RESUMO
BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe systemic genodermatosis lacking therapies beyond supportive care for its extensive, life-limiting manifestations. OBJECTIVES: To report the safety and preliminary responses of 10 patients with RDEB to bone marrow transplant (BMT) with post-transplant cyclophosphamide (PTCy BMT) after reduced-intensity conditioning with infusions of immunomodulatory donor-derived mesenchymal stromal cells (median follow-up 16 months). METHODS: BMT toxicities, donor blood and skin engraftment, skin biopsies, photographic and dynamic assessments of RDEB disease activity were obtained at intervals from pre-BMT to 1 year post-BMT. RESULTS: Related donors varied from haploidentical (n = 6) to human leucocyte antigen (HLA)-matched (n = 3), with one HLA-matched unrelated donor. Transplant complications included graft failure (n = 3; two pursued a second PTCy BMT), veno-occlusive disease (n = 2), posterior reversible encephalopathy (n = 1) and chronic graft-versus-host disease (n = 1; this patient died). In the nine ultimately engrafted patients, median donor chimerism at 180 days after transplant was 100% in peripheral blood and 27% in skin. Skin biopsies showed stable (n = 7) to improved (n = 2) type VII collagen protein expression by immunofluorescence and gain of anchoring fibril components (n = 3) by transmission electron microscopy. Early signs of clinical response include trends toward reduced body surface area of blisters/erosions from a median of 49·5% to 27·5% at 100 days after BMT (P = 0·05), with parental measures indicating stable quality of life. CONCLUSIONS: PTCy BMT in RDEB provides a means of attaining immunotolerance for future donor-derived cellular grafts (ClinicalTrials.gov identifier NCT02582775). What's already known about this topic? Severe, generalized recessive dystrophic epidermolysis bullosa (RDEB) is marked by great morbidity and early death. No cure currently exists for RDEB. Bone marrow transplant (BMT) is the only described systemic therapy for RDEB. What does this study add? The first description of post-transplant cyclophosphamide (PTCy) BMT for RDEB. PTCy was well tolerated and provided excellent graft-versus-host disease prophylaxis, replacing long courses of calcineurin inhibitors in patients receiving human leucocyte antigen-matched sibling BMT. What is the translational message? The PTCy BMT platform permits identification of a suitable related donor for most patients and for subsequent adoptive transfer of donor nonhaematopoietic cells after establishment of immunological tolerance.
Assuntos
Transplante de Medula Óssea/métodos , Ciclofosfamida/administração & dosagem , Epidermólise Bolhosa Distrófica/terapia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Mesenquimais/métodos , Condicionamento Pré-Transplante/métodos , Adolescente , Biópsia , Transplante de Medula Óssea/efeitos adversos , Criança , Pré-Escolar , Seleção do Doador/métodos , Epidermólise Bolhosa Distrófica/imunologia , Epidermólise Bolhosa Distrófica/patologia , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Masculino , Qualidade de Vida , Índice de Gravidade de Doença , Pele/imunologia , Pele/patologia , Transplante Homólogo/efeitos adversos , Transplante Homólogo/métodos , Resultado do Tratamento , Adulto JovemRESUMO
Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.
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Transplante de Medula Óssea , Doença Enxerto-Hospedeiro/imunologia , Tolerância Imunológica/imunologia , Cinurenina/metabolismo , Linfócitos T Reguladores/imunologia , Triptofano/metabolismo , Animais , Células Cultivadas , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Técnicas In Vitro , Camundongos , Taxa de SobrevidaRESUMO
AIMS/HYPOTHESIS: We sought to establish if stem cells contained in cord blood cell allografts have the capacity to differentiate into insulin-expressing beta cells in humans. METHODS: We studied pancreases obtained at autopsy from individuals (n = 11) who had prior opposite-sex cord blood transplants to reconstitute haematopoiesis. Pancreatic tissue sections were stained first by XY-fluorescence in situ hybridisation and then insulin immunohistochemistry. Pancreases obtained at autopsy from participants without cord blood cell infusions served as controls (n = 11). RESULTS: In the men with prior transplant of female cord blood, there were 3.4 ± 0.3% XX-positive insulin-expressing islet cells compared with 0.32 ± 0.05% (p < 0.01) in male controls. In women with prior transplant of male cord blood cells we detected 1.03 ± 0.20% XY insulin-expressing islet cells compared with 0.03 ± 0.03 in female controls (p < 0. 001). CONCLUSIONS/INTERPRETATION: Cord blood stem cells have the capacity to differentiate into insulin-expressing cells in non-diabetic humans. It remains to be established whether these cells have the properties of beta cells.
