RESUMEN
Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.
Asunto(s)
Alelos , Perros/genética , Antígenos de Histocompatibilidad Clase I/genética , Polimorfismo Genético , Animales , Perros/inmunología , Técnicas de Genotipaje , Antígenos de Histocompatibilidad Clase I/inmunologíaRESUMEN
Developing a preclinical canine model that predicts outcomes for hematopoietic cell transplantation in humans requires a model that mimics the degree of matching between human donor and recipient major histocompatibility complex (MHC) genes. The polymorphic class I and class II genes in mammals are typically located in a single chromosome as part of the MHC complex. However, a divergent class I gene in dogs, designated dog leukocyte antigen-79 (DLA-79), is located on chromosome 18 while other MHC genes are on chromosome 12. This gene is not taken into account while DLA matching for transplantation. Though divergent, this gene shares significant similarity in sequence and exon-intron architecture with other class I genes, and is transcribed. Little is known about the polymorphisms of DLA-79 and their potential role in transplantation. This study was aimed at exploring the reason for high rate of rejection seen in DLA-matched dogs given reduced intensity conditioning, in particular, the possibility that DLA-79 allele mismatches may be the cause. We found that about 82% of 407 dogs typed were homozygous for a single, reference allele. Owing to the high prevalence of a single allele, 87 of the 108 dogs (â¼80%) transplanted were matched for DLA-79 with their donor. In conclusion, we have developed an efficient method to type alleles of a divergent MHC gene in dogs and identified two new alleles. We did not find any statistical correlation between DLA-79 allele disparity and graft rejection or graft-versus-host disease, among our transplant dogs.
Asunto(s)
Rechazo de Injerto/veterinaria , Enfermedad Injerto contra Huésped/veterinaria , Antígenos de Histocompatibilidad Clase I/inmunología , Prueba de Histocompatibilidad/veterinaria , Alelos , Animales , Cromosomas de los Mamíferos/inmunología , Perros , Exones , Expresión Génica , Rechazo de Injerto/inmunología , Enfermedad Injerto contra Huésped/inmunología , Histocompatibilidad , Antígenos de Histocompatibilidad Clase I/clasificación , Antígenos de Histocompatibilidad Clase I/genética , Homocigoto , Intrones , Leucocitos/inmunología , Leucocitos/metabolismo , Tipificación Molecular/métodos , Filogenia , Polimorfismo GenéticoRESUMEN
A retrospective analysis of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cell (G-PBMC) products harvested from healthy donors indicates significant variability in both the absolute number and relative proportion of CD34, CD3, and CD14 cells obtained. This report examined whether variations in the cellular composition of G-PBMC products correlated with clinical outcomes after myeloablative allogeneic transplantation. The numbers of CD34, CD3, and CD14 cells infused into 181 human leukocyte antigen (HLA)-identical sibling recipients were analyzed with respect to tempo of engraftment, acute graft-versus-host-disease (GVHD), clinical extensive chronic GVHD, overall survival, and disease relapse. Neither acute GVHD, overall survival, nor disease relapse was statistically significantly associated with CD34, CD3, or CD14 cell doses or the CD14 to CD3 ratio. CD3 and CD14 cell doses and CD14 to CD3 ratios did not correlate with the tempo of neutrophil and platelet engraftment. However, increasing CD34 cell numbers were significantly associated with accelerated neutrophil (P =.03) and platelet (P =.01) engraftment. Higher doses of CD34 cells (> 8.0 x 10(6)/kg) were also associated with a significantly increased hazard of clinical extensive chronic GVHD (HR = 2.3, 95% confidence interval [CI] 1.4-3.7, P =.001), but neither CD3 nor CD14 doses were statistically significantly associated with chronic GVHD. It was concluded that CD34 cell dose in G-PBMC grafts appears to affect both the engraftment kinetics and the development of clinical extensive chronic GVHD in HLA-identical sibling recipients but without a demonstrable impact on survival, relapse, and acute GVHD. Given the morbidity associated with extensive chronic GVHD, efforts to further accelerate engraftment in HLA-matched sibling transplants by increasing CD34 cell number in G-PBMC products may be counterproductive.
