RESUMEN
The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. The SERCA-LVAD trial was one of a program of AAV1/SERCA2a cardiac gene therapy trials including CUPID1, CUPID 2 and AGENT trials. Enroled subjects were randomised to receive a single intracoronary infusion of 1 × 1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients at 3 years follow up, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort which may help guide future trial design.
Asunto(s)
Insuficiencia Cardíaca , Corazón Auxiliar , Adulto , Estudios de Factibilidad , Terapia Genética , Vectores Genéticos/genética , Insuficiencia Cardíaca/terapia , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Adeno-associated virus serotype 1 (AAV1) has many advantages as a gene therapy vector, but the presence of pre-existing neutralizing antibodies (NAbs) is an important limitation. This study was designed to determine: (1) characteristics of AAV NAbs in human subjects, (2) prevalence of AAV1 NAbs in heart failure patients and (3) utility of aggressive immunosuppressive therapy in reducing NAb seroconversion in an animal model. NAb titers were assessed in a cohort of heart failure patients and in patients screened for a clinical trial of gene therapy with AAV1 carrying the sarcoplasmic reticulum calcium ATPase gene (AAV1/SERCA2a). AAV1 NAbs were found in 59.5% of 1552 heart failure patients. NAb prevalence increased with age (P=0.001) and varied geographically. The pattern of NAb titers suggested that exposure is against AAV2, with AAV1 NAb seropositivity due to crossreactivity. The effects of immunosuppression on NAb formation were tested in mini-pigs treated with immunosuppressant therapy before, during and after a single AAV1/SERCA2a infusion. Aggressive immunosuppression did not prevent formation of AAV1 NAbs. We conclude that immunosuppression is unlikely to be a viable solution for repeat AAV1 dosing. Strategies to reduce NAbs in heart failure patients are needed to increase eligibility for gene transfer using AAV vectors.
Asunto(s)
Anticuerpos Antivirales/inmunología , Dependovirus/genética , Dependovirus/inmunología , Vectores Genéticos/inmunología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Animales , Anticuerpos Neutralizantes/inmunología , Terapia Genética , Humanos , Modelos Animales , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Porcinos , Porcinos EnanosRESUMEN
We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.
Asunto(s)
Formación de Anticuerpos , Genes de Inmunoglobulinas , Transgenes , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Diversidad de Anticuerpos , Linfocitos B/citología , Linfocitos B/inmunología , Cromosomas Artificiales de Levadura/genética , Receptores ErbB/inmunología , Reordenamiento Génico de Linfocito B , Humanos , Hibridomas/inmunología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Interleucina-8/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de la Especie , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Interactions between products of the mouse W locus, which encodes the c-kit tyrosine kinase receptor, and the Sl locus, which encodes a ligand for c-kit receptor, which we have designated stem cell factor (SCF), have a critical role in the development of mast cells. Mice homozygous for mutations at either locus exhibit several phenotypic abnormalities including a virtual absence of mast cells. Moreover, the c-kit ligand SCF can induce the proliferation and maturation of normal mast cells in vitro or in vivo, and also can result in repair of the mast cell deficiency of Sl/Sld mice in vivo. We now report that administration of SCF intradermally in vivo results in dermal mast cell activation and a mast cell-dependent acute inflammatory response. This effect is c-kit receptor dependent, in that it is not observed when SCF is administered to mice containing dermal mast cells expressing functionally inactive c-kit receptors, is observed with both glycosylated and nonglycosylated forms of SCF, and occurs at doses of SCF at least 10-fold lower on a molar basis than the minimally effective dose of the classical dermal mast cell-activating agent substance P. These findings represent the first demonstration in vivo that a c-kit ligand can result in the functional activation of any cellular lineage expressing the c-kit receptor, and suggest that interactions between the c-kit receptor and its ligand may influence mast cell biology through complex effects on proliferation, maturation, and function.