Assuntos
Sangue Fetal/citologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adolescente , Adulto , Idoso , Diferenciação Celular/fisiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco , Transplante Homólogo , Adulto JovemRESUMO
Adoptive transfer of thymus-derived natural regulatory T cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft-versus-host disease (GVHD). TGFß induces Foxp3 expression and suppressive function in stimulated murine CD4+25- T cells, and these induced Treg (iTregs), like nTreg, suppress auto- and allo-reactivity in vivo. However, while TGFß induces Foxp3 expression in stimulated human T cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFß-dependent Foxp3 expression and induces a potent suppressor function in naive (CD4+ 25-45RA+) T cells. Rapa/TGFß iTregs are anergic, express CD25 at levels higher than expanded nTregs and few cells secrete IL-2, IFNγ or IL-17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFß induces suppressive function even in the presence of memory CD4+ T cells. A single apheresis unit of blood yields an average ~240 × 109 (range ~ 70-560 × 109) iTregs from CD4+25- T cells in ≤ 2 weeks of culture. Most importantly, Rapa/TGFß iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Camundongos Knockout , Sirolimo/farmacologiaRESUMO
During three field campaigns spectral actinic flux was measured from 290-500 nm under clear sky conditions in Alpine terrain and the associated O3- and NO2-photolysis frequencies were calculated and the measurement products were then compared with 1-D- and 3-D-model calculations. To do this 3-D-radiative transfer model was adapted for actinic flux calculations in mountainous terrain and the maps of the actinic flux field at the surface, calculated with the 3-D-radiative transfer model, are given. The differences between the 3-D- and 1-D-model results for selected days during the campaigns are shown, together with the ratios of the modeled actinic flux values to the measurements. In many cases the 1-D-model overestimates actinic flux by more than the measurement uncertainty of 10%. The results of using a 3-D-model generally show significantly lower values, and can underestimate the actinic flux by up to 30%. This case study attempts to quantify the impact of snow cover in combination with topography on spectral actinic flux. The impact of snow cover on the actinic flux was ~ 25% in narrow snow covered valleys, but for snow free areas there were no significant changes due snow cover in the surrounding area and it is found that the effect snow-cover at distances over 5 km from the point of interest was below 5%. Overall the 3-D-model can calculate actinic flux to the same accuracy as the 1-D-model for single points, but gives a much more realistic view of the surface actinic flux field in mountains as topography and obstruction of the horizon are taken into account.
