RESUMO
About 10-15% of patients with acquired aplastic anemia (AAA) have resistant/recurrent disease not eligible for standard treatment like hematopoietic stem cell transplantation and/or combined immunosuppression. We report a 17-year-old male with an 11 years history of AAA who, after two courses of immunosuppression, was red cell transfusion-dependent, severely thrombocytopenic, refractory to platelet transfusion, had iron overload and post-transfusion HCV infection. This patient achieved transfusion independence from platelets and normalized Hb after treatment with the anti-TNF agent Etanercept. Over a 12 months follow-up he experienced only transient increase of liver transaminases.
Assuntos
Anemia Aplástica/tratamento farmacológico , Anemia Refratária/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Imunossupressores/uso terapêutico , Receptores do Fator de Necrose Tumoral/uso terapêutico , Terapia de Salvação/métodos , Adolescente , Anemia Aplástica/fisiopatologia , Anemia Refratária/fisiopatologia , Ensaios Clínicos como Assunto , Etanercepte , Hepatite C/complicações , Humanos , MasculinoRESUMO
A bone marrow harvest is filtered either in the operating room, in the laboratory or during infusion to the patient. Filters are usually discarded. Little is known of haemopoietic progenitor cells (HPCs) trapped in the filters. The aim of the study was to evaluate HPC content in the filters and to assess the outcome of transplants with filter-discarded or filter-recovered cells. Haemopoietic progenitors were grown from filters of 19 marrow transplants. We then compared the outcome of 39 filter-recovered transplants from HLA-identical siblings (years 2001-2004) with a matched cohort of 43 filter-discarded marrow grafts (years 1997-2000). Filters contained on average 21% long-term culture-initiating cells (LTC-IC) and 15% fibroblasts colony-forming units (CFU-F) of the total progenitor cell content. Filter-discarded transplants had significantly more grade II-IV graft-versus-host disease (GvHD) (42 vs 15%, P=0.008) as compared to filter-recovered transplants, and more transplant-related mortality (TRM) (20 vs 3%, P=0.04). The actuarial survival at 5 years is 69 vs 87%, respectively (P=0.15). This study suggests that a significant proportion of LTC-IC is lost in the filters together with CFU-F. Recovery and add back of progenitors trapped in the filters may reduce GvHD and TRM.
Assuntos
Transplante de Medula Óssea/métodos , Filtração/métodos , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Adolescente , Adulto , Transplante de Medula Óssea/efeitos adversos , Células Cultivadas , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
OBJECTIVE: The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. MATERIALS AND METHODS: We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4). RESULTS: The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p < 0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p < 0.02 and p < 0.001 respectively). CONCLUSIONS: Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.
Assuntos
Técnicas de Cultura de Células/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células 3T3/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Células Estromais/fisiologia , TemperaturaRESUMO
OBJECTIVE: In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myeloreductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. MATERIALS AND METHODS: We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%-100% Ph-negative PBPC for a median time of 28 months (range 18-50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). RESULTS: Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%-100% to 0%-25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21-43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term-culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p < 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p < 0.03). CONCLUSION: We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.
