Colony-forming Unit-granulocyte And Macrophage Research Articles

Umbilical cord blood cell characteristics in very preterm neonates for autologous cell therapy of preterm-associated complications

BackgroundThere are emerging clinical evidence for umbilical cord blood mononuclear cells (UCBMNCs) intervention to improve preterm complications. The first critical step in cell therapy is to obtain high-quality cells. This retrospective study aimed to investigate the quantity and quality of UCBMNCs from very preterm infants (VPIs) for the purpose of autologous cell therapy in prevention and treatment of preterm complications.MethodsVery preterm infants (VPIs) born in Guangdong Women and Children Hospital from January 1, 2017, to December 8, 2022, from whom cord blood was successfully collected and separated for public or private banking, were enrolled. The UCBMNCs characters from route cord blood tests performed in cord blood bank, impact of perinatal factors on UCBMNCs, the relationship between UCBMNCs characteristics and preterm outcomes, and the correlation of UCBMNCs characteristics and peripheral blood cells in VPIs were analyzed.ResultsTotally, 89 VPIs underwent UCB collection and processing successfully. The median cell number post processing was 2.6 × 108. To infuse a dose of 5 × 107 cells/kg, only 3.4% of infants required a volume of more than 20 mL/kg, which exceeded the maximum safe volume limit for VPIs. However, when infusing 10 × 107 cells/kg, 25.8% of infants required a volume of more than 20 ml/kg volume. Antenatal glucocorticoids use and preeclampsia was associated with lower original UCBMNCs concentration. Both CD34+ hematopoietic stem cells (HSC) frequency and colony forming unit - granulocyte and macrophage (CFU-GM) number correlated negatively with gestational age (GA). UCBMNCs characters had no significant effect on preterm outcomes, whereas a significant positive correlation was observed between UCBMNCs concentration and total white blood cell, neutrophil, lymphocyte and PLT counts in peripheral blood.ConclusionUCBMNCs collected from VPIs was feasible for autologous cell therapy in improving preterm complications. Setting the infusion dose of 5 × 107 cells/kg guaranteed a safe infusion volume in more than 95% of the targeted infants. UCBMNCs characters did not affect preterm complications; however, the effect of UCBMNCs concentration on peripheral blood classification count should be considered when evaluating the immunomodulation of UCBMNCs transfusion.

Granulocyte colony-stimulating factor exacerbates hematopoietic stem cell injury after irradiation.

BackgroundExposure to a moderate to high dose of ionizing radiation (IR) not only causes acute radiation syndrome but also induces long-term (LT) bone marrow (BM) injury. The latter effect of IR is primarily attributed to the induction of hematopoietic stem cell (HSC) senescence. Granulocyte colony-stimulating factor (G-CSF) is the only treatment recommended to be given to radiation victims soon after IR. However, clinical studies have shown that G-CSF used to treat the leukopenia induced by radiotherapy or chemotherapy in patients can cause sustained low white blood cell counts in peripheral blood. It has been suggested that this adverse effect is caused by HSC and hematopoietic progenitor cell (HPC) proliferation and differentiation stimulated by G-CSF, which impairs HSC self-renewal and may exhaust the BM capacity to exacerbate IR-induced LT-BM injury.MethodsC57BL/6 mice were exposed to 4 Gy γ-rays of total body irradiation (TBI) at a dose-rate of 1.08 Gy per minute, and the mice were treated with G-CSF (1 μg/each by ip) or vehicle at 2 and 6 h after TBI on the first day and then twice every day for 6 days. All mice were killed one month after TBI for analysis of peripheral blood cell counts, bone marrow cellularity and long-term HSC (CD34-lineage-sca1+c-kit+) frequency. The colony-forming unit-granulocyte and macrophage (CFU-GM) ability of HPC was measured by colony-forming cell (CFC) assay, and the HSC self-renewal capacity was analyzed by BM transplantation. The levels of ROS production, the expression of phospho-p38 mitogen-activated protein kinase (p-p38) and p16INK4a (p16) mRNA in HSCs were measured by flow cytometry and RT-PCR, respectively.ResultsThe results of our studies show that G-CSF administration mitigated TBI-induced decreases in WBC and the suppression of HPC function (CFU-GM) (p < 0.05), whereas G-CSF exacerbated the suppression of long-term HSC engraftment after transplantation one month after TBI (p < 0.05); The increase in HSC damage was associated with increased ROS production, activation of p38 mitogen-activated protein kinase (p38), induction of senescence in HSCs.ConclusionOur findings suggest that although G-CSF administration can reduce ARS, it can also exacerbate TBI-induced LT-BM injury in part by promoting HSC senescence via the ROS-p38-p16 pathway.

