We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.
Human ; Leukocyte Count ; Measles/*blood ; Monocytes/*chemistry ; Neutropenia/etiology ; Neutrophils/*chemistry ; Receptors, Granulocyte Colony-Stimulating Factor/*blood ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/*blood

OBJECTIVETo observe serum contents of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in mice with marrow inhibition before and after acupuncture and moxibustion treatment, so as to discuss the molecular biological mechanisms of acupuncture and moxibustion on improving marrow inhibition and increasing white cells after chemotherapy.

METHODSEighty clean-grade male Kunming mice were selected and randomly divided into a normal group, a model group, an acupuncture group and a moxibustion group according to the weight, 20 cases in each one. Mice in the model group, acupuncture group and moxibustion group were injected with cyclophosphamide (CTX) to establish mice models of marrow inhibition, while mice in the normal group received intraperitoneal injection of 0.9% NaCl. Four hours after model establishment, mice in the acupuncture group and moxibustion group were treated with acupuncture or moxibustion at "Dazhui" (GV 14), "Geshu" (BL 17), "Shenshu" (BL 23) and "Zusanli" (ST 36), respectively. Mice in the normal group and model group were immobilized without any treatment. All the treatment was given once a day for consecutive 5 days. Mice blood samples were collected from caudal vein. With manual examination, the white blood cells in peripheral blood were measured on each day from model establishment to end of treatment. Enzyme linked immunosorbent assay (ELISA) method was used to measure the serum contents of GM-CSF and G-CSF 3 days and 5 days after treatment.

RESULTSCompared with the normal group, the white cells in the model group were all reduced at each time point (all P<0.05), and the serum contents of GM-CSF and G-CSF were significantly reduced (all P<0.05). Three days after treatment, compared with the model group, the white cells in the acupuncture group and moxibustion group were increased, and the difference in acupuncture group was significant (P<0.05); the serum contents of GM-CSF and G-CSF were significantly lifted (P<0.05). Four days after treatment, compared with the model group, the white cells in the acupuncture group and moxibustion group were increased (both P<0.05). Five days after treatment, compared with the model group, the white cells in the acupuncture group and moxibustion group were increased and close to the normal level; the serum contents of GM-CSF and G-CSF were significantly lifted (all P<0.05).

CONCLUSIONThrough increasing serum contents of GM-CSF and G-CSF in CTX mice, acupuncture and moxibustion could prompt maturation and proliferation of myeloid hematopoietic cells, which is benefit to the reconstruction of hematopoietic function and relieve the marrow inhibition caused by CTX, and thus lift peripheral white blood cells.

Acupuncture Therapy ; Animals ; Bone Marrow ; drug effects ; Cyclophosphamide ; adverse effects ; Granulocyte Colony-Stimulating Factor ; blood ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Male ; Mice ; Moxibustion

The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P <0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.
Adolescence ; Adult ; Cell Division/drug effects ; Child ; Female ; Granulocyte Colony-Stimulating Factor/therapeutic use ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Granulocyte Colony-Stimulating Factor/adverse effects ; Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology* ; Granulocyte-Macrophage Colony-Stimulating Factor/adverse effects ; Human ; Isoenzymes/metabolism ; Isoenzymes/biosynthesis ; Leukocyte Count ; Leukocytes/pathology ; Leukocytes/enzymology ; Leukocytes/drug effects ; Male ; Middle Age ; Neoplasms/enzymology ; Neoplasms/drug therapy ; Neoplasms/blood* ; Neutropenia/metabolism* ; Neutropenia/chemically induce ; Neutropenia/blood ; Tetrahydrofolate Dehydrogenase/metabolism* ; Tetrahydrofolate Dehydrogenase/biosynthesis