Assuntos
Hematopoese , Transplante de Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Adulto , Feminino , Sobrevivência de Enxerto , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Masculino , Pessoa de Meia-Idade , Cromossomo Filadélfia , Fatores de Tempo , Transplante AutólogoRESUMO
Allogeneic bone marrow transplant recipients maintain normal peripheral blood counts long term, suggesting durable support from engrafted stem cells. In order to investigate late hemopoietic reconstitution at the level of committed and early progenitors (LTC-IC), we studied 64 long-term survivors at a median interval of 6 years (range: 2-20) after allogeneic bone marrow transplant. CFU-GM and BFU-E numbers did not differ from normal controls; CFU-GEMM were found to be significantly decreased (1.2 +/- 0.2/10(5) vs 3.1 +/- 0.4, P = 0.001). The most remarkable defect was however, the low frequency of LTC-IC (3.2 +/- 0.6/10(6) vs 54.2 +/- 9.3, P = 0.0001) that did not improve with time and did not correlate with phase of the disease, conditioning regimen, CMV infections or GVHD. Number of infused cells and CFU-GM content of marrow grafts did not seem to influence the number of LTC-IC. This study documents a significantly reduced number of early progenitors in BMT patients despite normal numbers of committed progenitors and normal peripheral blood counts. This finding may suggest a permanent reduction of the stem cell reservoir after allogeneic bone marrow transplantation.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/patologia , Células-Tronco Hematopoéticas/patologia , Adolescente , Adulto , Anemia Aplástica/patologia , Anemia Aplástica/terapia , Estudos de Casos e Controles , Criança , Ensaio de Unidades Formadoras de Colônias , Feminino , Sobrevivência de Enxerto , Humanos , Leucemia/patologia , Leucemia/terapia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Transplante HomólogoRESUMO
INTRODUCTION: Limited information is available on the natural killer cell reconstitution after bone marrow transplantation and on the possible role of these cells in graft-versus-host-disease. MATERIALS AND METHODS: Blood samples were collected at different time intervals after transplantation. Lymphocytes were analyzed for informative markers by immunocytofluorimetric analysis. Natural killer cells derived from patients undergoing matched unrelated donor transplant were cloned by limiting dilution in the presence of phytohemoagglutinin and IL2. The natural killer cell clones were analyzed for cytolytic activity. RESULTS: In the nine patients analyzed undergoing transplantation from sibling donors, the majority of peripheral blood lymphocytes during the first 80 d after BMT were represented by T lymphocytes, while in the 15 patients undergoing matched unrelated donor transplant natural killer cells consistently outnumbered T lymphocytes. During the early phases after transplantation, most CD56+CD3- natural killer cells did not express CD16 which was expressed at later intervals. Analysis of the inhibitory receptors specific for HLA-class I molecules showed that CD94/NKG2A, specific for HLA-E, unlike normal donors, was expressed by all natural killer cells including the early appearing CD16-negative ones. Killer inhibitory receptors of the Ig superfamily were expressed late and in low percentages after transplantation and were always coexpressed with CD94/NKG2A. Natural killer-cell clones efficiently lysed the HLA-class I-negative cell lines K562 and 721-221. Natural killer-cell populations or clones isolated from patients with graft-versus-host-disease, failed to lyse donor or recipient derived phytohemoagglutinin-induced lymphoblasts. CONCLUSION: Our analysis shows that (1) recipients of matched unrelated donors transplants exhibit a high proportion of NK cells (2) all NK cells express CD94/NKG2A while the HLA-class I-specific inhibitory receptors of Ig superfamily appear at later stages and (3) donor NK cells do not lysed donor or recipient target cells.
Assuntos
Antígenos CD/imunologia , Transplante de Medula Óssea/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Teste de Histocompatibilidade , Células Matadoras Naturais/imunologia , Adolescente , Adulto , Alelos , Antígenos CD/análise , Técnicas de Cultura de Células/métodos , Células Clonais , Feminino , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunofenotipagem , Leucemia/sangue , Leucemia/imunologia , Leucemia/terapia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/terapia , Núcleo Familiar , Linfócitos T/imunologia , Fatores de Tempo , Doadores de TecidosRESUMO
We have previously demonstrated that Philadelphia negative (Ph-ve) hemopoietic cells can be collected by leukaphereses after an acute leukemia-like chemotherapy during the early hemopoietic recovery in patients with chronic myeloid leukemia (CML). In this study we have evaluated whether these collections contain very primitive hemopoietic cells defined as 'long-term culture initiating cells' (LTC-IC) and whether these cells belong to the Ph-positive or Ph-negative population. Twenty-eight out of 76 cytaphereses collected in 15 patients with CML proved to contain Ph-ve cells only (six patients), 21 showed only Ph+ve cells (five patients), and 27 a mixture of Ph+ve and Ph-ve cells (four patients). In cytaphereses containing Ph-ve cells only, we found variable numbers of LTC-ICs, more consistently when we mobilized patients in the first 3 months from diagnosis. In three cases cytogenetic analysis on LTC-ICs and CFU-GM confirmed results obtained on fresh samples. Ph-positive collections were devoid of LTC-ICs except for 2/21 samples. However, their cytogenetic analysis revealed a small number of Ph-negative progenitors. LTC-ICs were randomly detected in mixed (Ph+ve and Ph-ve) collections. In conclusion these data indicate that, in a consistent proportion of chronic myeloid leukemia patients, intensive chemotherapy is able to recruit Ph-ve LTC-ICs in to the peripheral blood. Moreover these data provide the biological basis for developing autografting programs with Ph-negative cells.
