Cultivation of the Erythrocytic Stages of Plasmodium berghei in Primary Bone Marrow Cells

1976 ◽  
Vol 62 (5) ◽  
pp. 657 ◽  
Author(s):  
Clarence A. Speer ◽  
Paul H. Silverman ◽  
Steven G. Schiewe
Keyword(s):  
Bone Marrow ◽  
Plasmodium Berghei ◽  
Bone Marrow Cells ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Marrow Cells

scholarly journals Jun Dimerization Protein 2 (JDP2), a Member of the AP-1 Family of Transcription Factor, Mediates Osteoclast Differentiation Induced by RANKL

2003 ◽  
Vol 197 (8) ◽  
pp. 1029-1035 ◽  
Author(s):  
Reimi Kawaida ◽  
Toshiaki Ohtsuka ◽  
Junichi Okutsu ◽  
Tohru Takahashi ◽  
Yuho Kadono ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Osteoclast Differentiation ◽  
Osteoclast Formation ◽  
Raw264.7 Cells ◽  
Tartrate Resistant Acid Phosphatase ◽  
Gene Promoters ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Marrow Cells

Osteoclasts are multinucleated cells that resorb bones, and are derived from hematopoietic cells of the monocyte/macrophage lineage. The receptor activator of NF-κB ligand (RANKL, also called ODF/TRANCE/OPGL) stimulates both osteoclast differentiation from osteoclast progenitors and activation of mature osteoclasts. To identify genes responsible for osteoclast differentiation, we used a molecular indexing technique. Here, we report a clone of one of these genes whose transcription is induced by soluble RANKL (sRANKL) in both the RAW264.7 cells of the mouse macrophage cell line and the mouse primary bone marrow cells. The predicted protein was found to be a mouse homologue of Jun dimerization protein 2 (JDP2), a member of the AP-1 family of transcription factors, containing a basic region-leucine zipper motif. Transient transfection experiments revealed that overexpression of JDP2 leads to activation of both tartrate-resistant acid phosphatase (TRAP) and cathepsin K gene promoters in RAW264.7 cells. Infection of mouse primary bone marrow cells with retroviruses expressing JDP2-facilitated sRANKL-mediated formation of TRAP-positive multinuclear osteoclasts. Importantly, antisense oligonucleotide to JDP2 strongly suppressed sRANKL-induced osteoclast formation of RAW264.7 cells. Our findings suggest that JDP2 may play an important role in the RANK-mediated signal transduction system, especially in osteoclast differentiation.

PU.1-mediated transcriptional regulation of prophenin-2 in primary bone marrow cells

Gene ◽  
2005 ◽  
Vol 352 ◽  
pp. 1-9 ◽  
Author(s):  
Balaji Ramanathan ◽  
J. Ernest Minton ◽  
Chris R. Ross ◽  
Frank Blecha
Keyword(s):  
Bone Marrow ◽  
Transcriptional Regulation ◽  
Bone Marrow Cells ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Marrow Cells

An In Vitro Library Screen to Rapidly Determine Activity Against Normal and Mutant Bone Marrow Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4916-4916
Author(s):  
Wan Xing Hong ◽  
Steven Chen ◽  
Jon Akutagawa ◽  
Michelle Arkin ◽  
Benjamin S. Braun
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Kinase Inhibitors ◽  
Bone Marrow Cells ◽  
Ras Signaling ◽  
Primary Cells ◽  
Primary Bone ◽  
Mutant Cells ◽  
Primary Bone Marrow ◽  
Marrow Cells

