Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs.

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.

Cytotoxic chemotherapy regimens that increase dose per cycle (dose intensity) by extending daily dosing from 5 consecutive days to 28 consecutive days and beyond.

Dose intensity, defined as dose administered per unit time, has emerged as a potentially important measurement of anticancer drug exposure and determinant of efficacy. There are several strategies for increasing dose intensity, one being a protracted daily dosing strategy without major dose reduction for toxicity. This strategy involves continued therapy during periods of recovery from reversible toxicity, and it inherently challenges our understanding that renewing tissues cannot repopulate (recover) in the continued presence of cytotoxic drug. We have tested this idea directly in a murine preclinical trial. Specifically, we have tested whether acutely myelotoxic doses of gemcitabine (i.p. injection, 6.0 mg/m2/day), acetyldinaline [CI-994; GOE 5549; PD 123 654; 4-acetylamino-N-(2'-aminophenyl)-benzamide, 150 mg/m2/day p.o.], and/or melphalan (i.p. injection, 7.2 mg/m2/day) can be tolerated for 28 consecutive days and whether suppressed bone marrow function recovers despite this protracted daily therapy. The three drugs all caused acute neutropenia and suppression of medullary hematopoiesis. Damage to progenitor populations exposed to acetyldinaline and gemcitabine was not as severe as that caused by melphalan, in which case absolute neutrophil count, mature progenitors (colony-forming unit granulocyte/macrophage), and immature progenitors (colony-forming unit-S) progressively declined to severely depressed levels. Marrow recovery was observed during continued daily treatment with acetyldinaline and gemcitabine but not melphalan, and marrow function completely recovered after finishing the 28-day course. Pharmacology studies proved that protracted therapy causes little, if any, change in cellular drug tolerance or systemic exposure.

In vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice.

OBJECTIVE: Intranasal inoculation of the i strain of the parvovirus minute virus of mice (MVMi) into immunodeficient SCID mice induces suppression of myeloid and erythroid progenitors in the bone marrow (BM) and lethal leukopenia. In the present study, we investigated whether the mouse megakaryocytic lineage was susceptible to MVMi. MATERIALS AND METHODS: In vitro and in vivo infections with purified MVMi were conducted and their effects on the megakaryocytic lineage studied. RESULTS: In vitro infection of BM cells showed a multiplicity of infection-dependent inhibition in the colony-forming ability of megakaryocytic progenitors (colony-forming unit megakaryocyte [CFU-MK]). Neutralization or heat inactivation of the virus abrogated this inhibition. Expression of the MVMi nonstructural-1 protein was detected in the in vitro infected and cultured megakaryocytic cells. In vivo, intranasal inoculation of a lethal dose of virus was incapable of producing significant thrombocytopenia, although an increase in mean platelet volume was observed. Significantly, in the BM of these animals, a progressive decrease in CFU-MK was noted from day 14 postinfection, with survival rates less than 1% by day 35 postinfection. At day 35 postinfection, intermediate megakaryocytic differentiation stages showed maintenance of the proportion and ploidy of cells and a moderate decrease in the total number of these cells per femoral BM. CONCLUSIONS: The results demonstrate that MVMi is capable of inhibiting the proliferative capacity of megakaryocytic committed progenitors both in vitro and in vivo. Moreover, the in vivo data show that depletion of BM CFU-MK is compensated by the system, and platelet counts in the peripheral blood are maintained close to normal values.

Latent hematopoietic stem cell toxicity associated with protracted drug administration.

OBJECTIVE: The protracted administration of near-conventional daily doses of chemotherapeutic agents is a strategy to increase dose intensity and, potentially, efficacy as well. However, protracted therapy carries the risk of damage to stem cells in proliferative tissues that are not targeted by intermittent schedules. Therefore, we have investigated the effects produced by the protracted administration of two anticancer drugs on hematopoietic stem cell function. MATERIALS AND METHODS: We used the competitive repopulating assay to assess stem cell damage caused by protracted daily drug treatment of mice. RESULTS: Treatment with acetyldinaline for 10 consecutive days mediated a modest effect on the short-term repopulating cells (STRCs) but spared the long-term repopulating cells (LTRCs). Gemcitabine for 10 days led to a modest decline in both the STRCs and LTRCs. Extending treatment with gemcitabine for 28 days resulted in more severe repopulating cell (RC) damage, which was much worse than in acetyldinaline-treated mice. As expected, melphalan for 10 or 28 days mediated a marked reduction in all of the RCs of treated mice. The analysis of the RCs from mice that were allowed a 1-year recovery period after completing the 28-day treatment with either acetyldinaline or gemcitabine showed normal levels of neutrophils and bone marrow (BM) progenitors. However, a reduction in the RCs was observed in both groups, with larger reductions in gemcitabine-treated mice. CONCLUSIONS: Our data show that protracted treatment with gemcitabine, but not acetyldinaline, of mice caused severe permanent damage to the stem cell components. Therefore, although 28-day therapy with acetyldinaline or gemcitabine appeared to be well tolerated at the level of peripheral blood and bone marrow progenitors, gemcitabine produces permanent stem cell damage when used in long-term administration regimens that should perhaps only be explored clinically with stem cell support available.