Assuntos
Citaferese , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Cromossomo Filadélfia , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologiaRESUMO
The study was devised to evaluate whether it was possible to collect Philadelphia-negative precursor cells in patients with chronic myeloid leukaemia. The approach was based on previous experience showing that complete remission (Ph-negative bone marrow cells) is rarely achieved after chemotherapy and is very short-lasting. We decided to explore whether it was possible to collect Ph-negative precursor cells in peripheral blood during the early phase of haemopoietic recovery. These data show that: the collection of Ph-negative precursor cells occurred in 12/16 (75%) patients mobilized within one year of diagnosis (group A) versus 12/33 (36%) in patients with a history of more than one year of disease (group B). Furthermore the numbers of Ph-negative precursor cells were significantly much higher at diagnosis. Ten patients mobilized at diagnosis were subsequently autografted with such Ph-negative precursor cells. Five of them remain Ph-negative from 4 to 12 months while the other five have percentages of Ph-positive cells in their marrow ranging from 20% to 70%. In this stage of the disease the procedure is safe and associated with a very good compliance. Occasional restoration of Ph-negative haemopoiesis could be observed up to 40 months after autograft, in patients of group B, but most of patients revert to Ph-positive haemopoiesis. in conclusion these data suggest that it is possible to restore Ph-negative haemopoiesis in 70% of patients mobilized at diagnosis. This percentage represent the highest one can obtain without allogeneic BMT, and this includes patients who never would have been cytogenetic responders to IFN-alpha. Whether and how long for Ph-negative status can be maintained is a matter for future observation and study.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Cromossomo Filadélfia , Adulto , Células Sanguíneas/ultraestrutura , Transplante de Medula Óssea , Protocolos Clínicos , Feminino , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Interferon-alfa/uso terapêutico , Leucaférese , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Transplante Autólogo , Transplante HomólogoRESUMO
'.the leukemic stem line is a small minority within the total cell mass;. when the leukemic stem line is not exceeding the normal stem cell numbers, its proliferation may still be under partial control.' LG Lajtha, Blood Cells 1981; 7: 45-62 We performed cytogenetic analysis on fresh bone marrow cells and on progenitor cell colonies in a patient who relapsed after allogeneic BMT for CML and was subsequently treated with donor lymphocyte infusions (DLI). Two Philadelphia-positive clones were identified at relapse. One clone displayed an additional chromosomal abnormality most likely induced by radio-chemotherapy and therefore arising in a single cell. This cell displays the characteristics of a stem cell, since it was able to support 20% of Ph-positive hemopoiesis for 5 months. If the progeny of a single Ph-positive stem cell account for 20% of hemopoiesis, a very low number of leukemic stem cells may sustain relapse after allogeneic BMT. This is in keeping with two observations: (1) at relapse, long-term culture initiating cells (LTC-IC) were all donor-derived and Ph-negative; (2) on average, the pace of the disease is very slow after relapse following allogeneic-BMT. Therefore, we hypothesize that a small number of leukemic stem cells may be involved in the initial events of relapse following BMT for CML.
