RESUMEN
BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.
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Asma , Interleucina-13 , Animales , Humanos , Ratones , Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Interleucina-4/genética , Pulmón , Proteínas , Nebulizadores y Vaporizadores , Receptores de Interleucina-4/inmunologíaRESUMEN
Scanning MS by MALDI MS imaging (MALDI-MSI) creates large volumetric global datasets that describe the location and identity of ions registered at each sampling location. While thousands of ion peaks are recorded in a typical whole-tissue analysis, only a fraction of these measured molecules are purposefully scrutinized within a given experimental design. To address this need, we recently reported new methods to query the full volume of MALDI-MSI data that correlate all ion masses to one another. As an example of this utility, we demonstrate that specific ion peak m/z signatures can be used to localize similar histological structures within tissue samples. In this study, we use the example of ion peak masses that are associated with tissue spaces occupied by airway bronchioles in rat lung samples. The volume of raw data was preprocessed into structures of 0.1 mass unit bins containing metadata collected at each sampling position. Interactive visualization in ParaView identified ion peaks that especially showed strong association with airway bronchioles but not vascular or parenchymal tissue compartments. Further iterative statistical correlation queries provided ranked indices of all m/z values in the global dataset regarding coincident distributions at any given X, Y position in the histological spaces occupied by bronchioles The study further provides methods for extracting important information contained in global datasets that previously was unseen or inaccessible.
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Iones , Imagen Molecular/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Peso Molecular , Ratas , Distribución TisularRESUMEN
Background: Pre-neutrophils, while developing in the bone marrow, transcribe the Inhba gene and synthesize Activin-A protein, which they store and release at the earliest stage of their activation in the periphery. However, the role of neutrophil-derived Activin-A is not completely understood. Methods: To address this issue, we developed a neutrophil-specific Activin-A-deficient animal model (S100a8-Cre/Inhba fl/fl mice) and analyzed the immune response to Influenza A virus (IAV) infection. More specifically, evaluation of body weight and lung mechanics, molecular and cellular analyses of bronchoalveolar lavage fluids, flow cytometry and cell sorting of lung cells, as well as histopathological analysis of lung tissues, were performed in PBS-treated and IAV-infected transgenic animals. Results: We found that neutrophil-specific Activin-A deficiency led to exacerbated pulmonary inflammation and widespread hemorrhagic histopathology in the lungs of IAV-infected animals that was associated with an exuberant production of neutrophil extracellular traps (NETs). Moreover, deletion of the Activin-A receptor ALK4/ACVR1B in neutrophils exacerbated IAV-induced pathology as well, suggesting that neutrophils themselves are potential targets of Activin-A-mediated signaling. The pro-NETotic tendency of Activin-A-deficient neutrophils was further verified in the context of thioglycollate-induced peritonitis, a model characterized by robust peritoneal neutrophilia. Of importance, transcriptome analysis of Activin-A-deficient neutrophils revealed alterations consistent with a predisposition for NET release. Conclusion: Collectively, our data demonstrate that Activin-A, secreted by neutrophils upon their activation in the periphery, acts as a feedback mechanism to moderate their pro-NETotic tendency and limit the collateral tissue damage caused by neutrophil excess activation during the inflammatory response.
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Virus de la Influenza A , Gripe Humana , Neumonía , Animales , Ratones , Humanos , Neutrófilos , Pulmón/patología , Neumonía/metabolismo , Gripe Humana/patología , Activinas/metabolismoRESUMEN
RATIONALE: Activin-A is up-regulated in various respiratory disorders. However, its precise role in pulmonary pathophysiology has not been adequately substantiated in vivo. OBJECTIVES: To investigate in vivo the consequences of dysregulated Activin-A expression in the lung and identify key Activin-A-induced processes that contribute to respiratory pathology. METHODS: Activin-A was ectopically expressed in murine lung, and functional, structural, and molecular alterations were extensively analyzed. The validity of Activin-A as a therapeutic target was demonstrated in animals overexpressing Activin-A or treated with intratracheal instillation of LPS. Relevancy to human pathology was substantiated by demonstrating high Activin-A levels in bronchoalveolar lavage (BAL) samples from patients with acute respiratory distress syndrome (ARDS). MEASUREMENTS AND MAIN RESULTS: Overexpression of Activin-A in mouse airways caused pulmonary pathology reminiscent of acute lung injury (ALI)/ARDS. Activin-A triggered a lasting inflammatory response characterized by acute alveolar cell death and hyaline membrane formation, sustained up-regulation of high-mobility group box 1, development of systemic hypercoagulant state, reduction of surfactant proteins SpC, SpB, and SpA, decline of lung compliance, transient fibrosis, and eventually emphysema. Therapeutic neutralization of Activin-A attenuated the ALI/ARDS-like pathology induced either by ectopic expression of Activin-A or by intratracheal instillation of LPS. In line with the similarity of the Activin-A-induced phenotype to human ARDS, selective up-regulation of Activin-A was found in BAL of patients with ARDS. CONCLUSIONS: Our studies demonstrate for the first time in vivo the pathogenic consequences of deregulated Activin-A expression in the lung, document novel aspects of Activin-A biology that provide mechanistic explanation for the observed phenotype, link Activin-A to ALI/ARDS pathophysiology, and provide the rationale for therapeutic targeting of Activin-A in these disorders.
