CCNU Toxicity After an Overdose in a Patient with Hodgkin's Disease

An overdose of CCNU (600 mg over a 15-d period) was unintentionally ingested by a patient with advanced Hodgkin's disease subjected to combination chemotherapy. A severe bone marrow depression occurred 3 weeks after the start of the CCNU treatment. The nadir of the platelet count was reached after 4 weeks and that of the granulocyte count after 5 weeks. At the nadir of the white blood cell count, colony-forming cells (CFU-C) were found in significantly reduced numbers in the bone marrow, and were not found at all in the peripheral blood; the amount of colony-stimulating activity (CSA) produced by peripheral blood cells was reduced. However, the cells producing CSA recovered earlier than the CFU-C, and the CSA peak value was reached about 1 week before the peak value for CFU-C in the bone marrow. Thus, in vivo CSA-producing cells appeared to be more resistant to CCNU than were CFU-C, and their recovery appeared to be a prerequisite for the recovery of CFU-C and myelopoietic cells.     

Studies on the regeneration of the CFU-C population in blood and bone marrow of lethally irradiated dogs after autologous transfusion of cryopreserved mononuclear blood cells

In a group of 8 lethally irradiated (1200 R) dogs, that were transfused autologously with cryopreserved mononuclear cells (MNC) derived from the peripheral blood by leucapheresis the concentration of colony-forming units in agar (CFU-C) in bone marrow and peripheral blood was estimated at regular intervals after irradiation and transfusion of MNC. The numbers of MNC transfused per kg body weight ranged from 0.32 x 10(9) to 1.63 x 10(9) with an incidence of CFU-C between 0.02 x 10(5) and 1.38 x 10(5). In 6 dogs the CFU-C levels in the bone marrow reached the normal pre-irradiation values between days 15 and 20. But in 2 dogs that had received the lowest CFU-C numbers the regeneration of the bone marrow CFU-C was markedly delayed. In general the time course of the bone marrow repopulation by CFU-C for single dogs was reflected by a corresponding regeneration pattern of the blood CFU-C. The time course of the curves for the blood CFU-C levels on the other hand was of the same kind as for the granulocyte values in the peripheral blood, thuations were seen in the blood CFU-C levels of single dogs before irradiation and after mononuclear leucocyte transfusion. Despite of such limitations the blood CFU-C content appeared to be a useful indicator of haematopoietic regeneration of the bone marrow.     

Inhibition in vivo of mouse granulopoiesis by cell-free activity derived from human polymorphonuclear neutrophils

Cell-free extracts from human polymorphonuclear neutrophils (PMN) inhibited rebound granulopoiesis in the bone marrow and spleen of mice pretreated with cyclophosphamide to remove endogenous PMN. Absolute numbers of granulocytemonocyte progenitor cells (CFU-C) and net endogenous colony-stimulating activity (CSA) production were found to be increased 3 days after cyclophosphamide in the bone marrow and 6 days after in the spleen. Administration of PMN extract to the drug- treated mice prior to rebound granulopoiesis substantially decreased CSA production and CFU-C but not spleen B lymphocyte colony-forming cells. In addition, mice treated with PMN extract had decreased levels of CSA in serum and in conditioned medium of marrow-free bone, heart, and lung cultures. Inhibition was reversed by injection of bacterial lipopolysaccharide. Extracts from PMN of patients with chronic myelogenous leukemia, inactive in vitro, had no effect in vivo. These results demonstrate that inhibitory activity derived from PMN can control granulopoiesis in vivo.     

Granulopoiesis in severe congenital neutropenia.

The pathogenesis of the granulopoietic failure in three children with severe congenital neutropenia was studied. Mature neutrophils were absent from both peripheral blood and bone marrow. Assay of bone marrow granulocyte colony-forming cells (CFU-C) in a methylcellulose tissue culture system using colony-stimulating activity (CSA) from peripheral blood leukocytes demonstrated normal or increased concentrations of CFU-C compared to those from marrows of 60 age-matched controls. Colonies were of normal size and by light microscopy appeared to contain granulocytes in all stages of maturation including the mature polymorphonuclear neutrophil. CFU-C from peripheral blood of two patients were normal. Production and activity of CSA from the patients' peripheral blood leukocytes and urinary CSA excretion were normal. No serum inhibitors against CFU-C or CSA could be demonstrated using both control and autologous marrow. The defect did not appear to be due to a lack of granulocytic stem cells, a reduction of humoral stimulators of granulopoiesis, nor the presence of an inhibitor as measured by these techniques.     

