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D Metcalf

Publications and source records attributed to D Metcalf.

At least 325 records · Page 18Linked to original sources
PubMed1981

The clonal culture in vitro of multipotential hemopoietic cells: problems and interpretations.

The ability to clone mixed hemopoietic colonies in vitro new permits hemopoietic populations to be more effectively assayed for multipotential precursor cells but it is unclear whether all CFU-S are detectable by the culture systems in current use. From the available data, some caution should be used in assuming that the in vitro culture systems give a direct estimate of the repopulating capacity of the population. Although the factors stimulating mixed colony formation have not been purified, it is now possible to analyze the early cellular events during commitment to specific hemopoietic lineages.

Aging↗
PubMed1981

Granulocyte-macrophage colony-stimulating factor produced by an inducible murine T-cell hybridoma: molecular properties and cellular specificity.

The properties of granulocyte-macrophage (GM) colony stimulating factor (CSF) produced by a mouse T-cell hybridoma (T19.1) as a result of stimulation by concanavalin A have been investigated. Stimulation of T19.1 cells (2 x 10(6)/ml) with concanavalin A (5 microgram/ml) in the absence of serum for 24 h led to the production of colony stimulating activity (100 ng/ml). The molecular and biological properties of the hybridoma GM-CSF were compared with similar molecules produced in vitro by mouse lung or muscle cells. When bone marrow cells were stimulated by T19.1 conditioned medium (CM) only colonies containing granulocytes (G), macrophages (M), or a mixture of both cell types developed. The distribution of colony types (G, G/M or M) was dependent on the concentration of T19.1 CM in the same way as the GM-CSF from mouse lung conditioned medium. Similarly GM-CSF from T19.1 CM was also able to stimulate the initial proliferation of other hemopoietic progenitor cells and induce differentiation in the myelo-monocytic leukemic cell line (WEHI3B). The apparent molecular weight of GM-CSF in T19.1 CM as determined by gel filtration on Ultrogel AcA44 was 24,000. Using high performance liquid chromatography, GM-CSF from T19.1 CM eluted at the same apparent molecular weight (32,000) on molecular sieve columns and acetonitrile concentration on reverse phase columns as the GM-CSF from mouse lung and muscle CM. The level of production of GM-CSF and the similarity to the molecule from mouse lung CM indicated that this continuous cell line should be useful for preparing GM-CSF in suitable quantities for structural analysis and in vivo testing.

Animals↗
PubMed1981

Isolation of murine fetal hemopoietic progenitor cells and selective fractionation of various erythroid precursors.

Hemopoietic progenitor cells (colony- and cluster-forming cells in semisolid agar) were purified from light density CBA murine fetal liver cells using fluorescein-conjugated pokeweed mitogen (PWM) and a rhodamine-conjugated antineutrophil serum sandwich (alpha N) and three-parameter fluorescence-activated cell sorting. All clonable progenitor cells were highly enriched (36-50-fold) in PWM-positive (greater than channel 15), alpha N-negative (less than channel 30) fractions with relatively high intensity (greater than 100) low angle light scatter. No separation was achieved between different types of progenitor cells (granulocyte-macrophage and erythroid colony-forming cells). The enriched fraction was a pure population of large, basophilic, undifferentiated blast cells, and in agar cultures stimulated with colony-stimulating factors, up to 90% of the enriched cells were hemopoietic progenitor cells capable of varying levels of clonal proliferation. Further fractionation based on increasing fluorescence with PWM separated into discrete populations, nonproliferative morphologically recognizable erythroid cells, late erythroid progenitor cells (day 2 CFU-E), and cells forming pure or mixed erythroid burst colonies. In addition, the majority of pluripotential hemopoietic stem cells (CFU-SS) were clearly separated from progenitor cells forming colonies in vitro. The present techniques provide suitable numbers of enriched progenitor cells for a variety of biological and biochemical studies.

Animals↗
PubMed1981

Control of hemopoietic cell proliferation and differentiation.

