Burst-forming Unit-erythrocyte Research Articles

Parvovirus B19 Infection in Human Bone Marrow Mesenchymal Stem Cells Affects Gene Expression of IL-6 and TNF-α and also Affects Hematopoietic Stem Cells Differentiation.

Parvovirus B19 (B19V) has been known to induce transient erythroid aplasia, cytopenia and aplastic anemia. This virus persists in bone marrow mesenchymal stem cells (HBMSCs) of some immunocompetent individuals several years after primary infection. In B19V infected erythroid progenitor cells, the virus induces transactivation of Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) gene expression. Due the critical role of HBMSCs in bone marrow niche and inhibitory effect of inflammatory cytokines on hematopoiesis, the aim of this study was to investigate the effect of B19V on IL-6 and TNF-α gene expression intransfected cells. In addition we assessed the clonogenicity potential of cord blood CD34+ stem cells that were co-cultured with infected cells. After 24 h of transfection, quantitative mRNA expression of IL-6 and TNF-α was evaluated and human cord blood CD34+ HSC were cultured with the transfected cells. At the end of 7days of culture, HSCs colony forming units (CFUs) assay was performed. Our findings demonstrated statistically significant (18.1 and 21.9 fold) increase of TNF-α and IL-6 gene expression respectively and decrease in burst forming unit-erythrocyte (BFU-E) and colony forming unit-erythrocyte (CFU-E) enumeration(p < 0.05). We concluded that, inducing inflammatory cytokines gene expression in B19V-infected HBMSCs might influence on bone marrow microenvironment and hematopoiesis.

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury.

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.

Effects of Angelica polysaccharide on erythrocyte immunity and marrow hematopoiesis in chickens

To investigate the effects of Angelica polysaccharide on erythrocyte immunity and marrow hematopoiesis in chickens, Angelica polysaccharide was used for the study of immunity and hematinic mechanism in order to provide basis for clinical reference. In this experiment, three gradient dosages (50, 100, 150 mg/kg body weight) of Angelica polysaccharide were drenched to the control group and the anemia groups, respectively. The anemia chickling model was made by abdominal injection of cyclophosphamide (CY) for 6 days (80 mg/kg·day).Red blood cell-C3b receptor (RBC-CR1) and red blood cell-immune complex (RBC-IC) rosette rates were measured and analyzed. Ectogenetic semi-solid culture medium of bone marrow hemopoietic progenitor cells was used to observe Angelica polysaccharide and separated serum fromAngelica polysaccharide treatment on the proliferation of colony-forming unit-erythrocyte (CFU-E), burst-forming unit-erythrocyte (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM). The results showed that Angelica polysaccharide can significantly increase RBC-CR1 rosette rate in the healthy chicken groups (p 0.05). At the same time, Angelica polysaccharide can restore the RBC- CR1 rosette rate and the RBC-IC rosette rate caused by cyclophosphamide to the normal level. Serum containing Angelica polysaccharide can significantly facilitate the proliferation on CFU-E (p<0.01), BFU-E (p<0.01) and CFU-GM (p<0.01), but Angelica polysaccharide had no more direct proliferation on CFU-E, BFU-E and CFU-GM. This indicated that Angelica polysaccharide had the proliferation on hemopoietic progenitor cells of marrow by the change of hemopoietic factor. Key words: Polysaccharide, chicken, erythrocyte, immunity, hematopoiesis.

The Neuropeptides Orexin a and B Have An Impact on Functional Properties of Human CD34+ Stem and Progenitor Cells.

