41 results on '"Ramshaw HS"'
Search Results
2. Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice
- Author
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Lee, EM, Yee, D, Busfield, SJ, McManus, JF, Cummings, N, Vairo, G, We, A, Ramshaw, HS, Powell, JA, Lopez, AF, Lewis, ID, McCall, MN, Lock, RB, Lee, EM, Yee, D, Busfield, SJ, McManus, JF, Cummings, N, Vairo, G, We, A, Ramshaw, HS, Powell, JA, Lopez, AF, Lewis, ID, McCall, MN, and Lock, RB
- Abstract
The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor α- chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31-41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug.
- Published
- 2015
3. The IL-3/IL-5/GM-CSF common receptor plays a pivotal role in the regulation of Th2 immunity and allergic airway inflammation
- Author
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Asquith KL, Ramshaw HS, Hansbro PM, Beagley KW, Lopez AF, and Foster PS
- Subjects
animal structures ,Immunology ,Granulocyte-Macrophage Colony-Stimulating Factor ,Dendritic Cells ,respiratory system ,Allergens ,Lymphocyte Activation ,Mice, Mutant Strains ,Asthma ,respiratory tract diseases ,Cytokine Receptor Common beta Subunit ,Eosinophils ,Mice ,Th2 Cells ,1107 Immunology ,Cell Movement ,Macrophages, Alveolar ,Respiratory Hypersensitivity ,Animals ,Cytokines ,Interleukin-3 ,Interleukin-5 ,Signal Transduction - Abstract
The eosinophil is a central effector cell in allergic asthma. Differentiation and function of eosinophils are regulated by the CD4 Th2 cytokines IL-3, IL-5, and GM-CSF, which all signal through a common beta receptor subunit (betac). Recent therapeutic approaches targeting IL-5 alone have not ablated tissue accumulation of eosinophils and have had limited effects on disease progression, suggesting important roles for IL-3 and GM-CSF. By using a mouse model of allergic airways inflammation, we show that allergen-induced expansion and accumulation of eosinophils in the lung are abolished in betac-deficient (betac-/-) mice. Moreover, betac deficiency resulted in inhibition of hallmark features of asthma, including airways hypersensitivity, mucus hypersecretion, and production of Ag-specific IgE. Surprisingly, we also identified a critical role for this receptor in regulating type 2 immunity. Th2 cells in the lung of allergen-challenged betac-/- mice were limited in their ability to proliferate, produce cytokines, and migrate to effector sites, which was attributed to reduced numbers of myeloid dendritic cells in the lung compartment. Thus, the betac plays a critical role in allergen-induced eosinophil expansion and infiltration and is pivotal in regulating molecules that promote both early and late phases of allergic inflammation, representing a novel target for therapy.
- Published
- 2008
4. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL- 6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts
- Author
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Peters, SO, primary, Kittler, EL, additional, Ramshaw, HS, additional, and Quesenberry, PJ, additional
- Published
- 1996
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5. Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status
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Ramshaw, HS, primary, Rao, SS, additional, Crittenden, RB, additional, Peters, SO, additional, Weier, HU, additional, and Quesenberry, PJ, additional
- Published
- 1995
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6. New approaches in the treatment of asthma.
- Author
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Lopez, AF, Ramshaw, Hs, Woodcock, Jm, Bagley, Cj, McClure, Bj, Hercus, Tr, and Lopez, Af
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- *
ASTHMA treatment , *EOSINOPHILIA - Abstract
Summary Asthma is a common and complex inflammatory disease of the airways that remains incurable. Current forms of therapy are long term and may exhibit associated side-effect problems. Major participants in the development of an asthma phenotype include the triggering stimuli such as the allergens themselves, cells such as T cells, epithelial cells and mast cells that produce a variety of cytokines including IL-5, GM-CSF, IL-3, IL-4 and IL-13 and chemokines such as eotaxin. Significantly, the eosinophil, a specialized blood cell type, is invariably associated with this disease. The eosinophil has long been incriminated in the pathology of asthma due to its ability to release preformed and unique toxic substances as well as newly formed pro-inflammatory mediators. The regulation of eosinophil production and function is carried out by soluble peptides or factors. Of these IL-5, GM-CSF and IL-3 are of paramount importance as they control eosinophil functional activity and are the only known eosinophilopoietic factors. In addition they regulate the eosinophil life span by inhibiting apoptosis. While one therapeutic approach in asthma is directed at inhibiting single eosinophil products such as leukotrienes or single eosinophil regulators such as IL-5, we believe that the simultaneous inhibition of more than one component is preferable. This may be particularly important with eosinophil regulators in that not only IL-5, but also GM-CSF has been repeatedly implicated in clinical studies of asthma. The fact that GM-CSF is produced by many cells in the body and in copious amounts by lung epithelial cells highlights this need further. Our approach takes advantage of the fact that the IL-5 and GM-CSF receptors (as well as IL-3 receptors) utilize a shared subunit to bind, with high affinity, to these cytokines and the same common subunit mediates signal transduction culminating in all the biological activities mentioned. By generating the monoclonal antibody BION-1 to... [ABSTRACT FROM AUTHOR]
- Published
- 2001
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7. Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC
- Author
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Erwin M. Lee, Samantha J. Busfield, Andrew D. Nash, Con Panousis, M Wong, Angel F. Lopez, Daniel Thomas, Andrew W. Roberts, S Z He, Mark Biondo, Louis Fabri, Souravi Ghosh, Gino Vairo, Hal Braley, Richard B. Lock, Hayley S. Ramshaw, Busfield, SJ, Biondo, M, Wong, M, Ramshaw, HS, Lee, EM, Ghosh, S, Braley, H, Panousis, C, Roberts, AW, He, SZ, Thomas, D, Fabri, L, Vairo, G, Lock, RB, Lopez, AF, and Nash, AD
- Subjects
Cancer Research ,Myeloid ,CD34 ,receptors ,Mice, SCID ,Protein Engineering ,killer cells ,Mice, Inbred NOD ,hemic and lymphatic diseases ,antibodies ,animal ,neoplasm transplantation ,humans ,Antibody-dependent cell-mediated cytotoxicity ,GPI-linked proteins ,humanized ,leukemia ,acute ,Myeloid leukemia ,Hematology ,erythroblastic ,Iinbred NOD ,animals ,Killer Cells, Natural ,Leukemia, Myeloid, Acute ,Leukemia ,female ,medicine.anatomical_structure ,Oncology ,Monoclonal ,Female ,myeloid ,mice ,IgG ,macaca fascicularis ,xenograft model antitumor assays ,Interleukin-3 Receptor alpha Subunit ,monoclonal ,Biology ,CD16 ,SCID ,Antibodies, Monoclonal, Humanized ,GPI-Linked Proteins ,interleukin-3 receptor alpha subunit ,medicine ,Animals ,Humans ,antibody-dependent cell cytotoxicity ,natural ,disease models ,Receptors, IgG ,Antibody-Dependent Cell Cytotoxicity ,protein engineering ,medicine.disease ,Xenograft Model Antitumor Assays ,Disease Models, Animal ,Macaca fascicularis ,Immunology ,Leukemia, Erythroblastic, Acute ,Neoplasm Transplantation ,Ex vivo - Abstract
Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor α-chain (IL-3 Rα/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (FcγRIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34+CD38−CD123+ LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient's own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML. Refereed/Peer-reviewed
- Published
- 2014
8. Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency
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Mark A. Guthridge, Frank C. Stomski, Anthony Wynshaw-Boris, Kazuhito Toyo-oka, Angel F. Lopez, Paul Q. Thomas, Sally Martin, Hayley S. Ramshaw, Pike See Cheah, Quenten Schwarz, Xiangjun Xu, P. Coyle, M. van den Buuse, Cheah, P-s, Ramshaw, HS, Thomas, PQ, Toyo-Oka, K, Xu, X, Martin, S, Coyle, P, Guthridge, MA, Stomski, F, van den Buuse, M, Wynshaw-Boris, A, Lopez, AF, and Schwarz, QP
- Subjects
Male ,Neurogenesis ,Hippocampus ,Motor Activity ,14-3-3ζ ,Synapse ,Cellular and Molecular Neuroscience ,DISC1 ,Mice ,synapse ,Cell Movement ,medicine ,Animals ,Genetic Predisposition to Disease ,Bipolar disorder ,Maze Learning ,Molecular Biology ,Cells, Cultured ,Mice, Knockout ,Neurons ,biology ,neurodevelopment ,neuropsychiatric disorder ,Brain ,Cognition ,Recognition, Psychology ,Sensory Gating ,medicine.disease ,schizophrenia ,Mice, Inbred C57BL ,Psychiatry and Mental health ,Disease Models, Animal ,14-3-3 Proteins ,Autism spectrum disorder ,Schizophrenia ,biology.protein ,Female ,Glutamatergic synapse ,Psychology ,Neuroscience - Abstract
Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3f causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3f-Deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3f-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3f. Our data provide the first evidence of a direct role for 14-3-3f deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3f as a central risk factor in the schizophrenia protein interaction network. Refereed/Peer-reviewed
