Number Of Colony-forming Cells Research Articles

Recovery of hematopoietic activity in bone marrow from human immunodeficiency virus type 1-infected patients during highly active antiretroviral therapy.

The mechanisms responsible for the hematopoietic failure in human immunodeficiency virus type 1 (HIV-1)-infected patients are still unknown. Several findings indicate that the in vitro proliferative potential of precursor cells from AIDS patients is reduced. The changes seen in bone marrow (BM) morphology and the defective BM functions associated with cytopenias have both been proposed as potential explanations. In patients treated with highly active antiretroviral therapy (HAART) an immune reconstitution associated with increased whole blood cell counts has been described. We have investigated the effects of HAART on the number of colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs), using long-term BM cell cultures (LTBMC) in a group of subjects with HIV-1 infection enrolled in an open study to evaluate the mechanisms of immune reconstitution during HAART. In each patient, the increase in colony growth was homogeneous, regardless of the type of hematopoietic progenitor cells assayed; in four subjects an increase in the most primitive progenitor cells (LTC-ICs) was observed. These findings were associated with the in vivo data showing increased numbers of BM mononuclear cells (BMMCs) after HAART and with a rise in peripheral CD4(+) T cell counts and decreased levels of plasma HIV-1 RNA. A decreased number of hematopoietic progenitor cells and/or a defective modulation of progenitor cell growth might be the cause of the hematological abnormalities in AIDS patients. Controlling HIV-1 replication by HAART could determine a restoration of stem cell activity, probably because of the suppression of factors that inhibit normal hematopoiesis.

The NUP98-HOXD13 fusion gene implicated in human leukemia induces myeloproliferation and blocks early b cell development in murine hematopoiesis

The nucleoporin gene NUP98 (11p15) has been reported to be fused to a number of genes including the homeobox HOX genes A9 and D13 in therapy-related AML. To investigate the role and effects that the fusion gene may have on hematopoiesis and in the development of leukemia, we used the MSCV retrovirus (GFP as a selectable marker) to express the NUP98-HOXD13 cDNA in murine bone marrow (BM). NUP98-HOXD13 (GFP+) transduced BM cells had a 1.8 fold increase (n = 5) in clonogenic colony forming cell numbers in vitro, compared to control GFP+ BM cells. Moreover, they yielded a 20 fold increase in day 12 CFU-S following 1 week in liquid culture. To assess the effect on hematopoiesis in vivo, mice were transplanted (TX) with transduced BM cells (n = 9). BM biopsy and peripheral blood analysis (PB) 12 weeks post-TX revealed a reduced level of circulating B220+ B cells (0.72 vs 2.47 × 106/ml) and a 25 fold reduction in B cell clonogenic progenitors in the BM. Two NUP98-HOXD13 mice sacrificed 18 weeks post-TX for further analysis manifested varying degrees of increased myelopoiesis. One mouse had clear signs of early myeloproliferative syndrome (MPS), including a marked increase in splenic CFC-GM (65 fold) and Gr-1+ cells (4 fold). The myeloproliferation was transplantable and progressive with 2 of 5 secondary mice becoming moribund by 10 weeks post-TX and MPS, as shown by increased PB counts, splenomegaly and myeloid cell infiltration in multiple organs. These results provide direct evidence of the potential of NUP98-HOXD13 to grossly perturb hematopoiesis.

Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor

Objective One of the mechanisms for mobilization of hematopoietic stem cells and progenitor cells is alternation of adhesion molecules. We investigated the mobilization of hematopoietic progenitor cells in blood by administration of anti-vascular cell adhesion molecule (VCAM)-1 antibody (Ab) in mice. Materials and Methods Twelve- to 14-week-old C57BL/6J mice were injected intravenously with anti-VCAM-1 Ab and anti-very late antigen (VLA)-4 Ab at a dose of 5 mg/kg for 2 days. Results The number of colony-forming cells (CFCs) in blood was increased 11.4-fold after anti-VCAM-1 Ab treatment, but the number of CFCs was not increased after treatment with anti-VLA-4 Ab. The number of colony-forming unit spleen (CFU-S) also was increased 21.6-fold in the peripheral blood by administration of anti-VCAM-1 Ab. The number of CFCs and CFU-S in the bone marrow of mice treated with anti-VCAM-1 Ab was decreased and that in the spleen also was decreased. On administration of recombinant human granulocyte colony-stimulating factor (125 μg/kg twice daily) with anti-VCAM-1 Ab, the numbers of CFCs and CFU-S were increased 141.8-fold and 439-fold, respectively. Conclusion These observations demonstrated that administration of anti-VCAM-1 Ab induced mobilization of hematopoietic progenitor cells into blood from bone marrow and spleen and that granulocyte colony-stimulating factor has synergistic effects on anti-VCAM-1 Ab-induced mobilization.

Volume-dependent collection of peripheral blood progenitor cells during large-volume leukapheresis for patients with solid tumours and haematological malignancies.

We investigated the efficacy of peripheral blood progenitor cell (PBPC) collection during large-volume leukapheresis (LVL) in patients with solid tumours and haematological malignancies (n = 18). The time- and volume-dependent harvest of leucocytes (WBC), mononuclear cells (MNC), CD34+ cells and colony-forming cells (CFU-GM) during LVL was analysed in six sequentially filled collection bags processing four times the patient's blood volumes. The amounts of leucocytes (WBC) and the purity of mononuclear cells (MNC%) did not show any significant changes during LVL. The percentage of CD34+ cells remained constant for the first three bags but consecutively decreased from initially 1.71% CD34+ cells in the beginning of LVL to finally 1.34% CD34+ cells (P = 0.02). The mean numbers of colony-forming cells (CFU-GM) decreased from 74 microL-1 to 59 microL-1 during LVL (P = 0.16). Furthermore, the comparison of volume-dependent PBPC collection for patients with high, medium and low total yields of CD34+ cells showed similar kinetics on different levels for the three groups. We concluded that - relative to the initial total amount of PBPC harvested - comparable numbers of progenitor cells can be collected during all stages of LVL with a slight decreasing trend processing four times the patient's blood volumes.

Administration of Flk2/Flt3 ligand induces expansion of human high-proliferative potential colony-forming cells in the SCID-hu mouse

The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells. Furthermore, higher percentages of CD33 + CD15 − cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies.

Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

AbstractHematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

In Vitro Hematopoietic and Endothelial Cell Development From Cells Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

In Vitro Hematopoietic and Endothelial Cell Development From Cells Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region

AbstractRecent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

Administration of daniplestim and granulocyte colony-stimulating factor for the mobilization of hematopoietic progenitor cells in nonhuman primates.

We investigated the effects of administering a high-affinity interleukin-3 receptor agonist (daniplestim) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of primitive hematopoietic progenitor cells into peripheral blood (PB). Groups of five rhesus monkeys were treated with 100 mg/kg of daniplestim for 5 days followed by 10 microg/kg of G-CSF for 5 days (D/G), daniplestim and G-CSF administered concurrently for 10 days (D+G), or G-CSF alone for 10 days. Phenotypic PB analysis indicated that the number of CD34+ cells in the G-CSF group had increased to 28 x 10(6)/L by Day 3 and then declined. In contrast, CD34+ cell counts of up to 68 x 10(6)/L were maintained until Day 10 in both the D/G and D+G groups. On Day 5, the total number of colony-forming cells in the PB had increased 15-fold in the D+G group and eightfold in both the D/G group and the G-CSF groups. By Day 7, the numbers of colony-forming units granulocyte/macrophage were comparable in all three groups, and 45-fold increases in the numbers of burst-forming units-erythroid and 12-fold increases in the numbers of multipotent colony-forming units were seen in both the D+G and the D/G groups. The frequency of circulating primitive progenitor cells in long-term stromal cultures was highest with D+G and lowest with G-CSF alone. These results indicate that the combination of daniplestim and G-CSF produces higher and more sustained levels of circulating stem cells than does G-CSF alone. D+G may offer advantages over D/G because it generates more long- and short-term clonogenic cells.

