Clonogenic potential and phenotypic analysis of CD34+ cells mobilized by different chemotherapy regimens.

BACKGROUND AND OBJECTIVE: Since limited data concerning quantitative and qualitative differences of CD34+ cells collected after different mobilization schedules are available, we investigated phenotype, proliferative capacity and primitive progenitor cell content of CD34+ cells mobilized with four different regimens. DESIGN AND METHODS: The number, phenotype, and progenitor cell content of CD34+ cells were investigated in 46 patients mobilized with cyclophosphamide (CY) 7 g/m(2) plus granulocyte colony-stimulating factor (G-CSF, 5 microg/kg) (CY7+G-CSF) (n=16), CY 4 g/m(2) plus G-CSF (CY4+G-CSF) (n=8), IVE [ifosphamide (2.5 g/m(2) for 3 d), etoposide (150 mg/m(2) for 3 d), epirubicin (100 mg/m(2) on day 1)] plus G-CSF (IVE+G-CSF) (n=9), or G-CSF (10 microg/kg) alone (n=13). RESULTS: The number of CD34+ cells collected per liter of processed blood was significantly higher in the CY7+G-CSF group than in the CY4+G-CSF and G-CSF groups (p 相似文献   

GM-CSF-based mobilization effect in normal healthy donors for allogeneic peripheral blood stem cell transplantation

It is important to optimize methods to mobilize hematopoietic stem cells into peripheral blood (PB) for successful allogeneic peripheral blood stem cell (PBSC) transplantation. Our primary intent was to investigate the role of GM-CSF for mobilization in normal healthy donors and to compare its efficacy in mobilizing stem cells alone, in concurrent combination and in sequential combination with G-CSF in this study. We analyzed the results of the PBSC harvest through large volume leukapheresis from 48 normal healthy donors mobilized by three different regimens including GM-CSF. Donors were assigned sequentially to one of the following regimens for mobilization: GM-CSF 10 microg/kg/day alone (group 1, n = 9); concurrent combination (group 2, n = 20) of G-CSF 5 microg/kg/day and GM-CSF 5 microg/kg/day; sequential combination (group 3, n = 19) of GM-CSF alone 10 microg/kg/day for 3 days followed by G-CSF alone 10 microg/kg/day for 2-3 days. The harvested CD34(+) cell count (P < 0.05) was statistically higher in group 3 than in group 1 or 2. Pre-collection WBC count in donors (P < 0.05), harvested MNC (P < 0.05) and CD3(+) cell count (P < 0.05) of group 2 or 3 were significantly higher than those of group 1. Recipients who received stem cells mobilized with combination regimens showed an earlier recovery of WBC and platelets count than those with GM-CSF alone. The incidence of acute graft-versus-host disease was not statistically different among three recipient groups. GM-CSF-based mobilization was well tolerated in normal healthy donors. The sequential combination regimen appears to be an excellent mobilization strategy and might be preferred as the optimal method in some clinical situations that need a higher number of stem cells.     

Significant mobilization of both conventional and regulatory T cells with AMD3100

In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4(+)/CD25(high)/CD127(low)/FoxP3(+) Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8(+) T cells were mobilized to a greater extent than CD4(+) T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8(+) effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.     

Low CXCR4 membrane expression on CD34(+) cells characterizes cells mobilized to blood

Summary:In this work, association between the presence and membrane density of CXCR4 and the effectiveness of mobilization was studied. Ninety G-CSF mobilized PBPC and 28 native BM (nBM) preparations obtained from healthy individuals for transplantation and BM obtained after G-CSF mobilized PBPC collection in 10 donors were investigated. Positivity for CD34, HLA-DR and CXCR4 were analysed in the three colour fluorescence. The cellular profile of PBPC differed from nBM preparations with respect to lower: (i) proportion of CD34(+) cells (0.64%+/-0.04 vs 0.92+/-0.07) and CD34(+)CXCR4(+) cells (0.30%+/-0.02 vs 0.61+/-0.07); (ii) contribution of CXCR4(+) cells to CD34(+) cells (52.2%+/-2.5 vs 62.2%+/-4.2); (iii) CXCR4 epitope density in CD34(+) cells (48.9+/-5.5 vs 94.7+/-10.4). PBPC yield for CD34(+) cells was correlated with the content of CD34(+) cells lacking CXCR4 in the leukapheresis product (R=0.38). In contrast, nBM harvested for transplantation was poor in CD34(+) cells if these cells were frequently CXCR4- (R=-0.49). The present study shows that CD34(+) cells mobilized to blood were characterized with a low proportion of CXCR4 and this associated with CD34(+) cell content in PBPC.     

