The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on primitive hematopoietic stem cell (PHSC) function and numbers, after chemotherapy

OBJECTIVE: Primitive hematopoietic stem cell function was assessed after cyclophosphamide with granulocyte-macrophage colony-stimulating factor (GM-CSF), with or without preadministration of interleukin-1, using competitive repopulation. METHODS: C57B6/J mice injected with one or four biweekly intravenous injections of cyclophosphamide, 200 mg/kg, received granulocyte-macrophage colony-stimulating factor, 1 microg, subcutaneously for 5 days, beginning 24 hours after cyclophosphamide. Alternatively, mice were injected with interleukin-1, 1 microg, 20 hours before administration of drug or drug and cytokine. Marrow obtained from mice sacrificed 4 weeks after the last dose of drug or drug and cytokine was used in competitive repopulation. RESULTS: Significant reductions in marrow repopulating ability occurred after a single dose of cyclophosphamide or multiple injections. Repopulating units (RU) were calculated, and both binomial and Poisson models for estimation of primitive hematopoietic stem cell (PHSC) numbers were used. RU were significantly diminished for all treatment groups when compared to controls. PHSC numbers were not significantly affected by either regimen of cyclophosphamide given alone. Addition of GM-CSF to cyclophosphamide, whether the latter was given in single or multiple doses, led to further, although insignificant, declines in repopulating ability, as well as PHSC and RU numbers. Interleukin-1 usage exacerbated the observed repopulating defect. There was evidence of replicative failure in individual cells, indicating a qualitative defect also. SUMMARY: Additive stem cell depletion and qualitative replicative defect occur after chemotherapy-cytokine usage. However, the replicative defect of PHSC seen after addition of GM-CSF is not significantly worse than that seen with cytotoxic drug use alone.     

Comparative effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) on priming peripheral blood progenitor cells for use with autologous bone marrow after high-dose chemotherapy

Two hematopoietic colony-stimulating factors, granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), have been shown to accelerate leukocyte and neutrophil recovery after high-dose chemotherapy and autologous bone marrow (BM) support. Despite their use, a prolonged period of absolute leukopenia persists during which infections and other complications of transplantation occur. We collected large numbers of peripheral blood (PB) progenitors after CSF administration using either G-CSF or GM-CSF and tested their ability to affect hematopoietic reconstitution and resource utilization in patients undergoing high-dose chemotherapy and autologous BM support. Patients with breast cancer or melanoma undergoing high-dose chemotherapy and autologous BM support were studied in sequential nonrandomized trials. After identical high-dose chemotherapy, patients received either BM alone, with no CSF; BM with either G-CSF or GM-CSF; or BM with G-CSF or GM-CSF and G-CSF or GM-CSF primed peripheral blood progenitor cells (PBPC). Hematopoietic reconstitution, as well as resource utilization, was monitored in these patients. The use of CSF- primed PBPC led to a highly significant reduction in the duration of leukopenia with a white blood cell (WBC) count under 100 and 200 cells/mL, and neutrophil count under 100 and 200 cells/mL with both GM- and G-CSF primed PB progenitor cells, compared with the use of the CSF with BM or with historical controls using BM alone. In addition, the use of CSF-primed PBPC resulted in a significant reduction in median number of antibiotics used, days in the Bone Marrow Transplant Unit, and hospital resources used. Patients receiving G-CSF primed PBPC also experienced a reduction in the median number of days in the hospital, red blood cell (RBC) transfusions, platelet transfusions, days on antibiotics, and discounted hospital charges. Phenotypic analysis of the CSF-primed PBPC indicated the presence of cells bearing antigens associated with both early and late hematopoietic progenitor cells. The use of CSF-primed PBPC can significantly improve hematopoietic recovery after high-dose chemotherapy and autologous BM support. In addition, the use of G-CSF-primed PBPC was associated with a significant reduction in hospital resource utilization, and a reduction in hospital charges.     

