuous presence of BSA. However, BPA could not be detected in the BSA. This observation was further supported by experiments in serum-free medium using human recombinant Epo, in which no BFU-e colony formation could be detected in the presence of BSA. From our investigations carried out at limited cell density and in serum-free medium, it could be concluded that the crude Epo preparation was the source of BPA.

Growth-inhibitory effect of 2-CdA on myeloid CFU-GM progenitors in normal human long-term bone marrow cultures and its compensation by G-CSF or IL-3.

As neutropenia is a common side effect of treatment with 2-chlorodeoxyadenosine (2-CdA), we investigated the myelosuppressive action of 2-CdA in Dexter-type human long-term bone marrow cultures (LTBMCs). LTBMCs were incubated with varying doses of 2-CdA (5 to 20 nM/L) during the first week. At 20 and 10 nM/L 2-CdA, we found a marked reduction in colony-forming unit-granulocyte/macrophage (CFU-GM) production throughout the culture period of 7 weeks (maximum reduction to 3.5% of untreated control cultures with 20 nM/L and to 27.2% with 10 nM/L, respectively). Even the lowest 2-CdA dose tested (5 nM/L) strongly reduced the number of CFU-GM progenitors during the first 3 weeks (maximum reduction to 52.4% of untreated controls), but this effect was transient, and values had recovered to normal within in 5 weeks. 2-CdA was also shown to cause a dose-dependent decrease in long-term culture-initiating cell (LTCIC) detections after 5 weeks in culture (49.6% of control cultures with 10 nM/L 2-CdA and 14% with 20 nM/L 2-CdA, respectively). When 2-CdA was added to LTBMCs initiated on preformed irradiated stromal feeder layers, similar results on CFU-GM production were obtained, indicating that the effects observed were not secondary to effects on the formation of a supportive layer. In addition, IL-6-concentrations in the supernatant of LTBMCs measured at various intervals after the addition of fresh medium with or without 2-CdA showed no significant decrease in cultures treated with 2-CdA. As neutropenia has been shown to be associated with a small but significant risk of fatal infection, we subsequently investigated the reversal potential of the 2-CdA effect by addition of recombinant human granulocyte colony-stimulating factor (rhG-CSF) or rh interleukin-3 (rhIL-3). The weekly addition of 100 ng/mL rhG-CSF counteracted the 2-CdA-mediated decrease in CFU-GM numbers during the entire period of 7 weeks, reaching statistical significance from weeks 3 to 7 (p < 0.05). Addition of rhIL-3 (100 ng/mL) showed an enhancement of CFU-GM output in 2-CdA-treated cultures that resulted in their numbers exceeding those in control cultures (without 2-CdA) from weeks 1 to 5 (p < 0.05) with a maximum increase of 5.1-fold over the parallel control value at week 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene rearrangement studies in the diagnosis of primary systemic and nodular primary localized cutaneous amyloidosis.

Difficulties may arise in the diagnosis of patients with clinical features suggestive of plasma cell dyscrasia-related amyloidosis (amyloidosis L), but without evidence of a paraprotein. We have employed gene rearrangement methodology to demonstrate the clonality of bone marrow cells not only in a patient with myeloma-associated systemic amyloidosis, but also in a patient with "primary" systemic amyloidosis without overt myeloma or a detectable paraprotein. Furthermore, we have shown the clonality of the amyloid-producing plasma cells within a skin nodule of a patient with primary localized cutaneous amyloidosis; by contrast, clonal rearrangement was not detected in bone marrow cells from this patient. This finding provides definitive proof that organ-limited nodular primary localized cutaneous amyloid deposits arise in relation to cutaneous plasmacytomas. Gene rearrangement studies may enable early diagnosis and initiation of treatment in patients with systemic amyloidosis L, as well as their differentiation from patients with organ-limited nodular cutaneous amyloidosis, who do not require aggressive therapy.

A comparison of the antiproliferative properties of recombinant human IFN-alpha 2 and IFN-omega in human bone marrow culture.