Transplantation of allogeneic CD34+ blood cells

Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA- identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.     

Correlation of hematopoietic progenitor cell count determined by the SE-automated hematology analyzer with CD34(+) cell count by flow cytometry in leukapheresis products

The yield of stem cell collection after mobilization is crucial for autologous peripheral blood stem cell (PBSC) transplantation. Quantitative determinations of CD34(+) cells using flow cytometry or stem cell culture have been used, but these methods require much time, technical experience, and expensive reagents. The automated hematology analyzer (Sysmex SE-9000trade mark, TOA, Japan) equipped with the Immature Information (IMI) channel for immature myeloid cells can detect IMI(+) cells within 90 sec. Detection is made possible by the combination of a special reagent system and direct current/radiofrequency biosensors. We studied the relation of IMI(+) cells and variable cell counts with CD34(+) cell yield in autologous stem cell harvest. In a series of 32 patients (median age, 44 years; M:F = 11:21), 184 leukaphereses were performed after mobilization regimens with chemotherapy and G-CSF or G-CSF alone. Full blood cell counts were enumerated on peripheral blood (PB) samples taken prior to each leukapheresis. Mononuclear cell (MNC) and IMI(+) cell counts by automated hematology analyzer and flow cytometry based CD34(+) cell yield were measured on the harvested product. The relationship among PB white blood cells (WBC), PB monocytes, IMI(+) cells, MNC, and CD34(+) cell yield in a single leukapheresis was estimated by Pearson correlation analysis. PB WBC count showed no correlation with CD34(+) cell yield in a single leukapheresis (r = 0.02, P = 0.81). PB monocyte count showed a weak correlation (r = 0.21, P = 0.01) and MNC in harvest also showed a weak correlation (r = 0.36, P = 0.0001) with CD34(+) cell yield. In contrast, CD34(+) cell yield correlated well with IMI(+) cell count (r = 0.68, P = 0.0001), and data could be fitted by a linear regression equation, y = 0.330 + 0.974x. IMI(+) cell assay by the automated hematology analyzer correlated well with the CD34(+) cell yield in a mobilized autologous stem cell harvest. The IMI(+) cell count might be used as a simple and efficient indicator of blood stem cell mobilization and collection.     

Mobilization, harvesting and selection of peripheral blood stem cells in patients with autoimmune diseases undergoing autologous hematopoietic stem cell transplantation

Peripheral blood stem cells (PBSC) were mobilized in 130 patients with autoimmune diseases undergoing autologous hematopoietic stem cell transplantation using cyclophosphamide 2 g/m(2) and either granulocyte colony-stimulating factor (G-CSF) 5 mcg/kg/day (for systemic lupus erythematosus (SLE) and secondary progressive multiple sclerosis, SPMS) or G-CSF 10 mcg/kg/day (for relapsing remitting multiple sclerosis (RRMS), Crohn's disease (CD), systemic sclerosis (SSc), and other immune-mediated disorders). Mobilization-related mortality was 0.8% (one of 130) secondary to infection. Circulating peripheral blood (PB) CD34(+) cells/microl differed significantly by disease. Collected CD34(+) cells/kg/apheresis and overall collection efficiency was significantly better using Spectra apheresis device compared to the Fenwall CS3000 instrument. Patients with SLE and RRMS achieved the lowest and the highest CD34(+) cell yields, respectively. Ex vivo CD34(+) cell selection employing Isolex 300iv2.5 apparatus was significantly more efficient compared to CEPRATE CS device. Circulating PB CD34(+) cells/microl correlated positively with initial CD34(+) cells/kg/apheresis and enriched product CD34(+) cells/kg. Mean WBC and platelet engraftment (ANC>0.5 x 10(9)/l and platelet count >20 x 10(9)/l) occurred on days 9 and 11, respectively. Infused CD34(+) cell/kg dose showed significant direct correlation with faster white blood cell (WBC) and platelet engraftment. When adjusted for CD34(+) cell/kg dose, patients treated with a myeloablative regimen had significantly slower WBC and platelet recovery compared to non-myeloablative regimens.     

Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation

Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA-4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony- stimulating factor (G-CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady- state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = - .86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = - .70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation.     

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.     

A combination of dexamethasone,cyclophosphamide, etoposide,and cisplatin is less toxic and more effective than high-dose cyclophosphamide for peripheral stem cell mobilization in multiple myeloma

BACKGROUND AND OBJECTIVES: The purpose of this study was to compare the efficacy and toxicity of two regimens for peripheral blood stem cell (PBSC) mobilization in multiple myeloma (MM) patients. DESIGN AND METHODS: From 1995 to 2001, 116 patients were enrolled in two high-dose programs including autologous transplantation, adopting two mobilizing regimens: 61 patients were mobilized with high-dose cyclophosphamide (HD-Cy) at 4 g/m2 (group I), and 55 patients with DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) (group II), both followed by granulocyte colony-stimulating factor (G-CSF 5 mg/Kg/day) started 48 hours after chemotherapy. RESULTS: The median number of CD34+ cells harvested was similar in the two groups (5.9 vs 5.82x106 cells/kg). The target of at least 4x106 cells/kg was reached in a higher percentage of patients in the DCEP group (75 vs 59%) (p=0.05). The proportion of poor mobilizers (<2x106 CD34+ cells/kg) was 21% with HD-Cy and 13% with DCEP (P=NS). In group I, 10 patients (16%) required packed red cell transfusions, 5 patients (8%) platelet support, and the majority of patients (87%) had a neutrophil count below 500/mL, whereas none did so in group II (p=0.0009, p=0.01, p=0.0009, respectively). Neutropenia-related fever occurred in 18% of patients in group I versus 0% in group II (p=0.0005). WHO grade >II extra-hematologic toxicities (microhematuria, cystitis, infections) were seen in 8 patients (13%) of group I vs 0 in group II (p=0.007). INTERPRETATION AND CONCLUSIONS: DCEP is a better tolerated and more effective regimen than HD-Cy for peripheral stem cell mobilization in MM patients assigned to high-dose therapy programs.     

Stem cell component therapy: supplementation of unmanipulated marrow with CD34 enriched peripheral blood stem cells

Eleven patients with hematologic malignancies and two with aplastic anemia were treated using unmanipulated marrow and immunoselected CD34+ blood cells. Donors began G-CSF (10 microg/kg) injections 1 day after undergoing bone marrow harvest. Blood stem cells were collected on day 5 of G-CSF. Peripheral blood lymphocytes were depleted via CD34-positive selection. If, after marrow and blood harvest, less than 2.0 x 10(6) CD34 cells/kg were mobilized, leukapheresis was repeated on day 6. Median time to an absolute neutrophil count greater than 500 microl was day 10; transfusion-independent platelet count greater than 20,000/microl was day 13; average hospital discharge was day 14; and average inpatient hospital charges were 101,870 US dollars. Acute GVHD grade II occurred in five of 13 patients. No patient developed grade III or IV acute GVHD. At a median follow-up of 10 months, no patient has developed extensive chronic GVHD. Allografts of unmanipulated bone marrow supplemented with G-CSF-mobilized and CD34 immunoselected blood cells may prevent an increased risk of GVHD while preserving the rapid engraftment kinetics of peripheral blood. Supplementation of marrow with CD34 enriched blood cells appears to result in rapid engraftment, early hospital discharge, lower inpatient charges, decreased regimen-related toxicity, and no apparent increase in GVHD.     

Stem cell factor in combination with filgrastim after chemotherapy improves peripheral blood progenitor cell yield and reduces apheresis requirements in multiple myeloma patients: a randomized, controlled trial.