Blood Colony-Forming Cells (CFU-C) and Leucocyte Colony-Stimulating Activity (CSA) in Patients with Neutrophilic Leucocytosis

The incidence of circulating colony-forming cells (CFU-C) and the ability of peripheral leucocytes to stimulate the colony formation (CSA) have been studied through a double layer agar culture system in 26 patients with neutrophilic leucocytosis and compared to the values obtained in 26 normal subjects. Both mean CFU-C incidence and mean leucocyte CSA of the whole group of patients were found significantly higher than normal, but considerable variation was observed among singular patients. The different patterns of blood CFU-C and leucocyte CSA are discussed. The combined evaluation of blood CFU-C and leucocyte CSA is found a useful tool to investigate the pathogenetic mechanisms of neutrophilic leucocytosis.     

Enhancing effect of cyclosporins A and G on bone marrow colony formation

The development of the number of colonies (cell colony-forming units, CFU-C) in soft agar from normal mouse bone marrow (BM) cells was enhanced 60% when total bone marrow cells (BMC) were preincubated for 1 h with either cyclosporin A (CsA) or cyclosporin G (CsG) before plating. Using cell fractionation techniques we found that the removal of macrophages enhanced CFU-C in noncyclosporin-treated BM and that cyclosporins mediated an additional enhancing effect. A similar enhancing effect on CFU-C in noncyclosporin-treated BM was obtained by depleting it of total T cells or Lyt-2.2+ cells. However, CFU-C growth in the residual BM population was no longer enhanced by cyclosporin. Conversely, removal of Lyt-1.2+ cells did not enhance CFU-C in noncyclosporin-treated BM, but the CFU-C in this population were enhanced by cyclosporin treatment. These results suggest that CsA and CsG can increase the cloning efficiency of normal mouse BMC, possibly by inhibiting an endogenous Lyt-2.2+ suppressor cell.     

Blood colony-forming cells (CFU-C) and leucocyte colony-stimulating activity (CSA) in patients with neutrophilic leucocytosis.

The incidence of circulating colony-forming cells (CFU-C) and the ability of peripheral leucocytes to stimulate the colony formation (CSA) have been studied through a double layer agar culture system in 26 patients with neutrophilic leucocytosis and compared to the values obtained in 26 normal subjects. Both mean CFU-C incidence and mean leucocyte CSA of the whole group of patients were found significantly higher than normal, but considerable variation was observed among singular patients. The different patterns of blood CFU-C and leucocyte CSA are discussed. The combined evaluation of blood CFU-C and leucocyte CSA is found a useful tool to investigate the pathogenetic mechanisms of neutrophilic leucocytosis.     

Lithium enhancement of megakaryocytopoiesis in culture: mediation via accessory marrow cells

To examine the effect of lithium (Li) on early megakaryocytopoiesis, murine marrow megakaryocytic (CFU-M) and granulocyte-macrophage (CFU-C) progenitors were assayed in vitro with and without addition of lithium chloride (LiCl) to culture. At 2 mM LiCl, the numbers of CFU-M- and CFU- C-derived colonies were increased to 146% +/- 8% and 128% +/- 6% of controls, respectively (p less than 0.005). Enumeration of megakaryocytes per colony showed a 78% increase of colonies (p less than 0.05) containing from 6 to 22 cells, suggesting an increased proliferative capacity of CFU-M in the presence of LiCl. Conditioned media from spleen cells cultured in the presence of both pokeweed mitogen (PWM-SCM) and 2 mM Li increased the numbers of CFU-M and CFU-C to 157% +/- 8% and 183% +/- 8%, respectively (p less than 0.025), compared to control cultures stimulated by PWM-SCM alone. Since the production of active colony-stimulating activities (CSA) from mitogen- stimulated conditioned media requires T lymphocytes, we hypothesized that the enhancement of the growth of early hematopoietic progenitors in marrow cultures was due to a Li-induced CSA production by accessory marrow cells, rather than a direct effect of Li on stem cells. To test this, cyclosporin-A (CyA), a T-lymphocyte function inhibitor known to suppress CSA production in PWM-SCM, was added to marrow cultures in the presence of 2 mM Li. CyA (3 micrograms/ml) abrogated the Li-induced enhancement of CFU-M and CFU-C growth, but had no effect on colony formation when added alone. The data suggest that the Li-induced enhancement of early megakaryocytopoiesis and granulocytopoiesis is due to local production of CSA(s) by an accessory cell population and requires the integrity of T-lymphocyte function.     

