RESUMO
Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.
Assuntos
Vírus Auxiliares/patogenicidade , Linfoma de Células T/microbiologia , Vírus da Leucemia Murina de Moloney/genética , Transfecção , Animais , Anticorpos Monoclonais , Antígenos CD/análise , Antígenos CD34 , Antígenos de Diferenciação/análise , Sequência de Bases , Transplante de Medula Óssea , Células Cultivadas , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Genoma Viral , Globinas/genética , Vírus Auxiliares/genética , Vírus Auxiliares/isolamento & purificação , Linfoma de Células T/sangue , Linfoma de Células T/patologia , Macaca mulatta , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/isolamento & purificação , Vírus da Leucemia Murina de Moloney/patogenicidade , Oligodesoxirribonucleotídeos , Fatores de Tempo , Replicação ViralRESUMO
Myeloid progenitor cells were highly purified from normal human bone marrow by positive immunoselection with high-affinity monoclonal antibodies linked to magnetic beads and were successfully infected in vitro with the human immunodeficiency virus type 1 (HIV-1). From 99 to 100 percent pure bone marrow cells expressing the CD34 phenotypic marker were obtained. These cells were devoid of mature myeloid or T cell surface and intracellular markers as analyzed by immunohistochemical staining and flow cytometry. HIV-1 particles were detected by supernatant reverse transcriptase activity and transmission electron microscopy 40 to 60 days after infection. Viral particles were predominantly observed assembling and accumulating from within intracellular membranes, while phenotypically the cells were observed to have differentiated into CD4+ monocytes. These studies have important implications in understanding the pathogenesis of HIV-1 as well as the possible cause of certain of the observed hematologic abnormalities in HIV-1 infection. They also indicate that the bone marrow may serve as a potentially important reservoir of HIV-1 in the body.
Assuntos
Células da Medula Óssea , HIV/fisiologia , Células-Tronco Hematopoéticas/microbiologia , Replicação Viral , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Superfície/análise , Membrana Celular/microbiologia , Separação Celular , Citometria de Fluxo , Imunofluorescência , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Fenótipo , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
This report presents results concerning the potential role of negative regulators in hematopoietic suppression observed in human immunodeficiency virus (HIV)-infected long-term cultures (LTC) of human bone marrow cells. Confluent stromal cell layers established from human bone marrow cells were exposed to HIV-1ADA, a monocytotropic strain of HIV-1. A progressive increase in the concentration of HIV-1 p24 antigen in cultures exposed to HIV-1ADA demonstrated that there was a productive infection. Cells from both noninfected and HIV-infected stromal cell layers produced factors that stimulated the proliferation of colony-forming units for granulocytes and macrophages (CFU-GM) from non-infected CD34+ cells. In contrast, when noninfected CD34+ cells were directly cocultured on intact stromal cell layers fewer CFU-GM and burst-forming units for erythroid cells (BFU-E) were detected in HIV-infected LTC than in noninfected LTC. One week after the addition of CD34+ cells, the number of CFU-GM in HIV-infected LTC in six of nine experiments was reduced compared to noninfected control LTC. In those six experiments, the number of CFU-GM was only 53 +/- 5% (SEM) of the number in noninfected LTC. The number of BFU-E in HIV-1-infected LTC was only 46 +/- 5% of the number in noninfected LTC (n = 5). There were fewer BFU-E in HIV-1-infected LTC, whether or not there was a reduced number of CFU-GM. Neutralizing antibody to tumor necrosis factor alpha (TNF-alpha) had no effect on the number of BFU-E in HIV-infected LTC. The number of BFU-E, however, was 2.1 +/- 0.2-fold greater (n = 3) in HIV-infected LTC incubated with neutralizing antibody to interferon-alpha. In HIV-infected LTC with decreased numbers of CFU-GM, the number of CFU-GM was approximately 2-fold greater after incubation of HIV-infected LTC with anti-interleukin-4 (IL-4). The effect of anti-TNF-alpha was variable, and anti-transforming growth factor-beta had no effect on the number of CFU-GM in HIV-infected LTC. After 2 weeks, the number of CFU-GM in HIV-infected LTC incubated with anti-IL-4 and anti-TNF-alpha was 2- to 4-fold greater than in untreated HIV-infected LTC. Antibody treatment did not promote an increase in the number of CFU-GM in noninfected LTC or in LTC in which CFU-GM numbers were not reduced after HIV infection.