Orexin receptors play a role in regulation of sleep-wake-rhythm, food intake and energy homeostasis and they were long thought to be exclusively expressed in the nervous system. During the last years orexin receptors are being identified in a growing number of peripheral tissues. We have earlier detected orexin receptor 1 and 2 expression on human CD34+ blood stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seem to be restricted to the central nerve system to this date. The main downstream signaling pathways of the orexin receptors include Ca2+-dependent signaling associated with activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways. In an attempt to investigate if the receptors are functionally active in CD34+ stem and progenitor cells, we used live cell calcium imaging and stimulated purified CD34+ stem and progenitor cells with orexin A and B. Upon stimulation a massive intracellular calcium release was seen which could not been detected using cells preincubated with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the selective OX1R-Antagonist SB334867 and CD34 negative cells. Additionally, upon stimulation with orexin A and B we found ERK (1/2) activation at a maximum 3 hours following incubation with orexin A whereas no effect was seen after stimulation with orexin B. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. So far, no effects of orexin stimulation on the proliferation and apoptosis of CD34+ cells were apparent. Remarkably, stimulation with orexin A and B led to a significantly higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors BFU-E (burst forming unit erythrocyte) and CFU-E (colony forming unit erythrocyte). A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significantly higher frequency of LTC-IC (long-term-culture initiating cells) indicating also a more immature phenotype of orexin-stimulated cells and a greater repopulating capacity. The selective orexin receptor antagonist SB-334867 abrogated these effects. No differences could be observed regarding the migration towards SDF-1 with and without stimulation with orexin A and B. Still, orexin A and B led to a decrease in the adhesive capacity of CD34+ stem and progenitor cells to fibronectin coated dishes. Since orexin receptors are coupled to inhibitory G-proteins (Gi/q) and stimulatory G-proteins (Gs) dependent on the tissue, we incubated CD34+ cells with the selective inhibitor of Gi – proteins pertussis toxin concurrently to stimulation with orexins and observed no differences in the adhesive capacity of CD34+ cells compared to the unstimulated controls suggesting coupling of the orexin receptor 1 and 2 to Gi – proteins rather than Gs-proteins in CD34+ cells. Given this functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using ELISA we did not find autocrine production of orexin by CD34+ cells whereas orexin could be detected in the serum obtained by bone marrow biopsies and peripheral blood pointing rather towards a humoral delivery of orexins to CD34+ cells. Taken together, our findings indicate a functional role of the orexin system in CD34+ stem and progenitor cells.

Sensitivity of human cord blood cells to tetrachloroethylene: cellular and molecular endpoints

The International Agency for Research on Cancer (IARC) currently lists tetrachloroethylene [perchloroethylene (PCE)] as being carcinogenic in animals. PCE is listed as possibly carcinogenic to humans upon occupational exposure. Human exposure to PCE can produce oesophageal cancer, cervical cancer, non-Hodgkin's lymphoma, urinary bladder cancer and leukemia. This work shows that PCE modulates the expression of some genes implicated in cancer induction, cell differentiation, cell-cycle progression, and the survival and clonogenic potential of human cord blood cells. After exposure to the compound, the modulated genes were involved in inflammatory responses as with the mitogen-activated protein kinase 14 (MPK 14), or in tumor and metastasis progression as with the matrix metalloproteinase 17 (MMP 17), in cell proliferation as with c-jun and c-fos, and moreover in the apoptotic process as with interferon alpha-inducible protein (IFI), BAX and BCL-2. Analysis of cord blood cells via flow cytometry showed that PCE treatment induced a statistically significant increase in necrosis after 24 h, while the clonogenicity of Human Colony-Forming Unit-Granulocyte/Macrophage (CFU-GM) and Burst-Forming Unit-Erythrocyte (BFU-E) progenitors did not change. In conclusion, our data showed that PCE affected various pathways involved in cancer induction, but its action on cell proliferation and differentiation is not yet clearly understood.

Ex Vivo Expansion of Hematopoietic Progenitors from CD34+-Selected Cells on Mouse Embryonic Fibroblast Feeders in a Non-Contact Co-Culture Using Matrigel or Fibronectin-Coated Inserts.

