scholarly journals PGE 2 pulsing of murine bone marrow cells reduces migration of daughter monocytes/macrophages in vitro and in vivo

2017 ◽  
Vol 56 ◽  
pp. 64-68 ◽  
Author(s):  
Terence A. McGonigle ◽  
Amy R. Dwyer ◽  
Eloise L. Greenland ◽  
Naomi M. Scott ◽  
Kevin N. Keane ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Murine Bone Marrow ◽  
Marrow Cells

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

Potent Cooperation Between NUP98-NSD1 and FLT3-ITD In AML Induction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3797-3797
Author(s):  
Angeliki Thanasopoulou ◽  
Alexandar Tzankov ◽  
Juerg Schwaller
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Latency Period ◽  
Histopathological Analysis ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract The NUP98-NSD1 fusion protein, product of the t(5;11)(q35;p15.5) chromosomal translocation, is an AML-associated cytogenetically silent genetic aberration, recently identified as the most frequent fusion in pediatric AML, generally associated with aggressive disease and poor prognosis. Interestingly, the vast majority (>70%) of the reported NUP98-NSD1-positive cases also carried an activating FLT3-ITD mutation suggesting functional cooperation. The purpose of this study was to search for experimental evidence of a functional cooperation between NUP98-NSD1 and FLT3-ITD in the transformation of murine hematopoietic cells in vitro and in vivo. Lineage surface marker-depleted murine bone marrow cells were transduced with either pMSCV-NUP98-NSD1-neo or pMSCV-FLT3-ITD-GFP or both expression constructs on fibronectin-coated plates. Serial colony formation assays in myeloid favoring medium and immunophenotypic analysis by flow cytometry indicated that retroviral expression of NUP98-NSD1 provided increased self-renewal capacity and impaired differentiation of murine bone marrow stem and progenitor cells. NUP98–NSD1 expressing cells displayed a typical myeloblastic morphology and co-expressed myeloid and early stem cell surface markers (CD34low/c-kit+/FcgR+/Gr-1+/ Mac-I+/B220-). Co-expression of FLT3-ITD resulted in high rates of cell proliferation, showed a more differentiated phenotype and concomitantly impaired the in vitro clonogenic capacity in methylcellulose cultures. Bone marrow cells expressing NUP98-NSD1 with or without FLT3-ITD were harvested from methylcellulose cultures and transplanted into sub-lethally irradiated syngeneic mice. All mice receiving cells co-expressing NUP98-NSD1 and FLT3-ITD developed AML that was transplantable into all secondary recipients. Myeloid leukemic blasts that co-expressed NUP98-NSD1 and FLT3-ITD were present in abundance both in BM preparations and in blood smears, and histopathological analysis showed widespread infiltration into solid organs. By contrast, no AML ever developed in mice receiving cells expressing only NUP98-NSD1. These mice, similar to mice receiving cells expressing FLT3-ITD only, developed signs of a chronic myeloproliferative disorder, characterized by expansion of Mac-1+/Gr-1+ BM cells with granulocytic/monocytic differentiation that in some cases caused severe distress after a latency period of more than one year. Intriguingly, upon injection with double transduced NUP98-NSD1 and FLT3-ITD progenitors rather different latency periods of the AML development were observed between different experiments. Interestingly, the latency periods could be correlated to the ratio of expression levels of FLT3-ITD to wildtype FLT3, with higher FLT3-ITD levels associated with a shorter latency. To further investigate the significance of aberrant FLT3 signaling, in vitro and in vivo transformed NUP98-NSD1 and NUP98-NSD1/FLT3-ITD cells were treated with a selective FLT3 tyrosine kinase inhibitor (PKC412). The higher sensitivity of cells co-expressing NUP98-NSD1 and FLT3-ITD to PKC412, compared to cells expressing NUP98-NSD1 only, indicated that proliferation and survival were dependent on FLT3-derived signals. Taken together, these observations demonstrate a potent cooperation between NUP98-NSD1 fusion and FLT3-ITD in leukemic transformation. However, neither the NUP98-NSD1 fusion protein nor the FLT3-ITD mutation alone was sufficient to induce AML. Moreover, the high sensitivity of NUP98-NSD1 and FLT3-ITD co-expressing leukemic blasts to FLT3 signaling inhibition suggests a possible therapeutic strategy to be further explored in this AML subgroup. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Erythroid cell growth from normal and W/WV murine bone marrow on macrophage-coated membranes

