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1.
Neoplasia ; 19(9): 661-671, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28753442

RESUMEN

Naratuximab emtansine (IMGN529) is an investigational antibody-drug conjugate consisting of a CD37-targeting antibody conjugated to the maytansine-derived microtuble disruptor, DM1. IMGN529 has shown promising preclinical and clinical activity in non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Due to the aggressive nature of the disease, DLBCL is often treated with combination therapies to maximize clinical outcomes; therefore, we investigated the potential of combining IMGN529 with both standard-of-care and emerging therapies against multiple oncology-relevant targets and pathways. The strongest enhancement in potency was seen with anti-CD20 antibodies, including rituximab. The combination of IMGN529 and rituximab was more potent than either agent alone, and this combinatorial benefit was associated with increased apoptotic induction and cell death. Additional studies revealed that rituximab treatment increased the internalization and degradation of the CD37-targeting antibody moiety of IMGN529. The combination of IMGN529 and rituximab was highly efficacious in multiple xenograft models, with superior antitumor efficacy seen compared to either agent alone or treatment with R-CHOP therapy. These findings suggest a novel mechanism whereby the potency of IMGN529 can be enhanced by CD20 binding, which results in the increased internalization and degradation of IMGN529 leading to the generation of greater amounts of cytotoxic catabolite. Overall, these data provide a biological rationale for the enhanced activity of IMGN529 in combination with rituximab and support the ongoing clinical evaluation of IMGN529 in combination with rituximab in patients with relapsed and/or refractory DLBCL.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Rituximab/farmacología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Ratones , Terapia Molecular Dirigida , Proteolisis , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Infect Immun ; 82(5): 2079-86, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24614661

RESUMEN

Infections with Streptococcus pneumoniae cause substantial morbidity and mortality, particularly in children in developing nations. Polysaccharide-conjugate vaccines provide protection against both invasive disease and colonization, but their use in developing countries is limited by restricted serotype coverage and expense of manufacture. Using proteomic screens, we recently identified several antigens that protected mice from pneumococcal colonization in a CD4(+) T cell- and interleukin-17A (IL-17A)-dependent manner. Since several of these proteins are lipidated, we hypothesized that their immunogenicity and impact on colonization are in part due to activation of Toll-like receptor 2 (TLR2), a receptor for lipoproteins. Here we show that lipidated versions of the antigens elicited significantly higher activation of both human embryonic kidney cells engineered to express TLR2 (HEK-TLR2) and wild-type (WT) murine macrophages than nonlipidated mutant antigens. Lipoprotein-stimulated secretion of proinflammatory cytokines was ∼10× to ∼100× lower in murine TLR2-deficient macrophages than in WT macrophages. Subcutaneous immunization of C57BL/6 mice with protein subunit vaccines containing one or two of these lipoproteins or protein fusion constructs bearing N-terminal lipid adducts elicited a robust IL-17A response and a significant reduction in colonization compared with immunization with alum alone. In contrast, immunization of Tlr2(-/-) mice elicited no detectable IL-17A response and no protection against pneumococcal colonization. These experiments suggest that the lipid moieties enhance the immunogenicity and protective efficacy of pneumococcal TH17 antigens through activation of TLR2. Thus, triggering TLR2 with an antigen-specific protein subunit formulation is a possible strategy for the development of a serotype-independent pneumococcal vaccine that would reduce pneumococcal carriage.


Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Lípidos/química , Infecciones Neumocócicas/prevención & control , Streptococcus pneumoniae , Receptor Toll-Like 2/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Portador Sano , Macrófagos/metabolismo , Ratones , Mutación , Receptor Toll-Like 2/genética
4.
Blood ; 119(1): 273-84, 2012 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-22072555

RESUMEN

Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.


Asunto(s)
Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Efecto Injerto vs Leucemia/inmunología , Inmunoterapia Adoptiva , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Transfusión de Leucocitos , Animales , Southern Blotting , ADN/genética , Proteínas de Unión al ADN/fisiología , Femenino , Citometría de Flujo , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Enfermedad Injerto contra Huésped/inmunología , Técnicas In Vitro , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Tasa de Supervivencia , Linfocitos T/inmunología , Donantes de Tejidos , Trasplante Homólogo
5.
Cancer Cell ; 19(4): 556-68, 2011 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-21481795

RESUMEN

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.


Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Proteínas de Fusión bcr-abl/química , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB C , Conformación Proteica , Proteínas Tirosina Quinasas/química
6.
Proc Natl Acad Sci U S A ; 106(11): 4372-7, 2009 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-19246396

RESUMEN

Systemic inflammation arising from the organismal distribution of pathogen-associated molecular patterns is a major cause of clinical morbidity and mortality. Herein we report a critical and previously unrecognized in vivo role for germinal center kinase (GCK, genome nomenclature: map4k2), a mammalian Sterile 20 (STE20) orthologue, in PAMP signaling, and systemic inflammation. We find that disruption of gck in mice strongly impairs PAMP-stimulated macrophage cytokine and chemokine release and renders mice resistant to endotoxin-mediated lethality. Bone marrow transplantation studies show that hematopoietic cell GCK signaling is essential to systemic inflammation. Disruption of gck substantially reduces PAMP activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) via reduced activation of the MAPK-kinase-kinases (MAP3Ks) mixed lineage kinases (MLKs)-2 and -3. Extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB) activation are largely unaffected. Thus, GCK is an essential PAMP effector coupling JNK and p38, but not ERK or NF-kappaB to systemic inflammation.


Asunto(s)
Inflamación/etiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Adrenomedulina/farmacología , Animales , Trasplante de Médula Ósea , Citocinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular , Quinasas del Centro Germinal , Células Madre Hematopoyéticas/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , FN-kappa B , Proteínas Serina-Treonina Quinasas/deficiencia , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
7.
Biochem Biophys Res Commun ; 370(2): 230-4, 2008 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-18359290

RESUMEN

This study was undertaken to interrogate cancer cell survival during long-term hypoxic stress. Two systems with relevance to carcinogenesis were employed: Fully transformed BJ cells and a renal carcinoma cell line (786-0). The dynamic of AMPK activity was consistent with a prosurvival role during chronic hypoxia. This was further supported by the effects of AMPK agonists and antagonists (AICAR and compound C). Expression of a dominant-negative AMPK alpha resulted in a decreased ATP level and significantly compromised survival in hypoxia. Dose-dependent prosurvival effects of rapamycin were consistent with mTOR inhibition being a critical downstream mediator of AMPK in persistent low oxygen.


Asunto(s)
Proteínas Quinasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Línea Celular Transformada , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Ribonucleótidos/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR
8.
Curr Protoc Pharmacol ; Chapter 14: Unit14.10, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22294220

RESUMEN

Human myeloproliferative diseases are common hematologic disorders characterized by clonal overproduction of maturing myeloid or erythroid cells, often caused by expression of a mutant, dysregulated tyrosine kinase (TK). These diseases can be accurately modeled in laboratory mice by the retroviral transfer of a mutant TK gene into murine hematopoietic stem and progenitor cells, followed by transplantation of these cells into irradiated recipient mice. This yields a model system for analyzing the molecular pathophysiology of these conditions and provides a platform for testing therapies, particularly molecularly targeted new chemical entities (NCEs). The Basic Protocol in this unit describes the preparation of mouse bone marrow cells to express the relevant human oncogene before transplanting them into irradiated recipient mice. An alternate protocol describes a similar technique that allows specific induction of lymphoproliferative disease by some TKs. Support protocols for generating and titering retroviral stocks are also included.


Asunto(s)
Trasplante de Médula Ósea/métodos , Modelos Animales de Enfermedad , Trastornos Mieloproliferativos/genética , Proteínas Tirosina Quinasas/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , Células de la Médula Ósea , Medios de Cultivo/farmacología , Citocinas/farmacología , Citometría de Flujo , Fluorouracilo/farmacología , Genes abl/genética , Células HEK293 , Humanos , Leucemia de Células B/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Trasplante de Neoplasias/métodos , Retroviridae , Manejo de Especímenes/métodos , Transfección/métodos , Células Tumorales Cultivadas
9.
J Vis Exp ; (10): 550, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18989403

RESUMEN

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.


