Source Of Colony-stimulating Activity Research Articles

Divergent regulation of 1,25-dihydroxyvitamin D3 on human bone marrow osteoclastogenesis and myelopoiesis

The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has influence over osteoclastogenesis and myelopoiesis, but the regulational mechanism is not well-defined. In this report, formation of osteoclast-like (OCL) cells from primitive myeloid colony-forming cells (PM-CFC) as mediated by 1,25(OH)2D3 was examined. Our results present in this report clearly show that 1,25(OH)2D3 dose-dependently stimulated OCL cell formation when added to suspension cultures of individually replated PM-CFC colonies. Marrow cells were plated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or the human bladder carcinoma cell line 5637 conditioned medium (5637 CM) as the source of colony-stimulating activity. The 1,25(OH)2D3 effect of osteoclast differentiation was associated with a concomitant decrease in clonogenic growth of myelopoietic progenitors in response to colony-stimulating activity. Secondly, the effect of adding the known stimulator of hematopoiesis, interleukin-1beta (IL-1beta) and/or 1,25(OH)2D3 on human myeloid colony growth was assessed. IL-1beta enhanced the formation of primitive myeloid colonies in response to GM-CSF by 160%. On the other hand, 1,25(OH)2D3 dose-dependently inhibited both GM-CSF- and 5637 CM-driven myeloid colony formation by as much as 90% at 100 nM. Addition of IL-1beta to GM-CSF-stimulated cultures dampened the inhibitory effect of 1,25(OH)2D3. The inhibition of myeloid clonogenic growth by 1,25(OH)2D3 was almost abolished (89%) by simultaneously adding anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha MoAb) to the culture medium. These results collectively suggest divergent roles for 1,25(OH)2D3 in osteoclastogenesis and myelopoiesis, promoting the differentiation of OCL cells from primitive myeloid cells but inhibiting the proliferation of later myeloid progenitor cells. This inhibition of myeloid progenitors may be mediated by TNF-alpha.

Granulocyte-Macrophage Colony-Forming-Unit Assay and Liquid Suspension Culture of Bone Marrow and Peripheral Blood in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia

Granulocyte-macrophage colony-forming-unit (CFU-GM)-assays and/or liquid suspension cultures of bone marrow (BM) and peripheral blood (PB), using human placental conditioned medium as a source of colony-stimulating activity, were performed in 125 children with newly diagnosed acute lymphoblastic leukemia (ALL). Unlike the previous small series which invariably showed poor BM granulocyte-macrophage colony and cluster formations, we found 3 growth patterns. A poor-growth pattern was seen in 101 patients. A low-growth pattern with BM colony and cluster formations in the lower range of the normal limits was observed in 6 patients. All 6 patients had low-risk ALL, and the in vitro growth may reflect the residual normal CFU-GM in BM. Thirdly an over-growth pattern was noted in one patient with refractory ALL. In 49 cases where liquid suspension cultures of BM were performed, 4 had a normal-growth pattern and 42 had apoptotic cells and/or few residual granulocytic/monocytic cells after 7 days of culture. However, in both B cell ALL cases and in 1 refractory T cell ALL case with hyperleukocytosis, repetitive liquid suspension cultures of BM and PB consistently revealed an extensive growth of lymphoblasts. Thus, an over-growth pattern in either the BM CFU-GM assay or liquid suspension culture of BM or PB in children with ALL at diagnosis may indicate a refractory or aggressive disease. A low-growth pattern in the BM CFU-GM assay in children with ALL at diagnosis probably predicts a favorable outcome.

Effects of L‐leucyl‐L‐leucine methyl ester on human marrow and protection of progenitor cells by IL‐1

