Rh Granulocyte Colony-stimulating Factor Research Articles

A Report of Fourcases of Severethrombocytopenia with Rh G-CSF Mobilized Allogeneic Peripheral Blood Stem Cell Transplant Donors

Background and Objective: Granulocyte colony-stimulating factor (G-CSF) is used to mobilize progenitor cells into the peripheral bloodin healthy donors. Thrombocytopenia is one of common side effects of G-CSFmobilization, but seriousthrombocytopenia is rare. Herein,we report serious thrombocytopeniain 4 allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) healthy donors induced by recombinant human G-CSF (rhG-CSF) mobilization. Methods: The donor received 10ug/kg/d G-CSF via subcutaneous injection.The blood parameterswere examined every day, Abdominal ultrasound detected the spleen size before, during, and after rhG-CSF mobilization. Results: From April 2015 to May 2020, 1127 donors received hematopoietic stem cell mobilization, the percentage of serious thrombocytopenia in healthy donors was 0.35%.The count of white blood cell increased to the 4-7 timesbasic level of (35.4x109/L)after rhG-CSF mobilization 4 days, the lowestcount ofplatelet was 22x109/Lafter single postharvest. After mobilization, length of spleen increased 0.6-1.3 cm (median 10.1 cm), thickness ofspleenincreased 0-1.3 cm (median 3.4 cm). There were no obvious bleeding symptomsafter mobilization in four cases of donor.The quality of stem cell collection was relatively poor in 3 of the 4 donors, the number of mononuclear cells (MNC) was 7.47-18.58×108/kg, and the value of CD34+ was 0.82-2.42×106/kg (CD34+ proportion: 0.11-0.16%). The quality of stem cell collection in the other 1 donor was satisfactory, the number of MNC was 9.29-11.75×108/kg, and the proportion of CD34+ was 0.26~0.38%. Platelet count of 4 donors recovered to the basal level 3-10 days after drug withdrawal. Conclusion:The severe thrombocytopenia caused by rhG-CSF mobilization healthy donor is very rare.while, the mechanismis unknown. Some studies suggested that it may be related to the insufficient expression of megakaryocytes proliferation-related genes, such as PF4 and PTFN4. Other studies indicated that rhG-CSF may aggravate the immune-mediated thrombocytopenia, which leads to hypersplenism and extensive activation of reticuloendothelial cells, thus increasing the consumption of platelets.In addition, the probability of severe thrombocytopenia was increased in allergic donors after rhG-CSF mobilization.In our report, although there was thrombocytopenia, mobilization was not affected.After the mobilization of rhG-CSF, platelets changes should be closely monitored to keep alert of donor bleeding tendency. While ensuring safe collection of hematopoietic stem cells by donors, patients should not be placed in extreme danger of not being able to receive allogeneic hematopoietic stem cells on time. Key words:Severe thrombocytopenia;Healthy donor; rhG-CSF; Mobilization; Allogeneic peripheral blood stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.

Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation

Granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) play key roles in regulating emergency granulopoiesis and inflammation, and are both negatively regulated by the inducible intracellular protein suppressor of cytokine signaling-3 (Socs3). Mice with Socs3 deleted specifically in hematopoietic cells succumb to severe neutrophil and macrophage-driven inflammation by 1 year of age, and responses to G-CSF are grossly exacerbated. In order to determine which elements of cellular responses to cytokines require Socs3, we have examined the differentiative and proliferative capacity of hematopoietic progenitor cells stimulated by G-CSF and IL-6. The differentiation of Socs3-deficient progenitor cells is skewed toward macrophage production in response to G-CSF or IL-6, whereas wild-type progenitor cells produce mainly neutrophils. The proliferative capacity of Socs3-deficient progenitor cells is greatly enhanced in response to G-CSF at all concentrations, but only at low concentrations for IL-6. Strikingly, synergistic responses to costimulation with stem cell factor and IL-6 (but not G-CSF) are lost at higher concentrations in Socs3-deficient progenitor cells. Cytokine-induced expression of transcriptional regulators including cebpb, Ets2, Bcl3, c-Myc, Jun, and Fosl2 are differentially regulated in Socs3-deficient cells. The tight regulation by Socs3 of signal transducer and activator of transcription 3 phosphorylation and gene transcription after cytokine receptor ligation significantly influences the fate of myeloid progenitor cells.