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/farmacología , Mastocitos/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Animales , Trasplante de Médula Ósea/fisiología , Células CHO , Cricetinae , Escherichia coli/genética , Fibrina/metabolismo , Factores de Crecimiento de Célula Hematopoyética/genética , Inflamación , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos , Ratones Mutantes , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit , Ratas , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes/farmacología , Factor de Células Madre , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Gene modification of hematopoietic stem cells (HSC) with antigen-specific, chimeric, or "universal" immune receptors (URs) is a novel but untested form of targeted immunotherapy. A human immunodeficiency virus (HIV) envelope-specific UR consisting of the extracellular domain of human CD4 linked to the zeta chain of the T cell receptor (CD4 zeta) was introduced ex vivo into murine HSC by retroviral transduction. After transplantation into immunodeficient SCID mice, sustained high level expression of CD4 zeta was observed in circulating myeloid and natural killer cells. CD4 zeta-transplanted mice were protected from challenge with a lethal dose of a disseminated human leukemia expressing HIV envelope. These results demonstrate the ability of chimeric receptors bearing zeta-signaling domains to activate non-T cell effector populations in vivo and thereby mediate systemic immunity.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Antígenos CD4/inmunología , Productos del Gen env/inmunología , VIH/inmunología , Proteínas de la Membrana/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Línea Celular , Citotoxicidad Inmunológica , Cartilla de ADN , Femenino , Productos del Gen env/biosíntesis , Supervivencia de Injerto/inmunología , Humanos , Masculino , Ratones , Ratones SCID , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa , Trasplante Heterólogo/inmunologíaRESUMEN
Mast cell development is a complex process that results in the appearance of phenotypically distinct populations of mast cells in different anatomical sites. Mice homozygous for mutations at the W or S1 locus exhibit several phenotypic abnormalities, including a virtual absence of mast cells in all organs and tissues. Recent work indicates that W encodes the c-kit tyrosine kinase receptor, whereas S1 encodes a c-kit ligand that we have designated stem cell factor (SCF). Recombinant or purified natural forms of the c-kit ligand induce proliferation of certain mast cell populations in vitro, and injection of recombinant SCF permits mast cells to develop in mast cell-deficient WCB6F1-S1/S1d mice. However, the effects of SCF on mast cell proliferation, maturation, and phenotype in normal mice in vivo were not investigated. We now report that local administration of SCF in vivo promotes the development of connective tissue-type mast cells (CTMC) in the skin of mice and that systemic administration of SCF induces the development of both CTMC and mucosal mast cells (MMC) in rats. Rats treated with SCF also develop significantly increased tissue levels of specific rat mast cell proteases (RMCP) characteristic of either CTMC (RMCP I) or MMC (RMCP II). These findings demonstrate that SCF can induce the expansion of both CTMC and MMC populations in vivo and show that SCF can regulate at least one cellular lineage that expresses c-kit, the mast cell, through complex effects on proliferation and maturation.
Asunto(s)
Células del Tejido Conectivo , Endopeptidasas/metabolismo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Mastocitos/citología , Piel/citología , Animales , División Celular , Femenino , Genotipo , Homocigoto , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Ratones , Ratones Mutantes , Membrana Mucosa/citología , Especificidad de Órganos , Ratas , Ratas Endogámicas , Proteínas Recombinantes/farmacología , Piel/efectos de los fármacos , Factor de Células MadreRESUMEN
Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.