Assuntos
Transplante de Medula Óssea , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Células-Tronco Neoplásicas/patologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Transfusão de Linfócitos , Recidiva , Transplante HomólogoRESUMO
INTRODUCTION: Normal immature hematopoietic progenitors are relatively well preserved in most patients newly diagnosed with chronic myeloid leukemia, but tend to decline rapidly with time. Such exhaustion could reflect a suppressive effect of the Philadelphia positive clone expansion and/or be induced by Interferon-alpha treatment. MATERIALS AND METHODS: A total of 51 CML patients were classified into three groups. Newly diagnosed untreated patients were group A (n=30). Of the 21 treated individuals with Interferon-alpha, for at least 12 months, 15 showed no cytogenetic response (group B) while six showed persisting major/complete response (group C). Patients belonging to groups A and B were mobilized with chemotherapy plus G-CSF while patients of group C received a short course of G-CSF only. RESULTS: Patients responding to IFN-alpha (group C) showed comparable numbers of bone marrow Ph- long-term culture initiating cells to those of newly diagnosed individuals (group A): 8.5 (<1-65)/10(6) MNC vs 10.5 (<1-30), while non-responders had markedly lower numbers: <1 (<1-5). The amount of Ph- LTC-IC collected was significantly lower in patients of group B 1.8 (0-325)x10(2)/kg than in patients of either group A 31.3 (0-952)x10(2)/kg (P<0.002) or group C 109 (8-259)x10(2)/kg (P<0.01). Interestingly, five patients of group B who had 100% Ph+ metaphases, but Ph- progenitors in their bone marrow, mobilized normal amounts of Ph(-) progenitors. CONCLUSION: These findings suggest that the decline of normal hematopoietic progenitors, currently observed in the majority of CML patients, is not induced by IFN-alpha treatment, but it is likely due to the expanding leukemic clone. They also indicate that normal hematopoietic reservoir is consistently preserved in patients given IFN-alpha early after diagnosis and achieving a stable cytogenetic response.
Assuntos
Interferon-alfa/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Cromossomo Filadélfia , Células-Tronco/efeitos dos fármacos , Adulto , Células da Medula Óssea/efeitos dos fármacos , Estudos de Casos e Controles , Contagem de Células , Análise Citogenética , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Interferon-alfa/administração & dosagem , Leucaférese , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologiaRESUMO
Twenty-three patients with chronic myelogenous leukemia in early chronic phase (ECP) and not previously treated with alpha-interferon (IFN-alpha) (10 patients), in ECP but pretreated with IFN-alpha (<12 months) (seven patients) and in late chronic phase (LCP) pretreated with IFN-alpha (>12 months) (six patients) underwent autografting with Philadelphia (Ph) chromosome-negative blood progenitor cells (BPCs) (20 patients), or partially/totally Ph-positive BPCs (three patients), previously mobilized during the early phase of recovery after aplasia induced by intensive chemotherapy. The conditioning regimen consisted of high-dose chemotherapy alone or followed by total body irradiation (TBI). Recombinant G-CSF was given after BPCs infusion on day +8. All patients in ECP not pretreated with IFN-alpha are alive and five of them are Ph-negative in the marrow after autografting. Six of seven patients autografted with Ph-negative BPCs in the group of ECP pretreated with IFN-alpha (<12 months) are alive and two of them are still Ph-negative in the marrow. In the same group, the only patient transplanted with partially Ph-positive BPCs, died of blastic transformation 2 months after reinfusion. Three patients (two patients autografted with Ph-negative BPCs