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Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 307 records · Page 17Linked to original sources
PubMed1983

Selective isolation of murine erythropoietin-responsive progenitor cells (CFU-E) with monoclonal antibodies.

Erythropoietin-responsive progenitor cells (CFU-E) from normal mouse foetal liver have been substantially purified on the basis of their differential binding to two monoclonal antibodies in conjunction with flow cytometry. This has allowed the formal identification of the foetal liver CFU-E as an early erythroid blast cell with a highly basophilic cytoplasm. It has also been possible to show a quantitative association of a membrane marker with proliferative potential within a single differentiation lineage. This is the first demonstration of such an association. Progenitor cells enriched in this way should allow study of the molecular mechanisms of erythropoietin action and also serve as a target for determining the cellular specificity of erythroid-transforming viruses.

Animals↗
PubMed1982

Autoinduction of differentiation in WEHI-3B leukemia cells.

Cells of the differentiation-responsive mouse myelomonocytic leukemia cell line WEHI-3B D+ form colonies in agar exhibiting a low frequency of spontaneous differentiation mainly in the macrophage pathway. Compared with undifferentiated colonies, spontaneously differentiating colonies have a reduced content of clonogenic cells and surviving clonogenic cells tend themselves to form differentiating colonies, both being characteristics of differentiated colonies induced by the regulator, granulocyte colony-stimulating factor, G-CSF. Colony crowding increased the frequency of spontaneously differentiating colonies and WEHI-3B D+ colony cells were shown to release material able to induce differentiation in WEHI-3B D+ colonies. Cells from spontaneously differentiating D+ colonies were not hyperresponsive to the induction of differentiation by G-CSF and did not release larger amounts of differentiation-inducing material than did cells from undifferentiated colonies. Cells of the differentiation-unresponsive WEHI-3B D-line produced similar amounts of differentiation-inducing material to those produced by D+ cells. Apparently spontaneous differentiation in WEHI-3B D+ colonies seems most likely to be due to exposure of the colony-forming cell or its ancestors to a differentiation-inducing factor of WEHI-3B origin prior to culture in agar, the genetic program initiating differentiation being inherited by the progeny of the exposed cell.

Animals↗
PubMed1982

Regulator-induced suppression of myelomonocytic leukemic cells: clonal analysis of early cellular events.

Mouse WEHI-3B myelomonocytic leukemic cells were cloned in semi-solid agar in the presence of post-endotoxin serum (ES) or semi-purified differentiation factor (DF). At the 2-cell, 4-cell and 8-32-cell stage, individual cells were washed and recloned in control cultures to test for clonogenicity and the ability to form differentiating progeny. With increasing clone size, an increasing proportion of treated cells either failed to proliferate or generated differentiating colonies of reduced size. The observed suppression indicated that both ES and DF exert irreversible effects on leukemic stem cell self-replication within one or two cell cycles. The stem cell suppression observed was markedly asymmetric and most often involved premature differentiation and death of progeny of the affected cells rather than immediate death of the cell. The results are consistent with the possibility that the regulator DF irreversibly modifies one or more of the newly synthesised daughter chromatids of myeloid leukemic cells during the induction of stem cell suppression and differentiation.

Animals↗
PubMed1982

Enhancement of human blood eosinophil cytotoxicity by semi-purified eosinophil colony-stimulating factor(s).

Purified human blood eosinophils, when incubated in human placental conditioned medium (a source of colony-stimulating factors) [CSF]) demonstrate an enhanced ability to damage antibody- or complement-coated schistosomula. This enhancement represents a 4- to 10-fold increase of eosinophil schistosomicidal ability and a 10-fold lowering of the threshold for antibody or complement required in the killing reaction. The activity that enhances eosinophil cytotoxicity and the eosinophil colony-stimulating activity in the placental conditioned medium are eluted in the same fraction (CSF-alpha) after chromatography on Sephadex G-100 and phenyl-Sepharose columns, suggesting that these two activities might be associated with the same molecule. CSF-alpha enhances the adherence step of the killing reaction: antibody-coated larvae were frequently found covered by several layers of eosinophils in tubes containing CSF-alpha. Such a degree of adherence was rarely seen in control tubes lacking CSF-alpha. This enhancement of the eosinophil adherence is detectable 45-60 min after addition of CSF-alpha to the culture. It is not affected by washing the cells after a short time of preincubation with CSF-alpha, and it occurs in the absence of protein synthesis, whereas colony-stimulating activity requires continuous protein synthesis and ceases when CSF is removed from the culture. Finally, CSF-alpha enhances the temperature-dependent reaction that insures the irreversibility of eosinophil attachment to schistosomula. These observations suggest that eosinopoietic factors could be responsible for some of the modified properties of blood eosinophils in eosinophilic individuals.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1982

Genetic predictors of patient response and side effects in the treatment of rheumatoid arthritis with a high dose nonsteroidal antiinflammatory drug regimen.

