Impaired neutrophil maturation in truncated murine G-CSF receptor–transgenic mice

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 2990-2995 ◽  
Author(s):  
Tetsuo Mitsui ◽  
Sumiko Watanabe ◽  
Yoshihiro Taniguchi ◽  
Sachiyo Hanada ◽  
Yasuhiro Ebihara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Acute Myelocytic Leukemia ◽  
Wild Type ◽  
Maturation Arrest ◽  
Mutant Receptor ◽  
Cell Counts ◽  
Marrow Cells

Abstract Severe congenital neutropenia (SCN) is a hematopoietic disorder characterized by neutropenia in peripheral blood and maturation arrest of neutrophil precursors in bone marrow. Patients with SCN may evolve to have myelodysplastic syndrome or acute myelocytic leukemia. In approximately 20% of SCN cases, a truncation mutation is found in the cytoplasmic region of the granulocyte colony-stimulating factor receptor (G-CSFR). We then generated mice carrying murine wild-type G-CSFR and its mutants equivalent to truncations at amino acids 718 and 731 in human G-CSFR, those were reported to be related to leukemic transformation of SCN. Although numbers of peripheral white blood cells, red blood cells, and platelets did not differ among mutant and wild-type G-CSFR transgenic (Tg) mice, both of the mutant receptor Tg mice had one third of peripheral neutrophil cell counts compared with wild-type receptor Tg mice. The mutant receptor Tg mice also showed impaired resistance to the infection with Staphylococcus aureus. Moreover, bone marrow of these Tg mice had an increased percentage of immature myeloid cells, a feature of SCN. This maturation arrest was also observed in in vitro cultures of bone marrow cells of truncated G-CSFR Tg mice under G-CSF stimulation. In addition, clonal culture of bone marrow cells of the truncated G-CSFR Tg mice showed the hypersensitivity to G-CSF in myeloid progenitors. Our Tg mice may be useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.

scholarly journals Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2148-2159 ◽  
Author(s):  
Harshal H. Nandurkar ◽  
Lorraine Robb ◽  
David Tarlinton ◽  
Louise Barnett ◽  
Frank Köntgen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Null Mutation ◽  
Wild Type ◽  
Normal Numbers ◽  
Interleukin 11 ◽  
Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

scholarly journals Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2148-2159 ◽  
Author(s):  
Harshal H. Nandurkar ◽  
Lorraine Robb ◽  
David Tarlinton ◽  
Louise Barnett ◽  
Frank Köntgen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Null Mutation ◽  
Wild Type ◽  
Normal Numbers ◽  
Interleukin 11 ◽  
Marrow Cells

Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

The Effect of Rara Haploinsufficiency in a Mouse Model of Acute Promyelocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3475-3475
Author(s):  
John S. Welch ◽  
Timothy Ley
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Promyelocytic Leukemia ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Maturation Arrest ◽  
Marrow Cells

