RESUMO
The present study was carried out to determine which component of the polycythemic strain of Friend erythroleukemia virus (FV-P)--Friend helper virus [Friend murine leukemia virus (F-MuLV)] of the replication-defective spleen focus-forming virus (SFFV)--is responsible for inducing erythropoietin-independent erythropoiesis and expansion of the erythroid compartment in infected mice. F-MuLV and SFFV were cloned in SC1 cells to give a cell line 643/22N, which released only F-MuLV and SC204, a nonproducer cell line carrying SFFV. On superinfection with specific helper viruses, SC204 yielded further cell lines, which released SFFV in conjunction with 1) Friend, 2) Moloney, or 3) Gross helper viruses. With the use of an in vitro colony formation assay to monitor erythropoiesis in the bone marrow of inbred DBA/2J mice infected with any of the virus preparations containing SFFV plus helper virus, the erythroid precursor cell population was found to become erythropoietin-independent and amplified; adult mice infected with any of the helper viruses alone did not develop any symptoms of Friend disease. Thus the biologic activity of the FV-P complex was unaffected by replacement of the F-MuLV with an unrelated helper virus (Moloney or Gross). These results indicated that the SFFV component of FV-P is responsible for modifying erythroid differentiation in adult mice.
Assuntos
Eritropoese , Vírus da Leucemia Murina de Friend/fisiologia , Leucemia Experimental/patologia , Animais , Diferenciação Celular , Linhagem Celular , Transformação Celular Neoplásica , Ensaio de Unidades Formadoras de Colônias , CamundongosRESUMO
The hematopoietic stem cell (CFU-S) and granulocyte precursor cell (CFU-C) populations have been assayed in the spleen, blood, and bone marrow of DBA/2 mice at various times after infection with the myeloproliferative sarcoma virus (MPSV). Beginning between 7 and 19 days after virus infection, the number of CFU-S showed a steady, parallel increase in the blood and spleen, reaching a maximum at both sites by days 25-30. At the maximum, in the spleen the concentration of CFU-S was 10 times greater than that in the blood, and the total number of CFU-S was over 100 times greater than that of normal animals. During the same period, in the bone marrow the number of CFU-S decreased to one-half of normal. Nevertheless, the CFU-S from MPSV-infected animals differentiated normally in the spleens of irradiated, normal recipient mice (except for some hyperplasia of the erythroid component of spleen colonies). The CFU-C content of the bone marrow, spleen, and blood paralleled the CFU-S content of these organs: The CFU-S and CFU-C populations changed almost synchronously after MPSV infection. In the terminal stage of the MPSV-induced disease, a variable proportion of the CFU-C population acquired the ability to differentiate in the absence of added colony-stimulating factor.
Assuntos
Granulócitos , Células-Tronco Hematopoéticas , Vírus do Sarcoma Murino , Animais , Medula Óssea/patologia , Contagem de Células , Ensaio de Unidades Formadoras de Colônias , Fatores Estimuladores de Colônias , Fêmur , Camundongos , Camundongos Endogâmicos DBA , Tamanho do Órgão , Baço/patologia , Viroses/sangue , Viroses/etiologiaRESUMO
Erythropoietin-like activity (EpLA) is one of several hematopoietic growth factors found in spleen cell-conditioned medium. Conditioned media obtained from both dissociated tissues and cloned cell lines were examined in order to define the cellular source of EpLA. The growth factor was only found in supernatants of tissues that contained T-lymphocytes. An examination of media conditioned by cloned cell lines representative of the major cell types in spleen (T- and B-lymphocytes, macrophages) confirmed the association between T cells and EpLA production. Limiting dilution analyses established that normal T-lymphocyte clones produced EpLA, but this property was not correlated with a specific functional T-cell subset. On stimulation with PMA, the T-cell lymphoma cell line, EL-4, proved to be the most potent producer of EpLA found to date.