Adult pluripotent stem cells can be characterized as cells with both the capacity to self-renew and the ability to differentiate into distinct hematopoietic cell lineages and also into other tissue types. Because of the important course to stem cell research and clinical application the development of ex vivo culture systems is crucial for an effective expansion of these hematopoetic progenitor cells. After positive selection, CD34+-cells from peripheral G-CSF stimulated blood of healthy donors were cultured for 2 weeks in a serum-free medium with 2% of cord blood serum and the addition of early-acting cytokines (SCF, IL-3, Flt3-ligand, LIF, and TPO). The cells were grown in 6-well inserts on a feeder layer of mouse embryonic fibroblastic cells (MEF). Both cell types were separated from each other by a microporous membrane allowing only diffusion of soluble factors like cytokines but not cell migration. Additionally, the membrane was coated with Matrigel, a basement membrane matrix equivalent (MG), or with fibronectin (FN). Cultures were sampled at days 4, 7, 10 and 14 for cell count, colony-forming units-assay and immunophenotyping by flow cytometry. After two weeks of culture, the highest proliferation of stem cells was found in the control (medium without MEF, Matrigel and FN; 364-fold) followed by MEF- and FN-cultures (341- and 306-fold, respectively). The lowest clonal production was estimated in the cultures with MG (MG alone: 124,4-fold and MEF+MG: 48,2-fold, respectively). Examining the cells for colony-forming units the highest number of colony-forming unit-granulocyte-erythrocyte-macrophage (CFU-GEM), burst-forming unit-erythrocyte (BFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM) and colony-forming unit-erythrocyte (CFU-E) were found in cultures with MEF and MEF+FN. We found multipotential colonies (CFU-GEM and BFU-E) at the highest expansion rate with FN (13,3-fold) and MEF (8,2-fold) at the fourth day of culture and with both additives in a MEF+FN-culture at the seventh day (17,2-fold). In addition to these results, there were sporadic undifferentiated progenitor cell colonies in MEF+MG-culture up to the day 14, despite an initially lower growth rate than that on MEF alone. The percentages of CD34+-cells estimated by flow cytometry were downregulated continiously during the culture period. At day 7, the highest number of CD34+-cells was found in the cultures with MG (46,0%) and MEF+MG (42,0%). The maximum number of CD34+-cells expressing no CD38 but CD61 was found at day 4 in FN culture (20,4%) and MG culture (15,9 %), respectively. In summary, we recognized in cultures with MG as a factor similar to the natural microenvironment a correlation between the diminished proliferation rate of stem cells in favor of an increased generation of multipotential colonies combined with their delayed differentiation into granulocyte-macrophage and erythrocyte colonies. Appropiate feeder cells as well as matrix proteins like Matrigel or fibronectin may helpful in improving the ex vivo expansion of hematopoietic progenitor cells.

Improvement of the precision in CFU-GM and BFU-E counting by flow cytometry-based standardization of short-term culture assays.

The counting of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units erythrocyte (BFU-E) provides substantial in vitro information about the graft quality after peripheral stem cell transplantation (PBSCT). By using different techniques for culturing and scoring, high inter- and intralaboratory coefficients of variation (CV) are frequently reported. We minimized the imprecision by using flow cytometry-based incorporation of constant numbers of CD34(+) cells per culture dish instead of the formerly used mononuclear cells. Our results show acceptable CVs for CFU-GM (12.3%) and for BFU-E (13.3%) based on this seeding technique, which contributes to fulfilling the demands of a quality assurance system in stem cell laboratories.

Autologous peripheral blood stem cells harvesting in Kaohsiung Medical University Hospital.

We report our clinical experience in autologous peripheral blood stem cells harvesting. A total of 40 patients with 112 apheresis procedures were analyzed, 88 with Cobe system and 24 with MCS3P system. Our results revealed that there was no significant difference in efficiency of CD34(+) cell harvesting between both apheresis systems, but the Cobe system had more nucleated cells collected and less red cell contaminated in the final PBSCs collections. The percentage of CD34(+) cells collected decreased significantly following the first day's harvesting (p = 0.026). There was a good correlation between the percentage of CD34(+) cells in PBSCs and colony forming units-granulocyte macrophage (CFU-GM) or burst forming unit-erythrocyte (BFU-E)(r = 0.909, p < 0.0001; r = 0.788, p < 0.0001, respectively). However, it was negatively correlated with the patient's age. The CD34(+) cells collected in patients with acute leukemias were also higher than those patients with other solid tumors. Ten cases (13%) with 15 apheresis procedures experienced side effects like numbness, nausea, fever, or headache etc. The Cobe system seemed to have higher frequency of side effects than that of MCS3P system (16% vs 4%). From our results, we concluded that both COBE and MCS3P system have similar efficiency and all patients could tolerate the apheresis procedures in peripheral blood stem cells harvesting. The CD34(+) cell can be used as a good parameter to estimate the amount of stem cells collected. The patient's age and disease pattern were significant factors influencing the CD34(+) stem cells collection in autologous peripheral blood stem cells harvesting.

CFU-Mk content of immunoselected CD34+ peripheral blood progenitor cells, evaluated with an adapted serum-free methylcellulose assay, is predictive of platelet lineage reconstitution in children with solid tumors.