RESUMO
BACKGROUND: Rapid quantitative recovery of NK cells but slower recovery of T-cell subsets along with frequent viral infections are reported after umbilical cord blood (UCB) compared with matched sibling donor (MSD) hematopoietic cell transplantation (HCT). However, it remains unclear whether increased propensity for viral infections is also a result of slower recovery of virus-specific immunity after UCB as compared to MSD HCT. OBJECTIVES: We examined the differences in the function of virus-specific peripheral blood mononuclear cells (PBMC) after UCB (N=17) vs. MSD (N=9) using previously collected patient blood samples at various time points after HCT. METHODS: Interferon-gamma (IFN-γ) enzyme-linked immune absorbent spot (ELISpot) assay was used to quantify the PBMC frequencies that secrete IFN-γ in response to 11 immunopeptides from 5 common viruses. We included the patients who received the same reduced intensity conditioning regimen without ATG, no systemic glucocorticoids and had no relapse or acute/chronic graft-versus-host disease within 1 year after HCT. RESULTS: The CMV-reactive PBMC frequencies were higher in CMV seropositive vs. seronegative patients after HCT. Among CMV seropositive patients, the frequency of CMV-reactive PBMC was lower after UCB compared to MSD throughout one year of HCT. We observed no differences in virus-specific PBMC responses towards HHV6, EBV, BK, and adenovirus antigens between UCB and MSD. CONCLUSION: Our data demonstrate that the reconstitution of CMV-specific immunity is slower in CMV seropositive recipients of UCB vs. MSD HCT in contrast to other viruses which had similar recoveries. These study findings support implementation of more potent prophylactic strategies for preventing CMV reactivation in CMV seropositive patients receiving UCB HCT.
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One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.
Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética/métodos , Lentivirus/genética , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Anemia de Fanconi/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mitomicina/farmacologia , Transdução GenéticaRESUMO
Aplastic anemia is a rare disease in children that is most commonly idiopathic and less often a hereditary disorder. Hereditary bone marrow failure (BMF) syndromes, however, should be considered both in children and in adults before any attempt at treatment. Precise diagnosis is important because it will modify prognostic treatment options and the results of bone marrow transplantation. In this review, we will report recent results of treatment of Fanconi anemia and other hereditary BMF syndromes.
Assuntos
Anemia Hipoplástica Congênita/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Transplante de Células-Tronco Hematopoéticas/métodos , Anemia Hipoplástica Congênita/genética , Pré-Escolar , Humanos , Estimativa de Kaplan-Meier , Agonistas Mieloablativos/uso terapêutico , Relações entre Irmãos , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodosRESUMO
Purine analogs are often used for conditioning preceding allogeneic hematopoietic stem cell transplantation (HCT). We prospectively tested fludarabine (Flu) 40 mg/m(2)/day x 5 days vs cladribine (Clad) 10 mg/m(2)/day x 5 days plus oral busulfan (1 mg/kg q6 h x 2 days) and total body irradiation 200 cGy in 32 recipients of matched sibling and unrelated donor (URD) HCT. Patients were similar in age (median 52 years), diagnosis, extensive pre-HCT therapy (56 vs 63%), and high-risk disease status (81 vs 93%). Neutrophil engraftment was prompt (median 11 vs 12 days), but early graft failure using Clad halted randomization. Platelet recovery was prompt (median Flu 18 vs Clad 24 days). Graft-versus-host disease (GVHD) after Flu vs Clad was similar; (acute grade II/IV 56 vs 69%, P=0.26; chronic 50 vs 31%, P=0.27). Nonrelapse mortality (Flu 25 vs Clad 38%, P=0.47) and progression-free survival at 3 years were similar as well. Multivariate analyses showed slightly, but not significantly lower relative risk (RR) of neutrophil engraftment with Clad (RR 0.6 (95% CI 0.2-1.3) P=0.16) and with URD RR 0.4 (0.2-1.0) P=0.04). Older patients with advanced hematologic malignancies achieve satisfactory outcomes using either of these reduced intensity conditioning regimens.