The regulatory role of interleukin 2-responsive T lymphocytes on human marrow granulopoiesis

The effect of recombinant interleukin 2 (IL2) on marrow CFU-C colony formation was evaluated to define the role for T lymphocytes in human marrow granulopoiesis. The colony-stimulating factor (CSA) used in our experiments was found to contain IL2. IL2 depletion from CSA resulted in a reduction in CFU-C colony proliferation. Addition of exogenous IL2 caused an increase in CFU-C colony numbers in a dose-dependent manner. This increase could be prevented by anti-Tac, a monoclonal antibody (MoAb) to the IL2 receptor. Moreover, anti-Tac in the absence of exogenous IL2 resulted in an overall decrease in colony numbers. Depletion of either adherent cells or T lymphocytes abolished the effect of IL2 and anti-Tac on colony growth. In the presence of IL2, re- addition of T lymphocytes to the T-depleted marrow or adherent cells to adherent cell-depleted marrow resulted in a significant increase in CFU- C colony numbers, whereas no significant effect was found when IL2- depleted CSA was used. Although T lymphocytes were not themselves essential for CFU-C colony growth, our studies indicate that IL2 and IL2-responsive T cells can regulate in vitro granulopoiesis.     

Mechanism of canine cyclic hematopoiesis: the role of prostaglandin E in feedback regulation

Prostaglandin E inhibits granulocyte-macrophage colony formation in vitro in man and mouse, suggesting that it plays a role in feedback regulation of granulocyte production in vivo. Therefore, we examined the role of PGE in normal canine hematopoiesis and its potential role in the pathogenesis of cyclic hematopoiesis in grey collie dogs. The prostaglandin synthesis inhibitors indomethacin and ibuprofen (10(-5) M) increased CFU-C growth to 194 and 160% of control, respectively, while PGE2 addition caused a dose-dependent inhibition of bone marrow CFU-C growth in both normal and grey collie dogs. These concentrations of indomethacin and ibuprofen decreased bone marrow cell elaboration of PGE measured by radioimmunoassay to less than 5% of control values. The levels of PGE in leukocyte conditioned medium prepared from grey collies correlated with the number of monocytes in the conditioning cell suspension (r = 0.78, n = 10, p less than 0.05) so that PGE production per monocyte was no different in normal and grey collie dogs. The effect of PGE2 upon CFU-C was to inhibit formation of macrophage, but not neutrophil colony subtypes. These findings make aberrant PGE-mediated inhibition of precursor cells an unlikely mechanism to cause cyclic hematopoiesis, and show that PGE produced by monocytes acts as a feedback inhibitor for precursor cells destined to produce monocytes but not for those destined to form neutrophils.     

Enrichment for CFU-C from murine and human bone marrow using soybean agglutinin

Mouse bone marrow and spleen cells agglutinated by soybean agglutinin (SBA) or peanut agglutinin (PNA) were previously shown to be enriched for spleen colony-forming cells (CFU-S) and sufficiently depleted of graft-versus-host reaction producing cells to allow hematologic reconstitution of lethally irradiated allogeneic recipient mice. A similar enrichment for cells capable of forming colonies in soft agar culture (CFU-C) has now been found in the SBA-agglutinated fraction of mouse bone marrow cells, in contrast to the finding that in human bone marrow the majority of the CFU-C are in the fraction not agglutinated by SBA. Cytofluorometric studies with fluorescein-labeled SBA (FITC- SBA) revealed that the majority of both mouse and human bone marrow cells bind the lectin. Experiments mixing the human marrow fractions separated by SBA reveal that true enrichment for CFU-C is achieved in the unagglutinated fraction, as opposed to a possible depletion of a suppressor cell population. Granulocytic, monocytic, and mixed cell colonies were all enriched in the SBA-unagglutinated cell fraction from human bone marrow.     

Oscillations of granulocytic and megakaryocytic progenitor cell populations in cyclic neutropenia in man.

