RESUMO
In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced.
Assuntos
Anemia Aplástica/metabolismo , Anemia Aplástica/patologia , Células-Tronco Mesenquimais/metabolismo , Adolescente , Adulto , Idoso , Anemia Aplástica/diagnóstico , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Casos e Controles , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Humanos , Imunofenotipagem , Leucócitos Mononucleares , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
OBJECTIVE: Bone marrow from aplastic anemia (AA) patients shows reduced numbers in long-term culture (LTC)-initiating cell (LTC-IC) assays. The LTC-IC assay is based on assumptions of the culture kinetics of normal hematopoietic stem cells (HSC), which are not necessarily justified in a disease state. We therefore undertook a detailed examination of the kinetics of quiescent HSC from AA patients in LTC. METHODS: Colony formation by quiescent HSC in LTC was tested by pretreating control (n=6) and AA bone marrow (n=7) with 5-fluorouracil. Secondly, we manipulated normal samples to inoculate cultures with proportions of CD34+ cells similar to those from AA samples. We obtained enough CD34+ cells to reconstitute one AA sample to "normal" levels. RESULTS: Patient cells showed altered kinetics with rapid proliferation and premature termination of LTC. In vivo, decreased numbers of HSC may induce rapid proliferation and differentiation; a similar phenomenon could explain the observations in culture. We therefore manipulated normal samples to contain a proportion of CD34+ HSC similar to that in AA samples. Although absolute numbers of secondary colonies in LTC were reduced, the kinetics of culture were not altered. However, when AA CD34+ HSC were reconstituted to "normal" levels, the cultures still demonstrated early termination. CONCLUSIONS: The kinetics of LTC are not affected by CD34+ HSC number. However, quiescent HSC derived from patients with AA have qualitative differences from normal cells, as reflected by distinct kinetics in long-term culture. This has implications for the interpretation of the LTC-IC assay with AA samples.
Assuntos
Anemia Aplástica/patologia , Células da Medula Óssea/patologia , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/patologia , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Divisão Celular , Resistência a Medicamentos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Cinética , Valores de ReferênciaAssuntos
Anemia Aplástica/diagnóstico , Adulto , Anemia Aplástica/complicações , Anemia Aplástica/terapia , Transfusão de Componentes Sanguíneos/métodos , Exame de Medula Óssea/métodos , Transplante de Medula Óssea/métodos , Diagnóstico Diferencial , Medicina Baseada em Evidências/métodos , Feminino , Teste de Histocompatibilidade , Humanos , Imunossupressores/uso terapêutico , Pessoa de Meia-Idade , Infecções Oportunistas/complicações , Infecções Oportunistas/prevenção & controle , Gravidez , Complicações Hematológicas na Gravidez/terapiaRESUMO
Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.
Assuntos
Anemia Aplástica/patologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos CD34 , Apoptose , Células da Medula Óssea/patologia , Estudos de Casos e Controles , Células Cultivadas , Humanos , Regulação para Cima/efeitos dos fármacos , Receptor fas/biossíntese , Receptor fas/imunologiaRESUMO
Aplastic anemia is associated with quantitative and functional abnormalities in the hematopoietic stem cell compartment. Currently, one of the most primitive human hematopoietic progenitor cells that can be functionally assayed in vitro is the long-term culture-initiating cell (LTC-IC). This assay identifies primitive cells that are capable of producing colonies after five weeks of long-term culture using a limiting dilution method. Previous investigators have demonstrated a significant reduction in the frequency of LTC-IC in bone marrow mononuclear cells (BMMC) isolated from aplastic anemia patients when compared to normal donors. However, immunosuppression of hematopoiesis is a prevalent feature of aplastic anemia. Therefore, we assayed the frequency of LTC-IC in cells expressing the CD34 antigen isolated from bone marrow, a population highly enriched for LTC-IC, of both aplastic anemia patients that had received immunosuppressive therapies (IST, n=13) and normal donors (n=28), thereby minimizing continued immunosuppression by T-cells and regulation by other autologous CD34- cells. We describe a significant reduction of LTC-IC frequency in purified CD34+ populations from aplastic patients (P>0.0001), thus demonstrating a functional difference between this phenotypically defined cell population from patients and normal donors.