Stem cell factor (SCF) has been shown to synergize with filgrastim to mobilize CD34(+) cells into the peripheral blood. To determine if addition of SCF to chemotherapy and filgrastim reduces the number of leukaphereses required to achieve a target yield of 5 x 10(6) CD34(+) cells/kg, 102 patients with multiple myeloma were randomized to receive mobilization chemotherapy with cyclophosphamide (4 g/m(2)) and either SCF (20 micrograms/kg/d) combined with filgrastim (5 micrograms/kg/d) or filgrastim alone (5 micrograms/kg/d), administered daily until leukaphereses were completed. After collection, patients were treated with myeloablative therapy supported by autologous peripheral blood progenitor cell (PBPC) infusion and filgrastim (5 micrograms/kg/d). There was a significant difference between the treatment groups in the number of leukaphereses required to collect 5 x 10(6) CD34(+) cells/kg (median of 1 v 2 for SCF + filgrastim and filgrastim alone, respectively, P =.008). Patients receiving the combination of SCF plus filgrastim had a 3-fold greater chance of reaching 5 x 10(6) CD34(+) cells/kg in a single leukapheresis compared with patients mobilized with filgrastim alone. The median CD34(+) cell yield was significantly increased for the SCF group in the first leukapheresis (11.3 v 4.0 x 10(6)/kg, P =.003) and all leukaphereses (12.4 v 8.2 x 10(6)/kg, P =.007). Total colony-forming unit-granulocyte-macrophage (CFU-GM) and mononuclear cell counts were also significantly higher in the SCF group in the first leukapheresis and in all leukaphereses. As expected for patients mobilized to an optimal CD34(+) cell yield, the time to engraftment was similar between the 2 treatment groups. Cells mobilized with the combination of SCF plus filgrastim were thus considered effective and safe for achieving rapid engraftment. Treatment with SCF plus filgrastim was well tolerated, with mild to moderate injection site reactions being the most frequently reported adverse events. There were no serious allergic-like reactions to SCF. The addition of SCF to filgrastim after cyclophosphamide for PBPC mobilization resulted in a significant increase in CD34(+) cell yield and a concomitant reduction in the number of leukaphereses required to collect an optimal harvest of 5 x 10(6) CD34(+) cells/kg.     

Difficult stem cell mobilization despite adequate CD34+ cell dose predicts shortened progression free and overall survival after autologous HSCT for lymphoma

Hematopoietic growth factors alone or in combination with myelosuppressive chemotherapy are used to mobilize peripheral blood stem cells for autologous transplantation. To identify characteristics of successful mobilization with granulocyte colony-stimulating factor (G-CSF) alone and to study the impact of immediate chemotherapy mobilization following G-CSF mobilization, we treated 175 chemotherapy sensitive lymphoma patients with G-CSF (G) mobilization and leukapheresis followed by chemotherapy plus G-CSF (CG) mobilization and leukapheresis and then autologous transplantation. Patients with stage I/II disease at diagnosis and < or =5 years from diagnosis were more likely to mobilize successfully with G-CSF alone (G). CG mobilization led to superior stem cell yields compared to the preceding mobilization with G (median 2.37 vs 1.37 ( x 10(6)CD34+ cells/kg); P<0.0001). Patients (n=58, 33%) with successful G-CSF mobilization (> or =2 x 10(6) CD34+ cells/kg) had quicker platelet recovery and improved progression free and overall survival compared to patients who had adequate collection only after chemotherapy mobilization or to those who failed to collect an adequate graft with either type of mobilization. The poor clinical outcome of patients with difficult mobilization using either method identifies them as a high-risk group who might benefit from alternative therapies.     

Autologous stem cell transplantation in multiple myeloma after VAD and EDAP courses: a high incidence of oligoclonal serum Igs post transplantation

Thirty-seven patients with multiple myeloma (stage II and III, 65% increased beta2-microglobulin level) were prospectively treated with a median of 3.7 VAD courses (range 2-8) followed by cyclophosphamide (6 g/m2) in conjunction with G-CSF (5 microg/kg filgrastrim (n = 14), or 3.5 microg/kg lenograstrim (n = 22)), and peripheral stem cell (PSC) isolation. After regeneration this was followed by one EDAP course and high-dose melphalan (HDM, 200 mg/m2) in combination with re-infusion of PSC. Adequate stem cell mobilization was obtained with both G-CSF regimens. A median of 41x10(6) CD34+ cells/kg (range 4.5-161) was collected in a median of 1.6 leukapheresis procedures following filgrastrim (n = 14) and 24x10(6) CD34+ cells/kg (range 2. 3-80) in a median of 1.7 leukapheresis procedures following lenograstrim (n = 22) which indicated no significant difference (P = 0.24) between both G-CSF regimens. A rapid hematological recovery was obtained after HDM with reinfusion of a median of 9.3x10(6) CD34+ cells/kg. After the total courses the overall response was 84% with a complete remission rate of 30%. Currently the median overall survival is 44.0 months (95% CI 38.9-49.1) with a median follow-up of 33 months (range 3-51) and a median event-free survival of 29.0 months (95% CI 25.3-32.7) (n = 33). Post transplantation a high incidence of oligloclonal serum immunoglobulins (Igs) was observed. In 73% of the patients new oligoclonal or monoclonal serum bands were noticed 3 months post transplantation. IgG-lambda and IgG-kappa bands predominated. In 48% of the cases the oligoclonal Igs disappeared after a median follow-up of 22 months (range 8-36), whereas in 52% of the cases the oligoclonal Igs persisted with a median follow-up of 31 months (range 21-45), which did not correlate with a significant difference in overall, and event-free survival between both subgroups.     