Granulocyte Colony-Stimulating Factor (G-CSF) for Cardio- and Cerebrovascular Regenerative Applications

The cytokine granulocyte colony-stimulating factor (G-CSF) is produced by numerous cell types including immune and endothelial cells. G-CSF binding to its receptor G-CSF-R which belongs to the cytokine receptor type I family depends on the interaction of alpha-helical motifs of the former and two fibronectin type III as well as an immunoglobulin-like domain of the latter. It activates several signalling transduction pathways including PI3K/Akt, Jak/Stat and MAP kinase, thereby promoting survival, proliferation, differentiation and mobilisation of haematopoietic stem and progenitor cells. Accordingly, recombinant human (rh)G-CSF has been extensively used in clinical haematology and oncology to enable bone marrow transplantation or to treat chemotherapy-associated neutropenia. Using animal models it has been recently shown that G-CSF, alone or in combination with other cytokines such as stem cell factor (SCF), causes an accumulation of bone marrow-derived cells in the infarcted heart which, however, do not differentiate into cardiac cells. Nevertheless, since beneficial effects on structural and functional properties were observed in animal models of cardiac, brain and hindlimb ischaemia other mechanisms of G-CSF action must be operative. Recent evidence suggests paracrine effects mediated by the immigrated bone marrow-derived cells and/or direct effects of the cytokine on resident G-CSF-R expressing cells. In both cases these may include promotion of cellular survival, proliferation and differentiation. First clinical studies in patients with myocardial infarction, heart failure and stroke have been accomplished and are reviewed in this paper.

Administration of granulocyte colony-stimulating factor to recipients followed by intra-bone marrow–bone marrow transplantation accelerates acceptance of allogeneic bone marrow cells in mice

We have recently established a novel method for bone marrow transplantation: intra-bone marrow–bone marrow transplantation (IBM–BMT), by which the rapid recovery of donor-derived hematopoiesis can be expected even when reduced radiation doses are used. In this paper, we examine, using mice, whether the combination of pretreatment of recipients with granulocyte-colony-stimulating factor (G-CSF) and IBM–BMT can induce a more rapid recovery of donor-derived hematopoiesis than IBM–BMT alone. We first pretreated recipients with recombinant human (rh) G-CSF (250 μg/kg/day) for 5 consecutive days (days −6 to −2). On day −1, the recipients were irradiated, and IBM–BMT was carried out on day 0. On day 12, we performed colony-forming units of spleen (CFU-S) assays. The combination of G-CSF pretreatment and IBM–BMT augmented the CFU-S counts, the weight of spleens, and the numbers of donor-derived hematopoietic cells. We next analyzed the mechanisms underlying these effects of G-CSF and found that (i) G-CSF induces Th2 polarization, which can prevent graft rejection, and (ii) G-CSF augments natural suppressor activity, which suppresses graft rejection. The combination of G-CSF pretreatment and IBM–BMT can produce the rapid recovery of donor-derived hematopoiesis and suppress graft rejection. This method would lighten the burden on patients in allogeneic BMT.

Long‐lasting partial remission by Interferon‐alpha treatment in a child with essential thrombocythemia

Essential thrombocythemia (ET) is a clonal myeloproliferative disorder characterized by sustained thrombocytosis, isolated hyperplasia of megakaryocytic lineage, and association with thrombotic or bleeding episodes. It is extremely rare in childhood and frequently presents without evident clinical signs. We describe a 3-year-old girl with severe headache and dizziness suffering from ET, who was treated with Interferon-alpha-2a (IFN) based on the potent effect of this agent to inhibit myeloid colonies induced by phytohemagglutinin A stimulated leukocyte conditioned medium (PHA-LCM). Bone-marrow-derived mononuclear cells of this patient did not exhibit spontaneous colony formation but responded to recombinant human (rh) erythropoietin (EPO), rh granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, and stem-cell factor in addition to PHA-LCM. After 65 months of in vivo IFN treatment, the patient experienced a sustained partial remission with platelet counts varying between 400 and 600 x 10(3)/microl.