Assuntos
Anemia Aplástica/patologia , Adolescente , Adulto , Antígenos CD34 , Medula Óssea/patologia , Estudos de Casos e Controles , Contagem de Células , Técnicas de Cultura de Células , Divisão Celular , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/patologia , Humanos , Pessoa de Meia-IdadeRESUMO
Aplastic anaemia seems to be predominantly a defect of the stem cell rather than the stroma, though abnormalities of the microenvironment may co-exist. There is highly suggestive evidence that the stem cell is the target of an immune attack, though the main evidence remains the response to immunosuppression with antilymphocyte globulin and cyclosporin. The stem cell defect remains even after recovery of the peripheral blood counts and the AA marrow is a fertile environment for the emergence of abnormal clones, particularly PNH. However, it has recently become apparent that there is an overlap with the myelodysplastic syndromes and clones of monosomy 7 and trisomy 8 amongst others are not uncommon in aplastic anaemia. Recent work has suggested that the emergence of a clone of monosomy 7 cells carries a poor prognosis, whereas trisomy 8 has a good prognosis particularly in response to cyclosporin. However, the setting in which monosomy 7 arises may affect the phenotypic expression. The immune targeting of stem cells may be associated with increased apoptosis in aplastic anaemia, in part mediated by fas expression, but not exclusively. Understanding the pathophysiology of AA should help to improve and perhaps target therapy.
Assuntos
Anemia Aplástica/patologia , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/etiologia , Células Clonais/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Imunossupressores/uso terapêuticoAssuntos
Anemia Aplástica/imunologia , Doenças Autoimunes/imunologia , Anemia Aplástica/terapia , Doenças Autoimunes/terapia , Células Clonais , Regiões Determinantes de Complementaridade/genética , Antígenos HLA-DR/análise , Subtipos Sorológicos de HLA-DR , Humanos , Imunossupressores/uso terapêutico , Polimorfismo Genético , Linfócitos T/imunologiaRESUMO
Hematological cancers and non-malignant hematological disorders are biologically diverse conditions and are treated differently. We compared the pattern of EBV infections following allogeneic stem cell transplantation between the above two groups of hematological disorders. Eighty-three transplants were evaluated over a consecutive 7-year period at a single center. No difference was found in the incidence of post-transplant EBV infections between the two groups, though a higher median peak viral load was noted in the non-malignant group (P=0·04). Pre-transplant immunosuppressive therapy with antithymocyte globulin (ATG) significantly increased the risk of post-transplant EBV infections (P=0·04) in the non-malignant group patients. No significance was found for prior cytotoxic chemotherapy among the malignant group of patients. Alemtuzumab based conditioning was not associated with an increased risk for EBV infections in either of the groups. Treatment with two or more courses of ATG was found to be significantly associated with post-transplant EBV-related PTLD (P=0·01). Post-transplant EBV infections did not influence overall survival (non-malignant, P=0·66; malignant, P=0·41) in either of the subgroups. There were no deaths directly attributable to EBV infections.
Assuntos
Infecções por Vírus Epstein-Barr/etiologia , Doenças Hematológicas/cirurgia , Neoplasias Hematológicas/cirurgia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Adolescente , Adulto , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Soro Antilinfocitário/efeitos adversos , Soro Antilinfocitário/uso terapêutico , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Análise de Sobrevida , Transplante Homólogo , Adulto JovemRESUMO
The haematological indications for allogeneic stem cell transplantation can be broadly divided into non-malignant and malignant disorders. We compared the incidence and risk factors for post-transplant cytomegalovirus (CMV) infections between these two biologically diverse subgroups of haematological conditions. Out of 105 allogeneic transplants, 64 and 41 were for underlying non-malignant and malignant indications respectively. CMV infections were significantly more frequent (P=0.016) in the malignant subgroup. Pre-transplant recipient CMV seropositivity in both subgroups (negative versus positive; non-malignant,P=0.023; malignant, p<0.001), donor seropositivity (P=0.002) and acute graft-versus-host disease (GVHD) (P=0.02) in the non-malignant subgroup and > or =3 courses of previous cytotoxic therapy (P=0.023) in the malignant subgroup were found to be associated with an increased risk of CMV infections. On multivariate analysis, donor seropositivity in the non-malignant patients (negative versus positive,P=0.022; odds ratio: 0.18) and recipient seropositivity in patients with malignancies (negative versus positive; P=0.001, odds ratio: 0.01) were identified to be significant factors for risk of CMV infection.