Assuntos
Eritropoetina/análise , Substâncias de Crescimento/análise , Linfócitos T/citologia , Animais , Linhagem Celular , Células Clonais/análise , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Baço/citologiaRESUMO
It has been suggested that oestriol protects against breast cancer, because in some experiments on uterine growth it is only weakly active, and partially inhibits the effects of oestradiol-17beta. When its effects are measured 24 h after a single injection, oestriol behaves as a typical impeded oestrogen with low potency and a flat dose-response line. This does not result from failure to stimulate certain critical stages of growth but from failure to sustain the products of growth. We found that oestriol induced all phases of uterine growth including DNA synthesis and cell division. It was as effective as oestradiol in stimulating early increases in protein synthesis and uterine weight, and half as effective in stimulating epithelial cells to replicate DNA and divide. However, epithelial cell numbers did not increase after a single injection of oestriol because cell death rate increased at the same time as mitotic rate, apparently as a result of the more rapid loss of oestriol from the uterus. Repeated injections of oestriol prevented premature cell death and produced as much uterine hypertrophy and hyperplasia as oestradiol-17beta. These results support the thesis that the oestrogenic potency of a substance is largely determined by the duration of its occupation of receptors. Thus in situations of continuous production, (e.g. pregnancy) oestriol would be as active as oestradiol and unlikely to exert any significant 'buffering' or protective action. The findings are also discussed in relation to a new model for the regulation of cell proliferation.
Assuntos
Estradiol/farmacologia , Estriol/farmacologia , Útero/efeitos dos fármacos , Animais , Castração , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , Demecolcina/toxicidade , Endométrio/metabolismo , Células Epiteliais , Epitélio/metabolismo , Feminino , Camundongos , Mitose/efeitos dos fármacos , Biossíntese de Proteínas , Timidina/metabolismo , Útero/metabolismoRESUMO
The mutagenic activity of cigarette smoke condensates (CSC) made from tobacco before and after removal of protein was assessed by the Ames Salmonella assay in bacterial strains TA98 and TA100. Removal of protein and peptides from flue-cured tobacco via water extraction followed by protease digestion reduced the mutagenicity of the resultant CSC by 80% in the TA98 strain and 50% in the TA100 strain. Similarly, reductions of 81% in TA98 and 54% in TA100 were seen following water extraction and protease digestion of burley tobacco. The significant reductions in Ames mutagenicity following protein removal suggest that protein pyrolysis products are a principal contributor to the genotoxicity of CSC as measured in this assay.
Assuntos
Mutagênicos/farmacologia , Nicotiana/química , Plantas Tóxicas , Salmonella/efeitos dos fármacos , Salmonella/genética , Fumaça , Endopeptidases/química , Testes de Mutagenicidade , Nitrogênio/análise , Nitrogênio/química , Proteínas de Plantas/isolamento & purificaçãoAssuntos
Estradiol/farmacologia , Estriol/farmacologia , Útero/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Estradiol/metabolismo , Estriol/metabolismo , Feminino , Injeções Subcutâneas , Camundongos , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Útero/efeitos dos fármacosRESUMO
Haematopoiesis is a useful model system for studying differentiation and the regulation of precursor cell populations intermediate between the multipotential stem cell and terminally differentiated end cells. For many years, erythropoietin (Epo) was recognized as the hormone which controls red cell production in vivo. Although other substances are now known to be required during the initial stages of erythropoiesis, late erythroid differentiation is regarded as strictly Epo-dependent. This concept is supported by the recent demonstration that the addition of Epo alone to serum-free bone marrow cell cultures is sufficient to stimulate the CFU-E (colony-forming unit-erythroid-a late erythroid precursor cell approximating to a proerythroblast) to complete differentiation into mature erythrocytes. However, the data reported here indicate that mature erythroid colonies (indistinguishable from those formed by CFU-E + Epo) are formed when adult bone marrow cells are grown for 2 d in methyl cellulose cultures containing spleen cell-conditioned medium (SCM) but no added EPO. SCM is a rich source of growth factors and initial observations suggested that its 'Epo-like' activity could be attributed to: (1) Epo, or (2) a factor which enhances the activity of small amounts of Epo in the culture medium, or (3) a factor(s) distinct from Epo which is also capable of stimulating late erythroid differentiation. The experiments reported here, which include a partial characterization of the 'Epo-like' activity, support the latter interpretation.