Characterization of chemotherapy mobilized peripheral blood progenitor cells for use in autologous stem cell transplantation.

Twenty patients were treated with chemotherapy to mobilize progenitors into the blood. Peripheral blood stem cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34+ cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU-GM by 24-48 h. Peak levels of CD34+ cells ranged from 0.6-5% and coincided with the peak increase in CFU-GM. Mobilized CD34+ cells contained subsets expressing CD33, CD13, CD45RA, CD38, HLA-DR, CD61 and CD41. Subsets of CD34+ cells expressing CD33, CD13, or CD45RA represent committed myeloid progenitors. In contrast to bone marrow CD34+ cells, few mobilized CD34+ cells expressed CD71, CD7, CD19 or CD10. Prompt engraftment of granulocytes greater than 500 x 10(6)/l at a median of 13 days and platelets greater than 50 x 10(9)/l at a median of 15 days was observed in patients reconstituted with mobilized cells. These data indicate that CD34+ cells mobilized during recovery from chemotherapy are predominantly myeloid in phenotype and contain few actively proliferating cells or cells with lymphoid phenotypes.     

The chemokine GRObeta mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GRObeta (GRObeta(Delta4)/CXCL2(Delta4)) or the combination of GRObeta(Delta4) plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF-mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GRObeta-mobilized grafts. PBSCs mobilized by GRObeta(Delta4) alone or with G-CSF contained significantly more Sca-1(+)-c-kit(+)-lineage(-) (SKL) cells and more primitive CD34(-)-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GRObeta(Delta4)-mobilized SKL cells adhered better to VCAM-1(+) endothelial cells compared with G-CSF-mobilized cells. GRObeta(Delta4)-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1alpha in vitro that was associated with higher CD26 expression. However, GRObeta(Delta4)-mobilized SKL and c-Kit(+) lineage(-) (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GRObeta(Delta4)-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GRObeta(Delta4)-mobilized cells is less dependent on the SDF-1alpha/CXCR4 axis.     

Etoposide (VP-16) plus G-CSF mobilizes different dendritic cell subsets than does G-CSF alone

Dendritic cells (DCs) are antigen-presenting cells that are critical to the generation of immunologic tumor responses. Myeloid DCs (DC1) express myeloid antigen CD11c; lymphoid DCs (DC2) express CD123(+) and are CD11c(-). Analysis of DC subsets from peripheral blood progenitor cells (PBPC) collected from normal donors mobilized with G-CSF shows a predominance of DC2 cells. Whether PBPCs mobilization by chemotherapy yields different subsets of DCs has not been studied. We analyzed DC subsets in apheresis products from 44 patients undergoing autologous stem cell transplantation from 6/00 to 5/01. Patients received either G-CSF alone (10 microg/kg per day, n=11) or etoposide (2 g/m(2)) plus G-CSF (n=33) for progenitor cell mobilization. The patients were apheresed for 2-10 days (median 3) to reach a minimum of 2.0 x 10(6) CD34(+) cells/kg. Patients receiving G-CSF alone mobilized significantly more total DC2s than did those receiving etoposide plus G-CSF (median 6.2 x 10(6)/kg vs 2.9 x 10(6)/kg, P=0.001). The DC2/DC1 ratio was also significantly different in the two groups, with the G-CSF group having a higher ratio (median 1.2 vs 0.4, P<0.001). We conclude that the combination of chemotherapy plus G-CSF yields different mobilized dendritic cell subsets than does G-CSF alone.     