Engraftment syndrome following autologous hematopoietic stem cell transplantation: definition of diagnostic criteria

Engraftment syndrome (ES) is an increasingly reported complication of hematopoietic stem cell transplantation (HSCT). In order to better characterize the clinical criteria for the diagnosis of ES, we retrospectively analyzed 125 autologous HSCT recipients. ES was first defined as the presence of noninfectious fever plus skin rash. Patients with and without these findings were compared (univariate and multivariate analyses) regarding the presence of weight gain, hypoalbuminemia, pulmonary infiltrates, diarrhea, neurological manifestations and jaundice. The variables that are significantly more frequent in patients with fever and skin rash were incorporated in the definition criteria. The final diagnostic criteria were noninfectious fever plus any of the following: skin rash, pulmonary infiltrates or diarrhea. The incidence of ES was 20%. The single risk factor for ES by multivariate analysis was a diagnosis other than Hodgkin's disease (odds ratio 6.17, 95% confidence interval 1.38-27.78). Patients with ES received empirical antifungal therapy more frequently than patients without the syndrome (40 vs 19%, P=0.03), and had a longer duration of hospitalization (P=0.0007). The prospective application of these diagnostic criteria may have a favorable impact on the early diagnosis of the syndrome, with the initiation of corticosteroids and a reduction in the unnecessary use of antimicrobial agents.     

Systemic capillary leak syndrome after granulocyte colony-stimulating factor (G-CSF)

Capillary leak syndrome (CLS) commonly occurs in the intensive care setting. CLS is seen in conditions such as septic shock or may result from conditions such as multitrauma and pancreatitis, which result in the systemic inflammatory response syndrome (SIRS). We present two cases in which both patients suffered with CLS, which we believe was caused following administration of granulocyte colony-stimulating factor, to our knowledge not described in the intensive care patient previously. We discuss how these patients management differs from other intensive care unit patients with CLS and how it is important to diagnose this condition early in haematological oncology cases.     

Improvement of pneumonia and arthritis in Felty's syndrome by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF)

Summary A 63-year old man with Felty's syndrome and pneumonia of unknown origin was treated with GM-CSF. Granulocyte counts increased and arthritis-related symptoms improved under GM-CSF. Pneumonia was treated effectively with antibiotics only during or after GM-CSF application. This suggests, that antibiotic-resistent infections can be treated effectively in patients with Felty's syndrome when granulocyte counts are raised by GM-CSF.     

Granulocyte colony-stimulating factor (G-CSF) increases neutrophil migration across vascular endothelium independent of an effect on adhesion: comparison with granulocyte-macrophage colony-stimulating factor (GM-CSF)

Granulocyte colony-stimulating factor (G-CSF) increases neutrophil counts, and enhances and primes many neutrophil functions, implicating a role for this growth factor in host defence. This study investigated whether G-CSF is able to directly influence the transendothelial migration of neutrophils, and how such effects might be related to other effects on neutrophil adhesive properties. G-CSF, like GM-CSF, increased surface levels of the adhesive receptor, CD11b/CD18, but down-regulated L-selectin expression on neutrophils. Unlike GM-CSF, however, G-CSF had no effect on neutrophil adhesion to endothelium. Despite the lack of effect on neutrophil adhesion, G-CSF was able to produce significant enhancement of neutrophil transmigration across unstimulated endothelium in vitro . When used at an optimal concentration of 100 ng/ml, G-CSF increased neutrophil migration to 217 ± 19% of baseline levels ( P <0.001, n  = 10). This effect was similar to that previously demonstrated for GM-CSF (which increased migration to 271 ± 40%, P <0.001, n  = 12). G-CSF-induced transmigration, like GM-CSF induced migration, was independent of concentration gradients, suggesting that these are not simply chemotactic effects. G-CSF differs from GM-CSF, however, in that although GM-CSF inhibited neutrophil migration across IL-1-activated endothelium (33 ± 8% inhibition, n  = 6, P <0.01), G-CSF had no effect on neutrophil migration across IL-1 activated endothelium. Hence G-CSF, despite having no effect on neutrophil adhesion to endothelium, is a powerful stimulator of transmigration, and, unlike GM-CSF, does not inhibit cell movement across inflamed endothelium. These results suggest that G-CSF is able to influence neutrophil recruitment into local infective sites, and, further, that G-CSF mobilized cells would be competent to migrate into tissues in response to inflammatory stimuli.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) secretion by adherent monocytes measured by quantitative immunoassays.