Clinical, laboratory, genetic, and radiologic studies were evaluated for 18 patients with rheumatoid arthritis who were treated for a mean of 16.6 months with a regimen involving supplementary aspirin and piroxicam, an investigational, nonsteroidal antiinflammatory agent. Although improvement in disease activity was seen, progression was evident on successive radiographs. Disease activity was not associated with the presence of any of the genetic markers. Peptic ulcers developed in 33% of patients, all of whom had type O blood. ABO blood typing may therefore be useful in patients with rheumatoid arthritis before consideration of therapy with potentially ulcerogenic drugs.

Adult↗
PubMed1982

Clonal analysis of progenitor cell commitment of granulocyte or macrophage production.

Granulocyte-macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte-macrophage stimulus, GM-CSF, or by the predominantly macrophage stimulus, M-CSF. After 24 hours, paired daughter cells of granulocyte-macrophage colony-forming cells (GM-CFC) were separated by micromanipulation and one cultured in GM-CSF, the other in M-CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and day 3 clones were cross-transferred to GM-CSF or M-CSF recipient cultures, irreversible commitment was more obvious. Most M-CSF-initiated clones exhibited irreversible commitment to macrophage formation in GM-CSF cultures and a high proportion of GM-CSF-initiated clones continued to produce granulocyte progeny after transfer to M-CSF. The results indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC respectively to granulocyte or macrophage production. While for women GM-CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions. Calculations from the data indicated that two-thirds of GM-CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM-CSF and M-CSF.

Animals↗
PubMed1982

Production of monocyte/macrophage colony-stimulating factor by preadipocyte cell lines derived from murine marrow stroma.

Three preadipocyte cell lines that have been independently derived from bone marrow stroma (Lanotte et al, 1982) have been tested for their capacity to produce granulocyte, macrophage, and erythroid colony-stimulating factors (CSFs). All elaborated colony-stimulating material that was active upon adult mouse marrow granulocyte/macrophage colony-forming cells (M-CFC) but not foetal liver GM-CFC. The major activity was characterised as a monocyte-macrophage colony-stimulating factor (M-CSF), and the pattern of colony stimulation was similar to that seen after addition of highly purified L-cell CSF. Furthermore, the stimulating activity was specifically neutralised by rabbit anti-L cell CSF antibodies. No evidence was found for stimulation of multipotential or erythroid colony-forming cells, only few granulocytic colonies were detected, and the stimulating activity had no mouse strain restriction. All cell lines produced large quantities of M-CSF; however, the production was found to be modulated during the adipogenesis process. A peak in M-CSF production corresponded to the period of growth arrest after confluence of the stromal cells was reached and when adipocyte maturation was at an early stage. A marked depression in M-CSF secretion was associated with the final steps of adipocyte maturation.

Adipose Tissue↗
PubMed1982

Effects of GM-CSF deprivation on precursors of granulocytes and macrophages.

Culture of C57BL bone marrow cells in the absence of GM-CSF led to a loss of recoverable granulocyte-macrophage colony-forming cells of 2% per hour. The rate of loss of progenitor cells in cultures of CBA fetal liver cells was 5-6% per hour. Surviving colony-forming cells exhibited a normal responsiveness to GM-CSF but generated smaller colonies than normal when subsequently stimulated by GM-CSF. Transfer of washed individual day-3 granulocyte-macrophage colony cells to cultures lacking GM-CSF indicated that most cells were unable to survive or proliferate in the absence of GM-CSF. Death of transferred cells was rapid and invariable when the cells were from macrophage-forming colonies. However some cells from 40-70% of granulocyte-forming colonies were able to undergo one or two divisions in the absence of GM-CSF. This phenomenon was seen most often with cells from colonies where matching colony cells exhibited a higher-than-average proliferative capacity in parallel stimulated cultures. The results indicate the difficulty that will be encountered in obtaining valid metabolic data from unstimulated populations of granulocyte-macrophage precursor cells. The ability of some granulocyte precursor cells to exhibit limited proliferation following GM-CSF deprivation suggests that significant amounts of GM-CSF may be bound to or be internalized in some precursor cells and result in cell division in the absence of GM-CSF from culture medium.