Study on Biological Effect Protection of Electromagnetic Radiation Shielding Fabric

In this study, electromagnetic radiation (EMR) shielding fabric was coated by Carbonyl Iron and Nickel powder. X-ray diffraction (XRD) measure was applied to test the existence of Carbonyl Iron and Nickel. In order to study protective performance of EMR shielding fabric for living body, tests on biological effect protection were proceeded respectively using EMR shielding fabric, stainless steel fiber blend fabric and common fabric of the same specification. The results showed that colony forming unit-granulocyte and macrophage (CFU-GM), which expressed the ability of the mice’s hematopoietic function, were obviously improved after using shielding fabric. And all the other biological characterizations of the life that covered the EMR shielding fabric and stainless steel fiber blend fabric were better than that that covered common fabric. Moreover, The EMR shielding fabrics had better protective effect than pure stainless steel fiber blended fabric. The parameters of the Carbonyl Iron and Nickel coated and the stainless steel fiber were the significantly factors to affect the shielding property.

Protective effect of Dammarane Sapogenins against chemotherapy-induced myelosuppression in mice

Chemotherapy is the most common way to treat malignancies, but myelosuppression, one of its common side-effects, is a formidable problem. The present study described the protective role of dammarane sapogenins (DS), an active fraction from oriental ginseng, on myelosuppression induced by cyclophosphamide (CP) in mice. DS was orally administered at different dosages (37.5, 75, and 150 mg/kg) for 10 d after CP administration (200 mg/kg intraperitoneally). The results showed that DS increased the number of white blood cells (WBC) on day 3 and day 7 (P < 0.05), such that WBC levels were increased by 105.7 ± 29.5% at 75 mg/kg of DS on day 3 (P < 0.05, compared with the CP group). Similar results were observed in red blood cells and platelets in DS-treated groups. The colony-forming assay demonstrated that the depressed numbers of CFU-GM (colony-forming unit-granulocyte and macrophage), CFU-E (colony-forming unit-erythroid), BFU-E (burst-forming unit-erythroid), CFU-Meg (colony-forming unit-megakaryocyte) and CFU-GEMM (colony-forming unit-granulocyte, -erythrocyte, -monocyte and -megakaryocyte) induced by CP were significantly reversed after DS treatment. Moreover, the ameliorative effect of DS on myelosuppression was also observed in the femur by hematoxylin/eosin staining. In DS-treated groups, ConA-induced splenocyte proliferation was enhanced significantly at all the doses (37.5, 75, 150 mg/kg) on day 3 at the rate of 50.3 ± 8.0%, 77.6 ± 8.5% and 44.5 ± 8.4%, respectively, while lipopolysaccharide-induced proliferation was increased mainly on day 7 (P < 0.01), with an increased rate of 39.8 ± 5.6%, 34.9 ± 6.6% and 38.3 ± 7.3%, respectively. The thymus index was also markedly increased by 70.4% and 36.6% at 75 mg/kg on days 3 and 7, respectively, as compared with the CP group. In summary, DS has a protective function against CP-induced myelosuppression. Its mechanism might be related to stimulating hematopoiesis recovery, as well as enhancing the immunological function.

Low Dose IGFBP1, IGFBP2 and ANGPTL3 Coordinately Stimulate Ex Vivo Expansion of Human Umbilical Cord Blood (UCB) Hematopoietic Stem Cells (HSCs).

