The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1434-1434
Author(s):  
Jianhui Wang ◽  
Ruimin Pan ◽  
Michael A. Nardi ◽  
Zongdong Li
Keyword(s):  
Nadph Oxidase ◽  
In Vitro Culture ◽  
Conflicts Of Interest ◽  
Signal Pathways ◽  
Mouse Bone Marrow ◽  
Colony Forming Units ◽  
Sequential Activation ◽  
12 Lipoxygenase ◽  
Hiv 1

Abstract Abstract 1434 We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation. Disclosures: No relevant conflicts of interest to declare.

The inhibition effect of anti-GPIIIa49–66 antibody on megakaryocyte differentiation

2011 ◽  
Vol 106 (09) ◽  
pp. 484-490 ◽  
Author(s):  
Michael Nardi ◽  
Ruimin Pan ◽  
Jianhui Wang ◽  
Zongdong Li
Keyword(s):  
Nadph Oxidase ◽  
In Vitro Culture ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Colony Forming Units ◽  
Cord Blood Cd34 ◽  
Sequential Activation ◽  
12 Lipoxygenase ◽  
Hiv 1

SummaryWe previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49–66 epitope. Anti-GPIIIa49–66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)- HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49–66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti- GPIIIa49–66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49–66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49–66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49–66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49–66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 μM). We therefore conclude that the anti-GPIIIa49–66 antibody inhibits MK differentiation through β3 integrin signalling independent of ROS release.

In vitro p53 and/or Rb antisense oligonucleotide treatment in association with growth factors induces the proliferation of peripheral hematopoietic progenitors

1995 ◽  
Vol 108 (3) ◽  
pp. 1287-1293
Author(s):  
T. Mahdi ◽  
A. Brizard ◽  
C. Millet ◽  
P. Dore ◽  
J. Tanzer ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Types ◽  
Suppressor Gene ◽  
Antisense Oligodeoxynucleotide ◽  
Signal Pathways ◽  
Semisolid Medium ◽  
Gm Csf ◽  
Colony Forming Units ◽  
Macrophage Colony

In this work we intended to determine whether p53 and/or retinoblastoma (Rb) tumor suppressor genes are involved at specific stages in the process of in vitro human peripheral stem cell hematopoiesis. Mononuclear peripheral blood cells were depleted of adherent cells and T lymphocytes (A-T-PMCs). Cells were then cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types. A-T-PMCs were exposed to p53 and/or Rb sense, scrambled DNA and antisense oligodeoxynucleotides. p53 and/or Rb antisenses (but not their senses or scrambled DNA) treatment of A-T-PMCs resulted in a significantly increase in the number of granulocyte/macrophage colony-forming units (CFU-GM) in the presence of interleukin-3 (IL-3) and/or granulocyte/macrophage colony-stimulating factor (GM-CSF). After antisense treatment, blast forming units/erythroblasts (BFU-E) derived from A-T-PMCs cultured in the presence of IL-3 + erythropoietin (Epo) were also increased whereas colony forming units/erythroblasts (CFU-E) were not markedly affected in the presence of Epo only. Megakaryocytic colony (CFU-Meg) formation from A-T-PMCs in the presence of interleukin-6 (IL-6) + IL-3 + Epo was also increased after antisense oligodeoxynucleotide treatment. These results are consistent with the hypothesis that p53 and Rb tumor suppressor gene products are involved in the control of distinct signal pathways in different peripheral progenitor cells.

Non-Competitive Protein Phosphatase-1 Inhibitors Prevent Sickling of SS RBCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4038-4038
Author(s):  
Jerod Hairston ◽  
Keon Combi ◽  
Altreisha Foster ◽  
Bak Kim ◽  
Victor R. Gordeuk ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Protein Phosphatase 1 ◽  
Cell Protein ◽  
Trypan Blue Exclusion ◽  
Future Design ◽  
Small Molecular Inhibitors ◽  
Hiv 1