Hemopoietic populations offer by far the best available model systems for analyzing many aspects of the fundamental mechanisms controlling cell proliferation and differentiation. Semisolid cloning systems of high plating efficiency exist for normal and leukemic hemopoietic populations, and in these culture systems both proliferation and differentiation occur under defined culture conditions. Furthermore, the various differentiated end cells differ so extremely in morphology, membrane markers, and functional activity that a wide variety of parameters is available for monitoring differentiation. For the erythroid and granulocyte-macrophage systems, specific progenitor cells can be stimulated to generate large clones of differentiating progeny using the purified glycoprotein regulators erythropoietin, GM-CSF, and M-CSF. Exploitation of these systems will provide information on the events leading to proliferation and differentiation that should have general relevance for many other cell systems.

Animals↗
PubMed1981

Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia.

Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4-5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.

Animals↗
PubMed1980

Characterization of a serum factor stimulating the differentiation of myelomonocytic leukemic cells.

Culture of WEHI-3B myelomonocytic leukemic cells in semi-solid agar medium containing serum from mice injected with endotoxin serum (ES) led to the development of maturing granulocytes and macrophages in most leukemic colonies. ES contains high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), a regulator known to stimulate differentiation of these leukemic cells, but an antiserum which neutralized greater than 85% of the GM-CSF in ES did not suppress the differentiation-inducing activity of ES on WEHI-3B cells. The active factor in endotoxin serum stimulating differentiation in WEHI-3B leukemic cells (GM-DF) was separated from most of the GM-CSF by gel filtration using Ultrogel AcA44. The residual CSF associated with the GM-DF appeared to stimulate selectively granulocytic colonies. Disproportionation of GM-DF and GM-CSF was observed in ES fractions obtained using concanavalin-A/Sepharose chromatography: none of the GM-DF bound to this matrix, whereas 40% of the GM-CSF bound and was eluted with competing alpha methylglucopyranoside. Although no separation of GM-CSF and GM-DF was obtained using DEAE-Sepharose, non-isoelectric focusing in amphoteric buffers indicated charge differences between the differentiation factor and several sub-species of GM-CSF. Sequential purification of GM-DF from ES using 40 - 70% ammonium sulfate precipitation gel filtration and phenyl-Sepharose chromatography resulted in a 25-fold purification. In all fractionation procedures used, a sub-species of GM-CSF, stimulating granulocyte colony formation, was consistently associated with partially purified GM-DF, but some subspecies of GM-CSF clearly lacked any capacity to induce differentiation in the leukemic cells. The observations suggest that the factor in post-endotoxin serum most efficient in enforcing differentiation in myelomonocytic leukemic cells may be a subset of GM-CSF molecules with a selective capacity to stimulate granulocyte colony formation by normal cells.

Animals↗
PubMed1980

Growth of factor-dependent hemopoietic precursor cell lines.

Cell lines have been produced from long-term cultures of mouse bone marrow that require a factor, present in WEHI-3 conditioned medium (CM) or in spleen CM, for their sustained growth. The cell lines were obtained from nonvirus-treated cultures, are nonleukemic, maintain a normal karyotype, and form colonies showing granulocyte maturation when plated in soft agar. Granulocyte/macrophage (GM) colony-stimulating factor is not the inductive moiety involved in the maintenance of proliferation of these cells. It is suggested that the cell lines represent a self-renewing population of cells ancestral to GM colony-forming cells, which may be responding to a hitherto unrecognized regulator.

Animals↗
PubMed1980

Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. I. Isolation of pluripotent hemopoietic stem cells and granulocyte-macrophage progenitor cells.