The kinetics of GM-CSF and G-CSF secretion by purified adherent human monocytes were studied by quantitative immunoassays. Interleukin-1 (IL-1); 4-40 ng/ml and E. coli lipopolysaccharide (LPS); 0.1-1.00 ng/ml, were the most effective stimuli and induced dose-dependent secretion of both GM-CSF and G-CSF. Secretion of newly synthesized CSF was detectable 3-6 hours after stimulation and continued for approximately 24 h. Twenty minutes pulse exposure to LPS was sufficient to induce half maximum secretion of GM-CSF, and after 24-36 h the adherent monocytes could not be restimulated. Neither GM-CSF nor TNF could down-regulate the secretion of GM-CSF. IL-3 induced a minor secretion of GM-CSF whereas TNF, G-CSF, M-CSF and IFN-gamma were unable to induce GM-CSF secretion. In addition to LPS and IL-1, GM-CSF and to a minor degree TNF induced G-CSF secretion. Enriched T lymphocytes secreted GM-CSF, but not G-CSF, after stimulation with PHA or staphylococcal enterotoxin A (SEA), whereas LPS and IL-1 were without stimulatory effects. We also noted that enriched T lymphocytes added to LPS-stimulated adherent monocytes at ratios of 1:10 or more inhibited, in a dose-dependent fashion, GM-CSF secretion by 13-55%. These findings add new quantitative data on CSF secretion by human monocytes.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjunct to autologous hemopoietic stem cell transplantation for lymphoma.

OBJECTIVE: To determine the hemopoietic effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients having autologous hemopoietic stem cell transplantation for Hodgkin or non-Hodgkin lymphoma. DESIGN: Placebo or GM-CSF was administered after bone marrow or peripheral blood stem cell transplantation or both in a randomized, double-blind phase III trial by daily intravenous infusion (10 micrograms/kg body weight) until absolute neutrophil counts reached greater than or equal to 1000/mm3 on 3 consecutive days. SETTING: Bone marrow transplantation unit in a university hospital. PATIENTS: Sixty-nine consecutive patients with Hodgkin or non-Hodgkin lymphoma received GM-CSF (36 patients) or placebo (33 patients). MEASUREMENTS AND MAIN RESULTS: Patients who received GM-CSF achieved absolute neutrophil counts greater than or equal to 500/mm3 (median, 12 compared with 16 days, P = 0.02) and absolute neutrophil counts greater than or equal to 1000/mm3 (median, 15 compared with 24 days, P less than 0.001) more quickly than patients who received placebo. Multivariate analysis indicated that use of GM-CSF, peripheral blood stem cells, and unpurged bone marrow were the strongest predictors for early neutrophil recovery greater than 500/mm3. Bacterial infections were significantly reduced in the GM-CSF group (P = 0.04). Delayed engraftment (neutrophils less than 500/mm3 at day 30) occurred in 26% and 17% of the placebo and GM-CSF groups, respectively, and correlated with the absence of detectable myeloid progenitor cells (colony-forming units-granulocyte macrophage, CFU-GM) (P less than 0.001) in marrow aspirate specimens obtained on day 15. Time to platelet independence, duration of hospital stay, severe adverse reactions, relapse, and disease-free survival rates did not differ significantly between the two groups. CONCLUSIONS: Administration of GM-CSF after autologous hemopoietic stem cell transplantation in patients with lymphoma resulted in accelerated myeloid recovery, particularly in patients who received peripheral blood stem cells and nonpurged bone marrow, and was associated with a decreased incidence of bacterial infections.     

Engraftment syndrome following hematopoietic stem cell transplantation.

During neutrophil recovery following hematopoietic stem cell transplantation, a constellation of symptoms and signs including fever, erythrodermatous skin rash, and noncardiogenic pulmonary edema often occur. These clinical findings have usually been referred to as engraftment syndrome, or, reflecting the manifestations of increased capillary permeability, capillary leak syndrome. While described most often following autologous stem cell transplantation, a similar clinical syndrome has been observed followed allogeneic stem cell transplantation. Distinction from graft-versus-host disease in the allogeneic setting however, has been difficult. Recent experience with non-myeloablative conditioning for stem cell transplantation, however, reveals that an engraftment syndrome independent of GVHD may occur. In some cases, this engraftment syndrome may be a manifestation of a host-versus-graft reaction (graft rejection). While cellular and cytokine interactions are believed to be responsible for these clinical findings, a distinct effector cell population and cytokine profile have not been defined. Engraftment syndromes are likely associated with an increased transplant-related mortality, mostly from pulmonary and associated multi-organ failure. Corticosteroid therapy is often dramatically effective for engraftment syndrome, particularly for the treatment of the pulmonary manifestations. A proposal for a more uniform definition of engraftment syndrome has been developed in order to allow for a reproducible method of reporting of this complication and for evaluating prophylactic and therapeutic strategies.     

