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1.
Gene Ther ; 27(12): 579-590, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32669717

RESUMO

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.


Assuntos
Insuficiência Cardíaca , Coração Auxiliar , Adulto , Estudos de Viabilidade , Terapia Genética , Vetores Genéticos/genética , Insuficiência Cardíaca/terapia , Humanos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
2.
Gene Ther ; 23(3): 313-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26699914

RESUMO

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.


Assuntos
Anticorpos Antivirais/imunologia , Dependovirus/genética , Dependovirus/imunologia , Vetores Genéticos/imunologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Animais , Anticorpos Neutralizantes/imunologia , Terapia Genética , Humanos , Modelos Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos , Porco Miniatura
3.
J Immunol ; 161(1): 375-84, 1998 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-9647246

RESUMO

TCR- and IgG-binding Fc receptors (Fc gamma R) mediate a variety of critical biologic activities including cytolysis via the structurally related zeta- and gamma-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR zeta-chain. Such zeta-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both zeta- and gamma-CIRs to activate the cytolytic function of two distinct classes of Fc gamma R-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing zeta- and gamma-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both zeta- and gamma-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the zeta-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.


Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Proteínas de Membrana/biossíntese , Neutrófilos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Receptores de Antígenos de Linfócitos T/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/imunologia , Adulto , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Testes Imunológicos de Citotoxicidade , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/síntese química , Humanos , Células Matadoras Naturais/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos SCID , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/imunologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/genética , Transdução Genética/imunologia
4.
Nat Genet ; 15(2): 146-56, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9020839

RESUMO

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.


Assuntos
Formação de Anticorpos , Genes de Imunoglobulinas , Transgenes , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Diversidade de Anticorpos , Linfócitos B/citologia , Linfócitos B/imunologia , Cromossomos Artificiais de Levedura/genética , Receptores ErbB/imunologia , Rearranjo Gênico do Linfócito B , Humanos , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Interleucina-8/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade da Espécie , Fator de Necrose Tumoral alfa/imunologia
5.
J Exp Med ; 184(6): 2261-9, 1996 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-8976181

RESUMO

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.


Assuntos
Transplante de Medula Óssea/imunologia , Antígenos CD4/imunologia , Produtos do Gene env/imunologia , HIV/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Citotoxicidade Imunológica , Primers do DNA , Feminino , Produtos do Gene env/biossíntese , Sobrevivência de Enxerto/imunologia , Humanos , Masculino , Camundongos , Camundongos SCID , Neutrófilos/fisiologia , Reação em Cadeia da Polimerase , Transplante Heterólogo/imunologia
6.
Immunol Lett ; 52(1): 45-52, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8877418

RESUMO

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.


Assuntos
Citotoxicidade Imunológica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Fator de Células-Tronco/farmacologia , Timo/citologia , Antígenos CD/análise , Diferenciação Celular , Células Cultivadas , Criança , Sinergismo Farmacológico , Humanos , Células Matadoras Ativadas por Linfocina , Fenótipo , Proteínas Proto-Oncogênicas c-kit/análise , Timo/efeitos dos fármacos , Timo/imunologia
7.
Blood ; 85(1): 74-9, 1995 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-7528575

RESUMO

Grey collie dogs have cyclic fluctuations in their blood cell counts caused by a regulatory defect of hematopoietic stem cells. To examine the role of stem cell factor (SCF) or its receptor in this disorder, we investigated the stimulatory effects of recombinant canine SCF (rc-SCF) on in vitro marrow cultures, cloned and sequenced the grey collie SCF gene, and treated three grey collies with rc-SCF, either alone or in combination with recombinant canine granulocyte colony-stimulating factor (rcG-CSF). Colony-forming unit granulocyte-macrophage formation from grey collie or normal dog marrow showed similar dose-response curves for rc-SCF. Cloning and sequencing the SCF gene for two grey collies showed no evidence of mutations in the coding region of the SCF gene. Treatment with rc-SCF (10 to 100 micrograms/kg/d) did not induce neutrophilia except at the highest dose (100 micrograms/kg/d), but daily rc-SCF abrogated the neutropenic periods in doses of 20 micrograms/kg/d or greater. Combination of rc-G-CSF (0.5 to 1.0 microgram/kg/d) with rc-SCF treatment (20 to 50 micrograms/kg/d) suggested a synergistic effect, ie, the neutrophil levels on combined therapy were higher than the sum of the levels when these two cytokines were given separately. Long-term treatment of these dogs with rc-SCF in doses of 10 to 30 micrograms/kg/d was generally well tolerated, suggesting that SCF may be useful as a therapy for some chronic hypoproliferative disorders of hematopoiesis.


