Receptor-mediated clearance of G-CSF derivative nartograstim in bone marrow of rats

1995 ◽  
Vol 269 (1) ◽  
pp. E1-E9 ◽  
Author(s):  
T. Kuwabara ◽  
T. Uchimura ◽  
H. Kobayashi ◽  
S. Kobayashi ◽  
Y. Sugiyama
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Maximal Velocity ◽  
Rate Dependent ◽  
Elimination Mechanism ◽  
Total Body

To clarify the role of the granulocyte colony-stimulating factor (G-CSF) receptor in the nonlinear elimination of a recombinant human G-CSF derivative, nartograstim (NTG), the accompanying changes in the in vivo NTG total body clearance at steady state (CLss) or the early-phase tissue uptake clearance (CLuptake) in rats were compared with the change in the number of G-CSF receptors in bone marrow. The infusion rate-dependent decrease in CLss in control rats confirmed the existence of a saturable elimination mechanism for NTG. The Michaelis-Menten constant (Km) and maximal velocity for this saturable process were estimated to be 107 pM and 15.5 pmol.h-1.kg-1, respectively. The Km for this saturable process was comparable with the dissociation constant (Kd) for the specific binding of NTG to bone marrow cells. After administration of excess NTG, the CLuptake of tracer amounts of 1251-NTG by bone marrow and spleen, which corresponds to the receptor density in the tissues, was reduced at 2 h but gradually recovered. This change in CLuptake corresponds well to the change in the in vitro NTG-binding capacity in each isolated cell. This reduction in CLuptake might be due to the downregulation of G-CSF receptors on the cell surface. On the other hand, the saturable CLss in cyclophosphamide-treated rats was 17% of that in control rats, whereas the saturable CLss in rats given NTG repeatedly was twofold greater than in controls, which is associated with the upregulation of G-CSF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombopoietin-Induced Stimulation of Megakaryocyte- Enriched Bone Marrow Cultures

1979 ◽  
Author(s):  
K. L. Kellar ◽  
B. L. Evatt ◽  
C. R. McGrath ◽  
R. B. Ramsey
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Bone Marrow Cells ◽  
Rabbit Plasma ◽  
Bone Marrow Cultures ◽  
Plasma Fractions ◽  
Marrow Cultures ◽  
Stimulation Of

Liquid cultures of bone marrow cells enriched for megakaryocytes were assayed for incorporation of 3H-thymidine (3H-TdR) into acid-precipitable cell digests to determine the effect of thrombopoietin on DNA synthesis. As previously described, thrombopoietin was prepared by ammonium sulfate fractionation of pooled plasma obtained from thrombocytopenic rabbits. A control fraction was prepared from normal rabbit plasma. The thrombopoietic activity of these fractions was determined in vivo with normal rabbits as assay animals and the rate of incorporation of 75Se-selenomethionine into newly formed platelets as an index of thrombopoietic activity of the infused material. Guinea pig megakaryocytes were purified using bovine serum albumin gradients. Bone marrow cultures containing 1.5-3.0x104 cells and 31%-71% megakaryocytes were incubated 18 h in modified Dulbecco’s MEM containing 10% of the concentrated plasma fractions from either thrombocytopenic or normal rabbits. In other control cultures, 0.9% NaCl was substituted for the plasma fractions. 3H-TdR incorporation was measured after cells were incubated for 3 h with 1 μCi/ml. The protein fraction containing thrombopoietin-stimulating activity caused a 25%-31% increase in 3H-TdR incorporation over that in cultures which were incubated with the similar fraction from normal plasma and a 29% increase over the activity in control cultures to which 0.9% NaCl had been added. These data suggest that thrombopoietin stimulates DNA synthesis in megakaryocytes and that this tecnique may be useful in assaying thrombopoietin in vitro.

scholarly journals Bone Marrow-Derived CD44+Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Yanzhu Lu ◽  
Junchao Xing ◽  
Xiaolong Yin ◽  
Xiaobo Zhu ◽  
Aijun Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Bone Repair ◽  
Bone Marrow Cells ◽  
Complex Model ◽  
Host Cells ◽  
Immunofluorescent Staining ◽  
Jnk Pathway