Asunto(s)
Factores Estimulantes de Colonias/farmacología , Granulocitos/fisiología , Leucemia/patología , Proteínas Recombinantes/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/genética , ADN/metabolismo , Escherichia coli/genética , Genes , Factor Estimulante de Colonias de Granulocitos , Humanos , Leucemia Mieloide/patología , Ratones , PlásmidosRESUMEN
Mast cell development in mice is critically regulated by stem cell factor (SCF), the term used here to designate a product of fibroblasts and other cell types that is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit. However, the factors which regulate the size of mast cell populations in primates are poorly understood. Here we report that the subcutaneous administration of recombinant human SCF (rhSCF) to baboons (Papio cynocephalus) or cynomolgus monkeys (Macaca fascicularis) produced a striking expansion of mast cell populations in many anatomical sites, with numbers of mast cells in some organs of rhSCF-treated monkeys exceeding the corresponding values in control monkeys by more than 100-fold. Animals treated with rhSCF did not exhibit clinical evidence of mast cell activation, and discontinuation of treatment with rhSCF resulted in a rapid decline of mast cell numbers nearly to baseline levels. These findings are the first to demonstrate that a specific cytokine can regulate mast cell development in primates in vivo. They also provide the first evidence, in any mammalian species, to indicate that the cytokine-dependent expansion of tissue mast cell populations can be reversed when administration of the cytokine is discontinued.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/farmacología , Mastocitos/citología , Animales , Esquema de Medicación , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/administración & dosificación , Humanos , Inyecciones Subcutáneas , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Yeyuno/citología , Yeyuno/efectos de los fármacos , Pulmón/citología , Pulmón/efectos de los fármacos , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Macaca fascicularis , Mastocitos/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Especificidad de Órganos , Papio , Proto-Oncogenes Mas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Factor de Células Madre , Estómago/citología , Estómago/efectos de los fármacos , Tráquea/citología , Tráquea/efectos de los fármacosRESUMEN
Accumulating evidence suggests that c-kit and its ligand, stem cell factor (SCF), play an important role in the regulation of at least three lineages of stem cell growth and possibly in leukemogenesis, while only limited data are available that suggest possible involvement of c-kit/SCF in the development of human solid tumors such as lung cancer. We have recently reported that c-kit is aberrantly expressed almost exclusively in small-cell lung cancer (SCLC) among various types of solid tumors. The present study revealed that c-kit protein ectopically expressed in SCLC is indistinguishable from that in leukemia cell lines with megakaryocytic characteristics with respect to amount, molecular size, and autophosphorylation status in response to recombinant human SCF. Furthermore, significant chemotactic response as well as moderate in vitro cell growth was induced in SCLC cell lines by the addition of recombinant human SCF, suggesting that c-kit/SCF may play an important biological role in the development of SCLC. Our extensive search for activating mutations naturally occurring in the c-kit gene revealed an amino acid substitution in the transmembrane domain of an SCLC cell line, although the functional consequences of this variant allele are yet to be determined.
Asunto(s)
Carcinoma de Células Pequeñas/genética , Quimiotaxis/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/farmacología , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas/análisis , Carcinoma de Células Pequeñas/química , Carcinoma de Células Pequeñas/patología , División Celular/efectos de los fármacos , Humanos , Leucemia/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-kit , Proteínas Recombinantes/farmacología , Análisis de Secuencia , Factor de Células Madre , Células Tumorales CultivadasRESUMEN
