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Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 379 records · Page 21Linked to original sources
PubMed1977

Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin.

Cells from CBA fetal mouse liver formed pure or mixed erythroid colonies in semisolid agarculture after stimulation by medium conditioned by pokeweed mitogen-stimulated mouse spleen cells. In general shape, the erythroid colonies resembled typical 7-day single or multiple (burst) colonies. However one-third to one-half contained, in addition to erythroid cells, macrophages and neutrophils and, less commonly, megakaryocytes or eosinophils. Culture of micro manipulated single colony-forming cells showed these erythroid colonies to be clones. Colony-forming cells declined in frequency with advancing fetal age, but low numbers were detectable in adult bone marrow. Assays of spleen conditioned medium in polycythemic mice failed to detect erythropoietin; the cloning system may detect a fetal type of erythropoietin-independent, erythropoietic cell since few were detected in adult marrow.

Animals↗
PubMed1977

In-vitro cloning techniques for hemopoietic cells: clinical applications.

Proliferating populations of neutrophils, monocytes, eosinophils, erythroid cells, and T-lymphocytes from normal subjects or patients with various diseases can now be analysed by colony formation in semisolid cultures. These cultures accurately determine the number and proliferative activity of the precursor cells of each population and can also be used to monitor the levels of specific regulatory factors (for example, erythropoietin, colony-stimulating factor) in the serum or urine of such patients. Studies using semisolid cultures have shown that the leukemic cells in chronic and acute myeloid leukemia remain dependent on the normal regulator, granulocyte-macrophage colony-stimulating factor, for proliferation. The cultures have proved valuable in the prognostic assessment of acute leukemic patients and in monitoring impending changes in the clinical status of patients with acute or chronic myeloid leukemia or myeloproliferative disorders.

B-Lymphocytes↗
PubMed1977

Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells.

Medium conditioned by human placental tissue was found to stimulate granulocytic and monocytic colony formation by human marrow cells in semisolid agar cultures. The colony-stimulating activity of unfractionated conditioned medium was equivalent to the activity of standard peripheral blood underlayers. Placentas were a reliable source of active material, and one placenta provided enough material to stimulate 5,000-10,000 cultures of normal or leukemic cells. The colony-stimulating factor in human placental conditioned medium (CSFHPCM) was concentrated and purified 1800-fold using ammonium sulfate precipitation, calcium phosphate gel absorption, DEAE-cellulose batch absorption, gel filtration on Sephadex G-150, and polyarcylamide gel gel electrophoresis. The active factor behaved on gel filtration as a macromolecule with an apparent molecular weight of 30,000 daltons. The active factor in placental conditioned medium was not dependent on the presence of adherent marrow cells with endogenous colony-stimulating activity.

Bone Marrow↗
PubMed1977

Serum half-life and organ distribution of radiolabeled colony stimulating factor in mice.

Granulocyte-macrophage colony stimulating factor (GM-CSF) from mouse lung conditioned medium has recently been purified. This GM-CSF was radioiodinated using the chloramine T method, but dimethylsulfoxide was needed to prevent the loss of biological activity during the iodination. 125I-labeled GM-CSF was isolated from the reaction mixture by gel filtration and affinity chromatography with concanavalin A-Sepharose. One hour after intravenous injection of 125I-labeled GM-CSF almost 80% of the radioactivity was still localized in the serum. 125I-labeled GM-CSF was cleared from the blood of C57BL mice with a serum half-life of 7.3 +/- 1.3 hours. Six hours after intravenous injection 125I-GM-CSF appeared to be distributed throughout most of the tissues of C57BL mice. There was no selective concentration of 125I-labeled GM-CSF in the bone marrow, but levels in the kidney were three times higher than expected from the distribution of 125I-labeled mouse serum albumin. 125I detected in the blood 6 hours after injection of 125I-labeled GM-CSF was precipitable with trichloroacetic acid and bound to concanavalin A-Sepharose. 125I detected in the urine was not macromolecular and appeared to result from the degradation of 125I-labeled GM-CSF.

Animals↗
PubMed1977

Colony formation by human T lymphocytes in agar medium.

An improved method is described for growing human T-lymphocyte colonies in agar medium containing phytohaemagglutinin (PHA) and sheep red blood cells (SRBC). Cluster and colony growth was obtained when blood mononuclear cells were plated directly in the agar-medium (one-step procedure) or after incubation of cells in liquid medium with PHA (two-step procedure). In the one-step procedure approximately 1 per 100 cells plated formed a cluster containing four to fifty cells. In the two-step procedure 1 per 20 cells plated formed a cluster or a colony (more than fifty cells). The proliferating cells were shown to be sheep-erythrocyte rosette-forming cells (E-RFC). Optimal proliferation was dependent on the presence of phagocytic cells in the cell suspensions cultured. No growth occurred in cultures depleted of E-RFC. Detailed studies of the cycle, velocity sedimentation, and density of the cells plated showed that the majority of cluster- and colony-forming cells were small non-cycline lymphocytes with a sedimentation velocity of 4 mm/hr, and a density between 1-069 and 1-077 g/cm3.