The concentration of granulocytic progenitor cells in the bone marrow of 2 patients with cyclic neutropenia was assessed at intervals during the cycle. Syncroneous oscillations were observed for cells forming neutrophilic and megakaryocytic colonies in diffusion chambers in vivo (CFU-D), suggesting a close relationship between the two cell types. However, an almost reverse relationship was observed between CFU-D and cells forming colonies in agar culture in vitro (CFU-C), which cycled out of phase with CFU-D. This supports the concept that CFU-D and CFU-C denote different cell types.     

Bone Marrow Fibroblastoid Colony-Forming Cells (F-CFC) in Aplastic Anaemia: Colony Growth and Stimulation of Granulocyte-Macrophage Colony-Forming Cells (GM-CFC)

S ummary . Colonies of fibroblast-like cells have been grown from the mononuclear cell fractions of bone marrow aspirated from normal individuals and patients with aplastic anaemia. Some of the characteristics of the fibroblastoid cells have been determined and their granulocyte-macrophage colony-stimulating activity (CSA) in semi-solid agar culture has been used as a functional test of their influence on granulopoiesis.
The incidence and growth rates of fibroblastoid colony-forming cells (F-CFC) from aplastic patients'bone marrows were not markedly different from normal either before or after treatment by allogeneic bone marrow transplantation or with antilymphocyte globulin (ALG).
Confluent monolayers of fibroblastoid cells grown from normal marrow were, on the whole, poor stimulators of granulocyte-macrophage colony-forming cells (GM-CFC) and CSA was not detected in the supernatant medium. Fibroblastoid monolayers derived from many of the aplastic bone marrows studied were efficient stimulators of GM-CFC but, like the monolayers grown from normal cells, did not release CSA into the culture medium.
Addition of methylprednisolone (MP) to the cultures had little effect on fibroblastoid cell growth, induced fat-accumulation by some of the fibroblastoid colonies comprising the monolayer and reduced the abilities of the monolayers to stimulate GM-CFC.     

Lithium therapy of children with chronic neutropenia

We treated five children with chronic neutropenia using lithium carbonate and studied the effect in vivo on granulopoiesis. Granulocyte precursors (CFU-C) from blood and marrow, and colonystimulating activity (CSA) from peripheral blood leukocytes, were assayed in a methylcellulose tissue culture system. Three patterns of response to lithium were seen. In patients with aplastic anemia (one acquired and two Fanconi's aplastic anemia) despite increased colony-stimulating activity, CFU-C numbers remained very low and the neutropenia persisted. In a patient with Kostmann neutropenia colony-stimulating activity, and blood and marrow CFU-C numbers increased, but the agranulocytosis was unchanged. An impressive therapeutic effect was seen in one patient with idiopathic neutropenia with low colony-stimulating activity who responded to lithium with an increase in colony-stimulating activity and CFU-C resulting in persisting normal neutrophil counts. Lithium appears useful in treating a select group of neutropenic patients in whom colonystimulating activity production is responsive to lithium, and the granulocytic progenitor compartment is capable of producing mature neutrophils.     

Lymphocytes from patients receiving lithium do not inhibit CFU-C growth

Lymphocyte subset levels and function were examined in 12 patients on lithium therapy and in 11 healthy hospital personnel. Co-culture of allogeneic human bone marrow cells with monocyte-depleted lymphocyte preparations revealed that CFU-C formation was significantly reduced (mean 43% inhibition) in the presence of normal lymphocytes but not with the patients' lymphocytes (less than 5% inhibition). This did not reflect numerical changes in lymphocyte subsets, since these were similar for control and lithium subjects. T colony formation was significantly depressed in the patient group (P less than 0.05), whereas B colony numbers were similar in both groups (P greater than 0.1). The possible role of HLA-incompatibility affecting CFU-C growth was investigated in co-culture experiments, using lymphocytes from HLA-identical twins, one of whom was receiving lithium. In four separate co-culture experiments, the inhibitory effect was shown with lymphocytes from the non-lithium twin but was not demonstrated by the lithium subject. Addition of lithium in vitro to co-cultures of normal marrow and lymphocytes was found to negate the inhibitory phenomenon in a dose-related manner. It is postulated that granulocytosis induced by the administration of lithium may be a manifestation of changes in a lymphocytic control system.     