- Published
- 2011
9. Targeting the Human β c Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.
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Yip KH, McKenzie D, Ramshaw HS, Chao J, McClure BJ, Raquet E, Kraushaar T, Röder J, Maxwell M, Alhamdoosh M, Hammet A, Fong JH, Zeglinski K, Monaghan K, Pant H, Grimbaldeston MA, Vairo G, Wilson NJ, Owczarek CM, Hercus TR, Lopez AF, and Tumes DJ
- Subjects
- Animals, Cytokines, Eosinophils, Humans, Interleukin-3 metabolism, Interleukin-5 metabolism, Mice, Mice, Transgenic, Dermatitis, Contact, Granulocyte-Macrophage Colony-Stimulating Factor metabolism
- Abstract
Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (β
c ) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit βc , a property that has made βc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of βc has precluded testing of inhibitors of human βc in mouse models. To overcome this problem, we developed a human βc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human βc instead of mouse βc . Human βc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human βc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human βc receptor transgenic mouse as a unique platform to test the inhibitors of βc in vivo., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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10. Role of the β Common (βc) Family of Cytokines in Health and Disease.
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Hercus TR, Kan WLT, Broughton SE, Tvorogov D, Ramshaw HS, Sandow JJ, Nero TL, Dhagat U, Thompson EJ, Shing KSCT, McKenzie DR, Wilson NJ, Owczarek CM, Vairo G, Nash AD, Tergaonkar V, Hughes T, Ekert PG, Samuel MS, Bonder CS, Grimbaldeston MA, Parker MW, and Lopez AF
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- Cytokines genetics, Gene Expression Regulation physiology, Humans, Inflammation metabolism, Sepsis metabolism, Signal Transduction, Cytokines classification, Cytokines metabolism
- Abstract
The β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the βc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2018
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11. Corrigendum: 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function.
- Author
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Schoenwaelder SM, Darbousset R, Cranmer SL, Ramshaw HS, Orive SL, Sturgeon S, Yuan Y, Yao Y, Krycer JR, Woodcock J, Maclean J, Pitson S, Zheng Z, Henstridge DC, van der Wal D, Gardiner EE, Berndt MC, Andrews RK, James DE, Lopez AF, and Jackson SP
- Abstract
This corrects the article DOI: 10.1038/ncomms12862.
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- 2017
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12. High CD123 levels enhance proliferation in response to IL-3, but reduce chemotaxis by downregulating CXCR4 expression.
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Wittwer NL, Brumatti G, Marchant C, Sandow JJ, Pudney MK, Dottore M, D'Andrea RJ, Lopez AF, Ekert PG, and Ramshaw HS
- Abstract
High expression of the α chain of the interleukin-3 receptor (IL-3Rα; CD123) is a hallmark of acute myeloid leukemia (AML) leukemic stem cells (LSCs). Elevated CD123 expression is part of the diagnostic immunophenotyping of myeloid leukemia, and higher expression is associated with poor prognosis. However, the biological basis of the poorer prognosis is unclear, and may include heightened IL-3 signaling and non-cell autonomous interactions with the bone marrow (BM) microenvironment. We used TF-1 cells expressing different levels of CD123 and found elevated CD123 levels amplified the proliferative response to exogenous IL-3 and maintained viability in reducing IL-3 concentrations. This was associated with stronger activation of STAT5, Akt, and extracellular signal-regulated kinase 1/2 in vitro. Surprisingly, in vivo e14.5 fetal liver cells transduced with retroviral constructs to express high CD123 failed to engraft in syngeneic recipients. In exploring the underlying mechanism for this, we found that CXCR4, a key molecule involved in LSC/BM interactions, was specifically downregulated in CD123 overexpressing cells in a manner dependent on IL-3 signaling. CXCR4 downregulation was sufficient to alter the chemotactic response of hematopoietic cells to stromal derived factor-1 (SDF-1). Thus, we propose that the overexpression of CD123 in AML LSC dictates their location by altering CXCR4/SDF-1 interaction in the BM, raising the possibility that this mechanism underpins the egress of BM AML LSC and more mature cells into the circulation., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.
- Published
- 2017
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13. miR-155 as a potential target of IL-3 signaling in primary AML cells.
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Sadras T, Kok CH, Perugini M, Ramshaw HS, and D'Andrea RJ
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- Cell Transformation, Neoplastic, Gene Expression Regulation, Leukemic, Humans, Interleukin-3 Receptor alpha Subunit genetics, Leukemia, Myeloid, Acute genetics, Tumor Cells, Cultured, Up-Regulation, Interleukin-3 metabolism, Leukemia, Myeloid, Acute metabolism, MicroRNAs metabolism, Signal Transduction
- Abstract
miR-155 has emerged as one of the key microRNAs (miRNAs) involved in normal and malignant myelopoiesis, and high expression of this miRNA has been flagged as a strong independent prognostic marker in Acute Myeloid Leukemia (AML). While elevated expression of miR-155 has been associated with FLT3-ITD mutations, other mechanisms which may regulate expression of this miRNA in AML remain largely unknown. Here, we present new evidence that miR-155 may be a prime target of IL-3 signaling in primary AML cells. This finding, together with the increasingly apparent role for miR-155 in oncogenesis, and the upregulation of the IL-3 receptor alpha subunit in AML, lead us to propose this pathway may significantly contribute to the leukemic transformation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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14. MicroRNA-194 Promotes Prostate Cancer Metastasis by Inhibiting SOCS2.
