Recombinant human granulocyte colony-stimulating factor repairs the abnormalities of neutrophils in patients with myelodysplastic syndromes and chronic myelogenous leukemia

We examined the in vitro effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) on neutrophil anomalies in 20 patients with myelodysplastic syndromes (MDS) and eight patients with chronic myelogenous leukemia (CML). Neutrophil alkaline phosphatase (NAP) activity was determined in nine MDS patients and eight CML patients by a scoring method. NAP scores were decreased in six of the nine patients with MDS and in all of the patients with CML. In all patients with these diseases, NAP scores increased by incubating the blood with rhG- CSF. An increase in NAP scores by rhG-CSF was observed even at a concentration of 1 U/mL in patients with MDS but was observed only at higher concentrations (1,000 to 10,000 U/mL) in patients with CML. Significant increases in NAP scores occurred at 12 hours' incubation in patients with MDS, whereas the increase was more gradual in patients with CML. This time course difference was thought to be due mainly to the difference in cell populations of circulating myeloid cells between MDS patients and CML patients. Induction of NAP activity by rhG-CSF in patients with both these diseases was suppressed by the addition of inhibitors of RNA or protein synthesis. Neutrophil superoxide anion (O2- ) production induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was determined in the other 11 patients with MDS. This neutrophil function was decreased in seven of the 11 patients with MDS, normal in two patients, and increased in two patients. Preincubation with rhG-CSF caused a significant increase in fMLP-induced O2- production in nine of the 11 patients with MDS. rhG-CSF enhanced this neutrophil function in a time- and dose-dependent manner, and maximal stimulation was observed at 2,000 to 4,000 U/mL of rhG-CSF and at five to ten minutes' incubation. The present results show that rhG-CSF is able to repair at least in part the neutrophil anomalies in these patients, and our data, especially for patients with MDS, suggest the clinical usefulness of rhG-CSF for this preleukemic disorder.     

In vivo activation of human neutrophil functions by administration of recombinant human granulocyte colony-stimulating factor in patients with malignant lymphoma

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered (50 to 800 micrograms/m2) once daily as a half-hour intravenous (IV) infusion for 14 days to seven patients with malignant lymphoma. In all patients, administration of rhG-CSF not only ameliorated the decrease in absolute neutrophil count after the cytotoxic chemotherapy but also enhanced superoxide (O2-) release in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The priming effect of rhG-CSF on neutrophil O2- release was rapid (evident within 6.5 hours) and sustained at least for 24 hours after a single IV administration of rhG-CSF. The responsiveness to further in vitro challenge of rhG-CSF was lost or reduced in neutrophils isolated after rhG-CSF treatment, indicating that neutrophils already primed in vivo by rhG-CSF are desensitized to this factor. In contrast to the results obtained with FMLP, when phorbol myristate acetate (PMA) was used as stimulus, no consistent enhancement of O2- release was observed, suggesting that rhG-CSF modulates the signal transduction pathways linked to FMLP receptors rather than increases the components of the O2- producing enzyme complexes. Administration of rhG-CSF also rapidly (evident within 15 minutes) caused an increase in expression of neutrophil C3bi-receptors that was sustained for at least 24 hours after a single IV administration of rhG- CSF. Pharmacokinetic study of rhG-CSF showed a half-life (t1/2) of 114 min. These findings show that rhG-CSF is a potent activator for neutrophil functions both in vivo and in vitro.     

Bilineage response in refractory aplastic anemia patients following long-term administration of recombinant human granulocyte colony-stimulating factor.

