RESUMEN
Chronic kidney disease (CKD) is characterized by inflammation and fibrosis in the kidney. Renal biopsies and estimated glomerular filtration rate (eGFR) remain the standard of care, but these endpoints have limitations in detecting the stage, progression, and spatial distribution of fibrotic pathology in the kidney. MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo both in clinical and preclinical studies. However, these imaging studies have not systematically identified fibrosis particularly deeper in the kidney where biopsy sampling is limited, or completed an extensive analysis of whole organ histology, blood biomarkers, and gene expression to evaluate the relative strengths and weaknesses of MRI for evaluating renal fibrosis. In this study, we performed DTI in the sodium oxalate mouse model of CKD. The DTI parameters fractional anisotropy, apparent diffusion coefficient, and axial diffusivity were compared between the control and oxalate groups with region of interest (ROI) analysis to determine changes in the cortex and medulla. In addition, voxel-based analysis (VBA) was implemented to systematically identify local regions of injury over the whole kidney. DTI parameters were found to be significantly different in the medulla by both ROI analysis and VBA, which also spatially matched with collagen III immunohistochemistry (IHC). The DTI parameters in this medullary region exhibited moderate to strong correlations with histology, blood biomarkers, hydroxyproline, and gene expression. Our results thus highlight the sensitivity of DTI to the heterogeneity of renal fibrosis and importance of whole kidney noninvasive imaging.NEW & NOTEWORTHY Chronic kidney disease (CKD) can be characterized by inflammation and fibrosis of the kidney. Although standard of care methods have been limited in scope, safety, and spatial distribution, MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo. In this study, we performed DTI in an oxalate mouse model of CKD to systematically identify local kidney injury. DTI parameters strongly correlated with histology, blood biomarkers, hydroxyproline, and gene expression.
Asunto(s)
Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Fibrosis , Ratones Endogámicos C57BL , Insuficiencia Renal Crónica , Animales , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/diagnóstico por imagen , Masculino , Oxalatos/metabolismo , Riñón/patología , Riñón/diagnóstico por imagen , Riñón/metabolismo , RatonesRESUMEN
Glomeruli number and size are important for determining the pathogenesis of glomerular disease, chronic kidney disease, and hypertension. Moreover, renal injury can occur in specific cortical layers and alter glomerular spatial distribution. In this study, we present a comprehensive structural analysis of glomeruli in a model of Adriamycin (doxorubicin) nephropathy. Glomeruli are imaged (micro-CT at 10 × 10 × 10 µm3) in kidney specimens from C57Bl/6 mouse cohorts: control treated with saline ( n = 9) and Adriamycin treated with 20 mg/kg Adriamycin ( n = 7). Several indices were examined, including glomerular number, glomerular volume, glomerular volume heterogeneity, and spatial density at each glomerulus and in each cortical layer (superficial, midcortical, and juxtamedullary). In the Adriamycin-treated animals, glomerular number decreased significantly in the left kidney [control: 8,298 ± 221, Adriamycin: 6,781 ± 630 (mean ± SE)] and right kidney (control: 7,317 ± 367, Adriamycin: 5,522 ± 508), and glomerular volume heterogeneity increased significantly in the left kidney (control: 0.642 ± 0.015, Adriamycin: 0.786 ± 0.018) and right kidney (control: 0.739 ± 0.016, Adriamycin: 0.937 ± 0.023). Glomerular spatial density was not affected. Glomerular volume heterogeneity increased significantly in the superficial and midcortical layers of the Adriamycin cohort. Adriamycin did not affect glomerular volume or density metrics in the juxtamedullary region, suggesting a compensatory mechanism of juxtamedullary glomeruli to injury in the outer cortical layers. Left/right asymmetry was observed in kidney size and various glomeruli metrics. The methods presented here can be used to evaluate renal disease models with subtle changes in glomerular endowment locally or across the entire kidney, and they provide an imaging tool to investigate diverse interventions and therapeutic drugs.
Asunto(s)
Doxorrubicina , Glomeruloesclerosis Focal y Segmentaria/diagnóstico por imagen , Glomérulos Renales/diagnóstico por imagen , Microtomografía por Rayos X , Algoritmos , Animales , Sulfato de Bario/administración & dosificación , Medios de Contraste/administración & dosificación , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/patología , Interpretación de Imagen Asistida por Computador , Glomérulos Renales/patología , Masculino , Ratones Endogámicos C57BL , Valor Predictivo de las PruebasRESUMEN
Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.