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Das R, Gregory PA, Fernandes RC, Denis I, Wang Q, Townley SL, Zhao SG, Hanson AR, Pickering MA, Armstrong HK, Lokman NA, Ebrahimie E, Davicioni E, Jenkins RB, Karnes RJ, Ross AE, Den RB, Klein EA, Chi KN, Ramshaw HS, Williams ED, Zoubeidi A, Goodall GJ, Feng FY, Butler LM, Tilley WD, and Selth LA
- Subjects
- Animals, Cell Line, Tumor, Epithelial-Mesenchymal Transition, GATA2 Transcription Factor physiology, Humans, MAP Kinase Signaling System physiology, Male, Mice, Neoplasm Invasiveness, Neoplasm Metastasis, Prostatic Neoplasms genetics, STAT3 Transcription Factor physiology, Suppressor of Cytokine Signaling Proteins physiology, MicroRNAs physiology, Prostatic Neoplasms pathology, Suppressor of Cytokine Signaling Proteins antagonists & inhibitors
- Abstract
Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021-34. ©2016 AACR ., (©2016 American Association for Cancer Research.)
- Published
- 2017
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15. Targeting sphingosine kinase 1 induces MCL1-dependent cell death in acute myeloid leukemia.
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Powell JA, Lewis AC, Zhu W, Toubia J, Pitman MR, Wallington-Beddoe CT, Moretti PA, Iarossi D, Samaraweera SE, Cummings N, Ramshaw HS, Thomas D, Wei AH, Lopez AF, D'Andrea RJ, Lewis ID, and Pitson SM
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Amino Alcohols pharmacology, Animals, Bone Marrow Cells drug effects, Bone Marrow Cells metabolism, Bone Marrow Cells pathology, Caspase Inhibitors pharmacology, Caspases genetics, Caspases metabolism, Cell Death drug effects, Cell Line, Tumor, Female, Humans, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute mortality, Leukemia, Myeloid, Acute pathology, Lysophospholipids metabolism, Mice, Mice, Inbred NOD, Molecular Targeted Therapy, Myeloid Cell Leukemia Sequence 1 Protein genetics, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Phosphotransferases (Alcohol Group Acceptor) genetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Protein Kinase Inhibitors pharmacology, Quinolines pharmacology, Receptors, Lysosphingolipid genetics, Receptors, Lysosphingolipid metabolism, Signal Transduction, Sphingosine analogs & derivatives, Sphingosine metabolism, Survival Analysis, Xenograft Model Antitumor Assays, Gene Expression Regulation, Leukemic, Leukemia, Myeloid, Acute drug therapy, Myeloid Cell Leukemia Sequence 1 Protein antagonists & inhibitors, Phosphotransferases (Alcohol Group Acceptor) antagonists & inhibitors, Receptors, Lysosphingolipid antagonists & inhibitors
- Abstract
Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34
+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML., (© 2017 by The American Society of Hematology.)- Published
- 2017
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16. 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function.
- Author
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Schoenwaelder SM, Darbousset R, Cranmer SL, Ramshaw HS, Orive SL, Sturgeon S, Yuan Y, Yao Y, Krycer JR, Woodcock J, Maclean J, Pitson S, Zheng Z, Henstridge DC, van der Wal D, Gardiner EE, Berndt MC, Andrews RK, James DE, Lopez AF, and Jackson SP
- Abstract
The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)-GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function.
- Published
- 2016
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17. Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.
- Author
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Lim GE, Piske M, Lulo JE, Ramshaw HS, Lopez AF, and Johnson JD
- Subjects
- 14-3-3 Proteins metabolism, Animals, Blood Glucose metabolism, Glucagon-Like Peptide-1 Receptor metabolism, Glucose Intolerance metabolism, Glucose Tolerance Test, Homeostasis physiology, Insulin blood, Mice, Mice, Knockout, Phosphorylation, Proto-Oncogene Proteins c-akt metabolism, 14-3-3 Proteins genetics, Enteroendocrine Cells metabolism, Glucagon-Like Peptide 1 metabolism, Glucagon-Like Peptide-1 Receptor genetics, Glucose Intolerance genetics, Insulin-Secreting Cells metabolism
- Abstract
Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal β-cell responsiveness to glucose when compared with wild-type littermate controls. In contrast with our observations after an ip glucose bolus, glucose tolerance was significantly improved in 14-3-3ζKO mice after an oral glucose gavage. This improvement in glucose tolerance was associated with significantly elevated fasting glucagon-like peptide-1 (GLP-1) levels. 14-3-3ζ knockdown in GLUTag L cells elevated GLP-1 synthesis and increased GLP-1 release. Systemic inhibition of the GLP-1 receptor attenuated the improvement in oral glucose tolerance that was seen in 14-3-3ζKO mice. When taken together these findings demonstrate novel roles of 14-3-3ζ in the regulation of glucose homeostasis and suggest that modulating 14-3-3ζ levels in intestinal L cells may have beneficial metabolic effects through GLP-1-dependent mechanisms.
- Published
- 2016
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18. Jak2V617F driven myeloproliferative neoplasm occurs independently of interleukin-3 receptor beta common signaling.
- Author
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Vu T, Austin R, Kuhn CP, Bruedigam C, Song A, Guignes S, Jacquelin S, Ramshaw HS, Hill GR, Lopez AF, and Lane SW
- Subjects
- Animals, Bone Marrow drug effects, Bone Marrow immunology, Bone Marrow pathology, Hematologic Neoplasms genetics, Hematologic Neoplasms pathology, Humans, Interleukin-3 genetics, Janus Kinase 2 genetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Myeloproliferative Disorders genetics, Myeloproliferative Disorders pathology, Receptors, Interleukin-3 genetics, STAT5 Transcription Factor genetics, STAT5 Transcription Factor immunology, Signal Transduction, Thrombopoietin pharmacology, Gene Expression Regulation, Neoplastic, Hematologic Neoplasms immunology, Interleukin-3 immunology, Janus Kinase 2 immunology, Myeloproliferative Disorders immunology, Receptors, Interleukin-3 immunology
- Published
- 2016
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19. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.
- Author
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Kular J, Scheer KG, Pyne NT, Allam AH, Pollard AN, Magenau A, Wright RL, Kolesnikoff N, Moretti PA, Wullkopf L, Stomski FC, Cowin AJ, Woodcock JM, Grimbaldeston MA, Pitson SM, Timpson P, Ramshaw HS, Lopez AF, and Samuel MS
- Subjects
- Animals, Epidermis metabolism, Mice, 14-3-3 Proteins metabolism, Cell Proliferation physiology, Homeostasis physiology, Signal Transduction physiology, Wound Healing physiology, rho-Associated Kinases metabolism
- Abstract
ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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20. 14-3-3ζ coordinates adipogenesis of visceral fat.
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Lim GE, Albrecht T, Piske M, Sarai K, Lee JTC, Ramshaw HS, Sinha S, Guthridge MA, Acker-Palmer A, Lopez AF, Clee SM, Nislow C, and Johnson JD
- Subjects
- 14-3-3 Proteins metabolism, 3T3-L1 Cells, Animals, Autophagy genetics, CCAAT-Enhancer-Binding Protein-delta metabolism, CCAAT-Enhancer-Binding Proteins genetics, CCAAT-Enhancer-Binding Proteins metabolism, Cyclin-Dependent Kinase Inhibitor p27 genetics, Cyclin-Dependent Kinase Inhibitor p27 metabolism, Flow Cytometry, Gene Expression Profiling, Hedgehog Proteins metabolism, Immunoblotting, In Vitro Techniques, Kruppel-Like Transcription Factors genetics, Kruppel-Like Transcription Factors metabolism, Mice, Mice, Knockout, Microscopy, Fluorescence, NIH 3T3 Cells, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Obesity metabolism, PPAR gamma genetics, PPAR gamma metabolism, Real-Time Polymerase Chain Reaction, Signal Transduction, Zinc Finger Protein Gli3, 14-3-3 Proteins genetics, Adipogenesis genetics, Intra-Abdominal Fat metabolism, Obesity genetics
- Abstract
The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27(Kip1). Indeed, concomitant knockdown of p27(Kip1) or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27(Kip1) abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis.
