RESUMEN
RMP1-14 is a monoclonal antibody that targets the murine PD-1 protein, and has been used extensively to probe the effects of PD-1 inhibition in preclinical murine models. However, to date, no quantitative analytical methods have been published for RMP1-14. To evaluate its anti-tumor activity in BALB/c mice inoculated with CT26.WT murine colon cancer cells, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify RMP1-14 in BALB/c mouse K3EDTA plasma was developed and validated. The methodology used a signature peptide (GFYPPDIYTEWK) as a surrogate for RMP1-14 quantitation and an isotopically labeled analog of the signature peptide as the internal standard. Initial method development focused on a hybrid LC-MS/MS assay involving Protein G immunoprecipitation, but this strategy was abandoned due to lack of selectivity. The final validated method consisted of dilution with Tris-buffered saline, trypsin digestion, and desalting using micro solid-phase extraction. Analytical run time was 3.50 min, and the method demonstrated linearity between 0.500 and 50.0 µg/mL of intact RMP1-14. Accuracy, precision, and robustness were all acceptable, and the method was demonstrated to be comparable to a commercially available fit-for-purpose enzyme-linked immunosorbent assay (ELISA) capable of measuring RMP1-14. The validated method was used to generate pharmacokinetic parameters from tumor-bearing BALB/c mice dosed with RMP1-14 at either 2.50 or 7.50 mg/kg. Overall, the validated method represents a novel tool that can be used to evaluate RMP1-14 activity in future immuno-oncology studies.
Asunto(s)
Anticuerpos Monoclonales/sangre , Cromatografía Liquida/métodos , Receptor de Muerte Celular Programada 1/inmunología , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Calibración , Límite de Detección , Ratones , Ratones Endogámicos BALB CRESUMEN
ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient-derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Receptores ErbB/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ADAM/inmunología , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cetuximab/administración & dosificación , Cetuximab/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Femenino , Células HCT116 , Células HT29 , Humanos , Ratones Endogámicos DBA , Ratones Desnudos , Neoplasias/inmunología , Neoplasias/metabolismo , Resultado del TratamientoRESUMEN
Considerable evidence points to important roles for inflammation in Alzheimer's disease (AD) pathophysiology. Epidemiological studies have suggested that long-term nonsteroidal anti-inflammatory drug (NSAID) therapy reduces the risk for Alzheimer's disease; however, the mechanism remains unknown. We report that a 9-month treatment of aged R1.40 mice resulted in 90% decrease in plaque burden and a similar reduction in microglial activation. Ibuprofen treatment reduced levels of lipid peroxidation, tyrosine nitration, and protein oxidation, demonstrating a dramatic effect on oxidative damage in vivo. Fibrillar ß-amyloid (Aß) stimulation has previously been demonstrated to induce the assembly and activation of the microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leading to superoxide production through a tyrosine kinase-based signaling cascade. Ibuprofen treatment of microglia or monocytes with racemic or S-ibuprofen inhibited Aß-stimulated Vav tyrosine phosphorylation, NADPH oxidase assembly, and superoxide production. Interestingly, Aß-stimulated Vav phosphorylation was not inhibited by COX inhibitors. These findings suggest that ibuprofen acts independently of cyclooxygenase COX inhibition to disrupt signaling cascades leading to microglial NADPH oxidase (NOX2) activation, preventing oxidative damage and enhancing plaque clearance in the brain.
Asunto(s)
Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/prevención & control , Antiinflamatorios no Esteroideos/farmacología , Ibuprofeno/farmacología , NADPH Oxidasas/antagonistas & inhibidores , Péptidos beta-Amiloides , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Ibuprofeno/uso terapéutico , Masculino , Ratones , Ratones Transgénicos , Microglía/enzimología , Microglía/metabolismo , Microglía/patología , Monocitos/metabolismo , NADPH Oxidasas/fisiología , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Placa Amiloide , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-vav , Transducción de Señal/fisiologíaRESUMEN
Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Abeta homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Abeta from the brain. The endolytic degradation of Abeta peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Abeta degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Abeta load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Abeta from the brain and suggest that LXR agonists may represent a novel therapy for AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/farmacología , Microglía/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Envejecimiento , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Animales Recién Nacidos , Conducta Animal/efectos de los fármacos , Benzoatos/farmacología , Bencilaminas/farmacología , Encéfalo/patología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática/métodos , Receptores X del Hígado , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Receptores Nucleares Huérfanos , Fragmentos de Péptidos/metabolismo , Placa Amiloide/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de TiempoRESUMEN
Removal of apoptotic cells is an essential process for normal development and tissue maintenance. Importantly, apoptotic cells stimulate their phagocytosis by macrophages while actively suppressing inflammatory responses. Growth arrest specific gene 6 (Gas6) is involved in this process, bridging phosphatidylserine residues on the surface of apoptotic cells to the Axl/Mer family of tyrosine kinases which stimulate phagocytosis. Animals with mutations or loss of these receptors exhibit phenotypes reflective of impaired phagocytosis and a hyperactive immune response. We report that Gas6 induces phagocytosis in microglia through a novel non-classical phagocytic mechanism. Gas6 stimulates a type-II-related phagocytic response, but requires Vav phosphorylation and Rac activation, distinguishing it from the classical type II mechanism. Importantly, Gas6 suppressed lipopolysaccharide-induced expression of the inflammatory molecules IL-1beta and iNOS. Gas6 inhibited iNOS expression through suppression of promoter activity. The present data provide direct evidence for the role of Gas6 receptors in mediating an anti-inflammatory response to ligands found on apoptotic cells with the simultaneous stimulation of phagocytosis. These data provide a mechanistic explanation for the phenotype observed in animals lacking Axl/Mer receptors.