Reduced mobilization of CD34+ stem cells in advanced human immunodeficiency virus type 1 disease

Granulocyte colony-stimulating factor (r-met Hu G-CSF; filgrastim; 10 microgram/kg/day for 7 days) was used to mobilize CD34+stem cells into the peripheral blood of human immunodeficiency virus type 1 (HIV-1)-infected individuals and a group of HIV-1-uninfected donors as a measure of immunologic reserve in HIV-1-infected people. G-CSF mobilized CD34+ cells of HIV-1-infected individuals with cell counts >500 CD4+ cells/mm3, as well as in HIV-1-uninfected donors. In contrast, CD34 cell mobilization was significantly blunted in HIV-1-infected individuals with cell counts <500 CD4+ cells/mm3 (<200 cell days vs. >650 cell days, P<.0005, compared with the >500 CD4+ cell cohort). At least 1.75x10(7) CD34 cells were harvested by leukapheresis from patients in each study cohort. CD34+ cell viability and the ability to differentiate precursor cells into myeloid and erythroid progenitor cells were not affected by HIV-1 infection.     

A randomized comparison of once versus twice daily recombinant human granulocyte colony-stimulating factor (filgrastim) for stem cell mobilization in healthy donors for allogeneic transplantation

To evaluate the schedule dependency of granulocyte colony-stimulating factor (G-CSF) (filgrastim) for stem cell mobilization, we conducted a randomized comparison in 50 healthy donors, with one subcutaneous daily injection of 10 microg/kg G-CSF (n = 25) compared with twice injections daily of 5 microg/kg G-CSF (n = 25). The two groups were well balanced for age, body weight and sex. G-CSF application was performed on an out-patient basis and leukapheresis was started in all donors on day 5. The most frequent side-effects of G-CSF were mild to moderate bone pain (88%), mild headache (72%), mild fatigue (48-60%) and nausea (8%) without differences between the two groups. The CD34(+) cell count in the first apheresis was 5.4 x 10(6)/kg donor weight (range 2.8-13.3) in the 2 x 5 microg/kg group compared with 4.0 x 10(6)/kg (range 0.4-8.8) in the 1 x 10 microg/kg group (P = 0.007). The target of collecting > 3.0 x 10(6) CD34(+) cells/kg donor weight with one apheresis procedure was achieved in 24/25 (96%) donors in the 2 x 5 microg/kg group and in 17/25 (68%) donors in the 1 x 10 microg/kg group. The target of collecting > 5.0 x 10(6) CD34(+) cells/kg in the first apheresis was achieved in 64% in the 2 x 5 microg/kg group, but in only 36% in the 1 x 10 microg/kg group. The progenitor cell assay for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) was higher in the 2 x 5 microg/kg group than in the 1 x 10 microg/kg group (7.0 vs. 3.5 x 10(5)/kg, P = 0.01; 6.6 vs. 5.0 x 10(5)/kg; P = 0.1). Administering G-CSF (filgrastim) at a dosage of 5 microg/kg twice daily rather than 10 microg/kg once daily is recommended; this leads to a higher CD34(+) cell yield and requires fewer apheresis procedures without increasing toxicity or cost.     

The VAD chemotherapy regimen plus a G-CSF dose of 10 microg/kg is as effective and less toxic than high-dose cyclophosphamide plus a G-CSF dose of 5 microg/kg for progenitor cell mobilization: results from a monocentric study of 82 patients

A study was conducted to compare the efficiency and toxicity of two peripheral blood stem cell (PBSC) mobilization procedures for newly diagnosed patients with multiple myeloma. Patients from group 1 (n=51) were treated by high-dose cyclophosphamide (HD-CY) plus G-CSF (5 microg/kg/day), and the second group (n=31) by VAD regimen plus G-CSF administration (10 microg/kg/day). Successful mobilization, defined by a minimal count of 2.5 x 10(6) CD34(+) cells/kg collected, was achieved in 96 and 90% of patients in groups 1 and 2, respectively (P=0.15). The mean peripheral blood CD34(+) cells concentration and the mean CD34(+) cells/kg collected were higher in group 2 than in the group 1 (P=0.05). The mean number of leukaphereses necessary to collect a count of 2.5 x 10(6) CD34(+) cells/kg was reduced in group 2 compared to group 1. Adverse events, blood products consumption and time spent in the hospital were significantly greater after HD-CY. In conclusion, VAD plus a G-CSF dose of 10 microg/kg administration seems preferential to HD-CY plus a G-CSF dose of 5 microg/kg for PBSC collection because of equivalent or better efficiency in stem cell mobilization, strong favorable toxicity profile and reduced cost.