Blood ◽  
1977 ◽  
Vol 50 (5) ◽  
pp. 857-866
Author(s):  
BJ Torok-Starb ◽  
NS Wolf ◽  
DR Boggs
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Bone Marrow Cells ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Cellulose Acetate Membranes ◽  
Coated Membranes ◽  
Marrow Cells

Cellulose acetate membranes (CAM) placed in the peritoneal cavity of mice develop a macrophage layer capable of supporting in vivo hematopoietic colonies from intraperitoneally injected bone marrow cells. Modifications allowing for routine morphologic identification of colonies showed that both erythrocytic (E) and granulocytic (G) colonies occur with a consistent E:G ratio of 0.19 +/- 0.037. Stimulating recipients by bleeding or phenylhydrazine injection did not produce a significant change in the total number of colonies and a reduction in granulocytic colonies so that the E:G ratio significnatly increased. Hypertransfusion of donor animals had no effect on the number of erythroid colonies that grew on CAM of average recipients. The total colony-forming ability of bone marrow cells from genetically anemic W/WV mice was found not to differ from that of normal +/+ littermates; however, the E:G ratio of W/WV marrow in bled recipients was significantly lower (p less than 0.01) then that of +/+ marrow. These studies suggest that a CAM system supports an erythroid progenitor which is not affected by hypotransfusion of the donor animal, yet is dependent upon erythropoietin for colony formation, and that it is defective in the W/WV mouse.

Comparison of the transport of chlorozotocin and CCNU in L1210 leukemia and murine bone marrow cells in vitro

1983 ◽  
Vol 11 (3) ◽  
Author(s):  
Philip Lazarus ◽  
JudithSt Germina ◽  
Maurice Dufour ◽  
Greg Palmer ◽  
Deborah Wallace ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
L1210 Leukemia ◽  
Murine Bone Marrow ◽  
Marrow Cells

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 Observations on the binding and interaction of radioiodinated colony- stimulating factor with murine bone marrow cells in vivo

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 408-420 ◽  
Author(s):  
G Pigoli ◽  
A Waheed ◽  
RK Shadduck
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Cell Interaction ◽  
Peritoneal Macrophages ◽  
Colony Stimulating Factor ◽  
Murine Bone Marrow ◽  
Stimulating Factor ◽  
Marrow Cells

Abstract Radioiodinated L-cell-derived colony-stimulating factor (CSF) was used to characterize the binding reaction to murine bone marrow cells. The major increment in cell-associated radioactivity occurred over 24 hr incubation at 37 degrees C, but virtually no binding was observed at 4 degrees C. The reaction was saturable with approximately 1 ng/ml of purified CSF. Unlabeled CSF prevented the binding, whereas a number of other hormones and proteins did not compete for CSF uptake. Further specificity studies showed virtually no binding to human bone marrow, which is unresponsive to this form of murine CSF. Minimal CSF uptake was noted with murine peritoneal macrophages, but virtually no binding was detected with thymic, lymph node, liver, or kidney cells. The marrow cell interaction with tracer appeared to require a new protein synthesis, as the binding was prevented by cycloheximide or puromycin. Preincubation of marrow cells in medium devoid of CSF increased the degree of binding after 1 hr exposure to the tracer. This suggests that CSF binding sites may be occupied or perhaps decreased in response to ambient levels of CSF in vivo. Approximately 70% of the bound radioactivity was detected in the cytoplasm at 24 hr. This material was partially degraded as judged by a decrease in molecular weight from approximately 62,000 to 2 peaks of approximately 32,000 and approximately 49,000, but 72% of the binding activity was retained. After plateau binding was achieved, greater than 80% of the radioactivity released into the medium was degraded into biologically inactive peptides with molecular weights less than 10,000. These findings suggest that the interaction of CSF with marrow cells is characterized by binding with subsequent internalization and metabolic degradation into portions of the molecule that are devoid of biologic activity.

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