A scheme is presented whereby pluripotent hemopoietic stem cells (PHSC) from rat bone marrow can be enriched 320-fold with the aid of the fluorescence- activated cell sorter. This scheme is based on the observations that PHSC are strongly positive for Thy-1 antigen (upper 10th percentile); have light- scattering properties (size distribution) between those of bone marrow lymphocytes and myeloid progenitor cells; and are relatively resistant to cortisone. It is estimated that PHSC may constitute 80 percent of the cells isolated according to these parameters. Candidate PHSC are described at the light and electron microscopic levels. At least two populations of accessory cells appear to influence the number and/or the nature of the hemopoietic colonies that form in the in vivo spleen colony-forming unit assay. Putative amplifier cells are strongly Thy-1(+) and cortisone sensitive; putative suppressor cells are weakly Thy-1(+) and cortisone resistant. Three subsets of granulocyte (G) -macrophage (M) progenitor cells (in vitro colony-forming cells [CFC]) are identified on the basis of relative fluorescence intensity for Thy-1 antigen: G-CFC are strongly Thy-l(+); M-CFC are weakly Thy-l(+); and cells that produce mixed G and M CFC have intermediate levels of Thy-1. GM-cluster-forming cells and mature G and M are Thy-1(-). The results suggest that G-CFC are bipotential cells that give rise to G and M-CFC; and that the latter produce mature M through a cluster- forming cell intermediate. Thy-1 antigen is also demonstrated on members of the eosinophil, megakaryocyte, erythrocyte, and lymphocyte cell series in rat bone marrow. In each instance, the relative concentration of Thy-1 antigen is inversely related to the state of cellular differentiation.

Animals↗
PubMed1980

Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. II. Isolation of terminal deoxynucleotidyl transferase-positive cells.

A method is described by which highly enriched populations of viable terminal deoxynucleotidyl transferase-positive (TdT+) cells can be isolated from rat bone marrow by use of the fluorescence-activated cell sorter. Such cells have been postulated to be progenitors of thymocytes and, possibly, of B lymphocytes, and may serve as the targets of neoplastic transformation in acute lymphoblastic leukemia. The separation procedure is based on differences in relative low-angle light scatter and relative fluorescence intensity for Thy-1 antigen between TdT+ cells and other lymphohemopoietic cell populations in bone marrow. Simultaneous sorting of bone marrow cells according to these two parameters resulted in a mean 87% purification of TdT+ cells. The morphological characteristics of the isolated TdT+ cells are described at the light and electron miscroscopic levels.

Animals↗
PubMed1980

Clonal extinction of myelomonocytic leukemic cells by serum from mice injected with endotoxin.

Culture of WEHI-3B myelomonocytic leukemic cells in semi-solid agar medium containing post-endotoxin serum led to the development of maturing granulocytes and macrophages in most leukemic colonies. Colony size was consistently increased but the colony content of colony-forming cells (stem-cell self-replication) was markedly reduced. Serial recloning of WEHI-3B colony cells in the continuous presence of post-endotoxin serum led to clonal extinction of the leukemic cells in five of seven experiments. These effects of post-endotoxin serum on WEHI-3B cells were not seen in clonal cultures of 10 other tumor lines. Serum with the capacity to induce differentiation in WEHI-3B cells could be induced by the injection of as little as 0.1 microgram endotoxin and by purified bacterial cell-wall preparations. Serum activity reached peak levels 3 - 6 h after endotoxin injection and returned to preinjection levels within 48 h.

Animals↗
PubMed1980

Granulocyte/macrophage-, megakaryocyte-, eosinophil- and erythroid-colony-stimulating factors produced by mouse spleen cells.