Assuntos
Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Baço/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Baço/citologia , Estimulação QuímicaRESUMO
Supernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2-day erythroid (CFU-E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin-like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of gamma-interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin-2 (IL-2), IL-1, and colony stimulating factor 1 (CSF-1) did not support CFU-E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep-responsive CFU-E population in fetal liver is sensitive to EpLA.
Assuntos
Eritropoetina/análise , Baço/análise , Animais , Cromatografia Líquida de Alta Pressão , Fatores Estimuladores de Colônias/análise , Durapatita , Eletroforese em Gel de Poliacrilamida , Eritropoetina/farmacologia , Substâncias de Crescimento/análise , Fatores de Crescimento de Células Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hidroxiapatitas , Interferon gama/análise , Camundongos , Peso MolecularRESUMO
Marrow cells from 5-fluorouracil (5-FU)-treated mice formed few or no mixed erythroid colonies when plated in semisolid medium with interleukin 3 (IL 3) and erythropoietin (Ep) alone. When conditioned medium (CM) from plastic-adherent marrow or thymus cells was also included, however, growth of mixed erythroid colonies was strongly stimulated. Both IL 3 and the accessory activity (AA) had to be present at the initiation of the cultures for growth to occur. AA was also produced by a cloned immortalized line (95/1.7) of fibroblastoid marrow cells that lacked macrophage-specific cell surface markers. Colony-stimulating factor-1 (CSF-1) was also released, but not granulocyte colony-stimulating activity. When 95/1.7 CM was analyzed by gel filtration, AA eluted with an apparent size of 35 kd and separated completely from the CSF-1. Biologic assays failed to detect IL 1 or IL 3 activity in 95/1.7 CM. Growth of mixed erythroid colonies from 5-FU-treated marrow is thus stimulated by adherent marrow cell-derived factors that appear distinct not only from the known CSFs including IL 3, but also from IL 1.
Assuntos
Medula Óssea/fisiologia , Adesão Celular , Substâncias de Crescimento/fisiologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Interleucina-3/farmacologia , Animais , Células da Medula Óssea , Células Cultivadas , Meios de Cultura/farmacologia , Sinergismo Farmacológico , Eritropoese/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fatores de Crescimento de Células Hematopoéticas , Camundongos , Camundongos Endogâmicos CBA , Timo/citologia , Timo/fisiologiaRESUMO
A simple one-step isolation technique significantly enriched mouse fetal liver cells that respond to interleukin 3 (IL-3), a multilineage hematopoietic growth factor. The fetal liver cell subpopulation isolated with monoclonal antibody AA4 contained 50- to 100-fold higher frequencies of multipotential (CFU-mix) or restricted (CFU-G/M, BFU-E) erythroid/myeloid precursors as well as precursors that differentiate to become mature B lymphocytes [CFU-mix = erythroid and myeloid colony-forming unit(s); CFU-G/M = CFU-granulocyte/macrophage; BFU-E = burst-forming unit-erythroid]. The B-lymphocyte precursors could be cloned in single-cell cultures when IL-3-containing supernatants were present. Growth of these clones was supported by purified IL-3 but not by purified IL-2. Stable growth has been maintained for greater than 6 mo in the presence of IL-3. Such clones express on their cell surface low amounts of class I major histocompatibility complex antigens and high amounts of AA4, GF1, and leukocyte common glycoprotein 200 antigens. They lack detectable rearrangements of their Ig-encoding genes [joining region heavy and light (kappa, lambda) chain genes], even after subcloning, but maintain their capacity to differentiate to mature B lymphocytes committed to multiple Ig specificities.
