RESUMO
BACKGROUND: In a single-center retrospective donor versus no-donor comparison, we investigated if allogeneic stem cell transplantation (alloSCT) can improve the dismal course of poor-risk chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: All patients with CLL who were referred for evaluation of alloSCT within a 7-year time frame and had a donor search indication according to the EBMT criteria or because of Richter's transformation were included. Patients for whom a matched donor could be found within 3 months (matches) were compared with patients without such a donor (controls). Primary end point was overall survival measured from the 3-month landmark after search initiation. RESULTS: Of 105 patients with donor search, 97 (matches 83; controls 14) were assessable at the 3-month landmark. Matches and controls were comparable for age, gender, time from diagnosis, number of previous regimens, and remission status. Disregarding if alloSCT was actually carried out or not, survival from the 3-month landmark was significantly better in matches versus controls [hazard ratio 0.38, 95% confidence interval (CI) 0.17-0.85; P = 0.014]. The survival benefit of matches remained significant on multivariate analysis. CONCLUSION: This study provides first comparative evidence that alloSCT may have the potential to improve the natural course of poor-risk CLL as defined by the EBMT criteria.
Assuntos
Leucemia Linfocítica Crônica de Células B/terapia , Transplante de Células-Tronco , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Risco , Doadores de Tecidos , Transplante Homólogo , Resultado do TratamentoRESUMO
Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.
Assuntos
Células Dendríticas/imunologia , Interleucina-12/genética , Interleucina-4/farmacologia , Animais , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interferon gama/farmacologia , Interleucina-12/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Proteínas Recombinantes/farmacologia , Baço/imunologia , Linfócitos T/imunologia , Timo/imunologiaRESUMO
There is increasing evidence that endothelial dysfunction is involved in refractoriness of acute GvHD (aGvHD). Here we investigated the hypothesis that another endothelial complication, transplant-associated thrombotic microangiopathy (TMA), contributes to the pathogenesis of aGvHD refractoriness. TMA was retrospectively assessed in 771 patients after allogeneic stem cell transplantation (alloSCT). Incidences of TMA and refractory aGvHD were correlated with biomarkers of endothelial damage obtained before alloSCT for patients receiving or not receiving statin-based endothelial prophylaxis (SEP). Diagnostic criteria for TMA and refractory aGvHD were met by 41 (5.3%) and 76 (10%) patients, respectively. TMA was overrepresented in patients with refractory aGvHD (45.0 vs 2.3% in all other patients, P<0.001). TMA independently increased mortality. Elevated pretransplant suppressor of tumorigenicity-2 and nitrates along with high-risk variants of the thrombomodulin gene were associated with increased risk of TMA. In contrast, SEP abolished the unfavorable outcome predicted by pretransplant biomarkers on TMA risk. Patients on SEP had a significantly lower risk of TMA (P=0.001) and refractory aGvHD (P=0.055) in a multivariate multistate model. Our data provide evidence that TMA contributes to the pathogenesis of aGvHD refractoriness. Patients with an increased TMA risk can be identified pretransplant and may benefit from pharmacological endothelium protection.
Assuntos
Endotélio Vascular , Doença Enxerto-Hospedeiro , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Transplante de Células-Tronco , Microangiopatias Trombóticas , Doença Aguda , Adolescente , Adulto , Idoso , Aloenxertos , Intervalo Livre de Doença , Endotélio Vascular/lesões , Endotélio Vascular/metabolismo , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/complicações , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/mortalidade , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Taxa de Sobrevida , Microangiopatias Trombóticas/tratamento farmacológico , Microangiopatias Trombóticas/etiologia , Microangiopatias Trombóticas/metabolismo , Microangiopatias Trombóticas/mortalidadeRESUMO
Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.