Animals↗
PubMed1982

Sources and biology of regulatory factors active on mouse myeloid leukemic cells.

The action of serum or cells in enforcing differentiation in mouse myelomonocytic leukemic cells was monitored in agar cultures of WEHI-3B leukemic cells. The repeated intravenous injection of 5 micrograms endotoxin initially increased serum differentiating activity but after the third injection responses to further injections decreased markedly. Congenitally athymic (nude) mice exhibited normal rises in serum differentiating activity when injected with endotoxin but C3H HeJ mice failed to respond to challenge with purified lipid A. Whole body irradiation up to 1,200 rads did not increase serum differentiating activity but did not suppress responses to challenge injection of endotoxin. Coculture of WEHI-3B cells with peritoneal cells from normal or irradiated BALB/c mice caused marked granulocytic differentiation in WEHI-3B colonies. This effect was not seen if leukemic cells were cultured with thymus, spleen, or bone marrow cells. The serum halflife of the factor in postendotoxin serum enforcing differentiation of WEHI-3B cells was shown to be 1.5-2.3 hr.

Animals↗
PubMed1982

Biochemical and functional characterization of mature progeny purified from a myelomonocytic leukemia cell line.

Comparative studies were performed on the cloned myelomonocytic leukemia cell line, WEHI-3B, and a subcloned line, WEHI-3BM6. WEHI-3BM6 cells were less responsive than WEHI-3B cells to differentiation factor (DF) present in the sera of mice injected with endotoxin (endotoxin serum, ES). WEHI-3BM6 cells produced only 8% monocytes after 6 days of incubation with ES compared with 68% monocytes produced by WEHI-3B cells. In the presence of ES the rate of differentiation of both cell lines was enhanced by the addition of actinomycin D (5 ng/ml) such that after 2 days of stimulation 62% of the cells were mature monocytes. Lysozyme content as well as the expression of alpha-napthyl acetate esterase were also increased by actinomycin D. As indicated by the shifts in modal fluoresence levels (209 vs 63), differentiating WEHI-3B cells showed an increase in the binding of an anti-neutrophil serum compared with untreated WEHI-3B cells. The binding of anti-neutrophil antibodies allowed the sorting of the mature monocytes from the blast cells in DF-treated WEHI-3B cells achieving a purity of 78%. Electrophoretic analysis of radiolabelled proteins from the cell extracts of WEHI-3B-derived monocytes showed close similarities to normal murine peritoneal macrophages and distinct differences from the protein profiles of purified murine peritoneal polymorphs. A protein of 75,000 mol, wt present in the polymorphs was absent from the WEHI-3B monocytes.

Animals↗
PubMed1982

Refractory Cushing's disease caused by multinodular ACTH-cell hyperplasia.

A patient with pituitary-dependent hypercortisolism, unresponsive to resection of nodules in the anterior lobe, is described. Histochemical stains of the nodules showed multiple, focal, cellular expansions of the fibrovascular stroma. Transitions between normal and expanded adenohypophysial acini were present. Immunoperoxidase stains for ACTH and other pituitary hormones revealed that these multiple foci contained an excess of ACTH-positive cells. Less than 10% of the cells in these foci were negative for ACTH and positive for other hormones. Serial sections showed that these foci of predominantly ACTH-producing acini were not connected. Clinical, morphological, and immunohistochemical data indicated that ACTH-cell hyperplasia caused Crushing's disease in this patient. Pathologic study of individual cases should concentrate on determining whether hyperplasia or adenoma exist at the time of surgical exploration of the pituitary gland, since this determination is important to proper treatment. Tentative criteria to recognize ACTH-cell hyperplasia are: 1. Multiple foci of ACTH laden cells. 2. A minor subpopulation of cells of alternate hormone series. 3. Expansion without destruction of acini in the adenohypophysis.

Adenoma↗
PubMed1981

Induction of differentiation in murine myelomonocytic leukemia cells by the serum of patients with acute myeloid leukemia and other diseases.