Assuntos
Transplante de Medula Óssea/métodos , Bussulfano/administração & dosagem , Cladribina/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Agonistas Mieloablativos/administração & dosagem , Condicionamento Pré-Transplante/métodos , Vidarabina/análogos & derivados , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica , Terapia Combinada , Feminino , Sobrevivência de Enxerto/efeitos dos fármacos , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/terapia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Estudos Prospectivos , Condicionamento Pré-Transplante/efeitos adversos , Transplante Homólogo/efeitos adversos , Transplante Homólogo/métodos , Vidarabina/administração & dosagem , Irradiação Corporal TotalRESUMO
Gonadal failure is a health and quality-of-life concern in hematopoietic cell transplant (HCT) survivors. While ovarian dysfunction is nearly universal following myeloablative (MA) conditioning, the risk is unclear after reduced-intensity conditioning (RIC). Gonadotropin-releasing hormone agonists decrease ovarian failure rates following conventional chemotherapy, but little is known about its effectiveness with HCT. We investigated the impact of leuprolide on ovarian function after MA conditioning and monitored ovarian function after RIC in this descriptive pilot study. Post-menarchal females <50 years undergoing HCT with adequate baseline ovarian function (follicle-stimulating hormone (FSH) level <40 mIU/mL and normal menstruation) were eligible. Prior to MA conditioning, leuprolide was administered. Those undergoing RIC were observed. FSH was measured at various time points. Seventeen women aged 12-45 years were evaluated (7 in the intervention group and 10 in the observation group). Compared to the historical high rate of ovarian failure after MA conditioning, 3 of 7 evaluable Lupron recipients had ovarian failure at a median of 703 days post transplant. Ovarian failure occurred in 1 of 10 recipients of RIC at a median follow-up of 901 days. In conclusion, leuprolide may protect ovarian function after MA conditioning. Additionally, RIC with cyclophosphamide, fludarabine and low-dose TBI has a low risk of ovarian failure.
Assuntos
Hormônio Liberador de Gonadotropina/agonistas , Transplante de Células-Tronco Hematopoéticas/métodos , Leuprolida/uso terapêutico , Ovário/efeitos dos fármacos , Condicionamento Pré-Transplante/métodos , Adolescente , Adulto , Criança , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Liberador de Gonadotropina/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Pessoa de Meia-Idade , Agonistas Mieloablativos , Testes de Função Ovariana , Ovário/fisiologia , Projetos Piloto , Adulto JovemRESUMO
PURPOSE: Allogeneic bone marrow transplantation (BMT) is an option for some patients with chronic myelogenous leukemia (CML). We retrospectively evaluated the effect of various risk factors observed at diagnosis and at transplantation on survival, event-free survival (EFS), and relapse after BMT. PATIENTS AND METHODS: Seventy-nine patients with CML in chronic phase (CP) were treated with cyclophosphamide and total body irradiation followed by BMT. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (CsA) in most instances or CsA plus the use of lymphocyte-depleted bone marrow (BM). RESULTS: Survival at 4.5 years was 52%. Stratified by age and GVHD prophylaxis, the actuarial survival was 65% (95% confidence interval [CI], 47% to 78%) in patients aged less than 30 years receiving unmanipulated BM, 33% (95% CI, 12% to 56%) in patients greater than or equal to 30 years old receiving unmanipulated BM, and 38% (95% CI, 14% to 63%) in patients greater than or equal to 30 years old receiving lymphocyte-depleted BM. In univariate analysis, patient age (greater than or equal to 30 years) and the use of lymphocyte-depleted BM negatively influenced EFS. When stratified by age and GVHD prophylaxis, however, ABO incompatibility, cytomegalovirus (CMV) seropositivity, and chronic GVHD significantly reduced the probability of EFS. Factors that have been associated with early death in nontransplanted patients (ie, sex, spleen size, blast and platelet counts at presentation) were not predictive of long-term survival outcome after BMT. CONCLUSIONS: The data suggest that (1) BMT should be offered early after diagnosis to all patients with CML in CP who have compatible sibling donors regardless of prognostic factors at presentation, (2) GVHD remains the principal cause of mortality after BMT in patients receiving CsA, and (3) T-cell depletion by the physical separation method of counterflow elutriation (CE) is associated with a significant risk of relapse.
