A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.     

Recombinant human granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes--a phase I/II trial

In a phase I/II study, 11 patients with myelodysplastic syndromes (MDS) and severe transfusion-dependent cytopenia were treated with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to investigate the effects of rhGM-CSF on normal hematopoiesis and leukemic cells. The treatment schedule included dose escalation from 15 micrograms/m2 to 150 micrograms/m2 administered by continuous intravenous (IV) infusion for seven to 14 days and was repeated after a two-week treatment-free interval. The blood leukocyte counts increased dose dependently by 130% to 1,800% in ten patients; a rise of monocytes and eosinophils occurred in seven and six patients, respectively. No sustained increase in reticulocytes or platelets was observed. Lymphocyte counts increased in all patients affecting both T- helper and T-suppressor cells; however, the lymphocytes were not activated as analyzed by the expression of the interleukin-2 receptor. In four of the patients, all with greater than 14% blast cells in the bone marrow, the percentage of bone marrow blast cells increased during treatment with rhGM-CSF. Cytogenetic data indicated induction of both proliferation and differentiation of the leukemic clones by rhGM-CSF. Toxic side effects were minor with slight fever, phlebitis at the infusion site, and bone pain in the minority of patients. In conclusion, rhGM-CSF effectively stimulates hematopoiesis in vivo in patients with myelodysplastic syndromes. However, as the leukemic cell population can be stimulated in patients with a higher initial blast cell count, the combination of rhGM-CSF with other differentiation- inducing or cytotoxic agents has to be considered.     

Long-term follow-up of patients who received recombinant human granulocyte-macrophage colony stimulating factor after autologous bone marrow transplantation for lymphoid malignancy

Twenty-seven patients with lymphoid neoplasia who underwent autologous bone marrow transplant (BMT) and who had received recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) were followed in order to examine the potential long-term consequences of rhGM-CSF. rhGM-CSF (15-240 micrograms/m2/day) was given daily either for 14 or 21 days after marrow infusion. All surviving patients who remained in remission had stable marrow graft function. The actuarial survival rate was 45% and the relapse incidence was 50% at a median of 774 days after autologous BMT. These findings suggest that treatment with rhGM-CSF does not have profound adverse long-term consequences.     

In vitro and in vivo activity of human recombinant granulocyte-macrophage colony-stimulating factor in dogs

Although it is well documented that human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production and functional activity of human and nonhuman primate granulocytes and macrophages, relatively little is known about its effects on cells obtained from other species. The molecular cloning of the complementary DNA for human GM-CSF has made it possible to determine the cross-reactivity of the purified recombinant human material (rhGM-CSF) on cells of other species. The results presented herein show that specific receptors for human GM-CSF exist on dog bone marrow cells and mature circulating dog granulocytes. The number of the receptors and the apparent binding affinity of the rhGM-CSF to its receptors on granulocytes were similar to those observed either on human or monkey cells. In cultures of dog bone marrow cells, rhGM-CSF was capable of promoting colony formation in a dose-dependent manner. Human GM-CSF also primed dog granulocytes for increased production of reactive oxygen metabolites in response to either phorbolmyristic acetate-or zymosan-activated dog serum. In vivo, s.c. administration to healthy dogs of rhGM-CSF in daily doses of 15, 50, or 150 micrograms/kg body weight over a period of 7-20 days induced a dose-dependent rise of up to a maximum of a fourfold increase in peripheral WBC counts. The rise in WBC counts was mainly due to elevated neutrophil levels, but an increase in the numbers of monocytes and eosinophils was also observed. However, the rhGM-CSF-induced leukocytosis in dogs was not as dramatic as that observed in nonhuman primates. In all rhGM-CSF-treated dogs, circulating platelet counts dropped to nadir levels of about 20%-30% of normal numbers. Dogs that were treated with 150 micrograms/kg rhGM-CSF developed specific antibodies after about 10-12 days of treatment. These antibodies were able to neutralize the effect of rhGM-CSF in in vitro assays. In vivo WBC counts began to decline when specific antibodies developed, but they never dropped below normal levels. Taken together, the results suggest that human GM-CSF does not appear to exhibit absolute species specificity.     