Assuntos
Doenças do Cão/tratamento farmacológico , Doenças Hematológicas/veterinária , Hematopoese , Fatores de Crescimento de Células Hematopoéticas/uso terapêutico , Animais , Sequência de Bases , Medula Óssea/patologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Cães , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Granulócitos/citologia , Doenças Hematológicas/tratamento farmacológico , Fatores de Crescimento de Células Hematopoéticas/administração & dosagem , Fatores de Crescimento de Células Hematopoéticas/genética , Macrófagos/citologia , Masculino , Dados de Sequência Molecular , Mutação , Neutropenia/prevenção & controle , Proteínas Recombinantes/uso terapêutico , Fator de Células-Tronco
8.
Blood ; 84(3): 847-52, 1994 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-7519080

RESUMO

The prognostic significance of c-kit receptor expression on leukemic blast cells was determined in 122 children with acute myeloid leukemia (AML) entered onto Childrens Cancer Group protocol 213. Clinical and laboratory characteristics as well as outcome were analyzed according to the percentage of blast cells expressing c-kit receptors and the relative number of c-kit receptors per cell as determined by indirect immunofluorescence. c-kit receptor expression was strongly associated with the expression of the CD34 antigen. However, contrary to findings in adult patients with AML, c-kit receptor expression by childhood AML blast cells was not predictive of a poor response to therapy.


Assuntos
Leucemia Mieloide/diagnóstico , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator Estimulador de Colônias/metabolismo , Doença Aguda , Adolescente , Adulto , Fatores Etários , Antígenos CD/metabolismo , Antígenos CD34 , Antígenos de Superfície/metabolismo , Criança , Pré-Escolar , Humanos , Imunofenotipagem , Lactente , Recém-Nascido , Leucemia Mieloide/metabolismo , Contagem de Leucócitos , Prognóstico , Proteínas Proto-Oncogênicas c-kit , Análise de Sobrevida
9.
Blood ; 83(11): 3146-51, 1994 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-7514901

RESUMO

To test whether primitive hematopoietic stem cells (PHSCs) are stimulated by Steel (SI) factor (c-kit ligand) in vivo, donor mice were studied after three or seven daily injections of SI factor. PHSC activity was measured as long-term erythroid and lymphoid competitive repopulating ability. Cells to be tested (usually marrow or spleen cells from treated donors) were mixed with untreated competitor marrow that produces erythrocytes and lymphocytes that are genetically distinguishable from the donors by differences in hemoglobin (Hb) and glucosephosphate isomerase (GPI) markers. These cell mixtures were injected into lethally irradiated hosts, and after 111 to 293 days, functional abilities of donor PHSC populations were assessed and expressed as percentages of donor-type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. A striking increase in splenic PHSC activity occurred after seven daily injections of SI factor, with a much smaller increase after three daily injections. Both three and seven daily injections of SI factor slightly reduced marrow PHSC activity. Rapid cycling greatly increases PHSC vulnerability to 5-fluorouracil (5FU). To test whether SI factor stimulates PHSCs into rapid cycling, donor mice were given a dose of 5FU in addition to SI factor. The increase in splenic PHSCs after 7 days of treatment with SI factor occurred to a similar degree whether donors were or were not treated with 5FU on day 8. However, a dose of 5FU on day 4 of the SI factor treatments almost totally prevented the increase in splenic PHSC activity. Apparently this increased activity requires PHSC cycling throughout the period of SI factor treatment.


Assuntos
Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Fator de Células-Tronco
10.
Zhonghua Zhong Liu Za Zhi ; 16(2): 93-7, 1994 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-7523053

RESUMO

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.