Assuntos
Transplante de Medula Óssea , Leucemia Mieloide de Fase Crônica/cirurgia , Adolescente , Adulto , Análise de Variância , Transplante de Medula Óssea/métodos , Criança , Pré-Escolar , Terapia Combinada , Ciclofosfamida/uso terapêutico , Ciclosporina/uso terapêutico , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Leucemia Mieloide de Fase Crônica/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Análise de Sobrevida , Irradiação Corporal TotalRESUMO
PURPOSE: To test the use of unrelated donor bone marrow transplantation (URD BMT) to cure children with high-risk acute leukemias. PATIENTS AND METHODS: Between June 1985 and December 1994, 50 children with acute leukemia (15 acute myelogenous leukemia [AML], 35 acute lymphoblastic leukemia [ALL]; 22 greater than second complete remission [CR]) received BMT from a URD at the University of Minnesota. Ages ranged from 0.9 to 17.5 years (median, 8.8). Median follow-up is 2.1 years (range, 1 to 7.3). Thirty patients (60%) received bone marrow fully matched at HLA-A,B and DRB1; 20 (40%) received bone marrow with a major or minor mismatch at a single HLA-A or B locus. RESULTS: The median time to neutrophil engraftment was day 24 (range, 14 to 42 days) in those receiving matched and day 25 (range, 15 to 32 days) in those receiving mismatched marrow (P = .35). The incidence of grades III to IV graft-versus-host disease (GVHD) was 23% (95% confidence interval [CI], 7% to 39%) in matched and 32% (95% CI, 8% to 52%) in HLA-mismatched patients (P = .57). The incidence of chronic GVHD was 50% (95% CI, 28% to 72%) in matched and 57% (95% CI, 23% to 91%) in mismatched patients (P = .80). Disease-free survival for patients with ALL is 37% (95% CI, 21% to 53%) at 1 year and 30% (95% CI, 15% to 46%) at 2 years; for patients with AML, 53% (95% CI, 28% to 78%) at 1 year and 33% (95% CI, 6% to 60%) at 2 years. CONCLUSION: URD BMT is an effective treatment for children with poor-prognosis acute leukemia and should be considered for all high-risk patients. Early referral of patients is strongly recommended.
Assuntos
Transplante de Medula Óssea , Leucemia Mieloide Aguda/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Transplante de Medula Óssea/efeitos adversos , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Doença Enxerto-Hospedeiro/etiologia , Humanos , Lactente , Masculino , Prognóstico , Risco , Índice de Gravidade de Doença , Análise de Sobrevida , Transplante Homólogo , Resultado do TratamentoRESUMO
The clinical efficacy of allogeneic bone marrow transplantation has been limited by difficulties in locating HLA-matched donors, and the occurrence of graft failure, severe graft-versus-host disease (GVHD), and opportunistic infections. Placental or umbilical cord blood may overcome some of these problems. Phase I-II trials of umbilical cord blood transplants (UCBT) show an overall engraftment rate of approximately 90%, with a median time to neutrophil recovery of 26 days. Nucleated cell, dose, and CFU-GM and CD34+ content were inversely correlated with the time to neutrophil recovery (P < .01 for each). The overall probability of grade II-IV GVHD was approximately 40%, despite high degrees of HLA disparity between donor and recipient. In analysis of data at two institutions, the only factors predictive of survival were age and cell dose within the context of HLA 0-2 antigen disparities. There was no significant effect of HLA on the likelihood or speed of engraftment, the risk of grade II-IV acute GVHD, or survival. UCBT offers several potential advantages over bone marrow transplants for reconstitution of hematopoiesis, including the shorter interval to transplant, the absence of donor attrition, the absence of harvest complications, and the extremely low risk of cytomegalovirus transmission.