Stimulation of hematopoiesis in patients with bone marrow failure and in patients with malignancy by recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotential hematopoietin. To assess the toxicity and biological activity of recombinant human GM-CSF (rhGM-CSF) in vivo, 25 patients with malignancy or bone marrow failure were treated with rhGM-CSF (specific activity approximately 5 x 10(7) U/mg) as part of a phase 1 trial. The treatment was administered by continuous intravenous (IV) infusion daily for 2 weeks at fixed dose levels and repeated after a 2- week rest period. Over the entire dose range tested (15 to 500 micrograms/m2/d), rhGM-CSF treatment was associated with dramatic increases (two- to 70-fold) in total leukocyte counts, which consisted predominantly of neutrophils, bands, eosinophils, and monocytes. Furthermore, six of the 14 patients with one or more cytopenias that received at least two cycles of treatment had multilineage responses characterized by twofold or greater increases in platelet count to a level above 100,000, twofold or greater increases in corrected reticulocyte count, and a reduced requirement for red cell transfusions. Three of these patients became independent of both red cell and platelet transfusions for 17 to 37 weeks of follow-up. Treatment was associated also with an increase in bone marrow cellularity and frequency of cycling progenitor cells. The treatment was well tolerated; side effects included constitutional symptoms and bone pain. These results demonstrated that rhGM-CSF has a significant impact on hematopoiesis in patients with advanced malignancy and also in patients with bone marrow failure.     

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.     

Sequential administration of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for malignant lymphoma: a phase I/II multicenter study

Preclinical studies of recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) have shown enhancement of multilineage hematopoiesis when administered sequentially. This study was designed to evaluate the safety, tolerability, and biologic effects of sequential administration of rhIL- 3 and rhGM-CSF after marrow ablative cytotoxic therapy and autologous bone marrow transplantation (ABMT) for patients with malignant lymphoma. Thirty-seven patients (20 patients with non-Hodgkin's lymphoma and 17 patients with Hodgkin's disease) received one of four different treatment regimens before ABMT. Patients were entered in one of four study groups to receive rhIL-3 (2.5 or 5.0 micrograms/kg/day) administered by subcutaneous injection for either 5 or 10 days starting 4 hours after the marrow infusion. Twenty-four hours after the last dose of rhIL-3, rhGM-CSF (250 micrograms/m2/d as a 2-hour intravenous infusion) administration was initiated. rhGM-CSF was administered daily until the absolute neutrophil count (ANC) was > or = 1,500/microL for 3 consecutive days or until day 27 posttransplant. The most frequent adverse events in the trial included nausea, fever, diarrhea, mucositis, vomiting, rash, edema, chills, abdominal pain, and tachycardia. Three patients were removed from the study because of chest, skeletal, and abdominal pain felt to be probably related to study drug. Four patients died during the study period because of complications unrelated to either rhIL-3 or rhGM-CSF. The median time to recovery of neutrophils (ANC > or = 500/microL) and platelets (platelet count > or = 20,000/microL) was 14 and 15 days, respectively. There were fewer days of platelet transfusions than seen in historical control groups using rhGM-CSF, rhG-CSF, or rhIL-3 alone. In addition, there were fewer days of red blood cell transfusions compared with historical controls using no cytokines or rhGM-CSF. These data indicate that the sequential administration of rhIL-3 and rhGM-CSF after ABMT is safe and generally well-tolerated and results in rapid recovery of multilineage hematopoiesis.     

Recombinant human granulocyte-macrophage colony-stimulating factor plus recombinant human erythropoietin may improve anemia in selected patients with myelodysplastic syndromes

The purpose of this study was to improve erythropoiesis in patients with anemia due to myelodysplastic syndromes (MDS). We treated 13 patients first with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 6 weeks, then with recombinant human erythropoietin (rhEpo) and rhGM-CSF for the next 12 weeks. Five patients had refractory anemia (RA), 3 refractory anemia with ringed sideroblasts (RAS), and 5 refractory anemia with excess of blasts (RAEB). Ten patients were transfusion-dependent at the time of inclusion. Eleven patients completed this phase II study. Five responded with an increase in hemoglobin level (3 patients) or a reduction in transfusion requirement (2 patients). We registered no response in the remaining 6 patients during treatment. Patients responding to combined treatment had relatively low concentrations of plasma Epo and plasma ferritin before treatment with rhEpo and a normal karyotype throughout the study. Long-term bone marrow cultures did not predict the response. Still, responders seemed to have a higher number of colony-forming progenitors than non-responders. In conclusion, combined therapy with rhGM-CSF and rhEpo may stimulate hematopoiesis and correct or improve anemia in some patients with MDS. © 1993 Wiley-Liss, Inc.     