Stem cell factor (SCF) is a new growth factor acting on early hematopoietic progenitor and stem cells. In our experiments human recombinant SCF stimulated short-term proliferation of accessory cell-depleted acute myeloid leukemia (AML) cells in 13/14 cases, as determined by 3H-thymidine (3H-TdR) incorporation and cell counts. Stimulatory activity was significantly greater than in the presence of GM-CSF and was comparable to that of granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and 5637 cell line supernatant (SN). Conversely, the ability of SCF to induce primary colony formation by AML clonogenic cells (CFU-L) was lower than that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 5637 SN in all but four cases. However, SCF potentiated the stimulatory effect of GM-CSF, G-CSF, and IL-3 on both 3H-TdR incorporation and colony formation. In a 7-day liquid culture SCF enhanced CFU-L recovery in all cases to a significantly greater extent than the other growth factors. A further increment was obtained by combinations of SCF with GM-CSF, G-CSF, or IL-3, and this was significantly more effective than 5637 SN. SCF did not induce leukemic cell differentiation. Human recombinant SCF is therefore highly efficient in stimulating AML cell proliferation and expanding the CFU-L pool. It was not, however, able to support long-term growth of AML cells (beyond 2-7 weeks) in five cases tested.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/farmacología , Leucemia Mieloide/patología , Antígenos de Diferenciación Mielomonocítica/análisis , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Medios de Cultivo , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Técnicas In Vitro , Interleucina-3/farmacología , Proteínas Recombinantes , Factor de Células Madre , Células Tumorales CultivadasRESUMEN
The M07e megakaryoblastic leukemia cell line is strictly dependent on either interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for continuous growth. This study shows that recombinant human stem-cell factor (rhSCF) can completely replace these lymphokines in supporting the continued propagation of M07e cells mostly by eliciting GM-CSF secretion in this target. In fact, in short-term proliferation assays the stimulatory activity of SCF is blocked about 75% by a GM-CSF-specific serum. In addition, we could detect GM-CSF expression by SCF-stimulated M07e cells, both at the protein and mRNA levels. In contrast, SCF does not induce transcripts for any other cytokine to which M07e cells are responsive, including IL-2, IL-3, IL-4, and IL-6. Overall, these data show that the ability of SCF to support the growth of this megakaryocytic cell line is mediated mostly by the induction of an autocrine loop of activation involving GM-CSF production. The finding that SCF can stimulate GM-CSF secretion also in an IL-2-dependent T-lymphoblastic leukemia cell line indicates that SCF can act on cells of both myeloid and lymphoid lineages, and that the ability to induce cytokines in target cells represents an important aspect of its mechanism of action.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Leucemia Megacarioblástica Aguda/patología , División Celular/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia de Células T/metabolismo , Leucemia de Células T/patología , Proteínas Recombinantes/farmacología , Factor de Células Madre , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Thrombocytopenia is an important clinical problem for many acquired immunodeficiency syndrome (AIDS) patients. Recently, the utility of recombinant cytokines in alleviating the hematopoietic complications of AIDS and AIDS therapy has been evaluated. The newly cloned cytokine stem cell factor (SCF) has been demonstrated to be a potent regulator of hematopoietic progenitor cell proliferation. Therefore, we evaluated the ability of SCF to alleviate thrombocytopenia caused by infection with LP-BM5 murine leukemia virus (mLV) in a murine model of AIDS (MAIDS). In addition, we evaluated the effects of SCF on previously demonstrated azidothymidine (AZT)-induced elevations of platelet counts. SCF was administered to normal or LP-BMS-infected C57BL/6 mice in combination with oral AZT for up to 1 month and effects on platelet, megakaryocyte (MK), and megakaryocyte colony-forming cell (CFU-MK) numbers were evaluated. SCF alone significantly increased the number of circulating platelets in thrombocytopenic MAIDS mice by 53%. SCF also significantly elevated platelet levels by 29% in normal mice. AZT elevated platelet counts 100% in normal and 50% in MAIDS mice. AZT and SCF increased platelet counts in an additive manner. SCF alone was a potent inducer of splenic CFU-MK in both MAIDS and normal mice, increasing splenic CFU-MK 13- to 15-fold at day 15 as compared with untreated controls. By day 30, however, the numbers of splenic CFU-MK had returned to control levels. In infected mice, AZT alone increased the number of splenic CFU-MK. SCF administered to AZT-treated MAIDS mice did not further enhance these increases. In contrast, in normal mice, AZT decreased splenic CFU-MK numbers. In AZT-treated mice, SCF enhanced the numbers of splenic CFU-MK 90-fold at day 8. In MAIDS mice, the number of bone marrow CFU-MK was significantly increased by SCF treatment at all time points. SCF significantly affected the total number of femoral CFU-MK in AZT-treated mice only at day 15. In normal mice, SCF or SCF and AZT in combination increased the total number of bone marrow CFU-MK five-fold at day 8, but failed to induce changes in the total number of femoral CFU-MK after that. These results indicate that SCF elevates platelet levels in both thrombocytopenic MAIDS and normal mice and profoundly affects CFU-MK proliferation. Combinations of SCF and AZT may be further explored to enhance the therapeutic effectiveness of these two drugs in alleviating thrombocytopenia.