Asunto(s)
Antígenos CD34/análisis , Enfermedad Injerto contra Huésped/epidemiología , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Histocompatibilidad , Enfermedad Aguda , Complejo CD3/análisis , Recuento de Células , Enfermedad Crónica , Femenino , Antígenos HLA/análisis , Neoplasias Hematológicas/terapia , Células Madre Hematopoyéticas/inmunología , Humanos , Cinética , Recuento de Leucocitos , Receptores de Lipopolisacáridos/análisis , Masculino , Neutrófilos , Núcleo Familiar , Recuento de Plaquetas , Recurrencia , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
A prospective randomized trial has shown that there is a survival advantage for allogeneic transplant recipients who received granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood mononuclear cells (GPBMC) versus those who received bone marrow (BM) as a source of stem cells. The biological basis for this advantage is not clear and may be attributable to qualitative as well as quantitative differences in the CD34 cells, T cells, and/or the monocytes transplanted. To begin to address this issue, gene expression patterns in CD34 cells isolated from these 2 stem cell sources were compared to identify functional pathways that may distinguish these 2 populations. CD34 cells were isolated to purity from the BM and peripheral blood stem cells of multiple healthy donors. (The complete data set will be available at http://parma.fhcrc.org/lgraf upon publication.) Two separate RNA preparations from pooled samples from both sources were analyzed by Affymetrix Oligonucleotide Array chips for expression of over 6400 human genes. Comparative analyses among the samples showed that a small set of 28 sequences increased and 38 sequences decreased in expression more than 3-fold in both of the GPBMC samples compared to those in BM samples. More highly expressed genes include several for nuclear proteins and transcriptional factors. Functional categorization of the genes decreased in expression indicated sequences influential in cell cycle progression, in agreement with the recognized quiescence of circulating CD34 cells. Multiple transcriptional regulators and chemokines were also found to be decreased. These data emphasize that in addition to increased numbers of CD34 cells, G-CSF mobilization also results in significant qualitative changes. Whether they impact engraftment remains to be determined.
Asunto(s)
Células Sanguíneas/metabolismo , Células de la Médula Ósea/metabolismo , Perfilación de la Expresión Génica/métodos , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Adulto , Antígenos CD34/análisis , Biotinilación , Células de la Médula Ósea/efectos de los fármacos , ADN Complementario/genética , Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/clasificación , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , ARN Mensajero/genética , Técnica de SustracciónAsunto(s)
Anemia Aplásica/virología , Proteínas del Envoltorio Viral/metabolismo , Anemia Aplásica/epidemiología , Anemia Aplásica/etiología , Médula Ósea/virología , Citomegalovirus/química , ADN Viral/análisis , ADN Viral/metabolismo , Humanos , Prevalencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
Clinical observations show that older patients do not tolerate high-dose chemoradiotherapy as well as younger patients. It is unclear whether this is due to age-related differences in their responses to hematopoietic injury or to differential toxicities to other organs. In the present study, 6 young (0.5 years) and 6 elderly (8 years) dogs were challenged with 7 repeated nonlethal doses of 50 or 100 cGy total body irradiation (TBI) each (total 550 cGy), and 21 days of recombinant canine granulocyte-colony stimulating factor (rcG-CSF) after the last TBI dose. Recoveries of absolute neutrophil, platelet, and lymphocyte counts after each TBI dose, responses to rcG-CSF treatment, and telomere lengths in neutrophils were compared before and after the study. No differences were found in recoveries of neutrophils, platelets, or in responses to rcG-CSF among young and old dogs. In contrast, recoveries were suggestively worse in younger dogs. After rcG-CSF, platelet recoveries were poor in both groups compared with previous platelet recoveries (P <.01). Consequently, 2 old and 3 young dogs were euthanized because of persistent thrombocytopenia and bleeding. At the study's completion, marrow cellularities and peripheral blood counts of the remaining young and elderly dogs were equivalent. The telomere lengths in both groups were significantly reduced after the study versus beforehand (P =.03), but the median attritions of telomeres were not different. It was concluded that aging does not appear to affect hematopoietic cell recoveries after repeated low-dose TBI, suggesting that poor tolerance of radiochemotherapy regimens in older patients may be due to nonhematopoietic organ toxicities rather than age-related changes in hematopoietic stem cells reserves.