AbstractAbstract 1546Ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may overcome the obstacle of low cell dose for UCB transplantation in adults. Insulin like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin like proteins (ANGPTLs) can further enhance the ex vivo expansion of HSCs when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-ligand (FL). Current doses of IGFBPs and ANGPTLs are in the range of 100∼500ng/ml, but these concentrations may not be optimal and high concentrations could be costly for clinical use. In order to determine the optimal dosage of IGFs, IGFBPs and ANGPTLs, 4×105cells/mL of cryopreserved clinical UCB was inoculated in serum-free Stemspan® medium supplied with standard basal cytokine combination of 100ng/ml SCF, 50ng/ml FL and 100ng/ml TPO on an MSCs stromal layer and with individually varied doses of IGFBP1, IGFBP2, IGF2 and ANGPTL3 in the range of 0∼200ng/ml. In order to determine optimal cytokine combination, complete permutation was carried out after establishing the optimal dosage of each cytokine. On day 7, the same amount of Stemspan® medium with the indicated cytokine combination was replenished to the culture system. Cord blood cells were harvested after 12 days ex vivo culture and assayed for total cell count, cell surface phenotype (viability determined by CD45/AnnV/7AAD staining, primitive progenitor determined by CD45/CD34/CD38/CD90 staining) and functional studies (Colony-forming unit-granulocyte and macrophage (CFU-GM) was determined by methylcellulose colony culture). Paradoxically, the highest expansion of CD34+CD38-CD90+ primitive progenitor was at a low dose of 20ng/ml for IGFBP1, IGFBP2, IGF2 and ANGPTL3 when concentrations of 0, 20, 50, 100 and 200 ng/ml were studied (Fig. 1A). Based on this results the cytokine dosage range was narrowed down to 0∼50ng/ml and experiments (Fig. 1B) showed that the optimal cytokines dosages were 20ng/ml of IGFBP2 and ANGPTL3, 15ng/ml IGFBP1 and 10ng/ml IGF2, which could stimulate 13.0±1.1 fold, 13.3±2.4 fold, 11.0±0.8 fold and 14.3±2.1 fold expansion of CD34+CD38-CD90+ primitive progenitor compared to 6.8±0.2 fold with standard cytokine control (p =0.01). Studying multiple permutations, combination “ABD” comprising 15ng/ml IGFBP1, 20ng/ml IGFBP2 and 20ng/ml ANGPTL3 had the highest expansion of CD34+CD38-CD90+ primitive progenitor (27.7±2.2 fold compared to 8.5±1.1 fold with standard cytokines, p =0.01), was found to be superior to all other combinations, including combinations “A”, “B”, “BC”, “BD”, “CD” and “ABCD”, which could stimulate over 2 fold expansion of primitive progenitor compare to control (Figure 1C). Interestingly, despite expansion of primitive CD34+CD38-CD90+ cells, there was no further enhancement of the expansion of total cells and general progenitors compare to control (data not shown), suggesting that the cyokine cocktail enhanced only the earliest progenitors. In conclusion, IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38-CD90+ primitive progenitor at low dosage, and the optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3. Further in vivo experimentation is in progress to verify the effect of our optimized cytokine combination culture system on ex vivo expansion of cryopreserved unselected clinical UCB HSCs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Preparation of chitosan-polyaspartic acid-5-fluorouracil nanoparticles and its anti-carcinoma effect on tumor growth in nude mice