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- 2015
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21. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders.
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Xu X, Jaehne EJ, Greenberg Z, McCarthy P, Saleh E, Parish CL, Camera D, Heng J, Haas M, Baune BT, Ratnayake U, van den Buuse M, Lopez AF, Ramshaw HS, and Schwarz Q
- Subjects
- 14-3-3 Proteins deficiency, Animals, Disease Models, Animal, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins genetics, Dopamine Plasma Membrane Transport Proteins metabolism, Female, Gene Expression, Lateral Ventricles metabolism, Lateral Ventricles pathology, Male, Maze Learning, Mice, Mice, Inbred BALB C, Mice, Knockout, Mossy Fibers, Hippocampal metabolism, Pyramidal Cells metabolism, Schizophrenia metabolism, Schizophrenia pathology, Signal Transduction, 14-3-3 Proteins genetics, Mossy Fibers, Hippocampal pathology, Pyramidal Cells pathology, Schizophrenia genetics, Schizophrenia physiopathology, Spatial Memory
- Abstract
Sequencing and expression analyses implicate 14-3-3ζ as a genetic risk factor for neurodevelopmental disorders such as schizophrenia and autism. In support of this notion, we recently found that 14-3-3ζ(-/-) mice in the Sv/129 background display schizophrenia-like defects. As epistatic interactions play a significant role in disease pathogenesis we generated a new congenic strain in the BALB/c background to determine the impact of genetic interactions on the 14-3-3ζ(-/-) phenotype. In addition to replicating defects such as aberrant mossy fibre connectivity and impaired spatial memory, our analysis of 14-3-3ζ(-/-) BALB/c mice identified enlarged lateral ventricles, reduced synaptic density and ectopically positioned pyramidal neurons in all subfields of the hippocampus. In contrast to our previous analyses, 14-3-3ζ(-/-) BALB/c mice lacked locomotor hyperactivity that was underscored by normal levels of the dopamine transporter (DAT) and dopamine signalling. Taken together, our results demonstrate that dysfunction of 14-3-3ζ gives rise to many of the pathological hallmarks associated with the human condition. 14-3-3ζ-deficient BALB/c mice therefore provide a novel model to address the underlying biology of structural defects affecting the hippocampus and ventricle, and cognitive defects such as hippocampal-dependent learning and memory.
- Published
- 2015
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22. Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice.
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Lee EM, Yee D, Busfield SJ, McManus JF, Cummings N, Vairo G, Wei A, Ramshaw HS, Powell JA, Lopez AF, Lewis ID, McCall MN, and Lock RB
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- Adoptive Transfer, Animals, Cytarabine pharmacology, Daunorubicin, Female, Gene Expression, Humans, Immunoglobulin Fc Fragments chemistry, Immunoglobulin Fc Fragments immunology, Interleukin-3 Receptor alpha Subunit genetics, Interleukin-3 Receptor alpha Subunit immunology, Killer Cells, Natural drug effects, Killer Cells, Natural immunology, Killer Cells, Natural pathology, Leukemia, Myeloid, Acute immunology, Leukemia, Myeloid, Acute mortality, Leukemia, Myeloid, Acute pathology, Mice, Survival Analysis, Transplantation, Heterologous, Xenograft Model Antitumor Assays, Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal, Humanized pharmacology, Antineoplastic Agents pharmacology, Immunocompromised Host, Interleukin-3 Receptor alpha Subunit antagonists & inhibitors, Leukemia, Myeloid, Acute therapy
- Abstract
The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor α-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31-41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug., (Copyright© Ferrata Storti Foundation.)
- Published
- 2015
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23. A Phase 1 study of the safety, pharmacokinetics and anti-leukemic activity of the anti-CD123 monoclonal antibody CSL360 in relapsed, refractory or high-risk acute myeloid leukemia.
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He SZ, Busfield S, Ritchie DS, Hertzberg MS, Durrant S, Lewis ID, Marlton P, McLachlan AJ, Kerridge I, Bradstock KF, Kennedy G, Boyd AW, Yeadon TM, Lopez AF, Ramshaw HS, Iland H, Bamford S, Barnden M, DeWitte M, Basser R, and Roberts AW
- Subjects
- Adult, Aged, Aged, 80 and over, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal adverse effects, Antibodies, Monoclonal pharmacokinetics, Antineoplastic Agents administration & dosage, Antineoplastic Agents adverse effects, Antineoplastic Agents pharmacokinetics, Cell Proliferation drug effects, Female, Gene Expression Regulation, Leukemic, Humans, Interleukin-3 metabolism, Interleukin-3 Receptor alpha Subunit antagonists & inhibitors, Interleukin-3 Receptor alpha Subunit genetics, Interleukin-3 Receptor alpha Subunit metabolism, Male, Middle Aged, Recurrence, Treatment Outcome, Young Adult, Antibodies, Monoclonal therapeutic use, Antineoplastic Agents therapeutic use, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute pathology
- Abstract
Acute myeloid leukemia (AML) blasts express high levels of interlekin-3 (IL-3) receptor-α (CD123). CSL360 is a recombinant, chimeric immunoglobulin G1 (IgG1), anti-CD123 monoclonal antibody (MoAb) that neutralizes IL-3 and demonstrates anti-leukemic activity in vitro. This phase 1 study assessed safety, pharmacokinetics and bioactivity of weekly intravenous CSL360 for 12 weeks in 40 patients with advanced AML across five dose levels (0.1-10.0 mg/kg). Other than mild infusion reactions, CSL360 was well tolerated. The maximal tolerated dose was not reached. The half-life was 4.9 days, and the area under the curve (AUC) and maximum concentration (Cmax) increased proportionally with dose. Doses ≥ 3.0 mg/kg resulted in complete saturation and down-regulation of CD123 and abolition of ex vivo proliferative responsiveness to IL-3, indicating adequate blockade of IL-3 signaling. Two patients responded, with one remaining in complete remission after 17 doses. CSL360 bound CD123 specifically, but did not induce anti-leukemic activity in most patients. While safe, MoAb blockade of CD123 function is insufficient as a therapeutic strategy.
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- 2015
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24. Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC.
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Busfield SJ, Biondo M, Wong M, Ramshaw HS, Lee EM, Ghosh S, Braley H, Panousis C, Roberts AW, He SZ, Thomas D, Fabri L, Vairo G, Lock RB, Lopez AF, and Nash AD
- Subjects
- Animals, Antibodies, Monoclonal, Humanized immunology, Antibody-Dependent Cell Cytotoxicity immunology, Disease Models, Animal, Female, GPI-Linked Proteins immunology, Humans, Killer Cells, Natural immunology, Leukemia, Erythroblastic, Acute immunology, Leukemia, Myeloid, Acute immunology, Macaca fascicularis, Mice, Inbred NOD, Mice, SCID, Neoplasm Transplantation, Protein Engineering, Receptors, IgG immunology, Xenograft Model Antitumor Assays, Antibodies, Monoclonal, Humanized pharmacology, Interleukin-3 Receptor alpha Subunit immunology, Leukemia, Erythroblastic, Acute drug therapy, Leukemia, Myeloid, Acute drug therapy
- Abstract
Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor α-chain (IL-3 Rα/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (FcγRIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34(+)CD38(-)CD123(+) LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient's own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML.