BACKGROUND: In vitro generated cells from cord blood (CB) CD34+ cells increase cell dose and may reduce the severity and the duration of pancytopenia after transplantation. But safe engraftment for adolescents and adults is still not predictable and standardized. We attempted to establish a clinically application of in vitro generated cells by investigating the use of cytokines for the culture of CB CD34+ cells. METHODS: CD34+ cells, purified from four separate human CB units by magnetic bead selection, were cultured in IMDM with several cytokines. Differentiation of the in vitro generated cells has been confirmed by flowcytometry with specific erythroid, myeloid and megakaryocytic lineage surface markers. And apoptosis of these cells also was analysed with annexin V staining and morphologic analysis under the electron microscopic examination was done, simultaneously. RESULTS: Purified CB CD34+ cells were expanded with differentiation from CD38- to CD38+ expression with time. CB CD34+ cells could be terminally differentiated into erythroid (GPA+) and myeloid (CD33+/CD15+) lineage cells without apoptosis (annexin-V - ) in the presence of EPO and G-CSF, respectively. Megakaryocytic differentiation was partially arrested in early stage due to apoptosis in the presence of TPO. Morphological examination using electron microscope revealed all stages of erythroid and myeloid lineage cells without apoptosis, and apoptotic megakaryocytic lineage cells of early stage. But we could observe the small number of fully maturated platelets. CONCLUSION: CB CD34+ cells were terminally differentiated to erythroid, myeloid, and megakaryocytic lineage cells with or without apoptosis by several cytokines.
Adolescent ; Adult ; Annexin A5 ; Apoptosis* ; Cytokines* ; Fetal Blood* ; Granulocyte Colony-Stimulating Factor ; Humans ; Pancytopenia

PURPOSE: Proper speed of distraction is critical for successful new bone formation in distraction osteogenesis. The purpose of this study was to evaluate the effect of granulocyte-colony stimulating factor (G-CSF) on formation of new blood vessels and new bones in the rat model of tibial distraction osteogenesis (DO) to develop enhancement method of bone formation while increasing the distraction speed. MATERIALS AND METHODS: Forty two rat-tibial DO models were included in this study, and were divided into 3 groups; group I (rapid distraction), group II (rapid distraction with G-CSF), and group III (slow distraction). The amount of bone formation and relative blood flow were analyzed by sequential radiographs and laser Doppler perfusion imaging (LDPI). Blood sampling was done before G-CSF injection, at 2 weeks and 5 weeks after G-CSF injection and surface expression such as Scal-1+ and C-kit+ of endothelial progenitor cells (EPCs) was analyzed by fluorescence-activated cell sorter (FACS) for the effects of G-CSF in inducing mobilization of EPCs. RESULTS: The amount of new bone formation in the distraction gap on serial radiographs was higher during the consolidation period in groups II and III than in group I but, the difference was not significant (p>0.05). The relative blood flow in the distraction gap in groups II and III increased more significantly than in group I (p<0.05). FACS analysis showed an increased EPCs fraction after G-CSF injection. CONCLUSION: We demonstrated that G-CSF administration ameliorated bone formation and blood flow during rapid distraction in the rat model of tibial distraction osteogenesis. We think that G-CSF has an effect on mobilization of EPCs resulting in an increase in the blood flow.
Animals ; Blood Vessels ; Granulocyte Colony-Stimulating Factor ; Osteogenesis ; Osteogenesis, Distraction ; Perfusion Imaging ; Rats ; Stem Cells

BACKGROUND: Transplantation of allogeneic peripheral blood stem cells (PBSCs) may have advantage over bone marrow transplantation with regards to the speed of hematopoietic and immunologic recovery. Recently to overcome the need for multiple leukaphereses to collect enough PBSCs for autologous transplantation, large volume leukaphereses (LVL) are used to process multiple blood volumes per session. Experience with this technique in healthy individuals after mobilization with colony stimulating factor (CSF) is limited. We have investigated the efficacy and safety of LVL for the collection of G-CSF and GM-CSF mobilized PBSCs from healthy donors. METHODS: This study was done on 40 healthy donors who were mobilized with G-CSF and GM-CSF for allogeneic peripheral blood stem cells transplantation (allo-PBSCT). After 5 days of mobilization treatment, PBSCs were collected by LVL with Fenwal CS-3000 Plus (Baxter Co, USA). In LVL, heparin was administered in addition to ACD-A. Patients underwent of LVL procedures daily to obtain a target cell dose of >or= 4x10(8)/kg MNCs and >or= 6x10(6)/kg CD34+ cells. RESULTS: 66 LVL procedures were done on 40 donors. Of these donors, 31 (77.5%) reached the collection target with one leukapheresis. The product per LVL contained a mean 5.79+/-2.47 10(8)MNCs/kg and 11.6+/-10.62x10(6) CD34+ cells/kg respectively. Mean percentages of MNC were 79.88+/-22.15% and collection efficiencies of MNCs were inversely related to the starting MNC count (r=-0.536, P<0.001). After LVL, although none of the donors exhibited bleeding complications, platelets decreased from 187.4+/-52.68x10(3)/microL to 74.88+/-13.7x10(3)/microL and activated partial thromboplastin time (APTT) prolonged from 29.13+/-3.77 seconds to 67.51+/-54.26 seconds. CONCLUSION: We conclude that LVL after mobilization treatment with G-CSF and GM-CSF in normal healthy donors was tolerable and efficient methods for PBSCs collection, but long-term risk of adverse effects in normal donors needs to be carefully addressed by individual institutions as well as national and international registries.
Autografts ; Blood Volume ; Bone Marrow Transplantation ; Colony-Stimulating Factors ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hemorrhage ; Heparin ; Humans ; Leukapheresis* ; Partial Thromboplastin Time ; Registries ; Stem Cells* ; Tissue Donors ; Transplantation, Autologous