Assuntos
Efeito Enxerto vs Leucemia , Imunomodulação , Leucemia Prolinfocítica de Células T/terapia , Neoplasia Residual/diagnóstico , Receptores de Antígenos de Linfócitos T/análise , Transplante de Células-Tronco/métodos , Adulto , Idoso , Células Clonais/imunologia , Rearranjo Gênico do Linfócito T/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinética , Leucemia Prolinfocítica de Células T/diagnóstico , Pessoa de Meia-Idade , Neoplasia Residual/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Transplante HomólogoRESUMO
Although the gastrin-releasing peptide-preferring bombesin receptor (GRPR) has been implicated in memory formation, the underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus. Male Wistar rats received bilateral infusions of bombesin (BB) into the dorsal hippocampus immediately after inhibitory avoidance (IA) training. Intermediate doses of BB enhanced, whereas a higher dose impaired, 24-h IA memory retention. The BB-induced memory enhancement was prevented by pretraining infusions of a GRPR antagonist or inhibitors of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) kinase and protein kinase A (PKA), but not by a neuromedin B receptor (NMBR) antagonist. We next further investigated the interactions between the GRPR and the PKA pathway. BB-induced enhancement of consolidation was potentiated by coinfusion of activators of the dopamine D1/D5 receptor (D1R)/cAMP/PKA pathway and prevented by a PKA inhibitor. We conclude that memory modulation by hippocampal GRPRs is mediated by the PKC, MAPK, and PKA pathways. Furthermore, pretraining infusion of BB prevented beta-amyloid peptide (25-35)-induced memory impairment, supporting the view that the GRPR is a target for the development of cognitive enhancers for dementia.
Assuntos
Hipocampo/fisiologia , Memória , Receptores da Bombesina/fisiologia , Peptídeos beta-Amiloides/farmacologia , Animais , Bombesina/farmacologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Hipocampo/efeitos dos fármacos , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C/fisiologia , Ratos , Ratos Wistar , Receptores da Bombesina/agonistas , Receptores de Dopamina D5/agonistas , Transdução de SinaisRESUMO
The antigen-presenting capacity of dendritic cells (DCs) makes them attractive potential cellular adjuvants for vaccination strategies. Currently, most in vitro culture systems for the production of these DCs include serum. However, this is undesirable because serum contains growth factors that vary between individuals and could affect DC development. Unless the patient's own serum is used, foreign antigens and the risk of infection will detract from the usefulness of these cells in clinical strategies. In this study we investigated the production of DCs from CD34+ progenitor cells of cancer patients or normal donors under serum-free conditions. We have established a model system for the investigation of DC development and maturation. Dendritic cells that developed from myeloid precursors accumulated after 2 weeks in an intermediate CD1a , CD80-, CD83-, CD86- stage. Intermediate DCs adhered to plastic surfaces, expressed Birbeck granules, and were negative for CD2 and CD14. In the presence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha, interleukin-4 promoted the development of these stages. Spontaneous maturation of intermediate DCs into fully activated DCs expressing CD83 and costimulatory molecules occurred asynchronously over the ensuing 2 to 3 weeks. This maturation involved increased expression of CD80, CD83, CD86, CMRF-44, HLA-A, -B, -C, and -DR as well as downregulation of CD1a and CD11b. Activated DCs are characterized by the lack of adherence to plastic surfaces and the absence of Birbeck granules. By day 28, these cells were nonphagocytic, potent antigen-presenting cells with an irreversible phenotype. This serum-free system offers advantages in that the process of differentiation and maturation of committed DCs is extended over a period of more than 28 days, allowing investigators to study the effects of individual cytokines or other supplements during distinct phases of DC development in a defined environment.
Assuntos
Técnicas de Cultura de Células/métodos , Células Dendríticas/citologia , Células-Tronco Hematopoéticas/citologia , Adulto , Antígenos CD34 , Diferenciação Celular , Meios de Cultura Livres de Soro , Citometria de Fluxo , HumanosRESUMO
To elucidate factors contributing to the effectiveness of allogeneic hematopoietic stem cell transplantation (alloHCT) in high-risk CLL, immune interventions, GvHD and clinical outcome of 77 consecutive patients allografted for CLL were analyzed. Immune modulation (immunosuppression tapering, rituximab-augmented donor lymphocyte infusions) was guided by minimal residual disease (MRD) monitoring and commenced at a median of 91 (22-273) days after alloHCT, resulting in a probability of being event free and MRD-negative 1 year after transplant of 57% (84% in those encountering chronic GvHD). Patients who were event free and MRD-negative at the 12-month landmark had a 4-year PFS of 77% and largely remained durably MRD-negative if MRD clearance had occurred subsequent to immune modulation. Three-year overall survival, PFS, relapse incidence and non-relapse mortality of all 77 patients were 69, 57, 26 and 24%, respectively. Survival was not affected by EBMT risk category but by active disease at alloHCT, which could not be overcome by intensification of conditioning. Twenty-three patients who experienced relapse post alloHCT had a survival of 56% at 2 years after CLL recurrence. In conclusion, MRD-guided immune modulation after alloHCT for high-risk CLL can provide durable MRD clearance in more than half of the patients.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodos , Adulto , Idoso , Erradicação de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Recidiva , Estudos Retrospectivos , Resultado do TratamentoRESUMO