Assuntos
Infecções por Citomegalovirus/epidemiologia , Doenças Hematológicas/cirurgia , Neoplasias Hematológicas/cirurgia , Transplante de Células-Tronco Hematopoéticas , Infecções Oportunistas/epidemiologia , Complicações Pós-Operatórias/epidemiologia , Adolescente , Adulto , Transplante de Medula Óssea/efeitos adversos , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Infecções por Citomegalovirus/transmissão , Suscetibilidade a Doenças , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/epidemiologia , Doenças Hematológicas/complicações , Neoplasias Hematológicas/complicações , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Hospedeiro Imunocomprometido , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Incidência , Doadores Vivos , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Estudos Prospectivos , Reoperação , Fatores de Risco , Transplante Homólogo/efeitos adversos , Ativação Viral , Adulto JovemRESUMO
Thrombopoietin (TPO) is the key cytokine involved in thrombopoiesis, and is the endogenous ligand for the thrombopoietin receptor that is expressed on the surface of platelets, megakaryocytes, and megakaryocytic precursors. First-generation thrombopoietic agents were recombinant forms of human TPO, and their development was discontinued after prolonged thrombocytopenia due to neutralizing auto-antibodies cross-reacting with endogenous TPO was observed. Second-generation thrombopoiesis-stimulating molecules are now available, which have unique pharmacological properties and no sequence homology to endogenous TPO. Two of these new agents, romiplostim and eltrombopag, have already completed phase III trials in primary immune thrombocytopenia and have been granted marketing authorization for use in this disease. Phase II and III trials with these novel drugs are ongoing in other conditions characterized by thrombocytopenia, such as chemotherapy, chronic liver disease, and the myelodysplastic syndromes. Most of the other second-generation thrombopoietic growth factors are in early phase clinical development.
Assuntos
Trombopoetina/farmacologia , Animais , Benzoatos/uso terapêutico , Humanos , Hidrazinas/uso terapêutico , Pirazóis/uso terapêutico , Receptores Fc/uso terapêutico , Receptores de Trombopoetina/agonistas , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/tratamento farmacológico , Trombopoese/efeitos dos fármacos , Trombopoetina/uso terapêuticoRESUMO
Persistent thrombocytopenia may be the consequence of chronic infections with hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Helicobacter pylori, and should be considered in the differential diagnosis of primary immune thrombocytopenia (ITP). Studies have shown that on diagnosis of infections, treatment of the primary disease often results in substantial improvement or complete recovery of the thrombocytopenia. In patients with thrombocytopenia due to HCV-related chronic liver disease, the use of eltrombopag, a thrombopoietin receptor agonist, normalizes platelet levels, thereby permitting the initiation of antiviral therapy. Antiviral therapy with highly active antiretroviral therapy for HIV has aided in platelet recovery, with a corresponding decrease in circulating viral load. Thrombocytopenia in the absence of other disease symptoms requires screening for H. pylori, especially in countries such as Japan, where there is a high prevalence of the disease and the chances of a platelet response to eradication therapy are high.