BACKGROUND: The use of colony stimulating factor (CSF) has increased to mobilize hematopoietic progenitor cells for allo-PBSCT. The most effective mobilization regimen has not yet defined. The authors analyzed the results of the mobilized PBSC collection through large volume leukapheresis from 38 normal healthy donors using three different regimens, namely, a single regimen with GM-CSF (Leucogen ), a concurrent use of GM-CSF and G-CSF (Leucostim ), and a sequential regimen with GM-CSF followed by G-CSF. METHODS: This study was done on 38 healthy donors from Sep. 1998 to Jan. 2001. One donor was mobilized with G-CSF alone, 9 with GM-CSF alone, 20 with concurrent regimen and 8 with sequential regimen. After 5 days of mobilization treatment, PBSCs were collected by large volume leukapheresis through femoral vein catheter. We compared the results of each collected progenitor cells and observed the side effects. RESULTS: The average WBC count before apheresis was 22.6+-11.0x103/uL and circulating CD34+cell percent was 1.31+-2.24%. Total 66 times with an average of 1.46+-0.61 of largevolume leukapheresis were performed for the 37 donors. The mean collected MNC count was 4.61+-2.77x108/kg, CD3+cell count was 2.95+-1.82x108/kg and CD34+cell count was 9.76+-12.42x106/kg. A significant side effect observed after large volume leukapheresis was thrombocytopenia showing decrease from 199.1+-52.2 to 80.7+-25.2x103/uL without any bleeding tendency. The mean collected MNC counts provoed to be significantly higher in combination groups with GM-and G-CSF than GM-CSF alone (P<0.05). The CD34+cell counts showed to be statistically higher in a sequential group compared to the concurrent and single regimen groups (P<0.05). CONCLUSION: A mobilization protocol with combination regimens of GM-CSF and G-CSF seemed to be superior to a single regimen with GM-CSF. Large volume leukapheresis through femoral vein catheter after mobilization with combination regimens of GM-and G-CSF in normal healthy donors was safe and proved to be an excellent. method to harvest stem cells.
Blood Component Removal ; Catheters ; Colony-Stimulating Factors ; Femoral Vein ; Granulocyte Colony-Stimulating Factor* ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Hematopoietic Stem Cells ; Hemorrhage ; Humans ; Leukapheresis* ; Stem Cells* ; Thrombocytopenia ; Tissue Donors*