Sera from patients with leukemia, lymphomas or other diseases were tested for their capacity to induce differentiation in colonies produced in semi-solid agar by the mouse myelomonocytic leukemia, WEHI-3B. Of 949 samples tested, higher than normal differentiating activity was detected in patients with infections (31% of sera tested) and with acute myeloid leukemia in relapse (35% of sera tested). The higher activity of the latter sera was correlated with the concurrent presence of infections in the relapse leukemic patients. Elevated differentiating activity was also observed in sera from patients with neutrophil levels below 1,000/microliter even if infections were not present. The study indicates that patients with acute myeloid leukemia are able to elevate serum differentiating activity in response to infections or neutropenia and that the serum differentiating factors could play a role in suppressing myeloid leukemic populations by enforcing differentiation in the leukemic cells.

Animals↗
PubMed1981

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation.

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF beta and CSF alpha) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSF beta died between the fifth and tenth day of culture after transfer to dishes with CSF alpha or CSF beta or to dishes with no stimulus. Cells in clusters initiated with CSF alpha proliferated significantly between the fifth and tenth day of culture when transferred to CSF alpha or CSF beta but not when transferred to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.

Bone Marrow Cells↗
PubMed1981

Biochemical properties of differentiation factors for murine myelomonocytic leukemic cells in organ conditioned media--separation from colony-stimulating factors.

A number of different organs and tissues from untreated or endotoxin-injected mice were surveyed for their ability to produce conditioned medium that could induce differentiation of the myelomonocytic leukemic cell line WEHI-3B. All organs were able to do this but were much more effective from endotoxin-injected animals. Production of the differentiation factor (DF) was dependent on protein synthesis and occurred throughout the 7-day incubation period. DF produced by the different organs in vitro and found in vivo in endotoxin serum were indistinguishable from each other by several fractionation procedures but could be distinguished from the majority of granulocyte-macrophage and macrophage colony-stimulating factors (GM- and M-CSFs). DF was precipitated by 55% ammonium sulfate whereas CSF required 85% saturation. DF did not bind to Concanavalin A-Sepharose whereas CSF did. DF eluted later than CSF from Phenyl-Sepharose columns. The dissociated molecular weight of DF was 21,000-29,000 and was not greatly affected by neuraminidase treatment. There was no evidence that DF was glycoprotein in nature although this cannot yet be ruled out. Although the DF activity could be clearly separated from the bulk of GM-CSF and M-CSF activities, there was always residual CSF activity associated with the DF. This CSF was distinct from GM- and M-CSF in its biochemical properties, in the fact that it only stimulated a subset of colony-forming cells and that upon dilution it stimulated exclusively granulocytic colonies to develop. In two steps (ammonium sulfate precipitation and Phenyl-Sepharose chromatography) it was possible to selectively enrich the differentiation activity over the CSF activity by 21-fold.

Animals↗
PubMed1981

Surface antigens on normal and leukaemic human cells detected by monoclonal antibodies.

Surface antigens were analyzed on normal human marrow and chronic myeloid leukaemic cells using 4 monoclonal mouse anti-human antibodies. The fluorescence-activated cell sorter was used to quantify the binding of each antibody to different subpopulations of cells, and sorted fractions were cultured in agar-medium to assay for granulocyte-macrophage and eosinophil precursors. All cells in the granulocyte series including colony-forming cells bound a similar quantity of an antibody to the human leucocyte common antigen. This antibody did not bind to cells in the erythroid series. A monoclonal antibody to antigen present on brain, lymphocytes and granulocytes (and almost certainly homologous to the W3/13 antigen of the rat) bound to the cells in the order: blast greater than promyelocytes and myelocytes greater than granulocytes. The third monoclonal antibody was directed against a determinant of the leucocyte common antigen present predominantly on B lymphocytes and absent from the myeloid series. The fourth antibody, directed against the human homologue of Thy-1, reacted with less than 1% of marrow cells, none of which appeared to be granulocyte or eosinophil progenitors. The leucocoyte common antigen and the brain-lymphocyte-granulocyte-antigen were also present on colony- and cluster-forming cells from a patient with chronic myeloid leukemia. Using the low angle and wide angle light scatter properties of CML blood cells, 7-fold enrichment was obtained for progenitor cells from chronic myeloid leukaemia. With the monoclonal antibodies up to 4-fold enrichment was obtained.

Animals↗