and one patient with Ph-positive BPC) in the group of LCP pretreated with IFN-alpha >12 months are alive but Ph-positive after autografting. The other three patients of the same group died of procedure-related toxicity (two patients) and blastic transformation (one patient). Seventeen patients (10/10 ECP not pretreated with IFN-alpha; 5/7 ECP pretreated with IFN-alpha and 2/6 LCP pretreated with IFN-alpha) of 23 autografted patients were treated with IFN-alpha +/- IL-2. Toxicities after autografting were mostly related to myelosuppression, particularly thrombocytopenia. All patients of the two groups pretreated with IFN-alpha developed febrile episodes during the aplastic phase following BPCs reinfusion. No patient autografted in ECP and those not pretreated with IFN-alpha developed febrile episodes. This is also probably due to the use of i.v. antibiotic and antimicotic prophylaxis when neutrophils were < or = 1 x 10(9)/l after autografting. Greater toxicity was observed in patients pretreated with IFN-alpha, being lethal in two cases in LCF. In conclusion, the "in vivo' manipulation approach employed in our institution is a safe procedure and it results in a high collection of Ph-negative cells in the blood if the cells are harvested: (1) in early chronic phase; (2) in early phase of recovery after chemotherapy-inducing aplasia; (3) in patients not extensively pretreated with IFN-alpha. The data presented here have shown encouraging trends in chronic phase of CML and offer new perspective for patients without an HLA-identical donor or for patients who do not respond to IFN-alpha.
Assuntos
Anemia Aplástica/terapia , Transplante de Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide de Fase Crônica/terapia , Adulto , Anemia Aplástica/etiologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Crise Blástica/mortalidade , Medula Óssea/patologia , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Terapia Combinada , Etoposídeo/administração & dosagem , Etoposídeo/efeitos adversos , Feminino , Humanos , Ifosfamida/administração & dosagem , Ifosfamida/efeitos adversos , Fatores Imunológicos/uso terapêutico , Interferon-alfa/uso terapêutico , Leucaférese , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/radioterapia , Leucemia Mieloide de Fase Crônica/sangue , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/patologia , Leucemia Mieloide de Fase Crônica/radioterapia , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Células Neoplásicas Circulantes , Cromossomo Filadélfia , Análise de Sobrevida , Trombocitopenia/etiologia , Trombocitopenia/terapia , Transplante Autólogo , Resultado do Tratamento , Irradiação Corporal Total/efeitos adversosRESUMO
In vitro priming of T cell with horse antilymphocyte globulin (HALG) results in cytokine release, and this has been associated with its clinical efficacy in patients with severe aplastic anaemia (SAA). Rabbit antithymocyte globulin (RATG) has been studied less extensively. In this study we compare the in vitro priming effect of HALG and RATG on purified normal marrow T cells: end-points of the study were 1) levels of TNF-alpha (TNF-alpha), IFN-gamma (IFN-gamma) GM-CSF in T cell supernatants, and 2) effect of T cell supernatants on colony formation with or without exogenous GM-CSF TNF-alpha, IFN-gamma and GM-CSF levels were comparable for HALG, RATG and phytohaemagglutinin (PHA). T cell supernatants showed comparable enhancement of colony formation in the presence of recombinant human GM-CSF (rhGM-CSF) and supported colony forming unit granulomacrophage (CFU-GM) growth in the absence of growth factor. This study shows that horse and rabbit derived ALG/ATG and PHA have a comparable in vitro priming effect on T cells: both agents should probably be tested for their clinical efficacy in SAA patients.