Osteoclast-Mediated Bone Resorption Is Stimulated During Short-Term Administration of Granulocyte Colony-Stimulating Factor But Is Not Responsible for Hematopoietic Progenitor Cell Mobilization

AbstractThe cellular and molecular mechanisms responsible for hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) into peripheral blood after administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) are still unknown. In this study we show that high concentrations of soluble calcium induce the detachment of BM CD34+ HPC adherent on fibronectin, a major component of BM extracellular matrix. Because G-CSF has been shown to induce osteoporosis in patients with congenital neutropenia and in G-CSF–overexpressing transgenic mice, we hypothesized that short-term G-CSF administration may be sufficient to induce bone resorption, resulting in the release of soluble calcium in the endosteum leading in turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced after 3 days of G-CSF administration. Furthermore, in patients mobilized with G-CSF either alone or in association with stem cell factor or interleukin-3, the reduction of serum osteocalcin is significantly correlated with the number of HPC mobilized in peripheral blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of bone resorption, gradually elevated during the time course of G-CSF administration until day 7 after cessation of G-CSF, showing a simultaneous stimulation of bone degradation during G-CSF–induced HPC mobilization. In an in vivo murine model, we found that the number of osteoclasts was dramatically increased paralleling the elevation of DPyr after G-CSF administration. When pamidronate, an inhibitor of osteoclast-mediated bone resorption, was administered together with G-CSF in mice, the G-CSF–induced increase of DPyr levels was completely abolished whereas the numbers of colony-forming cells mobilized in peripheral blood were not decreased, but unexpectedly increased relative to the numbers elicited by G-CSF alone. Collectively, our data therefore show that short-term administration of G-CSF induces bone degradation by a simultaneous inhibition of bone formation and an enhanced osteoclast-mediated bone resorption. This increased bone resorption is inhibited by pamidronate without reducing G-CSF–induced HPC mobilization, suggesting that the activation of bone resorption after G-CSF administration is not the direct cause of HPC mobilization as initially hypothesized, but a parallel event.© 1998 by The American Society of Hematology.

Osteoclast-Mediated Bone Resorption Is Stimulated During Short-Term Administration of Granulocyte Colony-Stimulating Factor But Is Not Responsible for Hematopoietic Progenitor Cell Mobilization

The cellular and molecular mechanisms responsible for hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) into peripheral blood after administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) are still unknown. In this study we show that high concentrations of soluble calcium induce the detachment of BM CD34+ HPC adherent on fibronectin, a major component of BM extracellular matrix. Because G-CSF has been shown to induce osteoporosis in patients with congenital neutropenia and in G-CSF–overexpressing transgenic mice, we hypothesized that short-term G-CSF administration may be sufficient to induce bone resorption, resulting in the release of soluble calcium in the endosteum leading in turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced after 3 days of G-CSF administration. Furthermore, in patients mobilized with G-CSF either alone or in association with stem cell factor or interleukin-3, the reduction of serum osteocalcin is significantly correlated with the number of HPC mobilized in peripheral blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of bone resorption, gradually elevated during the time course of G-CSF administration until day 7 after cessation of G-CSF, showing a simultaneous stimulation of bone degradation during G-CSF–induced HPC mobilization. In an in vivo murine model, we found that the number of osteoclasts was dramatically increased paralleling the elevation of DPyr after G-CSF administration. When pamidronate, an inhibitor of osteoclast-mediated bone resorption, was administered together with G-CSF in mice, the G-CSF–induced increase of DPyr levels was completely abolished whereas the numbers of colony-forming cells mobilized in peripheral blood were not decreased, but unexpectedly increased relative to the numbers elicited by G-CSF alone. Collectively, our data therefore show that short-term administration of G-CSF induces bone degradation by a simultaneous inhibition of bone formation and an enhanced osteoclast-mediated bone resorption. This increased bone resorption is inhibited by pamidronate without reducing G-CSF–induced HPC mobilization, suggesting that the activation of bone resorption after G-CSF administration is not the direct cause of HPC mobilization as initially hypothesized, but a parallel event.© 1998 by The American Society of Hematology.