In murine models, L-leucyl-L-leucine methyl ester (LLME) is effective as an ex vivo purging agent for the prevention of graft-versus-host disease. However, reduction of progenitor cells by LLME raises concerns about the engraftment potential of LLME-treated marrow. Therefore, the effects of LLME on human marrow were investigated. Concentrations of LLME from 0.005 mM to 5 mM were used to examine the effects of LLME on marrow cells. Concentrations of LLME less than or equal to 0.05 mM had no effect on recovery of nucleated marrow cells, mononuclear cells (MNC), myeloid cells or monocytes. At 0.5 mM, nucleated marrow cell recovery was 25%, and MNC recovery was 93%. As determined by cytochemical stains, 0.5 mM LLME eliminated myeloid cells and monocytes. At 0.005 mM, LLME had little effect on marrow progenitor cells; progenitor cell recovery with 0.05 mM LLME was 35% for granulocyte-macrophage colony forming units (CFU-GM) and 23% for erythroid burst forming units (BFU-E) (p less than 0.05 compared to 0.005 mM). At higher LLME concentrations, recoveries of both CFU-GM and BFU-E were less than 1%. To determine if earlier hematopoietic cells remained after treatment with LLME, progenitor cell experiments were repeated with 0.5 mM LLME using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) or recombinant human interleukin 3 (rhIL-3) plus or minus recombinant human mast cell growth factor (rhMGF) as sources of colony-stimulating activity. In this system, both IL-3 and GM-CSF induced rare progenitor cells (less than 6/5 x 10(4) cells plated).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of hydrocortisone on the survival and cluster and colony forming characteristics of normal human granulocyte‐macrophage progenitors

This study was performed to determine the colony and cluster forming ability of granulocyte-macrophage (CFU-GM) progenitors of normal human blood low density cells cultured in a liquid culture system in the presence and absence of physiological doses of hydrocortisone (Hc). The CFU-GM recovered from the liquid cultures were assayed in soft agar medium. The results of the assays indicated that time-related development of clusters and colonies over 1-16 days, proliferative responsiveness to a source of colony stimulating activity, number of cells developed per colony, and the cellular composition of clusters and colonies produced from CFU-GM recovered from 14-day-old liquid cultures with 1.0 microM Hc, were all similar to those that developed from the normal human blood low density cells. However, a higher fraction of the CFU-GM in day 14 liquid cultures with 1.0 microM Hc were in DNA synthesis phase compared with the CFU-GM from the peripheral blood. This study confirmed the results of previous studies showing lower numbers of recognizable neutrophilic granulocytes and improved survival/proliferation of CFU-GM at day 14 in liquid cultures with 1.0 microM Hc compared with cultures without Hc. The present results suggest that the normal human blood CFU-GM which persists and proliferates under the influence of Hc in a liquid culture system is similar in ontogeny to the blood CFU-GM, and that the recovery of CFU-GM from liquid cultures under the influence of Hc appears to be exerted through stimulation of proliferation and controlled differentiation.

Characterization of clonogenic cells in refractory anemia with excess of blasts (RAEB-CFU): Response to recombinant hematopoietic growth factors and maturation phenotypes

The proliferative and maturation abilities of bone marrow progenitors in patients with refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) have previously been investigated in vitro using impure sources of colony stimulating activity. Here we report studies that were concerned with defining growth factor responses of RAEB progenitors (RAEB-CFU) in colony culture using pure hematopoietic growth factors. Marrow cells of 10 RAEB patients were cultured with recombinant IL3, GM-CSF, G-CSF, M-CSF and EPO. Factor dependent colony growth of four patients was examined in detail cytologically. The analysis revealed notable deficiencies in the colony forming spectrum as compared with normal marrow: although granulocytic colonies were formed in all of these four RAEB cases, macrophage colonies could not be induced in 1 4 cases and cosinophilic and erythroid colony formation could not be propagated in 2 4 cases with the proper stimuli. These findings are indicative of the intrinsic incapabilities of RAEB-CFU to mature along certain differentiation pathways in response to the growth factor. We then determined the surface phenotypes of RAEB-CFU using MoAbs Vim-2 (myelomonocytic) and BI3C5 (CD34) following dual labeling and fluorescence activated cell sorting and subsequent culture of the separately sorted BI3C5 + Vim-2 + , BIC5 + Vim-2 − , BI3C5 - Vim- 2+ and BIC5 − Vim-2 − cells. In normal marrow most clonogenic cells were recovered from the BI3C5 + Vim-2 − fraction. In contrast, in RAEB marrow increased proportions of the colony forming cells were BIC5 + Vim-2 + , BI3C5 − Vim-2 + , or BI3C5 −. The altered distribution of surface immunophenotypes of RAEB-CFU provides further evidence for the imbalance of maturation in the progenitor cell compartment. The results are discussed in view of the concept that the inabilities of the RAEB hematopoietic precursors to mature in response to the hematopoietic growth factors are partial and variable, but may culminate in a progressive loss of the differentiation competence of the progenitors when leukemia evolves.