Sequential in vivo treatment with two recombinant human hematopoietic growth factors (interleukin-3 and granulocyte-macrophage colony-stimulating factor) as a new therapeutic modality to stimulate hematopoiesis: results of a phase I study.

In a phase I study, the sequentially administered combination of recombinant human interleukin-3 (rhIL-3) and rhGM-CSF was compared with treatment with rhIL-3 alone in 15 patients with advanced tumors but normal hematopoiesis. Patients were initially treated with rhIL-3 for 15 days. After a treatment-free interval, the patients received a second 5-day cycle of rhIL-3 at an identical dosage, immediately followed by a 10-day course of rhGM-CSF, to assess the toxicity and biologic effects of this sequential rhIL-3/rhGM-CSF combination. rhIL-3 doses tested were 125, and 250 micrograms/m2, whereas rhGM-CSF was administered at a daily dosage of 250 micrograms/m2. Both cytokines were administered by subcutaneous (SC) bolus injection. rhIL-3/rhGM-CSF treatment was more effective than rhIL-3 but equally effective to each other in increasing peripheral leukocyte counts, especially neutrophilic and eosinophilic granulocyte counts. In contrast, both modes of cytokine therapy raised the platelet counts to the same degree. rhIL-3/GM-CSF treatment was more effective than rhIL-3 in increasing the number of circulating hematopoietic progenitor cells BFU-E and CFU-GM. High-dose rhIL-3, but not low-dose rhIL-3, was as effective as the rhIL-3/rhGM-CSF combinations in increasing the number of circulating CFU-GEMM. The increase in absolute neutrophil counts correlated with the increase in the number of circulating CFU-GM. Side effects, mainly fever, headache, flushing, and sweating, were generally mild, but in two patients the occurrence of chills, rigor, and dyspnea after initiation of GM-CSF treatment necessitated dose reduction and discontinuation, respectively. These results indicate that sequential treatment with rhIL-3 and rhGM-CSF is as effective as single-factor treatment with rhIL-3 in stimulating platelet counts, whereas the effect of combination therapy on neutrophil counts and circulating progenitor cells is superior.     

Effect of mast cell growth factor (c-kit ligand) on clonogenic leukemic precursor cells.

Mast cell growth factor (MGF), the ligand for the c-kit receptor, has been shown to be a hematopoietic growth factor that preferentially stimulates the proliferation of immature hematopoietic progenitor cells (HPC). We studied the effect of MGF on the in vitro growth of clonogenic leukemic precursor cells in the presence or absence of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or erythropoietin (EPO). Leukemic blood and bone marrow cells from patients with various types of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) in chronic phase, as well as bone marrow samples from patients with myelodysplastic syndromes (MDS) were studied. MGF as a single factor did not induce significant colony formation by clonogenic leukemic precursor cells. In the presence of IL-3 and/or GM-CSF, MGF weakly stimulated the colony formation by clonogenic precursor cells from patients with AML. In contrast, in the presence of IL-3 and/or GM-CSF, MGF strongly induced both size and number of leukemic colonies from patients with CML in chronic phase. Furthermore, in the presence of EPO, MGF strongly stimulated erythroid colony formation by CML precursor cells. Cytogenetic analysis of the colonies showed that all metaphases after 1 week of culture were derived from the leukemic clone. In patients with MDS, MGF strongly stimulated myeloid colony formation in the presence of IL-3 and/or GM-CSF (up to fourfold), and erythroid colony formation in the presence of EPO (up to eightfold). Not only the number, but also the size of the colonies increased. In the presence of MGF, the percentage of normal metaphases increased in three patients tested after 1 week of culture compared with the initial suspension, suggesting that the normal HPC were preferentially stimulated compared with the preleukemic precursor cells. In the absence of exogenous EPO and in the presence of 10% human AB serum, MGF in the presence of IL-3 and/or GM-CSF induced erythroid colony formation from normal bone marrow and patients with MDS or CML, illustrating that MGF greatly diminished the EPO requirement for erythroid differentiation. These results indicate that MGF may be a candidate as a hematopoietic growth factor to stimulate normal hematopoiesis in patients with acute myeloid leukemia, or with myelodysplastic syndromes.     