Assuntos
Infecções por HIV/complicações , Infecções por Helicobacter/complicações , Hepatite C/complicações , Púrpura Trombocitopênica Idiopática/etiologia , Antibacterianos/uso terapêutico , Antivirais/uso terapêutico , Doença Crônica , Infecções por HIV/tratamento farmacológico , Infecções por Helicobacter/tratamento farmacológico , Hepatite C/tratamento farmacológico , Humanos , Púrpura Trombocitopênica Idiopática/terapiaRESUMO
Aplastic anaemia (AA) in man is an often fatal disease characterized by pancytopenia of the peripheral blood and aplasia of the bone marrow. AA is a toxic effect of many drugs and chemicals (e.g. chloramphenicol, azathioprine, phenylbutazone, gold salts, penicillamine and benzene). However, there are no widely used or convenient animal models of drug-induced AA. Recently, we reported a new model of chronic bone marrow aplasia (CBMA = AA) in the busulphan (BU)-treated mouse: eight doses of BU (10.50 mg/kg) were administered to female BALB/c mice over a period of 23 days; CBMA was evident at day 91/112 post-dosing with significantly reduced erythrocytes, platelets, leucocytes and nucleated bone marrow cell counts. However, mortality was high (49.3%). We have now carried out a study to modify the BU-dosing regime to induce CBMA without high mortality, and investigated the patterns of cellular responses in the blood and marrow in the post-dosing period. Mice (n = 64/65) were dosed 10 times with BU at 0 (vehicle control), 8.25, 9.0 and 9.75 mg/kg over 21 days and autopsied at day 1, 23, 42, 71, 84, 106 and 127 post-dosing (n = 7-15); blood and marrow samples were examined. BU induced a predictable bone marrow depression at day 1 post-dosing; at day 23/42 post-dosing, parameters were returning towards normal during a period of recovery. At day 71, 84, 106 and 127 post-dosing, a stabilized, late-stage, nondose-related CBMA was evident in BU-treated mice, with decreased erythrocytes, platelets and marrow cell counts, and increased MCV. At day 127 post-dosing, five BU-treated mice showed evidence of lymphoma. In this study, mortality was low, ranging from 3.1% (8.25 mg/kg BU) to 12.3% (9.75 mg/kg BU). It is concluded that BU at 9.0 mg/kg (or 9.25 mg/kg) is an appropriate dose level to administer (10 times over 21 days) to induce CBMA at approximately day 50-120 post-dosing.
Assuntos
Anemia Aplástica/patologia , Células da Medula Óssea/patologia , Bussulfano , Modelos Animais , Agonistas Mieloablativos , Anemia Aplástica/mortalidade , Animais , Apoptose , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Linfoma/induzido quimicamente , Linfoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/patologia , Neoplasias Esplênicas/induzido quimicamente , Neoplasias Esplênicas/patologia , Coloração e Rotulagem , Timo/efeitos dos fármacos , Timo/patologia , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/patologiaRESUMO
Mitomycin (MMC), like many antineoplastic drugs, induces a predictable, dose-related, bone marrow depression in man and laboratory animals; this change is generally reversible. However, there is evidence that MMC may also cause a late-stage or residual bone marrow injury. The present study in female CD-1 mice investigated the haematological and bone marrow changes induced by MMC in a repeat dose study lasting 50 days. Control and MMC-treated mice were dosed intraperitoneally on eight occasions over 18 days with vehicle, or MMC at 2.5 mg/kg, autopsied (n = 6-12) at 1, 7, 14, 28, 42 and 50 days after the final dose and haematological changes investigated. Femoral nucleated bone marrow cell counts and levels of apoptosis were also evaluated and clonogenic assays carried out; serum levels of FLT3 ligand (FL) were assessed. At day 1 post-dosing, MMC induced significant reductions in RBC, Hb and haematocrit (HCT) values, and there were decreases in reticulocyte, platelet, and femoral nucleated cell counts (FNCC); neutrophil, lymphocyte and monocyte values were also significantly reduced. On days 7 and 14 post-dosing, all haematological parameters showed evidence of a return towards normal values, but at these times, and at day 28, values for RBC and FNCC remained significantly reduced in comparison with controls. At days 42 and 50 post-dosing, many haematological parameters in MMC-treated mice had returned to control levels; however, there remained evidence of late-stage effects on RBC, Hb and HCT values, and FNCC also continued to be significantly decreased. Results for granulocyte-macrophage colony-forming units and erythroid colonies showed a profound decrease immediately post-dosing, but a return to normal values was evident at day 50. Serum FL concentrations demonstrated very significant increases in the immediate post-dosing period, but a return to normal was seen at day 50 post-dosing; a relatively similar pattern was seen in the number of apoptotic femoral marrow nucleated cells. The histopathological examination of kidney tissues from MMC animals at day 42 and 50 post-dosing showed evidence of hydronephrosis with cortical glomerular/tubular atrophy and degeneration. It is therefore concluded that MMC administered on eight occasions over 18 days to female CD-1 mice at 2.5 mg/kg induced profound changes in haematological and bone marrow parameters in the immediate post-dosing period with a return to normal levels at day 50 post-dosing; however, there was evidence of mild but significant late-stage/residual effects on RBC and FNCC, and on cells of the erythroid lineage in the bone marrow.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Células da Medula Óssea/patologia , Células-Tronco Hematopoéticas/patologia , Mitomicina/administração & dosagem , Animais , Antibióticos Antineoplásicos/efeitos adversos , Apoptose , Células da Medula Óssea/efeitos dos fármacos , Contagem de Células , Esquema de Medicação , Contagem de Eritrócitos , Feminino , Fêmur , Hematócrito , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hemoglobinas/análise , Rim/efeitos dos fármacos , Rim/patologia , Proteínas de Membrana/sangue , Camundongos , Camundongos Endogâmicos , Mitomicina/efeitos adversosRESUMO