Granulocyte/macrophage colony-forming units from cord blood of premature and full-term neonates: its role in ontogeny of human hemopoiesis.

It is still uncertain whether the yolk sac is the only origin of hemopoietic stem cells during fetal development. We studied the level of circulating granulocyte/macrophage colony-forming unit (CFU-GM) from the 23rd week of gestation to fullterm, trying to delineate its role in the ontogeny of human hemopoiesis. Cord blood samples were collected from 45 healthy premature neonates and 91 healthy full-term neonates. CFU-GM assays were performed using a single agar layer system with human placental conditioned medium as a source of colony stimulating activity. The mean numbers of colonies produced from the cord blood samples of the premature and full-term neonates were 116 +/- 185 (SD) and 96 +/- 113/2 x 10(5) mononuclear cells, respectively. There was no statistical difference between the numbers of colonies of the two groups (Mann-Whitney test, p greater than 0.05). These results indicate that the level of circulating CFU-GM from the 23rd week of gestation to full term is constantly high and add credence to the assumption that CFU-GM are produced not only from the yolk sac but also from other hemopoietic sites.

Antileukemic effect of recombinant tumor necrosis factor alpha in vitro and its modulation by alpha and gamma interferons

The effect of recombinant human tumor necrosis factor alpha (rTNF-alpha) on human myelogenous leukemia clonogenic cells growing either in semisolid media or in suspension cultures was studied and compared with the effect on normal granulocyte-macrophage progenitors (GM-CFC). Exposure of cells to a range of rTNF-alpha doses including pharmacologically achievable plasma concentrations revealed a large heterogeneity in the response of leukemic clonogenic growth to rTNF-alpha. Only one of 13 specimens was highly resistant to rTNF-alpha. Eight of ten leukemic samples were significantly more sensitive than were normal GM-CFC, particularly within the in vivo achievable dose range (1 x 10(0) to 1 x 10(2) ng/mL). No significantly increased inhibition of either normal or leukemic clonogenic growth could be achieved by increasing the rTNF-alpha concentration above 250 ng/mL. Proliferation of leukemic clonogenic cells (L-CFC) was studied in suspension cultures. In five cases the clonogenic cells were significantly inhibited by rTNF-alpha while in one case no inhibition was observed. The inhibition of L-CFC growth by rTNF-alpha was dose dependent between 1 x 10(0) and 1 x 10(2) ng/mL. In suspension cultures, the TNF effect on L-CFC was a function of time of exposure, particularly with low concentrations of TNF. A remarkably higher inhibition of L-CFC as compared with the total leukemic population was observed in suspension cultures. Stimulation of L-CFC growth by rTNF-alpha was not observed. Normal GM-CFC were inhibited by alpha and gamma interferons (INF-alpha, -gamma) in a dose-related manner, with higher sensitivity of colonies than clusters. The response of GM-CFC to combination of recombinant IFNs and TNF was influenced by the size of clones scored and the source of colony-stimulating activity. The response of L-CFC to recombinant IFN-alpha and/or -gamma was highly variable, and sensitivity to one of the lymphokines did not predict for sensitivity to another. The response of L-CFC to combinations of rTNF-alpha and either IFN-alpha or IFN-gamma was complex, varying from synergistic to additive and indifferent. In three of six specimens, IFN-gamma acted antagonistically with rTNF-alpha, a phenomenon not observed with IFN-alpha. These observations suggest that the action of rTNF-alpha in acute myelogenous leukemia could be exploited therapeutically and the dose-time-response relationship should be considered in designing treatment schedules.(ABSTRACT TRUNCATED AT 400 WORDS)

Antileukemic Effect of Recombinant Tumor Necrosis Factor α in vitro and Its Modulation by α and γ Interferons