We compared the antiproliferative effects in vitro of recombinant preparations of interferon-alpha 2c (IFN-alpha 2) and IFN-omega on the formation of colonies from bone marrow progenitor cells. Both IFNs led to statistically indistinguishable dose-dependent inhibitory effects when tested on bone marrow cells derived from 8 normal donors or from 7 patients with chronic myelogenous leukemia (CML). With both IFNs, the cells from CML patients appeared slightly but not significantly more sensitive to inhibition than the cells from normal donors. These results suggest that under some circumstances, IFN-omega may prove an effective treatment for CML, for example, in those becoming resistant to IFN-alpha 2 because of the formation of neutralizing antibodies.

Gemcitabine--a novel immunosuppressive agent--prevents rejection in a rat cardiac transplantation model.

BACKGROUND: 2',2' -difluorodeoxycytidine (dFdC, gemcitabine) is a pyrimidine antimetabolite with antineoplastic activity against a wide range of solid tumors. The immunosuppressive activities of this compound have not been described to date. METHODS: The in vitro effects on activated T lymphocytes were studied with a lymphocyte colony-forming assay in a microagar culture system. Heart transplantations were performed in the fully allogeneic Lewis/ Brown Norway combination. dFdC was administered once daily at various dosages from the time of surgery until day 50. RESULTS: Phytohemagglutinin-induced lymphocyte proliferation was inhibited 50% by dFdC at a concentration of 3.25+/-0.9 nmol/L. Allografts of untreated animals survived for 7.5 (7-8) days and those with 25, 50, and 75 microg/kg body weight dFdC for 7.3 (7-8), 9.3 (8-10), and 16.3 (10-38) days, respectively. Treatment with 100 or 125 microg/kg body weight of dFdC, however, prolonged allograft survival until day 152.8 (129-178). Dose-dependent leukopenia was the main toxicity. CONCLUSIONS: DFdC is a new immunosuppressive agent that can successfully prevent cardiac rejection in a rat transplantation model.

Differential effects of cladribine and gemcitabine on erythroid and granulocytic progenitors from patients with chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal neoplastic disease that originates in a pluripotent stem cell. Selection of normal progenitors by graft-purging may improve the outcome after autologous transplantation. In our methylcellulose assays, the nucleoside analogs cladribine (2-CdA) and gemcitabine (dFdC) showed more prominent inhibitory effects on CML than normal bone marrow (BM) progenitors. For dFdC, however, long-term incubations were necessary to achieve complete inhibition. Deoxycytidine kinase, the key enzyme of both 2-CdA and dFdC metabolisms, was only partially responsible for this differential sensitivity. We suggest that 2-CdA and dFdC might be helpful in purging of CML BM cells before autologous BM transplantation. Further studies on more primitive cells are warranted.

Progressive preleukemia presenting amegakaryocytic thrombocytopenic purpura: association of the 5q- syndrome with a decreased megakaryocytic colony formation and a defective production of Meg-CSF.

The authors present a patient with the typical clinical picture of an acquired amegakaryocytic thrombocytopenic purpura. After 16 months of observation, the patient developed acute myelomonocytic leukemia. During the preleukemic phase and after progression to overt leukemia, serial in-vitro analyses of megakaryocytic, granulocytic, erythrocytic and T-lymphocytic colony growth were carried out in a microagar culture system. At presentation, a marked diminution of CFU-M was observed, whereas CFU-E, BFU-E, CFU-C and CFU-TL were in the normal range. The CFU-M number remained at its low level during the whole observation period. The CFU-C number declined steadily during the preleukemic period, while BFU-E, CFU-E and CFU-TL remained constant until January 1985 when the patient developed AML. After progression to overt leukemia, a distinct reduction became evident in all colony-forming cells. Cytogenetic studies performed during the preleukemic phase indicated the presence of a 5q- chromosome. The authors submit evidence here that the patient was not only characterized by defective megakaryocytic colony formation but also by a deficiency of functional megakaryocyte colony-stimulating activity. No humoral or cellular inhibitors of CFU-M colony formation were found. It is concluded that in preleukemia with a 5q- chromosome the megakaryocytic cell lineage may be involved in the process that precedes overt leukemia at an earlier time than cells of granulocytic and erythrocytic lineages. In addition, it is shown here that megakaryocytopoiesis during the preleukemic period can be characterized by two different defects: first, an intrinsic megakaryocytic stem cell defect and, second, a deficiency of functional megakaryocytic colony-stimulating activity.