Assuntos
Linfócitos B/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Células Clonais/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imunoglobulina M/análise , Imunoglobulinas/genética , Interleucina-3 , Fígado/embriologia , Fígado/imunologia , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Seven mesothelioma cell lines, established from patients with pleural mesothelioma, exhibited substantial heterogeneity regarding in vitro morphology and growth characteristics. Media conditioned by these cell lines and by MeT5A normal mesothelial cells were examined for (i) colony formation on human bone-marrow cells, (ii) hematopoietic growth-factor content and (iii) mitogenic activity on mesothelioma cells. Colony-stimulating activity was produced only by the ZL34 cell line. Analysis of conditioned media by ELISA revealed that all mesothelioma cell lines constitutively produced IL-6, while the MeT5A normal mesothelial cells did not; in addition, GM-CSF and G-CSF were detected in the supernatant of the ZL34 cell line. Using a 3H-thymidine incorporation assay, we showed that all mesothelioma cell lines produced mitogenic activity in the culture supernatant, in contrast to the MeT5A normal mesothelial cells. The mitogenic effect of the hematopoietic growth factors detected in mesothelioma culture supernatants was tested on mesothelioma cells and on MeT5A normal mesothelial cells: IL-6, GM-CSF and G-CSF did not stimulate any DNA synthesis. Our results suggest that these hematopoietic growth factors do not act as autocrine growth factors. A common feature of this panel of mesothelioma cell lines is the production of IL-6; although the biological significance of the aberrant production of cytokines by mesotheliomas remains unclear, IL-6 might be involved in paraneoplastic syndromes such as thrombocytosis.
Assuntos
Interleucina-6/análise , Mesotelioma/química , Neoplasias Pleurais/química , Fator Estimulador de Colônias de Granulócitos/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Humanos , Mesotelioma/patologia , Neoplasias Pleurais/patologia , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Adult susceptible mice (DBA/2J) infected with MPSV (myeloproliferative sarcoma virus), a defective RNA tumour virus, develop splenomegaly and progressive disruption of the haematologic system culminating in death. The present study was specifically directed toward determining the effects of the virus on erythroid differentiation. Early and late precursor cells (erythroid burst-forming units; BFU-E and colony-forming units; CFU-E, respectively) were evaluated by the ability of bone marrow and spleen cells to form colonies of fully differentiated erythroid cells in vitro. MPSV caused substantial modification of both the BFU-E and CFU-E populations in the bone marrow and spleen of infected animals. Changes were detected in the CFU-E population preceding any significant increase in spleen weight. In the bone marrow, the proportion of CFU-E cells increased almost twofold by days 5-10 after virus infection but decreased by day 15. In the spleen, CFU-E frequency rose 40-fold by days 10-15 and then declined steadily prior to death. At the peak of CFU-E expansion, a small proportion of the population appeared to be erythropoietin (Ep) independent, although there was no evidence of a complete switch to Ep-independence which occurs in Friend virus-induced erythroleukemia. Dose-response curves showed that none of these data could be explained in terms of a changing responsiveness to Ep. However, evidence is presented that indicates that BFU-E from MPSV-infected animals lose or have a reduced requirement for burst-promoting activity (BPA) relative to normal cells although their progeny still need Ep for terminal erythroid differentiation.
Assuntos
Eritropoese , Vírus do Sarcoma Murino/fisiologia , Sarcoma Experimental/fisiopatologia , Animais , Medula Óssea/patologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Eritrócitos/patologia , Eritropoetina/farmacologia , Células-Tronco Hematopoéticas/patologia , Camundongos , Camundongos Endogâmicos DBA , Sarcoma Experimental/patologia , Baço/patologiaRESUMO
A structure-activity relationship study of human interleukin-3 (huIL-3) was performed by functional analysis of huIL-3 deletion and substitution variants combined with epitope mapping of huIL-3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL-3 variants was accomplished by defining their capacity to compete with wild-type huIL-3 for binding to the huIL-3 receptor and to induce the proliferation of the huIL-3 dependent cell line M-O7. HuIL-3 variants with either 14 amino acids (aa) deleted from the N-terminus or eight aa from the C-terminus retained full biological activity in vitro. An huIL-3 variant, with 18 N-terminal aa deleted, exhibited a greater than 7-fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C-terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C-terminal region. Computer-aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha-helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10-fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL-3 molecule can be constructed with two active sites in close proximity.