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells.

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion. Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.

In vitro toxicity of ET-743 and aplidine, two marine-derived antineoplastics, on human bone marrow haematopoietic progenitors. comparison with the clinical results.

Ecteinascidine-743 (ET-743) and aplidine are two marine-derived antineoplastics currently in phase II development. With the aim of evaluating whether in vitro haematopoietic studies can predict the toxicity of these two drugs in patients, human bone marrow (BM) samples were incubated with these drugs under conditions which mimicked the administration exposures used in the clinics. As it was observed in different cancer cell lines, ET-743 was more toxic on an equimolar basis in human hematopoietic progenitors (inhibitory concentration reducing the viability to 50% after 24 h exposures; IC50(24h): 10-50 nM) compared with doxorubicin (IC50(24h) values: 280-460 nM), used as a control anticancer drug. In contrast to the high haematotoxic effects observed for ET-743, similar IC values were obtained for aplidine (IC50(24h): 150-530 nM) compared with doxorubicin. For both ET-743 and aplidine, the megakaryocytic progenitor was the most sensitive, compared with the other haematopoietic progenitors (IC50 values were 3- to 5-fold lower in the CFU-Megs compared with the CFU-GMs). The observation that the Cmax observed in patients treated with the aplidine maximum tolerated dose (MTD) (7.1 nM) was 21-75 fold lower than the IC50(24h) value observed for the different haematopoietic progenitors is highly consistent with the lack of haematotoxicity observed in patients treated with this drug. In the case of ET-743, differences between the Cmax value corresponding to the MTD (2.6 nM) and the in vitro IC50 values corresponding to the different progenitors were much lower (4-19-fold), also consistent with the haematotoxicity that was observed in patients treated at recommended doses (RDs) and MTDs. Although CFU-Megs were more sensitive than CFU-GM progenitors to ET-743 in vitro, clinical data showed that neutropenic events were more frequent than thrombocytopenic episodes. Aiming to further improve the predictive value of in vitro IC values corresponding to the different haematopoietic progenitors, additional refinement parameters derived from pharmacokinetic and animal studies are proposed.

Preserved long-term repopulation and differentiation properties of hematopoietic grafts subjected to ex vivo expansion with stem cell factor and interleukin 11.

BACKGROUND: The ex vivo expansion of hematopoietic grafts has been proposed as an efficient procedure for improving the hematological recovery of recipients. The fate of the long-term repopulating cells during the ex vivo manipulation of the graft is, however, a critical issue in ex vivo expansion protocols and ultimately will define the applicability of this new technology in hematopoietic transplants. METHODS: The repopulating ability of mouse hematopoietic samples was determined by means of bone marrow (BM*) competition assays, using congenic strains that express the pan-leukocyte Ly-5.1 and Ly-5.2 antigens. The generation of potential changes in the repopulating properties of human hematopoietic samples subjected to ex vivo expansion was determined by comparing the engraftment of fresh and ex vivo-manipulated CD34+ cord blood cells in irradiated nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. RESULTS: Under our optimized conditions of mouse BM incubation (stem cell factor plus interleukin-11, either with or without macrophage inflammatory protein-1alpha or Flt3 ligand), both the short-term and the mid-term repopulating ability of the ex vivo-expanded samples were significantly improved when compared with fresh samples. In the long-term, no changes in the repopulation and differentiation properties of the graft were observed as a result of the ex vivo expansion process. As deduced from the analysis of NOD/SCID mice transplanted with fresh and ex vivo expanded human CD34+ cord blood cells, the in vitro stimulation mediated by SCF/IL-11/FLT3L was capable of preserving the ability of the grafts to repopulate the lympho-hematopoiesis of recipients for at least 3 months. CONCLUSION: These results indicate that under our optimized conditions of ex vivo expansion, the amplification of the hematopoietic progenitors responsible for the short- and mid-term repopulating properties of the graft can take place without compromising the long-term lympho-hematopoietic repopulating properties.