Asunto(s)
Envejecimiento , Hematopoyesis , Irradiación Corporal Total/efectos adversos , Animales , Células de la Médula Ósea/citología , Perros , Factor Estimulante de Colonias de Granulocitos/farmacología , Recuento de Leucocitos , Recuento de Linfocitos , Neutrófilos/ultraestructura , Recuento de Plaquetas , Proteínas Recombinantes , Estrés Fisiológico/sangre , Estrés Fisiológico/etiología , Telómero/ultraestructura , Trombocitopenia/etiologíaRESUMEN
Toxicities have limited the use of allogeneic hematopoietic cell transplantation (HCT) to younger, medically fit patients. In a canine HCT model, a combination of postgrafting mycophenolate mofetil (MMF) and cyclosporine (CSP) allowed stable allogeneic engraftment after minimally toxic conditioning with low-dose (200 cGy) total-body irradiation (TBI). These findings, together with the known antitumor effects of donor leukocyte infusions (DLIs), led to the design of this trial. Forty-five patients (median age 56 years) with hematologic malignancies, HLA-identical sibling donors, and relative contraindications to conventional HCT were treated. Immunosuppression involved TBI of 200 cGy before and CSP/MMF after HCT. DLIs were given after HCT for persistent malignancy, mixed chimerism, or both. Regimen toxicities and myelosuppression were mild, allowing 53% of eligible patients to have entirely outpatient transplantations. Nonfatal graft rejection occurred in 20% of patients. Grades II to III acute graft-versus-host disease (GVHD) occurred in 47% of patients with sustained engraftment. With median follow-up of 417 days, survival was 66.7%, nonrelapse mortality 6.7%, and relapse mortality 26.7%. Fifty-three percent of patients with sustained engraftment were in complete remission, including 8 with molecular remissions. This novel allografting approach, based on the use of postgrafting immunosuppression to control graft rejection and GVHD, has dramatically reduced the acute toxicities of allografting. HCT with the induction of potent graft-versus-tumor effects can be performed in previously ineligible patients, largely in an outpatient setting. Future protocol modifications should reduce rejection and GVHD, thereby facilitating studies of allogeneic immunotherapy for a variety of malignancies. (Blood. 2001;97:3390-3400)
Asunto(s)
Envejecimiento , Efecto Injerto vs Tumor , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas , Adulto , Anciano , Causas de Muerte , Ciclosporina/administración & dosificación , Ciclosporina/efectos adversos , Ciclosporina/uso terapéutico , Femenino , Rechazo de Injerto , Prueba de Histocompatibilidad , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/uso terapéutico , Leucemia Linfocítica Crónica de Células B/terapia , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Leucemia Mieloide Aguda/terapia , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapia , Ácido Micofenólico/administración & dosificación , Ácido Micofenólico/efectos adversos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapéutico , Neutrófilos , Recuento de Plaquetas , Inducción de Remisión , Tasa de Supervivencia , Linfocitos T/inmunología , Acondicionamiento Pretrasplante , Irradiación Corporal Total/efectos adversosRESUMEN
Stable mixed donor/host hematopoietic chimerism can be uniformly established in dogs conditioned with 200 cGy TBI before dog leukocyte antigen (DLA)-identical marrow transplantation and immunosuppressed with a short course of mycophenolate mofetil (MMF) and cyclosporine (CSP) after the transplantation. A further decrease in the TBI dose to 100 cGy or the elimination of MMF in this model results in graft rejection. Here we asked whetherthe addition of G-CSF-mobilized peripheral blood mononuclear cells (G-PBMC) to marrow grafts would enhance donor engraftment in dogs conditioned with 100 cGy TBI and given postgrafting immunosuppression with CSP alone. Using this model, 7 of 9 dogs given only marrow cells rejected their grafts within 8 to 17 weeks after transplantation. In contrast, the addition of unmodified G-PBMC to marrow grafts resulted in stable mixed donor/host chimerism in 5 of 8 dogs studied (P = .06). However, addition of the CD3-depleted fraction of G-PBMC, which contained both CD34 cells and CD14 cells, resulted in engraftment in only 1 of 7 recipients. We conclude that adding G-PBMC to marrow grafts replaced the requirement of MMF and 100 cGy of TBI, and that CD3 cells were required to facilitate engraftment of marrow cells in DLA-identical recipients, whereas the additional CD34 cells present in G-PBMC were not sufficient for this effect.