Assuntos
Fatores de Crescimento de Células Hematopoéticas/farmacologia , Leucemia Promielocítica Aguda/patologia , Animais , Divisão Celular , DNA de Neoplasias/biossíntese , Sinergismo Farmacológico , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Ratos , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco
11.
Blood ; 83(4): 916-25, 1994 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-7509212

RESUMO

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP-I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I-/II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA-SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.


Assuntos
Mastócitos/citologia , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator Estimulador de Colônias/genética , Deleção de Sequência , Animais , Anticorpos Monoclonais/farmacologia , Células da Medula Óssea , Moléculas de Adesão Celular/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Concanavalina A , Meios de Cultivo Condicionados , Endopeptidases/biossíntese , Endopeptidases/metabolismo , Fibroblastos/fisiologia , Imunofluorescência , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Cinética , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/fisiologia , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/fisiologia , Fenótipo , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-kit , Ratos , Ratos Mutantes , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Fator Estimulador de Colônias/biossíntese , Baço/imunologia , Baço/fisiologia , Fator de Células-Tronco , Fatores de Tempo
12.
AIDS ; 8(2): 193-6, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7519020

RESUMO

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.


Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Doenças da Medula Óssea/induzido quimicamente , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-3/farmacologia , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/efeitos dos fármacos , Ganciclovir/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Granulócitos , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Macrófagos , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Zidovudina/farmacologia
14.
Nat Immun ; 12(6): 293-301, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7505667

RESUMO

Steel factor (S1F), also known as stem cell factor, is a potent growth stimulator of hemopoietic progenitor cells. In the context of transplantation of hemopoietic cells to irradiated allogeneic hosts, natural killer (NK) cells exert restrictive control on hemopoietic cell proliferation, and are regularly found in elevated concentration in areas of intense hemopoiesis. The present study was designed to examine the effects with time of S1F in vivo on the numbers of NK cells, identified by the presence of the NK 1.1 surface molecule, in the spleen and bone marrow. Throughout the first 3 days of S1F exposure, NK cell numbers, in spite of rapid (1 day) and significant increases in the other hemopoietic cell lineages, did not change in either the spleen or the bone marrow. However, NK cells were increased 2-fold in both organs by 7 days of S1F exposure. At this time, immature granuloid and erythroid cells and the large lymphoid cells in the spleen had more than doubled their respective control numbers and in the bone marrow, immature granuloid cells increased by 47% of control levels. The presence of a late, but not early, influence of S1F on NK cells of the spleen and bone marrow suggests an indirect effect of S1F on this lineage, occurring only when S1F-stimulated hemopoiesis becomes sufficiently intense, providing, thus, an abundance of NK cell targets.


Assuntos
Medula Óssea/imunologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Baço/imunologia , Animais , Antígenos/análise , Antígenos Ly , Antígenos de Superfície , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Cruzamentos Genéticos , Hematopoese/efeitos dos fármacos , Lectinas Tipo C , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Proteínas/análise , Baço/citologia , Fator de Células-Tronco
15.
Blood ; 82(2): 436-44, 1993 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-7687159

RESUMO

Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB-02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose-dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c-kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.


Assuntos
Fatores de Crescimento de Células Hematopoéticas/farmacologia , Leucemia Eritroblástica Aguda/patologia , Proteínas Proto-Oncogênicas/metabolismo , Anticorpos Monoclonais , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Eritropoetina/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Humanos , Interleucina-3/farmacologia , Leucemia Eritroblástica Aguda/metabolismo , Peso Molecular , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-kit , RNA Mensageiro/metabolismo , Fator de Células-Tronco , Células Tumorais Cultivadas
16.
Blood ; 82(2): 445-55, 1993 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-7687160

RESUMO

We have examined the effects of administration of stem cell-factor (SCF) on the number and distribution of pluripotent hematopoietic stem cells (PHSC) in normal mice. Using the competitive repopulation assay we found that in vivo administration of SCF increases the absolute number of PHSC per mouse threefold. The increased numbers of PHSC are found in the peripheral blood and spleen of the SCF-treated animals. The spleen and peripheral blood stem cells completely repopulated the erythroid, myeloid, and lymphoid lineages of irradiated or W/Wv hosts, similar to bone marrow PHSC. PHSC from the peripheral blood of SCF-treated mice have a lineage marker-negative, c-kit-positive phenotype that is indistinguishable from that of bone marrow PHSC. The increase in the absolute number of spleen PHSC is associated with efficient gene transfer to these cells without prior treatment with 5-fluorouracil. This is a US government work. There are no restrictions on its use.