Ontogeny-associated changes in the cytokine responses of primitive human haemopoietic cells.

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34+ CD38- cord blood (CB) cells as was obtained in similar cultures of adult human CD34+ CD38- bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34+ CD38- CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.

Overgrowth of a leukemic culture by a minor CD34+ population

We have investigated the differentiation potential of blast cells in a case of acute myeloid leukemia which comprised a majority CD34− population and a minor (2%) CD34+ fraction. Blasts were cultured for 2 weeks in a combination of cytokines—c-Kit ligand, interleukin 3 and granulocyte–macrophage colony-stimulating factor (SIGm mix)—together with all- trans retinoic acid or 1 α,25-dihydroxy vitamin D 3. Maturation of blasts was assessed by morphology on Romanowsky-stained slides, changes in surface CD markers and clonogenic culture. After 7 days of culture of unseparated blasts in SIGm, most maturation was monocytic, but with retinoic acid 63% of blasts had matured into granulocytes. Vitamin D 3 enhanced monocytic differentiation, with 60% of cells becoming monocytic. The percentage of CD14 and CD15 positive cells decreased over 7 days in SIGm (from 62% to 17% and from 76% to 39% for CD14 and CD15, respectively). CD14+ cell numbers were maintained, or recovered, in cultures supplemented with vitamin D 3 (59% at day 7), and CD15+ cell numbers, too, remained unchanged in the presence of retinoic acid (67%) or vitamin D 3 (66%). Aberrant markers CD7 and CD56 declined under any conditions. When separated, both the CD34− and CD34+ fractions showed similar changes in morphology and surface maturation markers, suggesting that these two populations may be closely related. However, only a few CD34+ cells expressed the aberrant markers present on the majority blast population. The CD34− population declined in culture while the CD34+ fraction rapidly expanded. This probably reflects the difference in progenitor content; high numbers of colony-forming cells were concentrated in the CD34+ subpopulation. We conclude that both CD34− and CD34+ populations can differentiate but only the CD34+ fraction proliferates. Primitive clonogenic CD34+ cells from this patient may generate occasional aberrant CD34+ blasts which could then differentiate into the accumulating aberrant CD34− blast population.

FLT-3 ligand mobilizes hematopoietic primitive and committed progenitor cells into blood in mice.

We investigated the effects of the administration of FLT-3 ligand (FL) on mobilization of primitive and committed progenitor cells in mice. C57bl/6J mice were injected subcutaneously with FL once a day for 5 d at doses of 20, 100 and 200 microg/kg. After the collection of peripheral blood, we determined the number of white blood cells (WBCs) with the differential counts. The formation of colony-forming cells (CFCs) in peripheral blood, bone marrow and spleen was evaluated. Although the administration of FL, 20 microg/kg, did not stimulate leukocytosis, its administration at doses of 100 and 200 microg/kg increased the number of WBC up to 1.7- and 2.4-fold, respectively. Committed progenitor cells were mobilized into the peripheral blood dose-dependently and the number of CFCs was increased up to 5.5-fold by the administration of FL at 200 microg/kg on d 5. The number of CFCs in the bone marrow increased, but not dose-dependently. The number of CFCs in the spleen also increased up to 32-fold at a dose of 200 microg/kg FL. Mobilized peripheral blood mononuclear cells were transplanted into lethally irradiated mice and the number of CFU-S (d 12) was scored. A dose-dependent mobilization of CFU-S (d 12) into peripheral blood was also observed. These observations suggest that FL can mobilize hematopoietic primitive and committed progenitor cells into the peripheral blood of mice and those cells mobilized by FL may be applicable to peripheral blood stem cell transplantation.