Assuntos
Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/epidemiologia , Doenças Hematológicas/mortalidade , Doenças Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Probabilidade , Taxa de SobrevidaRESUMO
Umbilical cord blood (UCB) is an attractive potential alternative to bone marrow (BM) as a source of hematopoietic progenitor cells since the number of progenitors in UCB is similar or even greater than that in normal BM. It was the aim of the present study to analyze the degree of immaturity of UCB progenitor cells. UCB mononuclear (MNC) and/or CD34+ cells were tested for surface antigen phenotype, expression of cytokines receptor, effect of stem cell factor (SCF) on colony growth, resistance to mafosfamide and replating potential. We have found that 34.9 +/- 3.4% and 77.9 +/- 2.6% of UCB CD34+ cells did not express CD38 and CD45RA antigens, respectively, suggesting that UCB contains a high proportion of immature progenitor cells. By means of three-color analysis, the receptor for SCF was detected on the majority of the CD34+ HLA-DR+ subpopulation; in fact, 81.8% +/- 4.3% of CD34+ HLA-DR+ cells were defined as SCF(low) and 8.1 +/- 1.5% as SCF(high). Colony growth of MNC and CD34+ cells was enhanced by the addition of SCF to methylcellulose mixture, resulting in a statistically significant increase in CFU-GM and CFU-GEMM but not in BFU-E numbers. UCB progenitor cells showed a higher resistance to mafosfamide treatment, in comparison to BM; the addition of SCF to the culture medium resulted in a statistically significant increase in mafosfamide concentration required to inhibit 95% of colony growth (P < or = 0.05). Moreover, as shown by single colony transfer assays, the presence of SCF in primary cultures promoted a significantly higher replating potential for both untreated (42 +/- 3.3% vs 21 +/- 4.6%, P < or = 0.018) and mafosfamide-treated samples (62 +/- 5.6% vs 44 +/- 6.1%, P < or = 0.018). In conclusion, UCB is a source of progenitor cells with immature characteristics in terms of surface antigen expression, distribution of SCF receptor, resistance to mafosfamide and replating potential. Therefore, UCB progenitor cells represent an ideal candidate population for experimental programs involving gene transfer and ex vivo stem cell expansion.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Antígenos CD34/análise , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacologia , Imunofluorescência , Antígenos HLA-DR/análise , Células-Tronco Hematopoéticas/imunologia , Humanos , Recém-Nascido , Fenótipo , Receptores de Fatores de Crescimento/análise , Fator de Células-Tronco/farmacologiaRESUMO
Natural suppressor cell activity (NSCA) has been ascribed to a subset of cells present in human and murine hematopoietic tissues which can suppress a variety of lymphocyte responses without MHC restriction. We investigated NSCA in lymphocyte-depleted rat bone marrow (BM) which is used as a model for prevention of graft vs host disease (GVHD) following allogeneic BM transplantation (BMT). The T-cell depleted fraction obtained after elutriation contained higher levels of NSCA than the unseparated BM. Further separation of this graft fraction by discontinuous Percoll gradient centrifugation revealed high levels of radiosensitive NSCA in the low density (less than 1.070) fraction which represented 0.5% of the original BM population. These cells were of blast morphology, stained intensely with a dansylated derivative of cyclosporine A (dans CsA) and weakly expressed macrophage/granulocyte antigens and non-specific esterase (NSE). These cells were initially non-adherent but proliferated in culture to produce intensely NSE positive, adherent, phagocytic cells of macrophage morphology. We conclude that the highly suppressive, radiosensitive cell present in rat BM may be of early progenitor or monocyte lineage. The grafting of natural suppressor (NS) cells and progenitor cells may affect graft/host immunoregulation and their characterization may provide insight into GVH biology and graft rejection.
Assuntos
Células da Medula Óssea , Linfócitos T Reguladores/fisiologia , Animais , Antígenos de Superfície/análise , Transplante de Medula Óssea , Separação Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Ciclosporinas/farmacologia , Fibronectinas/metabolismo , Doença Enxerto-Hospedeiro/prevenção & controle , Histocitoquímica , Teste de Cultura Mista de Linfócitos , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Linfócitos T Reguladores/enzimologiaRESUMO
We have developed a simple three flow-rate, fixed rotor speed, counterflow centrifugal elutriation (CCE) procedure that permits the isolation of an engraftable lymphocyte-depleted (greater than 98%) fraction from ACI rat bone marrow. The different cell fractions were characterized by morphology, alloreactivity in mixed lymphocyte culture and limiting dilution analysis, colony-forming capacity, and their capacity to reconstitute hematopoiesis and effect a graft-versus-host reaction in lethally irradiated allogeneic hosts. After CCE fractionation of ACI rat marrow, transplantation of the lymphocyte-depleted marrow fraction resulted in sustained engraftment without evidence of clinical or histologic acute graft-versus-host disease (GVHD). CCE fractionation of rat bone marrow may be a useful preclinical model for studying lympho-hematopoietic and immune reconstitution after transplantation with lymphocyte-depleted donor marrow, as well as for studying the role of lymphocyte subpopulations on engraftment, acute GVHD, and leukemia relapse in syngeneic and allogeneic bone marrow transplantation.