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Células da Medula Óssea , Citocinas/fisiologia , Eritropoese , HIV-1/fisiologia , Hematopoese , Antígenos CD/análise , Antígenos CD34 , Medula Óssea/virologia , Células Cultivadas , Humanos , Interferon-alfa/fisiologia , Interleucina-4/fisiologia , Células Estromais/virologia , Fator de Crescimento Transformador beta/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação Mielomonocítica/fisiologia , Células da Medula Óssea , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Antígenos CD34 , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos , Imunotoxinas , Técnicas In Vitro , Ricina/administração & dosagem , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Fatores de TempoRESUMO
This report presents the results of studies using long-term bone marrow cultures (LTBMC) of human bone marrow cells to investigate the effect of HIV-1 on in vitro hematopoiesis. Confluent stromal cell layers established from human bone marrow cells were irradiated to eliminate residual hematopoietic progenitor cells and exposed to HIV-1ADA or to HIV-1IIIB, monocytotropic and lymphocytotropic strains of HIV-1, respectively. A productive infection did not develop in cultures exposed to HIV-1IIIB but did for cultures exposed to HIV-1ADA as there was a progressive increase in HIV-1 p24 antigen. Stromal cell layers infected with HIV-1ADA were also cocultured with autologous CD34+ bone marrow cells. Four days, 1, 2, and 3 weeks later, the number of colony-forming units granulocyte/macrophage (CFU-GM) in non- and HIV-infected LTBMC was determined. The number of CFU-GM increased during the first week in both non- and HIV-infected LTBMC. One week after the coculture of CD34+ cells with stromal cell layers infected with HIV-1ADA, the number of CFU-GM in six out of eight experiments was reduced compared to noninfected control LTBMC. In those six experiments, the number of CFU-GM was 53 +/- 6% standard error of the mean (SEM) of the number in noninfected LTBMC. A reduced number of CFU-GM was observed in the nonadherent fraction of HIV-infected LTBMC for at least 2 weeks. These results demonstrate that some cells in the stromal cell layers of LTBMC were targets for HIV-1 and that HIV-infected stromal cell layers suppressed or delayed the production of CFU-GM.
Assuntos
Células da Medula Óssea , Infecções por HIV/fisiopatologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Antígenos CD/análise , Antígenos CD34 , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Substâncias de Crescimento/farmacologia , Humanos , Técnicas In Vitro , Fatores de TempoRESUMO
We have examined the capacity of highly purified human CD34+ marrow cell isolates from unrelated, HLA-mismatched donors to establish in vitro hematopoiesis on recipient marrow stromal cells in 2-stage hematopoietic long-term marrow cultures (H-LTMC). HLA-typing of both peripheral blood mononuclear cells and CD34+ marrow cells was performed for both HLA class I and HLA class II antigens for eight healthy individuals. Significant antigenic mismatches for these molecules ranged from three to six antigens for each recipient-donor pair. Comparison of MHC antigen expression by peripheral blood cells and CD34+ marrow cell isolates confirmed the presence of identical HLA-A, -B, and -C, and -DR specificities on the surface of these cells. Typing of -DQ specificities, however, was not consistently reactive on CD34+ cells. The > or = 20% plating efficiency of purified CD34+ cells for BFU-E, CFU-GM, and CFU-MIX allowed us to use inoculum doses of 10(3), 10(4), and 10(5) cells to determine the efficiency of allogeneic CD34+ cells in achieving in vitro engraftment and the establishment of hematopoiesis in H-LTMC. Engraftment of adherent BFU-E, CFU-GM, and CFU-MIX was equally efficient for autologous and allogeneic CD34+ cells. In vitro hematopoiesis reflected by the cumulative recoveries of progenitor cells over time was also equivalent for allogeneic and autologous CD34+ cells. These results demonstrate that highly purified, HLA-mismatched CD34+ marrow cells proliferate and establish in vitro hematopoiesis as efficiently as autologous cells in marrow derived stromal cell cultures and confirm that interactions between stromal cells and highly purified CD34+, DR-, and CD34+, DR+ marrow cell isolates are not MHC-restricted.