Assuntos
Anemia Aplástica/terapia , Células da Medula Óssea/imunologia , Complexo CD3/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Interferon gama/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Soro Antilinfocitário/uso terapêutico , Separação Celular , Hematopoese/imunologia , Cavalos , Humanos , Fito-Hemaglutininas/farmacologia , Coelhos , Valores de Referência , Linfócitos T/imunologiaRESUMO
p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French-American-British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in approximately 70% inhibition of cell proliferation as assessed by [(3)H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Apoptose/imunologia , Leucemia Mieloide/imunologia , Receptores Imunológicos/imunologia , Doença Aguda , Animais , Anticorpos Monoclonais/imunologia , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais CultivadasRESUMO
Elevated white blood cell counts are frequently found in patients with chronic myeloid leukaemia (CML). Although some studies have disclosed that bone marrow of CML patients may contain some normal Philadelphia-negative early progenitor cells, it has been assumed that the dramatic increase of white blood cells was entirely related to the leukaemic cell expansion. In this study we attempted to quantify the number of normal and leukaemic progenitor cells in the bone marrow and peripheral blood of newly diagnosed CML patients. Bone marrow and peripheral blood cells of eight newly diagnosed CML patients were analysed for clonogenic colony-forming cells (CFC) and very early progenitor cells, i.e. long-term culture initiating cells (LTC-IC). The leukaemic (Ph-positive) or normal (Ph-negative) origin of progenitor cells was revealed by cytogenetic analysis performed on single colonies arising from in-vitro assays. In 6/8 patients the marrow CFC frequency ranged from 400 to 9300/10(6) mononuclear cells (MNC), 0-50% being Philadelphia chromosome negative; the LTC-IC frequency ranged from 0 to 11/10(6) MNC, and were 80-100% Ph-negative. The corresponding absolute values into peripheral blood were: CFC = 1-35.5 x 10(3)/ml, 0-50% Ph-negative, and LTC-IC = 0-2.5 x 10(3)/ml, 0-100% Ph-negative. In one patient, no LTC-IC were detected in either the marrow or the peripheral blood. In conclusion, in the peripheral blood of some CML patients, the number of normal LTC-IC is more than 3 times the number of leukaemic progenitor cells, and is much higher (50 times) than the corresponding value found in normal subjects in steady state (2.5/ml v 124/ml). These data support the concept that leukaemic 'stem cells', with respect to normal ones, may be considerably fewer than previously thought. In addition it is shown that at the beginning of CML high numbers of normal LTC-IC are spontaneously mobilized into the blood. Finally, the presence of Ph-negative early progenitors into the blood may represent a potential source of normal stem cells available for autografting providing they can be separated from leukaemic cells.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Células-Tronco/patologia , Adulto , Medula Óssea/patologia , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-IdadeRESUMO
We investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P < .00001) both for IS and BMT patients. Their number did not improve with time and was not affected by transplant or treatment-related variables. When IS patients were compared with BMT we found comparable numbers of CFU-GEMM (P = .8) and LTC-IC (P = .9), but lower numbers of BFU-E and CFU-GM (P = .05 and P = .004, respectively), suggestive of a persistent suppressive mechanism. These data indicate that LTC-IC numbers are severely reduced in BMT and IS patients, contradicting the common belief that the former are fully reconstituted as compared with the latter. In addition, the number of mature cells and committed progenitors does not seem to reflect the real size of the hematopoietic reservoir and few stem cells may be sufficient to guarantee normal hematopoiesis long term.