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Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/fisiopatología , Diafragma/fisiopatología , Unión Neuromuscular/fisiopatología , Proteínas Tirosina Quinasas Receptoras/agonistas , Esclerosis Amiotrófica Lateral/patología , Animales , Diafragma/patología , Modelos Animales de Enfermedad , Activación Enzimática/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Unión Neuromuscular/patología , Superóxido Dismutasa-1/genéticaRESUMEN
Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.
Asunto(s)
Artritis Experimental/inmunología , Citocinas/inmunología , Inflamación/inmunología , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Artritis Experimental/metabolismo , Artritis Experimental/prevención & control , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Células Cultivadas , Citocinas/metabolismo , Femenino , Citometría de Flujo , Células HEK293 , Miembro Posterior/efectos de los fármacos , Miembro Posterior/inmunología , Miembro Posterior/patología , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Osteoartritis/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Inhibidores Enzimáticos/farmacocinética , Glioblastoma/metabolismo , Indazoles/farmacocinética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirimidinas/farmacocinética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Sulfonamidas/farmacocinética , Tiofenos/farmacocinética , Animales , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Indazoles/farmacología , Ratones , Ratones Desnudos , Pirimidinas/farmacología , Sulfonamidas/farmacología , Tiofenos/farmacología , Distribución TisularRESUMEN
Joint and skeletal development is highly regulated by extracellular matrix (ECM) proteoglycans, of which chondroitin sulfate proteoglycans (CSPGs) are a major class. Despite the requirement of joint CSPGs for skeletal flexibility and structure, relatively little is understood regarding their role in establishing joint positioning or in modulating signaling and cell behavior during joint formation. Chondroitin sulfate synthase 1 (Chsy1) is one of a family of enzymes that catalyze the extension of chondroitin and dermatan sulfate glycosaminoglycans. Recently, human syndromic brachydactylies have been described to have loss-of-function mutations at the CHSY1 locus. In concordance with these observations, we demonstrate that mice lacking Chsy1, though viable, display chondrodysplasia and decreased bone density. Notably, Chsy1(-/-) mice show a profound limb patterning defect in which orthogonally shifted ectopic joints form in the distal digits. Associated with the digit-patterning defect is a shift in cell orientation and an imbalance in chondroitin sulfation. Our results place Chsy1 as an essential regulator of joint patterning and provide a mouse model of human brachydactylies caused by mutations in CHSY1.
Asunto(s)
Tipificación del Cuerpo , Desarrollo Óseo , Huesos/enzimología , Braquidactilia/genética , Glicosiltransferasas/metabolismo , Articulaciones/embriología , Animales , Densidad Ósea , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Glucuronosiltransferasa , Glicosiltransferasas/genética , Humanos , Ratones , Enzimas Multifuncionales , N-Acetilgalactosaminiltransferasas , EmbarazoRESUMEN
Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Modelos Moleculares , Receptores de Complemento/genética , Animales , Artritis Experimental/complicaciones , Resorción Ósea/etiología , Inactivadores del Complemento , Cristalización , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Ratones , Receptores de Complemento/químicaRESUMEN
Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1ß and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Benzamidas/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Células Mieloides/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Agammaglobulinemia Tirosina Quinasa , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Benzamidas/química , Benzamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ratones , Células Mieloides/inmunología , Células Mieloides/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/farmacología , Proteínas Tirosina Quinasas/uso terapéutico , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Anticuerpos Antiidiotipos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Tumores Neuroendocrinos/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/inmunología , Adenocarcinoma/genética , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Modelos Animales de Enfermedad , Quimioterapia Combinada , Ingeniería Genética , Indoles/uso terapéutico , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Ratones , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirroles/uso terapéutico , Carcinoma Pulmonar de Células Pequeñas/genética , Sunitinib , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1beta, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome.