Conclusions of a national multicenter intercomparative study of in vitro cultures of human hematopoietic progenitors.

With the aim of developing a standardized program of clonogenic cultures, a multicenter intercomparative study of human CFU-GM, BFU-E and CFU-GEMM cultures was conducted. Aliquots of fresh mononuclear cord blood cells, as well as uniform culture materials and instructions for cell culture and for colony scoring were distributed to 28 national laboratories involved in hematopoietic research and transplantation. High interlaboratory coefficients of variation (CV) in the reported number of progenitors were found in our first intercomparative study (range 67-231%). To investigate the relevance of colony scoring in variations of the reported colony numbers, participants were invited to attend a meeting where a single culture dish was scored. In this case, the CVs ranged from 31% to 81%. A subsequent intercomparative assay was then conducted, and significant reductions in the inter-laboratory CVs were obtained with respect to the first study (CVs for colonies grown with two different media: CFU-GMs, 48% and 55%; BFU-Es, 70% and 62%; CFU-GEMMs, 70% and 51%; respectively). In most instances CVs were not significantly different from those obtained in the single plate scoring study, suggesting that the scoring process was the most relevant parameter accounting for variations in the reported colony numbers.

Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

AM218, a new polyanionic polysaccharide, induces radioprotection in mice when administered shortly before irradiation.

We have shown that the polyanionic polysaccharide AM218 improves the survival rate of the potentially lethally irradiated mice. This radioprotective effect was highly dependent on the administration schedule, the most efficient protocol being that in which the drug was given shortly before irradiation. The haematopoietic implications in the pharmacological action of AM218 were confirmed by the improved recovery in the three peripheral blood lineages observed in the AM218-treated mice. However, because of a marked increase observed in the number of white blood cells during the period of highest mortality of the control irradiated mice, effects on the neutrophil lineage may account for the effects mediated by AM218 in the irradiated mice. Both in vitro and in vivo treatment with AM218 before irradiation improved the survival rate of CFU-GM progenitors, while no effects were observed on the CFU-S pool. This led us to postulate that the improved survival rate of the committed progenitors, at least the granulocyte-macrophage progenitors, accounts for the radioprotective capacity of AM218.

In vitro hematotoxicity of Aplidine on human bone marrow and cord blood progenitor cells.

Aplidine is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans. In experimental animals, Aplidine mediated an in vivo inhibitory effect in a number of tumor cell types. In humans, Aplidine is currently used in phase I clinical trials. Aiming to predict the hematotoxicity of Aplidine in humans, samples from human bone marrow (BM) and cord blood (CB) were exposed in vitro to increasing concentrations of the drug and then assayed for the clonogenic ability of myeloid (CFU-GM), erythroid (BFU-E), megakaryocitic (CFU-Meg) and pluripotent (CFU-Mix) hematopoietic progenitors. We investigated whether predictions of the hematotoxicity of Aplidine based on bone marrow (BM) cultures were reproduced when a more readily available source of human hematopoietic cells, cord blood cells, was used in experiments involving 24-h exposures. Although hematopoietic progenitors derived from bone marrow were generally more sensitive than those derived from cord blood, differences on the IC50, IC70 and IC90 varied within a relatively small range of 1.6-6.2-fold. Moreover, data obtained from cord blood cultures confirmed the observation made in bone marrow assays indicating that the myeloid (CFU-GM) and the erythroid (BFU-E) progenitors were the least sensitive to Aplidine. Regardless of the origin of the hematopoietic progenitors (bone marrow or cord blood) the toxicity of Aplidine in human hematopoietic progenitors (IC50: 150-2250 nM) was lower than that observed in previous studies with tumoral cell lines.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

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Does the granulocyte-macrophage colony-forming unit content in ex vivo-expanded grafts predict the recovery of the recipient leukocytes?