Abstract 3475 Poster Board III-412 Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation, which leads to expression of the fusion protein that initiates this disease (PML-RARA), the creation of a small and inconsistently expressed RARA-PML fusion protein, and haploinsufficiency for both RARA and PML. While alternative translocations and fusion proteins have been associated with APL, RARA appears to be a necessary member of each (e.g. PLZF-RARA, NPM-RARA, STAT5b-RARA, NuMA-RARA). Furthermore, RARA activity is important for myeloid maturation. We have therefore explored the effect of RARA haploinsufficiency in the murine Cathepsin-G PML-RARA (mCG-PR) model of APL. We crossed RARA+/− mice with mCG-PR mice, all on a C57/B6 background. Both mCG-PR and the mutant RARA allele were observed at normal Mendelian ratios. At 8 weeks, mCG-PR x RARA+/− mice exhibited normal peripheral blood counts, spleen sizes and total bone marrow cells. Bone marrow cells from 8-week old mCG-PR x RARA+/− and mCG-PR mice both exhibited increased self-renewal in methylcellulose replating assays, with increased average cells per colony in mCG-PR x RARA+/− and mCG-PR colonies compared to wild type (28,470 ± 6,000 and 15,400 ± 2,375 vs 7,700 ± 630, p = 0.0001 and 0.0003 following the initial plating). Five months after bone marrow transplantation at a 1:9 ratio with competitor Ly5.1 bone marrow cells, mCG-PR derived cells have engrafted and expanded in four recipients to 10%, 12.4%, 13.8% and 15.2% of peripheral blood cells, while mCG-PR x RARA+/− derived cells have more robustly expanded to 8.7%, 20.2%, 24.5%, and 30.3% of peripheral blood cells. A cohort of 29 mCG-PR x RARA+/− and 20 mCG-PR mice was subjected to a tumor watch. With an average follow up of 10 months, we have observed AML arise in 9 mCG-PR x RARA+/− mice and 11 mCG-PR mice (an additional 20 and 9 mice in each respective cohort remain at risk of leukemia and will be subsequently evaluated). Leukemia arising from mCG-PR mice exhibited leukocytosis, splenomegaly and variable myeloid differentiation arrest in the peripheral blood and spleen, as measured by manual differential counts and cKit/CD11b expression in the Gr1+ compartment. Leukemia from two mice had marked promyelocytic maturation arrest, but 5 others retained differentiation, with 30-60% bands and ring-formed neutrophils in the spleen, which correlated with similar percentages of Gr1+/cKit-/CD11b+ cells (4 mice died with splenomegaly noted during necropsy and were not further characterized). AML arising in mCG-PR x RARA+/− mice displayed similar variability in maturation arrest, but these mice had a trend towards lower peripheral blood WBC counts compared to mCG-PR AML (mean WBC 28,000/mcl ± 26,000 vs 82,000/mcl ± 71,000/mcl, p = 0.06) and larger spleen size (mean 1,425 mg ± 475 mg vs 1,017 mg ± 193 mg, p = 0.03). In addition, 6/9 mCG-PR x RARA+/− mice with AML had marked cervical lymphadenopathy caused by infiltrating AML cells. Only 1/11 mCG-PR mice with AML displayed lymphadenopathy, which previously has been observed only rarely with this mouse model (Westervelt et al, Blood 2003). Both mCG-PR x RARA+/− and mCG-PR AML cells responded to ATRA with increased maturation in vitro and in vivo, a shift from cKit+/CD11b- to cKit-/CD11b+ expression, a loss of self renewal capacity, and improved survival compared to untreated controls, as expected. In sum, mCG-PR x RARA+/− mice are different from mice with mCG-PR alone. RARA haploinsufficiency further perturbs hematopoiesis in mCG-PR mice, accelerating an early competitive advantage conferred by PR expression, and possibly altering the pattern of homing and peripheralization of AML cells. Lower peripheral white blood cell counts in these animals more accurately reproduce the relatively low white counts of most patients with APL. These data strongly suggest that RARA haploinsufficiency contributes to the overall phenotype of APL initiated by PML-RARA, and thus is a relevant mutation created by the t(15;17) translocation. Disclosures: Welch: Cephalon: Research Funding; Eisai: Research Funding.

Kras Plays An Important Role In Generating Differentiated Blood Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2451-2451 ◽  
Author(s):  
Alisa Damnernsawad ◽  
Guangyao Kong ◽  
Yangang Liu ◽  
Yuan-I Chang ◽  
Jingfang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Background Kras is a small GTPase essential for mouse embryonic development. Although Kras-/- fetal liver cells reconstitute recipient mice indistinguishably from wild-type cells, chimeric mice generated from injection of Kras-/- embryonic stem cells into wild-type blastocysts show little contribution of knockout cells to hematopoietic tissues even when these cells contribute to all the other tissues to a high degree. These results suggest that Kras plays an important role in adult hematopoiesis. However, early embryonic lethality of Kras-/- mice prevents further investigation of Kras functions in adulthood. To overcome this problem, we generated Kras conditional knockout mice (Krasfl/fl), which allow the deletion of Kras by the Cre recombinase in desired tissues and at desired developmental stages. Method We used two transgenic Cre lines, Mx1-Cre and Vav-Cre, to knockout Kras in adult hematopoietic system. The Mx1 promoter is induced by interferon signaling, which can be triggered by injections of polyinosinic-polycytidylic acid (pI-pC). The Vav promoter drives Cre expression specifically in fetal liver hematopoietic cells since E11.5 as well as in adult hematopoietic tissues. Both Cre lines efficiently deleted Kras expression in above 95% of hematopoietic cells as judged by single hematopoietic stem cell (HSC) genotyping. Results obtained from these two different Cre lines were essentially same. Results We found that the frequency and absolute number of Kras-/- HSCs, multipotent progenitors (MPPs), LSK (Lin- Sca1+ cKit+) cells, myeloid progenitors and common lymphoid progenitors are comparable to wild-type control cells. Consistent with this observation, cytokine signaling in Kras-/- hematopoietic stem/progenitor cells (HSPCs, Lin- cKit+) is indistinguishable from control HSPCs. In contrast, the percentage of CD19+ B-cells is moderately but significantly reduced in Kras-/- spleens and concomitantly cytokine-evoked ERK1/2 activation is greatly reduced in differentiated blood cells. To determine whether Kras plays an important role in regulating HSC functions, we performed a competitive bone marrow reconstitution assay using CD45.2+ control or Kras-/- bone marrow cells mixed together at ratios 1:1 and 3:1 with congeneic competitor cells (CD45.1+ bone marrow cells). Kras-/- bone marrow cells show significantly reduced long-term reconstitution in recipient mice compared to control cells (10% vs 45%). The reduced reconstitution is persistent in the secondary and tertiary recipients. However, detailed analysis in primary and secondary recipients revealed that the frequency of Kras-/- HSCs and MPPs is comparable to that of control cells and Kras-/- progenitor cells are also largely normal, indicating that Kras is dispensable for adult HSC functions but might play an important role in generating differentiated blood cells. The reduced generation of myeloid cells is further validated in an in vitro culture assay, in which we quantitatively measured the myeloid cell production from Lin- progenitor cells. Conclusions Our results indicate that loss-of-Kras could be compensated by other Ras isoforms in adult HSCs. However, in mature blood cells, Kras deficiency results in greatly reduced cytokine-evoked ERK1/2 activation. Under a stressed condition (e.g. competitive bone marrow transplantation), the generation of Kras-/- blood cells is defective. Taken together, our study reveals a novel and unique function of Kras in regulating adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals BAP1 Loss Results in EZH2-Dependent Transformation in Myelodysplastic Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 713-713
Author(s):  
Lindsay M LaFave ◽  
Wendy Béguelin ◽  
Richard Koche ◽  
Matt Teater ◽  
Efthymia Papalexi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Chip Sequencing ◽  
Cell Counts ◽  
Marrow Cells