Asunto(s)
Trasplante de Médula Ósea/métodos , Complejo CD3 , Movilización de Célula Madre Hematopoyética/métodos , Leucocitos Mononucleares/trasplante , Ácido Micofenólico/análogos & derivados , Acondicionamiento Pretrasplante/métodos , Animales , Antígenos CD34 , Complejo CD3/sangre , Ciclosporina/administración & dosificación , Perros , Supervivencia de Injerto , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Inmunosupresores/administración & dosificación , Leucocitos Mononucleares/efectos de los fármacos , Receptores de Lipopolisacáridos , Modelos Animales , Ácido Micofenólico/administración & dosificación , Quimera por Trasplante , Irradiación Corporal TotalRESUMEN
Laminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed alpha, beta, and gamma chains. Combinations of 11 characterized laminin subunits (alpha 1-alpha 5, beta 1-beta 3, and gamma 1-gamma 3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase-polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34(+) cell lines. Antibodies against the beta 1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells. (Blood. 2000;96:4194-4203)
Asunto(s)
Médula Ósea/química , Laminina/aislamiento & purificación , Isoformas de Proteínas/aislamiento & purificación , Western Blotting , Células de la Médula Ósea/química , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Sueros Inmunes , Integrina beta1/inmunología , Integrina beta1/metabolismo , Laminina/química , Laminina/genética , Laminina/farmacología , Mitosis/efectos de los fármacos , Familia de Multigenes , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Subunidades de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/químicaRESUMEN
Cord blood (CB) stem cell transplantations have been associated with delayed hematopoietic engraftment. This has most likely been due to the limited numbers of hematopoietic short-term repopulating cells in CB. Ex vivo expansion of CB has been attempted, and expansion of CD34-enriched CB has been successful; however, CD34 enrichment procedures are in general associated with substantial cell loss. Thus, we have studied culture conditions for expansion of nonenriched CB. Nonenriched CB cells were cultured for 21 days in the presence of conditioned medium from the HS-5 stromal cell line and FLT3-L or alternatively in the presence of FLT3-L, stem cell factor (SCF), megakaryocite growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF) (FSMG), either on fibronectin fragment CH-296-coated dishes or on uncoated dishes. With all four culture conditions, the number of mononuclear cells initially decreased until day 7 and then increased until the end of the expansion cultures. Overall expansion using HS-5 and FLT3-L resulted in superior expansion of MNC and CFU-C (44-/34-fold) for both cultures with and without CH-296 compared to FSMG (18-/17-fold). Expansion on CH-296 was less efficient than expansion on tissue culture-treated wells without CH-296 for both conditions. We then studied the best time for transduction on nonenriched CB. In contrast to enriched CD34 cells, we found for both conditions, HS-5/FLT3-L and growth factor cocktail, higher transduction efficiencies when cells were transduced on day 7 as compared to day 2. Gene transfer rates up to 45% were achieved with both conditions, which corresponded with the increased number of cells in S phase on day 7 compared to day 2. We conclude that HS-5 and FLT-3L allow efficient expansion and transduction of nonenriched CB.
