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1.
J Immunol ; 202(6): 1885-1894, 2019 03 15.
Article de Anglais | MEDLINE | ID: mdl-30710044

RÉSUMÉ

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123+ leukemic stem or initiating cells (LSC), resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.


Sujet(s)
Carcinogenèse , Leucémie myéloïde , Protéines nucléaires , Tests d'activité antitumorale sur modèle de xénogreffe , Animaux , Cellules souches hématopoïétiques , Humains , Souris , Souris de lignée NOD , Souris SCID , Nucléophosmine
2.
Cell Mol Immunol ; 10(4): 284-5, 2013 Jul.
Article de Anglais | MEDLINE | ID: mdl-23686228
3.
Cell Mol Immunol ; 9(3): 215-24, 2012 May.
Article de Anglais | MEDLINE | ID: mdl-22425741

RÉSUMÉ

Humanized mice are immunodeficient animals engrafted with human hematopoietic stem cells that give rise to various lineages of human blood cells throughout the life of the mouse. This article reviews recent advances in the generation of humanized mice, focusing on practical considerations. We discuss features of different immunodeficient recipient mouse strains, sources of human hematopoietic stem cells, advances in expansion and genetic modification of hematopoietic stem cells, and techniques to modulate the cytokine environment of recipient mice, in order to enhance reconstitution of specific human blood lineage cells. We highlight the opportunities created by new technologies and discuss practical considerations on how to make best use of the widening array of basic models for specific research applications.


Sujet(s)
Survie cellulaire , Hématopoïèse , Transplantation de cellules souches hématopoïétiques , Animaux , Cytokines/immunologie , Cytokines/métabolisme , Génie génétique , Hématopoïèse/génétique , Hématopoïèse/immunologie , Humains , Immunomodulation , Souris , Souches mutantes de souris , Souris SCID , Plan de recherche , Niche de cellules souches , Transplantation hétérologue
4.
ACS Nano ; 6(1): 81-8, 2012 Jan 24.
Article de Anglais | MEDLINE | ID: mdl-22176729

RÉSUMÉ

The ability to control the timing and order of release of different therapeutic drugs will play a pivotal role in improving patient care and simplifying treatment regimes in the clinic. The controlled sequential release of a broad range of small and macromolecules from thin film coatings offers a simple way to provide complex localized dosing in vivo. Here we show that it is possible to take advantage of the structure of certain nanomaterials to control release regimes from a scale of hours to months. Graphene oxide (GO) is a two-dimensional charged nanomaterial that can be used to create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. Protein-loaded polyelectrolyte multilayer films were fabricated using layer-by-layer assembly incorporating a hydrolytically degradable cationic poly(ß-amino ester) (Poly1) with a model protein antigen, ovalbumin (ova), in a bilayer architecture along with positively and negatively functionalized GO capping layers for the degradable protein films. Ova release without the GO layers takes place in less than 1 h but can be tuned to release from 30 to 90 days by varying the number of bilayers of functionalized GO in the multilayer architecture. We demonstrate that proteins can be released in sequence with multi-day gaps between the release of each species by incorporating GO layers between protein loaded layers. In vitro toxicity assays of the individual materials on proliferating hematopoietic stem cells (HSCs) indicated limited cytotoxic effects with HSCs able to survive for the full 10 days of normal culture in the presence of Poly1 and the GO sheets. This approach provides a new route for storage of therapeutics in a solid-state thin film for subsequent delivery in a time-controlled and sequential fashion.


Sujet(s)
Préparations à action retardée/administration et posologie , Graphite/composition chimique , Cellules souches hématopoïétiques/effets des médicaments et des substances chimiques , Nanocapsules/composition chimique , Protéines/administration et posologie , Survie cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Diffusion , Cellules souches hématopoïétiques/cytologie , Humains , Test de matériaux , Nanocapsules/administration et posologie , Protéines/composition chimique
5.
PLoS One ; 6(4): e18382, 2011 Apr 29.
Article de Anglais | MEDLINE | ID: mdl-21559522

RÉSUMÉ

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.