Assuntos
Antígenos CD34/análise , Células da Medula Óssea , Células-Tronco/imunologia , Células Estromais/imunologia , Doadores de Tecidos , Adesão Celular , Comunicação Celular , Células Cultivadas , Teste de Histocompatibilidade , Humanos , Células-Tronco/citologia , Células Estromais/citologiaRESUMO
In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11-10). The Lof(11-10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11-10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11-10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11-10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed. Copyright 1997 S. Karger AG, Basel
RESUMO
Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from human bone marrow by transfecting a plasmid containing the SV40 large T-antigen and isolating foci exhibiting a transformed phenotype. The Lof(11-10) cell line was characterized to determine its susceptibility to HIV infection, to identify its cytokine production profile, and to test the ability of conditioned media from this line to support CD34+ cell differentiation in the presence and absence of HIV expression. Nine cytokines were detected by RT-PCR and ELISA analysis. Conditioned media obtained from the Lof(11-10) cell line was able to support CD34+ celle differentiation. However, because the Lof(11-10) cells are not infectible by HIV, molecular clones of HIV were introduced into these cells by transfection. There was no qualitative difference in the levels of cytokine production between HIV-expressing and control Lof(11-10) cells. Furthermore, conditioned media derived from HIV-expressing and control Lof(11-10) cells added to bone marrow-derived CD34+ progenitor cells yielded similar colony formation in methylcellulose assays. Our data suggest that HIV infection of the cytokine-producing cells within the bone marrow microenvironment, as represented by the Lof(11-10) cell line, results in both normal cytokine production and hematopoiesis in spite of HIV expression. This report adds to the evidence against stromal cells being a significant target of HIV and establishes a system for comparison with more relevant models. Copyright 1995 S. Karger AG, Basel
Assuntos
Linfócitos B/imunologia , Imunoglobulina D/imunologia , Anticorpos Anti-Idiotípicos , Antígenos T-Independentes/imunologia , Diferenciação Celular , Células Clonais/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/imunologia , Baço/imunologiaRESUMO
Procedures are detailed for the rapid isolation of representative cell membrane antigens with protein A-bearing staphylococci as an adsorbent for IgG antibodies complexed with the antigens. Cell surface membrane proteins were radioiodinated and solubilized in nonionic detergent. Specific antisera were subsequently added and the immune complexes precipitated by addition of the staphylococcal adsorbent and low speed centrifugation. The antigens isolated included surface immunoglobulins from mouse and human lymphocytes, human beta-microglobulin and HL-A alloantigens, mouse H-2 alloantigens, and the murine leukemia virus glycoprotein gp 70. Rabbit, sheep, goat, and mouse antisera were all effective for the specific phase of the precipitation reaction. The surface membrane immunoglobulins of mouse splenic lymphocytes and human peripheral blood lymphocytes differed with respect to class composition and protein A reactivity. Mouse lymphocyte surface immunoglobulins were nonreactive with protein A, whereas a high proportion of human lymphocyte surface immunoglobulins of different classes bound directly to the staphylococci. In sequential immunoprecipitation studies the prior isolation of one antigen had no appreciable effect on the subsequent recovery of another antigen. Adsorption of antigen-antibody complexes is quantitative when protein A sites are provided in excess over antiserum IgG sites, and this obviates the need for equivalence point titrations for optimal precipitation necessary with alternative double antibody techniques.