Asunto(s)
Ciclo Celular/fisiología , Sangre Fetal/citología , Sustancias de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/farmacología , Técnicas de Cultivo de Célula/métodos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos/farmacología , Proteínas Fluorescentes Verdes , Hematopoyesis , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Recién Nacido , Kanamicina Quinasa/análisis , Kanamicina Quinasa/genética , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Factor de Células Madre/farmacología , Células del Estroma/citología , Trombopoyetina/farmacología , TransfecciónRESUMEN
Donor-derived hematopoiesis was assessed in 17 patients who received allogeneic marrow grafts from HLA-matched siblings between 1971 and 1980. Complete blood counts were normal or near normal in all patients except one. Chimerism analyses, using either dual-color XY-chromosome fluorescence in situ hybridization (FISH) or analysis of variable number tandem repeat loci, indicated that 15 out of 16 patients had greater than 97% donor-derived hematopoiesis, whereas 1 patient had indeterminate chimerism. All 12 recipients of grafts from female donors exhibited polyclonal hematopoiesis by X-linked clonal analysis with the use of molecular probes. Of the 17 recipients, 9 exhibited a less than 1.0-kilobase shortening of granulocyte telomere length compared with their respective donors, according to terminal restriction fragment analysis or flow-FISH with a fluorescein-labeled peptide nucleic acid probe. These data suggest that under standard transplantation conditions, the stem cell proliferative potential is not compromised during hematopoietic reconstitution. (Blood. 2000;96:3991-3994)
Asunto(s)
Trasplante de Médula Ósea , Hematopoyesis/fisiología , Telómero/ultraestructura , Adolescente , Adulto , Trasplante de Médula Ósea/normas , División Celular/fisiología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Granulocitos/ultraestructura , Humanos , Hibridación Fluorescente in Situ , Masculino , Núcleo Familiar , Polimorfismo de Longitud del Fragmento de Restricción , Factores Sexuales , Quimera por Trasplante , Trasplante Homólogo , Cromosoma X/ultraestructuraRESUMEN
The gradual disappearance of host antidonor isohemagglutinins after major ABO-mismatched hematopoietic stem cell (HSC) allografts has been attributed to the gradual destruction of host plasma cells by graft-versus-host effects. To corroborate this hypothesis, we retrospectively analyzed results from 383 major or major/minor ABO-mismatched unrelated and related HSC allografts performed between 1983 and 1998. All patients were conditioned by high-dose pretransplant therapy and given methotrexate/cyclosporine for graft-versus-host disease (GvHD) prophylaxis. Of the 383 patients, 155 had HLA-matched related and 228 had unrelated grafts. We asked whether unrelated recipients experienced a more rapid disappearance of isohemagglutinins than related recipients, and whether, within the groups of related and unrelated recipients, the titer disappeared faster in patients with GvHD than in those without GvHD. The median time to reach undetectable antidonor IgG and IgM titers was significantly shorter in unrelated recipients (46 versus 61 days; P =.016). In addition, related recipients with GvHD had a 2. 2-fold increased likelihood (1.12-4.39,95% CI; P =.02) of reaching undetectable titers within 100 days than patients without GvHD. The persistence of antidonor isohemagglutinins led to significantly increased red blood cell (RBC) transfusion requirements in the ABO-mismatched related patients compared with ABO-matched counterparts. However, time to neutrophil and platelet engraftment, incidence of GvHD, and survival were not influenced by ABO incompatibility. In conclusion, our results corroborate the hypothesis that the rate of disappearance of antidonor isohemagglutinins after ABO-mismatched allogeneic HSC grafts is influenced by the degree of genetic disparity between donor and recipient, suggesting a graft-versus-plasma cell effect.
Asunto(s)
Tipificación y Pruebas Cruzadas Sanguíneas , Trasplante de Médula Ósea , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Hemaglutininas/inmunología , Adolescente , Adulto , Anciano , Biomarcadores , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/sangre , Hemaglutininas/sangre , Humanos , Lactante , Masculino , Persona de Mediana Edad , Células Plasmáticas/inmunología , Valor Predictivo de las Pruebas , Trasplante HomólogoRESUMEN
A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1alpha (IL-1alpha) and IL-1beta completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1beta was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1-treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 +/- 3 v 151 +/- 12/well, respectively; GFP intensity was 535 +/- 165 v 6,516 +/- 652/well, respectively, in 4 separate experiments). At day 21, when IL-1beta-treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1beta, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1beta effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1-induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1beta-treated and nontreated HS-27a cells has identified some candidate molecules.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Interleucina-1/farmacología , Replicación Viral/efectos de los fármacos , Células de la Médula Ósea/virología , Células Cultivadas , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/fisiología , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/genética , Regiones Promotoras Genéticas , Células del Estroma/efectos de los fármacos , Células del Estroma/virologíaRESUMEN