In a preliminary study we have shown that optimally collected human umbilical cord (HUC) blood cells grow significantly better in long term cultures (LTC) than normal adult marrow cells (NBM) p = 0.0007. The LTC findings are supported by the observation in clonogenic assay that a similar number of GM-CFC colonies can be grown from HUC blood and NBM mononuclear cells (MNC). Also there is a trend towards a higher proportion of primitive erythroid (BFU-E) and primitive megakaryocyte colonies (MK-CFC containing greater than 20 cells) in HUC blood compared with NBM. We suggest that further work on the feasibility of a HLA typed, cryopreserved HUC blood bank as a source of unrelated haemopoietic 'stem' cells for clinical transplantation is indicated.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas , Contagem de Células Sanguíneas , Coleta de Amostras Sanguíneas , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Estudos de Viabilidade , Transplante de Tecido Fetal , Transplante de Células-Tronco Hematopoéticas , Humanos , Recém-NascidoRESUMO
Although reduced intensity conditioning (RIC) before allografting is associated with low treatment-related morbidity and mortality, graft-versus-host disease (GVHD) remains a significant complication of hematopoietic stem cell transplantation (HSCT). T cell depletion (TCD) has been successfully used in conventional allotransplantation to reduce the incidence of GVHD, but was associated with an increased rate of engraftment failure. In a small cohort of six patients at high risk of developing GVHD we have determined whether sustained engraftment could be achieved using reduced intensity conditioning and T cell depletion in combination. All patients engrafted and 5/6 developed high levels (i.e. > or =95%) of donor chimerism, even though mismatched related or matched unrelated donors were used. Only one patient developed acute GVHD, as he received donor lymphocyte infusions (DLI) for relapse. In summary, TCD might be a useful prophylactic tool in RIC allogeneic HSCT. Although TCD after RIC might be associated with high relapse rate, as 5/6 patients are not in remission, this combined strategy might be appropriate for patients with less aggressive malignant or non-malignant diseases in which high transplant-related morbidity and mortality is not acceptable.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Depleção Linfocítica , Quimeras de Transplante , Condicionamento Pré-Transplante , Transplante Homólogo , Vidarabina/uso terapêutico , Irradiação Corporal Total , Adulto , Estudos de Coortes , Progressão da Doença , Estudos de Viabilidade , Feminino , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/terapia , Humanos , Transfusão de Linfócitos , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Doadores de Tecidos , Resultado do Tratamento , Vidarabina/análogos & derivadosRESUMO
The combination of azacitidine and donor lymphocyte infusions (DLI) as first salvage therapy for relapse after allogeneic transplantation (allo-HSCT) was studied in 30 patients with acute myeloid leukemia (AML; n=28) or myelodysplastic syndromes (MDS; n=2) within a prospective single-arm multicenter phase-II trial. Treatment schedule contained up to eight cycles azacitidine (100 mg/m(2)/day, days 1-5, every 28 days) followed by DLI (from 1-5 × 10(6) to 1-5 × 10(8) CD3(+)cells/kg) after every second azacitidine cycle. A median of three courses azacitidine (range 1-8) were administered, and 22 patients (73%) received DLI. Overall response rate was 30%, including seven complete remissions (CRs, 23%) and two partial remissions (7%). Five patients remain in CR for a median of 777 days (range 461-888). Patients with MDS or AML with myelodysplasia-related changes were more likely to respond (P=0.011), and a lower blast count (P=0.039) as well as high-risk cytogenetics (P=0.035) correlated with the likelihood to achieve CR. Incidence of acute and chronic graft-versus-host disease was 37% and 17%, respectively. Neutropenia and thrombocytopenia grade III/IV occurred during 65% and 63% of treatment cycles, while infections were the most common grade III/IV non-hematological toxicity. Azacitidine and DLI as salvage therapy is safe, induces long-term remissions and may become an alternative for patients with AML or MDS relapsing after allo-HSCT.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Transplante de Células , Leucemia Mieloide Aguda/terapia , Linfócitos/citologia , Síndromes Mielodisplásicas/terapia , Terapia de Salvação , Transplante de Células-Tronco , Adulto , Idoso , Terapia Combinada , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/cirurgia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/cirurgia , Recidiva , Transplante HomólogoRESUMO
Respiratory syncytial virus (RSV) usually causes self-limiting upper respiratory tract infections, but can be associated with severe lower respiratory tract infection disease (LRTID) in infants and in patients with hematologic malignancies. We have analyzed the risk factors and the measures for containment within an outbreak of nosocomial RSV infections in a hematology and SCT unit. A total of 56 patients were affected (53 RSV-A and 3 RSV-B) including 32 transplant patients (16 allogeneic and 16 autologous). Forty (71%) of the 56 patients suffered from LRTID and 14 (35%) of the patients with LRTID subsequently died. However, because of concomitant infections with fungal and bacterial pathogens, the impact of RSV on the fatal outcome was difficult to assess. Multivariate analysis showed that low levels of IgG were significantly associated with fatal outcome (P=0.007), treatment with oral ribavirin represented a protective factor (P=0.02). An extremely protracted viral shedding was observed in this cohort of patients (median=30.5 days, range: 1-162 days), especially pronounced in patients after allogeneic transplantation (P=0.002). Implementation of rigorous isolation and barrier measures, although challenged by long-term viral carriers, was effective in containment of the outbreak.