The clinical observation of G-CSF used in supplementary chemotherapy of cancer patients

2614 Background: To evaluate the effectiveness of different doses of G-CSF(granulocyte-colony stimulating factor) used to improve the condition of leukopenia after routine chemotherapy in cancer patients. Methods: 50 cancer patients (34 male, 16 female) who suffered stage III-IV cancer got leukopenia were treated with different doses of Granocyte (rh G-CSF manufactured by Chugai Pharmaceutical CO., LTD) after combined chemotherapy. The patients'average age is 56(48∼60) years old. They were randomized into two groups. During the stage of leukopenia,Group A with 25 patients were injected with Granocyte 100ug Bid subcutaneously after each cycle of chemotherapy for 7 to 14 days. Group B with 25 patients were injected with Granocyte150ug Bid subcutaneously for 7 to 14 days. The routine peripheral blood examinations were observed. Results: It took 7 to 10 days to treat leukopenia in Group A and took 5 to 7 days in Group B. The race of leucocyte recovery in group B is quicker than in group A. It has significantly statistical difference between two groups. Leucocyte count does not descent again after ascending and without infection. Conclusions: Granocyte(the only glycosylated rh G-CSF) is able to reduce the recovery time of functional bone marrow, decrease the time of leukopenia and prevent from infection. However, it cannot ameliorate the range of leukopenia. The recovery race of leukopenia has relationship with the dose of G-CSF. No significant financial relationships to disclose.

Clinical study of low dose and short term rhG-CSF on neutropenia induced by chemotherapy

Objective To Study the effect of low dose and rh GCSF(Recombinant Human Granulocyte Colony Stimulating Factor) on leukepenia and nenutropenia induced by chemotherapy in a randomized clinical trial.Methods The total patients entrolled were 108 in number and were randominzed into A B and C group(36patients each).In group A rhG CSF was given for 3 day at the dose of 75μg/dS.C. In group B rh GCSF was given for 6 days at the dose of 75μg/d S.C I n group C rhG CSF was given for 5 day at the dose of 75μg/d S.C. Results Iow dose and short term rhG CSF remarkably increased the count of WBC and ANC and lowored the number of days with WBC4 0×10 9/l,ANC50% by cancer chemotherapy with minimal side effects.In group B is was higher than A.C group for the effect.Conclusion Low dose and short term rhG CSF were effective,B group had the hest effect.

A Murine Stromal Cell Line Promotes the Expansion of CD34hlgh+-Primitive Progenitor Cells Isolated from Human Umbilical Cord Blood in Combination with Human Cytokines

The in vitro expansion of CD34− cells is important for clinical applications such as transplantation and gene therapy with CD34+ cells isolated from human umbilical cord blood. In the present study, we developed a xenogenic coculture system involving HUCB-CD34+ cells and a murine stromal cell line, HESS-5 cells, in the presence of recombinant human (rh) cytokines. We examined the effects of combinations of cytokines, such as rh-IL-3, rh-SCF, rh-granulocyte colony-stimulating factor (G-CSF), rh-granulocyte-macrophage-CSF and h-erythropoietin (EPO), on the expansion of CD34hlgh- cells and colony-forming progenitor cells (CFCs). The proliferation of CD34high+ cells and CFCs was dramatically promoted on coculture with HESS-5 cells, and the expansion ratio of the CD34hlgh+ cells showed good correlation with that of high-proliferative potential colony-forming cells (HPP-CFCs). The most potent combination of cytokines in this xenogenic coculture system for the expansion of CD34high+ cells and HPP-CFCs was rh-IL-3 and rh-SCF. The proliferation of CD34high+ cells was supported in the presence of HESS-5 cells with direct cell contact, but not observed in the indirect coculture involving a microporous membrane. Furthermore, we developed a unique coculture method, designated as the bilayer coculture method, involving CD34+ cells and HESS-5 cells using a microporous membrane. This expansion system will be applicable to the expansion of the primitive progenitor cells of HUCB-CD34− cells and is worthy of consideration for the clinical application of HUCB-CD34− cells.

Interactions of Erythropoietin, Granulocyte Colony-Stimulating Factor, Stem Cell Factor, and Interleukin-11 on Murine Hematopoiesis During Simultaneous Administration

AbstractWe investigated how in vivo effects of single hematopoietic cytokines change if given in combination for a prolonged time. Mice were treated with every combination of recombinant human (rh) erythropoietin (EPO), rh granulocyte colony-stimulating factor (G-CSF), recombinant rat (rr) stem cell factor (SCF), and rh interleukin (IL)-11 by continuous infusion over 7 days (full factorial design with three dose levels for each cytokine). Burst-forming unit–erythroid (BFU-E), colony-forming unit–erythroid (CFU-E), and colony-forming unit–granulocyte-macrophage (CFU-GM) were determined in bone marrow and spleen, reticulocytes, hematocrit, granulocytes, and thrombocytes in the peripheral blood. An analysis of variance (ANOVA) and multiple comparison of means was used to evaluate the data. For several cell types, cytokine effects superimposed in an additive way if combined. However, in a large number of circumstances, nonadditive pairwise interactions were found. They differed in type and magnitude involving high-dose saturation, high-dose antagonistic effects, and even effect reversals (qualitative interactions). Hence, in general, it was not possible to foresee the combination effects on the basis of existing knowledge of single effects. On the other hand, the cytokine network was robust and no system hazards were observed under multiple cytokine combinations. The results illustrate that the cytokine network has nonlinear dynamic properties in vivo with dose-response characteristics of one cytokine being continuously modified by other cytokines.

Interactions of Erythropoietin, Granulocyte Colony-Stimulating Factor, Stem Cell Factor, and Interleukin-11 on Murine Hematopoiesis During Simultaneous Administration

We investigated how in vivo effects of single hematopoietic cytokines change if given in combination for a prolonged time. Mice were treated with every combination of recombinant human (rh) erythropoietin (EPO), rh granulocyte colony-stimulating factor (G-CSF), recombinant rat (rr) stem cell factor (SCF), and rh interleukin (IL)-11 by continuous infusion over 7 days (full factorial design with three dose levels for each cytokine). Burst-forming unit–erythroid (BFU-E), colony-forming unit–erythroid (CFU-E), and colony-forming unit–granulocyte-macrophage (CFU-GM) were determined in bone marrow and spleen, reticulocytes, hematocrit, granulocytes, and thrombocytes in the peripheral blood. An analysis of variance (ANOVA) and multiple comparison of means was used to evaluate the data. For several cell types, cytokine effects superimposed in an additive way if combined. However, in a large number of circumstances, nonadditive pairwise interactions were found. They differed in type and magnitude involving high-dose saturation, high-dose antagonistic effects, and even effect reversals (qualitative interactions). Hence, in general, it was not possible to foresee the combination effects on the basis of existing knowledge of single effects. On the other hand, the cytokine network was robust and no system hazards were observed under multiple cytokine combinations. The results illustrate that the cytokine network has nonlinear dynamic properties in vivo with dose-response characteristics of one cytokine being continuously modified by other cytokines.

Bedeutung des Rezeptors und der Signaltransduktion des Granulozyten-Kolonie-stimulierenden Faktors (G-CSF) beim Ovarialkarzinom

Rapid development of resistance in initially chemosensitive ovarian carcinomas is observed in 80% of all patients. One consequence of chemotherapy is myelosuppression, which can be avoided by applying granulocyte-colony stimulating factor (G-CSF). This prompts the question whether G-CSF could play a role in resistance development. A precondition for this is the presence of G-CSF-receptor on these tumour cells. Epithelial tumours like ovarian cancer, express considerably different forms of G-CSF-receptor. Clinically, this is of relevance for tumour initiation and progression. Concluding the results so far, rh (recombinant human) G-CSF could be a growth factor for some ovarian cancer cells. The different efficiency of G-CSF-receptor is possibly caused by the condition of the receptor : While wild-type (wt) G-CSF-receptor could lead to proliferation after ligation with its ligand, signal transduction by G-CSF receptor with a variant C-terminus is presumably not possible. Probably only 30% of all ovarian cancers have a wt-G-CSF receptor, 30% express a variant form and the rest are G-CSF-receptor negative. The expression of wt-G-CSF receptor is not obligatory for functional signal transduction.

Effect of delayed treatment with recombinant human granulocyte colony-stimulating factor on survival and plasma cytokine levels in a non-neutropenic porcine model of Pseudomonas aeruginosa sepsis.

Neutrophils are of great importance for the host's defense against invading organisms. Granulocyte colony-stimulating factor (G-CSF) has been used to augment both the neutrophil number and function, and its prophylactic administration has proved beneficial in animal models of sepsis. However, pretreatment with G-CSF is not practical under clinical conditions. We therefore investigated the effect of recombinant human (rh)G-CSF, administered only after infection, on the survival rate as well as the hemodynamic and cytokine response of the animals. Chronically catheterized conscious pigs were challenged with Pseudomonas aeruginosa (8 x 10(7) colony-forming units kg(-1) x h(-1) for 120 h (control group, n = 10). Animals in the G-CSF group (n = 7) also received rhG-CSF (5 microg kg(-1) x day(-1)), the first dose being given 3 h after beginning bacterial infusion. The mortality rate was 50% (5/10) and 29% (2/7) in the control and G-CSF groups, respectively (p = NS, control vs. G-CSF group). Fever, severe pulmonary hypertension, and a hyperdynamic response were recorded in all of the animals. In spite of a prompt and significant recovery from the initial leukopenia (p < .05 vs. control group), the animals of the G-CSF group showed no significant differences in the parameters investigated from those of the controls. Compared with the survivors, the interleukin-1 receptor antagonist was markedly elevated in all nonsurvivors after 6 h of sepsis (p < .05). These data suggest that treatment with rhG-CSF after the onset of bacterial sepsis might not significantly improve the chances of survival for non-neutropenic patients.

Mature and immature myeloid cells decrease the granulocyte colony-stimulating factor level by absorption of granulocyte colony-stimulating factor.

We studied the effects of polymorphonuclear neutrophils (PMN) and immature myeloid cells on the granulocyte colony-stimulating factor (G-CSF) level in vitro to better understand the regulatory mechanisms of neutropoiesis. Intact normal PMN decreased the G-CSF level after incubation with recombinant human (rh) G-CSF in a time- and dose-dependent manner. The percent reduction decreased as the concentration of rhG-CSF increased. However, the cell-free PMN-conditioned medium (PMN-CM) did not decrease the G-CSF level. The intact PMN also decreased the granulocyte-macrophage (GM)-CSF level after culture with rhGM-CSF, but did not affect the monocyte (M)-CSF level after culture with rhM-CSF. Normal bone marrow (BM) immature neutrophilic cells and G-CSF-dependent acute myeloid leukemic cells (OCI/AML la) also decreased the G-CSF level, whereas K-562 cells, which have no detectable G-CSF receptors, did not affect it. Phenylarsine oxide (PhAsO), an inhibitor of endocytosis of ligand receptor complex, abrogated this decreasing effect of intact PMN and OCI/AML la cells. These findings suggest that mature and immature myeloid cells negatively regulate neutropoiesis by, at least in part, decreasing the G-CSF level probably through receptor-mediated continual absorption and metabolism of G-CSF.

Defective polymorphonuclear leukocyte functions in children receiving chemotherapy for cancer are partially restored by recombinant human granulocyte colony-stimulating factor in vitro.

Granulocyte colony-stimulating factor (G-CSF) has important direct and priming effects on different functions of normal mature polymorphonuclear leukocytes (PMNL). Previous study has shown an alteration in respiratory burst and bactericidal activities of PMNL harvested from children with cancer treated with chemotherapy. The present study evaluates the possibility that recombinant human (rh) G-CSF could correct these defective functions in vitro. Free radical formation in defective PMNL was enhanced by rhG-CSF to a level similar to that found in normal PMNL primed by rhG-CSF. The defective bactericidal activity against Escherichia coli and Staphylococcus aureus was also corrected. This bactericidal activity was not different from that observed in normal PMNL primed by rhG-CSF. In conclusion, correction of the altered free radical-formation pathway by rhG-CSF in these cells contributed to the restoration of normal bactericidal activity against both gram-positive and gram-negative microorganisms.

Kinetics of serum granulocyte-colony stimulating factor (G-CSF) concentration and G-CSF receptor expression during g-csf treatment of cyclophosphamide-treated mice

We analyzed the kinetics of leukocyte number, serum granulocyte colony stimulating factor (G-CSF) concentration, and G-CSF receptor expression in mice after recombinant human (rh) G-CSF treatment. Intraperitoneal (i.p.) injection with 200mg/kg of cyclophosphamide (CP) induced a transient decrease of leukocyte number in peripheral blood and spleen. Daily i.p. inoculation of a low dose of rhG-CSF (1 μg/kg/day) from day 1 of CP injection for 8 days resulted in a significant increase of spleen cell number from day 5 to day 8, while serum rhG-CSF concentration decreased to an undetectable level on day 7. Furthermore, daily i.p. inoculation of high dose rhG-CSF (100 μg/kg/day) for 5 days to CP-injected mice induced a significant increase of leukocytes from day 3 to day 6 in both peripheral blood and spleen, and the increase was higher than that observed after low dose G-CSF treatment. In the course of the high dose rhG-CSF treatment, however, serum rhG-CSF concentration decreased from day 3, which is earlier than the decrease of serum rhG-CSF seen after low dose rhG-CSF treatment. A reverse transcription-polymerase reaction analysis of mRNA expression showed that spleen cells from high dose rhG-CSF-treated mice on day 4 and day 6 expressed more than 10 times higher levels of membrane G-CSF receptors than did those obtained before rhG-CSF treatment. All these results suggest that decrease of serum G-CSF during daily G-CSF treatment does not always indicate neutralization of G-CSF, but implies that the inoculated G-CSF is still stimulating granulocytes and their precursors by binding to membrane G-CSF receptors which are up-regulated by G-CSF treatment. This observation is important when we measure pharmacokinetics of G-CSF in patients chronically injected with G-CSF.

Combined Use of Growth Factors to Stimulate the Proliferation of Hematopoietic Progenitor Cells after Autologous Bone Marrow Transplantation for Lymphoma Patients

We studied the kinetic response and concentration of bone marrow (BM) progenitor cells of patients with lymphoid malignancies submitted to autologous bone marrow transplantation (ABMT), treated with a granulocyte-colony-stimulating factor (G-CSF)/interleukin-3 (IL-3) combination. The results were compared with those of lymphoma patients receiving the same pretransplant conditioning regimen followed by G-CSF alone. Recombinant human (rh)G-CSF was administered as a single subcutaneous (s.c.) injection at the dose of 5 micrograms/kg/day from day + 1 after reinfusion of autologous stem cells, while rhIL-3 was added from day +6 at the dose of 10 micrograms/kg/day s.c. (overlapping schedule). In both groups (i.e. G-CSF- and G-CSF/IL-3-treated patients), cytokine administration was discontinued when the absolute neutrophil count was > 0.5 x 10(9)/l of peripheral blood for 3 consecutive days. Following treatment with the CSF combination, the percentage of marrow CFU-GM and erythroid progenitors (BFU-E) in the S phase of the cell cycle increased from 9.3 +/- 2 to 33.3 +/- 12% and from 14.6 +/- 3 to 35 +/- 6%, respectively (p < 0.05). The number of actively cycling megakaryocyte progenitors (CFU-MK and BFU-MK) also increased. Conversely, G-CSF augmented the proliferative rate of CFU-GM (22.6 +/- 6% compared to a baseline value of 11.5 +/- 3%; p < 0.05) but not of BFU-E, CFU-MK or BFU-MK, and the increase in S-phase CFU-GM was significantly lower than that observed in the posttreatment samples of patients receiving IL-3 in addition to G-CSF. The absolute number of both CFU-GM and BFU-E/ml of BM was significantly augmented after treatment with G-CSF/IL-3 but not G-CSF alone. Similarly, administration of the cytokine combination resulted in a higher number of CD34+ cells and their concentration was significantly greater than that observed in the posttreatment samples of G-CSF patients. We also investigated the responsiveness to CSFs, in vitro, of highly enriched CD34+ cells, collected after priming with G-CSF in vivo (i.e. after 5 days of G-CSF administration). Our results demonstrated that pretreatment with G-CSF modified the response of BM cells to subsequent stimulation with additional CSFs. When the hematological reconstitution of patients treated with G-CSF/ IL-3 was compared to that of individuals receiving G-CSF alone, the addition of IL-3 resulted in a significant improvement in granulocyte and platelet recovery, a lower transfusion requirement and shorted hospitalization. In conclusion, our results indicate that in vivo administration of two cytokines increases the proliferative our results indicate that in vivo administration of two cytokines increase the proliferative our results rate and concentration of BM progenitor cells better than G-CSF alone and support a role for growth factor combinations for accelerating hematopoietic recovery after high-dose chemotherapy.

A SYNERGISTIC INCREASE IN TRANSPLANTABLE PERIPHERAL BLOOD STEM CELLS IN MICE BY CO-ADMINISTRATION OF RECOMBINANT HUMAN INTERLEUKIN 6 AND RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR

We examined the effects of co-administration of recombinant human (rh) IL-6 (10 micrograms/day) and rh granulocyte colony-stimulating factor (G-CSF) (0.35 micrograms/day) on the number of peripheral blood cells and peripheral progenitor cells in mice. Among blood cells counts, only white blood cells were synergistically enhanced by co-administration of rhIL-6 and rhG-CSF. Moreover, it was found that co-administration of rhIL-6 and rhG-CSF also caused a marked synergistic increase in the number of peripheral blood progenitor cells. Namely, in combination with rhG-CSF, which alone induced a 170-fold increase in peripheral granulocyte-macrophage colony-forming units (CFU-GM) on day 14, rhIL-6 synergistically increased the number of CFU-GM to more than 1600-fold higher than the number in control mice. Administration of rhIL-6 alone induced a 46-fold increase in CFU-GM. Similar synergistic increases of other hematopoietic progenitors, such as colony-forming units in spleen and megakaryocyte colony-forming units in blood, were also observed in mice co-administered rhIL-6 and rhG-CSF. The survival rate of lethally irradiated recipient mice transplanted with mononuclear cells from 100 microliters of blood from mice administered rhIL-6 and/or rhG-CSF was examined. When mononuclear cells from mice co-administered rhIL-6 and rhG-CSF were injected, survival rate at day 100 was 92%. In contrast, recipient mice transplanted with mononuclear cells from mice administered either rhIL-6 or rhG-CSF alone showed a survival rate of 31% or 46%, respectively, although transplantation of mononuclear cells from control mice failed to rescue any lethally irradiated recipient mice. These results suggest that co-administration of rhIL-6 and rhG-CSF may be useful for peripheral blood stem cell transplantation.

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.

A Randomized, Placebo-Controlled Trial of Recombinant HumanGranulocyte 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.