To prepare chitosan-polyaspartic acid-5-fluorouracil (CTS-Pasp-5Fu) nanoparticles and investigate its anti-carcinoma effect and toxicity. CTS-Pasp-5Fu nanoparticles were synthesized by ionic gelatification. Male BABL/c nude mice were injected with SGC-7901 gastric carcinoma cell line mass to establish a human gastric carcinoma model. They were randomly allocated into 4 groups: CTS-Pasp-5Fu (containing 5-Fu 1.25 mg/kg), 5-Fu (1.25 mg/kg), CTS-Pasp and normal saline groups. Tumor weight was measured and assay of colony forming unit-granulocyte and macrophage (CFU-GM) was performed. The structural change of cells and tissues was observed and the Bax and Bcl-2 genes were detected. Compared with normal saline, the inhibition rates of tumor growth for the CTS-Pasp, 5-Fu and CTS-Pasp-5Fu groups were 5.58%, 58.69% and 70.82%, respectively. The tumor inhibition rates for the CTS-Pasp, 5-Fu and CTS-Pasp-5Fu groups were 5.09%, 65.3% and 72.79%, respectively. There was a significant decrease in the number of CFU-GM formation and increase of total bilirubin, and alanine aminotransferase in the 5-Fu group, but no change in those of the other three groups. There was no change in white blood cell count and creatinine among the four groups. Pathological section of liver and nephridial tissues showed that the damage in the 5-Fu group was more severe than that in the CTS-Pasp-5Fu group. 5-Fu and CTS-Pasp-5Fu groups could both down-regulate the Bcl-2 expression and up-regulate the Bax expression to different extent, and the accommodate effect of CTS-Pasp-5Fu was more obvious than 5-Fu. The tumor inhibition rate of CTS-Pasp-5Fu nanoparticles is much higher than that of 5-Fu alone.

Elimination of CD13 positive cells — an approach to prevent transmission of HCMV in bone marrow transplantation

The effects of native thrombospondin (TSP), an 18 kd recombinant protein comprising residues 1–174 of TSP (TSP1–174) with heparin-binding domain and a fusion protein comprising residues 559–669 of TSP (TSP559–669) on murine hematopoiesis, were studied by using different in vitro culture systems. TSP by itself did not show an inhibitory effect on colony-forming unit-megakaryocyte (CFU-MK) growth in a serum-free agar system and on the growth of colony-forming unit-granulocyte and macrophage (CFU-GM) in a plasma clot system. It was, however, found that in the plasma clot culture system when using aplastic anemia serum as the source of thrombopoietin or C-Mpl ligand (TPO), TSP and TSP1–174, but not TSP559–669, were able to inhibit the growth of CFU-MK from unfractionated and lineage negative (Lin-) bone marrow cells in a dose-dependent manner. A statistically significant suppression was seen at 1 μg/ml of TSP and 5 μg/ml of TSP1–174. This inhibitory effect of TSP was further found in both the serum-free agar system and the plasma clot system without aplastic anemia serum, where megakaryocyte colony growth was stimulated by recombinant TPO, basic fibroblast growth factor (bFGF), or interleukin-3 (IL-3). In a methylcellulose system, where a combination of stem cell factor (SCF), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were used, TSP inhibited the growth of colony-forming unit-granulocyte-erythroblast, megakaryocyte, and macrophage (CFU-GEMM) but not CFU-GM and burst-forming unit-erythroblast (BFU-E). Interestingly, this inhibitory effect of TSP on megakaryocyte colony growth could be counteracted by Fraxiparin, a low-molecular-weight heparin. These results demonstrate that TSP is a negative modulator of megakaryocytopoiesis and suggest that its inhibitory effect is at least partially mediated by N-terminal heparin-binding domain.