Abstract Abstract 4916 Background: Aberrant signal transduction plays a central role in the pathogenesis of MDS/MPN, as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the central role of Ras signaling in the pathogenesis of JMML, at this time there are no signal transduction inhibitors with established efficacy in JMML. A screen of inhibitors has the potential to reveal potential therapeutic strategies and inform efforts to treat other neoplasms driven by hyperactive Ras signaling, both in the hematopoietic system and elsewhere. Aim: To investigate novel therapeutic options for JMML by utilizing a novel, reproducible system for rapid screening in primary cells. Innovations include using flow cytometry to isolate a highly clonogenic, disease-relevant “PreGM” population of primary bone marrow cells that recapitulate the abnormal growth pattern characteristic of JMML and unsorted bone marrow, the use of a genetically engineered mouse model, and the development of automated microscopy protocols. Method: Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 and wildtype mice were utilized in the screens. PreGM cells, identified as Lineage lo/- Sca1- c-kit+ CD34+ CD16/32- CD105- CD150-, were purified from harvested bone marrow using flow cytometry. The purified PreGM cells were sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1X (5 μg/ml, approx. 10 μM for most compounds) to 10−7X (5×10−7 μg/ml). The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions (at 37°C, 98% humidity and 5% CO2) in 80% IMDM, 20% FBS and saturating dose of 10ng/ml of GM-CSF. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 (GE). A total of 94 different inhibitors were screened using this method. The screen included a negative control (DMSO) and cytotoxic positive controls (Cytarabine, Adriamycin and Gemcitabine). Compound families included cyotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. The majority of compounds were either FDA approved drugs or agents used in recent clinical trials. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. Results: Primary bone marrow cells were harvested from a total of 28 mice, 18 wild type (WT) and 10 Mx1-Cre, KrasD12. PreGM growth was quantified and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc’ package from the R Project for Statistical Computing. Out of 94 candidates tested in this screen, none were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells. Neither were any of the drugs found to be comparatively toxic to WT cells or to have significantly higher IC50s in mutant PreGM cells in comparison to WT cells. Some compounds of interest included Vorinostat, an epigenetic inhibitor, which was found to have robust inhibitory activity against both mutant and WT cells. It has comparable IC50s in mutant and WT cells with a calculated IC50 of 0. 0480X (std. error: 0. 135) in Mx1-Cre, KrasD12 cells and 0. 0244X (std. error: 0. 0293) in WT cells. Conclusion: None of the 94 compounds used in the screen were found to preferentially inhibit mutant or WT cell growth, indicating that Kras mutant cells have similar drug sensitivities to normal cells over a broad range of mechanistic approaches. These findings suggest that it may be difficult to find “synthetic lethal” opportunities for drugs that are selectively toxic to primary cells driven by hyperactive Ras signaling. Disclosures: No relevant conflicts of interest to declare.

Growth enhancing effect of LBL-assembled magnetic nanoparticles on primary bone marrow cells

2016 ◽  
Vol 59 (11) ◽  
pp. 901-910 ◽  
Author(s):  
Xuan Liu ◽  
Jie Zhang ◽  
Shijia Tang ◽  
Jianfei Sun ◽  
Zhichao Lou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Magnetic Nanoparticles ◽  
Bone Marrow Cells ◽  
Enhancing Effect ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Marrow Cells

Familial Mastocytosis: Identification Of KIT K509I Mutation and Its In Vitro Sensitivity To Imatinib, Dasatinib and PKC412

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5267-5267 ◽  
Author(s):  
Paula De Melo Campos ◽  
João Agostinho Machado-Neto ◽  
Adriana Silva Santos Duarte ◽  
Renata Scopim-Ribeiro ◽  
Flavia Fonseca de Carvalho Barra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Bone Marrow Cells ◽  
Systemic Mastocytosis ◽  
History Of ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Apoptosis Ratio

Abstract Background Mast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug. Aims To characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors. Patients and methods Four individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter]), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture. Results Case 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen. Conclusions We herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013. Disclosures: No relevant conflicts of interest to declare.

STAT1 Promotes Megakaryocytic Differentiation in a GATA-1-Independent Manner.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1157-1157
Author(s):  
Zan Huang ◽  
John D. Crispino
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Independent Manner ◽  
Megakaryocytic Differentiation ◽  
Brdu Labeling ◽  
Primary Bone ◽  
Stat1 Signaling ◽  
Primary Bone Marrow

Abstract GATA-1 is a critical transcription factor that governs megakaryocyte development and differentiation. GATA-1-deficient megakaryocytes proliferate excessively and fail to undergo terminal differentiation, likely as a consequence of an aberrant gene expression program. In our studies to identify key signal transduction pathways that are dysregulated in the absence of GATA-1, we observed that STAT1, a gene of unknown function in megakaryocytes, was significantly downregulated in GATA-1-deficient megakaryocytes. Surprisingly, ectopic expression of STAT1, or IRF-1, a downstream effector of STAT1 signaling, promoted megakaryocytic differentiation of G1ME cells, a GATA-1-null and TPO-dependent erythromegakaryocytic cell line. Both STAT1- and IRF-1-transduced G1ME cells, but not GFP-expressing control cells, displayed multiple markers of terminal differentiation, including expression of CD42, as well as a significant fraction of polyploid cells. BrdU labeling assays confirmed that these transduced cells continued to effectively synthesize DNA, suggesting that STAT1 signaling switches these cells from mitosis to endomitosis. STAT1 and IRF-1 also modulated TPO signaling evidenced by enhanced STAT3 and STAT5 phosphorylation. Since STAT1 signaling is activated by IFN-g, we further investigated whether IFN-g-treatment also led to polyploidization of G1ME cells. IFN-g effectively promoted polyploidization of both G1ME cells and primary bone marrow-derived CD41+ cells in the absence of TPO. Finally, overexpression of STAT1 in primary bone marrow cells isolated from GATA-1 knockdown mice also promoted polyploidization in the absence of TPO. Taken together, these data reveal a GATA-1-independent mechanism by which STAT1 signaling promotes megakaryocyte differentiation.

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