Assuntos
Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controle , Surtos de Doenças/prevenção & controle , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Adolescente , Adulto , Idoso , Infecção Hospitalar/virologia , Feminino , Alemanha/epidemiologia , Hematologia , Departamentos Hospitalares , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Estudos Retrospectivos , Fatores de Risco , Transplante de Células-Tronco , Adulto JovemRESUMO
Acute GVHD (aGVHD) remains a major cause of mortality in patients undergoing allo-SCT. In particular, the outcome of those patients who fail first-line therapy with glucocorticosteroids is poor. Preliminary reports suggested that the purine analogue pentostatin might be effective for treatment of steroid-refractory aGVHD. Here, we report on our single-center experience with pentostatin in this condition. From 2005 to 2008, a total of 24 consecutive patients, who had undergone first-line salvage treatment for steroid-refractory aGVHD of the gastrointestinal tract with pentostatin, were identified from 301 patients allografted during that period and retrospectively analyzed. Response to treatment, defined as CR or very good PR (VGPR), was observed in nine patients (38%), with a median time to response of 10 days. Although pentostatin was associated with only moderate myelosuppressive toxicity, if any, 2-year survival was only 17% with five of the nine responders dying from infection (four patients) or recurrent GVHD (one patient). We conclude that pentostatin is a moderately effective therapy for steroid-refractory aGVHD, showing response and outcome rates similar to other clinically used regimens.
Assuntos
Doença Enxerto-Hospedeiro/tratamento farmacológico , Pentostatina/uso terapêutico , Esteroides/farmacologia , Adulto , Antineoplásicos , Feminino , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Estudos Retrospectivos , Terapia de Salvação/métodos , Resultado do Tratamento , Adulto JovemRESUMO
Thrombotic microangiopathy (TMA) has been described as a complication of bone marrow or stem cell transplantation. It is usually associated with high dose therapy for autologous transplantation or myeloablative conditioning in the allogeneic setting. Here we report three cases of TMA after reduced intensity conditioning and allogeneic peripheral blood stem cell transplantation. All three patients had high dose Melphalan therapy with autologous stem cell support preceding the allogeneic transplantation for several weeks, which may have contributed to endothelial damage and subsequent development of TMA.
Assuntos
Síndrome Hemolítico-Urêmica/etiologia , Mieloma Múltiplo/complicações , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Púrpura Trombocitopênica Trombótica/etiologia , Antineoplásicos/efeitos adversos , Endotélio Vascular/patologia , Humanos , Mieloma Múltiplo/terapia , Transplante de Células-Tronco de Sangue Periférico/métodos , Radioterapia Adjuvante/efeitos adversos , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodos , Transplante Autólogo , Transplante HomólogoRESUMO
BACKGROUND: Non-myeloablative allogeneic stem cell transplantation followed by immunomodulatory therapies is considered a potentially curative approach in the treatment of multiple myeloma and most effective in a minimal residual disease setting. PATIENTS AND METHODS: The aim of this study was to find the most sensitive real-time PCR assay (TaqMan), based on the IGH rearrangement, to quantify the tumour load of 11 patients with multiple myeloma after non-myeloablative allogeneic transplantation. Patient-allele specific primers (ASO) and the TaqMan probe were derived from CDR2 and CDR3 hypervariable regions of IGH, while consensus primers were located within the FR3 and FR4/JH regions. Four different approaches of primer combinations were tested. RESULTS: ASO-forward and -reverse primers together with the clone-specific TaqMan probe were the most sensitive approach compared with the JH (P=0.071) or the FR3 consensus primer (P <0.001). The detection limit amounted to 1/10(4)-1/10(5) cells. Consecutively, 120 samples from 11 patients prior and post allogeneic transplantation were analysed. Three patients reached complete clinical remission accompanied by molecular remission. Disease progression or relapse was seen in six patients. In five, molecular progressive disease was detected prior to the clinical diagnosis of progression or relapse. CONCLUSION: Patient-specific real-time IGH-PCR provides the opportunity for earlier treatment intervention.