Immunoselected CD34+ peripheral blood progenitor cell (PBPC) transplantation is now frequently used to support autologous hematopoiesis after myeloablative therapy, its feasability having been proved by several groups. However, we and others observed delayed platelet recovery. We hypothesized that immunoselection processing might induce selective loss of megakaryocyte progenitors, or a decrease in their proliferation. We used a colony-forming units megakaryocyte (CFU-Mk) assay to evaluate these consequences and predict platelet recovery in patients. In CD34+ PBPCs from 10 children with solid tumors, we observed no selective loss in CFU-Mk numbers during immunoselection processing and no impairment of clonogenicity. The CFU-Mk yield (59.2 +/- 11.3%) was at least similar to the CD34+ yield (44.2 +/- 3.8%). We assessed the predictive value of CFU-Mk numbers infused for recovery of platelet lineage. We found an inverse correlation between the time taken to reach a platelet count greater than 50 x 10(9)/L and only the CFU-Mk dose (r = -0.71; p = 0.022) among the different type of progenitors, including colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythrocyte (BFU-E) and colony-forming units-mixed (CFU-Mix). These findings suggest that CFU-Mk number could be used as sole predictive functional parameter for platelet reconstitution in children after immunoselection of CD34+ cells, in particular for low CD34+ cell dose, and thus as an indicator for initial quality of hematopoietic cells before in vitro expansion.

HLA-DR inhibits granulocytic differentiation without inducing apoptosis of CD34 cells

Hematopoietic progenitors express HLA-DR molecules. However the significance of HLA-class II molecules on CD34+ cells remains unknown. The primary role of HLA-class-II molecules is antigen presentation although a second role, that of signal transduction, has been established in B cells. The role of HLA-DR in hematopoiesis was examined by determining the ability of CD34+ progenitor cells to differentiate to “Colony Forming Unit Granulocyte-Macrophage” (CFU-GM) and “Burst Forming Unit Erythrocyte” (BFU-E) in the presence of anti-HLA-DR monoclonal antibody. We observed a reduction in the number of CFU-GM which was due in part to down regulation of granulocyte rather than monocyte differentiation. These observations suggest that HLA-DR signals can regulate myelopoiesis. We point out especially the role of the HLA-DR molecule in the switch of CFU-GM between granulocyte or monocyte lineages. Although HLA-DR mediated apoptosis has been described in mature B lymphocytes apoptosis of CD34+ cells was excluded as a mechanism.

Macrophage-Inflammatory Protein-3β/EBI1-Ligand Chemokine/CKβ-11, a CC Chemokine, Is a Chemoattractant with a Specificity for Macrophage Progenitors Among Myeloid Progenitor Cells

AbstractChemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3β/EBI1-ligand chemokine/CKβ-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3β attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3β attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3β, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3β mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-γ, and TNF-α. Taken together, our findings suggest that MIP-3β is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.

Human Hematopoiesis in SCID Mice Implanted With Human Adult Cancellous Bone

The persistence of hematopoietic cells from human adult cancellous bone fragments implanted subcutaneously into CB-17 scid/scid mice was studied. Recipient mice received either no pretreatment (control group) or pretreatment with 3 Gy total-body irradiation and anti-asialo GM1 sera (ASGM1; pretreated group) before implantation. Pretreated severe combined immunodeficient (SCID) mice implanted with human bone were subsequently given ASGM1 every 7 days for the duration of the experiments. At 12 weeks postimplantation, flow cytometry of cells from pretreated and control animal tissues detected human CD45+ cells in the mouse spleen (mean, 7.8% and 3.4% positive cells, pretreated and control animals, respectively), bone marrow (BM; mean, 16.5% and 4.8% positive cells, respectively), and blood (mean, 5.5% and < 2% positive cells, respectively), and in the implanted human bone (73% and 8.9% positive cells, respectively). At 12 weeks, pretreated mice had human granulocyte-macrophage colony-forming cells (GM-CFC) and burst-forming units-erythrocyte (BFU-E) in the implanted human bone in the murine BM and in some of the spleens. The spleens also had extensive infiltration of human B cells and macrophages. Mean serum levels of human IgG in pretreated animals were 14 micrograms/mL during weeks 6 to 12, compared with trace levels (< 1 microgram/mL) in control mice. Bone from patients with acute myeloblastic leukemia (AML) was also implanted in pretreated SCID mice, and retrieved at 8 weeks for analysis. Comparison of preimplantation and implanted samples showed that the original histology was maintained, and massive infiltration of human CD68+ cells was observed in the mice spleens and BM. Implantation of AML bone in SCID mice facilitates analysis of in situ AML cell interaction with stromal cells in the leukemic state, and therapies against AML can be tested in this system, especially the selective killing of AML cells in the presence of other BM cells. Furthermore, this model requires no exogenous administration of cytokines to maintain human hematopoiesis with both normal or AML bone. Because the structure and function of both normal and diseased human adult bone is maintained, this animal model should facilitate investigation of both normal human hematopoiesis and hematopoietic malignancies.