Assuntos
Interleucina-3/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Simulação por Computador , Epitopos/imunologia , Humanos , Interleucina-3/genética , Interleucina-3/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Recombinant human interleukin-3 (rhuIL-3) variants were generated by site-directed mutagenesis and expression in Escherichia coli. Amino acid deletions and substitutions were made in the previously identified epitopes of two huIL-3-specific neutralizing monoclonal antibodies (mAbs). The rhuIL-3 variants were analyzed for their ability to bind to the IL-3 receptor and to induce the proliferation of the human IL-3-dependent cell line M-O7. Several deletion mutants spanning the epitopes of these neutralizing mAbs indicated the importance of residues Pro33 and Leu34 for biological activity. Further, substitution of Pro33 with Asn (Asn33) showed an enhanced proliferative activity (4-fold) and a moderate increase in receptor binding (2-fold) compared to wild-type (wt) rhuIL-3. The most remarkable change, however, was seen with variant Gly33, which showed a 14-fold increase in promoting the growth of M-O7 cells without a significant modification in its receptor binding capacity. In contrast, substitution of Leu34 with Gly (Gly34) yielded an IL-3 variant that had a 25-fold decreased receptor binding capacity and proliferative activity, while Glu34 had properties similar to wild-type rhuIL-3. Analysis of the binding of these variants to different rhuIL-3-specific monoclonal antibodies suggested that no major modification had occurred in their conformations. These results indicate that both residues, Pro33 and Leu34, play a critical role in modulating the activity of rhuIL-3.
Assuntos
Interleucina-3/farmacologia , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Ligação Competitiva , Western Blotting , DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Interleukin-11 (IL-11), a pleiotropic cytokine originally isolated from a primate bone marrow stromal cell line, has been shown to stimulate T-cell-dependent B-cell maturation, megakaryopoiesis, and various stages of myeloid differentiation, but to inhibit adipogenesis. Because stromal cells are essential for the maintenance of early hematopoietic progenitor cells in long-term culture, we investigated the effects of IL-11 on multipotent and erythroid precursors from murine bone marrow in vitro in suspension and semisolid cultures. Our results show that in the presence of IL-3 or c-kit ligand (KL), IL-11 has profound stimulatory effects on primitive multilineage hematopoietic progenitors, pre-CFC(multi), as well as on precursors representing various stages of erythroid differentiation observable in vitro, including CFC(multi), BFU-E, and CFU-E. In addition, the combination of KL with IL-11 also stimulated highly proliferative erythroid progenitors that yield remarkable macroscopic erythroblast colonies in culture. These results indicate that IL-11 is likely to play a pivotal role in early hematopoiesis and at multiple stages of erythropoiesis.
Assuntos
Eritropoese/efeitos dos fármacos , Interleucinas/farmacologia , Animais , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/fisiologia , Eritropoetina/farmacologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Técnicas In Vitro , Interleucina-11 , Interleucina-3/farmacologia , Camundongos , Fator de Células-TroncoRESUMO
The luminal epithelium of the mouse uterus was removed simply and rapidly by opening the uterine horns longitudinally and 'vortexing' them in buffer with glass balls. This procedure, which requires no specialized apparatus, was developed for use with uteri in different hormonal states, and 2 uterine horns/tube, 5 glass balls and vortexing for 2 min are suggested for routine use. Under these conditions, 79--100% of the cells were removed, yielding epithelial fractions of 65--90% purity. The suspension of cell constituents recovered contained intact nuclei which could be further purified. Extensive histological examination demonstrated that the other uterine tissues were minimally damaged.
Assuntos
Técnicas Histológicas , Útero/citologia , Animais , Células Epiteliais , Feminino , CamundongosRESUMO
The novel stromal cell factor, IL-11, has been reported to have diverse effects on the lymphopoietic and myeloid/erythroid cells in vitro. These include expansion of T cell-dependent Ig-secreting B cells, proliferation and differentiation of megakaryocytic progenitors and of a variety of myeloid and erythroid precursor cells, and multiplication of pluripotential hematopoietic progenitors. In addition, IL-11 inhibits adipogenesis in vitro. In vivo administration of IL-11 elevated the number of circulating neutrophils and platelets and increased the number of megakaryocytes in the spleens of normal mice.