Asunto(s)
Retroviridae/crecimiento & desarrollo , Retroviridae/genética , Transfección/métodos , Replicación Viral/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animales , Trasplante de Médula Ósea , Línea Celular Transformada , Vectores Genéticos , Humanos , Ratones , Oncogenes , Infecciones por Retroviridae/genética
10.
Infect Immun ; 72(3): 1257-64, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-14977926

RESUMEN

The opportunistic protozoan Toxoplasma gondii is a prototypic Th1-inducing pathogen inducing strong gamma interferon (IFN-gamma) cytokine responses that are required to survive infection. Intracellular signaling intermediate STAT1 mediates many effects of IFN-gamma and is implicated in activation of T-bet, a master regulator of Th1 differentiation. Here, we show that T. gondii-infected STAT1-null mice fail to upregulate the IFN-gamma-dependent effector molecules inducible nitric oxide synthase (iNOS), IGTP, and LRG-47, which are required for mice to survive infection. Both T-bet and interleukin-12 receptor beta2 (IL-12Rbeta2) failed to undergo normal upregulation in response to T. gondii. Development of IFN-gamma-producing CD4(+) and CD8(+) T lymphocytes was severely curtailed in the absence of STAT1, but a substantial level of STAT1-independent non-T-cell-derived IFN-gamma was induced. Absence of STAT1 also resulted in increased IL-4, Arg1, Ym1, and Fizz1, markers of Th2 differentiation and alternative macrophage activation. Together, the results show that T. gondii induces STAT1-dependent T-lymphocyte and STAT1-independent non-T-cell IFN-gamma production, but that effector functions of this type 1 cytokine cannot operate in the absence of STAT1, resulting in extreme susceptibility to acute infection.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Interferón gamma/biosíntesis , Toxoplasmosis Animal/etiología , Transactivadores/fisiología , Animales , Secuencia de Bases , Citocinas/biosíntesis , ADN Complementario/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , GTP Fosfohidrolasas/biosíntesis , Proteínas de Unión al GTP/biosíntesis , Inmunidad Innata , Macrófagos/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Factor de Transcripción STAT1 , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/fisiopatología , Transactivadores/deficiencia , Transactivadores/genética
12.
Infect Immun ; 71(5): 2577-83, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12704131

RESUMEN

Toxoplasma gondii is an opportunistic intracellular parasite. Infection with the high-virulence T. gondii strain RH induces inflammatory cytokine overproduction and uncontrolled apoptosis in lymphoid organs. Here, we show by fluorescent terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and binding of fluorescein isothiocyanate-conjugated VAD-FMK, an irreversible pan-caspase inhibitor, that parasite-triggered apoptosis occurs among CD4(+), CD8(+), B220(+), Gr-1(+), and NK1.1(+) splenic populations. Caspases 8 and 9 were activated during infection, implicating cell surface death receptors and mitochondria in apoptosis. Induction of apoptosis was absent among all cell populations in both interleukin-12 (IL-12) p40- and Fas ligand (FasL)-negative mice. STAT-1 phosphorylation correlated with onset of apoptosis during infection, but in the absence of IL-12 p40 and functional FasL, activation of this transcription factor failed to occur. The results demonstrate T. gondii-induced activation of multiple apoptotic pathways, dependent upon both IL-12 p40 and FasL, that may play a role in the lethal pathology of infection.


Asunto(s)
Apoptosis , Proteínas de Unión al ADN/metabolismo , Interleucina-12/fisiología , Glicoproteínas de Membrana/fisiología , Subunidades de Proteína/fisiología , Toxoplasmosis/patología , Transactivadores/metabolismo , Animales , Proteína Ligando Fas , Interferón gamma/farmacología , Subunidad p40 de la Interleucina-12 , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fosforilación , Factor de Transcripción STAT1 , Toxoplasmosis/metabolismo
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