Thrombospondin, a negative modulator of megakaryocytopoiesis

The effects of native thrombospondin (TSP), an 18 kd recombinant protein comprising residues 1–174 of TSP (TSP 1–174) with heparin-binding domain and a fusion protein comprising residues 559–669 of TSP (TSP 559–669) on murine hematopoiesis, were studied by using different in vitro culture systems. TSP by itself did not show an inhibitory effect on colony-forming unit-megakaryocyte (CFU-MK) growth in a serum-free agar system and on the growth of colony-forming unit-granulocyte and macrophage (CFU-GM) in a plasma clot system. It was, however, found that in the plasma clot culture system when using aplastic anemia serum as the source of thrombopoietin or C-Mpl ligand (TPO), TSP and TSP 1–174, but not TSP 559–669, were able to inhibit the growth of CFU-MK from unfractionated and lineage negative ( Lin-) bone marrow cells in a dose-dependent manner. A statistically significant suppression was seen at 1 μg/ml of TSP and 5 μg/ml of TSP 1–174. This inhibitory effect of TSP was further found in both the serum-free agar system and the plasma clot system without aplastic anemia serum, where megakaryocyte colony growth was stimulated by recombinant TPO, basic fibroblast growth factor (bFGF), or interleukin-3 (IL-3). In a methylcellulose system, where a combination of stem cell factor (SCF), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were used, TSP inhibited the growth of colony-forming unit-granulocyte-erythroblast, megakaryocyte, and macrophage (CFU-GEMM) but not CFU-GM and burst-forming unit-erythroblast (BFU-E). Interestingly, this inhibitory effect of TSP on megakaryocyte colony growth could be counteracted by Fraxiparin, a low-molecular-weight heparin. These results demonstrate that TSP is a negative modulator of megakaryocytopoiesis and suggest that its inhibitory effect is at least partially mediated by N-terminal heparin-binding domain.

Comparison of toxicity induced by T-2 toxin on human and rat granulo-monocytic progenitors with an in vitro model

T-2 toxin is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and their derivatives. In man as well as in animals, T-2 toxin has been shown to induce alimentary intoxication and, among others, haematological symptoms. Granulo-monocytic progenitors from human umbilical cord blood on the one hand and granulo-monocytic progenitors from rat bone marrow on the other, were cultured in the presence of T-2 toxin (from 10(-7) to 10(-10) M) for 14 days. A study of concentration and effect relationships showed a strong and rapid effect of T-2 toxin on rat colony forming unit-granulocyte and macrophage (CFU-GM) between 5 x 10(-9) M and 10(-9) M. On the other hand, human CFU-GM were able to grow in the presence of the same T-2 toxin concentrations. IC50 were determined on day 7, 10 and 14. They were, respectively, 1.6 x 10(-9) M; 3.6 x 10(-9) M; 1.4 x 10(-9) M for human cells, and 2.2 x 10(-9) M; 3.3 x 10(-9) M; 2.6 x 10(-9) M for rat cells. The present study was prompted by the need to define precisely the cytotoxic and inhibitory T-2 toxin concentrations for rat and human CFU-GM. It is particularly relevant for the investigation of cellular T-2 toxin targets and in order to elucidate the mechanism of trichothecene haematotoxicity.

In vitro toxicity of trichothecenes on human haematopoietic progenitors.

The culture of human haematopoietic progenitors, Colony-Forming-Unit Granulocyte and Macrophage (CFU-GM), has been performed in the presence of four trichothecenes, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), and deoxynivalenol (DON). Our results showed that trichothecenes were cytotoxic for human haematopoietic progenitors. This work and the analysis of results described in the literature allowed us to propose that the haematologic lesions observed during human intoxication could be due to a destruction of haematopoietic progenitors such as granulocytic and macrophage colony-forming cells.

Lindane haematotoxicity confirmed by in vitro tests on human and rat progenitors.

Blood dyscrasias such as aplastic anaemia and leukopenia are described following the use of lindane for agricultural purposes or against ectoparasites in animal and human health. In order to determine the involvement of lindane in these effects, an in vitro model of haematotoxicity evaluation has been used. Culture of haematopoietic progenitors, Colony Forming Unit-Granulocyte and Macrophage (CFU-GM), have been performed in the presence of lindane with increasing concentrations. Results showed that lindane was cytotoxic for human progenitors. They were one thousand times more sensitive to the lindane than rat CFU-GM. This cytotoxicity was observed with lindane concentrations similar to those measured in human blood in cases of acute intoxication and in fat tissues of exposed populations.