Abstract Abstract 4038 Poster Board III-974 Protein phosphatase-1 (PP1) has been implicated in the regulation of KCC (K:Cl) transporters, which transport K+ and Cl- ions from red blood cells (RBCs) and in the setting of sickle cell disease may contribute to RBC dehydration and sickling. We have studied host cell protein phosphatase-1 (PP1) in the context of HIV-1 replication and designed novel small molecule non-competitive inhibitors of PP1 that are efficient in the inhibition of HIV-1 but not toxic for cultured cells. We analyzed the effect of our novel non-competitive PP1 inhibitors and the conventional competitive PP1 inhibitor, ocadaic acid, on the sickling of hemoglobin SS RBCs in vitro. We cultured hemoglobin SS RBCs overnight at 1% O2 in the presence of the PP1 inhibitors and then photographed the RBCs and counted the percentage of sickled RBCs. We found that the non-competitive PP1 inhibitor, 1E7-04 prevented RBC sickling by 40% at 10 mM concentration. The 1E7-04 was not toxic at 10 mM concentration for cultured CEM T cells as determined by trypan blue exclusion assay using an automatic cell counter. Our study suggests that small molecular inhibitors of PP1 might be candidates for the future design of anti-sickling drugs. Acknowledgments. This work was supported by NHLBI grant U54HL090508-02; NHLBI grant R25 HL003679-08 from the National Institute of Helath and The Office of Research on Minority Health and by U.S. Civilian Research & Development Foundation grant. Disclosures: No relevant conflicts of interest to declare.

Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2321-2321
Author(s):  
Brandon K Hadland ◽  
Barbara Varnum-Finney ◽  
Irwin D. Bernstein
Keyword(s):  
Notch Signaling ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Signal Pathways ◽  
Mouse Bone Marrow ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Notch Activation

Abstract Abstract 2321 An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC. Disclosures: Bernstein: Seattle Genetics, Inc.: Consultancy.

Passage-Dependent Cancerous Transformation Of Human Mesenchymal Stem Cell Of Adipose Origin During In Vitro Culture

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4862-4862
Author(s):  
Jung Ah Kim ◽  
Qute Choi ◽  
Kyong Ok Im ◽  
Ji Seok Kwon ◽  
Si Nae Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Telomere Length ◽  
Conflicts Of Interest ◽  
Bac Clone ◽  
Interphase Cell ◽  
Cytogenetic Changes ◽  
Chromosomal Changes

Abstract Introduction In vitro culture of adult human mesenchymal stem cells (hMSCs) can induce cancerous transformation, depending on environmental factors. To evaluate the passage dependent chromosomal changes of hMSCs toward malignant transformation, we passaged adipose origin hMSC to 9th passage and analyzed cytogenetic change, molecular cytogenetic changes, and telomere length variations on every passage. Methods On each passage, in situ karyotyping was performed on 3 separate batches with subsequent Giemsa staining. Karyotyping was analyzed using Metafer system (MetaSystems, Altlussheim, Germany). For analysis of nonproliferating interphase cell, each chromosome were counted with centromere fluorescent in situ hybridization (FISH) using Same Day OligoFISH™(Cellay Inc., Cambridge, Massachusetts, USA). Telomere length was analyzed using FISH technique with a Cy3-lableled Telomere peptide nucleic acid (PNA) FISH kit (DakoCytomation Denmark A/S, Glostrup, Denmark). To confirm the chromosomal translocation appeared by in situ karyotyping, we made home-brew FISH probe with bacterial artificial chromosome(BAC) clone and quantitated the proportion of abnormal cells by interphase FISH. Results On 5th passage, translocation and polysomy of chromosome 7 and 9 appeared, and on 6th passage, additional translocation t(6; 10) appeared. ISCN Karyotypes of chromosomal changes from 5th passage to 7th passage were 48,XX,+7,t(7;22)(q11.22;q13.3),+9[4]/46,XX[21] → 47,XX,+7[2]/47,XX,t(6;10)(q21;q25.1),+7[2]/46,XX[13] → 48,XX,+7,t(7;22)(q11.22;q13.3),+9[6]/ 47,XX,+7[5]/46,XX[9]. Telomere length was decreased on late passages. Fusion signal of t(7;22) on passage 5(fig 1) and that of t(6;10) on passage 6(fig 2) were confirmed by BAC clone. Conclusions The behavior of late passage (from passage 5) follows a cytogenetic profile similar to that of transformed cancer cells. Cytogenetic abnormalities which were not observed in earlier passage, showed up and disappeared, but eventually persisted during passages. We suggest in vitro environment cause hMSCs to undergo cancer-like cytogenetic changes. It is of great importance to test safeguards for clinical applications of human stem cells manufactured in vitro. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Amin Hadi ◽  
Abbas Rastgoo ◽  
Maryam Eskandarian ◽  
Nooshin Haghighipour ◽  
Azam Bolhassani
Keyword(s):  
Gene Transfection ◽  
Therapeutic Vaccine ◽  
Cyclic Stretch ◽  
Mouse Bone Marrow ◽  
Transfection Efficacy ◽  
Protein Boost ◽  
Mechanical Methods ◽  
Protein Immunization ◽  
Hiv 1

An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.

