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D Metcalf

Publications and source records attributed to D Metcalf.

At least 361 records · Page 20Linked to original sources
PubMed1979

Peritoneal cell population of mice infected with Mesocestoides corti as a source of eosinophils.

A prominent feature of the inflammatory cellular response in the peritoneal cavity of Mesocestoides corti-infected mice is a marked and sustained increase in the number of eosinophils. In intact mice, the total number of nucleated cells in the peritoneal cavity rises from less than 5 X 10(6) to more than 50 X 10(6) and, at certain time points, in excess of 50% of these cells are eosinophils. Peritoneal eosinophils are absent in infected hypothymic nude (nu/nu) mice of three genotypes, and eosinophils counts can be elevated in infected nude mice by injection of peripheral lymphoid cells or thymocytes. The peritoneal cells of M. corti-infected mice are a convenient starting cell population for eosinophil purification.

Animals↗
PubMed1979

Two cases of carcinoma of the lung characterized by a bone marrow agar culture pattern resembling acute myeloid leukemia.

In vitro agar culture patterns of bone marrow cells in acute myeloid leukemia may show several growth patterns, including cultures where no colonies or clusters develop, cultures with varying numbers of clusters and no colonies, or colony and cluster formation with an extremely high ratio of clusters to colonies. Twelve cases of carcinoma of the lung are described, of which two show an in vitro growth pattern of cluster formation alone, characteristic of that seen in acute myeloid leukemia. The remaining ten patients showed slightly reduced colony numbers compared to normal.

Bone Marrow↗
PubMed1979

Separation of functionally distinct human granulocyte-macrophage colony-stimulating factors.

Human placental conditioned medium (HPCM) contans colony-stimulating factors (CSFs) required for the growth in vitro of neutrophilic granulocyte-macrophage (GM) and eosinophilic (EO) progenitor cells from human bone marrow. Fractionation of CSFs in HPCM was achieved by manipulation of the elution conditions on a column of phenyl-Sepharose. After equilibration of the phenyl-Sepharose column at high ionic strength (1 M ammonium sulfate), all of the CSF bound; one species of GM-CSF (alpha) and all of the elutable EO-CSF were eluted from the column simply by reducing the salt concentration, whereas the second species of GM-CSF (beta) was free of EO-CSF and was eluted only by increasing the concentration of tehylene glycol in the elution buffer. The two GM-CSFs were functionally distinct. GM-CSF alpha preferentially stimulated colony formation by day 14 of culture, and there was a decreased proportion of neutrophil colonies and increased proportion of macrophage colonies as the strength of the stimulus was decreased; GM-CSF beta, on the other hand, preferentially stimulated colony formation by day 7 of culture, and the proportion of neutrophil colonies was high (average 80%) and independent of the concentration of GM-CSF beta. GM-CSF alpha and GM-CSF beta were indistinguishable on the basis of apparent molecular size on tel filtration columns (molecular weight 30,000), charge properties on isoelectric focusing beds (isoelectric point, 4.9), and were not related to each other as a sialoglycoprotein is related to its asialo form. Adherent cell removal of the target bone marrow cells (to remove colony-stimulating cells) suggested that both GM-CSFs acted directly rather than by stimulating the production of GM-CSF. Mixing and titration experiments indicated that the differences in functional specificities of the two GM-CSFs (and the lack of EO-CSF associated with GM-CSF beta) were not due to the presence of specific inhibitory molecules or lower absolute levels of CSF in one fraction relative to the other. These two species of GM-CSF should be useful in separately enumerating subpopulations of different GM-progenitor cells inhuman hemopoietic disorders.

Bone Marrow Cells↗
PubMed1979

Detection and analysis of human granulocyte--monocyte precursors using semi-solid cultures.

The in vitro cloning of haemopoietic precursors is a rapidly growing field. The data reviewed above and the current practical applications of the techniques can be expected to increase quite rapidly in the next decade. Despite the technical problems of tissue culture and the special problems associated with culturing human cells, it is clear that these procedures can be effectively applied at the clinical level. The value of the data obtained will vary directly with the quality of the culture techniques. Any centre undertaking these techniques must be prepared to properly equip the culture laboratory, appoint a full-time staff member for the work and maintain a constant surveillance of the quality of the culture work.

Agar↗
PubMed1978

Membrane receptors of mouse leukocytes. II. Sequential expression of membrane receptors and phagocytic capacity during leukocyte differentiation.