The formation of mature haemopoietic cells is controlled by hormones that specifically stimulate the progenitor cells of the granulocyte/macrophage, eosinophil, megakaryocyte and erythroid pathways. PWMSC medium (pokeweed-mitogen-stimulated spleen-cell-conditioned medium) is known to contain the biological activities that control the clonal proliferation of these four progenitor cells in vitro in semi-solid agar cultures. In this study the molecular properties of these biological activities were characterized, and all four colony-stimulating factors appear to be associated with glycoproteins. These factors were precipitated between 50 and 80%-satd. (NH(4))(2)SO(4) and could be concentrated by ultrafiltration over a 10000-mol.wt.-cut-off hollow-fibre membrane. Megakaryocyte- and erythroid-colony-stimulating factors were lost when the conditioned medium was dialysed at low ionic strength (<0.03m). Neither asialo- nor sialo-erythropoietin was detectable in concentrated PWMSC medium or in the fractions purified from it by gel filtration on Sephadex G-150. The factors bound to concanavalin A-Sepharose were eluted with alpha-methyl-d-glucopyranoside (0.10m). Analysis by gel filtration on Sephadex G-150 indicated that the apparent molecular-weight distributions of all colony-stimulating factors were identical (37000). Treatment with neuraminidase did not alter the biological activities of any of these factors, but when the molecular weights were analysed, after neuraminidase treatment, on Sepharose CL-6B in the presence of guanidine hydrochloride (6m) all were eluted with a mol.wt. of 24000. Although the apparent molecular weights of the different factors were identical, charge differences were detectable by isoelectric focusing on thin-layer granulated gels. There appeared to be considerable charge heterogeneity associated with each factor, as all were focused over 2-4 pH units. The maximum activity of the granulocyte/macrophage-colony-stimulating factor on isoelectric focusing was at pH4.8, whereas the maximum activity for the eosinophil-colony-stimulating factor was at pH5.8. The erythroid- and megakaryocyte-colony-stimulating activities were detected in the pH ranges 4.8-5.8 and 4.6-7.1 respectively. Chromatographic differences between the granulocyte/macrophage- and eosinophil-colony-stimulating factors were also detected by hydrophobic chromatography at low ionic strength (0.15m-NaCl) on Cibacron Blue-Sepharose and at high ionic strength [2m-(NH(4))(2)SO(4)] on phenyl-Sepharose. Eosinophil-colony-stimulating factor bound more strongly than the other factors to both matrices. The megakaryocyte- and erythroid-colony-stimulating activities were always associated with those for granulocytes/macrophages and eosinophils. Preparations highly enriched for eosinophil-colony-stimulating factor were also obtained by DEAE-cellulose chromatography. An overall purification of 100-fold for all of the factors was achieved with the present techniques, and, although differences were observed, only granulocyte/macrophage-stimulating factors and a small proportion of the eosinophil-stimulating factors could be completely separated from the others. Our results are consistent with the existence of separable factors for granulocyte/macrophage and eosinophil stimulation, but the megakaryocyte- and erythroid-stimulating activities were always associated with the granulocyte/macrophage- and eosinophil-stimulating activities. Thus there may be one molecule that is able to stimulate all four colony types or four very similar molecules that are difficult to separate.

Animals↗
PubMed1980

Differential expression of lectin receptors during hemopoietic differentiation: enrichment for granulocyte-macrophage progenitor cells.

Molecular changes occur at the surface of hemopoietic cells during differentiation from progenitor cells to mature granulocytes and macrophages. The differential expression of surface carbohydrate residues has been probed using lectins and the results used to purify normal mouse granulocyte-macrophage progenitor cells. Ten different lectins were screened for selective interaction with mouse hemopoietic colony-forming cells (CFCs), using agglutination or a quantitative analysis of the number of fluoresceinated lectin molecules bound per cell using a fluorescence activated cell sorter (FACS). Pokeweed mitogen (PWM), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), and peanut agglutinin (PNA) preferentially bound to CFCs so that it was possible to enrich 4 to 10-fold for these progenitor cells by sorting for the highly fluorescent cells. Further analysis of the low and high angle light scattering characteristics of the CFCs indicated that these cells were polydisperse, but could be enriched ten-fold by selecting for cells with high intensity low angle (0 degrees) scatter and low intensity high angle (90 degrees) scatter. PWM gave the best enrichment (10 to 15-fold) for adult bone marrow CFCs, for CFCs from fetal sources (fetal liver, fetal blood), and for CFCs from the spleens of mice injected previously with outer membrane lipoprotein from E. coli. Three parameter sorting for CFC using the FACS (low angle scatter, high angle scatter, and PWM-fluorescence) resulted in large enrichment factors (16 to 50-fold) for CFCs from all the above sources. Over 7% of the cells sorted from bone marrow, 10% of the cells sorted from post-lipoprotein spleen, and 28% of the cells sorted from fetal peripheral blood were hemopoietic CFCs. Ninety percent of the cells in these fractions had the morphology of blast cells or myelocytes. Thus, it was possible to identify the morphological characteristics of the hemopoietic progenitor cells. Screening of other developmental systems using quantitation of fluorescence with lectins should prove of general value for the purification of selected differentiation states.

Animals↗
PubMed1980

Cellular and molecular basis of the increased splenic hemopoiesis in mice treated with bacterial cell wall components.