Assuntos
Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/citologia , Animais , Medula Óssea/química , Células da Medula Óssea , Contagem de Células , DNA/análise , Resistência a Medicamentos/genética , Células Precursoras Eritroides/citologia , Fluoruracila/farmacologia , Granulócitos/citologia , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Polietilenoglicóis , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-kit , Proteínas Recombinantes/farmacologia , Baço/citologia , Fator de Células-Tronco , Timo/química , Transfecção , Irradiação Corporal Total
17.
Immunology ; 79(2): 325-30, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7688344

RESUMO

Human foetal liver cells are an enriched source of mast cell progenitors that complete their differentiation and mature in response to stem cell factor, the ligand for Kit, in liquid culture. These mast cells are Kit+, metachromatic with toluidine blue+, tryptase+, histamine+ and show ultrastructure features of mast cells. Using a panel of monoclonal antibodies (mAb) against different cell-surface antigens (33 mAb were used), the cell-surface phenotype of human stem cell factor-dependent foetal liver-derived mast cells was examined by flow cytometry. Consistent with previous reports on tissue-derived mast cells, those derived from foetal liver in vitro expressed HLA class I, CD9, CD29, CD33, CD43, CD45 and Kit. Unlike mast cells dispersed from tissue, a high expression of CD13 was found. Also, these in vitro-derived mast cells express little, if any, high-affinity IgE receptor. However, small amounts of mRNA for the alpha-chain in foetal liver-derived mast cells compared to KU812 cells (a human basophil-like cell line) could be detected by Northern blotting. Full expression of Fc epsilon RI may require additional growth factor(s).


Assuntos
Antígenos de Superfície/análise , Fatores de Crescimento de Células Hematopoéticas/imunologia , Fígado/embriologia , Mastócitos/imunologia , Northern Blotting , Células Cultivadas , Humanos , Fígado/imunologia , RNA Mensageiro/análise , Receptores de IgE/análise , Receptores de IgE/genética , Proteínas Recombinantes/imunologia , Fator de Células-Tronco
18.
Cancer Res ; 53(7): 1709-14, 1993 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-7680956

RESUMO

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.


Assuntos
Carcinoma de Células Pequenas/genética , Quimiotaxia/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/análise , Carcinoma de Células Pequenas/química , Carcinoma de Células Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Humanos , Leucemia/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit , Proteínas Recombinantes/farmacologia , Análise de Sequência , Fator de Células-Tronco , Células Tumorais Cultivadas
19.
Leukemia ; 7(2): 235-40, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7678880

RESUMO

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.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Leucemia Megacarioblástica Aguda/patologia , Divisão Celular/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco
20.
Blood ; 81(3): 656-60, 1993 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-7678995

RESUMO

Stem cell factor (SCF) is a recently described factor active in the early stages of hematopoiesis. It can exist in membrane-bound form and in proteolytically released soluble form. The levels and nature of SCF in human serum are described. As determined by an enzyme-linked immunosorbent assay performed for 257 samples, SCF level in serum averaged 3.3 +/- 1.1 ng/mL. The serum SCF was partially purified by immunoaffinity chromatography and analyzed by glycosidase treatments in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the SCF has N-linked and O-linked carbohydrate and corresponds to the soluble form, at or about 165 amino acids in length. The findings suggest functional importance for soluble SCF in humans.


Assuntos
Fatores de Crescimento de Células Hematopoéticas/sangue , Adulto , Fatores Etários , Células Cultivadas , Cromatografia de Afinidade , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática/métodos , Eritropoetina/farmacologia , Feminino , Fatores de Crescimento de Células Hematopoéticas/isolamento & purificação , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Humanos , Imunoglobulina G , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia , Valores de Referência , Caracteres Sexuais , Fator de Células-Tronco
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