Agar↗
PubMed1977

B lymphocyte colony-forming cells in the SJL/J mouse.

Mercatoethanol-induced B lymphocyte cloning in semi-solid agar has been used to study lymphocyte colony formation by cells from the SJL/J mouse thymus. From the 3rd month of life, the SJL/J mouse thymus. From the 3rd month of life, the SJL thymus develops an increasing frequency of cells forming B lymphocyte colonies in agar. The peak frequency in 6- to 12-month-old mice was one colony per 1000 to 2000 cultured thymus cells. In contrast, 10 to 100 times lower frequencies were found in the thymus of five other inbred mouse strains. The rise in B lymphocyte colony-forming cells correlated well with the age-related rise in Ig-positive cells and approximately 50% of the colony cells reacted with anti-micron-serum indicating the B lymphocyte nature of the colony cells. Colony-forming cells from the thymus showed higher sensitivity than colony-forming spleen cells to cortisol and irradiation. Cell transfer experiments and thymus grafting suggested that the increased frequency of colony-forming cells in the thymus is caused by development of special thymus-seeking B lymphocytes in ageing SJL/J mice. Finally, B lymphocyte colony-forming cells were found to be more frequent in the thymus, spleen, and lymph nodes from healthy aged mice than in lymphoid organs from mice with spontaneous reticulum cell tumors.

Age Factors↗
PubMed1977

Normal human bone marrow cultures in vitro: cellular composition and maturation of the granulocytic colonies.

The analysis of single haemopoietic colonies grown in methylcellulose and in agar was performed at intervals by a cytocentrifugation method. Correlation was established between morphology of the whole colonies and their cellular content. Three main cell lines predominated: neutrophils, macrophages, eosinophils; a few colonies contained a pure population of basophil-like granulocytes. Development was followed from myeloblasts to polymorphs, both being present in most of the colonies. Cumulated results showed that (1) the proliferating compartment remained quite large till day 14, with 58% of cells in S phase, and rapidly decreased after day 16, and that (2) the polymorphs rapidly disappeared from the culture medium. Differentiation proceeded at different rates from one colony to the other, thus suggesting heterogeneity between colony forming cells (CFC). Neutrophil colonies appeared and lysed more rapidly than did eosinophil colonies. Macrophages arose from large immature cells with many promyelocyte features; such cells were present in mixed colonies, containing both neutrophils and macrophages. It is very likely that granulopoiesis results from the development of distinct committed CFC. This work was carried out using normal human bone marrow and may be a useful tool for studying pathological material in the future.

Adult↗
PubMed1977

Eosinophil colony formation in semisolid cultures of human bone marrow cells.

A method for in situ staining and scoring of eosinophil colonies and clusters in the whole culture dish has been developed for agar cultures of human marrow cells. Cultures stimulated by human placental conditioned medium were found to develop the same proportion of eosinophil, neutrophil, and monocyte colonies as cultures stimulated by conventional peripheral white cell underlayers. Eosinophil colonies and clusters always contained pure populations of eosinophils, and no mixed colonies of eosinophils with other cells were observed. Eosinophil colony formation was delayed in onset relative to neutrophil or monocyte colony formation, but by day 14 eosinophil colonies comprised 20% of all colonies. Eosinophil colonies were absent in cultures of marrow cells from patients with acute myeloid leukemia in relapse, but eosinophil colonies and clusters were found with higher than normal frequency in cultures from acute leukemic patients in remission. A high frequency of eosinophil colonies and clusters was also observed in cultures from three patients with idiopathic thrombocytopenic purpura. The new staining procedure should permit a wide range of studies on eosinophil precursor populations in the marrows of patients with various diseases.

Bone Marrow Cells↗
PubMed1977

Radiation-induced enlargement of granulocytic and macrophage progenitor cells in mouse bone marrow.

The peak sedimentation velocity of C57BL mouse bone marrow progenitors of granulocytes and macrophages (GM-colony-forming cells, GM-CFC's) increased from 4.3 mm/h to 7-8 mm/h by 2 days after 250 rad whole body irradiation and slowly returned to normal over the next 3 weeks. Ppreliminary irradiation and/or endotoxin injection did not prevent this radiation-induced change. Some change in sedimentation velocity was seen with as little as 100 rad irradiation. Neither buoyant density nor cell cycle changes could account for the sedimentation velocity data which therefore indicate a major volume increase in the GM-CFC's. This size enlargement affected all subpopulations of GM-CFC's which consequently maintained their size relationship with one another.