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/farmacología , Síndrome de Inmunodeficiencia Adquirida del Murino/fisiopatología , Animales , Células de la Médula Ósea , Quimioterapia Combinada , Femenino , Hematopoyesis/efectos de los fármacos , Leucocitos/efectos de los fármacos , Megacariocitos , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas/efectos de los fármacos , Bazo/citología , Factor de Células Madre , Células Madre/efectos de los fármacos , Zidovudina/farmacologíaRESUMEN
The cDNA for human stem cell factor (hSCF) has been cloned and expressed in mammalian and bacterial hosts and recombinant protein purified. We have examined the stimulatory effect of recombinant human SCF (rhSCF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors (CSFs) and erythropoietin (rhEpo). RhSCF alone resulted in no significant colony formation, however, in the presence of rhGM-CSF, rhG-CSF or rhIL-3, rhSCF stimulated a synergistic increase in colony numbers. In addition, increased colony size was stimulated by all combinations. The morphology of cells in the colonies obtained with the CSFs plus rhSCF was identical to the morphology obtained with rhGM-CSF, rhG-CSF or rhIL-3 alone. RhEpo also synergised with rhSCF to stimulate the formation of large compact hemoglobinized colonies which stained positive for spectrin and transferrin receptor and had a morphological appearance consistent with normoblasts. RhSCF stimulation of low density non-adherent, antibody depleted, CD34+ cells suggests that rhSCF directly stimulates progenitor cells capable of myeloid and erythroid differentiation.
Asunto(s)
Médula Ósea/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Eritropoyetina/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Hematopoyesis/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Animales , Células de la Médula Ósea , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Sinergismo Farmacológico , Eritrocitos/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/ultraestructura , Histocitoquímica , Humanos , Receptores de Transferrina/metabolismo , Proteínas Recombinantes/farmacología , Espectrina/metabolismoRESUMEN
We have investigated the effect of stem cell factor (SCF) alone and in combination with interleukin-3 (IL-3) or interleukin-6 (IL-6) on the proliferation and maintenance of primitive hemopoietic progenitor cells. Results from liquid preculture of either unfractionated bone marrow cells or lineage (Lin)-c-kit+ cells indicates that the combination of SCF + IL-3 results in the greatest expansion of total nucleated cell numbers; however, the combination of SCF + IL-6 results in the greatest expansion of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Morphologic examination confirmed the increase in immature cells after culture with SCF + IL-6 and, therefore, this combination is deemed superior for the expansion of primitive cells in liquid culture. Reconstitution assays using congenic mice (Ly5) revealed that cultured cells in the presence of SCF + IL-6 contained stem cells that were capable of reconstituting the hemopoiesis in lethally irradiated mice. Although it remains unclear whether SCF + IL-6 directly supports the self-renewal of stem cells, SCF + IL-6 is a powerful tool for manipulating primitive hemopoietic cells in vitro.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Interleucina-6/farmacología , Proteínas Proto-Oncogénicas/análisis , Animales , Células de la Médula Ósea , Diferenciación Celular , División Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Factores de Crecimiento de Célula Hematopoyética/administración & dosificación , Interleucina-3/farmacología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit , Ratas , Factor de Células Madre , Irradiación Corporal TotalRESUMEN
We have previously defined a subset of High Proliferative Potential Colony Forming Cells (HPP-CFC), derived from murine marrow purified for early progenitors expressing the Stem Cell Antigen (SCA+) and lacking terminal lineage markers (Lin-), which are responsive to multiple cytokines in combination. Stem Cell Factor (SCF), a multipotent growth factor which is the ligand for c-kit, synergizes with these multiple factor combinations to increase colony number and size. SCF is thus a potent mitogen with direct action on early hematopoietic progenitor cells. These data are consistent with a model of stromal control of hematopoiesis via elaboration of multiple cytokines in locally high concentration, with SCF playing a central role.