BACKGROUND: Peripheral blood progenitor cells (PBPC) mobilized by hematopoietic growth factors such as G-CSF or GM-CSF are increasingly being used instead of bone marrow to allow hematopoietic reconstitution after myeloablative therapy for variety of malignancies. Ex vivo expansion of PBPC with growth factors leads marked increase in CFU-GM and CD34+ cells. To define the influence of G-CSF and stem cell factor alone and in combination on in vitro culture of PBPC, and to address the question of optimal duration of exposure with growth factors and the effects of G-CSF according to dosages, mobilized progenitors were incubated in liquid media containing autologous serum, stem cell factors and different dose of G-CSF. After 1, 7 and 10 day culture, viable cells were collected and innoculated to methylcellulose media, CFU-GM assay and evaluation of CD33 and CD34 positive cells were done. METHOD: PBPC were obtained from 10 patients by apheresis using COBE Spectra after chemotherapy with or without G-CSF. After Ficoll Hypaque separation, viable 2x106 PBPC were incubated in each 6 sets of RPMI media containing 10% autologous serum and addition of 100ng/mL of stem cell factor, 1,000U/mL of G-CSF, 5,000U/mL of G-CSF, 100ng/mL stem cell factor+1,000U/mL of G-CSF, 100ng/mL stem cell factor+5,000U/mL G-CSF in each culture flask and control group which didn' t contain any growth factor. After 1, 7 and 10 day of culture, viable cells were collected and 1x105 cells were seeded in methylcellulose media containing PHA-LCM and were cultured in duplicate. After 14 day incubation, aggregated with over 50 cells were scored as colony. And 1 day and 10 day of culture of control group and 10 day culture of stem cell factor+5,000U/mL G-CSF group, 1x105 cells were also collected for evaluation of CD33 and CD34 positive cells using flow cytometry. RESULT: CFU-GM were significantly increased even in 1 day exposure with combination of stem cell factor and G-CSF and there showed synergistic effect of stem cell factor and G-CSF. Seven day exposure with growth factor also represented similar increase in CFU-GM. In 10 day exposure of PBPC with growth factor showed significant increase in CFU-GM except 1,000ng/mL G-CSF group. The peak increase of CFU-GM was noted on 7 day culture with G-CSF+stem cell group and on 10 day culture of stem cell group. Number of CD33 & CD34 positive cells were increased in growth factor group and most of them were CD33+ CD34+ cells. There revealed significant positive correlation between CD34+ cells and day 14 CFU-GM. CONCLUSION: G-CSF and stem cell factor act synergistically and their action on ex vivo expansion of PBPC was prominent even in 1 day exposure with stem cell factor and G-CSF. CD34+ cells were also increased under the effect of growth factors and showed good positive corelation with CFU-GM.
Blood Component Removal ; Bone Marrow ; Diatrizoate ; Drug Therapy ; Ficoll ; Flow Cytometry ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells* ; Humans ; Intercellular Signaling Peptides and Proteins* ; Methylcellulose ; Stem Cell Factor ; Stem Cells

BACKGROUND: Peripheral blood progenitor cells collected from normal donors after granulocyte- colony stimulating factor (G-CSF) treatment are increasingly used for allogeneic transplantation. Previously, we investigated the mobilization kinetics of CD34+/Thy-1dim progenitor cells during subcutaneous administration of G-CSF in normal donors (Transfusion 1997;37:406-10). Although it is considered to be a relatively safe procedure, there are still uncertainties about the most efficient method of progenitor cell mobilization. Due to the short elimination half-life of G-CSF of about 3-4 hours we considered the subcutaneous administration of G-CSF once or twice daily might be suboptimal. The aim of the present study is to evaluate the mobilization kinetics of CD34+ cells during continuous intravenous (IV) administration of G-CSF in normal donors. METHODS: Fifteen healthy donors were enrolled in this study. The median age was 38 years (range, 20-56). G-CSF (Filgrastim, 10 microgram/kg/day) was administered for 4 consecutive days through continuous IV infusion. Then, we collected PBPC on the day following the 4th dose of G-CSF using a blood cell separator. For measurement of complete blood counts and CD34+ cell levels, peripheral blood sampling was performed immediately before the administration of G-CSF (steady-state) and after 4, 8, 24, 48, 72, 96, 120 hours of continuous IV administration of G-CSF. RESLUTS: After continuous IV administration of G-CSF, the WBC counts increased up to day 5 and reached approximately 8.4-fold above the steady-state level. Changes in the granulocyte count were similar to those in WBC counts. The number of lymphocytes increased up to day 4 (2.7-fold above the steady-state level), but no further increase occurred on day 5. Although there were considerable variations among the healthy donors, the statistical peaks of CD34+ cell levels were consistently observed on day 3 or day 4. Up to the fourth day of G-CSF treatment, the circulating CD34+ cells expanded by 25-fold. The percentage and absolute number of CD34+ cells significantly increased on day 3 (0.55 +/- 0.09%, 51.12 +/- 24.83x103/mL) and day 4 (0.47 +/- 0.09%, 46.66 +/- 24.93x103/mL), compared with steady-state values (0.06 +/- 0.09%, 2.03 +/- 5.69x103/mL). CONCLUSION: Our results showed that continuous IV administration of G-CSF apparently results in more rapid mobilization of CD34+ cells at least 24-48 hours compared with daily subcutaneous administration of G-CSF in normal donors.
Administration, Intravenous* ; Blood Cell Count ; Blood Cells ; Colony-Stimulating Factors ; Granulocyte Colony-Stimulating Factor* ; Granulocytes ; Half-Life ; Humans ; Kinetics* ; Lymphocytes ; Stem Cells ; Tissue Donors* ; Transplantation, Homologous

Adult ; Aged ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; blood ; physiopathology ; Ventricular Function, Left