Gene Transfer Into Human Bone Marrow Hematopoietic Cells Mediated by Adenovirus Vectors

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli β-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, β-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-β-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38–), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.

The selective uptake of benzoporphyrin derivative mono-acid ring A results in differential cell kill of multiple myeloma cells in vitro.

High-dose chemotherapy (HDCT) and autologous bone marrow/blood stem cell transplantation are an effective combination for treating a number of malignant disorders. The contamination of the autograft by malignant cells may be a reason for recurrences in spite of this treatment, for instance, in multiple myeloma. Therefore, we evaluated the use of photodynamic therapy (PDT) using the photosensitizer benzoporphyrin derivative mono-acid ring A (BPD-MA) on multiple myeloma cells in comparison to the components of the normal bone marrow (NBM) and peripheral blood apheresis product. Flow cytometry was used to measure differential BPDMA uptake of NBM components: namely lymphocytes, monocytes, granulocytes and enriched hematopoietic stem cell (CD34+) populations and also the multiple myeloma cell lines OCI-MY7 and OCI-MY4. When each population was measured individually, the order of uptake was [OCI-MY7/MY4] > [CD34+] > [granulocytes] = [monocytes] >> [lymphocytes]. Further, clonogenic assay was used to demonstrate surviving fractions for OCI-MY7, OCI-MY4 and NBM in vitro. The LD90 for OCI-MY7 and OCI-MY4 was between 10 and 20 ng/mL BPD-MA whereas this concentration did not show any significant cell kill for the colony-forming units-granulocyte/macrophage (CFU-GM) and burst-forming units-erythrocyte (BFU-E). When the NBM was "contaminated" with multiple myeloma cells in vitro, the LD90 for OCI-MY7 in this cell mixture was shifted to between 40 and 80 ng/mL BPD-MA. However, at 40 ng/mL BPD-MA at least 50% of normal CFU-GM and BFU-E colonies survived. For CFU-GM and BFU-E derived from the enriched CD34+ cell population, BPDMA up to a concentration of 80 ng/mL did not significantly reduce the surviving fractions. We have observed a 3-4 log therapeutic window with differential cell kill when comparing multiple myeloma cell lines to the components of the NBM and apheresis product in vitro. We conclude, that BPD-MA is a molecule potentially useful as an ex vivo purging agent.

Gamma delta-T cell-receptor-positive lymphocytes inhibit human hematopoietic progenitor cell growth in HIV type 1-infected patients.

In severe HIV infection, the majority of patients exhibit signs of hematopoietic deficiency including anemia, leukopenia, and thrombocytopenia. Besides other pathophysiological mechanisms, the disturbed helper/suppressor ratio of T-lymphocytes suggests that alterations within T cell subpopulations may have a suppressive effect on HIV-associated hematopoiesis. Since a delta TCS-1- and mostly CD-8-positive subpopulation of cytotoxic T-lymphocytes expressing the gamma delta-receptor is increased in peripheral blood and bone marrow of HIV-infected persons, it was the aim of this study to investigate the role of gamma delta-positive cells in HIV-associated bone marrow deficiency. The number of bone marrow-derived pluripotent colony-forming units (CFU-GEMM), burstforming units-erythrocyte (BFU-E), and colony-forming units-granulocyte-monocyte (CFU-GM) of HIV-1-positive patients was significantly (p < 0.05) increased after depletion of CD-8-positive, gamma delta-positive, and delta TCS-1-positive T-lymphocytes. In contrast, the depletion of these subpopulations had no stimulatory effect in healthy controls. Further experiments identified direct cellular contact between effector and hematopoietic progenitor cells and the production of interferon-gamma and tumor necrosis factor-alpha as the mechanisms mediating the suppressive effect of the delta TCS-1-positive cells in HIV-positive patients.

Sequential Changes in Stem Cell Markers in Peripheral Blood and Leukapheresis Samples after Injections of Recombinant Human Granulocyte Colony-stimulating Factor in Patients with Urogenital Malignant Solid Tumors: A Preliminary Study

In order to evaluate the mobilization effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on peripheral blood stem cells (PBSCs), rhG-CSF was given to patients with urogenital malignancy before chemotherapy. Markers for the stem cells, such as colony forming unit-granulocyte/macrophage (CFU-GM) and burst forming unit-erythrocyte (BFU-E), were sequentially monitored in peripheral blood and leukapheresis samples. Five patients, including a 13-year-old boy, were given 5 micrograms/kg rhG-CSF subcutaneously: the pediatric case for four consecutive days and the adult cases for six consecutive days (53-72 years of age). None of the patients had received chemotherapy within the four weeks prior to the start of the rhG-CSF series. PBSC collections were performed on the fifth day in the pediatric case and on the fifth and seventh days in the adult cases. Progenitor cells were monitored by methyl-cellulose cell culture techniques. CFU-GM on day 5 of the rhG-CSF series in peripheral blood increased 14- to 53-fold compared with samples taken immediately before the series. CFU-GM in the leukapheresis products on day 5 was greatest (70 x 10(3)/kg) in the pediatric case and least (14 x 10(3)/kg) in the oldest patient's case. The totals of the CFU-GM collected by two phereses in the adult cases were 21-73 x 10(3)/kg and the totals of CD34 positive cells were 0.6 to 1.4 x 10(6)/kg. The data suggest rhG-CSF to induce sufficient PBSCs for bone marrow rescue into the peripheral blood without any preceding chemotherapy. The patient's age may, however, be a contributory factor in using this method.

In vitro improvement of bone marrow‐derived hematopoietic colony formation in HIV‐positive patients by alpha‐D‐tocopherol and erythropoietin

The majority of patients with progressive HIV infection develop a severe hematopoietic failure which is aggravated by the hematotoxic effect of azidothymidine (AZT) treatment. Since it was shown in a mouse model that alpha-D-tocopherol (vitamin E derivative) antagonizes the inhibitory influence of AZT on the growth of burst-forming units-erythrocyte (BFU-E), it was the aim of this study to investigate whether alpha-D-tocopherol and high dosages of erythropoietin (EPO) increase the hematopoietic colony-forming capacity of bone marrow cells from patients with progressive HIV disease and especially if they reverse the inhibitory effects of AZT. The data demonstrate that tocopherol (1-100 mumol/l) significantly increases the growth of BFU-E and colony-forming units granulocyte-monocyte (CFU-GM) from HIV-infected patients. This stimulatory effect is dose-dependent (maximum at 30-100 mumol/l) and only occurs when the agent is present from the beginning of the cultures. EPO (5-10 U/ml) also augments the numbers of BFU-E from HIV-infected patients. Tocopherol equally ameliorates the growth of BFU-E and CFU-GM from the HIV-positive cohort in the presence of AZT (10-100 mumol/l). For healthy controls, no such increase was observed, either with tocopherol or with higher dosages of EPO. In conclusion, both tocopherol and EPO partially reverse the myelosuppressive action of AZT in HIV-positive patients.

Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis

Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45- RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU- GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst- forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.

Rapid Discrimination of Early CD34+ Myeloid Progenitors Using CD45-RA Analysis

Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45-RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU-GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst-forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.

Preclinical Analysis of Cytokine Therapy in the SCID-hu Mouse

AbstractA severe combined immunodeficient (SCID)-hu mouse model implanted with human fetal bone was used to assess the effects of various recombinant human (rh) hematopoietic growth factors, administered either alone or in combination, on human hematopoiesis in vivo. Treatment with rh granulocyte colony-stimulating factor (G-CSF) elicited the expansion of mature neutrophilic granulocyte populations in human marrow. Administration of rh interleukin-3 (IL-3) induced significant increases of eosinophilic granulocyte and burst-forming unit, erythrocyte (BFU-E) activity. The rhIL-6 did not cause significant changes in the subpopulations of human hematopoietic cells within the grafts, but did increase the number of colony-forming unit, granulocyte-macrophage and BFU-E. Pretreatment with rhIL-3 followed by rh erythropoietin (Epo) administration enhanced Epo-induced human erythropoiesis significantly. No synergistic effects on myelopoiesis were observed using sequential treatment with rhIL-3 followed by rhG-CSF. Instead, these factors seemed to work independently, with rhG-CSF increasing the percentage of neutrophils and rhIL-3 increasing the percentage of eosinophils. When administered simultaneously with rhEpo, rhIL-6 showed dose-dependent inhibitory effects on in vivo Epo-induced human erythropoiesis. The rhIL-6 also caused a reduction in the percentage of human neutrophils induced by rhG-CSF. These results suggest that the SCID-hu mouse provides a useful small animal model to assess the in vivo effects of hematopoietic growth factors on human hematopoiesis.