Bifunctional effects of tumor necrosis factor alpha (TNF alpha) on the growth of mature and primitive human hematopoietic progenitor cells: involvement of p55 and p75 TNF receptors

Tumor necrosis factor alpha (TNF alpha) has previously been reported to have both inhibitory and stimulatory effects on hematopoietic progenitor cells. Specifically, TNF alpha has been proposed to stimulate early hematopoiesis in humans. In the present study we show that TNF alpha, in a dose-dependent fashion, can potently inhibit the growth of primitive high proliferative potential colony-forming cells (HPP-CFCs) stimulated by multiple cytokine combinations. Using agonistic antibodies to the p55 and p75 TNF receptors or TNF alpha mutants specific for either of the two TNF receptors, we show that both receptors can mediate this inhibition. In contrast, the potent stimulation of interleukin-3 (IL-3) plus granulocyte-macrophage colony- stimulating factor (GM-CSF) induced HPP-CFC colony formation observed at low concentrations of TNF alpha (2 ng/mL) was only a p55-mediated event. Moreover, the stimulatory effects of TNF alpha on GM-CSF or IL-3- induced colony formation, as well as the inhibition of G-CSF-induced colony growth, were also exclusively signaled through the p55 TNF receptor. Taken together, our results suggest that the inhibitory effects of TNF alpha on primitive bone marrow progenitor cells are mediated through both p55 and p75 TNF receptors, whereas the p55 receptor exclusively mediates the bidirectional effects on more mature, single factor-responsive bone marrow progenitor cells as well as stimulation of IL-3 plus GM-CSF-induced HPP-CFC colony growth.     

c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.     

Colony-stimulating activity in the serum of patients with hemopoietic malignancies after intensive chemotherapy/radiotherapy: its augmentation by GM-CSF in vivo and interleukin 4 in vitro.

Colony-stimulating activity (CSA) in the serum of patients with hematological malignancies increased substantially after intensive therapy with cyclophosphamide/busulfan, cyclophosphamide/total body irradiation, or melphalan/total body irradiation. This was not dependent on patients receiving allogeneic bone marrow transplantation (ABMT) or autologous bone marrow rescue (ABMR). In 44 of 62 patients CSA was maximum approximately 7 days after chemotherapy/radiotherapy, whereas in 18 of 62 patients CSA was maximum between 9 and 20 days after therapy and decreased thereafter. The time course of CSA was not dependent on disease and was not affected by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) given as a continuous infusion for 14 days after therapy; however, serum from patients receiving rhGM-CSF produced significantly more colonies from donor bone marrow than serum from patients who did not receive the cytokine (p = 0.013). Despite the early peak in CSA in the majority of patients, there was no correlation between the time at which CSA was maximum and the return of patients' neutrophils to 500/microliters. Recombinant human interleukin 4 (IL-4) increased the number of granulocyte-macrophage colony-forming unit colonies, principally granulocyte colony-forming unit colonies, from normal bone marrow exposed to patients' serum after intensive therapy and antibody to GM-CSF reduced colony numbers. The results suggest that after intensive therapy granulocyte colony-stimulating factor (G-CSF) as well as GM-CSF is released into the serum and, in addition to acting directly with G-CSF, IL-4 may stimulate mononuclear cells to produce and/or release G-CSF.     