In Vitro Colony Studies in 87 Patients with Acute Nonlymphoblastic Leukemia

ABSTRACT. Colony-forming cells (CFU-C) in peripheral blood and bone marrow and colony-stimulating activity (CSA) in mononuclear peripheral white blood cells were studied at diagnosis in 87 patients with acute nonlymphoblastic leukemia (ANLL). Absence of CFU-C in peripheral blood was more frequent in patients who did not enter remission than in those who did, and survival was significantly shorter in CFU-C-negative than in CFU-C-positive patients. No correlation was found between CFU-C in the bone marrow and frequency of remission or survival time. Absence of CSA was significantly more frequent in patients who did not enter remission than in those who did. Only 4 of 28 patients who lacked CSA entered remission. Survival was significantly longer in CSA-positive than in CSA-negative patients. Thus, CSA synthesis in peripheral mononuclear blood cells appears to be a valuable prognostic factor in ANLL.     

Proliferative state of normal in vitro colony-forming cells during development of L5222 rat leukemia and their reaction to chemotherapy

In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.     

The exceptional responsiveness of certain human myeloid leukemia cells to colony-stimulating activity.

We have studied the marrow cells from a patient with acute myeloid leukemia (AML) for their responsiveness to colony-stimulating activity (CSA) in vitro. The AML cells were stimulated by CSA to rapid and extended growth in liquid culture. In the absence of CSA, the majority of cells died. CSA also stimulated the clonal growth of AML cells, and the minimum requirement for CSA was one-tenth to one-fiftieth that required to stimulate the growth of normal marrow CFU-C. CSA for AML cells was eluted from Sephacryl S-200 columns in fractions that represented an apparent molecular weight of 45,000 daltons. This fraction also produced optimal stimulation of normal human marrow. During remission, the patient's marrow cells did not grow in liquid culture and produced normal numbers of granulocytic and erythroid colonies in response to CSA and erythropoietin. Extended culture of the AML cells resulted in cell differentiation evidenced by decreasing proliferative capacity and by morphological and histochemical changes. These studies indicate that certain AML cells are extraordinarily responsive to CSA, an in vitro mediator of normal granulopoiesis.     

Derivatives of benzo(c)fluorene. XI. Effect of Benfluron on hemopoietic stem cells

The effect of Benfluron-- 5-(2-N,N-dimethylamino-ethoxy)-7-oxo-7H-benzo(c)fluorene hydrochloride--on hemopoietic stem cells was tested by the production of spleen cell colonies (CFU-S) in irradiated mice following application of bone marrow cells and by the production of hemopoietic colonies (CFU-C) in semisolid agar. The reduced numbers of CFU-S were found in mice applied Benfluron or Benfluron-treated bone marrow cells. The increased numbers of CFU-S, however, were found in mice receiving bone marrow cells from Benfluron-treated donors. The production of CFU-C was decreased after 2 h incubation of bone marrow cells with 0.5-5 micrograms/ml of Benfluron, and fully inhibited at higher concentrations.     

Improved Plasma Culture System for Production of Erythrocytic Colonies In Vitro: Quantitative Assay Method for CFU-E

An improved plasma culture system is described for the production of erythrocyticcolonies by mammalian adult hemopoieticcells in vitro under the influence of erythropoietin. The concentration of fetal calfserum in the medium used for dilution ofthe cells was critical for erythrocyticcolony formation when low numbers ofcells were plated. Optimal concentrationswere found for plasma, fetal calf serum,bovine serum albumin, and L-asparaginein the culture medium, and the colony-forming efficiency was shown to dependon the concentration of erythropoietin.With erythropoietin at plateau concentration, the number of erythrocytic colonies produced was directly proportionalto the number of bone marrow or spleencells plated, over a wide range of cell concentrations. Colony numbers per cultureconformed to a Poisson distribution. Thus,the improved plasma culture system maybe used for the quantitative assay ofCFU-E. The method is rapid (2 days), reliable, convenient, and inexpensive. Sincethe improved plasma culture system alsosupports granulocytic colony formationby bone marrow cells in the presence ofconditioned medium (CSA), and the number of granulocytic colonies produced isproportional to the number of cells plated,the same hemopoietic cell suspensionscan be simultaneously assayed for CFU-Eand CFU-C under virtually identical conditions.

Submitted on February 19, 1974 Accepted on April 9, 1974