Development of acute inflammatory arthritis by granulocyte-macrophage colony-stimulating factor during autologous stem cell transplantation for cryoglobulinemia

Hematopoietic growth factors (HGFs) are now known to influence the function of mature myeloid cells as well as their traditional roles as regulators of hematopoiesis. It became apparent that they could take part in inflammatory and immune responses by activating monocytes/macrophages to release mediators of such responses. We describe a 53-year-old male who developed acute inflammatory arthritis by granulocyte-macrophage colony-stimulating factor (GM-CSF) following autologous stem cell transplantation for cryoglobulinemia. Arthritis improved dramatically soon after GM-CSF was withdrawn and steroid therapy was admitted. This case may lead to further attention for this potential problem since HGFs are frequently used in a variety of clinical settings.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in serum in bone marrow transplanted patients.

Colony-stimulating factors (CSF) are being increasingly used to accelerate hematopoietic recovery after bone marrow transplantation. To study the endogenous serum levels of CSF in bone marrow transplanted patients we have used immunoassays measuring granulocyte-macrophage colony-stimulating factor (GM-CSF) with a sensitivity of 0.10 ng/ml and granulocyte colony-stimulating factor (G-CSF) with a sensitivity of 0.05 ng/ml. Serum samples, taken from the conditioning treatment until engraftment, were analysed in 13 patients receiving allogeneic transplants and in eight patients receiving autologous transplants. Ten patients had acute myeloid leukemia, seven acute lymphoblastic leukemia, one acute undifferentiated leukemia, two non-Hodgkin's lymphoma and one multiple myeloma. Samples were taken 1-2 times before transplantation and 1-2 times per week after transplantation (median of 46 days in allotransplant recipients and 32 days in autotransplant recipients); 17% of the allogeneic transplanted patients and 35% of the autologous transplanted patients had detectable levels of G-CSF. In both types of transplantation the G-CSF concentrations were low: median 0.06 (range 0.05-0.14) and 0.08 (range 0.05-0.40) ng/ml respectively. GM-CSF was detected only in one analysed sample in all patients. There was no evidence of increased CSF levels related to engraftment or documented infections.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in serum during induction treatment of acute leukaemia

Granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are increasingly used to stimulate granulopoiesis in neutropenic patients but in most cases without any knowledge of the endogenous CSF-levels. With the purpose to define serum levels of GM-CSF and G-CSF during induction chemotherapy and haematological reconstitution in patients with acute leukaemia we have used enzyme-linked immunosorbent assay (ELISA) techniques to measure these growth factors in 18 patients with acute myeloid leukaemia (AML) and eight patients with acute lymphoblastic or undifferentiated leukaemia (ALL/AUL). G-CSF above 0.05 ng/ml was detected in 54% of the analysed AML samples, median 0.29 (range 0.05-2.80) ng/ml; and in 40% of analysed ALL/AUL samples, median 0.09 (range 0.05-3.00) ng/ml. In patients with AML there was a clear correlation between an elevated serum concentration of G-CSF and documented infections. On the other hand, 15/18 of the patients with acute myeloid leukaemia and 8/8 patients with ALL/AUL had non-detectable levels of GM-CSF (less than 0.10 ng/ml). Two patients had measurable levels of GM-CSF in all samples, median 0.71 (range 0.26-1.18) ng/ml and in these patients the levels successively decreased during and after chemotherapy and did not increase in response to infections. In normals detectable levels of GM-CSF were found in 2/35 individuals and G-CSF in 0/10 individuals.     

Retinoic acid acts to neutralize the inhibitory effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on alkaline phosphatase activity of neutrophils that is induced by granulocyte colony-stimulating factor (G-CSF).

Granulocyte colony-stimulating factor (G-CSF) is known to act on the neutrophilic granulocytes from chronic myelogenous leukemia (CML) patients to induce neutrophil alkaline phosphatase (NAP) activity. Gamma-interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to suppress NAP induction with G-CSF. We confirmed that this inhibitory effect of GM-CSF is accompanied by the decrease of the NAP mRNA level. Moreover, we found that the simultaneous addition of retinoic acid completely neutralized this inhibitory effect of GM-CSF. Recovery of the NAP activity brought about by the retinoic acid was also accompanied by the increase of NAP mRNA level. These results indicate that retinoic acid neutralizes the inhibitory effect of GM-CSF on the induction of NAP activity through the change of the NAP mRNA level.     

Clinical outcomes of multiple myeloma patients who undergo autologous hematopoietic stem cell transplant with G-CSF or G-CSF and plerixafor mobilized grafts

Impact of Plerixafor (P) mobilized stem cells on immune reconstitution, such as absolute lymphocyte count at day 30 (ALC30), and on long-term outcomes of Multiple Myeloma (MM) patients undergoing autologous stem cell transplant (ASCT) has not been well established. We evaluated total of 469 patients mobilized with G-CSF (G) alone, and 141 patients mobilized with G-CSF plus plerixafor (G+ P). Patients only received plerixafor if they had peripheral blood CD34⁺ blood count <20/μL on first planned day of collection. Primary endpoint, ALC30, was 1.3 K/μL (range, 0.1-4.5) and 1.2 K/μL (range, 0.1-5.1) for G and G + P, respectively (P =. 61). The median PFS was 2.5 years (95% CI, 2.1-3.2) and 2.8 years (95% CI, 2.0-3.3) for G and G + P, respectively (HR: 1.13; 95% CI, 0.84-1.50; P = .42). The median OS was 6.1 years (95% CI, 4.6-NR) for G group compared to 3.7 years (95% CI, 3.2-NR) for the G + P group (HR: 1.64; 95% CI, 1.12-2.40; P = .01). The median follow-up time for OS was 2.53 years (95% CI, 2.13-2.99) and 1.59 years (95% CI, 1.17-2.02) for G and G+ P group, respectively. In this large retrospective analysis of MM patients mobilized with G-CSF vs G-CSF + P, there was no significant difference in lymphocyte recovery or PFS. There was an overall survival difference in patients who were poor mobilizers and could not be mobilized with G-CSF alone.     

Harvesting and enrichment of hematopoietic progenitor cells mobilized into the peripheral blood of normal donors by granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF: potential role in allogeneic marrow transplantation

To explore the use of stem/progenitor cells from peripheral blood (PB) for allogeneic transplantation, we have studied the mobilization of progenitor cells in normal donors by growth factors. Normal subjects were administered either granulocyte-macrophage colony-stimulating factor (GM-CSF) at 10 micrograms/kg/d, or G-CSF at 10 micrograms/kg/d, or a combination of G- and GM-CSF at 5 micrograms/kg/d each, administered subcutaneously for 4 days, followed by leukapheresis on day 5. Mononuclear cells expressing CD34 (CD34+ cells) were selectively enriched by affinity labeling using Dynal paramagnetic microspheres (Baxter Isolex; Baxter Healthcare Corp, Santa Ana, CA). The baseline CD34+ cells in peripheral blood before mobilization was 0.07% +/- 0.05% (1.6 +/- 0.7/microL; n = 18). On the fifth day after stimulation (24 hours after the fourth dose), the CD34+ cells were 0.99% +/- 0.40% (61 +/- 14/microL) for the 8 subjects treated with G-CSF, 0.25% +/- 0.25% (3 +/- 3/microL, both P < .01 v G-CSF) for the 5 subjects administered GM-CSF, and for the 5 subjects treated with G- and GM-CSF, 0.65% +/- 0.28% (41 +/- 18/microL, P < .5 v GM-CSF). Parallel to this increase in CD34+ cells, clonogenic assays showed a corresponding increase in CFU- GM and BFU-E. The total number of CD34+ cells collected from the G-CSF group during a 3-hour apheresis was 119 +/- 65 x 10(6) and was not significantly different from that collected from the group treated with G- and GM-CSF (101 +/- 35 x 10(6) cells), but both were greater than that from the group treated with GM-CSF (12.6 +/- 6.1 x 10(6); P < .01 for both comparisons). Analysis of the CD34+ subsets showed that a significantly higher percentage of cells with the CD34+/CD38- phenotype is found after mobilization with G- and GM-CSF. In the G-CSF group, immunomagnetic selection of CD34+ cells permitted the enrichment of the CD34+ cells in the apheresis product to 81% +/- 11%, with a 48% +/- 12% yield and to a purity of 77% +/- 21% with a 51% +/- 15% recovery in the G- and GM-CSF group. T cells were depleted from a mean of 4.5 +/- 2.0 x 10(9) to 4.3 +/- 5.2 x 10(6) after selection, representing 99.9% depletion. We conclude that it is feasible to collect sufficient numbers of PB progenitor cells from normal donors with one to two leukapheresis procedures for allogeneic transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engraftment syndrome after allogeneic hematopoietic cell transplantation in adults