Asunto(s)
Antígenos/fisiología , Células de la Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Animales , Sinergismo Farmacológico , Interleucina-1/farmacología , RatonesRESUMEN
OBJECTIVE: To determine whether the early-acting hematopoietic growth factors stem-cell factor (SCF) or interleukin-3 (IL-3), are able to overcome the bone-marrow suppressive effects of cytokines or drugs involved in the hematologic abnormalities that accompany HIV-1 infection. DESIGN: In vitro colony formation assays of normal human bone-marrow cells exposed to the myelosuppressive drugs, zidovudine, interferon-alpha (IFN-alpha) and ganciclovir, or the myelosuppressive cytokines, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta (TGF-beta), implicated in HIV dysmyelopoiesis. RESULTS: SCF (10 ng/ml) enhanced the numbers of erythroid (BFU-E) colonies in the presence of zidovudine or ganciclovir (P < 0.05) and myeloid [colony-forming unit granulocyte macrophage (CFU-GM)] colonies in the presence of ganciclovir or IFN-alpha (P < 0.05) relative to controls. IL-3 (10 ng/ml) also improved erythroid colony numbers in the presence of zidovudine (P < 0.05) and CFU-GM in the presence of IFN-alpha (P < 0.05). Neither factor consistently altered the inhibition of TGF-beta or TNF-alpha. The 50% inhibitory concentration (IC50) of the myelosuppressive agents was altered in only one setting, using IL-3 in the presence of zidovudine. CONCLUSIONS: These data suggest that SCF or IL-3 may have a therapeutic application in overcoming hematopoietic abnormalities associated with drugs commonly used in the care of AIDS patients. However, they may have less capacity to overcome the bone-marrow inhibitory effects of the endogenous cytokines TNF-alpha and TGF-beta.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Enfermedades de la Médula Ósea/inducido químicamente , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , División Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/efectos de los fármacos , Ganciclovir/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granulocitos , Humanos , Interferón alfa-2 , Interferón-alfa/farmacología , Macrófagos , Proteínas Recombinantes/farmacología , Factor de Células Madre , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Zidovudina/farmacologíaRESUMEN
Stem cell factor (SCF) is a newly described hematopoietic growth factor that stimulates the growth of primitive hematopoietic progenitors and mast cells. Since the osteoclast precursor is hematopoietic in origin, we tested SCF for its capacity to stimulate the formation of osteoclast-like multinucleated cells (MNC) in long-term human marrow cultures. These MNC express an osteoclast phenotype and form resorption lacunae on calcified matrices. Addition of SCF alone (0.1 pg/ml to 100 ng/ml) to long-term marrow cultures did not increase MNC formation. However, treatment of these cultures sequentially with SCF for 1 week followed by 1,25-(OH)2D3 for the second and third weeks of culture significantly enhanced MNC formation. [3H]Thymidine incorporation studies showed that SCF increased the proliferation of MNC precursors. These data suggested that SCF was acting on early MNC precursors. We then tested the capacity of SCF to stimulate the formation of colonies of committed precursors for osteoclast-like MNC. SCF (20 pg/ml to 20 ng/ml) enhanced osteoclast precursor formation in unfractionated bone marrow mononuclear cells but was unable to increase osteoclast precursor formation when a highly purified population of hematopoietic precursors was used as the target cells for SCF. These data suggest that SCF works in concert with other factors produced by nonhematopoietic marrow cells to increase the precursor pool for osteoclasts and that other factors, such as 1,25-(OH)2D3, complete the differentiation process to the mature osteoclast.