Deregulated Hoxa1 Expression in Murine Bone Marrow Results in An MDS-Like Phenotype

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2423-2423
Author(s):  
Jean Hendy ◽  
Stewart A Fabb ◽  
Meaghan Wall ◽  
Lorraine J Gudas ◽  
Grant A. McArthur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cell ◽  
Insertional Mutagenesis ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Proliferative Potential ◽  
Mouse Bone Marrow ◽  
Post Transplant

Abstract Abstract 2423 Homeobox (Hox) genes have been shown to play critical roles in normal and aberrant hematopoiesis, however, little is known about the role of Hoxa1. Unlike most Hox genes, Hoxa1 is expressed as two different isoforms: full length Hoxa1 (FL-Hoxa1) and a truncated form, Hoxa1-T, resulting from alternative splicing within exon 1 of the gene. The two isoforms are expressed differentially in immature hematopoietic cells. Overexpression of FL-Hoxa1 and Hoxa1-T in mouse bone marrow (BM) cells significantly increased, and significantly decreased, respectively, cell proliferation in vitro compared to control (MXIE) transduced BM cells. These findings suggested that Hoxa1-T may negatively regulate FL-Hoxa1. We therefore generated a mutant Hoxa1 (muHoxa1) that expresses FL-Hoxa1 but was no longer capable of generating Hoxa1-T by changing the AGC serine to a TCT serine at the splice site of Hoxa1-T. Hoxa1 levels in muHoxa1-overexpressing BM were significantly higher than those in FL-Hoxa1 BM. Furthermore, BM cells overexpressing muHoxa1 had higher proliferative potential in vitro than FL-Hoxa1-overexpressing BM. Their in vivo potential was therefore assessed. At 12 weeks post-transplant, all primary transplant recipients had similar %GFP expression in the peripheral blood (PB), being approximately 10%. At this time point all recipients of muHoxa1-overexpressing BM cells displayed thrombocytopenia (mean ± SEM PB platelets (x 106/ml): MXIE= 1062 ± 75; FL-Hoxa1= 1053 ± 146; muHoxa1= 681 ± 71*, *P<0.05 vs MXIE). Secondary transplants were performed into irradiated and non-irradiated recipients. Strikingly, the %GFP+ve cells were markedly increased in the PB of recipients of muHoxa1 BM (%GFP: MXIE: 0.29 ± 0.06; FL-Hoxa1: 3.33 ± 1.38; muHoxa1: 27.46 ± 8.35*; *P<0.05 vs MXIE and FL-Hoxa1). All secondary recipients of muHoxa1 BM developed myeloid neoplasias, resembling myelodysplastic syndrome (MDS). The thrombocytopenia persisted (PB platelets (x 106/ml): MXIE: 923 ± 42; FL-Hoxa1: 812 ± 38; muHoxa1: 388 ± 108*; *P<0.001 vs MXIE and FL-Hoxa1) and secondary recipients of muHoxa1 BM developed anemia (PB Hb (g/L): MXIE: 143 ± 2.6; FL-Hoxa1: 146 ± 2.6; muHoxa1: 117 ± 5.2*, *P<.0005 vs MXIE and FL-Hoxa1). The PB leukocyte counts in the majority of muHoxa1 recipients were unchanged compared to MXIE and FL-Hoxa1 recipients, however, muHoxa1 PB cells were predominantly granulocytes. These neoplasms also occurred in non-irradiated recipients of muHoxa1 BM, although they had a much longer latency. Interestingly, 40% of the non-irradiated recipients developed acute myeloid leukemia between 6 and 12 months post-transplant. Importantly, integration site and cytogenetic analysis demonstrated that the malignant phenotype was not due to co-operating insertional mutagenesis or chromosomal instability in the transduced cells. The PB anemia was accompanied by a significant two-fold reduction in Ter119+ cells in BM of muHoxa1-overexpressing cells (GFP+ve= 16.3± 5.3%, GFP-ve= 34.8 ± 5.1%, P<0.05). This was due to an accumulation of the cells in early stages of erythroid differentiation, with increased proportions of proerythroblasts (GFP+ve: 36.6 ± 7.6%; GFP-ve: 8.6 ± 1.3% P<0.002) and basophilic erythroblasts (GFP+ve: 34.3 ± 3.4%; GFP-ve: 13.2 ± 3.5%, P<0.001) in muHoxa1 Ter119+ cells. The block in erythroid differentiation was also accompanied by significant alterations in the proportions of immature progenitors in recipients of muHoxa1 BM. GFP+ve cells were detectable in HSC and myeloid progenitor cell subsets, however, there was a significant increase in the proportion of megakaryocyte erythroid progenitors (MEPs) within the lineage negative, c-kit+, Sca-1 negative progenitor cell fraction (GFP+ve: 42.9 ± 2.1%; GFP-ve: 8.2 ± 1.3%, P<0.01). Significantly reduced GATA-1 expression was observed in both the GFP+ve proerythroblasts (400-fold) and MEPs (100-fold) compared to GFP-ve populations sorted from the same mice (P<0.001). Taken together, these results suggest that overexpression of FL-Hoxa1 in the absence of Hoxa1-T results in the development of MDS. This is partly due to an accumulation of MEPs and impaired differentiation of erythrocytes, both of which have significantly downregulated expression of GATA-1. Given the striking similarities in hematological phenotype to human patients with MDS, this novel mouse model will be invaluable in identifying the mechanisms contributing to this disease. Disclosures: No relevant conflicts of interest to declare.