- Published
- 2014
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25. Interleukin-3-mediated regulation of β-catenin in myeloid transformation and acute myeloid leukemia.
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Sadras T, Perugini M, Kok CH, Iarossi DG, Heatley SL, Brumatti G, Samuel MS, To LB, Lewis ID, Lopez AF, Ekert PG, Ramshaw HS, and D'Andrea RJ
- Subjects
- Animals, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Cell Line, Transformed, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic pathology, Gene Expression Regulation, Leukemic genetics, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Humans, Interleukin-3 genetics, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute pathology, Mice, Neoplasm Proteins genetics, Neoplasms, Experimental genetics, Neoplasms, Experimental metabolism, Neoplasms, Experimental pathology, Transcription Factor 4, Transcription Factors genetics, Transcription Factors metabolism, beta Catenin genetics, Cell Transformation, Neoplastic metabolism, Interleukin-3 metabolism, Leukemia, Myeloid, Acute metabolism, Neoplasm Proteins metabolism, Signal Transduction, Wnt Signaling Pathway, beta Catenin metabolism
- Abstract
Aberrant activation of β-catenin is a common event in AML and is an independent predictor of poor prognosis. Although increased β-catenin signaling in AML has been associated with oncogenic translocation products and activating mutations in the FLT3R, the mechanisms that activate β-catenin in AML more broadly are still unclear. Here, we describe a novel link between IL-3 signaling and the regulation of β-catenin in myeloid transformation and AML. In a murine model of HoxB8 and IL-3 cooperation, we show that β-catenin protein levels are modulated by IL-3 and that Cre-induced deletion of β-catenin abolishes IL-3-dependent growth and colony formation. In IL-3-dependent leukemic TF-1.8 cells, we observed increased β-catenin protein levels and nuclear localization in response to IL-3, and this correlated with transcriptional induction of β-catenin target genes. Furthermore, IL-3 promoted β-catenin accumulation in a subset of AML patient samples, and gene-expression profiling of these cells revealed induction of WNT/β-catenin and TCF4 gene signatures in an IL-3-dependent manner. This study is the first to link β-catenin activation to IL-3 and suggests that targeting IL-3 signaling may be an effective approach for the inhibition of β-catenin activity in some patients with AML., (© 2014 Society for Leukocyte Biology.)
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- 2014
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26. Monoclonal antibody targeting of IL-3 receptor α with CSL362 effectively depletes CML progenitor and stem cells.
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Nievergall E, Ramshaw HS, Yong AS, Biondo M, Busfield SJ, Vairo G, Lopez AF, Hughes TP, White DL, and Hiwase DK
- Subjects
- ADP-ribosyl Cyclase 1 metabolism, Adult, Aged, Animals, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing immunology, Antibodies, Neutralizing pharmacology, Antigens, CD34 metabolism, Cell Line, Female, Humans, Interleukin-3 Receptor alpha Subunit metabolism, Killer Cells, Natural immunology, Killer Cells, Natural pathology, Leukemia, Erythroblastic, Acute immunology, Leukemia, Erythroblastic, Acute pathology, Male, Mice, Mice, Inbred NOD, Mice, SCID, Middle Aged, Stem Cells immunology, Stem Cells metabolism, Stem Cells pathology, Antibodies, Monoclonal immunology, Interleukin-3 Receptor alpha Subunit immunology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive immunology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Receptors, Interleukin-3 immunology
- Abstract
Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.
- Published
- 2014
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27. Immune insufficiency during GVHD is due to defective antigen presentation within dendritic cell subsets.
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Markey KA, Koyama M, Kuns RD, Lineburg KE, Wilson YA, Olver SD, Raffelt NC, Don AL, Varelias A, Robb RJ, Cheong M, Engwerda CR, Steptoe RJ, Ramshaw HS, Lopez AF, Vega-Ramos J, Lew AM, Villadangos JA, Hill GR, and MacDonald KP
- Subjects
- Adoptive Transfer, Animals, Bone Marrow Cells immunology, Bone Marrow Cells pathology, Bone Marrow Transplantation immunology, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes pathology, Cross-Priming immunology, Graft vs Host Disease pathology, Histocompatibility Antigens Class II immunology, Inflammation immunology, Inflammation pathology, Interferon-gamma metabolism, Isoantigens immunology, Mice, Mice, Transgenic, Peptides immunology, T-Lymphocytes, Regulatory immunology, T-Lymphocytes, Regulatory pathology, Tumor Necrosis Factor-alpha metabolism, Antigen Presentation immunology, Dendritic Cells immunology, Dendritic Cells pathology, Graft vs Host Disease immunology, Immunosuppression Therapy
- Abstract
Alloreactivity after transplantation is associated with profound immune suppression, and consequent opportunistic infection results in high morbidity and mortality. This immune suppression is most profound during GVHD after bone marrow transplantation where an inflammatory cytokine storm dominates. Contrary to current dogma, which avers that this is a T-cell defect, we demonstrate that the impairment lies within conventional dendritic cells (cDCs). Significantly, exogenous antigens can only be presented by the CD8(-) cDC subset after bone marrow transplantation, and inflammation during GVHD specifically renders the MHC class II presentation pathway in this population incompetent. In contrast, both classic and cross-presentation within MHC class I remain largely intact. Importantly, this defect in antigen processing can be partially reversed by TNF inhibition or the adoptive transfer of donor cDCs generated in the absence of inflammation.
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- 2012
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28. The GM-CSF receptor family: mechanism of activation and implications for disease.
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Hercus TR, Broughton SE, Ekert PG, Ramshaw HS, Perugini M, Grimbaldeston M, Woodcock JM, Thomas D, Pitson S, Hughes T, D'Andrea RJ, Parker MW, and Lopez AF
- Subjects
- Chronic Disease, Hematologic Neoplasms pathology, Humans, Inflammation pathology, Myeloid Cells metabolism, Myeloid Cells pathology, Signal Transduction, Granulocyte-Macrophage Colony-Stimulating Factor metabolism, Hematologic Neoplasms metabolism, Inflammation metabolism, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor metabolism
- Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.
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- 2012
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29. Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency.
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Cheah PS, Ramshaw HS, Thomas PQ, Toyo-Oka K, Xu X, Martin S, Coyle P, Guthridge MA, Stomski F, van den Buuse M, Wynshaw-Boris A, Lopez AF, and Schwarz QP
- Subjects
- 14-3-3 Proteins genetics, Animals, Brain metabolism, Cell Movement genetics, Cell Movement physiology, Cells, Cultured, Disease Models, Animal, Female, Hippocampus metabolism, Hippocampus pathology, Male, Maze Learning physiology, Mice, Mice, Inbred C57BL, Mice, Knockout, Motor Activity physiology, Neurogenesis genetics, Neurons metabolism, Neurons physiology, Recognition, Psychology physiology, Sensory Gating physiology, 14-3-3 Proteins physiology, Brain growth & development, Genetic Predisposition to Disease genetics, Hippocampus growth & development, Neurogenesis physiology, Schizophrenia genetics
- Abstract
Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.
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- 2012
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30. Molecular basis of cytokine receptor activation.