Abstract The effect of recombinant human tumor necrosis factor alpha (rTNF- alpha) on human myelogenous leukemia clonogenic cells growing either in semisolid media or in suspension cultures was studied and compared with the effect on normal granulocyte-macrophage progenitors (GM-CFC). Exposure of cells to a range of rTNF-alpha doses including pharmacologically achievable plasma concentrations revealed a large heterogeneity in the response of leukemic clonogenic growth to rTNF- alpha. Only one of 13 specimens was highly resistant to rTNF-alpha. Eight of ten leukemic samples were significantly more sensitive than were normal GM-CFC, particularly within the in vivo achievable dose range (1 x 10(0) to 1 x 10(2) ng/mL). No significantly increased inhibition of either normal or leukemic clonogenic growth could be achieved by increasing the rTNF-alpha concentration above 250 ng/mL. Proliferation of leukemic clonogenic cells (L-CFC) was studied in suspension cultures. In five cases the clonogenic cells were significantly inhibited by rTNF-alpha while in one case no inhibition was observed. The inhibition of L-CFC growth by rTNF-alpha was dose dependent between 1 x 10(0) and 1 x 10(2) ng/mL. In suspension cultures, the TNF effect on L-CFC was a function of time of exposure, particularly with low concentrations of TNF. A remarkably higher inhibition of L-CFC as compared with the total leukemic population was observed in suspension cultures. Stimulation of L-CFC growth by rTNF- alpha was not observed. Normal GM-CFC were inhibited by alpha and gamma interferons (INF-alpha, -gamma) in a dose-related manner, with higher sensitivity of colonies than clusters. The response of GM-CFC to combination of recombinant IFNs and TNF was influenced by the size of clones scored and the source of colony-stimulating activity. The response of L-CFC to recombinant IFN-alpha and/or -gamma was highly variable, and sensitivity to one of the lymphokines did not predict for sensitivity to another. The response of L-CFC to combinations of rTNF- alpha and either IFN-alpha or IFN-gamma was complex, varying from synergistic to additive and indifferent. In three of six specimens, IFN- gamma acted antagonistically with rTNF-alpha, a phenomenon not observed with IFN-alpha. These observations suggest that the action of rTNF- alpha in acute myelogenous leukemia could be exploited therapeutically and the dose-time-response relationship should be considered in designing treatment schedules.(ABSTRACT TRUNCATED AT 400 WORDS)

Megakaryocyte colony formation in chronic myeloid leukemia and myelofibrosis

Colony formation by megakaryocytic progenitors from the blood or bone marrow was studied in 22 patients with chronic myeloid leukemia (CML) and in 17 patients with idiopathic myelofibrosis (MF). Thirteen of the 22 CML patients showed megakaryocytic colony formation, when PHA-LCM and plasma of a patient with aplastic anemia were used as a source of colony stimulating activity. Twelve of these 13 patients also showed spontaneous megakaryocytic growth, i.e. colony formation when PHA-LCM was omitted and normal plasma was used instead of aplastic plasma. All the untreated CML patients exhibited both stimulated and spontaneous growth. Each patient without any megakaryocytic colony formation had recently received cytotoxic treatment. Thirteen of the 17 patients with MF grew megakaryocytic colonies and ten of these 13 patients also showed spontaneous megakaryocytic growth. The colony numbers were roughly similar in the stimulated and non-stimulated cultures. The present study shows that spontaneous megakaryocytic colony formation, previously shown to be common in PV and ET, is also seen in many patients with CML and MF.

Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor.

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.

Effects of hypoxia on megakaryocytopoiesis and granulopoiesis

Previous studies have reported that exposure of mice to hypoxic conditions results in a decrease in blood platelets. To further explore the effect of hypoxia on megakaryocytopoiesis, megakaryocytes and their progenitor cells (CFU-M) were studied in hypoxic mice. Mice were exposed to hypoxia by enclosure in cages covered with dimethyl-silicone rubber membranes for up to 10 d. At various times during the hypoxic and ex-hypoxic periods the total megakaryocytes and CFU-M were determined in the humerus and spleen. CFU-M were assayed in the soft gel colony forming assay using pokeweed mitogen-stimulated spleen cell conditioned medium (PWCM) as a source of colony stimulating activity. After 10 d of hypoxia, packed red cell volume (PRCV) increased to 148% of control levels and blood platelets decreased to 40% of controls. Total megakaryocytes and CFU-M per humerus decreased to 18% and 50% of controls respectively. 4 d into the ex-hypoxic phase, PRCV was still increased at 128% of controls while marrow megakaryocytes and CFU-M increased to normal levels. Platelet recovery was somewhat slower, returning to normal by d 6. In contrast to the findings in the marrow, total spleen megakaryocytes and CFU-M increased to about 3- and 5-fold of control levels respectively by 6 d of hypoxia. During the exhypoxic phase, CFU-M decreased to normal on d 4, followed by a rebound of 3-fold control values on d 8. Spleen megakaryocytes decreased more slowly, returning to normal by d 10. A marked granulocytosis was observed during the hypoxic phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Colony formation by megakaryocytic progenitors in essential thrombocythaemia.