Analysis of four mature cell markers on mouse bone marrow leukocytes grown in vitro, demonstrated a distinct sequence of marker appearance during the terminal phases of granulocytic cell differentiation. A similar pattern of marker expression was also suggested by analysis of mature neutrophils and macrophages isolated from normal tissues. Among cultured neutrophils, receptors for the Fc portion of IgG (FcR) were first expressed on myelocytes and metamyelocytes, and then subsequently on more mature cells. Morphologically mature colony neutrophils (polymorphs) from agar cultures contained only FcR and complement receptor type two (CR(2)) (C3d receptor), and lacked both complement receptor type one (CR(1)) (C3b receptor) and the capacity to ingest latex, bacteria, or iron particles. Neutrophils from 2 and 3 wk liquid media cultures of marrow cells differed from agar grown neutrophils in that they had phagocytic capacity (particle ingestion) [Pi] in addition to FcR and CR(2). Furthermore, in the 4th and 5th wk of these continuous liquid cultures, CR(1) was also expressed, completing the surface marker profile of normal blood neutrophils. Based on these studies, the following order of appearance of these four markers on cells from the myelocytic series was proposed: FcR {arrow} FcR CR(2) {arrow} FcR CR(2) Pi {arrow} FcR CR(2) Pi CR(1). Differential studies of tissue leukocytes containing these same markers revealed that a heterogeneity existed among morphologically mature neutrophils. Even though 95 percent of blood polymorphs contained all four markers, the same was true of only half of spleen polymorphs and only 20 percent of bone marrow polymorphs. Cells of the monocyte-macrophage series were studies in parallel with neutrophils. Cultured marrow monocytes acquired the four mature cell markers so rapidly that the order of receptor appearance could not be determined. However, it was found that CR2 was lost during the terminal phase of monocyte maturation into activated macrophages.

Animals↗
PubMed1978

Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium.

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.

Animals↗
PubMed1978

Regulation of hemopoietic cell differentiation and proliferation.

Differentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakaryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin. The rate of proliferation of the GM-progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM-CSF (10(-11) M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte-macrophage differentiation appears to be related to a) the concentration of GM- CSF and b) the different sensitivity of a subpopulation of monocyte colony-forming cells which are responsive to GM-CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM-CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM-CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of 125I-labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Cell Differentiation↗
PubMed1978

Separation of mouse bone marrow cells using wheat germ agglutinin affinity chromatographyy.

Mouse bone marrow cells were fractionated on columns of wheat germ agglutinin-Sepharose 6MB (WGA-Sepharose) and conditions established for specific binding and cell enrichment in the eluted fractions. A small proportion (7%) of the cells applied did not bind to the column and 60% of these were lymphocyte-like cells. Twice as many cells were eluted from the WGA-Sepharose column using N-acetyl-D-glucosamine (GlcNAc), and 80% of these cells were polymorphonuclear granulocytes and metamyelocytes. These cells were only released from the matrix in the presence of GlcNAc at high buffer flow rates (greater than 4 ml/min). Approximately 3 x 10(8) bone marrow cells bound to 1 ml of WGA-Sepharose. At least 5 min were required for 90% binding of the cells and elution of the cells with GlcNAc required nearly 20 min at 24 degrees. At 37 degrees the elution of cells with ClcNAc was much faster (less than 5 min) and a much larger percentage of cells (40-50%) was specifically eluted. Sodium azide (0.02%, w/v) did not prevent cells binding to WGA-Separose or alter the distribution of cells eluted by GlcNAc, but did slightly increase cell yields. Elution of cells with different concentrations of GlcNAc gave fractions enriched in different cell types. Analysis of cell fraction with a fluorescence-activated cell sorter showed that the lymphocyte subpopulation which failed to bind to WGA-Sepharose had been depleted of cells with a high density of immunoglobulin on their surface. The lymphocytes with a high density of surface immunoglobulin were recovered in the cells released from WGA-Sepharose using mechanical agitation.

Acetylglucosamine↗
PubMed1978

Sources and nature of granulocyte-macrophage colony stimulating factor in fetal mice.

At the earliest stages of fetal hepatic hemopoiesis in CBA mice (11-12 days gestation), colony stimulating activity could be found only in peripheral blood, yolk-sac fluid and media conditioned by yolk-sacs (YSCM). The colony stimulating factor (GM-CSF) from YSCM was able to be concentrated by absorption to DEAE-cellulose and subsequent elution. Titration of this material produced a sigmoid dose-response curve in agar cultures of adult CBA bone marrow cells. Unlike the high proportion of granulocyte colonies stimulated by the GM-CSF from mouse lung conditioned medium, all concentrations of YSCM produced a high proportion of macrophage colonies after 7 days of incubation. Mixing experiments eliminated the possibility that a specific inhibitor preventing granulocyte differentiation was present in YSCM. Fetal liver cells were relatively unresponsive to YSCM, but their ability to respond increased with gestational age. When stimulated by YSCM, fetal liver colony forming cells from mice of all gestational ages produced more than 90% macrophage colonies after 7 days of incubation. The experimental data suggest that the proliferation and differentiation of granulocyte and macrophage precursors in the early fetal liver could be controlled by a fetal type of GM-CSF favoring macrophage production.