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Lopez AF, Hercus TR, Ekert P, Littler DR, Guthridge M, Thomas D, Ramshaw HS, Stomski F, Perugini M, D'Andrea R, Grimbaldeston M, and Parker MW
- Subjects
- Cell Survival physiology, Humans, Models, Molecular, Protein Multimerization, Receptors, Cytokine genetics, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor metabolism, Receptors, Interleukin-3 metabolism, Receptors, Interleukin-5 metabolism, Signal Transduction, Cytokine Receptor Common beta Subunit metabolism, Cytokines metabolism, Receptors, Cytokine metabolism
- Abstract
Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell. Receptor binding triggers a cascade of intracellular signaling events, including reversible phosphorylation of receptor subunits and associated signaling molecules, leading to multiple biological responses, with the prevention of apoptosis or "cell survival" being a key cellular function that underpins all others. Many chronic inflammatory diseases and a number of haematological malignancies are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine receptor signaling, highlighting their importance in disease. A major step in understanding how these cytokine receptors function is to elucidate their three dimensional structure and to relate this to the many signaling pathways emanating from their receptors. We have recently solved the structure of the human GM-CSF receptor complexed to GM-CSF which revealed distinct forms of receptor assembly: a hexamer that comprises two molecules each of GM-CSF, GM-CSF receptor alpha chain and GM-CSF receptor beta chain; and an unexpected dodecamer in which two hexameric complexes associate through a novel site 4. This latter form is necessary to bring JAK2 molecules sufficiently close together to enable full receptor activation. In this review we focus on the most recent insights in cytokine receptor signaling, and in receptor assembly. The stage is now set to link distinct forms of cytokine receptor assembled structures to specific forms of cytokine receptor signaling and function. Armed with this knowledge it may be possible to map distinct cytokine receptor signaling pathways from the cell surface to the cell nucleus which may themselves become new therapeutic targets., ((c) 2010 IUBMB IUBMB Life.)
- Published
- 2010
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31. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells.
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Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, Wilkinson L, Guthridge MA, Thomas D, Barry EF, Boyd A, Gearing DP, Vairo G, Lopez AF, Dick JE, and Lock RB
- Subjects
- Adult, Aged, Aged, 80 and over, Animals, Antibodies, Monoclonal therapeutic use, Antigens, CD34 metabolism, Bone Marrow metabolism, Cell Line, Cell Movement drug effects, Cell Proliferation drug effects, Female, Hematopoietic Stem Cells metabolism, Humans, Interleukin-3 Receptor alpha Subunit immunology, Intracellular Signaling Peptides and Proteins metabolism, Leukemia, Myeloid, Acute immunology, Male, Mice, Mice, Inbred NOD, Mice, SCID, Middle Aged, Neoplastic Stem Cells metabolism, Stem Cell Transplantation, Transplantation, Heterologous, Tumor Burden, Antibodies, Monoclonal pharmacology, Interleukin-3 Receptor alpha Subunit antagonists & inhibitors, Leukemia, Myeloid, Acute therapy, Neoplastic Stem Cells drug effects
- Abstract
Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.
- Published
- 2009
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32. Sphingosine kinase regulates the rate of endothelial progenitor cell differentiation.
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Bonder CS, Sun WY, Matthews T, Cassano C, Li X, Ramshaw HS, Pitson SM, Lopez AF, Coates PT, Proia RL, Vadas MA, and Gamble JR
- Subjects
- Animals, Bone Marrow Cells cytology, Bone Marrow Cells physiology, Cell Proliferation, Cells, Cultured, Colony-Forming Units Assay, Endothelial Cells metabolism, Mice, Mice, Knockout, Phenotype, Phosphotransferases (Alcohol Group Acceptor) genetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Proprotein Convertases physiology, Serine Endopeptidases physiology, Stem Cells metabolism, Cell Differentiation genetics, Endothelial Cells physiology, Phosphotransferases (Alcohol Group Acceptor) physiology, Stem Cells physiology
- Abstract
Circulating endothelial progenitor cells (EPCs) are incorporated into foci of neovascularization where they undergo differentiation to mature endothelial cells (ECs). We show here that the enzyme sphingosine kinase-1 (SK-1) regulates the rate and direction of EPC differentiation without effect on the hematopoietic compartment. EPCs have high levels of SK-1 activity, which diminishes with differentiation and is, at least partially, responsible for maintaining their EPC phenotype. EPCs from SK-1 knockout mice form more adherent EC units and acquire a mature EC phenotype more rapidly. Conversely, EPCs from mice overexpressing SK-1 in the EC compartment are retarded in their differentiation. Exogenous regulation of SK-1 levels in normal EPCs, by genetic and pharmacologic means, including the immunomodulating drug FTY720, recapitulates these effects on EC differentiation. SK-1 knockout mice have higher levels of circulating EPCs, an exaggerated response to erythropoietin-induced EPC mobilization, and, in a mouse model of kidney ischemia reperfusion injury, exhibit a recovery similar to that of ischemic mice administered exogenous EPCs. Thus, SK-1 is a critical player in EPC differentiation into EC pointing to the potential utility of SK-1 modifying agents in the specific manipulation of endothelial development and repair.
- Published
- 2009
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33. The Shc-binding site of the betac subunit of the GM-CSF/IL-3/IL-5 receptors is a negative regulator of hematopoiesis.
- Author
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Ramshaw HS, Guthridge MA, Stomski FC, Barry EF, Ooms L, Mitchell CA, Begley CG, and Lopez AF
- Subjects
- Animals, Binding Sites, Bone Marrow Cells metabolism, Cell Survival genetics, Cells, Cultured, Cytokine Receptor Common beta Subunit genetics, Cytokine Receptor Common beta Subunit metabolism, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Humans, Inositol Polyphosphate 5-Phosphatases, Liver embryology, Liver metabolism, Mice, Mice, Knockout, Phosphatidylinositol 3-Kinases metabolism, Phosphoproteins metabolism, Phosphoric Monoester Hydrolases metabolism, Phosphorylation, Point Mutation, Shc Signaling Adaptor Proteins, Src Homology 2 Domain-Containing, Transforming Protein 1, Transduction, Genetic, Adaptor Proteins, Signal Transducing metabolism, Cytokine Receptor Common beta Subunit chemistry, Cytokine Receptor Common beta Subunit physiology, Hematopoiesis genetics
- Abstract
Tyrosine and serine phosphorylation of the common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is widely viewed as a general mechanism that provides positive inputs by coupling the receptor to signaling pathways that stimulate several cellular functions. We show here that despite the known action of Tyr577 in beta(c) to recruit Shc-PI-3 kinase (PI3K) pathway members, Tyr577 plays, surprisingly, a negative regulatory role in cell function, and that this is mediated, at least in part, through the uncoupling of SH2-containing inositol 5'-phosphatase (SHIP) from beta(c). Fetal liver cells from beta(c)/beta(IL-3)(-/-) mice expressing human GM-CSF receptor alpha chain and beta(c) Tyr577Phe mutant showed enhanced colony formation and expansion of progenitor cells in response to GM-CSF. Dissection of these activities revealed that basal survival was increased, as well as cytokine-stimulated proliferation. As expected, the recruitment and activation of Shc was abolished, but interestingly, Gab-2 and Akt phosphorylation increased. Significantly, the activation of PI3K was enhanced and prolonged, accompanied by loss of SHIP activity. These results reveal a previously unrecognized negative signaling role for Tyr577 in beta(c) and demonstrate that uncoupling Shc from cytokine receptors enhances PI3K signaling as well as survival and proliferation.
- Published
- 2007
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34. Chronic myelomonocytic leukemia requires granulocyte-macrophage colony-stimulating factor for growth in vitro and in vivo.