Cloned human stromal cell lines representing functionally distinct cellular components of the marrow microenvironment were generated to serve as tools for identifying gene products that regulate hematopoiesis. Oligonucleotide arrays, or "gene chips" were used to provide a comprehensive comparison of gene expression among the cell lines. One line, designated HS-5, was found to secrete large amounts of cytokines, and conditioned media from this line was found to support the ex vivo expansion of both immature and mature progenitors. In contrast, a second line, designated HS-27a, does not secrete known cytokines but does support cobblestone area formation by CD34+/38lo cells. HS-27a, but not HS-5, was also found to express hJagged1, a ligand for Notch1, which may function to influence cell fate decisions of hematopoietic precursors. Both cell lines are currently being used to identify other gene products that regulate hematopoiesis and to generate reagents that will allow more formal evaluation of the putative role of hJagged1 in hematopoietic cell fate decisions.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Citocinas/genética , Regulación de la Expresión Génica , Animales , Línea Celular , Células Clonales , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Células del Estroma/citología , Células del Estroma/fisiologíaAsunto(s)
Células de la Médula Ósea/citología , Células Madre Hematopoyéticas/citología , Leucopoyesis , Proteínas/fisiología , Células del Estroma/citología , Antígenos CD/biosíntesis , Antígenos CD11/biosíntesis , Proteínas de Unión al Calcio , Comunicación Celular , Línea Celular , Células Madre Hematopoyéticas/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Leucocitos/citología , Proteínas de la Membrana , Proteínas/genética , Proteínas Serrate-Jagged , Células U937RESUMEN
A 50-year-old woman who was retrospectively diagnosed with an early asymptomatic myelodysplastic syndrome (MDS) served as a haemopoietic stem cell donor for her HLA-identical sister who had chemotherapy-refractory non-Hodgkin's lymphoma. The MDS of the donor was classified as refractory anaemia (RA) and cytogenetically characterized by deletion of the long arm of chromosome 20 [del(20q)]. Donor cell engraftment in marrow and peripheral blood was analysed over a period of 5 months after transplant using conventional cytogenetics, fluorescence in situ hybridization, and variable number of tandem repeats. Neutrophil counts >0.5 x 109/l and platelet counts >20 x 109/l were reached promptly on days 12 and 24, respectively. Throughout the period of observation the percentage of cells with the del(20q) abnormality in the recipient's marrow and peripheral blood was comparable to the proportion of these cells in the donor. These data indicate that the abnormal clone was capable of homing to the marrow, proliferating, differentiating, and therefore contributing to haemopoiesis in a relatively efficient manner. This implies that MDS progenitor cells may not have homing and growth deficiencies, a finding that has particular relevance for autologous transplantation in MDS patients where tumour cells potentially contaminate the graft.
Asunto(s)
Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas/métodos , Síndromes Mielodisplásicos/sangre , Adulto , Células de la Médula Ósea/patología , Diferenciación Celular , División Celular , Deleción Cromosómica , Cromosomas Humanos Par 20/genética , Células Clonales , Femenino , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Donantes de TejidosRESUMEN
Myelodysplastic syndromes (MDS) caused by a clonal hematopoietic stem cell disorder progress to either overt leukemia or cytopenia, which leads to lethal infection or bleeding. Although several clinical trials have attempted to reverse cytopenia by using hematopoietic growth factors (HGF), success has been limited due in part to a limited understanding of the role of HGF in MDS progression. The FLT3 ligand, which binds to and activates the FLT3 receptor, does not have a stimulatory effect on hematopoietic cells, but can synergize with other HGF to support the expansion of both immature and committed progenitors. Using ELISA technology we measured endogenous serum levels in 93 patients with MDS: 29 RA, 1 RARS, 31 RAEB, 23 RAEBt, 9 CMML. 48.3% of RA patients' sera had significantly elevated FLT3 ligand levels ranging from 404 to 5735 pg/ml, whereas none of the RAEB, RAEBt, or CMML patients sera had levels different from controls. No significant correlation was found between FLT3 ligand levels and peripheral blood counts, bone marrow cellularity, age, cytogenetic abnormalities, or survival. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS, which may represent an appropriate response to a decreased number of normal progenitors, or alternatively a dysregulated HGF system.