Use of recombinant human granulocyte-macrophage colony-stimulating factor in graft failure after bone marrow transplantation

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was evaluated in 37 patients with marrow graft failure after allogeneic (n = 15), autologous (n = 21), or syngeneic (n = 1) bone marrow transplantation. rhGM-CSF was administered by 2-hour infusion at doses between 60 and 1,000 micrograms/m2/d for 14 or 21 days. At doses of less than 500 micrograms/m2, rhGM-CSF was well-tolerated and did not exacerbate graft-versus-host disease in allogeneic transplant recipients. No patient with myelogenous leukemia relapsed while receiving rhGM-CSF. Twenty-one patients reached an absolute neutrophil count (ANC) greater than or equal to 0.5 x 10(9)/L within 2 weeks of starting therapy while 16 did not. None of seven patients who received chemically purged autologous marrow grafts responded to rhGM-CSF. The survival rates of GM-CSF-treated patients were significantly better than those of a historical control group.     

Antagonistic effects of interleukin 6 and G-CSF in the later stage of human granulopoiesis in vitro.

The effect of recombinant human interleukin 6 (rhIL-6) on the in vitro growth of human bone marrow myeloid progenitors (granulocyte-macrophage colony-forming units, CFU-GM) was investigated. Recombinant human IL-6 by itself did not induce colony formation. When rhIL-6 at various concentrations was added to the CFU-GM colony cultures containing recombinant human granulocyte colony-stimulating factor (rhG-CSF) or recombinant human granulocyte-monocyte/macrophage colony-stimulating factor (rhGM-CSF), rhIL-6 significantly suppressed the colony formation induced by rhG-CSF, but not by rhGM-CSF. This suppressive effect of rhIL-6 on rhG-CSF-induced, but not rhGM-CSF-induced colony formation was confirmed by using an MY10(+)-cell-enriched population. Neither interleukin 3 nor interleukin 1 alpha suppressed the growth of myeloid progenitors. The preincubation of bone marrow cells with rhIL-6 for a short time (30 min) resulted in a reduction of colonies induced by rhG-CSF, but not by rhGM-CSF. The suppressive effect of rhIL-6 on rhG-CSF-induced colony formation was not observed when the cells were preincubated together with rhG-CSF at a high ratio of rhG-CSF to rhIL-6. The rhIL-6-mediated suppressive effect was further confirmed by blocking the effect by the anti-IL-6 antibody. These results suggest antagonistic interaction between IL-6 and G-CSF in the later differentiation of myeloid progenitors.     

Mofarotene (Ro 40-8757) inhibits hematopoiesis in vitro by preventing maturation from primitive progenitor cells

The effect of the arotinoid mofarotene (Ro 40-8757; 4-[2-[p-[(E)- 2(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)- propenyl]phenoxy]ethyl]morpholine) on stromal cell-mediated hematopoiesis was examined in murine long-term bone marrow cultures. Whether added at week 2 to regenerating cultures or at week 4 to plateau-phase cultures, mofarotene strongly inhibited total cell production in a dose-dependent manner. Progenitor cell production was also inhibited, but to a lesser extent. When added at the initiation of culture, 1 mumol/L mofarotene did not affect formation of the adherent layer, but production of total nucleated cells and progenitors was inhibited over the next 10 weeks by 95% and 96%, respectively. However, after mofarotene treatment ceased, progenitor cell levels began increasing immediately, and cell production reached plateau levels comparable with those of control cultures within 4 weeks. Hematopoiesis was maintained for 14 more weeks, indicating that long-term culture- initiating cells survived the treatment. Assays of spleen colony- forming units (CFU-S) in the adherent layers showed an enrichment of day-13 CFU-S relative to the more mature day-9 CFU-S. Mofarotene did not inhibit colony formation by bone marrow cells stimulated by exogenous growth factors and did not decrease production of growth factors by stromal cells in the cultures, as determined by functional assays and by mRNA levels. These results suggest that mofarotene blocks differentiation of very primitive progenitors, inhibiting production of more mature hematopoietic elements.     

