Studies on inhibitors of bone marrow colony formation in normal human sera and during a viral infection

Various methods are compared for removing inhibitors of bone marrow colony stimulating activity from the serum of normal individuals. The best method proved to be chloroform treatment of sera, which is easily performed on a large scale. Chloroform treatment had no effect in itself on the colony-stimulating factor (CSF) and no additional inhibitors were detected after such treatment. Inhibitors were characterized by preparative ultracentrifugation. After fractionation, most of the inhibitory activity was recovered in the light (LDL) and very light density lipoprotein (VLDL) fractions. Treatment with specific anti human β-lipoprotein serum removed most of the inhibitory activity. Increased CSF levels have been reported to accompany the acute phase of infectious diseases. Application of the chloroform treatment to sera from patients with mumps showed that the mean CSF in these sera did not differ significantly from that found in sera from normal individuals. High CSF levels in acute infections therefore seem to reflect variations in inhibitor levels rater than a true increase in CSF.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

Activated charcoal diminishes the lot difference of fetal bovine sera in erythroid colony formation of human bone marrow cells

Using normal bone marrow as target cells, we assayed the colony-forming efficiency of early and late erythroid progenitor cells and granulocyte-macrophage progenitor cells using several different lots of fetal bovine serum (FBS). There was a marked difference in the ability of these sera to support colony formation, particularly in erythroid colony assays. When adsorbed by activated charcoal, all these sera supported erythroid colony formation more efficiently than before adsorption. There was no significant effect of charcoal adsorption of FBS on granulocyte-macrophage colony formation. Gel-filtration study showed that charcoal adsorption diminished low-molecular-weight fractions by less than 5000 Da. The inhibitory activity of this fraction was heat labile and Pronase sensitive. Concentrated samples obtained from these fractions inhibited erythroid colony formation in a dose-dependent manner. These results suggest that low-molecular-weight inhibitors that are relatively specific to erythropoiesis play a critical role in the lot differences of FBS for erythroid colony formation.     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.     

Depression of bone marrow colony formation in gold-induced neutropenia.

Bone marrow culture in semi-solid agar was used to assess the proliferative activity and the response to sodium aurothiomalate of the myeloid precursor cells from patients during and after recovery from neutropenia associated with the use of this drug. Colony formation was reduced during the neutropenia and returned to normal after recovery. The rheumatoid process itself did not impair colony formation even in patients with Felty''s syndrome. Sodium aurothiomalate inhibited colony formation by normal marrow in a dose-dependent manner. Bone marrow colonies from patients who had recovered from neutropenia induced by sodium aurothiomalate were not abnormally sensitive to the inhibitory effect of the drug in vitro. The metabolism of gold is probably altered in a small proportion of patients, which causes high local concentrations within the bone marrow leading directly to marrow depression.     

Antibody-mediated suppression of bone marrow colony formation in vitro.

Antisera to mouse brain reacts with hematopoietic stem cells in the mouse bone marrow. We have examined the effect of anti-mouse brain serum (AMBS) on the development of in vitro colonies from mouse bone marrow cells. The addition of 5% AMBS to the cultures markedly decreased the numbers of colonies formed to an average of 10% of the number obtained with normal rabbit serum. AMBS suppressed formation induced by colony stimulating factors (CSF) derived from three different sources; serum from endotoxin treated mice, mouse L-cell conditioned media, and human peripheral mononuclear cell conditioned media. The suppressive activity was quantitatively recovered in the IgG fraction of AMBS. Divalent F(ab')2 fragments were as effective as the intact IgG in decreasing colony formation. Fab fragments were not suppressive. These results suggest that colony formation is induced via a dynamic interaction between CSF and the progenitor cell membrane, and that antibody directed at cell membrane antigen(s) interferes with the generation of the induction signal.     

Synergistic interactions between recombinant human interleukin-3, GM-CSF and G-CSF in normal human marrow granulocyte-macrophage colony formation.