Asunto(s)
Proteínas Bacterianas/inmunología , Interacciones Huésped-Patógeno/inmunología , Macrófagos/microbiología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium marinum/crecimiento & desarrollo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/inmunología , Inflamación/microbiología , Interleucina-1beta/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Proteína con Dominio Pirina 3 de la Familia NLR , Fagosomas/inmunología , Cola (estructura animal)/microbiología , Tuberculoma/inmunología , Tuberculoma/microbiología , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Here, we have developed an automated image processing algorithm for segmenting lungs and individual lung tumors in in vivo micro-computed tomography (micro-CT) scans of mouse models of non-small cell lung cancer and lung fibrosis. Over 3000 scans acquired across multiple studies were used to train/validate a 3D U-net lung segmentation model and a Support Vector Machine (SVM) classifier to segment individual lung tumors. The U-net lung segmentation algorithm can be used to estimate changes in soft tissue volume within lungs (primarily tumors and blood vessels), whereas the trained SVM is able to discriminate between tumors and blood vessels and identify individual tumors. The trained segmentation algorithms (1) significantly reduce time required for lung and tumor segmentation, (2) reduce bias and error associated with manual image segmentation, and (3) facilitate identification of individual lung tumors and objective assessment of changes in lung and individual tumor volumes under different experimental conditions.
RESUMEN
Loss-of-function TREM2 mutations strongly increase Alzheimer's disease (AD) risk. Trem2 deletion has revealed protective Trem2 functions in preclinical models of ß-amyloidosis, a prominent feature of pre-diagnosis AD stages. How TREM2 influences later AD stages characterized by tau-mediated neurodegeneration is unclear. To understand Trem2 function in the context of both ß-amyloid and tau pathologies, we examined Trem2 deficiency in the pR5-183 mouse model expressing mutant tau alone or in TauPS2APP mice, in which ß-amyloid pathology exacerbates tau pathology and neurodegeneration. Single-cell RNA sequencing in these models revealed robust disease-associated microglia (DAM) activation in TauPS2APP mice that was amyloid-dependent and Trem2-dependent. In the presence of ß-amyloid pathology, Trem2 deletion further exacerbated tau accumulation and spreading and promoted brain atrophy. Without ß-amyloid pathology, Trem2 deletion did not affect these processes. Therefore, TREM2 may slow AD progression and reduce tau-driven neurodegeneration by restricting the degree to which ß-amyloid facilitates the spreading of pathogenic tau.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Atrofia/genética , Atrofia/metabolismo , Atrofia/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Receptores Inmunológicos/genética , Proteínas tau/genéticaRESUMEN
Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.
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Asma/tratamiento farmacológico , Inflamación Neurogénica/tratamiento farmacológico , Dolor/tratamiento farmacológico , Prurito/tratamiento farmacológico , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Canal Catiónico TRPA1/antagonistas & inhibidores , Adolescente , Adulto , Animales , Estudios de Cohortes , Modelos Animales de Enfermedad , Perros , Método Doble Ciego , Femenino , Cobayas , Voluntarios Sanos , Humanos , Isotiocianatos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Dolor/inducido químicamente , Prurito/inducido químicamente , Ratas , Ratas Sprague-Dawley , Canal Catiónico TRPA1/deficiencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Whole-body MRI combined with a semiautomated hierarchical multispectral image analysis technique was evaluated as a method for detecting viable tumor tissue in a murine model of metastatic breast cancer (4T1 cell line). Whole-body apparent diffusion coefficient, T(2), and proton density maps were acquired in this study. The viable tumor tissue segmentation included three-stage k-means clustering of the parametric maps, morphologic operations, application of a size threshold, and reader discrimination of the segmented objects. The segmentation results were validated by histologic evaluation, and the detection accuracy of the technique was evaluated at three size thresholds (15, 100, and 500 voxels). The accuracy was 88.9% for a 500-voxel size threshold, and the area under receiver operating characteristic curve was 0.84. The regions of segmented viable tumor tissue within the primary tumors were found mostly on the periphery of the tumors in agreement with the histologic findings. The presented technique was found capable of detecting metastases and segmenting the viable tumor from necrotic regions within tumors found in this model. It offers a noninvasive, whole-body, viable tumor tissue detection method for preclinical and potentially clinical applications such as tumor screening and evaluating therapeutic efficacy.