Colony formation by megakaryocytic progenitors (CFU-M) from the blood or bone marrow of 12 patients with essential thrombocythaemia was studied in vitro with the methyl cellulose assay. When the cultures were stimulated with plasma from a patient with aplastic anaemia and with phytohaemagglutinin stimulated conditioned medium (PHA-LCM), the patients showed a significant trend towards higher circulating CFU-M numbers when compared with the controls; three of the patients exceeded our normal range. In the bone marrow cultures there were no differences in the number or morphology of megakaryocytic colonies between the patients and the controls. When normal human plasma was the only source of colony stimulating activity, 11 out of 12 patients, but none of the controls, showed megakaryocytic colony formation. The same patients also had 'spontaneous' erythroid colony growth without the addition of exogenous erythropoietin into the cultures. Only one of the patients with essential thrombocythaemia (ET) had normal megakaryocytic and erythroid colony formation. The present study shows that, in most patients with ET 'spontaneous' CFU-M colony formation occurs in suboptimal culture conditions, a phenomenon obviously analogous to the spontaneous erythroid colony formation seen in the myeloproliferative disorders.

In Vitro Action of Tumor Necrosis Factor on Myeloid Leukemia Cells

We investigated the in vitro action of recombinant tumor necrosis factor alpha (TNF) on the clonal growth of normal and malignant myeloid cells. Clonogenic cells from six of nine myeloid leukemia cell lines were very sensitive to the effects of TNF with 50% of the colonies inhibited (ED50) by concentrations of TNF that ranged between 6 and 150 U/mL. A decrease in DNA, RNA, and protein synthesis and in cloning efficiency occurred within three hours of exposure of HL-60 promyelocytes to TNF. The TNF in combination with recombinant interferons produced an additive or synergistic inhibition of colony formation of HL-60 and THP-1 myelomonoblasts. Normal human CFU-GM are sensitive to TNF (ED50 between 100 and 50,000 U/mL), but their sensitivity to TNF depends on the source of colony stimulating factor (CSF) with T lymphocyte derived GM-CSF (recombinant or natural) partially protecting the CFU-GM from the suppression exerted by TNF (and interferons). In eight of 15 cases the clonogenic myeloid leukemia cells from patients with acute or chronic myeloid leukemia were more sensitive than normal CFU-GM using GM-CSF as a source of colony stimulating activity. Further studies showed that the action of TNF on myeloid leukemia cells probably can be only partially explained by differentiation. Our finding of a possible selective cytotoxicity to leukemic clonogenic cells by TNF suggests that TNF may have value in the therapy of some patients with myeloid leukemia.

In vitro action of tumor necrosis factor on myeloid leukemia cells

We investigated the in vitro action of recombinant tumor necrosis factor alpha (TNF) on the clonal growth of normal and malignant myeloid cells. Clonogenic cells from six of nine myeloid leukemia cell lines were very sensitive to the effects of TNF with 50% of the colonies inhibited (ED50) by concentrations of TNF that ranged between 6 and 150 U/mL. A decrease in DNA, RNA, and protein synthesis and in cloning efficiency occurred within three hours of exposure of HL-60 promyelocytes to TNF. The TNF in combination with recombinant interferons produced an additive or synergistic inhibition of colony formation of HL-60 and THP-1 myelomonoblasts. Normal human CFU-GM are sensitive to TNF (ED50 between 100 and 50,000 U/mL), but their sensitivity to TNF depends on the source of colony stimulating factor (CSF) with T lymphocyte derived GM-CSF (recombinant or natural) partially protecting the CFU-GM from the suppression exerted by TNF (and interferons). In eight of 15 cases the clonogenic myeloid leukemia cells from patients with acute or chronic myeloid leukemia were more sensitive than normal CFU-GM using GM-CSF as a source of colony stimulating activity. Further studies showed that the action of TNF on myeloid leukemia cells probably can be only partially explained by differentiation. Our finding of a possible selective cytotoxicity to leukemic clonogenic cells by TNF suggests that TNF may have value in the therapy of some patients with myeloid leukemia.

