Assuntos
Plaquetas/química , Proliferação de Células/efeitos dos fármacos , Imunoglobulinas/farmacologia , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/imunologia , Talidomida/análogos & derivados , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Humanos , Imunoglobulinas/química , Células Matadoras Naturais/patologia , Lenalidomida , Masculino , Mieloma Múltiplo/patologia , Talidomida/farmacologiaRESUMO
The correlation between the incidence of GVHD and the number of infused CD34(+) cells remains controversial for PBSC transplantation after a reduced-intensity-conditioning (RIC) regimen. We evaluated 99 patients transplanted with an HLA-identical sibling after the same RIC (2-Gy-TBI/fludarabine). Donor and recipient characteristics, donor's blood G-CSF-mobilized CD34(+) cell count, and number of infused CD34(+) and CD3(+) cells were analyzed as risk factors for acute and chronic GVHD There was a trend for an increased incidence of extensive chronic GVHD in the quartile of patients receiving more than 10 × 10(6) CD34(+) cells/kg (P = 0.05). Interestingly, the number of donor's blood CD34(+) cells at day 5 of G-CSF mobilization was closely associated with the incidence of extensive chronic GVHD, that is, 48% (95% CI: 28-68) at 24-months in the quartile of patients whose donors had the highest CD34(+) cell counts versus 24.3% (95% CI: 14-34) in the other patients (P = 0.007). In multivariate analysis, the only factor correlating with extensive chronic GVHD (cGVHD) was the donor's blood CD34(+) cell count after G-CSF (HR 2.49; 95% CI: 1.16-5.35, P = 0.019). This study shows that the incidence of cGVHD is more strongly associated with the donor's ability to mobilize CD34(+) cells than with the number of infused CD34(+) cells.
Assuntos
Antígenos CD34/imunologia , Doença Enxerto-Hospedeiro/imunologia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco de Sangue Periférico/métodos , Adulto , Idoso , Antígenos CD34/sangue , Doença Crônica , Doença Enxerto-Hospedeiro/sangue , Células-Tronco Hematopoéticas/metabolismo , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Transplante Homólogo , Adulto JovemRESUMO
Promising results of umbilical cord blood transplantation (UCBT) from unrelated donors have been reported in patients with hematologic disorders. These transplants, having potential to trigger beneficial donor-versus-recipient natural killer (NK) cell-mediated alloreaction, we have conducted the first extensive analysis of the phenotypic and functional properties of NK cells after UCBT. NK cells from 25 patients with high-risk hematologic malignancies were compared with cells derived from both healthy adult and CB cells. We found that following UCBT, NK cells display not only some phenotypic features associated with maturity but also unique characteristics that make them fully functional against leukemic blasts. We propose that this full functionality of alloreactive donor-derived NK may drive graft-versus-leukemia reactions after UCBT.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Efeito Enxerto vs Leucemia/imunologia , Neoplasias Hematológicas/terapia , Células Matadoras Naturais/imunologia , Adolescente , Adulto , Feminino , Hematopoese , Teste de Histocompatibilidade , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Doadores de Tecidos , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Adulto JovemRESUMO
This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.
Assuntos
Hematopoese , Músculo Esquelético/citologia , Músculo Esquelético/transplante , Animais , Antígenos de Superfície/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Humanos , Lentivirus/genética , Leucócitos Mononucleares/citologia , Macaca fascicularis , Macaca mulatta , Masculino , Modelos Animais , Transdução Genética , Irradiação Corporal TotalRESUMO
Ex vivo expansion is a new strategy for hematopoietic stem and progenitor cell transplantation based on cytokine-induced amplification to produce grafts of controlled maturity. If the cell cycle position of CD34(+) cells has been reported to govern their engraftment potential, the respective role of stem and progenitor cells in short- and long-term hematopoietic recovery remains debated. Studies focused on long-term engraftment potential suggest impairment when using cultured grafts, but the capacity to sustain short-term recovery is still controverted. The aim of this study was: A) to evaluate the consequences of cell cycle activation on short and long-term engraftment capacity, and B) to determine if cell cycle status of grafts could predict hematopoietic recovery. We showed in a nonhuman primate model of autologous peripheral blood stem and progenitor cell transplantation that cell cycle activation of CD34(+) cells in the presence of stem cell factor + FLT3-ligand + thrombopoietin + interleukin 3 (six days of culture) which induced G1 and S/G2/M cell amplification (G0: 6.1% +/- 2.8%; G0/G1: 64.2% +/- 7.2%; S/G2/M: 30.4% +/- 7.3% respectively of expanded CD34(+) cells on average) resulted in the acceleration of short-term granulocyte recovery. By contrast, G0/G1 and S/G2/M cell content of expanded grafts did not correlate with short- or long-term engraftment.