The network of interactions between human myelopoietic growth factors can lead to signal amplification that all regulate normal myelopoiesis. The present study identified synergistic interactions between recombinant human interleukin-3, GM-CSF and G-CSF on normal human marrow day 14 CFU-GM suggesting that the interactions between these human myelopoietic growth factors are mainly confined to the early stages of normal human myelopoiesis. The synergistic combinations of recombinant human interleukin-3 plus G-CSF and GM-CSF plus G-CSF warrant clinical trial for the recovery from cancer chemotherapy or radiotherapy-induced myelosuppression and for augmenting human defence against infections.     

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems

Summary Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first 2 weeks while an increasing number of monocytes and macrophages appeared in the media of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO₂ concentrations, i.e. 2%, 5%, 10%; similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. Supported by U.S.P.H.S. Grant CA-5834 from the National Cancer Institute. Department of Medicine A. Department of Cell Physiology Department of Immunology and Immunochemistry.     

Human bone marrow colony formation in diffusion chambers. Normal values.

In situ hybridization techniques have previously employed a series of manipulations to effect denaturation of chromosomal DNA and reannealing of DNA-RNA hybrids. This report presents a new protocol which combines the denaturation and reannealing processes. DNA is heated in a solution of 50% formamide, 50% 4 × SSC containing the RNA to be hybridized. After l h at 70 °C the preparation is slowly cooled to 37 °C over a period of 6 h and incubated at 37 °C for an additional 10 h. This technique eliminates the possibility of premature reannealing of the DNA while employing hybridization conditions which, in vitro, lead to accurate base pairing.     

The humoral nature of the colony-stimulating action of bone marrow cells on stromal colony formation in bone marrow cultures

In 24 hours adherent marrow cell cultures (AMCC) were represented by single stretched fibroblasts. In non-feeder-supplemented AMCC most of the CFU-f remained single fibroblasts or passed through 1-3 cell doublings [correction of dudlings]. The colony stimulating activity of irradiated marrow cells was found to be diffuse across the Millipore filter, which seems to indicate that haemopoietic marrow cells produce a colony stimulating factor which is required for triggering the CFU-f from the Go-period of the cell cycle into cell proliferation.     

Precursor cells of fibroblast colony forming unit in the normal and pathologic human bone marrow and in the peripheral blood

In many myeloproliferative disorders the microenvironmental compartment of bone marrow, as represented by fibroblastoid colony forming units (F-CFU) incidence, is impaired.     

Studies on colony formation in vitro by mouse bone marrow cells. I. Continuous cluster formation and relation of clusters to colonies

Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.     

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems.

Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first two weeks while an increasing number of monocytes and macrophages appeared in the media of of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO2 concentrations, i.e. 2%, 5%, 10% similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera.     

Effect of L-phenylalanine methyl ester on the colony formation of hematopoietic progenitor cells from human bone marrow

We have previously shown that L-phenylalanine methyl ester (PME) is capable of removing monocytes and enhancing the growth of hematopoietic colonies from human peripheral blood (PB) mononuclear cells (MNC). In the present study, we further compared the effect of PME on the colony formation of bone marrow (BM) and PB. Low density (less than or equal to 1.077 g/ml) MNC were obtained by Ficoll-diatrizoate density gradient centrifugation. Granulocyte/macrophage colony-forming units (CFU-gm) and erythroid burst-forming units (BFU-e) were cultured in agarose with conditioned media (CM) and/or interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage-CSF (GM-CSF). Treatment of BM MNC with 5 mM PME for 15 min at room temperature yielded a nucleated cell recovery of 44.8 +/- 5.0% (mean +/- SE; N = 8). CFU-gm were enriched 2.7-fold (range 2.0 to 4.8). Using CM or CM supplemented with G-CSF or GM-CSF has minimal effect on the enrichment. Leukocyte differentials revealed that 94.3 +/- 3.05% of the monocytes, as well as 91.2 +/- 1.60% of the cells in the neutrophilic maturation series were removed by PME. Incubation for 40 min in PME abolished CFU-gm formation. BFU-e were not enriched by the PME treatment. In contrast, 40 min incubation of PB MNC produced higher enrichment of CFU-gm than that obtained from 15 min of treatment, although lower cell recovery was obtained with the longer treatment time. In conclusion, we have demonstrated that phagocytic cells can be removed from BM or PB MNC by PME treatment.(ABSTRACT TRUNCATED AT 250 WORDS)