Abstract Recurrent somatic loss-of-function mutations in ASXL1 (Addition of sex combs-like 1) are common genetic events in a spectrum of myeloid malignancies and these alterations demarcate patients with poor outcome. ASXL1 forms a chromatin regulatory complex with the ubiquitin hydrolase BAP1 (BRCA1 associated protein-1), a protein that has been found to be transcriptionally repressed in MDS patients. These data are consistent with BAP1 having tumor suppressive activity in MDS; however, the mechanism by which disruption of the ASXL1-BAP1 axis leads to transformation is not well understood. We conditionally deleted Bap1 in the murine hematopoietic system utilizing Mx1-Cre (hereafter referred to as Bap1 KO). One hundred percent of mice with confirmed Bap1 deletion developed a fully penetrant MDS-like disease characterized by leukocytosis, anemia, and splenomegaly. Bap1 KO mice have an expansion of the granulocyte macrophage progenitor compartment (GMP; Lin- c-Kit+ Sca1- CD34+ Fcϒ+). Given the role of BAP1 in epigenetic regulation, we investigated the effect of Bap1 loss on chromatin state and transcriptional output. We first assessed epigenetic changes in Bap1 KO mice by performing histone mass spectometry in control and Bap1 KO hematopoietic stem and progenitor cells (HSPCs, c-Kit+ enriched). Bap1 loss increased H3K27me2/3 at the expense of H3K27me0/1. We confirmed that H3K27me3 was increased in Bap1 KO bone marrow cells by completing H3K27me3 ChIP-Sequencing in HSPCs. Enumeration of H3K27me3 peaks in Bap1 KO versus control cells indicated an increase in H3K27me3 domains (Figure A). We next overlaid RNA-Sequencing from GMP sorted Bap1 KO bone marrow cells with genes marked by H3K27me3, as indicated by ChIP-Sequencing. We found that Bap1 loss resulted in a global decrease in gene expression (68% downregulated, 657/968 genes, p-adj <0.01) and that increased H3K27me3 identified genes with reduced expression after Bap1 loss (NES=-1.39, FDR<0.001) (Figure A). Gene set enrichment analyses (GSEA) revealed that genes that were altered following depletion of Bap1 corresponded to differentiation, hematopoietic lineage specification, and proliferation pathways. Combined, these data suggest that Bap1 depletion results in increased H3K27me3 and represses gene targets implicated in normal and malignant hematopoiesis. Given the alterations in H3K27me3 in Bap1 KO mice, we investigated whether Bap1- deficient transformation could be rescued by abrogation of PRC2-mediated gene repression. We developed a genetic model with compound deletion of Bap1 and Ezh2, the catalytic component of the PRC2 complex. Co-deletion of Bap1 and Ezh2 resulted in a phenotypic rescue of Bap1 KO associated splenomegaly (spleen weights, Bap1 KO avg. 541.6 mg, Bap1/Ezh2 KO avg. 157.0 mg, p<0.005) (Figure B), leukocytosis (white blood cells counts, Bap1 KO avg. 51 K/uL, Bap1/Ezh2 KO avg. 8 K/uL, p <0.005), anemia (hematocrit, Bap1 KO avg. 28.2%, Bap1/Ezh2 KO avg. 46.0%, p<0.005). Importantly, the increased H3K27me3 levels in Bap1 KO mice were reduced in Bap1/Ezh2 KO mice (Figure B), suggesting that loss of Bap1 leads to Ezh2-dependent malignant transformation. EZH2 small molecule inhibitors have proven effective in EZH2-dependent models of B cell lymphoma. To determine if Ezh2 inhibition was efficacious in the setting of Bap1 loss, we treated a cohort of Bap1 KO mice with either vehicle (NaCMC) or 500 mg/kg EPZ011989, an EZH2 inhibitor with in vivo activity. Treatment of Bap1 KO mice for 16 days resulted in significant reduction of splenomegaly (spleen weights, vehicle avg. 522.0, EPZ011989 treated avg. 216.2, p<0.005) (Figure C) and anemia (white blood cell counts, vehicle avg. 61.7 K/uL, EPZ011989 treated avg. 14.5 K/uL, p <0.005), consistent with the phenotype of our genetic Bap1/Ezh2 compound deletion model. These data suggest that decreased BAP1 expression could serve as a biomarker for sensitivity to EZH2 inhibition. Figure 1. Figure 1. Disclosures Knutson: Epizyme, Inc: Employment. Campbell:Epizyme, Inc: Employment. Keilhack:Epizyme: Employment, Equity Ownership. Melnick:Janssen: Other: Research; ROCHE: Other: Research; Genentech: Speakers Bureau; Celgene: Consultancy; Eli Lilly: Consultancy; Epizyme: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