Synergistic action of recombinant-derived murine interleukin-1 on the augmentation of colony stimulating activity on murine granulocyte-macrophage hematopoietic stem cells in vitro.

This communication reports studies that were designed to investigate the role of recombinant-derived murine interleukin-1 on granulocyte macrophage (CFU-GM) progenitor stem cells in vitro. Interleukin-1 (IL-1, 5-25 units/ml culture) in a dose-dependent fashion, stimulated CFU-GM with a maximum effect at 25 units (147.3 +/- 6.4 colonies/10(5) nonadherent-derived marrow cells). At a higher concentration (50 units/ml) this stimulation was significantly reduced. IL-1, in combination with various types of conditioned media: pokeweed mitogen stimulated spleen cell, mouse lung and WEHI-3 cell, as a source of colony-stimulating activity (CSA), produced more CFU-GM when compared to controls (conditioned media alone). Furthermore, when assayed for their esterase activity, IL-1 increased both types of nonspecific and specific esterase content in CFU-GM colonies. Also, the actual ratio between neutrophilic and monocyte/macrophage colonies was reduced when compared to cultures stimulated in the presence of CSA, indicating that IL-1 increased myeloid differentiation. In the presence of indomethacin, an effective inhibitor of prostaglandin synthesis (1 microgram/ml), greater numbers of CFU-GM were present with IL-1 and/or CSA than without indomethacin. Cultures with anti-CSA demonstrated a reduced number of CFU-GM when plated in the presence of CSA but not with IL-1, demonstrating the specificity of IL-1 to stimulate CFU-GM in the presence of anti-CSA antibody. These results indicate a role for IL-1 in modulating granulopoiesis in vitro.

Spermine and spermidine are non-specific inhibitors of in vitro hematopoiesis

The polyamine spermine has been reported to be the inhibitor of in vitro erythropoiesis present in uremic serum. We have employed a panel of hematopoietic colony-forming assays to evaluate the specificity of the inhibitory activity. Spermine and its precursor spermidine when added to culture inhibited mouse and human erythroid (CFU-E and BFU-E), granulocyte-macrophage, and megakaryocyte colony growth in a non-specific, dose-dependent fashion. Erythroid and non-erythroid colony growth were equally sensitive to spermine- and spermidine-induced inhibition. Increasing concentrations in culture of erythropoietin and mitogen-stimulated leukocyte-conditioned medium, a source of colony-stimulating activity, failed to overcome the in vitro inhibition. Although anemia is characteristic of chronic renal failure (CRF), leukopenia and thrombocytopenia are not. Therefore, we conclude that the non-specific inhibitory activity of spermine and spermidine, as defined by in vitro colony assays, is either of no pathophysiologic significance in the anemia of CRF, or else there are unrecognized repair mechanisms in vivo which maintain granulopoiesis and thrombopoiesis at normal levels.

Studies of interferon as a regulator of hematopoietic cell proliferation.

The presence of interferon (IFN) in normal bone marrow and its abnormal production in aplastic anemia suggest that IFN may have normal regulatory roles and implicates them in the pathophysiology of bone marrow failure. We studied the effects of recombinant IFN (r-IFN) on hematopoietic colony formation in methylcellulose cultures of human bone marrow. Both recombinant IFN-gamma (r-IFN-gamma) and recombinant IFN-alpha (r-IFN-alpha) were potent suppressors of myeloid (CFU-C-derived) colony formation, with 50% inhibition occurring at 291 u/ml for r-IFN-gamma and 275 U/ml for r-IFN-alpha. Small amounts of r-IFN-gamma acted synergistically with r-IFN-alpha; as little as 5 U/ml of r-IFN-gamma increased inhibition of CFU-C-derived colony formation by r-IFN-alpha over threefold. Conversely, small amounts of r-IFN-alpha did not affect inhibition by r-IFN-gamma. Inhibition by r-IFN was highly dependent on culture conditions: reduction of the fetal calf serum concentration from 30% to 20%, a change that did not alter the plating efficiency of control cultures, significantly enhanced the action of r-IFN-gamma. Competition between positive hematopoietic factors and r-IFN was further demonstrated as increasing amounts of human placenta-conditioned media, used as a source of colony-stimulating activity, also partially blocked r-IFN inhibition. To determine if r-IFN could directly inhibit the proliferation of a progenitor cell, cells isolated from immature BFU-E-derived colonies, a population enriched for late erythroid progenitors and free of auxiliary cells, were tested; similar inhibition by r-IFN-gamma was observed with these isolated erythroid progenitors as with total bone marrow CFU-E. Although small amounts of r-IFN-gamma also increased inhibition of bone marrow CFU-E-derived colony formation by r-IFN-alpha, no synergy was demonstrable with isolated erythroid progenitor cells. Therefore, even though r-IFN can directly inhibit proliferation of a progenitor cell, auxiliary cells may be required for synergy between r-IFN-gamma and r-IFN-alpha.