We have investigated the leukocyte-repopulating-predictive value of granulocyte-macrophage colony-forming unit (CFU-GM) analyses in ex vivo-expanded versus fresh murine bone marrow (BM) grafts. After the transplantation of graded numbers of normal BM cells (from 15 to 5 x 10(3) CFU-GMs/mice), a dose-dependent increase in the recipient leukocytes was observed between the first and third weeks posttransplantation. During these stages, increases in the graft size of 100-fold improved the leukocyte counts up to 30-fold and shortened the leukopenia period by 5 to 11 days, depending on the leukocyte threshold considered. To investigate whether similar correlations could be established using ex vivo-expanded samples, the size of the CFU-GM population was maximized by means of the preactivation of the BM with 5-fluorouracil (9-day 5FU-BM), followed by 3 days of incubation with interleukin-1 plus stem cell factor. Under these conditions, the CFU-GM content of the ex vivo-expanded grafts was 73-fold higher than that observed in equivalent femoral fractions of normal fresh BM. When equivalent fractions of both graft types were transplanted, an improved leukocyte recovery was observed in mice transfused with the expanded grafts. However, the leukocyte values obtained after the transplantation of the ex vivo-expanded samples were not as high as expected, based on the number of transplanted CFU-GMs. Analyses performed during the second week posttransplantation showed that, in comparison with normal fresh BM, ex vivo-expanded grafts containing 6 to 50 times more CFU-GMs were required to generate a similar number of leukocytes. These results were confirmed in both the peripheral blood leukocytes and the myeloid Gr1+ cells, when similar numbers of CFU-GMs were transfused in the fresh and the ex vivo-expanded BM. The possibility that the preactivation of the ex vivo-expanded grafts with 5FU had a role in this effect was ruled out, because the leukocyte repopulation capacity of fresh 5FU-treated BM was as high as that observed in normal fresh BM which contained a similar number of CFU-GMs. Neither by extending the ex vivo incubation period nor by using other hematopoietic growth factor combinations was the functional capacity of the expanded grafts improved. The results presented in this study are consistent with the belief that ex vivo expansion procedures will be a useful tool for improving the hematologic recovery of patients who receive hematopoietic transplants. However, our data indicate that predicting the leukocyte repopulating capacity of ex vivo-expanded grafts according to correlations established with numbers of fresh CFU-GMs can lead to overestimations of their function, and therefore to unexpected and delayed hematopoietic engraftments.

The expression of Bcl-x is downregulated during differentiation of human hematopoietic progenitor cells along the granulocyte but not the monocyte/macrophage lineage.

Expression of the apoptosis inhibitory protein Bcl-x was studied in CD34+ hematopoietic precursor cells and in the promyelocytic leukemia cell line HL-60. The enriched population of CD34+ cells (more than 95%) was cultured in the presence of stem cell factor, interleukin-3 (IL-3), IL-6, and either granulocyte colony-stimulating factor or macrophage colony-stimulating factor to achieve granulocyte or monocyte/macrophage differentiation, respectively. The expression of Bcl-x increased in the early stages of both differentiation pathways. However, by day 21 of culture mature granulocytes were Bcl-x-negative, whereas monocytes/macrophages either maintained or increased the expression of Bcl-x. The pattern of Bcl-x expression in the differentiated CD34+ cells was similar to that observed in HL-60 cells differentiated along the granulocyte lineage (induced by incubation with retinoic acid), or along the monocyte/macrophage lineage (induced by incubation with phorbol diester). The bcl-x transcript predominant in HL-60 and CD34+ cells differentiated into monocytes/macrophages was bcl-xL. Although little is yet known regarding the functional significance of Bcl-x within the granulomonocytic compartment, marked changes in the pattern of its expression, as observed during granulomonocytic differentiation of HL-60 and CD34+ cells, are likely to alter the life span of mature granulocytes and monocytes/macrophages.

Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells.

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo-expanded CB cells, purified CD34(+) cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo-expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34(+) cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo-expanded versus the fresh CB cells (CD45(+) repopulation in NOD/SCIDs BM: 3. 7% +/- 1.2% v 26.2% +/- 5.9%, respectively, at 20 days posttransplantation; P <.005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% +/- 1.7% of CD45(+) cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo-expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45(+) and CD45(+)/CD34(+) cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo-expanded samples were found. The analysis of the engrafted CD45(+) cells showed that both the fresh and the in vitro-incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

Phenotypic and functional characteristics of hematopoietic cell lineages in CD69-deficient mice.

AIM/CD69 is the earliest leukocyte activation antigen and is expressed mainly by activated T, B, and natural killer (NK) cells. It is also constitutively expressed by platelets, by bone marrow myeloid precursors, and by small subsets of resident lymphocytes in the secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including Ca(++) flux, cytokine synthesis, and cell proliferation. To investigate the physiological relevance of CD69, we generated mice deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells. CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell receptor transgenic model demonstrated that these processes were not altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The B220(hi)IgM(neg) bone marrow pre-B cell compartment was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led to a slight increase in immunoglobulin (Ig) G2a and IgM responses to immunization with T-dependent and T-independent antigens. Nevertheless, CD69-deficient lymphocytes had a normal proliferative response to different T-cell and B-cell stimuli. Together, these observations indicate that CD69 plays a role in B-cell development and suggest that the putative stimulatory activity of this molecule on bone marrow-derived cells may be replaced in vivo by other signal transducing receptors.