Assuntos
Antígenos/isolamento & purificação , Proteínas de Bactérias/imunologia , Membrana Celular/imunologia , Técnicas de Imunoadsorção , Animais , Complexo Antígeno-Anticorpo , Cabras , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade/isolamento & purificação , Humanos , Imunoeletroforese , Imunoadsorventes/imunologia , Vírus da Leucemia Murina/imunologia , Camundongos , Coelhos , Receptores de Antígenos de Linfócitos B/isolamento & purificação , Ovinos , Baço/imunologia , Staphylococcus , Proteínas Virais/isolamento & purificaçãoRESUMO
The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a superior alternative to other methods of immune precipitation for the isolation of antigens. It exploits the high adsorption capacity for IgG molecules by protein A molecules on the cell walls of certain strains of staphylococci, along with the advantageous sedimentation properties of the bacteria. The interaction of immune complexes with the adsorbent was defined initially using a model system of bovine serum albumin with a high excess of rabbit anti-bovine serum albumin antibodies (IgG). The uptake of immune complexes under these conditions was extremely rapid, occurring within seconds, whereas maximum binding of free IgG was much slower. In addition, once bound the complexed antigen could not be displaced from the adsorbent either by large amounts of normal IgG or by extra free antibody. Antigen could be eluted almost completely from the inert adsorbent for analytic or preparative purposes with a variety of solvent systems, such as the detergent SDS in combination with urea and high temperature, and neutral salts with strong lyotropic salting in properties. The efficacy of the protein A-antibody adsorption technique was tested in direct comparisons with a conventional double antibody precipitation method for the isolation of mouse lymphocyte IgM. The bacterial adsorbent not only had a distinct advantage in speed of antigen isolation, but analyses by polyacrylamide gel electrophoresis in SDS also revealed consistently higher antigen recoveries, lower levels of background radioactivity, and an absence of other cell components which may nonspecifically bind to and complicate analyses using conventional immune precipitates.
Assuntos
Anticorpos Antibacterianos , Complexo Antígeno-Anticorpo , Antígenos/isolamento & purificação , Staphylococcus aureus/imunologia , Adsorção , Animais , Sítios de Ligação de Anticorpos , Imunoglobulina G/análise , Técnicas Imunológicas , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Soroalbumina Bovina/imunologiaRESUMO
Mice have recently been shown to have serum IgD. We have used affinity chromatography to partially purify mouse serum IgD, and have prepared a rabbit antiserum against this mouse Ig class. This antiserum, once adsorbed by IgD-depleted mouse serum bound to Sepharose, was isotype specific as determined by radioimmunoassay and fluorescence activated cell sorter analysis, and bound to the same splenic lymphocyte surface molecules as a hybridoma produced monoclonal anti-mouse delta antibody.
Assuntos
Anticorpos/imunologia , Imunoglobulina D/imunologia , Animais , Sítios de Ligação , Cromatografia de Afinidade , Soros Imunes/farmacologia , Imunoglobulina D/isolamento & purificação , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Baço/imunologiaRESUMO
To define conditions for improved efficiency of retroviral-mediated gene transfer and expression in primate progenitor cells, four rhesus monkeys were treated with a 200 mg/kg intravenous bolus of 5-fluorouracil (5-FU). The kinetics of hematopoietic suppression and recovery were assessed in peripheral blood, bone marrow mononuclear cells, and bone marrow cells fractionated in an albumin density gradient. Bone marrow mononuclear cells were transduced with N2, a retroviral vector carrying the bacterial neomycin phosphotransferase gene (NPT), which confers resistance to the otherwise toxic neomycin analogue, G418. Circulating colony-forming units-granulocyte-macrophage (CFU-GM) disappeared at 2 days. CFU-GM, transducible CFU-GM, CD34+ cells, and the percent of cells in cycle decreased at 3 days in unfractionated bone marrow cells and in a light density population known to be enriched for these progenitors and for stem cells. NPT activity in the light-density fraction, marginally detectable before treatment, disappeared at 3 days as well. At day 7 the CFU-GM plating efficiency, the CD34+ cell content, and the percentage of cells in cell cycle began to increase in the light-density fraction. The NPT assay became faintly positive again but the CFU-GM were not yet transducible, implying that it was an earlier progenitor population that was dividing and differentiating. By day 15, there was a marked rebound in all of the progenitors measured, and transduction efficiency assessed by G418R CFU-GM and NPT assay rebounded to several times pretreatment levels. The data suggest that CFU-GM are optimally transduced at 15 days but that earlier progenitors are more likely cycling and transducible before 5 days, a time when a gene transfer experiment would probably have the best chance to succeed.