We performed a retrospective study of the engraftment syndrome (ES) as defined by the Spitzer Criteria in adult patients undergoing allogeneic hematopoietic cell transplantation (HCT) for various hematological malignancies at a single institution, over a decade, and analyzed its relationship to acute GVHD; 217 patients underwent either myeloablative (38.7%) or reduced intensity (61.3%) HCT; 22.1% met the criteria for ES. Acute GVHD prophylaxis (P = 0.006) and transplants prior to 2006 (P < 0.0001) were significantly associated with a risk of ES in univariable analysis. Early aGVHD within 4 weeks of engraftment was significantly more common in the ES compared to the non ES cohort (21 vs. 8.3% respectively, P = 0.02). ES did not predict for future GVHD, as at day +180, the cumulative incidences of grades II–IV aGVHD (31 vs. 23%, P = 0.19) and of chronic GVHD at 2 years of engraftment (42 vs. 36%, P = 0.28) were not significantly different between the ES and non ES groups, respectively. No significant differences in NRM, overall survival and progression‐free survival were observed between the two groups. Although predictive of early aGVHD, ES occurred independently of GVHD in 79% of the patients. Survival outcomes should be evaluated in a larger randomized study to investigate if there is a correlation with ES. Am. J. Hematol. 89:698–705, 2014. © 2014 Wiley Periodicals, Inc.     

Polyethylene glycol (PEG)-modified granulocyte-macrophage colony-stimulating factor (GM-CSF) with conserved biological activity.

Polyethylene glycol (PEG) modification improves the pharmacological properties of proteins, usually extending plasma half-life and concomitantly increasing in vivo bioactivity, reducing both antigenicity and immunogenicity, and increasing solubility and resistance to proteolysis. Despite these established benefits, few PEG proteins are in use. Current coupling methods are either traumatic for the protein or involve lengthy and difficult procedures to activate monomethoxyPEG (MPEG). We have applied a new coupling method that allows coupling of MPEG directly to proteins under physiological conditions. Using this method with recombinant human (rh)granulocyte-macrophage colony-stimulating factor (GM-CSF) we were able to construct biologically active PEG-GM-CSF. Fast protein liquid chromatography (FPLC) and phase-partitioning confirmed the presence of PEG modification, and the former was used to fractionate modified and unmodified material. Bioactivity was measured in colony assays of normal human bone marrow cells and by tritiated thymidine uptake (of chronic myeloid leukemia cells and TF-1 cells). With both uptake and colony assays, using unfractionated material, we observed only a modest reduction in biological activity. Assays of FPLC-fractionated material confirmed that much of the bioactivity of the PEG-GM-CSF preparations was due to the modified species and any residual unmodified GM-CSF. Species uncontaminated by tresylmonomethoxyPEG (TMPEG; which was somewhat inhibitory in the thymidine uptake assay and eluted over a broad region of the FPLC profile) had no significant reduction in activity, but we cannot rule out the possibility that PEG-GM-CSF species eluting elsewhere in the profile had modest reduction of activity. Subcutaneous injection into mice confirmed the anticipated improved half-life in vivo and demonstrated a longer uptake from the injection site. This is, as far as we are aware, the first successful construction of PEG-GM-CSF with conserved biological activity.