scholarly journals Germline MPO Variants Predispose to Myeloid Neoplasia: Potential Mechanisms Suggested By In Vivo and in Vitro Studies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3662-3662
Author(s):  
Sunisa Kongkiatkamon ◽  
Laila Terkawi ◽  
Vera Adema ◽  
Yihong Guan ◽  
Metis Hasipek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Repair ◽  
Murine Model ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Human Leukemia ◽  
Risk Alleles ◽  
Myeloid Neoplasia

Abstract In recent decades, there has been a substantial increase in recognition of germline (GL) predisposition to MN. Some congenital blood disorders predispose to subsequent myeloid neoplasia (MN). Severe congenital neutropenia (SCN) and Fanconi anemia serving as prominent examples. There is emerging evidence that heterozygous variants of disease recessive conditions may constitute risk alleles for late developing MN. However, their low penetrance may preclude identification of such alterations. Our previous analysisof allelic burden among selected combined potential GL predisposition genes to MN identified that the myeloperoxidase (MPO) gene (17q22-23) carried the highest risk of pathogenic alterations (Li, S.T, Leukemia, 2020). GL MPO mutations cause MPO deficiency syndrome, one of the most common inherited disorder associated with phagocyte defects and variable clinical penetrance. Despite the high prevalence, inherited MPO deficiency patients are asymptomatic and may remain unrecognized. We investigated 3280 MN and bone marrow failure syndromes (BMF) pts including 1138 MDS, 260 MDS/MPN, 1661 AML and 221 AA for presence of MPO mutations. In total, 38 different germline MPO mutations were identified in 143 cases. With a stringent bioanalytic pipeline, 28 pathogenic/likely pathogenic variants from 100 MN patients were included in the study. Germline MPO variants were significantly enriched in MN compared to control population (294 vs. 125 per 10 4 individuals; P<.0001) with an odds ratio of 2 (95%CI=1.6-2.5, P<.0001) for AML and 1.8 (95%CI=1.4-2.4, P<.0001) for MDS. The most common pathogenic/likely pathogenic variant (46%; 46/100) was a 3' splice site of intron11 (c.2031-2A>C) followed by R569W (13/100), M519fs* (13/100) and Y173C (6/100) with none being biallelic. While no differences were found in distribution of -7/7q, del5q, or del20q, MPO variants carriers harbored less tri-8 compared to wild type counterparts (2% vs. 12%; P=.008). Only FLT3 and NRAS were significantly associated with MPO mutations. We then investigated the effects of MPO deficiency on hematopoietic function in murine model using competitive repopulation assays, whereby the difference in CD45.1/CD45.2 isotypes transplanted in to ROSA26 (tdTomato-EGFP) recipients assayed by flow cytometry allowed distinction of the 2 grafts. Mpo-/- cells gained over time proliferative advantage over normal murine bone marrow cells. Reverse combinations (graft mix vs recipient) also replicated this result. This effect was not due to increased HSC content in Mpo-/- marrow as there were no significant differences in LSK, CMP and MEPs cells. We then investigated the clonogenic consequences of Mpo-/- cells in the setting of H 2O 2 induced oxidative stress, after exposing to hydrogen peroxide. Mpo-/- cells increased clonogenic potential after second serially replating. This prompted us to investigate an MPO inhibitor, AZD5904 in human leukemia cells with different MPO expression (HL-60, high; K562, low). High MPO expressors retained higher cell viability following H 2O 2 and MPOi addition compared to those without MPOi (85±10 vs.59±8, P<.0001) while no change was observed in low MPO stage cells with or without MPOi (84±2 vs.89±10, P=0.7). In conclusion, for the first time we demonstrated that germline MPO variants constitute risk alleles for MN evolution and replicated the potential replicative advantage of MPO deficient cells in murine model and potential mechanisms of the MPO deficiency in vitro including activation of non-homologous DNA repair response and error-prone DNA repair favoring replication. Repeated cycles of stress hematopoiesis e.g., due to increased infection rate may provide conditions in which MPO variants contribute to the risk of MN. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy.

Improved Survival and Recovery of Hematopoiesis Following Lethal Radiation by Chloroquine-Mediated Activation of ATM

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2228-2228
Author(s):  
Yiting Lim ◽  
Mohammad Hedayati ◽  
Akil Merchant ◽  
Yonggang Zhang ◽  
Theodore DeWeese ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dose Rate ◽  
Cell Lines ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Chloroquine Treatment ◽  
Radiation Induced

Abstract Abstract 2228 Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p < 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p<0.05). Similarly, we found that in vivo chloroquine treatment significantly increased the recovery of bone marrow myeloid CFC (p=0.02), suggesting that it impacted myeloid progenitors. To further validate this finding, we transplanted bone marrow from LDR irradiated mice into lethally irradiated CD45 congenic recipient mice, and found a significant improvement in early engraftment (4.2% vs. 0.4% engraftment at 6 weeks post-transplant, p=0.015). Chloroquine has been found to protect cancer cell lines from LDR radiation in vitro by activating ATM, an essential DNA damage sensor. We examined the effect of chloroquine on ATM and treated unradiated lin- bone marrow cells with chloroquine in vitro (35 ug/ml, 2 hr). Compared to control cells, chloroquine treated cells expressed 2.5-fold more phosphorylated ATM suggesting that the activation of ATM by chloroquine abrogated the lethal effects of LDR radiation in hematopoietic progenitors. We confirmed that ATM was required for chloroquine-mediated radioprotection by studying ATM null mice. In contrast to wild type mice, chloroquine treatment failed to protect ATM null mice from LDR radiation (9 Gy total) with 8/13 (62%) and 9/13 (69%) of animals surviving in treated or non treated mice, respectively (p=0.86). These data suggest that chloroquine exerts a radioprotective effect from LDR radiation by activating ATM in vivo, and may represent a novel means of limiting acute bone marrow failure in the event of widespread environmental LDR radiation exposure. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.

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.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

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.

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