Assuntos
Interleucina-11 , Tecido Adiposo/efeitos dos fármacos , Animais , Doenças da Medula Óssea/induzido quimicamente , Doenças da Medula Óssea/fisiopatologia , Doenças da Medula Óssea/terapia , Ciclofosfamida/toxicidade , Feminino , Genes , Hematopoese/efeitos dos fármacos , Humanos , Fatores Imunológicos/uso terapêutico , Interleucina-11/genética , Interleucina-11/farmacologia , Interleucina-11/fisiologia , Interleucina-11/uso terapêutico , Linfócitos/citologia , Megacariócitos/efeitos dos fármacos , Camundongos , Proteínas Recombinantes/farmacologiaRESUMO
A sensitive and specific two-site ELISA was developed for human granulocyte-macrophage colony-stimulating factor (huGM-CSF) based on monoclonal antibodies (mAbs) which have been selected for high affinities and different epitope specificities. Using a cocktail of three mAbs, both for coating and, in their labeled form, for detection, a major increase in sensitivity was achieved (20-fold) compared to a two-site assay employing two different mAbs (one for coating and one for detection). The assay is as sensitive as the most sensitive biological assays. Recombinant mammalian cell expressed and natural huGM-CSF can be reliably detected down to 100 pg/ml (7 pmol/l). In contrast to conventional bioassays, the ELISA is highly specific for huGM-CSF and does not detect other human lymphokines. Results from quantification of recombinant and natural huGM-CSF in ELISA and bioassay correlate.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Anticorpos Monoclonais , Afinidade de Anticorpos , Especificidade de Anticorpos , Bioensaio , Epitopos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Sensibilidade e EspecificidadeRESUMO
The epitopes of neutralizing mAb were mapped in order to identify a receptor binding site on human IL-3 (huIL-3). To initiate this structure and activity analysis, four neutralizing mAb were selected on the basis of preventing rhuIL-3 stimulated proliferation of peripheral blood cells from a patient with chronic myelogenous leukemia (CML). In order to identify continuous epitopes, the neutralizing mAb were assayed in a solid-phase ELISA for their reactivity with either denatured rhuIL-3 or with the peptides generated by digestion of rhuIL-3 by using two different proteinases. Two of the neutralizing mAb recognized single fragments from both digestions. Amino acid (aa) sequence determination showed that these peptides overlap, defining a region of 22 aa (aa 29 to 50 of the mature rhuIL-3 protein). In a competition ELISA, the two continuous epitopes were shown to be linked to one another and to the two discontinuous epitopes, suggesting that all four neutralizing mAb bind to a discrete region of the IL-3 molecule, which might be involved in binding to the IL-3R.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/análise , Interleucina-3/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The human interleukin-3 gene was cloned in 1986 and the biochemical and biological properties of recombinant human interleukin-3 (rhuIL-3) protein were described. In this report we compare rhuIL-3 with nonrecombinant, natural huIL-3, purified from the supernatant of the human T cell leukemia line Jurkat. The main purification step, affinity chromatography, using a selected monoclonal anti-huIL-3 antibody, resulted in an approximately 40,000-fold enrichment of huIL-3. Combination of this step with ion-exchange and reverse-phase chromatography yielded natural huIL-3 of high purity (greater than 98%). A highly sensitive and specific sandwich ELISA, comprising two epitope-mapped monoclonal anti-huIL-3 antibodies, was used to quantitate huIL-3 during purification. Amino acid sequence determination revealed that the 38 N-terminal amino acids of Jurkat-derived huIL-3 are identical to the published sequence deduced from human fetal liver genomic DNA but differ in one residue from that derived from human T cell clones. The degree of glycosylation of Jurkat-derived huIL-3 was similar to Chinese hamster ovary cell-expressed rhuIL-3. Natural huIL-3 showed very similar biological activities to rhuIL-3 in proliferation and receptor binding assays utilizing huIL-3 responsive primary cells and cell lines, as well as in the human bone marrow colony assay.