- Author
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Ramshaw HS, Bardy PG, Lee MA, and Lopez AF
- Subjects
- Adult, Aged, Aged, 80 and over, Animals, Blast Crisis pathology, Cell Division drug effects, DNA Primers, Female, Granulocyte-Macrophage Colony-Stimulating Factor deficiency, Granulocyte-Macrophage Colony-Stimulating Factor genetics, Humans, Male, Mice, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, Transplantation, Heterologous, Tumor Cells, Cultured, Granulocyte-Macrophage Colony-Stimulating Factor pharmacology, Leukemia, Myelomonocytic, Chronic pathology
- Abstract
Objective: Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease with no effective treatments or cure. Several factors have been implicated in its pathogenesis. In the current study, we studied the dependence of CMML on granulocyte-macrophage colony-stimulating factor (GM-CSF)., Materials and Methods: We used in vitro colony assays in methylcellulose where CMML cells were tested in the presence or absence of the specific GM-CSF antagonist E21R. We also developed an in vivo model in which CMML cells were tested for their ability to engraft into immunodeficient mice transgenic for human GM-CSF., Results: Bone marrow cells from seven of seven patients with CMML formed spontaneous colonies that were sensitive to E21R treatment, with reduction in colony growth by up to 92%. E21R also inhibited colony formation by CMML patient cells stimulated by exogenously added GM-CSF but not interleukin-3. In in vivo experiments we observed engraftment of CMML cells (but not normal cells) in immunodeficient mice transgenic for human GM-CSF. None engrafted in nontransgenic mice. Cell dose escalation showed that the optimal number was 0.5 to 1 x 10(8) peripheral blood mononuclear cells per mouse, which is equivalent to an infusion of 0.2 to 3.6 x 10(6) CD34(+) cells. Time course experiments showed that maximal engraftment occurred 6 weeks after injection., Conclusions: These results demonstrate that in some CMML patients, GM-CSF produced by either autocrine or paracrine mechanisms is a major growth determinant. The results suggest that therapies directed at blocking this cytokine could control the growth of some CMML patients in vivo.
- Published
- 2002
- Full Text
- View/download PDF
35. Monoclonal antibody BB9 raised against bone marrow stromal cells identifies a cell-surface glycoprotein expressed by primitive human hemopoietic progenitors.
- Author
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Ramshaw HS, Haylock D, Swart B, Gronthos S, Horsfall MJ, Niutta S, and Simmons PJ
- Subjects
- Animals, Antibody Specificity, Antigens, CD analysis, Antigens, CD34 analysis, Breast Neoplasms, Cell Line, Cell Separation methods, Cryopreservation, Female, Flow Cytometry, HL-60 Cells, Hematopoietic Stem Cell Mobilization, Humans, Hybridomas, Jurkat Cells, K562 Cells, Membrane Glycoproteins immunology, Mice, Mice, Inbred BALB C, Multiple Myeloma, Tumor Cells, Cultured, Antibodies, Monoclonal, Bone Marrow Cells cytology, Hematopoietic Stem Cells cytology, Leukocytes cytology, Membrane Glycoproteins analysis, Stromal Cells cytology
- Abstract
Objective: The identification of cell-surface antigens whose expression is limited to primitive hematopoietic progenitor cells (HPC) is of major value in the identification, isolation, and characterization of candidate stem cells in human hemopoietic tissues. Based on the observation that bone marrow stromal cells and primitive HPC share several cell-surface antigens, we sought to generate monoclonal antibodies to HPC by immunization with cultured human stromal cells., Methods: BALB/c mouse were immunized with human bone marrow (BM)-derived stromal cells. Splenocytes isolated from immunized mice were fused with the NS-1 murine myeloma cell line and resulting hybridomas selected in HAT medium, then screened for reactivity against stromal cells, peripheral blood (PB), and BM cells., Results: A monoclonal antibody (MAb), BB9, was identified based on its binding to stromal cells, a minor subpopulation of mononuclear cells in adult human BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bound to a minor subpopulation of BM CD34(+) cells characterized by high-level CD34 antigen and Thy-1 expression, low-absent expression of CD38, low retention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cells, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stimulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstrated binding to CD34(+) cells from mobilized PB., Conclusion: Collectively, these data therefore demonstrate that MAb BB9 identifies an antigen, which is selectively expressed by hierarchically primitive human HPC and also by stromal cells.
- Published
- 2001
- Full Text
- View/download PDF
36. CD34+ cells and their derivatives contain mRNA for CD4 and human immunodeficiency virus (HIV) co-receptors and are susceptible to infection with M- and T-tropic HIV.
- Author
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Carr JM, Ramshaw HS, Li P, and Burrell CJ
- Subjects
- Antigens, CD analysis, CD4 Antigens genetics, DNA, Viral analysis, Gene Products, gag genetics, HIV metabolism, HIV Core Protein p24 analysis, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells virology, Humans, RNA, Messenger, Receptors, CCR1, Receptors, CCR2, Receptors, CCR5 genetics, Receptors, CXCR4 genetics, Receptors, Chemokine genetics, Receptors, Cytokine genetics, Receptors, HIV genetics, Antigens, CD34, CD4 Antigens metabolism, HIV physiology, Hematopoietic Stem Cells metabolism, Receptors, Cytokine metabolism, Receptors, HIV metabolism
- Abstract
Highly purified (>98%) CD34+ cells directly after isolation (D0) or 2 weeks in culture (D14) were CD4+ and contained mRNA for the T-tropic HIV co-receptor, CXCR-4, and minor co-receptor, CCR-2B. D14 but not D0 cells were RT-PCR positive for mRNA for the major M-tropic human immunodeficiency virus (HIV) co-receptor, CCR-5, and potential co-receptor, CCR-1. D14 and D0 cells were susceptible to T- (HXB2) and M-tropic HIV (Bal), showing greater virus production with Bal than HXB2, and with higher virus production levels in D14 compared to D0 cells. Seven days post-infection of D0 cells Bal DNA was present in CD14bright and CD14- fractions, suggesting D0 infection of diverse progenitor types. HXB2 DNA was detected in CD14bright cells alone indicating D0 infection of monocyte progenitors only. It is concluded that CD34+ cells and cultured derivatives are susceptible to M- and T-tropic HIV and this correlates in part with co-receptor expression at the mRNA level.
- Published
- 1998
- Full Text
- View/download PDF
37. Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand.
- Author
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Haylock DN, Horsfall MJ, Dowse TL, Ramshaw HS, Niutta S, Protopsaltis S, Peng L, Burrell C, Rappold I, Buhring HJ, and Simmons PJ
- Subjects
- ADP-ribosyl Cyclase, ADP-ribosyl Cyclase 1, Adult, Antigens, CD34 analysis, Antigens, Differentiation analysis, Bone Marrow Cells, Cell Cycle, Cell Separation, Cells, Cultured, Flow Cytometry, Hematopoietic Cell Growth Factors physiology, Hematopoietic Stem Cells physiology, Humans, Immunophenotyping, Membrane Glycoproteins, NAD+ Nucleosidase analysis, Retroviridae genetics, Transduction, Genetic, fms-Like Tyrosine Kinase 3, Antigens, CD, Erythropoiesis, Hematopoiesis, Hematopoietic Stem Cells cytology, Membrane Proteins physiology, Proto-Oncogene Proteins physiology, Receptor Protein-Tyrosine Kinases physiology
- Abstract
The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.
- Published
- 1997
38. Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts.