Asunto(s)
Algoritmos , Inteligencia Artificial , Neoplasias de la Mama/diagnóstico , Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Magnética/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Imagen de Cuerpo Entero/métodos , Animales , Neoplasias de la Mama/secundario , Línea Celular Tumoral , Aumento de la Imagen/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Complement pathway overactivation can lead to neuronal damage in various neurological diseases. Although Alzheimer's disease (AD) is characterized by ß-amyloid plaques and tau tangles, previous work examining complement has largely focused on amyloidosis models. We find that glial cells show increased expression of classical complement components and the central component C3 in mouse models of amyloidosis (PS2APP) and more extensively tauopathy (TauP301S). Blocking complement function by deleting C3 rescues plaque-associated synapse loss in PS2APP mice and ameliorates neuron loss and brain atrophy in TauP301S mice, improving neurophysiological and behavioral measurements. In addition, C3 protein is elevated in AD patient brains, including at synapses, and levels and processing of C3 are increased in AD patient CSF and correlate with tau. These results demonstrate that complement activation contributes to neurodegeneration caused by tau pathology and suggest that blocking C3 function might be protective in AD and other tauopathies.
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Enfermedad de Alzheimer/inmunología , Amiloidosis/inmunología , Complemento C3/metabolismo , Degeneración Nerviosa/inmunología , Tauopatías/inmunología , Enfermedad de Alzheimer/genética , Animales , Atrofia , Conducta Animal , Biomarcadores/metabolismo , Encéfalo/patología , Complemento C1q/metabolismo , Complemento C3/líquido cefalorraquídeo , Complemento C3/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Masculino , Ratones Transgénicos , Degeneración Nerviosa/genética , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/metabolismo , Sinapsis/metabolismoRESUMEN
Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.
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Homeostasis/inmunología , Inflamación/inmunología , Interleucinas/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Homeostasis/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.
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Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Activación Neutrófila , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Activación Enzimática , Inflamación/enzimología , Inflamación/genética , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
CRTh2 is expressed on immune cells that drive asthma pathophysiology. Current treatment options for severe asthma are inadequate and therapeutic antibody-mediated depletion of CRTh2-expressing cells represents a promising new therapeutic strategy. Here we report for the first time that CRTh2 is not only expressed on immune cells, but also on microvasculature in the central nervous system (CNS) and gastric mucosa in humans. Microvascular expression of CRTh2 raises a safety concern because a therapeutic antiCRTh2 antibody with enhanced depletion capacity could lead to vascular damage. To evaluate this safety risk, we characterized microvascular expression in human and in transgenic mice expressing human CRTh2 protein (hCRTh2.BAC.Tg) and found that CRTh2 is not localized to microvascular endothelium that is directly exposed to circulating therapeutic antibody, but rather, to pericytes that in the CNS are shielded from direct circulatory exposure by the blood-brain barrier. Immunohistochemical visualization of an intravenously administered antiCRTh2 antibody in transgenic mice revealed localization to microvascular pericytes in the gastric mucosa but not in the CNS, suggesting the blood-brain barrier effectively limits pericyte exposure to circulating therapeutic antibody in the CNS. Repeated dosing with a depleting antiCRTh2 antibody in hCRTh2.BAC.Tg mice revealed linear pharmacokinetics and no drug-related adverse findings in any tissues, including the CNS and gastric mucosa, despite complete depletion of CRTh2 expressing circulating eosinophils and basophils. Collectively, these studies demonstrate that the likelihood of drug-related CNS or gastrointestinal toxicity in humans treated with a therapeutic depleting antiCRTh2 antibody is low despite pericyte expression of CRTh2 in these tissues.
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Antiasmáticos/farmacología , Anticuerpos Monoclonales/farmacología , Asma/tratamiento farmacológico , Sistema Nervioso Central/efectos de los fármacos , Mucosa Gástrica/efectos de los fármacos , Pericitos/efectos de los fármacos , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Animales , Antiasmáticos/administración & dosificación , Antiasmáticos/farmacocinética , Antiasmáticos/toxicidad , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/toxicidad , Asma/inmunología , Asma/metabolismo , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Mucosa Gástrica/inmunología , Mucosa Gástrica/metabolismo , Humanos , Inyecciones Intravenosas , Ratones Endogámicos C57BL , Ratones Transgénicos , Pericitos/inmunología , Pericitos/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/inmunología , Receptores de Prostaglandina/metabolismo , Medición de Riesgo , Distribución TisularRESUMEN
Genetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non-small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner.