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Stem Cells, Vol. 14, No. 6, 678-689, November 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Effects of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor on Thrombocytopenia Induced by a New Myelosuppressive Chemotherapy Regimen in Mice

Hiromichi Akahori, Kazunori Shibuya, Masako Ozai, Masumi Ida, Koji Kabaya, Takashi Kato, Hiroshi Miyazaki

Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan

Key Words. Thrombopoietin • Megakaryocyte growth and development factor (MGDF) • Chemotherapy • Thrombocytopenia • Platelet • Neutropenia

Dr. Hiromichi Akahori, Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki, Gunma 370-12, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy-induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by I.V. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia; it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG-CSF had an addictive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Recently, curative remedial therapy for cancer has progressed marvelously by the development of new chemotherapeutic agents and/or new combination regimens. With many tumors, the outcome might be better if the agent doses could be increased [1]. However, the major adverse effect of most cancer chemotherapy is myelosuppression with resultant neutropenia and thrombocytopenia that remains a major dose-limiting toxicity of high-dose chemotherapy. The severe neutropenia and thrombocytopenia can result in severe clinical consequences such as sepsis and hemorrhage, respectively, which are major causes of death during myelosuppression. Administration of recombinant human (rHu) G-CSF to patients after cancer chemotherapy can reduce the duration and severity of neutropenia with resultant sepsis [2, 3]. On the other hand, platelet transfusion has been only supportive care for reduction of the duration and severity of thrombocytopenia after cancer chemotherapy, but this care is usually accompanied by complications such as alloimmunization [4].

Several cytokines such as interleukin 3 (IL-3) [5-7], IL-6 [6, 8-11], IL-11 [12, 13] and leukemia inhibitory factor [14, 15], which increase platelet counts in normal animals, have been evaluated for their efficacy on the duration and severity of thrombocytopenia induced by cancer chemotherapy and/or irradiation exposure in preclinical or clinical studies, but amelioration of clinically important thrombocytopenia has not yet been achieved.

Megakaryopoiesis leading to thrombopoiesis has long been thought to be regulated by lineage-specific humoral factor, called thrombopoietin (TPO). Recently, four groups, including ours, reported the identification, purification, and cloning of TPO, also called c-Mpl ligand from various species including humans [16-19]. Three of these groups [16-18] isolated TPO cDNA as the ligand for c-Mpl, which is a member of the cytokine receptor superfamily [20]. On the other hand, we purified rat TPO protein from plasma and sublethally irradiated rats without the use of Mpl by measuring the activity which stimulated the production of megakaryocytes from rat bone marrow megakaryocyte progenitor cells (colony forming units-megakaryocytes [CFU-MK]) and determined its partial amino acid sequences [19, 21]. Based on the sequence information, we cloned rat TPO cDNA and subsequently human TPO cDNA and genomic DNA [19, 22, 23]. We also reported that the expression of TPO and mRNA is detected in hepatocytes and some hepatoma cell lines [24].

Several recent in vitro studies have demonstrated that TPO regulates megakaryocytopoiesis and thrombopoiesis with lineage-dominant action. TPO supports the formation of megakaryocyte colonies from murine bone marrow cells and human CD34+ cells in semisolid cultures [19, 25-31]. TPO also serves as a potent megakaryocyte maturation factor, because megakaryocytes generated in liquid culture containing TPO exhibit a marked increase in ploidy classes and well-developed demarcation membranes in their cytoplasm [26, 27, 29, 31]. Several recent studies have demonstrated that TPO stimulates a marked increase in circulating platelet counts and in the numbers of marrow megakaryocytes and CFU-MK in both femur and spleen when administered to normal mice or nonhuman primates [18, 19, 32]. Several studies in myelosuppressed mice have demonstrated that TPO significantly reduced the severity and duration of thrombocytopenia [33, 34].

Several murine models of thrombocytopenia induced by cancer chemotherapeutic agents and/or irradiation exposure have been used to study the therapeutic efficacy of several cytokines [6, 9, 10, 13]. In this study, we have developed a new murine model of thrombocytopenia in which mice received different doses of mitomycin C (MMC) by I.V. injection for two consecutive days. This regimen produced a severe and prolonged duration of thrombocytopenia without rebound thrombocytosis. Using this model, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of rHuTPO derivatized with polyethylene glycol, alone or in combination with rHuG-CSF on thrombocytopenia in this model. This molecule has biological activity similar to full-length human TPO, indicating that the C-terminal portion of the molecule is not required for the biological activity [16, 18].


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
Male Balb/c mice, weighing approximately 24 g and 8 weeks of age, were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice were housed in autoclaved cages in the specific pathogen-free rooms and were given sterilized commercial rodent chow and water ad libitum. Animal rooms were maintained at a temperature of 21°-23°C and 50%-60% relative humidity. The light cycle was 12/12 hours beginning from 8:00 a.m. All experiments were approved by the Institutional Animal Care Use Committee of our laboratory.

Experimental Design
PEG-rHuMGDF, which was expressed in E. coli as truncated form including the N-terminal erythropoietin-like domain of human TPO and derivatized with polyethylene glycol on the N-terminus by reductive alkylation, was used in this study. rHuG-CSF, (Filgrastim; Kirin Brewery Co., Ltd.; Tokyo, Japan) expressed in E. coli as a nonglycosylated form, was also used. Mice were treated with i.v. injection of MMC (Kyowa Hakko Kogyo Co.; Tokyo, Japan) at 4 mg/kg/day on day –1 and 3 mg/kg/day on day 0.

From the next day (day 1) after MMC treatment, mice were given various s.c. doses of PEG-rHuMGDF alone or in combination with rHuG-CSF in a volume of 5 ml/kg daily for different consecutive days. In some experiments, injections of PEG-rHuMGDF into MMC-treated mice started from day 1 with different intervals of days.

Peripheral Blood Evaluations
Peripheral blood samples were obtained from retro-orbital plexus using 75 mm heparinized capillary tubes (Funakoshi Pharmaceutical Co.; Tokyo, Japan). CBCs were performed with Sysmex automatic micro-cell counter K-2000 (Toa Medical Electrics Co.; Kobe, Japan); white blood differentials were performed on smear preparations stained with Wright-Giemsa using a blood cell analyzer (MICROX HEG-70A, Omron; Tokyo, Japan).

Progenitor Cell Assay
Bone marrow cells were harvested from mice by flashing the femoral contents with {alpha}-medium (Flow Laboratories; McLean, VA). CFU-MK cultures were performed according to the method previously described [35] with minor modifications. Briefly, 3 x 10 bone marrow cells were plated in 1 ml aliquots of Iscove's modified Dulbecco's medium (Sigma; St. Louis, MO) containing 0.3% Noble agar (Difco; Detroit, MI), 10% fetal calf serum ([FCS]; GIBCO; Grand Island, NY), 2 mM glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), 50 µM 2-mercaptoethanol (MERCK; Darmstadt, Germany), and 50 ng recombinant murine (rm) IL-3 in a 35 mm tissue culture dish (Nunc; Naperville, IL). After 7 days of culture, agar disks were detached from the culture dishes, placed onto glass slides, and stained with acetylcholinesterase (AchE) according to the method described by Jackson [36]. Megakaryocyte colonies comprising three or more cells were counted as CFU-MK-derived colonies.

Cultures of colony-forming units of granulocyte/macrophage (CFU-GM) and mixed erythroid colony-forming cells (E-mix) were performed by a methylcellulose method. Briefly, 3 x 10 bone marrow cells were plated in 1 ml aliquots of {alpha}-medium containing 0.88% methylcellulose (Shinetsu Scientific Inc.; Tokyo, Japan), 30% FCS, 1% bovine serum albumin (Sigma), 50 µM 2-mercaptoethanol, 16 IU recombinant human erythropoietin (rHuEPO) and 10 ng rmIL-3 in a 35 mm tissue culture dish. After 7 days of culture, colonies of more than 50 cells consisting of granulocytes and/or macrophages were scored as CFU-GM and mixed erythroid colonies and pure erythroid burst colonies were counted as E-mix. Colony counts were performed using an inverted microscope.

The total number of hematopoietic progenitor cells per femur was calculated as follows: the number of colonies generated per dish was multiplied by the total number of cells obtained from one femur which was divided by the number of cells seeded per dish.

Measurement of the Number of Megakaryocytes in the Femur
Bone marrow cells were fixed at 1 x 105 cells per well in 100 µl of 0.1 M phosphate buffer containing 2.5% glutaraldehyde (Wako Pure Chem. Ind. Ltd.; Osaka, Japan) in 96-well immunoplates (Nunc) for 10 min at room temperature. After fixation, the plates were centrifuged at 120 g for 5 min and then the supernatant of each well was removed by aspiration taking care not to disturb the cell pellet. A volume of 100 µl of 0.1 M phosphate buffer was added to each well, and plates were washed as described above. The washing procedure was repeated twice and the cell pellets were stained for AchE. The number of AchE+ cells per well were counted as megakaryocytes using an inverted light microscope.

The total number of megakaryocytes per femur was calculated as follows: the number of AchE+ megakaryocytes per well was multiplied by the total number of cells obtained from one femur which was divided by the number of cells plated per well.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Effects of PEG-rHuMGDF Consecutive Administration
Each group comprising five MMC-treated mice received either vehicle (days 1-23), PEG-rHuMGDF at 1 µg/kg/day (days 1-23), 5 µg/kg/day (days 1-20) or 30 µg/kg/day (days 1-9) respectively. In vehicle-treated mice following MMC treatment, platelet counts began to decrease on day 6, reached a nadir of 4% of basal level on day 14, and retained approximately 70% of basal level on day 26. Treatment with PEG-rHuMGDF reduced the severity of thrombocytopenia and enhanced platelet recovery in a dose-dependent manner (Fig. 1Go). In mice treated with PEG-rHuMGDF at 1 µg/kg/day platelet counts decreased to 30% of basal level on day 14. Thereafter, platelet counts recovered to 60% and 70% of basal level on day 16 and day 26, respectively. In mice treated with PEG-rHuMGDF at 5 µg/kg/day, platelet counts were maintained within 80% to 130% of basal level during the study period. In mice treated with PEG-rHuMGDF at 30 µg/kg/day, thrombocytopenia was completely prevented. Platelet counts began to increase above basal level by day 5, and the maximum platelet counts, a two-fold increase over basal level, were observed on day 14.



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  		Figure 1. Effects of consecutive administration of PEG-rHuMGDF on thrombocytopenia induced by MMC treatment in mice. Mice received 4 mg/kg of i.v. MMC on day –1 and 3 mg/kg on day 0, respectively. Then, mice received PEG-rHuMGDF s.c. at either 1 µg/kg/day (days 1-23), 5 µg/kg/day (days 1-20) or 30 µg/kg/day (days 1-9), respectively, or vehicle (days 1-23) as a control. Each point represents the mean ± SE of platelet counts from five mice.

 
In the vehicle-treated mice, the number of CFU-MK in the femur markedly decreased on day 5 through day 14 (approximately 5% of basal level), and returned to basal level on day 26. In contrast, in mice treated with PEG-rHuMGDF at 30 µg/kg/day the number of CFU-MK was maintained over basal level during the study period (Fig. 2AGo). The number of megakaryocytes in the femur in the vehicle-treated mice also began to decrease by day 5, reached a nadir on days 8 through 14 (approximately 2% of basal level), and recovered to about 25% of basal level on day 26. In mice treated with PEG-rHuMGDF at 30 µg/kg/day, the number of megakaryocytes decreased to a nadir (about 20% of basal level) on day 5 and recovered to basal level on day 14 (Fig. 2BGo).



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  		Figure 2. Effects of consecutive administration of PEG-rHuMGDF on the number of CFU-MK (A) and megakaryocytes (B) in bone marrow in MMC-treated mice. Mice received either s.c. PEG-rHuMGDF (30 µg/kg/day on days 1-9) (solid bar), or vehicle (on days 1-23) (open bar) following MMC treatment, respectively. Each data represent the mean ± SE of the number of CFU-MK or megakaryocytes from five mice.

 
Treatment with MMC also resulted in a decrease in absolute neutrophil counts (ANC) in a two-phase manner. The first nadir was observed on day 5 and the second nadir on days 14 through 20 (Fig. 3Go). Treatment only with PEG-rHuMGDF at the highest dose (30 µg/kg/day) significantly reduced the severity of the first phase of neutropenia (p < 0.05, Dunnett multiple comparison test) (Fig. 3Go). Administration of PEG-rHuMGDF improved the severity of the second nadir in a dosedependent manner (Fig. 3Go). PEG-rHuMGDF at 5 µg/kg/day or more prevented the second phase of neutropenia.



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  		Figure 3. Effects of consecutive administration of PEG-rHuMGDF on neutropenia induced by MMC treatment in mice. Mice received PEG-rHuMGDF following MMC treatment as described in the legend to Figure 1Go. Each point represents the mean ± SE of absolute neutrophil counts form five mice.

 
A significant decrease in red cell counts (approximately 90% of control) was observed in vehicle-treated control mice on days 16 and 20 (p < 0.05, Dunnett multiple comparison test). Treatment with PEG-rHuMGDF improved anemia in a dose-dependent manner (Fig. 4Go). In mice treated with PEG-rHuMGDF at either 5 or 30 µg/kg/day, red cell counts were significantly increased to basal level on days 16 and 20 compared with the control value (p < 0.01, Dunnett multiple comparison test).



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  		Figure 4. Effects of consecutive administration of PEG-rHuMGDF on red cell counts after MMC treatment in mice. Mice received PEG-rHuMGDF following MMC treatment as described in the legend to Figure 1Go. Each point represents the mean ± SE of red cell counts from five mice.

 
Effects of PEG-rHuMGDF with Various Administration Protocols
We examined the hematological effects of PEG-rHuMGDF with various administration protocols in this model. Following MMC treatment, mice received PEG-rHuMGDF by s.c. injection starting from day 1 with the following protocols: 5 µg/kg/day for 23 consecutive days (total dosage: 115 µg/kg), 5 µg/kg/day on alternate days for 12 days (total dosage: 60 µg/kg) or 30 µg/kg/day on days 1 and 8 (total dosage: 60 µg/kg). All these protocols had similar effects on thrombocytopenia in the MMC-treated mice (Fig. 5Go). In all groups treated with PEG-rHuMGDF, platelet counts decreased to a minimum of approximately 30% of basal level on day 10. On day 14, platelet counts in control mice reached a nadir but in all groups treated with PEG-rHuMGDF, platelet counts recovered 60%-80% of basal level, and there-after were maintained over 60% of basal level until the end of the study.



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  		Figure 5. Effects of PEG-rHuMGDF with several administration protocols on thrombocytopenia induced by MMC treatment in mice. Following MMC treatment, mice received PEG-rHuMGDF by s.c. injection from day 1 with the following protocols: either 5 µg/kg/day for 23 consecutive days (Group A), 5 µg/kg/day on alternate days for 12 days (Group B) or 30 µg/kg/day on days 1 and 8 (once a week; Group C). Each point represents the mean ± SE of platelet counts from five mice.

 
Combination Therapy of PEG-rHuMGDF and rHuG-CSF
MMC-treated mice were given either 5 µg/kg/day of s.c. PEG-rHuMGDF for 13 consecutive days, 10 µg/kg/day of rHuG-CSF for 9 consecutive days, or a combination of PEG-rHuMGDF and rHuG-CSF, respectively. These cytokines were administered s.c. from the next day following MMC treatment (Fig. 6Go). In mice treated with PEG-rHuMGDF alone, platelet counts decreased to a minimum of about 60% of basal level on day 10 and were normalized on day 14. In mice treated with rHuG-CSF alone, platelet counts decrease to almost the same level as control mice on day 10 and remained at about 40% of basal level until day 26. Concurrent administration of both cytokines improved thrombocytopenia similarly to PEG-rHuMGDF alone. This combination showed an additive effect on enhancement of platelet counts over the baseline value after normalization.



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  		Figure 6. Effects of PEG-rHuMGDF, rHuG-CSF, or a combination of PEG-rHuMGDF and rHuG-CSF on thrombocytopenia induced by MMC treatment in mice. Mice were administered either s.c PEG-rHuMGDF (5 µg/kg/day on days 1-13), rHuG-CSF (10 µg/kg/day on days 1-9), or a combination of PEG-rHuMGDF and rHuG-CSF following MMC treatment, respectively. Each point represents the mean ± SE of platelet counts from five mice.

 
Treatment with PEG-rHuMGDF at 5 µg/kg/day had no effect on the severity of the first phase of neutropenia (Fig. 7Go). Treatment with either rHuG-CSF alone or in combination with PEG-rHuMGDF significantly accelerated the ANC recovery on day 5 during the first neutropenic period, compared with vehicle treatment (p < 0.05, Dunnett multiple comparison test). Each of the two factors or their combination prevented the second phase of neutropenia noted on day 14 through day 20.



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  		Figure 7. Effects of PEG-rHuMGDF, rHuG-CSF, or a combination of PEG-rHuMGDF and rHuG-CSF on neutropenia induced by MMC treatment in mice. Mice received cytokines following MMC treatment as described in the legend to Figure 6Go. Each point represents the mean ± SE of absolute neutrophil counts from five mice.

 
As shown in Table 1Go, the numbers of hematopoietic progenitor cells in bone marrow, such as CFU-MK, CFU-GM and E-mix, were markedly decreased on day 5 through day 10 in the MMC-treated control mice. Treatment with either PEG-rHuMGDF alone or in combination with rHuG-CSF reduced the decrease in the numbers of these progenitor cells and enhanced normalization.


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  		Table 1. Effects of PEG-rHuMGDF alone or in combination with rHuG-CSF on the numbers of hematopoietic progenitor cells in the femoral bone marrow
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
A murine model of thrombocytopenia induced by a combination regimen of low-dose irradiation and carboplatin has been used in several cytokine studies as well as PEG-rHuMGDF [13, 33]. This regimen can produce a severe and prolonged thrombocytopenia with myelosuppression and is thought to be a more useful regimen than the other previous regimens [6, 9, 10]. In this study, to evaluate the efficacy of PEG-rHuMGDF on the thrombocytopenia induced by chemotherapy alone, we developed a new murine model of thrombocytopenia induced by MMC which had similar severity and prolonged thrombocytopenia to a combined regimen of irradiation and carboplatin administration. This chemotherapy model was not fatal to mice.

Ulich et al. have shown that administration of rHuMGDF to the carboplatin-induced thrombocytopenic mice prevented the thrombocytopenia and normalized the megakaryocyte numbers in bone marrow [34]. In mice treated with a combination of irradiation and carboplatin administration, administration of PEG-rHuMGDF significantly reduced mortality and ameliorated the depth and duration of thrombocytopenia [33]. In this study, consecutive administration of PEG-rHuMGDF reduced the severity of thrombocytopenia and accelerated the recovery of platelet counts in a dose-dependent manner. In mice treated with PEG-rHuMGDF, the number of CFU-MK was maintained above basal level over the period of the study, and the number of megakaryocytes in the bone marrow recovered more quickly to basal level than those in vehicle-treated mice. These results suggested that administration of PEG-rHuMGDF stimulates the rapid recovery of the numbers of CFU-MK and megakaryocytes in bone marrow to facilitate platelet production n myelosuppressed mice. In this study, we also examined the therapeutic efficacy of three different administration protocols in ameliorating thrombocytopenia induced by MMC. Weekly administrations of PEG-rHuMGDF is able to improve thrombocytopenia similarly to daily injections. In other words, daily administration is not required to improve thrombocytopenia. The present results may provide some information on setting up effective administration protocols of PEG-rHuMGDF in treating thrombocytopenic patients. Previously, several cytokines have been shown to affect megakaryocytopoiesis and platelet production in myelosuppressed animals [6, 9, 10, 13]. The present data indicate that PEG-rHuMGDF is most effective in ameliorating thrombocytopenia in myelosuppressed mice among the cytokines so far tested.

In addition to the effects on thrombocytopenia, administration of PEG-rHuMGDF improved the neutropenia and anemia induced by MMC treatment. TPO has only minimal effects on the growth of the granulocyte progenitor cells in vitro [25]. However, recent in vivo studies have shown that a mild yet significant increase in the ANC was observed when normal nonhuman primates received rHuMGDF [32]. Administration of PEG-rHuMGDF lessened the leukopenic duration in myelosuppressed mice [33]. TPO enhances proliferation of erythroid progenitors in the presence of EPO but cannot produce the erythroid colony without EPO [37]. Kaushansky et al. have shown that TPO increases both the number and size of BFU-E-derived colonies in the presence of IL-3 or stem cell factor (SCF) and EPO [38]. They have also shown that TPO enhances erythroid recovery in myelosuppressed mice through the expansion of erythroid precursors. Zeigler et al. have reported that TPO stimulates a population of murine stem cells, in combination with other cytokines such as SCF or IL-3, to enhance myelopoiesis in vitro [39]. According to these results, TPO or PEG-rHuMGDF also affects the development of early progenitor cells in addition to the thrombopoietic activity, but alone has no influence on terminal differentiation in granulopoiesis and erythropoiesis. The increase in ANC and red cell counts in response to TPO, or PEG-rHuMGDF, administration in myelosuppressed animals may be the result of the synergistic effect of TPO or PEG-MGDF and endogenously increased other cytokines such as EPO, G-CSF, or IL-3.

Although concurrent administration of PEG-rHuMGDF and rHuG-CSF was effective in improving thrombocytopenia, there was no significant increase in platelet counts in mice treated with both factors compared with those in mice treated with PEG-rHuMGDF alone. Additionally, the combined effect of both factors showed no increase in the number of megakaryocyte progenitors in the bone marrow. However, this combination additively increased platelet counts above the baseline value after the platelet recovery. Such effect may be caused by the action of rHuG-CSF on proliferation of multipotential hematopoietic progenitor cells [40, 41]. The effect of co-administration of rHuG-CSF and PEG-rHuMGDF on neutropenia was similar to the case of thrombocytopenia. The combination resulted in a slight, but not significant, enhancement of ANC, compared with rHuG-CSF alone, over the scope of this study. There also was no collaboration of rHuG-CSF with PEG-rHuMGDF in increasing the number of granulocyte/macrophage progenitor cells. Although the combining effect of both factors was not observed under the present conditions, these results provide important information in future clinical applications of PEG-rHuMGDF in that the combination of PEG-rHuMGDF and rHuG-CSF is able to reduce the severity of thrombocytopenia and neutropenia and to accelerate the recovery of platelet counts and ANC without lessening the effect of either cytokine.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors gratefully acknowledge Ms. Yuko Nitta, Ms. Miyuki Kato, and Ms. Kazumi Takahashi for their excellent technical assistance.


   Footnotes
 
Provisionally accepted June 9, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received May 13, 1996; accepted for publication July 5, 1996.




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