This retrospective study evaluated the outcome of 18 patients with aplastic anaemia treated with a third course of anti-thymocyte globulin (ATG)-containing immunosuppressive therapy (IST). Of the 18 patients, seven had responded to one of the previous courses of ATG and 11 were refractory to both the previous courses. Self-limiting grade >/=3 liver toxicity was observed in three patients. Two patients had to discontinue ATG because of severe systemic side effects. The incidence and manifestations of serum sickness did not appear to be different during the three courses. All of the seven patients that previously responded to one of the courses responded to a third course. In contrast, of 11 patients refractory to the previous courses, only two had a transient partial response. The 3-yr event-free survival for the patients who had responded to one of the previous courses of ATG was significantly superior to that of patients refractory to both the previous courses of ATG (83% vs. 0%, P = 0.0001). For aplastic anaemia patients, a third course of ATG-containing IST is a reasonable option in previous responders. Patients refractory to previous two courses of ATG have a much lower response rate and may be suitable candidates for novel therapeutic options.
Assuntos
Anemia Aplástica/terapia , Soro Antilinfocitário/uso terapêutico , Imunossupressores/uso terapêutico , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Soro Antilinfocitário/administração & dosagem , Soro Antilinfocitário/efeitos adversos , Esquema de Medicação , Feminino , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Doença do Soro/etiologia , Análise de Sobrevida , Falha de Tratamento , Resultado do TratamentoRESUMO
Paroxysmal nocturnal haemoglobinuria (PNH) has a dual pathogenesis. PIG-A mutations generate clones of haemopoietic stem cells (HSC) lacking glycosylphosphatidylinositol (GPI)-anchored proteins and, secondly, these clones expand because of a selective advantage related to bone marrow failure. The first aspect has been elucidated in detail, but the mechanisms leading to clonal expansion are not well understood. We have previously shown that apoptosis and Fas expression in HSC play a role in bone marrow failure during aplastic anaemia. We have now investigated apoptosis in PNH. Ten patients were studied. Apoptosis, measured by flow cytometry, was significantly higher among CD34+ cells from patients compared with healthy controls. Fas expression was also increased. Cells that were stained for CD34, CD59 and apoptosis showed a significantly lower apoptosis in CD34+/CD59- compared with CD34+/CD59+ cells from the same patient. In three patients, staining for CD34, CD59 and Fas revealed lower Fas expression on CD34+/CD59- cells. Differential apoptosis of CD34+/CD59- HSC may be sufficient in itself to explain the expansion of PNH clones in the context of aplastic anaemia. In addition to demonstrating a basic mechanism underlying PNH clonal expansion, these results suggest further hypotheses for the evolution of PNH, based on the direct or indirect resistance of GPI-negative HSC to pro-inflammatory cytokines.
Assuntos
Apoptose , Glicosilfosfatidilinositóis/metabolismo , Hemoglobinúria Paroxística/patologia , Células-Tronco/patologia , Receptor fas/metabolismo , Adolescente , Adulto , Antígenos CD34/metabolismo , Antígenos CD59/metabolismo , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Hemoglobinúria Paroxística/imunologia , Humanos , Masculino , Células-Tronco/imunologiaRESUMO
Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haemopoietic stem cell (HSC). The pathogenetic link with bone marrow failure is well recognized; however, the process of clonal expansion of the glycosylphosphatidylinositol (GPI)-deficient cells over normal haemopoiesis remains unclear. We have carried out detailed analysis of the stem cell population in 10 patients with de novo haemolytic PNH using the long-term culture-initiating cells (LTC-IC) assay in parallel with measurements of CD34+ cells and mature haemopoietic progenitors, granulocyte-macrophage colony-forming unit (CFU-GM) and CFU-erythroid [burst-forming units erythroid (BFU-E) + CFU granulocyte/erythroid/macrophage/megakaryocyte (GEMM)]. All patients had hypercellular bone marrows with erythroid hyperplasia, normal blood counts or mild peripheral blood cytopenias, increased reticulocyte counts and evidence of deficient GPI-anchored proteins. We found a significant reduction in the LTC-IC frequency in the CD34+ compartment of PNH patients (mean 2, range 1.3-3.0; n=6) compared with normal donors (mean 13, range 5.2-45.5; n=21) (P<0.0001). Furthermore, there was a significant reduction in the erythroid compartment [CFU-E/105 bone marrow mononuclear cells (BMMC) and CFU-E/105 CD34+ cells] of PNH patients, but no significant difference in the granulocyte-monocyte precursors (CFU-GM/105 BMMC or CFU-GM/105 CD34+ cells) compared with normal donors, suggesting that there is a defect in the early stem cell pool in PNH patients without clinical or haematological evidence of bone marrow failure.