Ciprofloxacin immunomodulation of experimental antiphospholipid syndrome associated with elevation of interleukin‐3 and granulocyte‐macrophage colony‐stimulating factor expression

Objective To evaluate the immunomodulatory potential of ciprofloxacin in mice with experimental antiphospholipid syndrome (APS). Methods Ciprofloxacin or ceftazidime (control antibiotic) was given to mice with experimentally induced APS. The titers of autoantibodies, levels of cytokines, and number of cytokine-producing cells were determined by enzyme-linked immunosorbent assay. Myeloid progenitor cells were determined by granulocyte-macrophage colony-forming unit, and interleukin-3 (IL-3) messenger RNA (mRNA) was tested by Northern analysis. Results A decrease in the incidence of pregnancy loss and an improvement in the clinical manifestations of APS were noted in the mice treated with ciprofloxacin, compared with the mice given ceftazidime. The effect of ciprofloxacin was found to be associated with increased serum levels of IL-3 and with increased IL-3 mRNA transcription in the splenocytes. Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was documented by elevated titers in the sera and elevated numbers of colony-forming cells in the bone marrow. Conclusion Ciprofloxacin prevents the manifestations of experimental APS. This effect may be associated with increased IL-3 levels and GM-CSF expression.

Ciprofloxacin immunomodulation of experimental antiphospholipid syndrome associated with elevation of interleukin-3 and granulocyte-macrophage colony-stimulating factor expression.

To evaluate the immunomodulatory potential of ciprofloxacin in mice with experimental antiphospholipid syndrome (APS). Ciprofloxacin or ceftazidime (control antibiotic) was given to mice with experimentally induced APS. The titers of autoantibodies, levels of cytokines, and number of cytokine-producing cells were determined by enzyme-linked immunosorbent assay. Myeloid progenitor cells were determined by granulocyte-macrophage colony-forming unit, and interleukin-3 (IL-3) messenger RNA (mRNA) was tested by Northern analysis. A decrease in the incidence of pregnancy loss and an improvement in the clinical manifestations of APS were noted in the mice treated with ciprofloxacin, compared with the mice given ceftazidime. The effect of ciprofloxacin was found to be associated with increased serum levels of IL-3 and with increased IL-3 mRNA transcription in the splenocytes. Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was documented by elevated titers in the sera and elevated numbers of colony-forming cells in the bone marrow. Ciprofloxacin prevents the manifestations of experimental APS. This effect may be associated with increased IL-3 levels and GM-CSF expression.

凍結さい帯血細胞の洗浄による細胞回収率の検討

We investigated the effect of the removal of cryoprotectant after thawing of cryopreserved cord blood cells on their recovery rates. Cord blood was obtained at normal delivery.Removal of red blood cells and the concentration of white blood cells in cord blood were performed by use of hydroxyethyl starch. As cryoprotectants, dimethyl sulfoxide and dextran were added at the final concentration of 10% and 1%, respectively. After 3-6 months preservation in liquid nitrogen, cord blood units were thawed, and an equal volume of dextran/albumin solution was added as washing medium. After centrifugation, the cell pellet was resuspended in washing medium. Recovery rates of nucleated cells (NC) and colony-forming cells (CFC) after the washing procedure were 97.0±9.7% and 103.8±20.1%, respectively.Potassium and free hemoglobin were removed at 90.3% and 81.3%. Residual numbers of NC and CFC after the suspensions at room temperature for 2hr remained at 90.6±12.5% and 90.6±12.3%, respectively. These results indicate that the removal of cryoprotectant is effective in cord blood transplantation.

Both cycling and noncycling primitive progenitors continue to be mobilized into the circulation during the leukapheresis of patients pretreated with chemotherapy and G-CSF.

Colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) include a spectrum of progenitor types whose potential contributions to the haemopoietic recovery seen in patients transplanted with mobilized peripheral blood progenitor cells (PBPC) remains unclear. We evaluated both the number and cycling status of the circulating LTC-IC and CFC harvested from 12 patients treated with chemotherapy and G-CSF using a modified 6-week LTC-IC assay. The frequency of the LTC-IC and CFC in the mobilized PB samples were increased 45- and 750-fold, respectively. Interestingly, comparison of these values for PB samples, taken just prior to the start of the leukapheresis, with the progenitor content of the 3 h harvest, showed that, on average, the leukapheresis product contained 19 times more LTC-IC (P < 0.01) than had been detectable in the entire blood volume of the patients at the start of the collection, whereas the number of CFC collected was approximately the same as the number in the initial circulating pool of PBPC. Cycling studies showed many of the LTC-IC in the apheresis collections to be proliferating although not more so than in the steady-state marrow LTC-IC compartment (i.e. per cent kill of mobilized LTC-IC after 16 h in 3H-Tdr = 70 +/- 8%, n = 9). On the other hand, the majority of the CFC in the apheresis collections were initially quiescent (per cent kill after 16 h in 3H-Tdr = 37 +/- 6%, n = 12). These findings demonstrate the rapidity with which a primitive subset of LTC-IC may enter the circulation during the early phase of rebound haemopoiesis induced by chemotherapy plus G-CSF and provide evidence of differences in the mechanisms regulating LTC-IC and CFC mobilization.

Detection and Characterization of Primitive Malignant and Normal Progenitors in Patients With Acute Myelogenous Leukemia Using Long-Term Coculture With Supportive Feeder Layers and Cytokines

Analysis of the mitogenic activity of interleukin-3 (IL-3), Steel factor (SF ), and flt-3 ligand (FL) on acute myelogenous leukemia (AML) blasts using the short-term endpoints of proliferation in 3H-thymidine (3H-Tdr) incorporation assays or methylcellulose cultures (colony assays) showed that greater than 90% of samples contained cells that were responsive to one or more of these cytokines. With this information, culture conditions that were known to support normal long-term culture-initiating cells (LTC-IC) were tested, with or without supplements of one or more of these three growth factors, for their ability to support primitive progenitors from 10 cell samples from patients with AML. In all cases cytogenetically abnormal colony forming cells (CFC) were detected after 5 weeks when AML peripheral blood or marrow cells were cocultured on preestablished, normal human marrow feeders (HMF ) and/or Sl/Sl mouse fibroblast feeders and the number of CFC detected in these 5-week-old LTC maintained a linear relationship to the number of input AML cells. Limiting dilution analysis, performed on 6 of the 10 samples, showed the frequency of AML cells initiating LTC (AML LTC-IC) to be 5- to 300-fold lower than the frequency of AML-CFC in the same cell sample, whereas the average number of CFC produced per LTC-IC varied from 1 to 13. Surprisingly, in each case the concentration of cytogenetically normal LTC-IC detected in AML patient blood was at least 10-fold higher than that previously observed in the blood of normal individuals. “Mixed” mouse fibroblast feeders engineered to produce human G-CSF, IL-3, and SF did not enhance detection of AML LTC-IC but did increase the output of cytogenetically normal CFC from LTC of 3 of 4 patient samples. Supplementation of AML LTC with IL-3 and exogenously provided SF and/or FL increased the output of AML-CFC from 5-week-old LTC by greater than or equal to twofold with 5 of 9 patient samples, whereas in one case exogenous addition of FL reduced the output of malignant CFC from LTC. These studies show that conditions that support normal LTC-IC also allow a functionally analogous but rare AML progenitor cell type to be detected. In addition, differences in the responses of normal and leukemic cells to various cytokines active on normal LTC-IC were revealed. Further analysis of these differences may enhance our understanding of leukemogenesis and lead to observations that could be exploited therapeutically.