Animals↗
PubMed1978

In vitro cloning of hemopoietic cells.

Semisolid cloning systems are now available to detect the specific progenitor cells of neutrophilmacrophages, eosinophils, megakaryocytes and erythroid cells. Colony proliferation in vitro with the production of mature progeny requires stimulation by glycoprotein regulators specific for each hemopoietic class. Two of these, erythropoietin and GM-CSF have been purified. A new cloning system has been developed using spleen conditioned medium that detects multipotential hemopoietic cells in the mouse.

Animals↗
PubMed1978

Regulation of hemopoiesis.

The development of in vitro cloning systems for populations of hemopoietic and lymphoid cells has enabled a family of specific regulatory macromolecules to be detected and characterized. These macromolecules control proliferation and differentiation in hemopoietic cell populations. Studies have demonstrated the complexity of the spectrum of molecules which are involved in the regulation of each group of hemopoietic cells. Heterogeneous subpopulations of molecules exist for each regulatory function and more than one molecular form exists for the various regulator molecules. While the cellular origin of many of these regulators has not been clearly demonstrated, it is apparent that hemopoietic populations themselves can be significant sources of both stimulatory and inhibitory regulators. In particular, there are now clear examples of regulatory interactions which occur in both directions between hemopoietic and lymphoid populations. While in no case a complete analysis of the control systems has been achieved, maintenance of homeostasis in hemopoietic systems is now better understood than for most other cell populations in the body. Further, with existing techniques, and in particular, the exploitation of the full potential of the semi-solid cloning systems, it is realistic to expect that a complete analysis of hemopoietic regulation can now be accomplished.

Cell Differentiation↗
PubMed1978

Characterization of mouse fetal liver granulocyte-macrophage colony-forming cells using velocity sedimentation.

Fetal liver cells from CBA mice were separated by velocity sedimentation to determine the distribution and properties of granulocyte-macrophage colony-forming cells (GM-CFC). Twelve-day fetal liver GM-CFC separated into two peaks (s=7.7 mm/h, s=9.4 mm/h) in cultures stimulated by mouse lung conditioned medium (GM-CSF MLCM) or human urine (GM-CSFHU). Because fetal liver GM-CFC are of relatively light bouyant density, this finding indicates that fetal liver GM-CFC are much larger than corresponding cell in adult bone marrow (s=4.5 mm/h). By 18 days gestation more slowly sedimenting colony-forming cells were present (s=4.7 mm/h, s=6.0 mm/h). At all gestational ages, the most rapidly sedimenting (larger) colony-forming cells were more responsive to stimulation by GM-CSF MLCM than the smaller (slowly sedimenting) cells. Unlike the situation with adult marrow cells, velocity sedimentation achieved no segregation of GM-CFC according to the morphological type of colony produced.

Animals↗
PubMed1977

Purification and properties of colony-stimulating factor from mouse lung-conditioned medium.

Colony-stimulating factor, which specifically stimulates mouse bone marrow cells to proliferate in vitro and generate colonies of granulocytes, or macrophages, or both, was purified 3500-fold from mouse lung-conditioned medium. Analysis by discontinuous polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate indicated that there was a single protein component. All of the colony-stimulating activity was coincident with the protein band. The molecular weight of colony-stimulating factor estimated by gel filtration was approximately 29,000 and by electrophoresis approximately 23,000. The specific activity of purified colony-stimulating factor from mouse lung-conditioned medium bound to concanavalin A-Sapharose, indicating that it is a glycoprotein. The small percentage of colony-stimulating factor in mouse lung-conditioned medium which did not bind to concanavalin A-Sepharose appeared to represent molecules which lacked the carbohydrate moieties required for binding to this lectin. It was necessary to include low concentrations (less than 0.01%, v/v) of polymers such as gelatin and polyethylene glycol, or nonionic detergents such as Triton X-100, in all of the buffers used throughout the purification scheme, otherwise colony-stimulating factor was lost from solution. At high concentrations (greater than 20 mug/ml) the factor stimulated the formation of granulocytic, macrophage, and mixed colonies from C57BL mouse bone marrow cells. As the concentration of purified colony-stimulating factor was decreased, the frequency of colonies containing granulocytes also decreased. At low concentrations of colony-stimulating factor (less than 70 pg/ml) only macrophage colonies were stimulated.

Animals↗
PubMed1977

The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM-CSF appeared to stimulate RNA-synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM-CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM-CSF had no apparent effect on the uptake of 3H-uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 x 10(6) cells/ml but was slightly smaller at 0.1 and 40 x 10(6) cells/ml. Increasing concentration of GM-CSF (up to 2 X 105 units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml. GM-CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells. Autoradiographic studies showed that GM-CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM-CFS. Labeling in lymphoid-like cells was highly variable but the level of labeling did not appear to be influenced by GM-CSF.

Animals↗