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Células de la Médula Ósea , Calcitriol/farmacología , Factores de Crecimiento de Célula Hematopoyética/farmacología , Osteoclastos/citología , Análisis de Varianza , Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Humanos , Osteoclastos/efectos de los fármacos , Factor de Células MadreRESUMEN
Human recombinant stem cell factor (SCF) increases the viability and cell size of a subset of thymocytes in vitro, but does not independently induce phenotypic changes on thymocytes indicative of T cell differentiation. The SCF-responsive thymocytes have characteristics of large granular cells, that do not express T, B or NK cell-related antigens, and are primarily found in immature thymocyte subsets. These large granular thymocytes do not display cytotoxic activity. However, SCF acts synergistically with IL-2 in the generation of cytotoxic effector cells from thymocyte precursors. Synergy in cytotoxicity is observed to both NK-sensitive and NK-resistant targets. Studies of the SCF receptors on thymocytes show that receptors are expressed on mature 'bright' CD3+ cells, immature 'dim' CD3+ cells as well as CD3- cells. IL-2 increases the frequency of SCF receptor-positive cells in cultured thymocytes, which may explain its synergy with SCF in the generation of NK/LAK cytotoxicity. These data show that SCF enhances the functional development of thymic NK/LAK cells in vitro.
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Citotoxicidad Inmunológica , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-2/farmacología , Células Asesinas Naturales/inmunología , Factor de Células Madre/farmacología , Timo/citología , Antígenos CD/análisis , Diferenciación Celular , Células Cultivadas , Niño , Sinergismo Farmacológico , Humanos , Células Asesinas Activadas por Linfocinas , Fenotipo , Proteínas Proto-Oncogénicas c-kit/análisis , Timo/efectos de los fármacos , Timo/inmunologíaRESUMEN
Human or rodent bone marrow treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a CFU-GM assay yield predominantly granulocytic colonies. The specificity for granulocyte progenitors in vitro is also demonstrated in vivo by a five- to six-fold elevation in hamster peripheral blood neutrophils. Other cell types (monocytes, lymphocytes and eosinophils) remain stable. Analysis of mRNA from the bladder carcinoma cell line 5637 (1A6) shows the predominant species of mRNA codes for a mature protein of 174 amino acids. A small fraction of the mRNA can code for an alternative form of hG-CSF containing additional three amino acids between positions 35 and 36.
Asunto(s)
Factores Estimulantes de Colonias/genética , Granulocitos/fisiología , Animales , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Hematopoyesis/efectos de los fármacos , Humanos , Recuento de Leucocitos , Ratones , ARN Mensajero/genética , Proteínas Recombinantes/genética , Especificidad de la Especie , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Stem cell factor is a recently identified earliest-acting hematopoietic growth factor and a ligand for the c-kit proto-oncogen. Based on our recent observations that recombinant rat interleukin-3 (IL3), human interleukin-6 (IL6) and murine granulocyte-macrophage colony stimulating factor (GM-CSF) possessed different degrees of suppressive activities on the proliferation of LT 12 cell line derived from BNML rat leukemic model, SCF was evaluated alone and in combination with either IL3, IL6 or GM-CSF for effects on leukemopoiesis in vitro. The results indicated that SCF alone had suppressive effect on DNA synthesis and colony forming unit-leukemic blast (CFU-L) in LT12 cells. 100ng/ml of SCF caused substantial reduction in colony number and 3H-TdR uptake although this suppression was of lower magnitude than those induced by IL3, IL6 or GM-CSF. Enhanced suppression on the proliferation of LT12 cells was observed when SCF was used in combination with one of these three factors. Among these combinations, SCF+GM-CSF or SCF+IL6 resulted in more suppression on LT12 cells than SCF+IL3 did. Combination of SCF with two or three factors produced even more suppression. No apparent effect on the size of leukemic colony was seen. Furthermore, in growth kinetics study of LT12 cells in the presence of SCF production of LT12 cells declined. Thus, SCF appears to have divergent hematopoietic activities on BNML rat model: effective stimulation of granulopoiesis and weak suppression of leukemopoiesis.