Assuntos
Células da Medula Óssea , Depleção Linfocítica , Animais , Separação Celular , Centrifugação/métodos , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/prevenção & controle , Ratos , Ratos Endogâmicos Lew , Ratos EndogâmicosRESUMO
Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disease (PTLD) is a well-recognized complication of T cell-depleted (TCD) allogeneic bone marrow transplantation (BMT). Certain methods of TCD, such as counterflow elutriation (CE), have not been associated with an increased incidence of PTLD. Since CE depletes B cells as well as T cells, the hypothesis that CE depletes donor marrow of EBV-infected cells was tested in this study. Marrow samples from 70 donors were assayed by qualitative and semi-quantitative polymerase chain reaction (PCR) amplification to detect EBV DNA in four cellular fractions produced by elutriation: a small cell fraction (F70), two intermediate-sized cell fractions (F110 and F140), and a large cell (hematopoietic precursor-enriched, lymphocyte-depleted) rotor-off (R/O) fraction. The distribution of B cells in elutriation fractions was 21% (F70), 59% (F110), 18% (F140), and 2% (R/O). Qualitatively, the frequency of EBV DNA detected in fractions was 49% (F70), 57% (F110), 73% (F140), and 44% (R/O). The relative concentration of EBV DNA in each fraction was determined by semiquantitative PCR. The mean number of EBV DNA copies per 150,000 cells was 0.57 (F70), 4.6 (F110), 5.7 (F140), and 0.61 (R/O). Relative EBV DNA load in each fraction was calculated by multiplying the mean concentration of EBV DNA per fraction and mean mononuclear cell content in each fraction. The relative distribution of EBV DNA was 0.5, 58, 40, and 1.5% in the F70, F110, F140, and R/O fractions, respectively. No correlation between the amount of EBV in the graft and development of PTLD could be made, because only one of the 70 recipients developed PTLD and this was following autologous hematopoietic recovery. Although these results demonstrate that the majority of EBV-infected cells in marrow are separated from the TCD graft by counterflow elutriation, further studies are required to differentiate the role of this phenomenon from that of other potential factors, such as the amount of T cell depletion and recovery of EBV immunity in the pathogenesis of PTLD after BMT.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/virologia , Separação Celular/métodos , Infecções por Herpesviridae , Herpesvirus Humano 4 , Infecções Tumorais por Vírus , Transplante de Medula Óssea/efeitos adversos , DNA Viral/sangue , Infecções por Herpesviridae/transmissão , Herpesvirus Humano 4/genética , Humanos , Transtornos Linfoproliferativos/etiologia , Doadores de Tecidos , Infecções Tumorais por Vírus/transmissão , Carga ViralRESUMO
Naltrexone treatment, used to prevent relapse among former opioid addicts, is reported to have an extraordinary rate of noncompliance. Since activation of opioid receptors produces a sense of well-being, naltrexone's blockade of these receptors might produce dysphoria, which could contribute to noncompliance among addicts under treatment. To test this hypothesis, the authors administered naltrexone to four men who had been free of opioids for 9 to 44 months using a 6-week, placebo-controlled crossover design. One subject dropped out with abstinence-like symptoms, and two others reported mild but significantly greater dysphoria during naltrexone administration. The results suggest that naltrexone may induce mild dysphoria long after addicts stop using opioids.