Assuntos
Anemia Aplástica/patologia , Transplante de Medula Óssea/patologia , Medula Óssea/patologia , Hematopoese , Células-Tronco Hematopoéticas/patologia , Terapia de Imunossupressão , Adolescente , Adulto , Anemia Aplástica/terapia , Contagem de Células , Células Cultivadas , Criança , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Transplante HomólogoRESUMO
UNLABELLED: Transplant-related mortality (TRM) remains a major problem in older patients undergoing allogeneic haemopoietic stem cell transplants (HSCTs). We have therefore explored a less intensive conditioning in 33 patients with a median age of 52 years (range 43-60) transplanted from human leucocyte antigen (HLA)-identical siblings. The underlying disease was chronic myeloid leukaemia (n = 15), acute myeloid leukaemia (n = 6), myelodysplasia (n = 7) or a chronic lymphoproliferative disorder (n = 5); 15 patients (45%) had advanced disease. The regimen consisted of thiotepa (THIO; 10 mg/kg) on day -5 and cyclophosphamide (CY; 50 mg/kg) on days -3 and -2 (total dose 100 mg/kg). The source was bone marrow (BM) (n = 17) or granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) (n = 16), which were infused without manipulation. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and a short course of methotrexate. Mean time to achieve a neutrophil count of 0.5 x 109/l was 17 d (range 11-23) and full donor chimaerism was detected in 79% of patients by day 100. Acute GVHD grade III or IV occurred in 3% of patients. Chronic GVHD was seen in 45% of patients, with a significant difference for PB (69%) compared with BM transplants (23%) (P = 0.009). For BM grafts, the actuarial 2-year TRM was 6%, the relapse 56% and survival 87%; for PB grafts, these figures were, respectively, 27%, 33% and 68%. Twenty-five patients are alive at a median follow-up of 762 d (range 216-1615) and 20 patients (60%) remain free of disease. Thirteen patients (39%) received donor lymphocyte infusion (DLI) either for persisting or relapsing disease and six patients had complete remission. IN CONCLUSION: (i) patients up to the age of 60 years can be allografted with reduced intensity conditioning; (ii) the procedure was associated with a low transplant-related mortality, particularly for bone marrow grafts, because of a lower risk of chronic GVHD; and (iii) DLI were required after transplant in half the patients for persisting disease or relapse.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Síndromes Mielodisplásicas/cirurgia , Tiotepa/administração & dosagem , Condicionamento Pré-Transplante/métodos , Adulto , Ciclofosfamida/administração & dosagem , Esquema de Medicação , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/mortalidade , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Imunossupressores/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/cirurgia , Leucemia Mieloide/mortalidade , Leucemia Mieloide/cirurgia , Transtornos Linfoproliferativos/mortalidade , Transtornos Linfoproliferativos/cirurgia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/mortalidade , Taxa de Sobrevida , Transplante HomólogoRESUMO
BACKGROUND AND OBJECTIVE: We previously reported that patients with acquired severe aplastic anemia (SAA) treated with antilymphocyte globulin (ALG), 6-methylprednisolone, cyclosporin A (CyA) and granulocyte colony-stimulating factor (G-CSF) can mobilize peripheral blood hemopoietic progenitors (PBHP). The aim of the present study was to assess phenotypic and functional properties of these PBHP. METHODS: We studied seven patients who underwent 43 leukophereses (median 5) between day +30 and +80 following ALG, while in treatment with CyA and G-CSF. Mobilized peripheral blood hemopoietic progenitors were analyzed using surface markers, conventional assays for clonogenic cells (CFU-GM, BFU-E, CFU-GEMM) as well as the recently developed assay for long-term culture initiating cells (LTC-ICs). RESULTS: The proportion of CD34+ cells ranged between 0% and 5.4% (median 0.3%), CD34+DR between 0% and 3.5% (median 0.1%) and CD8+ cells between 3.3% and 56% (median 31%). When light density mononuclear cells (MNC) were plated in vitro, we could grow colony-forming units-granulo-macrophage (CFU-GM) (range 0-45/10(5) MNC; normal controls 21-200/10(5) MNC), burst-forming units-erythroid (BFU-E) (range 0-5/10(5) MNC; normal controls 0-6/10(5) MNC), multipotent colonies (CFU-GEMM) (range 0-3/10(5) MNC; normal controls 0-6/10(5) MNC) and high proliferative potential colony-forming cells (HPP-CFC) (range 0-3.4/10(5) MNC). We studied long-term culture-initiating cells (LTC-ICs) in 18 leukophereses from 4 patients; in 7/18 samples LTC-ICs were grown at low frequency (range 0.4-2/10(6) MNC) (normal controls 5-130/10(6) MNC), and in one patient in the absence of CFU-GM growth. The total yield of LTC-ICs in two patients was 7.64 and 10.5 x 10(2)/kg of body weight. INTERPRETATION AND CONCLUSIONS: This study suggests that cells with the phenotype and in vitro function of early hemopoietic progenitors are found, though in small numbers, in the peripheral blood of patients with SAA after treatment with immunosuppressants and prolonged G-CSF administration. Whether G-CSF-mobilized progenitors contribute to hemopoietic recovery in these patients remains to be determined.