The effect of recombinant human granulocyte-macrophage colony-stimulating factor on neutropenia and related morbidity in chronic severe neutropenia

STUDY OBJECTIVE: To define the clinical and hematologic effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on patients with chronic severe neutropenia. DESIGN: Open-label, phase II study of rhGM-CSF. SETTING: Inpatient hematology and surgery clinic at a university medical center. PATIENTS: Four consecutive patients with chronic severe neutropenia, which in two cases was complicated by severe infection, in one case by perianal fistula, and in one case by complete rectal prolapse. Two patients had chronic idiopathic neutropenia; one patient had congenital neutropenia (myelokathexis); and one patient had autoimmune neutropenia. INTERVENTIONS: The rhGM-CSF was given intravenously or subcutaneously at starting dosages of 150 to 1000 micrograms/m2 body surface area.d for 12 to 14 consecutive days. Two patients received a second course of daily rhGM-CSF treatment after a nontreatment interval of 14 to 20 days. MEASUREMENTS AND MAIN RESULTS: In all four patients, the absolute neutrophil counts increased from less than 0.25 x 10(9)/L to 3.2 to 19.2 x 10(9)/L within 2 weeks of beginning rhGM-CSF therapy. Two patients had life-threatening infections that resolved during therapy. The two other patients had major ano-rectal surgery during rhGM-CSF treatment and had no postoperative infections. CONCLUSIONS: In patients with chronic neutropenia, rhGM-CSF may increase neutrophil counts. This therapy may be a useful adjunct to antibiotic therapy for patients with infection and perioperatively for patients having anorectal surgery.     

Colony stimulating activity in the serum of patients with multiple myeloma is enhanced by interleukin 3: a possible role for interleukin 3 after high dose melphalan and autologous bone marrow transplantation for multiple myeloma

Sera from 36/37 multiple myeloma patients and 19/21 sera from patients with other solid or liquid tumours had granulocyte-macrophage colony stimulating activity (CSA) towards normal human donor bone marrow whereas 1/16 sera from normal donors had this activity. Unlike human rhGM-CSF and GM-CSF from 5637 (human bladder cell line) conditioned medium which is heat stable, CSA from serum is heat labile (56 degrees C/30 min). In multiple myeloma patients, CSA was detectable more than 2 years after treatment with 'high dose melphalan. Although multiple myeloma patients, at relapse, have sufficient CSA in their serum to produce maximal stimulation of GM-CFUc from normal donor bone marrow in vitro, their own GM population responds poorly. The results suggest that the failure of patients own bone marrow to respond to endogenous CSA may be due to damage to the stem cells of the marrow or the failure of precursor cells to respond to CSA. Addition of rhIL-3 to myelomatous serum increased the number of GM-CFUc from both normal and myelomatous bone marrow but did not stimulate the growth of MY-CFUc significantly. The results suggest that rhIL-3 may assist bone marrow recovery in multiple myeloma patients after intensive chemotherapy.     

Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice

The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11- treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.     

Tumor necrosis factor-alpha levels in long-term marrow cultures from patients with aplastic anemia: modulation by granulocyte-macrophage colony-stimulating factor.

We have previously shown that the levels of hematopoietic progenitors in long-term marrow cultures (LTMC) from patients with aplastic anemia (AA) are drastically reduced, as compared to normal LTMC. We have also reported that when LTMC from AA patients are supplemented with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) there is an increase in colony-forming cell (CFC) levels. However, such a stimulation is only transient and it is followed by an inhibition in CFC growth. Based on these observations, in the present study we have tested the hypothesis that the levels of tumor necrosis factor-alpha (TNF-alpha), an inhibitor of hematopoiesis, are increased in AA LTMC and that such levels are further increased after rhGM-CSF has been added to the cultures for several weeks. Accordingly, we have determined the levels of TNF-alpha in the supernatant of LTMC established from normal (n = 8) and AA (n = 6) bone marrow and in AA LTMC supplemented with rhGM-CSF (n = 6). At the time of culture initiation, TNF-alpha levels were below detection in all the samples analyzed. After 5 weeks of culture, TNF-alpha levels in normal LTMC were very low, with a median of 7.3 pg/mL. In contrast, AA LTMC contained higher levels of TNF-alpha (median of 49.6 pg/mL). In keeping with our hypothesis, addition of rhGM-CSF to AA LTMC resulted in a significant further increase of TNF-alpha levels (median of 135.4 pg/mL). Our results demonstrate an inverse correlation between reduced hematopoiesis in AA LTMC and increased levels of TNF-alpha in this culture system. Based on the results presented here, together with previous reports indicating that TNF-alpha is a potent inducer of apoptosis in hematopoietic progenitor cells, it seems reasonable to suggest that TNF-alpha is implicated in the pathophysiology of AA.