- Author
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Kittler EL, Peters SO, Crittenden RB, Debatis ME, Ramshaw HS, Stewart FM, and Quesenberry PJ
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Animals, Bone Marrow immunology, Bone Marrow Cells, Cells, Cultured, Coculture Techniques, Drug Resistance, Multiple genetics, Female, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells immunology, Humans, Interleukin-11 pharmacology, Interleukin-3 pharmacology, Interleukin-6 pharmacology, Male, Mice, Mice, Inbred BALB C, Polymerase Chain Reaction, RNA, Messenger biosynthesis, Recombinant Proteins pharmacology, Stem Cell Factor pharmacology, Transcription, Genetic, Y Chromosome, ATP Binding Cassette Transporter, Subfamily B, Member 1 biosynthesis, Bone Marrow Transplantation immunology, Cytokines pharmacology, Harvey murine sarcoma virus genetics, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Paclitaxel pharmacology
- Abstract
Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft.
- Published
- 1997
39. Stem cell transplantation in the normal nonmyeloablated host: relationship between cell dose, schedule, and engraftment.
- Author
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Rao SS, Peters SO, Crittenden RB, Stewart FM, Ramshaw HS, and Quesenberry PJ
- Subjects
- Animals, Bone Marrow Cells, Cell Movement, Female, Graft Survival, Hematopoietic Stem Cells cytology, Male, Mice, Mice, Inbred BALB C, Spleen cytology, Thymus Gland cytology, Time Factors, Hematopoietic Stem Cell Transplantation methods
- Abstract
In previous studies we have shown high rates of stable engraftment when 40 million male BALB/c cells were infused intravenously daily for 5 days (a total of 200 million cells) to normal nonmyeloablated female hosts. The present studies evaluate engraftment of male BALB/c bone marrow cells in female host marrow, spleen, and thymus 20-25 weeks after transplantation using varying cell dosages within a 5-day schedule. Engraftment in recipient mice was assessed by detection of male specific sequence in recipient DNA from each organ. When 40 million cells were given per daily injection for 1, 2, 3, 4, or 5 days, engraftment percentages in host marrow were 11 +/- 0.83, 20 +/- 2.0, 23 +/- 2.5, 32 +/- 6.3, and 39% +/- 5.7 (+/- standard error of mean), respectively, yielding engraftment percentages per million cells infused of 0.28, 0.25, 0.19, 0.20, and 0.20%, respectively. When levels of 2.5, 5, 10, 20, or 40 million cells were injected 5 times over a 5-day schedule into normal BALB/c female hosts, progressively increasing levels of engraftment from 3 +/- 0.6 to 39% +/- 5.7 were seen in host marrow. Highest levels of engraftment per million cells injected were obtained on days 1 and 2 of a 5-day schedule and with a level of 10 million cells given daily over 5 days. Engraftment profiles varied with spleen and thymus and percent engraftment was generally lower than for marrow. The present work indicates that regardless of cell level infused or number of infusions, rates of engraftment observed in marrow approached or exceeded the highest rates of engraftment estimated by theoretical calculations based on replacing host cells ("replacement model") or adding to host cells ("incremental model"). Engraftment in spleen and thymus was lower, but also at times approached or exceeded theoretical maxima. These data show extraordinary levels of engraftment in normal hosts, suggesting that rates in this competitive model are superior to those seen in irradiated hosts; alternatively, there may be selective repression of host stem cell proliferation and differentiation.
- Published
- 1997
40. High levels of engraftment with a single infusion of bone marrow cells into normal unprepared mice.
- Author
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Ramshaw HS, Crittenden RB, Dooner M, Peters SO, Rao SS, and Quesenberry PJ
- Subjects
- Animals, Blotting, Southern, Female, Male, Mice, Mice, Inbred BALB C, Bone Marrow Transplantation methods, Graft Survival
- Abstract
Repetitive infusion of 40 million male murine marrow cells (total 200 million cells) into normal unprepared female BALB/c hosts for 5 consecutive days results in high levels of engraftment at 1-25 months postinfusion, as determined by Southern blot analysis using a Y chromosome-specific probe. We investigated the importance of the schedule of injections in this engraftment model. Surprisingly, a single infusion of 200 x 10(6) male BALB/c bone marrow cells analyzed at 7-14 weeks postinfusion resulted in engraftment levels in individual female mice of over 50% with mean values of 25 +/- 2% for 44 individual transplant points. Engraftment levels in spleen and thymus were 14 +/- 1% and 18 +/- 3%, respectively. Including heparin in the infusion increased engraftment in marrow, spleen, and thymus. Administration of the cells over five or 10 separate infusions, rather than in one injection, did not increase engraftment levels. If the infused bone marrow cells seeded equally between host spleen, thymus, and bone marrow, and if all cells engrafted, the bone marrow engraftment seen here approaches the theoretical maximum. This suggests either a large number of available "niches" or the displacement of host marrow cells by infused marrow. The latter possibility is upheld by cell counts per tibia/femur and total seven-factor HPP-CFC/tibia, which were not increased. These data suggest that a single infusion of marrow homes quantitatively to spleen, thymus, and bone marrow, possibly displacing host cells in the process.
- Published
- 1995
41. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts.
- Author
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Peters SO, Kittler EL, Ramshaw HS, and Quesenberry PJ
- Subjects
- Animals, Cell Division drug effects, Cells, Cultured, Colony-Forming Units Assay, Female, Graft Survival, Male, Mice, Mice, Inbred BALB C, Spleen cytology, Stem Cell Factor, Thymus Gland cytology, Bone Marrow Cells, Hematopoietic Cell Growth Factors pharmacology, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells drug effects, Interleukin-11 pharmacology, Interleukin-3 pharmacology, Interleukin-6 pharmacology
- Abstract
Stimulatory cytokines may induce murine hematopoietic progenitor cells (HPCs) to survive, self-renew, proliferate, or differentiate. We studied the role of active cell cycling induced by the cytokines interleukin-3 (IL-3), IL-6, IL-11, and Steel factor (SF) on murine progenitor cell frequency and cell cycle status in an in vitro system and on engraftment potential in nonmyeloablated mice. Marrow exposure to IL-3, IL-6, IL-11, and SF in in vitro liquid culture maintained or expanded seven factor-responsive high and low proliferative potential colony-forming cells (HPP-CFC and LPP-CFC). The HPP-CFC and LPP-CFC were dormant at the initiation of culture, as determined by 3H-thymidine suicide. There was an increase in the number and proliferation of HPP-CFC and LPP-CFC at 48 hours; by 48 hours, 62% of HPP-CFC and 56% of LPP-CFC were killed by 3H-TdR exposure. In engraftment studies of cytokine-stimulated marrow cells into normal hosts, female BALB/c mice received the equivalent of 40 x 10(6) starting male marrow cells exposed to cytokines in vitro for 48 hours for 3 consecutive days and were sacrificed 8 weeks after transplantation. Control groups received either 40 x 10(6) male uncultured marrow cells, 40 x 10(6) starting marrow cells cultured in medium without growth factors for 48 hours, or phosphate-buffered saline (PBS) for 3 days. Engraftment of male cytokine-treated cells was analyzed by Southern blot analysis using the Y-chromosome-specific pY2-cDNA probe. There was minimal engraftment (approaching background levels) in marrow, spleen, and thymus of nonmyeloablated female recipients. Transplant recipients that had received uncultured marrow directly after sacrifice showed engraftment levels of 21% (11 mice; range = 8 to 44%) into marrow, of 9% (range = 0 to 22%) into spleen, and 13% (range = 2 to 43%) into thymus. We conclude that active cell cycling of marrow cells induced by cytokine stimulation is associated with an engraftment defect in the normal host.
- Published
- 1995
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