Recombinant interferon-alpha-2C in chronic myelogenous leukaemia: relationship of sensitivity of committed haematopoietic precursor cells in vitro (BFU-E, CFU-GM, CFU-Meg) and clinical response.

In an ongoing phase-II trial we aimed to predict clinical responsiveness of Philadelphia chromosome positive (Ph1+) chronic myelogenous leukaemia (CML) to recombinant IFN-alpha-2C (rIFN-alpha-2C) by pretesting in vitro. From five normal controls and 14 CML patients in chronic phase, bone marrow samples were taken before treatment and tested for antiproliferative activity by rIFN-alpha-2C, using a microagar culture system for BFU-E, CFU-GM, and CFU-Meg. Light-density nucleated bone marrow cells were stimulated for BFU-E and CFU-Meg colony formation with Alpha medium containing 20% serum obtained from a patient with severe aplastic anaemia. CFU-GM growth was induced with conditioned medium from the cell line GCT. In normal controls BFU-E, CFU-GM and CFU-Meg colony formation was inhibited by rIFN-alpha-2C in a dose-dependent manner. BFU-E proved to be the most sensitive cell lineage (IC50: 65; range: 53-116 U/ml) whereas CFU-GM was about 20 times less sensitive (IC50: 643; range: 480-897 U/ml). The sensitivity of CFU-Meg ranged between these two colony types with 50% growth inhibition at an IFN concentration of 160 (range: 68-246 U/ml). A heterogeneous response to rIFN-alpha-2C in vitro was seen in CML patients. Three of the 14 patients were 'resistant' to rIFN-alpha-2C in vitro with IC50 values for BFU-E, CFU-GM and/or CFU-Meg colony formation greater than 10(4) U/ml. Patients were subsequently treated with a daily dose of rIFN-alpha-2C of 5 x 10(6) U. Four patients achieved a complete and six achieved a partial haematological response. Of the four non-responders three rapidly progressed into blastic crisis. Thus it was seen that treatment failure to interferon was accompanied by IFN-resistance in vitro of BFU-E, CFU-GM and/or CFU-Meg colony formation by bone marrow precursors (p less than 0.01). These results suggest a predictive value of IFN-sensitivity testing in vitro in Ph1 + CML.

Studies of myelopoiesis in vitro on blood and bone marrow cells of patients with acute leukemia in long-term remission.

In vitro myelopoiesis in a group of ten patients with acute leukemia (6 AML, 1 APML, 3 ALL) in long-term remission has been studied. The patients remained in first stable remission for at least 4 years and maintenance therapy had been completed in all patients except one. In the patients, colony formation of bone marrow cells after 7 days of incubation was significantly increased compared to a representative control group. The proliferation of microclusters after 3 incubation days was also markedly enhanced, and accelerated proliferation of all aggregates (microclusters, macroclusters and colonies) could be demonstrated in culture with the patients' bone marrow cells. The use of autologous feeder layers and autologous serum showed no inhibitory effect on colony formation. The proportion of eosinophil colonies formed with the patients' bone marrow cells was in the normal range. In contrast to the high proliferation capacity of bone marrow precursor cells the CFU-c number of peripheral blood was significantly decreased in the patients' group. No significant correlation between CFU-c number of bone marrow and blood cells could be found. The colony stimulating activity of the patients' peripheral mononuclear cells was normal compared to healthy controls. We conclude from this study that even in long-term remission of acute leukemia certain in vitro abnormalities exist in myelopoietic proliferation and regulation.