Asunto(s)
Inflamación/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Microglía/efectos de los fármacos , Proteínas Oncogénicas/fisiología , Fagocitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Encéfalo/metabolismo , Línea Celular/efectos de los fármacos , Línea Celular/enzimología , Línea Celular/fisiología , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Lipopolisacáridos/toxicidad , Ratones , Microglía/enzimología , Microglía/fisiología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Fagocitosis/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética/efectos de los fármacos , Tirosina Quinasa c-Mer , Proteínas de Unión al GTP rac/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
Epidemiological evidence suggests that long term treatment with hydroxymethylglutaryl-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins have pleiotropic actions and act to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. In neuroblastoma cell lines, treatment with statins inhibits the membrane localization of Rho and Rab proteins at statin doses as low as 200 nm, without affecting cellular cholesterol levels. In addition, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of a subset of GTPases. The amyloid precursor protein (APP) is proteolytically cleaved to generate beta-amyloid (Abeta), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of Rab family proteins involved in vesicular trafficking. Moreover, inhibition of Rho family protein function reduces levels of APP C-terminal fragments due to enhanced lysosomal dependent degradation. Statin inhibition of protein isoprenylation results in decreased Abeta secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Abeta production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Prenilación de Proteína/efectos de los fármacos , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Animales , Toxinas Bacterianas/farmacología , Línea Celular Tumoral , Lovastatina/farmacología , Lisosomas/metabolismo , Ácido Mevalónico/farmacología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/análisis , Simvastatina/farmacología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.
RESUMEN
Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the alpha(6)beta(1)-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav(-/-) mice. Stimulation of Vav(-/-) microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.
Asunto(s)
Péptidos beta-Amiloides/fisiología , Microglía/metabolismo , Monocitos/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Estallido Respiratorio , Transducción de Señal , Animales , Antígenos CD36/biosíntesis , Antígeno CD47/biosíntesis , Humanos , Integrina alfa6beta1/metabolismo , Ratones , Ratones Transgénicos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been reported to reduce the risk of Alzheimer disease. We have shown previously that statins inhibit a beta-amyloid (Abeta)-mediated inflammatory response through mechanisms independent of cholesterol reduction. Specifically, statins exert anti-inflammatory actions through their ability to prevent the isoprenylation of members of the Rho family of small G-proteins, resulting in the functional inactivation of these G-proteins. We report that statin treatment of microglia results in perturbation of the cytoskeleton and morphological changes due to alteration in Rho family function. Statins also block Abeta-stimulated phagocytosis through inhibition of Rac action. Paradoxically, the statin-mediated inactivation of G-protein function was associated with increased GTP loading of Rac and RhoA, and this effect was observed in myeloid lineage cells and other cell types. Statin treatment disrupted the interaction of Rac with its negative regulator the Rho guanine nucleotide dissociation inhibitor (RhoGDI), an interaction that is dependent on protein isoprenylation. We propose that lack of negative regulation accounts for the increased GTP loading. Isoprenylation of Rac is also required for efficient interaction with the plasma membrane, and we report that statin treatment dramatically reduces the capacity of Rac to interact with membranes. These results suggest a mechanism by which statins inhibit the actions of Rho GTPases and attenuate Abeta-stimulated inflammation.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Proteínas de Unión al GTP rho/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/prevención & control , Animales , Línea Celular , Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Inflamación , Ratones , Microglía/efectos de los fármacos , Monocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Prenilación de ProteínaRESUMEN
Neuronal survival in developing animals is often dependent on afferent activity. In the posthatch chick, approximately 30% of the neurons in the avian cochlear nucleus, nucleus magnocellularis (NM) die following elimination of VIIIth nerve activity. The factors that influence death or survival of an individual NM neuron are largely unknown. Previous studies indicate that both cell death and cell survival mechanisms compete to determine cell fate. One factor that has been shown to suppress cell death cascades in a variety of systems is bcl-2. If this gene product plays a role in regulating cell survival following deafferentation, then one might expect the expression of this gene to be influenced by removal of afferent input. In the present study, in situ hybridization revealed a rapid and transient increase in bcl-2 mRNA in NM neurons following deafferentation. Enhanced bcl-2 mRNA expression was observed at 6 and 12 h following deafferentation, but not at 3 or 24 h. Surprisingly, the upregulation of bcl-2 mRNA was limited to a subpopulation (20-30%) of deafferented neurons corresponding to the number of NM neurons that eventually die following cochlea removal. The robust and rapid upregulation of this gene suggests that cell death cascades regulated by bcl-2 may be initiated following deafferentation.