5 patients with refractory aplastic anemia (AA) received long-term administration (2-11 + months) of recombinant human G-CSF (rhG-CSF) in doses from 250-500 micrograms/body/day by intravenous infusion or 75-300 micrograms/body/d by subcutaneous injection. All 5 evaluable patients showed a substantial increase in absolute neutrophil count (ANC) with a recovery of myeloid components in the bone marrow after 1 to 2 months of treatment. Interestingly, 2 out of the 5 patients showed a dramatic improvement in severe anemia after 2 to 4 months of treatment accompanying a recovery of erythroid components in the bone marrow. In addition, there was no serious infection before or during therapy. Long-term administration of rhG-CSF was well tolerated because of its minimal toxicity. Clonal assay revealed a recovery of myeloid progenitors in all patients and a recovery of erythroid progenitors in 3 out of the 5 patients. These results suggest that long-term administration of rhG-CSF at least mobilizes residual myeloid as well as erythroid progenitor cells and induces a bilineage response in severe refractory AA.     

Clinical effects of recombinant human granulocyte colony-stimulating factor in leukemia patients: a phase I/II study

A phase I/II study of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in 24 leukemia patients was conducted at our institute. Recombinant human G-CSF (50-200 micrograms/m2/day) was administered i.v. In seven allogeneic bone marrow transplantation (BMT) recipients, treatment with rhG-CSF was started 5 days after BMT. Neutrophils began to increase within 3 days after the start of rhG-CSF administration in five of seven patients. The mean duration necessary for recovery of neutrophils to greater than 500/microliters was 11.3 days after BMT with rhG-CSF; 26.8 days is the figure for recovery without rhG-CSF from Japanese historical data. In seven out of eight patients who received rhG-CSF administration after the first remission-induction chemotherapy, the neutrophil counts increased from less than 300/microliters to greater than 4000/microliters within 10 days. Blasts did not increase in all patients including four acute nonlymphocytic leukemia (ANLL) patients. Severe infections such as septicemia and pneumonia, which were unable to be controlled by antibiotics only, were successfully treated with rhG-CSF and antibiotics. rhG-CSF either stimulated or inhibited myeloid leukemic cells in some refractory cases. Mild bone pain occurred in one patient while receiving rhG-CSF i.v. rhG-CSF seems to have the ability to shorten the period of neutropenia, prevent infections after allogeneic BMT and remission-induction chemotherapy for acute leukemia, and support therapy for infections.     

Effects of rhG-CSF on neutrophil functions and bone marrow parameters in diabetic rats

Neutrophils have an important role in the host defense. The elevated serum glucose levels of diabetics affect traditional host defenses such as neutrophil counts and functions. The causes of these impairments are not clear. We aimed to investigate changes of peripheral neutrophil counts and functions and their relation with bone marrow cells in diabetic rats. Thirty-two rats were divided into four equal groups. Group 1 were controls and Groups 2 and 4 were made diabetic by a single intraperitoneal injection of streptozotocin. Granulocyte colony stimulating factor (G-CSF) was injected subcutaneously into Groups 3 and 4. White blood cell count, neutrophil counts and function and bone marrow cell count were determined. Peripheral blood cell counts, neutrophil phagocytosis index were decreased but neutrophil adhesivity index was not different in the diabetes-induced group. There was a difference in circulating white blood cell counts and neutrophil counts between the rhG-CSF treated and non-treated groups. The phagocytosis index of neutrophil in diabetic rats was significantly diminished by rhG-CSF treatment. A hyperplasia of early cells of the myeloid series in G-CSF treated groups was observed when compared with those of nontreated groups (p<0.001). A significant decrease was noted in the number of mature marrow segmented cells diabetic groups (p<0.001). Finally, G-CSF has been shown to cause neutrophilia by acting as a releasing factor for mature marrow neutrophils in diabetic rats. These results suggest that G-CSF may be used to improve nonspecific immunity in diabetic patients.     

Effect of recombinant human granulocyte colony-stimulating factor on white blood cell count and neutrophil functions in patients with malignant lymphoma

We studied the in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on white blood cell (WBC) count and neutrophil functions in nine patients with malignant lymphoma. The WBC count and absolute neutrophil count were rapidly increased without a nadir phase after chemotherapy. Neutrophil alkaline phosphatase (NAP) scores also markedly increased following chemotherapy in all patients. Phagocytosis of India ink and nitroblue tetrazolium (NBT) reduction were revealed tend to be increased, but not exceeded significantly to normal range. RhG-CSF repaired neutrophil function in patients with decreasing that. Thus, rhG-CSF may be useful for prevention and treatment of infection after chemotherapy.     