Assuntos
Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Transplante de Células-Tronco/métodos , Carga Tumoral , Adulto , Progressão da Doença , Humanos , Pessoa de Meia-Idade , Monitorização Imunológica/métodos , Mieloma Múltiplo/imunologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transplante Homólogo , Carga Tumoral/imunologiaRESUMO
CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.
Assuntos
Transplante de Medula Óssea/métodos , Células-Tronco Hematopoéticas/citologia , Antígenos CD/análise , Antígenos CD34 , Separação Celular/métodos , Células Cultivadas , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Citocinas/farmacologia , Humanos , Técnicas In Vitro , Neoplasias/terapia , Fatores de TempoRESUMO
Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two-stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.
Assuntos
Células Sanguíneas , Células da Medula Óssea , Ensaio de Unidades Formadoras de Colônias/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas , Antígenos CD/análise , Antígenos CD34 , Contagem de Células Sanguíneas , Contagem de Células , Células Cultivadas , Humanos , Recém-Nascido , LeucaféreseRESUMO
This study identifies type I IFNs as activating cytokines in a serum-free system in which human dendritic cells (DC) were generated from CD34+ progenitor cells. After 14 days of culture in GM-CSF, TNF-alpha, and IL-4, CD34+ progenitors gave rise to a population of large, immature DC expressing CD1a and CD11b but lacking CD14, CD80, CD83, CD86, and CMRF44. During the next 2 wk, this population spontaneously matured into nonadherent, CD1a(low/-), CD11b(low/-), CD14-, CD80+, CD83+, CD86+, CMRF44+ DC with high allostimulatory activity in the MLR. To examine which factors influenced this maturation, 25 different cytokines or factors were added to the immature DC culture. Only type I IFNs (alpha or beta) accelerated this maturation in a dose-dependent manner, so that after only 3 days the majority of large cells acquired the morphology, phenotype, and function characteristics of mature DC. Furthermore, supernatants from cultures containing spontaneously maturing DC revealed low levels of endogenous IFN production. Because of the similarity of the activation of DC in our culture system with the phenotypic and functional changes observed during Langerhans cells activation and migration in vivo, we investigated the effect of IFN-alpha on human Langerhans cell migration. IFN-alpha also activated the migration of human split skin-derived DC, demonstrating that this effect was not limited to DC derived in vitro from hemopoietic progenitor cells. DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Interferon Tipo I/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , Citocinas/farmacologia , Células Dendríticas/imunologia , Humanos , Imunofenotipagem , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Pele/citologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous beta(2)-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno/fisiologia , Peptídeos/imunologia , Peptídeos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Antígenos de Neoplasias , Ligação Competitiva , Diferenciação Celular , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/biossíntese , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Cinética , Antígeno MART-1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Cryopreserved human umbilical-cord (HUC) blood is an alternative to bone marrow as a source of haemopoietic "stem" cells for HLA-identical transplantation of children with leukaemia or Fanconi's anaemia. We have studied the in-vitro growth potential of HUC blood in clonogenic assays and in longterm haemopoietic cultures. Clonogenic assays showed that HUC blood produced as many haemopoietic-cell colonies as normal adult bone marrow and a higher proportion of primitive-cell colonies. In longterm culture on preformed irradiated marrow stroma, both progenitor-cell production and lifespan of cultures were significantly greater in HUC blood than in normal bone marrow (p = 0.0007). Our findings indicate that the quality and quantity of HUC-blood-derived haemopoietic "stem" cells are better than those of normal bone marrow. Therefore, single HUC-blood donations are probably sufficient for adults requiring transplantation for leukaemia and other haemopoietic disorders. Banking of HLA-typed HUC blood to facilitate transplantation of patients who lack a family donor should be considered.