Asunto(s)
Proteínas de la Membrana/sangre , Síndromes Mielodisplásicos/sangre , Adolescente , Adulto , Anciano , Anemia Refractaria/sangre , Anemia Refractaria/patología , Anemia Refractaria con Exceso de Blastos/sangre , Anemia Refractaria con Exceso de Blastos/patología , Biomarcadores , Biomarcadores de Tumor/sangre , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Hematopoyesis , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/complicaciones , Síndromes Mielodisplásicos/patología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/fisiología , Factor de Células Madre/fisiología , Tirosina Quinasa 3 Similar a fmsRESUMEN
Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) have been used increasingly to reconstitute hematopoiesis after myeloablative therapy in allogeneic transplantation. Compared with conventional bone marrow, faster engraftment is consistently observed with G-PBMC, with differences more pronounced in platelet than in neutrophil recovery. G-PBMC contain not only severalfold more CD34+ cells than bone marrow but also, on average, 50-fold more monocytes, which may stimulate stromal cell function and facilitate engraftment. Although G-PBMC also contain 10-fold more T cells, the incidence and severity of acute graft-vs.-host disease (GVHD) is no higher than that observed in allogeneic bone marrow transplantation. Hypothetically, these clinical observations can be explained by the direct effect of G-CSF on T cell function as demonstrated by polarization of T cells expressing the T helper type 2 (Th 2) cytokine interleukin (IL)-4 in the murine model. Alternatively, G-PBMC may contain cells that actively suppress donor T cell responsiveness. Recent reports indicate that the large number of CD14+ monocytes in G-PBMC can suppress donor T cell proliferation in vitro. This effect may be attributable to both the increased ratio of CD14+:CD3+ cells in G-PBMC and the evidence that CD14+ cells in G-PBMC have decreased expression of both B7.2 and HLA-DR. There is some indication that natural killer (NK) cell number and function may be augmented in G-PBMC, which could have a favorable impact on the graft-vs.-leukemia (GVL) effect. Therefore, both the CD34+ and accessory cell content of G-PBMC may be important in early engraftment by controlling acute GVHD and facilitating GVL.
Asunto(s)
Células Presentadoras de Antígenos/trasplante , Trasplante de Células Madre Hematopoyéticas , Células Presentadoras de Antígenos/clasificación , Células Presentadoras de Antígenos/inmunología , Células de la Médula Ósea/citología , Recuento de Células , Enfermedad Injerto contra Huésped/epidemiología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Inmunofenotipificación , Incidencia , Monocitos/clasificación , Subgrupos de Linfocitos T/trasplante , Trasplante Homólogo/efectos adversosRESUMEN
The application of ex vivo expansion to cell products pharmacologically purged in vitro may provide sufficient numbers of cells for rapid engraftment in a product with reduced tumor burden. To pursue this possibility we evaluated the effect of 4-hydroperoxycyclophosphamide (4-HC) treatment on granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSC) and their subsequent expansion potential. A small number of G-PBSC CD34+ cells are resistant to 4-HC and are phenotypically and functionally immature. 4-HC-resistant G-PBSC cells are CD34+ bright, CD38+/-, DR(lo), CD13(lo), CD33-, CD71-, and rhodamine dull. In six experiments, treating G-PBSC with 60 microg/mL of 4-HC at 37 degrees C for 30 minutes reduced the number of colony-forming units (CFUs) per 5000 CD34+ cells by 96.3% (from 1333 +/- 137 to 46.5 +/- 11). This purging also reduced the frequency of 5-week long-term culture initiating cells (LTC-ICs) from 1/39 (range 1/27 to 1/62) to <1/1680 (range 1/1180 to 1/2420). Ex vivo expansion cultures were used to compare the proliferative potential of treated and untreated CD34+ cells. These cells were cultured with either the HS-5 stromal cell line serum-deprived conditioned media supplemented with 10 ng/mL kit ligand (HS-5CM/KL) or a recombinant growth factor mix (GFmix) containing 10 ng/mL each of interleukin (IL)-1, IL-3, IL-6, KL, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and 3 U/mL of erythropoietin. Culturing untreated CD34+ G-PBSC with 10% HS-5CM/KL increased total nucleated cells by 460-fold after 15 days. Progenitors, which were measured as CFUs, also increased by 47-fold over the same period. More significantly, culturing the 4-HC-treated CD34+ cells with HS-5/KL increased CFUs 98-fold and the nucleated cells increased 4573-fold. The absolute number of CFUs present after expansion of the 4-HC-resistant cells with HS-5CM/KL was threefold higher than that detected before purging and significantly higher than that obtained with GFmix. These data indicate that G-PBSC contain a very immature pool of cells not detectable using the 5-week LTC-IC assay, but have extremely high proliferative potential. Additionally, pharmacological purging of G-PBSC greatly reduces mature cells while retaining an immature population. Also significant is the finding that supernatant from the HS-5 bone marrow stromal cell line plus KL can fully regenerate progenitors from the 4-HC-resistant CD34+ G-PBSC.