Hypereosinophilic syndrome treated with α-interferon and granulocyte colony-stimulating factor but complicated by nephrotoxicity

A 34-year-old male with hypereosinophilic syndrome (HES) and cardiac complications was treated with recombinant human α-interferon (rhIFN) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the attempt to suppress the eosinophilic cell clones and stimulate the neutrophil myelopoiesis in the bone marrow, respectively. After 1 month of pretreatment with rhIFN, rhG-CSF was administered daily for 22 days. Within a few days the combined treatment with rhIFN and rhG-CSF was followed by a marked increase in absolute neutrophil count but complicated by abdominal pain and an increase in plasma creatinine and blood urea nitrogen. The renal failure persisted when rhIFN therapy was stopped but resolved after discontinuing rhG-CSF. The pathogenesis of this reversible renal involvement needs further investigation. In that hematological improvement in vivo as well as in vitro followed the administration of rhIFN and rhG-CSF, a role for combined therapy with these cytokines may be advocated. However, caution with regard to kidney function should be taken with this combination therapy.     

A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: significant induction of peripheral and bone marrow neutrophilia

Host defenses in the human neonate are limited by immaturity in phagocytic immunity. Such limitations seem to predispose infected newborns to neutropenia from an exhaustion of the neutrophil reserve. Among the critical defects thus far identified in neonatal phagocytic immunity is a specific reduction in the capacity of mononuclear cells to express granulocyte colony-stimulating factor (G-CSF) after stimulation. However, the safety, pharmacokinetics, and biological efficacy of administration of recombinant human (rh)G-CSF to infected human newborns to compensate for this deficiency is unknown. Forty-two newborn infants (26 to 40 weeks of age) with presumed bacterial sepsis within the first 3 days of life were randomized to receive either placebo or varying doses of rhG-CSF (1.0, 5.0 or 10.0 micrograms/kg every 24 hours [36 patients] or 5.0 or 10.0 micrograms/kg every 12 hours [6 patients]) on days 1, 2, and 3. Complete blood counts with differential and platelet counts were obtained at hours 0, 2, 6, 24, 48, 72, and 96. Circulating G-CSF concentrations were determined at hours 0, 2, 6, 12, 14, 16, 18, 24, and 36. Tibial bone marrow aspirates were obtained after 72 hours for quantification of the bone marrow neutrophil storage pool (NSP), neutrophil proliferative pool, granulocyte progenitors, and pluripotent progenitors. Functional activation of neutrophils (C3bi expression) was determined 24 hours after rhG-CSF or placebo administration. Intravenous rhG-CSF was not associated with any recognized acute toxicity. RhG-CSF induced a significant increase in the blood neutrophil concentration 24 hours after the 5 and 10 micrograms/kg doses every 12 and 24 hours and it was sustained as long as 96 hours. A dose-dependent increase in the NSP was seen following rhG-CSF. Neutrophil C3bi expression was significantly increased at 24 hours after 10 micrograms/kg every 24-hour dose of rhG- CSF. The half-life of rhG-CSF was 4.4 +/- 0.4 hours. The rhG-CSF was well tolerated at all gestational ages treated. The rhG-CSF induced a significant increase in the peripheral blood and bone marrow absolute neutrophil concentration and in C3bi expression. Future clinical trials aimed at improving the outcome of overwhelming bacterial sepsis and neutropenia in newborn infants might include the use of rhG-CSF.     

Efficacy of recombinant human granulocyte colony-stimulating factor in a murine model of pneumococcal pneumonia: effects of lung inflammation and timing of treatment

The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a murine model of pneumococcal pneumonia was examined. Intranasal inoculations were 10(7) cfu/mouse (high inoculum) and 5 x 10(4) cfu/mouse (low inoculum) of Streptococcus pneumoniae, which induced severe or mild lung inflammation, respectively. With the low inoculum, rhG-CSF significantly improved survival when initiated 24 h or 10 min before, but not when initiated 24 h after, infection. Pretreatment with rhG-CSF significantly increased myeloperoxidase (MPO) activity in lungs 8 h after the infection and increased circulating neutrophil count 24, 48, and 72 h after infection. In contrast, rhG-CSF did not improve survival of animals infected with the high inoculum and did not increase MPO activity or neutrophil count in blood over those of sham-treated controls. These data strongly suggest that the severe inflammatory response typically observed in pneumococcal pneumonia recruits a maximum number of neutrophils in the lungs and thus masks the beneficial effect of rhG-CSF.     

Increased respiratory burst activity of neutrophils in patients with aplastic anemia: effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

The superoxide (O2-)-releasing capacity in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and the priming effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on FMLP-induced O2-release were investigated in neutrophils from 13 patients with aplastic anemia (AA). The O2(-)-releasing capacity of AA neutrophils (0.85 +/- 0.36 nmol/5 min/1 x 10(5) cells, n = 13) was significantly (p < 0.01) increased as compared with that of normal neutrophils (0.24 +/- 0.12 nmol/5 min/1 x 10(5) cells, n = 17). There was no close relationship between the O2(-)-releasing capacity and the peripheral blood neutrophil count or the plasma concentration of C-reactive protein. The plasma concentrations of G-CSF and GM-CSF were not elevated to the detectable levels (< 0.1 ng/ml and < 0.2 ng/ml, respectively) in all patients tested. FMLP-induced O2(-)-release was further enhanced by pretreatment of cells with rhG-CSF or rhGM-CSF for 10 min at 37 degrees C, except that no significant priming by rhG-CSF was observed in five patients. The priming effect of rhGM-CSF was consistently greater than that of rhG-CSF in all patients. The i.v. administration of rhGM-CSF (6 micrograms/kg body weight/day) to one patient resulted in an increase in neutrophil O2(-)-release stimulated by FMLP. These findings indicate that neutrophils from AA patients are already primed in vivo for enhanced release of O2- and that these neutrophil functions are further potentiated by rhG-CSF or rhGM-CSF.     

A successful case of congenital agranulocytosis treated with recombinant human granulocyte colony-stimulating factor]

A 1-month-old girl was admitted because of staphylococcal cellulitis of the buttock and the shoulder, and peripheral agranulocytosis. CBC on admission showed 10,100/microliters of WBC with 1% mature neutrophils, 5% monocytes, 6% eosinophils, 2% basophils and 86% lymphocytes. Bone marrow aspiration revealed maturation arrest of neutrophil precursors at the level of myelocyte. We treated the patient with subcutaneous rhG-CSF (Kirin-Amgen, Tokyo) for sequential 7-day course at the starting dose of 3 micrograms/kg, and increased weekly. The dose was escalated at the level of 18 micrograms/kg for 2 weeks subcutaneously, 8 days after effective dose of 18 micrograms/kg, the absolute neutrophil counts more than 1,000/microliters was attained, and bone marrow aspiration showed an increase of neutrophil precursors beyond the myelocyte level with maturation. Our case proved that both WBC and absolute neutrophil counts were increased parallel with the dose escalation of rhG-CSF. Shortly after the cessation of rhG-CSF, WBC and absolute neutrophil counts were decreased. No side effect was detected except for mild splenomegaly which was resolved after cessation of rhG-CSF. In methylcellulose culture with PHA-LCM, marrow cell of patient produced normal number of CFU-GM, and myeloid precursors could proliferate and differentiate to normal polymorphonuclear neutrophils, but rhG-CSF produced only small number of CFU-GM. Our case confirms that rhG-CSF is a new approach to control the life-threatening infection of congenital agranulocytosis.     

Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor

Peripheral blood mononuclear cells (PBMC) were collected after the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and used as the sole source of hematopoietic stem cells after myeloablative therapy with busulfan (Bu) and cyclophosphamide (Cy). These studies were performed in 12 patients with malignancies (4 non-Hodgkin's lymphoma, 5 breast cancer, 1 testicular carcinoma, 1 Wilm's tumor, and 1 undifferentiated carcinoma) who had bone or bone marrow disease or had low marrow cellularity. rhG-CSF (16 micrograms/kg/d) was administered for 5 to 7 days by subcutaneous injection and PBMC were collected for 2 to 5 days beginning on day 4 after initiation of rhG-CSF, using continuous-flow blood-cell separators that processed 10 to 12 L of whole blood. From a median of three collections, a mean of 24.0 x 10(8) (+/- 10.5 SD) total nucleated cells/kg containing 12.6 x 10(8) (+/- 4.5 SD) mononuclear cells/kg, 7.3 x 10(6) (+/- 4.3 SD) CD34+ cells/kg and 20.5 x 10(4) (+/- 28.1 SD) granulocyte-macrophage colony-forming units (CFU-GM)/kg were harvested and cryopreserved. After the administration of Bu 14 to 17 mg/kg and Cy 120 to 150 mg/kg, PBMC were thawed and infused. One patient received rhG-CSF after the infusion of PBMC and the remaining 11 patients did not receive postinfusion growth factors. Mean days to recovery of neutrophil levels of 0.1, 0.5, and 1.0 x 10(9)/L were 11.4 (range, 9 to 13), 12.7 (range, 10 to 15), and 13.6 (range, 11 to 16) and the mean day to platelet transfusion independence was 13.3 (range, 7 to 49). Time to recovery of neutrophils to 0.5 and 1.0 x 10(9)/L and platelets to 20 x 10(9)/L was more rapid than in historical patients treated with Bu and Cy who received marrow alone or marrow followed by the posttransplant administration of rh-G or GM-CSF. No graft failures have been observed with a follow-up of 4 to 12 months. These results indicate that PBMC collected after rhG-CSF lead to rapid hematopoietic recovery after myeloablative chemotherapy.     

Factor(s) responsible for the increase in alkaline phosphatase activity of postmitotic granulocytes from normal individuals and patients with chronic myeloid leukemia

Using postmitotic granulocytes (PMGs) with low neutrophil alkaline phosphatase activity (NAP activity), factor(s) having the capacity to increase their NAP activity were examined in vitro. A high activity of the factor was demonstrated in the cystic fluid of a human squamous cell carcinoma, which is known to produce a large amount of granulocyte- macrophage colony-stimulating factor (GM-CSF). The NAP-stimulating factor increased NAP values both in PMGs from normal bone marrow and PMGs from patients with chronic myeloid leukemia (CML), and NAP values in cells treated with the factor approached or rose above those of normal peripheral granulocytes after 48 hr of culture. The effect of the factor was specific in that the factor caused stimulation only in granulocytic series. These findings may indicate that increases in NAP activity reflect maturation or granulocytes and that low NAP activity of neutrophils derived from patients with CML is due to the immaturity of these cells. The relationship between the factor responsible for the increase in NAP activity and GM-CSF is also discussed.     

Neutrophil alkaline phosphatase levels after induction of hypersegmented neutrophil release into blood of the rat

Summary Hypersegmentation of nuclei was induced in rat circulating neutrophils after cyclophosphamide (CY) administration and neutrophil alkaline phosphatase (NAP) activity was measured. It was shown that changes in NAP levels and segmentation of neutrophil nuclei were parallel. Relationship between age of neutrophils and NAP levels is discussed.     

Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine- SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.     

A phase II trial of recombinant human granulocyte colony-stimulating factor in the myelodysplastic syndromes

We conducted a phase II study of the intravenous administration of a glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) for 7-14 d in 41 patients with the myelodysplastic syndromes (MDS). Administration of rhG-CSF elicited striking rises in both leucocyte and neutrophil counts in the majority of the patients irrespective of the FAB subtypes of MDS. The rises in neutrophil counts were dose dependent and 5 micrograms/kg/d of rhG-CSF yielded approximately an 8-fold increase in neutrophil counts. Leucocytes and neutrophil counts started to increase shortly after the first injection of 5 micrograms/kg, was maintained at significantly elevated levels during 14 d of treatment, and returned to the pretreatment levels within several days following discontinuation of rhG-CSF. The action of rhG-CSF was specific for neutrophils since leucocytosis was due exclusively to neutrophilic increase associated with an increased marrow myeloid maturation. There were no consistent changes in the monocyte, eosinophil, lymphocyte, platelet or reticulocyte counts. After treatment, the percentage of marrow blast cells was reduced in eight of 13 evaluable patients with refractory anaemia with an excess of blasts (RAEB) or RAEB in transformation (RAEB-t). No patients developed acute leukaemia during the treatment or in the immediate follow-up period. The treatment was well tolerated with only minimal toxicity. The results suggest that rhG-CSF is a safe and effective way to promptly improve neutropenia in MDS patients.     

Effect of stem cell factor with and without granulocyte colony-stimulating factor on neonatal hematopoiesis: in vivo induction of newborn myelopoiesis and reduction of mortality during experimental group B streptococcal sepsis.

Neonatal hematopoiesis and host defense are developmentally immature and under states of increased demand predispose the newborn to peripheral cytopenias and depletion of bone marrow storage pool reserves. We have previously demonstrated that recombinant human granulocyte colony-stimulating factor (rhG-CSF) can significantly modulate neonatal rat granulopoiesis and act synergistically with antibiotic therapy to reduce the mortality rate during experimental group B streptococcal sepsis. Stem cell factor (SCF) has been shown to stimulate early hematopoietic progenitor cells and, in the presence of lineage-specific CSFs, enhance committed progenitor cell proliferation. In the present study we examined the in vivo neonatal hematologic effects of recombinant rat (rr) SCF (14 days), simultaneous rrSCF + rhG-CSF (14 days), and sequential combination of rrSCF (7 days) + rhG-CSF (7 days). Sprague-Dawley newborn rats (less than or equal to 24 hours) were injected intraperitoneal (IP) x 14 days with the above combinations. rrSCF (0 to 200 micrograms/kg/d) had a negligible effect on the peripheral platelet count and absolute neutrophil count (ANC) but the diminution in the hematocrit during the first 10 days of treatment was less pronounced (P = .0001). However, the simultaneous use of rrSCF + rhG-CSF synergistically increased the circulating day 6 to 13 ANC (P = .001). Similarly, sequential rrSCF + rhG-SCF also had a synergistic significant effect during the second week of therapy on the circulating ANC (P = .01). The bone marrow neutrophil storage and proliferative pools were also significantly increased in newborn rats treated with rrSCF + rhG-CSF versus rhG-CSF (P = .02). The bone marrow and liver/spleen CFU-GM pool was unchanged; however, the CFU-GM proliferative rates were significantly increased in the rrSCF + rhG-CSF group (P = .04). rrSCF also induced a significant increase in the bone marrow and liver/spleen mast cell pool (P = .002). Lastly, rrSCF x 14 days +/- rhG-CSF significantly reduced the mortality rate at 48 and 120 hours after experimental group B streptococcus sepsis (P = .03 and .05, respectively). These data suggest that combination SCF + G-CSF therapy compared with G-CSF alone significantly increases the neonatal rat peripheral neutrophil count, bone marrow myeloid pools and proliferative rates, and induces a reduction in the mortality rate during experimental bacterial sepsis. SCF therapy may have future potential applications in the modulation of human neonatal hematopoiesis and host defense.     

Clinical study of rhG-CSF (KRN8601) in patients with myelodysplastic syndrome]

A clinical study of rhG-CSF (KRN8601) in patients with myelodysplastic syndrome (MDS) was performed to investigate the hematopoietic effects and the increase of neutrophils. The rhG-CSF was administered daily by intravenous infusion over 30 min. to 21 patients with MDS (PARA = 11, RAEB = 4, RAEB in T = 6). The dose was escalated stepwise from 50 to 400 microgram/m2 every week. Within one week to 26 days after commencement of rhG-CSF administration, the increases of absolute neutrophil counts in peripheral blood were observed in all patients. Treatment with rhG-CSF enhanced normal marrow myeloid cell differentiation and maturation in 3 of 9 PARA patients and in 3 of 4 RAEB patients. None of patients changed to acute leukemia attributable to rhG-CSF, but one of RAEB patient and two of RAEB in T patients progressed to leukemic phase in 21 days or two months after treatment. Minor side effects or abnormal laboratory findings were observed in 3 patients (14.3%). These results suggested that treatment with rhG-CSF was well tolerated and effective for improving the neutropenia between 50 to 400 micrograms/m2 in patients with MDS.     

Motility of rhG-CSF-induced neutrophils in patients undergoing chemotherapy: evidence for inhibition detected by image analysis

The motility of circulating neutrophils from seven patients affected by intermediate and high-grade non-Hodgkin's lymphoma was investigated before and after rhG-CSF administration (5μg/kg/d for 5 d subcutaneously) in the course of chemotherapy. Random motility and bacterial lipopolysaccharide-induced chemotaxis were studied by the micropore filter technique in a Boyden chamber. These functions were evaluated by a very sensitive technique, based on a computer-assisted image processing system, capable of giving several parameters about the kinetics of cell migration. Along with a significant increase in neutrophil number, a significant decrease both in random and stimulated motility was found. The kinetics of cell migration showed that the cells maintained the typical gaussian pattern of random motility. On the contrary, neutrophils were found to have lost the typical stimulated migration peak. These findings are consistent with a rhG-CSF-induced impairment of the directional movement, rather than of the ability of moving at random. These effects were found in patients who, in the same experimental conditions, had displayed an enhanced phagocytosis and phagocytosis-associated chemiluminescence along with an enhanced CD32 expression, not due to an aspecific cell manipulation. Two hypotheses may be taken into account: (i) an increased adhesiveness due to a direct or an indirect activity of the cytokine; (ii) an abnormality in the cytoskeleton maturation and/or rearrangement during the accelerated bone marrow transit of myeloid cells. These findings emphasize that rh-GCSF administration can modulate several functions which play an important role in host defence, and suggest the utility of carrying out further studies to investigate the optimum dosage both to correct neutrophil number and preserve neutrophil functional activities.     

In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro

Abstract: We recently showed that recombinant human granulocyte-colony stimulating factor (rhG-CSF) maintained the viability of human neutrophils in incubation for up to 72 hours. However, it is not known whether rhG-CSF can enhance neutrophil survival in in vivo situations. To clarify this issue, we investigated neutrophil survival in vitro following in vivo injection of rhG-CSF. Neutrophils were obtained from 4 pediatric patients with malignancies and healthy adult volunteers before and after rhG-CSF administration. Neutrophils obtained before rhG-CSF treatment started to undergo apoptosis after 24 h of incubation. In contrast, the survival of neutrophils drawn after rhG-CSF administration increased by approximately 24 h. Concomitantly, the appearance of typical ladder-like DNA fragmentation was delayed. Such an increase in neutrophil survival was inhibited by coincubation with either H 7 (10 μmol/1) or H 8 (20 μmol/1), which worked as protein kinase C inhibitors. Although our study did not measure neutrophil survival in vivo directly, it provides us with further evidence that rhG-CSF may function to prolong neutrophil life expectancy in vivo.