Assuntos
Células da Medula Óssea , Fluoruracila/farmacologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Hematopoese/fisiologia , Macaca mulatta/fisiologia , Retroviridae/fisiologia , Células-Tronco/citologia , Transfecção/fisiologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Feminino , Fluoruracila/administração & dosagem , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Hematopoese/efeitos dos fármacos , Injeções Intravenosas , Canamicina Quinase , Fosfotransferases/genética , Fosfotransferases/metabolismo , Retroviridae/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Supressão Genética/efeitos dos fármacos , Supressão Genética/fisiologia , Transfecção/efeitos dos fármacos , Transfecção/genéticaRESUMO
We have used an immunofluorescence inhibition assay to identify 2 BALB/c plasmacytomas, TEPC-1017 and TEPC-1033, that secrete large quantitites of IgD. Both TEPC-1017 and TEPC-1033 myeloma proteins bound to anti-kappa as well as hybridoma and heterologous anti-delta antibodies, but not to anti-mu, gamma, alpha, or lambda antibodies. Both myeloma proteins were purified by (NH4)2SO4 precipitation, ion exchange chromatography, gel filtration, and Staphylococcus aureus Protein A absorption. These IgD kappa myeloma proteins were used to prepare affinity purified rabbit antibodies to delta-chain and the TEPC-1017 and TEPC-1033 idiotypes. Native TEPC-1017 and TEPC-1033 both had mobilities between those of mouse IgA kappa dimers and trimers when analyzed by polyacrylamide gradient gel electrophoresis. Both IgD myeloma proteins broke down under mild reducing conditions into subunits with electrophoretic mobilities slightly slower than those of an IgA kappa monomer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced TEPC-1017 and TEPC-1033 demonstrated kappa-chains and heavy chains that co-migrated with alpha chain. These data suggested that secreted IgD contains 2 delta 2 kappa 2 subunits that are linked by an easily reducible disulfide bond. The kappa-chains of IgD secreted by TEPC-1017 and TEPC-1033 have apparent m.w. of approximately 63,000 daltons, whereas the apparent m.w. of intracytoplasmic delta-chain, intracytoplasmic delta-chain synthesized in the presence of tunicamycin, and the cellfree translation product of TEPC-1017 delta-chain mRNA are 54,000, 43,000, and 44,000 daltons, respectively. This is compatible with the interpretation that the delta-chain peptide has a leader sequence and is N-glycosylated during or shortly after peptide synthesis and is glycosylated further shortly before IgD secretion.
Assuntos
Imunoglobulina D , Proteínas do Mieloma/metabolismo , Plasmocitoma/metabolismo , Animais , Ascite/imunologia , Imunodifusão , Imunoglobulina D/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Proteínas do Mieloma/biossíntese , Proteínas do Mieloma/isolamento & purificação , Coelhos , Radioimunoensaio , Tunicamicina/farmacologiaRESUMO
It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação/análise , Células da Medula Óssea , Mastócitos/citologia , Células-Tronco/citologia , Antígenos CD34 , Linfócitos B/citologia , Basófilos/citologia , Diferenciação Celular/imunologia , Eosinófilos/citologia , Fibroblastos/fisiologia , Humanos , Depleção Linfocítica , Macrófagos/citologia , Células-Tronco/imunologia , Linfócitos T/citologiaRESUMO
Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).