Sujet(s)
Angiopoïétines/métabolisme , Antigènes CD34/biosynthèse , Antigènes CD/biosynthèse , Glycoprotéines/biosynthèse , Sous-unité gamma commune aux récepteurs des interleukines/génétique , Antigène AC133 , Protéines semblables à l'angiopoïétine , Animaux , Plaquettes/métabolisme , Sang foetal/métabolisme , Cytométrie en flux/méthodes , Humains , Souris , Souris de lignée NOD , Souris SCID , Souris transgéniques , Peptides
6.
Cell ; 144(2): 296-309, 2011 Jan 21.
Article de Anglais | MEDLINE | ID: mdl-21241896

RÉSUMÉ

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.


Sujet(s)
Régulation de l'expression des gènes , Réseaux de régulation génique , Hématopoïèse , Facteurs de transcription/métabolisme , Analyse de profil d'expression de gènes , Humains
7.
J Struct Biol ; 145(3): 295-306, 2004 Mar.
Article de Anglais | MEDLINE | ID: mdl-14960380

RÉSUMÉ

DNA-dependent protein kinase (DNA-PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA-PK (DNA-PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of approximately 4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA-PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA-PKcs protein and identifies regions of interest for further experimental studies.


Sujet(s)
Protéines de liaison à l'ADN/composition chimique , ADN/composition chimique , Protein-Serine-Threonine Kinases/composition chimique , Algorithmes , Motifs d'acides aminés , Animaux , Domaine catalytique , Altération de l'ADN , Réparation de l'ADN , DNA-activated protein kinase , Bases de données comme sujet , Électrons , Humains , Microscopie électronique , Protéines nucléaires , Phosphatidylinositol 3-kinases/composition chimique , Conformation des protéines , Structure secondaire des protéines , Structure tertiaire des protéines , Logiciel , VDJ recombinases/composition chimique
8.
DNA Repair (Amst) ; 3(1): 33-41, 2004 Jan 05.
Article de Anglais | MEDLINE | ID: mdl-14697757

RÉSUMÉ

Cellular life depends upon the preservation and transmission of genetic material. Double stranded DNA breaks (DSBs) cause catastrophic gene loss in cell division and must be promptly and accurately repaired. In eukaryotes DSBs may be repaired by either non-homologous end-joining (NHEJ), single strand annealing or homologous recombination (HR). Vertebrate NHEJ has been shown to depend upon the DNA-dependent protein kinase (DNA-PK) consisting of the phosphatidylinositol 3 (PI 3)-kinase like (PIKK) catalytic sub-unit (DNA-PKcs) and the DNA targeting factor Ku. Our analysis of recently completed genomes found several novel PIKKs in Anopheles gambiae and Drosophila melanogaster including a novel mosquito DNA-PKcs orthologue, the first non-vertebrate DNA-PKcs described to date. We also detected a DNA-PKcs fragment in the high quality EST set of Apis mellifera ligustica (honey bee) suggesting that DNA-PK is a far older and more important eukaryotic complex than previously thought.


Sujet(s)
Anopheles/enzymologie , Abeilles/enzymologie , Culicidae/enzymologie , Drosophila melanogaster/enzymologie , Protein-Serine-Threonine Kinases/métabolisme , Séquence d'acides aminés , Animaux , Anopheles/classification , Anopheles/génétique , Antigènes nucléaires/métabolisme , Arthropodes/enzymologie , Abeilles/génétique , Culicidae/génétique , DNA-activated protein kinase , Protéines de liaison à l'ADN/métabolisme , Drosophila melanogaster/génétique , Humains , Autoantigène Ku , Données de séquences moléculaires , Protéines nucléaires , Protein-Serine-Threonine Kinases/génétique , Similitude de séquences d'acides aminés , Vertébrés/génétique , Vertébrés/métabolisme
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