Assuntos
Granulócitos/metabolismo , Transplante de Células-Tronco Hematopoéticas , Interleucina-3/metabolismo , Proteínas de Membrana/metabolismo , Fator de Células-Tronco/metabolismo , Células-Tronco/metabolismo , Trombopoetina/metabolismo , Animais , Antígenos CD34/biossíntese , Ciclo Celular , Células Cultivadas , Citometria de Fluxo , Fase G1 , Fase G2 , Mitose , Papio , Fenótipo , Fase de Repouso do Ciclo Celular , Fatores de TempoRESUMO
BACKGROUND: Dexamethasone. cytarabine (ara-C), and cisplatin (DHAP) can be used effectively to treat patients with non-Hodgkin's lymphoma (NHL). We hypothesized that substitution of cisplatin by oxaliplatin (L-OHP) could result in less toxicity and greater efficacy. L-OHP is active in patients with lymphoma. It produces mild myelosuppression and is devoid of renal toxicity. We report on a phase II study of dexamethasone, high-dose ara-C, and L-OHP (DHAOx) used to treat patients with NHL who were previously treated with chemotherapy. PATIENTS AND METHODS: Fifteen patients were given DHAOx. They had failed to achieve a CR with initial chemotherapy or had recurrent disease. DHAOx consisted of dexamethasone, 40 mg/day (days 1 to 4): L-OHP, 130 mg/m2 (day 1); and ara-C, 2,000 mg/m2 every 12 h (day 2). Treatment was repeated every 21 days. RESULTS: Patients received a median of four courses of DHAOx. Myelosuppression and transient sensory peripheral neuropathy were the most prominent toxic effects. Serum creatinine levels did not increase in patients with normal renal function, nor in patients who had renal impairment before DHAOx. The median follow-up time from the start of DHAOx treatment was 17 months. Eight patients (53%) achieved a CR, and three patients (20%) had a PR. Responses were achieved by patients with lymphomas of various histologies that included mainly the follicular subtype, and by patients with and without resistance to prior chemotherapy. None of the eight responders have relapsed from CR at 4+. 6+, 14+, 15+, 19+, 20+, 24+, and 24+ months. They had various types of therapy after DHAOx. Disappearance of molecular markers was observed in all four patients who achieved a CR and whose tumor cells carried molecular abnormalities. CONCLUSION: DHAOx possesses characteristics of toxicity which compare favorably to those reported with DHAP, and it is useful as a salvage treatment for patients with NHL. Larger studies are required to establish the therapeutic potential of the regimen.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma não Hodgkin/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/análise , Citarabina/administração & dosagem , Dexametasona/administração & dosagem , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Humanos , Infusões Intravenosas , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/administração & dosagem , Recidiva , Resultado do TratamentoRESUMO
To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short-term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3-L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)-3 (50 ng/ml each); CD34+ cells, granulocyte-macrophage colony-forming units (GM-CFU) and megakaryocytic colony-forming units (MK-CFU) were amplified, respectively, 10.5-, 20.5- and 17.9-fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 x 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 x 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 x 109/l) and neutrophil (> 0.5 x 109/l) recovery occurred on days 8 (range 6-9) and 9 (range 6-11), respectively, compared with days 12 (range 10-15) and 14 (range 11-16); platelets recovered (> 20 x 109/l) on day 9 (range 7-12) compared with day 13 (range 11-15) in the control group (P < 0.05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow-up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long-term haematopoiesis.