Assuntos
Células-Tronco Hematopoéticas/patologia , Hemoglobinúria Paroxística/patologia , Adulto , Antígenos CD34/análise , Estudos de Casos e Controles , Contagem de Células , Células Cultivadas , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Feminino , Glicosilfosfatidilinositóis/deficiência , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinúria Paroxística/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estatísticas não ParamétricasRESUMO
Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.
Assuntos
Anemia Aplástica/terapia , Antígenos CD34/imunologia , Soro Antilinfocitário/uso terapêutico , Células-Tronco Hematopoéticas/metabolismo , Adolescente , Adulto , Idoso , Anemia Aplástica/imunologia , Soro Antilinfocitário/metabolismo , Estudos de Casos e Controles , Criança , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Ligação ProteicaRESUMO
Aplastic anaemia (AA) is characterized by hypocellular marrow, pancytopenia, and risk of severe anaemia, haemorrhage and infection. AA is often idiopathic, but frequently occurs after exposure to drugs/chemicals. However, the pathogenesis of AA is not clearly understood, and there are no convenient animal models of drug-induced AA. We have evaluated regimens of busulphan (BU) administration in the mouse to produce a model of chronic bone marrow aplasia showing features of human AA. Mice were given 8 doses of BU at 0, 5.25 and 10.50 mg/kg over 23 days; marrow and blood samples were examined at 1, 19, 49, 91 and 112 days after dosing. At day 1 post dosing, in mice treated at 10.50 mg/kg, nucleated marrow cells, CFU-GM and Erythroid-CFU were reduced. Similarly, peripheral blood erythrocytes, leucocytes, platelets and reticulocytes were reduced. At day 19 and 49 post dosing, there was a trend for parameters to return towards normal. However, at day 91 and 112 post dosing, values remained significantly depressed, with a stabilized chronic bone marrow aplasia. At day 91 and 112 post dosing, marrow cell counts, CFU-GM and Erythroid-CFU were decreased; marrow nucleated cell apoptosis and c-kit+ cell apoptosis were increased; peripheral blood erythrocyte, leucocyte, and platelet counts were reduced. We conclude that this is a model of chronic bone marrow aplasia which has many interesting features of AA. The model is convenient to use and has potential in several areas, particularly for investigations on mechanisms of AA pathogenesis in man.
Assuntos
Anemia Aplástica , Células da Medula Óssea , Bussulfano , Imunossupressores , Modelos Animais , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We report 30 'low-risk' patients with myelodysplasia (MDS) (defined as < 10% bone marrow blasts) who were treated with antithymocyte globulin (ATG). In total, 20 patients were evaluable at the study end-point (response to treatment at 6 months). The diagnosis in these 20 patients was refractory anaemia (RA) in 13, RA with excess blasts in four, and RA with ringed sideroblasts in three. Median age was 54.5 years (range, 31-73 years). There were two cases of secondary MDS. The bone marrow was hypocellular in eight cases and cytogenetics were abnormal in four cases. All patients received lymphoglobuline (horse ATG; Sangstat, France) at a dose of 1.5 vials/10 kg/day for 5 d. The treatment was well tolerated. Three patients in the study died (disease progression, invasive aspergillosis and lung carcinoma respectively); 10 out of 20 evaluable patients (50%) responded to treatment and became transfusion independent; eight out of 13 (62%) patients with RA responded. The median duration of response was 15.5 months (2-42+ months) at the time of analysis.