An analysis was made of the mechanisms responsible for the increased splenic hemopoiesis occurring in mice after the injection of the bacterial cell wall components lipid A and outer membrane lipoprotein. No evidence was obtained for the presence of functional lipid A receptors on hemopoietic precursor cells. Serum from lipid A-injected mice, on injection into normal mice, induced in the spleen an increased content of all hemopoietic progenitor cells. The magnitude of the response was dependent on the dose of lipid A used and the volume of serum transferred to the recipients. C3H/HeJ mice unresponsive to lipid A exhibited similar spleen changes when injected with active post-lipid A sera. Progenitor cells of all hemopoietic lineages, including multipotential hemopoietic stem cells, were involved in the response. The results suggest that a humoral factor mediates the lipid A-induced increase of splenic hemopoiesis.

Animals↗
PubMed1980

Clonal analysis of proliferation and differentiation of paired daughter cells: action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors.

Mouse granulocyte-macrophage progenitor cells were stimulated to divide by the granulocyte-macrophage colony-stimulating factor (GM-CSF). The two daughter cells were separated; one daughter was transferred to medium containing a high concentration of GM-CSF, the other to medium containing a low concentration. Daughter cell-derived clones in the presence of 2500 units of GM-CSF had average cell cycle times 3.5 +/- 2.5 (SEM) hr shorter than clones derived from the paired daughter cell stimulated by 50 units of GM-CSF. Final colony size achieved after stimulation by 50 units of GM-CSF was always smaller than that of colonies stimulated by 2500 units of GM-CSF. In 8 of 41 instances, colonies stimulated by 50 units of GM-CSF developed, or were composed only of, macrophage populations in contrast to the granulocytic composition of colonies derived from the paired daughter cell growing in the presence of 2500 units of GM-CSF. The regulator GM-CSF appears able to directly influence cell cycle times and the pathway of differentiation entered by many bipotential granulocyte-macrophage precursor cells.

Animals↗
PubMed1980

A clinical study of delirium in children and adolescents.

A group of 33 children and early adolescents with acute central nervous system disorders of toxic, metabolic, traumatic, and other types were studied in the hospital. They were compared with a matched control group, also in the hospital, who had no central nervous system disorder. Methods of study for both groups included neurological examination; mental status; electroencephalographic tracings (using developmental norms established here); neuropsychological and other techniques carried out during the acute disorder and repeated after its subsidence. Results distinguished significantly between the two groups, confirming the findings of Romano and Engel with adults. Some interesting differences related to childhood developmental level were noted: the occurrence of regression, the persistence of mild perceptual-motor and other abnormalities for some weeks even after complete recovery (involving potential learning difficulties), and the finding of a significant number of "soft" neurological signs in the control group.

Adolescent↗
PubMed1980

Isolation of mouse bone marrow neutrophils by light scatter and autofluorescence.

Murine bone marrow and blood cells have been analyzed and fractionated using an automated FACS II cell sorter. Using visible light scattered in the direction of (0 degrees) and perpendicular to (90 degrees) the laser beam it was possible to enrich for neutrophils (84%), immature myeloid cells (47%), and monocytes (78%). The enrichment for neutrophils was improved to 92% by using the light scattered by ultraviolet laser light (ca.360 nm). The autofluorescence at these wavelengths proved useful for obtaining further enrichment (to 97%). Indeed, three parameter sorting with 0 degrees and 90 degrees light scatter as well as autofluorescence also allowed the separation of lymphocytes (95%) and immature myeloid cells (89%). The same procedures could be applied for the isolation of neutrophils from mouse peripheral blood.

Animals↗
PubMed1980

An innovative approach to methadone detoxification.

An atmosphere can be created in which the user's anxiety during methadone or heroin detoxification is reduced via the facilitation of adaptive, druglike experiences provided through behavioral and psychological means. The switch to a nondrug life-style is promoted by the continued satisfaction of underlying needs which, through appropriate dosage, can lead to a higher probability of self-reliance abd abstinence. The addictive dependency on the nondrug, need-gratifying therapy is resolved through clinically monitored and graded frustrations.

Fantasy↗