Hematopoietic damage prior to PBSCT and its influence on hematopoietic recovery.
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BACKGROUND AND OBJECTIVE: Patients with malignancies receive chemotherapy to induce tumor remission which could damage hematopoiesis and adversely influence hematopoietic reconstitution after transplantation. In the present study we used a long-term culture (LTBMC) system and clonogenic assays to evaluate the marrow damage in patients selected to receive peripheral blood stem cell transplantation (PBSCT). DESIGN AND METHODS: Thirty-five patients - 20 with breast cancer (BC), 9 with non-Hodgkin's lymphoma (NHL) and 6 with Hodgkin's disease (HD) - were included. Bone marrow aspiration was performed one day prior to the initiation of the conditioning therapy. CFU-GM were cultured in methylcellulose with PHA-LCM. Delta assays of plastic adherent progenitor cells (PD) were performed according to Gordon's method. LTBMC were established for 5 weeks. RESULTS: There were fewer CFU-GM from all patient groups than from normal BM (p<0.05). In contrast, the number of immature progenitor cells (PD) was not decreased. The total number of CFU-GM produced by LTBMC patients was significantly reduced (p<0.05). The adherent layer from patients was often qualitatively different. In order to know whether the hematopoietic damage could affect hematopoietic reconstitution, we correlated culture data with time taken to reach peripheral cell counts. A negative correlation (r= - 0.71) was found between percentage of stromal layer and time taken to reach 20x10(9) platelets/L (tplat= 20x3-0.08% stromal layer). INTERPRETATION AND CONCLUSIONS: We can conclude that prior to PBSCT, hematopoietic function is impaired at both the level of committed progenitor cells and that of BM stroma. This damage could influence platelet recovery. (+info)
Erythropoietin and platelet production.
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BACKGROUND AND OBJECTIVE: Erythropoietin (Epo) is the primary growth factor for the red cell lineage but treatment with recombinant human Epo (rHuEpo) has been shown to increase platelet counts. In several animal species treatment with rHuEpo stimulated platelet production, but platelet counts tended to normalize after 1-2 weeks and large, chronic doses even caused thrombocytopenia. This paper aims to review the evidence about the effects of Epo on megakaryopoiesis. INFORMATION SOURCES: I examined the literature published in journals covered by Medline(R)a concerning the effects of Epo, hypoxia and iron deficiency on megakaryopoiesis and platelets. The reference list of each article was reviewed to try to identify further contributions. STATE OF THE ART: In vivo data have shown that moderate Epo stimulation, i.e. that produced by standard doses of rHuEpo, short-term hypoxia or moderate iron deficiency, causes a moderate elevation of platelet counts, whereas intense Epo stimulation, as produced by high doses of rHuEpo, prolonged hypoxia or severe iron deficiency, causes some degree of thrombocytopenia. In the latter case, there appears to be a diphasic response to Epo, the initial positive response (a stimulation of platelet production) being followed by thrombocytopenia. Contrarily to the thrombocytopenia due to increased platelet destruction induced by other growth factors, Epo-induced thrombocytopenia is the result of an inhibition of platelet production. CONCLUSION AND PERSPECTIVE: Stem-cell competition between erythroid and platelet precursors appears to be the cause of these phenomena in situations of prolonged, intense stimulation by Epo. In vitro data support the existence of a common erythrocytic and megakaryocytic precursor. It remains to be determined whether these effects of rHuEpo are a result of the dose itself or of the magnitude of the erythropoietic effect of that dose. It is not known whether a lower dose given in a patient with decreased marrow function would bring about the same biological effects as those induced by high doses of rHuEpo in the presence of a normal marrow function. Caution should be exercised before using high doses of hematopoietic growth factors. (+info)
Influence of recombinant human granulocyte colony-stimulating factor (filgrastim) on hematopoietic recovery and outcome following allogeneic bone marrow transplantation (BMT) from volunteer unrelated donors.
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Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF, filgrastim) on hematopoietic recovery and clinical outcome in patients undergoing allogeneic bone marrow transplantation (BMT) from volunteer unrelated donors (VUD) were analyzed retrospectively. Additionally, the influence of baseline patient and transplant characteristics on hematopoietic recovery was evaluated. From January 1994 to March 1996, 47 consecutive adult patients received VUD-BMT. GVHD prophylaxis was cyclosporin A/short course methotrexate/prednisolone, and in four patients additional ATG. Post-transplantation, cohorts of patients received rhG-CSF (5 microg/kg/day) (n = 22) or no rhG-CSF (n = 25) in a non-randomized manner. The patient groups with and without rhG-CSF were rather comparable with respect to baseline patient and transplant characteristics. Median time to neutrophil counts (ANC) >500/microl was 14 days with rhG-CSF vs 16 days without rhG-CSF (P = 0.048), to ANC >1000/microl was 15 vs 18 days (P = 0.084). Neutrophil recovery was accelerated in patients receiving more than the median MNC dose of 2.54 x 10(8)/kg with a median time to ANC >1000/microl of 13 days vs 19 days (P = 0.017). RhG-CSF did not influence platelet recovery and incidence of infectious complications. Incidence of acute GVHD II-IV was 50% with rhG-CSF and 28% without rhG-CSF (P = 0.144), but death before acute GVHD II-IV occurred in 9% of patients with and 20% of patients without rhG-CSF. The median follow-up time was 38 and 36 months in patients with and without rhG-CSF, respectively. Survival at 2 years post-transplant was 39% (95% confidence interval (18%, 60%)) in patients with rhG-CSF and 24% (95% confidence interval (7%, 41%)) in patients without rhG-CSF. Administration of rhG-CSF after VUD-BMT may lead to more rapid neutrophil recovery, but did not influence the incidence of infectious complications. Patients receiving rhG-CSF showed a slightly higher incidence of acute GVHD II-IV. Higher numbers of MNC in the marrow graft accelerated hematopoietic engraftment. (+info)
Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis.
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The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105. (+info)
Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis.
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Embryonic liver is a transient site for definitive hematopoiesis. Along with maturation of the bone marrow and spleen, hematopoietic cells relocate from the liver to their final destinations while the liver starts organizing its own structure and develops numerous metabolic functions toward adult. Recently, it was demonstrated that the signal exerted by oncostatin M (OSM) through gp130 plays a pivotal role in the maturation process of the liver both in vitro and in vivo. However, the molecular basis underlying the termination of embryonic hematopoiesis remains unknown. In this study, we report that primary culture of fetal hepatic cells from embryonic day 14.5 murine embryos supported expansion of blood cells from Lin-Sca-1(+)c-Kit+ cells, giving rise to myeloid, lymphoid, and erythroid lineages. Of interest, promotion of hepatic development by OSM and glucocorticoid strongly suppressed in vitro hematopoiesis. Consistent with these results, hepatic culture from the embryonic day 18.5 liver no longer supported hematopoiesis. These data together with the previous observations suggest that the signals exerted by OSM and glucocorticoid induce hepatic differentiation, which in turn terminate embryonic hematopoiesis and promote relocation of hematopoietic cells. (+info)
Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells.
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Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis. (+info)
Loss of FancC function results in decreased hematopoietic stem cell repopulating ability.
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Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue of FANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC -/- cells in vivo. Competitive repopulation assays were conducted where "test" FancC -/- or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC -/- BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 x 10(5) competitor cells. We determined that FancC -/- cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+ cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function. (+info)
Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.
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To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients. (+info)