Differences in the in vitro growth pattern of fresh and cryopreserved granulo-monopoietic precursors

A study of the in vitro growth model of human granulo-monopoietic precursors (CFU-GM) before and after cryopreservation using both leukocyte feeder layers and GCT conditioned medium as the source of colony stimulating activity (CSA) is reported. The number of colonies produced with fresh cells was linearly related to the amount of marrow seeded with both CSA sources, whereas after cryopreservation this was true with feeder layers, and with GCT only at relatively high cell concentrations. This might indicate the production of granulopoietic stimulators on the part of a second population that is at least partly resistant to freezing. It seems more likely, however, that these results depend mainly on a sublethal damage to CFU-GM induced by freezing, thus making the cells hyporesponsive to some forms of CSA, such as those contained in GCT conditioned medium.

Human bone marrow fibroblast proliferation and GM-CFC colony formation in patients with acute leukemia.

The proliferation of bone marrow (BM)-derived fibroblasts and granulocyte-macrophage progenitors (GM-CFC) was studied in culture in 23 patients with acute leukemia (AL). The number of GM-CFC colonies was significantly lower in patients with infiltrated BM when compared to those in remission: 17 +/- 12 vs. 125 +/- 35 colonies per 2 X 10(5) cells (p less than 0.005). The proliferation of fibroblasts derived from leukemic BM was significantly more active in acute nonlymphocytic (ANLL) than in acute lymphoblastic leukemia (ALL): 6.8 +/- 3.2 X 10(5) vs. 2.8 +/- 1.8 X 10(5) fibroblasts per flask (p less than 0.05). The reduced GM-CFC colony formation correlated significantly (r = 0.84; p less than 0.02) with the reduced fibroblast proliferation in patients with ALL but not in those with ANLL. Supernatants from fibroblast monolayers stimulated additionally normal GM-CFC cultured in the presence of a potent conditioned medium as the source of colony stimulating activity. Fibroblast supernatants derived from BM with leukemic infiltrates, stimulated the incorporation of 3H-thymidine by monolayers of normal bone marrow fibroblasts but had no effect on ANLL fibroblasts.

Myelopoiesis of human bone marrow cells in a micro-agar culture system: comparison of two sources of colony stimulating activity (CSA).

Human bone marrow cells were grown in a micro-agar culture system in the presence of human placenta (HPCM) and giant cell tumor conditioned media (GCT). The effects of HPCM and GCT conditioned media on linearity, growth dynamics, and morphological composition of colonies were studied after 7 and 14 days of incubation. Under the described conditions the dose-response curves for HPCM and GCT were different: on day 7 maximal stimulation was obtained with 2.5% HPCM and 20% GCT; on day 14 a maximal response was reached with 1.25% HPCM and 5% GCT. Both stimuli produced maximal growth after 7 days of incubation, followed by a rapid decrease in the number of formed colonies up to day 14. The morphological study of aggregates showed that after 7 days of incubation 80% pure granulocyte and 20% mixed granulocyte-macrophage colonies were found in the presence of both stimuli. However, on day 14 the incidence of granulocyte-macrophage colonies increased to 60%, whereas the percentage of pure granulocyte colonies decreased to 20%. The frequency of eosinophil colonies was relatively low (median 15%) with both stimuli. The described system can be applied successfully for studies of myelopoiesis in vitro. Both sources of colony stimulating activitites (CSA) employed had no significant difference in their ability to stimulate myelopoiesis.