Assuntos
Anemia Aplástica/sangue , Soro Antilinfocitário/administração & dosagem , Ciclosporina/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Células-Tronco Hematopoéticas/patologia , Imunossupressores/administração & dosagem , Adolescente , Adulto , Anemia Aplástica/tratamento farmacológico , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucaférese , Masculino , Pessoa de Meia-IdadeRESUMO
Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Cálcio/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Adenosina Difosfato Ribose/antagonistas & inibidores , Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/farmacologia , Antígenos CD34/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , ADP-Ribose Cíclica , Citarabina/farmacologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Líquido Intracelular/metabolismo , Sistemas do Segundo MensageiroRESUMO
Cytogenetic abnormalities and paroxysmal nocturnal haemoglobinuria (PNH) phenotype are frequent findings in aplastic anaemia patients treated with immunosuppressive therapy (IST). In this study we investigated whether the appearance of clonal haemopoiesis influences patient outcome and survival. 97 patients entered this study and were followed from the onset of the disease for a median follow-up (FU) of 53 months. 93% are alive, 56% achieved complete remission, 30% partial remission, both transfusion independent, and 14% did not respond. Three groups were identified: (A) patients without evidence of emerging clones (71/97); (B) patients who acquired chromosomal abnormalities (13/97); (C) patients who showed low expression of glycosyl phosphatidylinositol anchored proteins (GPI-AP) (PNH phenotype) at presentation or later (16/97). Three patients showed both PIG-AP deficiency and chromosomal abnormalities. The actuarial survival of patients without clonal haemopoiesis (n = 71) at 6 years was 95%, for patients with chromosomal abnormalities (n = 13), 88%, and for patients with PIG-AP deficiency (n = 16), 89%. There was no difference in the probability of becoming transfusion independent in the three groups (93%, 92% and 88% respectively). This study confirmed that a proportion of severe aplastic anaemia (SAA) patients exhibit clonal markers during the time after IST, often coexisting with cytogenetically or phenotypically normal haemopoiesis. There was no significant clinical impact of these abnormalities on transfusion independence and survival at the median follow-up of 4 years.
Assuntos
Anemia Aplástica/patologia , Hematopoese/fisiologia , Terapia de Imunossupressão/métodos , Adolescente , Adulto , Idoso , Anemia Aplástica/terapia , Criança , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Resultado do TratamentoRESUMO
We carried out studies to quantify Ph-negative progenitors both in steady state and during regeneration after chemotherapy and G-CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady-state bone marrow, Ph-negative long-term culture initiating cells (LTC-IC) and Ph-negative colony-forming-cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P<0.001 and P<0.02, respectively). The absolute number of mobilized Ph-negative progenitors was markedly higher in group A versus group B (P<0.02 for LTC-IC, P<0.003 for CFC). 12/16 newly diagnosed patients mobilized Ph-negative LTC-IC only and the yield was in the range of normal allogeneic donors. Overall the frequency of Ph-negative LTC-IC in the bone marrow predicted the yield of Ph-negative LTC-IC mobilized into peripheral blood (P<0.001). The bone marrow frequency of Ph-positive LTC-IC was considerably lower than the normal counterpart. Taken together, these findings suggest that normal progenitors are relatively well preserved in newly diagnosed CML patients, but tend to rapidly decline with time. This observation helps in the understanding of the pathogenesis of CML and has potential implications for autografting. The optimal time for a successful collection of Ph-negative circulating progenitors would appear to be soon after diagnosis.