Assuntos
Sintomas Afetivos/induzido quimicamente , Naloxona/análogos & derivados , Naltrexona/efeitos adversos , Transtornos Relacionados ao Uso de Opioides/reabilitação , Adulto , Ejaculação/efeitos dos fármacos , Emoções/efeitos dos fármacos , Humanos , Libido/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Naltrexona/farmacologia , Receptores Opioides/efeitos dos fármacosRESUMO
PURPOSE: To determine if the favorable outcomes after transplantation of matched sibling donor bone marrow in patients with chronic myelogenous leukemia can be achieved using bone marrow from an HLA-A,B/DRB1-matched unrelated donor. SUBJECTS AND METHODS: Between April 1983 and December 1997, 141 patients with chronic myelogenous leukemia in its first chronic phase received a bone marrow transplant from a matched sibling donor (n = 96) or an HLA-A,B/DRB1-matched unrelated donor (n = 45). The median age of matched sibling donor recipients was 38 years (range, 8 to 56 years) and of unrelated donor recipients was 35 years (range, 3 to 53 years; P = 0.03). The median follow-up was 6 years (range, 1 to 15 years) in matched sibling donor recipients and 5 years (range, 2 to 10 years) in unrelated donor recipients. RESULTS: There was no significant difference in the 5-year survival rates of matched sibling donor recipients [58%; 95% confidence interval (CI), 48% to 68%] and unrelated donor recipients (53%; 95% CI, 39% to 67%; P = 0.4). Among patients who underwent transplantation within 1 year after diagnosis, the 5-year survival rate of matched sibling donor recipients (76%; 95% CI, 65% to 87%) was not significantly different (P = 0.5) from that of unrelated donor recipients (70%; 95% CI, 52% to 88%). In multiple regression analysis, longer time from diagnosis to transplantation, T-cell depletion, and grades III or IV graft versus host disease were independently associated with poorer survival. Transplantation of unrelated donor bone marrow was not associated with mortality (relative risk, 1.1; 95% CI, 0.6 to 2.1; P = 0.7). CONCLUSIONS: Transplantation of bone marrow from a matched sibling donor or an HLA-A,B/DRB1-matched unrelated donor produces equivalent outcomes in patients with chronic myelogenous leukemia, particularly if the transplant takes place within 1 year after diagnosis.
Assuntos
Transplante de Medula Óssea/métodos , Antígenos HLA/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Adolescente , Adulto , Intervalo Livre de Doença , Seguimentos , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Antígenos HLA-DR/imunologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Fenótipo , Modelos de Riscos Proporcionais , Risco , Análise de Sobrevida , Fatores de Tempo , Doadores de Tecidos , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
PURPOSE: The syndrome of idiopathic hyperammonemia occurs in patients who have received high-dose cytoreductive therapy for the treatment of hematologic malignancy. It is characterized by abrupt alteration in mental status and respiratory alkalosis associated with markedly elevated plasma ammonium levels in the absence of any identifiable cause, and frequently results in intractable coma and death. Our goal was to survey clinical and pathologic manifestations of the disorder and discuss treatment options. PATIENTS AND METHODS: Plasma ammonium levels were measured in patients on the acute leukemia service or on the bone marrow transplant service at The Johns Hopkins Hospital, and a level more than twice normal was considered diagnostic of hyperammonemia. The syndrome was identified in nine patients; in eight, hyperammonemia occurred after administration of intensive cytoreductive therapy that resulted in profound leukopenia. The disorder occurred in the ninth patient two months after allogeneic bone marrow transplantation. RESULTS: Three of the nine patients survived an episode of idiopathic hyperammonemia; one patient subsequently died of leukemia and one of recurrent idiopathic hyperammonemia. The one long-term survivor is currently alive and well without neurologic sequelae 250 days after autologous bone marrow transplantation. CONCLUSION: Because neurologic function can deteriorate rapidly, early recognition of this disorder and close monitoring of the patient's neurologic status are critical.