In vitro study of pesticide hematotoxicity in human and rat progenitors

Some pesticides are hematotoxic and cause aplastic anemia, agranulocytosis, neutropenia, and thrombopenia. In order to evaluate hematopoietic progenitor cultures in the exploration of pesticide hematotoxicity, human and rat colony-forming unit-granulocyte and macrophage (CFU-GM) were cultured in the presence of different concentrations of pesticides, known to be either hematotoxic or innocuous for blood cells. The results were compared to the control culture of the same sample. Four insecticides (lindane, azinphos, mevinphos, parathion methyl), three herbicides, (2,4,5, T, bromacil, MCPA), and two fungicides (fosethyl-aluminum, DNOC) were tested and exhibited the following data: 1) Pesticides, known to be hematotoxic, inhibited the development of progenitors. Different phenomena were observed and suggested different mechanisms: cell destruction, block in mitosis, decrease or delay in mitosis. 2) Difference in sensitivity to molecules between human and rat progenitors was observed. Human progenitors were more sensitive to pesticides, except for 2,4,5 T.

Heat sensitivity, thermotolerance and protein synthesis of granulocyte and macrophage progenitors from mice and from long-term bone marrow cultures

Ex-vivo purging of leukaemic cells by single or fractionated heating during bone marrow transplantation for acute myeloid or lymphoblastic leukaemias may be possible since many leukaemic cells may be more sensitive to killing by heat than normal bone marrow colony-forming unit-granulocyte and macrophage (CFU-GM). In these studies we have compared heat response, thermotolerance response and heat shock protein (HSP) synthesis of bone marrow progenitors from intact mice or bone marrow obtained from 37 degrees C or 33 degrees C long-term bone marrow cultures (LTBMCs). Such studies were done to examine whether CFU-GM responses to heat are influenced by in vitro growth conditions. In terms of heat response CFU-GM progenitors from LTBMCs showed increased heat sensitivity when compared to CFU-GM from mice, with CFU-GM from 33 degrees C cultures being more heat-sensitive than those from 37 degrees C cultures. The kinetics of thermotolerance development and decay in CFU-GM were similar from all three systems. Thermotolerance was maximum at 3-9 h, began to decay by 24 h and was absent by 48 h from cells obtained from mice and LTBMCs grown at 37 degrees C. However, CFU-GM from 33 degrees C LTBMCs showed a delay in decay of thermotolerance. The kinetics of the development and decay of thermotolerance remained as above and was independent of incubation conditions whether bone marrow was left in situ or incubated in vitro in medium containing various colony-stimulating factors (CSF) or medium containing no CSF. The synthesis of HSPs was measured by one- and two-dimensional gel electrophoresis. Synthesis of HSP-70 kd was detected following a triggering dose of heat at 0-3 h for bone marrow heated in vivo and 0-6 h for bone marrow from LTBMCs. These studies indicate that CFU-GM heat and thermotolerance response was influenced by the temperature at which the cultures were maintained but not by other differences present in their microenvironment.

The response of human marrow colony-forming units-granulocyte and macrophage to inhibition by prostaglandin E and acidic isoferritins is associated with expression of MHC class II antigens and requires the participation of a CD8+ T lymphokine.

The in vitro expression of MHC class II Ag by human bone marrow colony-forming unit-granulocyte and macrophage (CFU-GM) and their proliferative response to negative growth regulators in agar culture are transient and can be modulated in vitro by 24-h suspension culture in the presence of PGE. Analysis of the participation of accessory cells in this phenomenon indicates that the ability of PGE to modulate CFU-GM MHC class II expression, the proportion of CFU-GM in S-phase of the cell cycle and the responsiveness of CFU-GM to inhibition in vitro by two negative growth regulators, acidic isoferritin inhibitory activity and PGE itself, requires the participation of CD8+ T lymphocytes. This effect is mediated by a lymphokine of m.w. 27,000 that we have purified to apparent homogeneity. This lymphokine possesses neither colony stimulatory nor inhibitory activity, is produced by both peripheral blood and bone marrow CD8+ T lymphocytes, as well as the CEM T-ALL cell line, and requires the obligatory presence of PGE for activity.