Animals↗
PubMed1977

Heterogeneity in human neutrophil, macrophage and eosinophil progenitor cells demonstrated by velocity sedimentation separation.

Progenitor cells of neutrophils, monocyte-macrophages, and eosinophils in human marrow were enumerated in agar cultures stimulated by placental conditioned medium or white cell underlayers. Fractionation of marrow populations by velocity sedimentation showed that the profiles of neutrophil and macrophage colony-forming cells shifted from a peak of 8-9 mm/hr in 7-day cultures to a peak of 6-7 mm/hr in 14-day cultures. This shift was due to degeneration of some early colonies formed by rapidly sedimenting cells and the delayed formation of colonies by slowly sedimenting cells. Eosinophil colony formation was delayed until the second week of incubation. Further evidence of heterogeneity was the observation that rapidly sedimenting colony forming cells were more responsive to stimulation than more slowly sedimenting cells. In the macrophage and eosinophil populations, cluster-forming cells were partially segregatable form colony-forming cells. The observed heterogeneity was similar to the described previously in the mouse and suggests that separate subpopulations of progenitor cells may exist within each hemopoietic family that could possibly give rise to functionally different progeny.

Cell Separation↗
PubMed1977

Eosinophil and neutrophil colony-forming cells in culture.

Clusters of cells obtained from a short-term culture of human bone marrow cells in methylcellulose were transplanted in cell-free plates containing conditioned medium. Of 2559 transplants, 439 gave small clusters, 223 gave large clusters, and 70 gave colonies. A better cloning efficiency was achieved from 4-day-old clusters, from aggregates containing 3 cells or more, and from large cells rather than from small cells. Cytocentrifugation could be performed on 215 large clusters or colonies. The population consisted of pure neutrophils in 36.3% and of pure macrophages in 36.8%. A mixture of neutrophils and macrophages was found in 23.2%, thus indicating that these two cell lines originated from the same committed cell (G-CFC). Eosinophils were found in 8 (3.7%) clusters as a pure population, and were never mixed with either neutrophils or macrophages in the other 207 clusters. Despite the small number of eosinophil colonies, one could suggest that using the in vitro technique the committed eosinophil colony-forming cells could be distinguished from the G-CFC.

Cells, Cultured↗
PubMed1976

Cancer research for small countries: why and how?

Small countries can, and in fact have a duty, to carry out cancer research. To be effective research must be carried out by teams of no fewer than eight to 10 full-time professional research workers, adequately funded on a relatively long-term basis.

Australia↗
PubMed1976

The nature of the cells generating B-lymphocyte colonies in vitro.

B-lymphocyte colonies are grown in semi-solid agar from mouse spleen or lymph node cells in the presence of mercaptoethanol with or without added sheep red cells. High levels of colony-forming cells were present in the spleen or normal mice and nu/nu (athymic) mice but colony-forming cells were rare in the thymus and not detected in activated T-lymphocyte populations. Colony-forming cells were theta-negative and most exhibited Fc receptors. Most colony-forming cells had the sedimentation velocity of small lymphocytes, were non-adherent and had a buoyant density similar to B-lymphocytes. Colony-forming cells were radiosensitive (Do60 rads) and sensitive to cortisone. Colony formation was potentiated by the addition of adherent spleen cells or peritoneal macrophages. It is concluded that most cells forming B-lymphocyte colonies are themselves characterisable as B-lymphocytes.

Animals↗
PubMed1976

Endotoxin-induced size change in bone marrow progenitors of granulocytes and macrophages.

Injection of 5 mug endotoxin to adult C57BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.

Animals↗
PubMed1976

Stimulation of B-lymphocyte colony formation in vitro by sera from patients with leukaemia or lymphoma.

Studies were made on the effects of 665 sera, from normal donors or patients with various diseases, on B-lymphocyte colony formation in agar by mouse spleen cells. Undiluted serum from most normal donors inhibited colony formation, but 43-53% of sera from patients with histiocytic lymphoma, lymphocytic lymphoma or Hodgkin's disease stimulated colony formation, serum activity correlating with the stage of the disease. Moderate colony-stimulating activity was observed with serum taken from patients with acute lymphoid or myeloid leukaemia following, but not prior to, chemotherapy. Colony stimulating activity was not correlated with the blood group of serum donors and could not be ascribed to the presence of endotoxin, red cells or mouse red cell haemagglutinins in the active sera. Elevated colony stimulating activity was not observed in sera from patients with non-neoplastic disorders ot haemopoiesis or with diseases of other organ systems.

Cell Division↗
PubMed1976

Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.

Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.

Animals↗