Assuntos
Encéfalo/irrigação sanguínea , Comunicação Celular , Endotélio Vascular/citologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD/análise , Antígenos CD34 , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Sinergismo Farmacológico , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Fator de Células-Tronco , SuínosRESUMO
Hemopoietic stem cell factor (SCF), which is the ligand for the proto-oncogene c-kit receptor (allelic with W locus) and the product of Sl locus of the mouse, has recently been cloned. The human homologue has also been cloned, and recombinant protein (human rSCF) expressed and purified to homogeneity. To determine the effect of human rSCF in the presence or absence of human rIL-3 on human bone marrow-derived mast cells and basophils, human CD34+ pluripotent progenitor cells, highly enriched (greater than 99%) from bone marrow mononuclear cells, were cultured over agarose surfaces (interphase cultures) in the presence of human rIL-3, human rIL-3 and increasing concentrations of human rSCF, or human rSCF alone. Over 3 to 4 wk, human rSCF acted synergistically with human rIL-3 at all concentrations, producing a three- to fivefold increase in total, mast cell, and basophil numbers over human rIL-3 alone when used at 100 ng/ml. The percentage of cell types in the human rIL-3 and human rIL-3 plus human rSCF cultures, however, remained the same, with basophils constituting 18 to 35% of the final cultured cells, and mast cells 3% or less of the final cell number. In the presence of human rSCF alone, the combined total percentage of mast cells and basophils was 0 to 1.0%, the majority of cells being macrophages. Mast cells cultured in human rIL-3 plus human rSCF, but not human rIL-3 alone, were berberine sulfate positive, suggesting the presence of heparin proteoglycans within granules. Electron microscopic examination of cultures supplemented with human rIL-3 and rSCF, but not human rIL-3 alone, revealed that after 3 wk in culture, mast cell granules contained tryptase and exhibited scroll, reticular, and homogeneous patterns as seen previously in CD34+/3T3 fibroblast cocultures. Thus, CD34+ cells cultured in the presence of both human rIL-3 and rSCF give rise to cultures containing increased numbers of basophils and mast cells, with the mast cells by ultrastructural studies showing evidence of maturation although the percentages of basophils and mast cells arising in these cultures remained unchanged.
Assuntos
Basófilos/citologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/citologia , Interleucina-3/farmacologia , Mastócitos/citologia , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Peptídeo Hidrolases/análise , Proto-Oncogene Mas , Fator de Células-Tronco , Fatores de TempoRESUMO
A case of acute, transient agranulocytosis and thrombocytopenia associated with ingestion of dipyrone is reported. This once widely used analgesic, which is now banned in the United States, was obtained by the patient as "aspirin" while traveling in Mexico. Studies of the effects of this patient's serum on purified CD34+ marrow cells, which were highly enriched for hematopoietic progenitors, showed not only a drug-dependent suppression of the in vitro growth of myeloid progenitors, as has been reported previously, but also a drug-dependent suppression of primitive multipotential progenitors (CFU-Mix) and erythroid progenitors (BFU-E). These findings indicate that autoimmune, antibody-hapten interactions which have been reported to occur in dipyrone- and aminopyrine-induced agranulocytosis are not restricted to the neutrophil lineage.
Assuntos
Agranulocitose/induzido quimicamente , Aminopirina/análogos & derivados , Autoanticorpos/fisiologia , Dipirona/efeitos adversos , Células-Tronco Hematopoéticas/fisiologia , Adulto , Agranulocitose/etiologia , Fenômenos Biomecânicos , Ensaio de Unidades Formadoras de Colônias , Dipirona/imunologia , Hipersensibilidade a Drogas/sangue , Hipersensibilidade a Drogas/complicações , Hipersensibilidade a Drogas/diagnóstico , Feminino , HumanosRESUMO
To determine the fate of Fc epsilon RI+ cells on fibroblasts in vitro, human bone marrow derived CD34+ cells were cultured in the presence of recombinant human interleukin 3 and recombinant human hematopoietic stem cell factor for 3 weeks, and Fc epsilon RI+ cells were purified by immunomagnetic selection. This enriched Fc epsilon RI+ cell population consisted of 92-94% basophils and 3-5% mast cells as determined by morphologic, immunohistochemical, and ultrastructural criteria. The Fc epsilon RI+ cells were then cocultured with 3T3 fibroblasts. Basophils decreased markedly by 1 week and were absent from cocultures by 2-3 weeks, while the mast cell numbers on the fibroblast monolayers remained constant. Ultrastructural examination of cocultures at 2 days demonstrated phagocytosis of basophils by fibroblasts. By 1 week, phagocytosed basophil membranes and granules gave fibroblasts the superficial appearance of mast cells by toluidine blue staining. Mast cells surviving in cocultures could be distinguished from granule-containing fibroblasts by IgE surface labeling and by ultrastructural demonstration of tryptase-positive granules. Thus, while mast cells remain viable in coculture with 3T3 fibroblasts, basophils do not survive and are internalized and degraded by the fibroblast monolayer.