Suppressing Hepcidin By Tocilizumab Effectively Improve Anemia in Inflammatory Diseases: Clinical Evidence and Basic Mechanisms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4006-4006
Author(s):  
Kazuyuki Yoshizaki ◽  
Soken-Nakazawa J Song ◽  
Hiroshi Kawabata
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Inflammatory Diseases ◽  
Hepatoma Cell ◽  
Signal Pathways ◽  
Rapid Reduction ◽  
Ameliorative Effect ◽  
Serum Hepcidin

Abstract Introduction Dysregulated production of hepcidin is implicated in anemia of inflammation (AI) and interleukin-6 (IL-6) is a major inducer of hepcidin production. Increased imflammatory cytokines, especially IL-6 is response for pathogenesis of multicentric Castleman's disease (MCD) and rheumatoid arthritis (RA). Patients and Method In this study we study role of hepcidin and IL-6 in AI patients with MCD or RA, by investigating the effect of tocilizumab, an anti-IL-6 receptor antibody, on the serum hepcidin and relationship between hepcidin and iron-related parameters. The mechanism involving inflammatory anemia in MCD and RA was further analyzed in vitro by studied the transcriptional regulation of hepcidin in hepatoma-derived cell lines in the presence of cytokines, antibodies against cytokines, inhibitors of signal pathways and other hematopoietic factors. Results Our data showed that treatment with tocilizumab resulted in a rapid reduction of serum hepcidin-25 in MCD and RA patients. Although treatment with anti-TNF-a antibody also resulted in decrease of serum hepcidin, compared to tocilizumab therapy it decrease by a smaller margin. Furthermore, long-term reductions of hepcidin-25 were observed in ten MCD cases for one year after the start of tocilizumab treatment, which was accompanied by progressive normalization of iron-related parameters and improved disease activity. In in vitro experiments, IL-6, but not TNF-a or IL-1,-induced upregulation of hepcidin mRNA in hepatoma cell lines was completely inhibited with tocilizumab, and partially by EPO as well as TNF-a, but enhanced by BMP4 and MCD patient’s serum. Discussion Our results suggest that, although multiple factors affect serum hepcidin levels, IL-6 plays an essential role in the induction of hepcidin. This accounts for the long-term ameliorative effect of IL-6 blockage with tocilizumab on anemia by inhibiting hepcidin production in MCD and RA patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of L-Asparaginase on Mouse Bone Marrow, Assayed by In Vitro Culture

Blood ◽  
1970 ◽  
Vol 36 (3) ◽  
pp. 385-389 ◽  
Author(s):  
CLARENCE H. BROWN ◽  
GEORGE P. CANELLOS ◽  
PAUL P. CARBONE
Keyword(s):  
Bone Marrow ◽  
In Vitro Culture ◽  
Rat Liver ◽  
Culture System ◽  
Human Subjects ◽  
Culture Technique ◽  
Mouse Bone Marrow ◽  
Therapeutic Doses ◽  
Marrow Cellularity

Abstract L-asparaginase administered in therapeutic doses to mice was found to reduce the total number of nucleated marrow cells and colony-forming cells when assayed 4 hours after administration by a methylcellulose bone marrow culture technique. Both marrow cellularity and the fraction of surviving IVCFC/femur were normal by 24 hours after injection, except with very large doses, at a time when there were sufficient quantities of L-asparaginase free or cellularly bound within the marrow to be toxic to the culture system. The enzyme could be removed from the marrow specimens with repeated washings. It is postulated that the bone marrow of the mouse is similar to the regenerating rat liver in that both have the ability to compensate for Lasparagine depletion, even in the presence of active L-asparaginase. The rarity of clinically observed myelosuppression in human subjects could be the result of a similar mechanism.

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