Assuntos
Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas/métodos , Papio , Animais , Antígenos CD34/imunologia , Técnicas de Cultura de Células , Criopreservação , Citometria de Fluxo , Mobilização de Células-Tronco Hematopoéticas/métodos , Interleucina-3/farmacologia , Contagem de Leucócitos , Masculino , Proteínas de Membrana/farmacologia , Contagem de Plaquetas , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Trombopoetina/farmacologia , Transplante AutólogoRESUMO
Recent progress in the culture of human megakaryocytes (MKs) has led to the capacity to produce platelets in vitro. This capability enables investigation into the possibility of modifying platelet structure and/or function by genetically altering the MK. To this end, a cDNA for the murine CD9 (mCD9) cell surface protein was introduced into MK progenitors by retrovirally mediated gene transfer and subsequently detected in cultured MKs with a monoclonal antibody (MoAb) that specifically recognizes the murine protein. CD34+ human peripheral blood or marrow progenitors, enriched by immunomagnetic bead selection, were cultured for 5 days in the presence of growth factors, including stem cell factor and thrombopoietin, to induce MK progenitors into the cell cycle. The stimulated cells were then cocultured with the mCD9 retroviral producer cell line for 3 days, followed by culture in serum-depleted medium for 3 to 7 additional days. Flow cytometry analysis using the anti-CD9 MoAb and TAB, a MoAb recognizing human GPIIb, revealed that a large proportion (40-100%) of the MKs expressed mCD9. To ascertain whether these cells were capable of producing mCD9+ platelets, flow cytometry analysis was performed at a time when proplatelets were observed in the culture. mCD9 was detected in up to 59% of the TAB+ platelet-sized particles. Because deteriorating MKs can produce platelet-sized particles in vitro, experiments were performed to determine whether mCD9+ TAB+ particles were functionally active. Addition of phorbol myristate acetate resulted in the redistribution of P-selectin (CD62) from the alpha granule to the platelet surface as detected by MoAbs S12 and G5 in three-color flow cytometry analyses. These studies showed that up to 76% of the mCD9+ TAB+ particles were functionally active. The data show that retrovirally mediated gene transfer is a viable approach for genetically altering MK progenitors, resulting in platelets that express heterologous proteins.
Assuntos
Antígenos CD/biossíntese , Antígenos CD/genética , Plaquetas/metabolismo , Expressão Gênica/genética , Técnicas de Transferência de Genes , Megacariócitos/metabolismo , Animais , Antígenos CD34/análise , Plaquetas/citologia , Linhagem Celular , Hematopoese , Humanos , Leucócitos Mononucleares/imunologia , Megacariócitos/citologia , Camundongos , Plasmídeos/genética , Ativação Plaquetária/efeitos dos fármacos , Retroviridae/genética , Acetato de Tetradecanoilforbol/farmacologia , Transdução GenéticaRESUMO
Regulation of megakaryocytopoiesis and platelet production is a complex phenomenon. It has been demonstrated that numerous pleiotropic cytokines act in vivo and in vitro on megakaryocytopoiesis. Historically, studies on the regulation of megakaryocytopoiesis were largely dominated by the concept of humoral regulation. Despite 35 years of work, the factor responsible for these effects (thrombopoietin, TPO) could not be purified to homogeneity. The proto-oncogene c-mpl has been demonstrated to encode a protein which is the receptor for a humoral cytokine regulating megakaryocytopoiesis. Its ligand was isolated by three independent groups. The purified protein was termed Mpl-L (Mpl-Ligand), thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF). Strong evidence that Mpl-L is the homeostatic regulator of platelet production has been provided by c-mpl or Mpl-L knock-out mice which have a severe but not lethal thrombocytopenia. In vitro experiments have indicated that the Mpl-L acts both as a proliferative and differentiative factor as many other CSF. At the unicellular level, Mpl-L acts on MK progenitors inducing their proliferation. Mpl-L induces polyploidisation of the MKs and cytoplasmic maturation leading to an in vitro platelet production. As a single cytokine Mpl-L is the most potent growth factor for the MK lineage in vitro. However, a combination of cytokines can totally replace the effects of Mpl-L both on proliferation of MK progenitors and their maturation. In addition, Mpl-L has a major effect on primitive hematopoietic progenitors.
Assuntos
Diferenciação Celular/fisiologia , Hematopoese/fisiologia , Megacariócitos/citologia , Animais , Diferenciação Celular/genética , Expressão Gênica/fisiologia , Hematopoese/genética , Humanos , Camundongos , Camundongos Knockout , Proto-Oncogene MasRESUMO
Granulocyte-colony stimulating factor (G-CSF) has been successfully administered to healthy subjects to mobilize peripheral blood stem cells (PBSC) for allogeneic transplantation. Adverse events are moderate. We report the first case of apparent reactivation of an alloantibody to a blood cell antigen (Jk(a)) after G-CSF administration to a healthy subject and its transmission to the PBSC transplant recipient; no concomitant reactivation of other alloantibodies was detected. This case raises questions on the effect of G-CSF on the immune system and its safety in healthy individuals.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Eritrócitos/imunologia , Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Isoanticorpos/imunologia , Adulto , Formação de Anticorpos/imunologia , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lenograstim , Proteínas Recombinantes/efeitos adversos , Condicionamento Pré-Transplante/efeitos adversosRESUMO
BACKGROUND: Reverse seroconversion to hepatitis B virus (HBV), i.e., HBV reactivation in patients with pretransplant antibodies to hepatitis B surface antigen (anti-HBs) and to hepatitis B core antigen (anti-HBc), is rarely re-ported after allogeneic bone marrow transplantation. METHODS: To determine this risk, we studied clinical outcome and serological changes in 37 patients with pretransplant anti-HBs and anti-HBc. RESULTS: In 33 cases, no change in HBV markers was observed in the posttransplant period. In four cases, anti-HBs and anti-HBc were lost, and hepatitis B surface antigen, hepatitis B e antigen, and HBV DNA emerged together with acute hepatitis, after cessation of immunosuppression. The actuarial risk of reactivation in the 37 patients was 20.5% (median follow-up 20 months). No reactivation occurred in patients with anti-HBs-positive donors. CONCLUSION: Although few cases of postallogeneic bone marrow transplantation reverse seroconversion to HBV have been reported, this study demonstrates that the actuarial risk is relatively high and suggests that donor vaccination might be proposed prophylactically or that HBs-specific immunoglobulin infusions might be warranted.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite B/imunologia , Imunossupressores/efeitos adversos , Ativação Viral , Adolescente , Adulto , Criança , Feminino , Anticorpos Anti-Hepatite B/análise , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Resultado do TratamentoRESUMO
Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 x 10(11)/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (< 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 x 10(11) platelets, the high dose between 6 and 8 x 10(11), and the very high dose > 8 x 10(11); for the children, the three doses corresponded to 2 to 4, 4 to 6, and > 6 x 10(11) platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 x 10(9)/L, respectively) than the medium dose (33 x 10(9)/L, P < .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P < .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 x 10(11) per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets. The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure.
Assuntos
Contagem de Plaquetas , Transfusão de Plaquetas , Doença Aguda , Adolescente , Adulto , Peso Corporal , Transplante de Medula Óssea , Criança , Feminino , Humanos , Leucemia Mieloide/terapia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Trombocitopenia/terapia , Fatores de Tempo , Resultado do TratamentoRESUMO
During megakaryocyte differentiation, the promegakaryoblast (immature megakaryocyte) increases its ploidy to a 2(x) DNA content by a poorly understood process called endomitosis. This leads to the formation of a giant cell, the megakaryocyte (MK), which subsequently gives rise to platelets. In this report, we show that endomitosis of human MKs is due to abortive mitosis. Human MKs were obtained by a two-step purification of CD34(+) blood or marrow precursors followed by in vitro culture in the presence of MK growth factors. Microscopic examination shows that a large number of centrosomes (up to 32) and centrioles are present in polyploid MKs. After nocodazole treatment, more than 20% of the MK are blocked in a typical pseudo-metaphase. Both spontaneous and nocodazole-induced endomitosis are associated with a breakdown of the nuclear envelope and possess a complex mitotic spindle composed of several asters. Spindle microtubules radiate from each aster, creating a spherical structure. At metaphase, expression of the kinetochore phosphoepitope recognized by the 3F3/2 antibody is lost, and the sister chromatids segregate moving toward the spindle poles. After limited segregation, the chromosomes decondense and the nuclear envelope reforms in the absence of cytokinesis, isolating all chromosomes in a single nucleus. It has been proposed that endomitosis could be due to an abnormal CDK1 activity or an absence of cyclin B1. Our results show that cyclin B1 can be detected in all MKs, including those with a ploidy of 8N or more. The cyclin B1 staining colocalizes with the mitotic spindle. Using flow cytometry, the level of cyclin B1 increased until 8N, but remained identical in 16N and 32N MKs. Cell sorting was used to separate the MKs into a 2N/4N and >4N population. Both cyclin B1 and CDK1 could be detected in the endomitotic polyploid MKs using Western blot analysis, and a histone H1 kinase activity was associated with immunoprecipitated cyclin B1. We conclude that endomitosis of human MKs is due to abortive mitosis, possibly due to alterations in the regulation of mitotic exit.
Assuntos
Megacariócitos/citologia , Mitose , Antígenos CD34/análise , Proteína Quinase CDC2/fisiologia , Diferenciação Celular , Separação Celular , Células Cultivadas , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Ciclina B/fisiologia , Ciclina B1 , Citometria de Fluxo , Humanos , Megacariócitos/efeitos dos fármacos , Nocodazol/farmacologia , Ploidias , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Trombopoetina/farmacologiaRESUMO
The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+ CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF- or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.
Assuntos
Antígenos CD34/análise , Plaquetas , Citocinas/farmacologia , Hematopoese , Células-Tronco Hematopoéticas , Megacariócitos , Sangue , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Células da Medula Óssea/imunologia , Células Cultivadas , Meios de Cultura Livres de Soro , Células-Tronco Hematopoéticas/imunologia , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Leucaférese , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , PolietilenoglicóisRESUMO
The discovery of the Mpl ligand (Mpl-L), also called thrombopoietin (TPO), has facilitated in vitro investigation of human megakaryocytopoiesis. By confocal microscopy, endomitosis appeared as abortive mitosis skipping late stages of mitosis. No telophase and cytokinesis were observed. A spherical multipolar spindle which limits chromatid segregation was observed. The nuclear envelope subsequently reformed isolating all chromatids in a single nucleus. Platelet shedding was ultrastructurally studied. Platelet release occurred after formation of long cytoplasmic extensions (proplatelet formation), constriction areas delineating platelet territories. Heterogeneity in platelet size may be determined by the length of these extensions. Pegylated-recombinant human megakaryocyte growth and development factor, a truncated form of Mpl-L, was the most efficient cytokine to produce proplatelet-bearing megakaryocytes (MKs) and platelets in vitro. However, functional platelets with a normal ultrastructure could be produced in the presence of a combination of other cytokines. Finally, we investigated whether the induction of MK differentiation by the MS-5 stromal cell lines is due to Mpl-L. MS-5 cells synthesized Mpl-L transcripts and a biologically active protein. When human CD34+ cells were grown in contact or noncontact cultures with MS-5 cells, MK differentiation was observed. Soluble Mpl receptor (sMpl-Fc) addition inhibited MK growth, suggesting that the MK-promoting activity was due to Mpl-L production. Marrow stromal cell lines derived from TPO-/- mice were also able to sustain MK growth. Despite the absence of any production of Mpl-L, the sMpl-Fc continued to inhibit MK differentiation. This result suggests that the sMpl has a direct inhibitory effect and may explain the divergent results in the literature concerning the precise role of Mpl-L on the MK terminal differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/farmacologia , Megacariócitos/efeitos dos fármacos , Trombopoetina/farmacologia , Animais , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Mitose/efeitos dos fármacos , Mitose/fisiologia , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Trombopoetina/biossíntese , Trombopoetina/genéticaRESUMO
The site and mechanism of platelet production by bone marrow megakaryocytes (MKs) has been the subject of extensive studies, but is still a matter of controversy. However, the recent discovery of the Mpl ligand (Mpl-l), also called megakaryocyte growth and development factor (MGDF) or thrombopoietin, has resulted in considerable progress in the understanding of the maturation of the MK lineage. To better understand the mechanism of platelet production, we examined the late stage of MK maturation by electron microscopy in cells cultured in the presence of Mpl-l. Human bone marrow CD34+ CD38+ cells, which contain late MK progenitors, were purified by flow cytometry and cultured in a serum-free liquid medium containing recombinant human Mpl-l (MGDF 10 ng/mL) for 7 days. In this system, the majority of cultured cells were large MKs with lobulated polyploid nuclei. The MKs displayed a smooth surface with harmonious cytoplasmic maturation and abundant, regularly distributed demarcation membranes and alpha-granules, and even some dense granules. Interestingly, approximately 30% of the MKs observed displayed morphologic evidence of platelet production: at optical microscopy, MKs formed long filamentous cytoplasmic extensions (proplatelets) that fragmented into platelet-sized particles. Moreover, flow cytometric analysis of this cultured cell population showed GPIIb-positive particles of the size of platelets. Electron microscopic observation showed that MKs producing platelets displayed thin pseudopods on the surface, and that the channels of the demarcation membrane system were dilated, allowing long strands of cytoplasm to extend from the cell periphery. These cytoplasmic strands displayed beading with constrictions separating platelet-sized segments; the more distal to the cell core, the smaller the fragments were. They eventually detached from the cell core into the culture medium either occasionally still elongated or, more often, separated into individual platelets. Parallel longitudinal and perpendicular microtubules were visualized in the constricted regions of these cytoplasmic strips; immunogold study of tubulin localization confirmed this subcellular distribution. On both sides of the constricted areas, vacuoles were noted, the fusion of which might have led to the detachment of individual platelets. Finally, in close proximity to the platelet-forming MKs, numerous microparticles were shed. Although some of these particles might correspond to transverse sections of pseudopods, this did not seem to be the case, since they were rarely seen around thrombin-stimulated MKs with surfaces bristled by numerous pseudopods. Flow cytometry showed that apart from shed cytoplasmic fragments of platelet size, numerous smaller particles strongly labeled for CD41 were also released by mature MKs. In conclusion, this study describes the ultrastructure of human platelet production in cultured MKs, involving the formation of proplatelets and the shedding of microparticles.
Assuntos
Plaquetas/ultraestrutura , Megacariócitos/efeitos dos fármacos , Trombopoetina/farmacologia , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Citoplasma/ultraestrutura , Citometria de Fluxo , Humanos , Megacariócitos/citologia , Megacariócitos/ultraestrutura , Microscopia Imunoeletrônica , Microtúbulos/ultraestrutura , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Previous studies have suggested that peripheral blood stem cell (PBSC) transplantation has an advantage over autologous bone marrow transplantation. OBJECTIVE: To compare the hematologic recovery and costs associated with PBSC transplantation with those associated with autologous bone marrow transplantation in patients receiving high-dose chemotherapy for solid tumors or lymphomas. DESIGN: Multicenter, randomized, controlled clinical trial. SETTING: French Federation of Cancer Centers, located in cancer facilities or public hospitals with transplantation units. PATIENTS: Children and adults with solid tumors or lymphomas who were candidates for high-dose chemotherapy. INTERVENTIONS: Bone marrow or filgrastim-mobilized PBSCs. MEASUREMENT: The major and point was the duration of thrombocytopenia (platelet count < 50 x 10(9)/L). An economic evaluation of both types of transplantation was done prospectively to measure costs and cost-effectiveness. RESULTS: 129 patients entered the trial; 64 had PBSC transplantation, and 65 had bone marrow transplantation. The median duration of thrombocytopenia was 16 days in the PBSC group and 35 days in the bone marrow group (P < 0.001). All of the other clinical end points studied (time to last platelet transfusion, duration of granulocytopenia, number of transfusion episodes, and duration of hospitalization) favored PBSC transplantation. A cost analysis showed that total cost was decreased by 17% in adults and 29% in children with PBSC transplantation; thus, PBSC transplantation was clearly more cost-effective than bone marrow transplantation for both platelet and granulocyte recovery. CONCLUSION: Transplantation of PBSCs is associated with more rapid hematologic recovery than is bone marrow transplantation after high-dose chemotherapy for solid tumors or lymphomas. Furthermore, global costs are lower and cost-effectiveness ratios are better with PBSC transplantation.