Background and Aims.Host-derived cells play crucial roles in the regeneration process of tissue-engineered constructs (TECs) during the treatment of large segmental bone defects (LSBDs). However, their identity, source, and cell recruitment mechanisms remain elusive.Methods.A complex model was created using mice by combining methods of GFP+bone marrow transplantation (GFP-BMT), parabiosis (GFP+-BMT and wild-type mice), and femoral LSBD, followed by implantation of TECs or DBM scaffolds. Postoperatively, the migration of host BM cells was detected by animal imaging and immunofluorescent staining. Bone repair was evaluated by micro-CT. Signaling pathway repressors including AMD3100 and SP600125 associated with the migration of BM CD44+cells were further investigated.In vitro, transwell migration and western-blotting assays were performed to verify the related signaling pathway.In vivo, the importance of the SDF-1/CXCR4-JNK pathway was validated by ELISA, fluorescence-activated cell sorting (FACS), immunofluorescent staining, and RT-PCR.Results.First, we found that host cells recruited to facilitate TEC-mediated bone repair were derived from bone marrow and most of them express CD44, indicating the significance of CD44 in the migration of bone marrow cells towards donor MSCs. Then, the predominant roles of SDF-1/CXCR4 and downstream JNK in the migration of BM CD44+cells towards TECs were demonstrated.Conclusion.Together, we demonstrated that during bone repair promoted by TECs, BM-derived CD44+cells were essential and their migration towards TECs could be regulated by the SDF-1/CXCR4-JNK signaling pathway.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

scholarly journals Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA

2020 ◽  
Vol 21 (11) ◽  
pp. 3774
Author(s):  
Giuliana Ascone ◽  
Yixuan Cao ◽  
Ineke D.C. Jansen ◽  
Irene Di Ceglie ◽  
Martijn H.J. van den Bosch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Bone Destruction ◽  
Mineral Dissolution ◽  
Long Bone ◽  
Osteoclast Precursors ◽  
Marrow Cells

Recently, it was shown that interleukin-1β (IL-1β) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn−/−) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C−), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31− Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn−/− mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn−/− mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn−/− cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn−/− osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

scholarly journals Effect of Zinc Supplementation on Renal Anemia in 5/6-Nephrectomized Rats and a Comparison with Treatment with Recombinant Human Erythropoietin

2019 ◽  
Vol 20 (20) ◽  
pp. 4985 ◽  
Author(s):  
Hui-Lin Feng ◽  
Yen-Hua Chen ◽  
Sen-Shyong Jeng
Keyword(s):  
Bone Marrow ◽  
Recombinant Human Erythropoietin ◽  
Zinc Supplementation ◽  
Bone Marrow Cells ◽  
Human Erythropoietin ◽  
Post Surgery ◽  
Rat Bone ◽  
Marrow Cells

Anemia is a severe complication in patients with chronic kidney disease (CKD). Treatment with exogenous erythropoietin (EPO) can correct anemia in many with CKD. We produced 5/6-nephrectomized rats that became uremic and anemic at 25 days post surgery. Injection of the anemic 5/6-nephrectomized rats with 2.8 mg zinc/kg body weight raised their red blood cell (RBC) levels from approximately 85% of the control to 95% in one day and continued for 4 days. We compared the effect of ZnSO4 and recombinant human erythropoietin (rHuEPO) injections on relieving anemia in 5/6-nephrectomized rats. After three consecutive injections, both the ZnSO4 and rHuEPO groups had significantly higher RBC levels (98 ± 6% and 102 ± 6% of the control) than the saline group (90 ± 3% of the control). In vivo, zinc relieved anemia in 5/6-nephrectomized rats similar to rHuEPO. In vitro, we cultured rat bone marrow cells supplemented with ZnCl2, rHuEPO, or saline. In a 4-day suspension culture, we found that zinc induced erythropoiesis similar to rHuEPO. When rat bone marrow cells were supplement-cultured with zinc, we found that zinc stimulated the production of EPO in the culture medium and that the level of EPO produced was dependent on the concentration of zinc supplemented. The production of EPO via zinc supplementation was involved in the process of erythropoiesis.

scholarly journals Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2859-2870 ◽  
Author(s):  
OJ Borge ◽  
V Ramsfjell ◽  
OP Veiby ◽  
MJ Jr Murphy ◽  
S Lok ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Calf Serum ◽  
Myeloid Cell ◽  
Promoting Effect ◽  
Multipotent Progenitors

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability-promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum-supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca-1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.

scholarly journals Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1060-1066 ◽  
Author(s):  
M Miura ◽  
CW Jackson ◽  
SA Lyles
Keyword(s):  
Bone Marrow ◽  
Plasma Concentration ◽  
Bone Marrow Cells ◽  
Single Cells ◽  
Rat Plasma ◽  
Whole Body ◽  
Growth Promoting ◽  
Marrow Cells

Abstract To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

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