Abstract 19419: Anti Atherogenic Effects of Micro RNA - 21

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Rihab E Hamed-Berair ◽  
Abhinav Agarwal ◽  
Marcin Wysoczynski ◽  
Srinivas D Sithu ◽  
Nalinie S Wickramasinghe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Interferon Gamma ◽  
Bone Marrow Cells ◽  
Plasma Cholesterol ◽  
Oxidized Ldl ◽  
White Blood Cells ◽  
Micro Rna ◽  
Chimeric Mice ◽  
Wild Type ◽  
Late Apoptosis

Micro RNA-21 (miR-21), an evolutionary conserved micro RNA has been implicated in the pathogenesis of restenosis, myocardial infarction and heart failure. However, little is known about the role of miR-21 in atherosclerosis. Our data show that in vitro, LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in bone marrow derived macrophages (BMDM). Feeding the LDL receptor-knockout (LDLR-KO) mice with western diet (WD, 8-20 weeks) increased the abundance of miR-21 in BMDM by 1.5-fold (P<0.05). Basally, BMDM isolated from miR-21-KO mice showed induction of TNF alpha, interferon gamma, M-CSF, RANTES, IP10 and LIF by (1.5-3.0-fold); increased early and late apoptosis (2-3-fold, P<0.05); and induced PDCD4 and PTEN. We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86) and decreased IL-10 formation as compared with WT BMDM. Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase 3. Characterization of miR-21-KO mice showed 30% decrease in white blood cells and neutrophils in KO mice. However, levels of circulating immune cells and common progenitor cells in bone marrow of miR-21-KO mice were comparable with wild type mice. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. This was accompanied by increased accumulation of macrophages in the non-necrotic areas of the lesion and decrease in lesional smooth muscle cells. Plasma cholesterol levels, and lesional collagen and T-cell levels in the miR-21 chimeric mice were comparable with wild type chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis by inhibiting macrophage apoptosis, necrosis and inflammation.

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 829-836 ◽  
Author(s):  
Timothy W. Austin ◽  
Suzan Salimi ◽  
Gabor Veres ◽  
Franck Morel ◽  
Heini Ilves ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Marrow Cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

scholarly journals Retroviral-Mediated Gene Transfer of gp91phox Into Bone Marrow Cells Rescues Defect in Host Defense Against Aspergillus fumigatus in Murine X-Linked Chronic Granulomatous Disease

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Helga Björgvinsdóttir ◽  
Chunjin Ding ◽  
Nancy Pech ◽  
Mary A. Gifford ◽  
Ling Lin Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Aspergillus Fumigatus ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Bone Marrow Cells ◽  
Superoxide Production ◽  
Granulomatous Disease ◽  
Wild Type ◽  
Marrow Cells

Abstract The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

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