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Activinas/metabolismo , Pulmón/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Receptores de Activinas Tipo II/uso terapéutico , Activinas/análisis , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Femenino , Proteína HMGB1/metabolismo , Humanos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Proteínas Recombinantes de Fusión/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Regulación hacia ArribaRESUMEN
Neutrophil extracellular traps (NETs) contribute to the pathophysiology of multiple inflammatory and autoimmune diseases. Targeting the NETosis pathway has demonstrated significant therapeutic potency in various disease models. Here, we describe a first-in-class monoclonal antibody (CIT-013) with high affinity for citrullinated histones H2A and H4, which inhibits NETosis and reduces tissue NET burden in vivo with significant anti-inflammatory consequences. We provide a detailed understanding of the epitope selectivity of CIT-013. Detection of CIT-013 epitopes in rheumatoid arthritis (RA) synovium provides evidence that RA is an autoimmune disease with excessive citrullinated NETs that can be targeted by CIT-013. We show that CIT-013 acts upon the final stage of NETosis, binding to its chromatin epitopes when plasma membrane integrity is compromised to prevent NET release. Bivalency of CIT-013 is necessary for NETosis inhibition. In addition, we show that CIT-013 binding to NETs and netting neutrophils enhance their phagocytosis by macrophages in an Fc-dependent manner. This is confirmed using a murine neutrophilic airway inflammation model where a mouse variant of CIT-013 reduced tissue NET burden with significant anti-inflammatory consequences. CIT-013's therapeutic activity provides new insights for the development of NET antagonists and indicates the importance of a new emerging therapy for NET-driven diseases with unmet therapeutic needs.
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Anticuerpos Monoclonales , Artritis Reumatoide , Enfermedades Autoinmunes , Trampas Extracelulares , Animales , Ratones , Antiinflamatorios , Anticuerpos Monoclonales/farmacología , Artritis Reumatoide/tratamiento farmacológico , Enfermedades Autoinmunes/tratamiento farmacológico , Epítopos/metabolismo , Histonas/metabolismo , Neutrófilos , Anticuerpos Antiproteína Citrulinada/farmacologíaRESUMEN
BACKGROUND: Angiopoietin (Ang)-1 and Ang-2, and their shared receptor Tie2, are expressed in rheumatoid arthritis (RA) synovial tissue, but the cellular targets of Ang signalling and the relative contributions of Ang-1 and Ang-2 to arthritis are poorly understood. OBJECTIVES: To determine the cellular targets of Ang signalling in RA synovial tissue, and the effects of Ang-2 neutralisation in murine collagen-induced arthritis (CIA). METHODS: RA and psoriatic arthritis (PsA) synovial biopsies were examined for expression of Tie2 and activated phospho (p)-Tie2 by quantitative immunohistochemistry and immunofluorescent double staining. Human monocyte and macrophage Tie2 expression was determined by flow cytometry and quantitative PCR. Regulation of macrophage intracellular signalling pathways and gene expression were examined by immunoblotting and ELISA. CIA was assessed in mice treated with saline, control antibody, prednisolone or neutralising anti-Ang-2 antibody. RESULTS: Expression of synovial Tie2 and p-Tie2 was similar in RA and PsA. Tie2 activation in RA patient synovial tissue was predominantly localised in synovial macrophages and was expressed by human macrophage. Ang-1 and Ang-2 stimulated activation of multiple intracellular signalling pathways, and cooperated with tumour necrosis factor to induce macrophage interleukin 6 and macrophage inflammatory protein 1α production. Ang-2 selectively suppressed macrophage thrombospondin-2 production. Ang-2 neutralisation significantly decreased disease severity, synovial inflammation, neo-vascularisation and joint destruction in established CIA. CONCLUSIONS: The authors identify synovial macrophages as primary targets of Ang signalling in RA, and demonstrate that Ang-2 promotes the pro-inflammatory activation of human macrophages. Ang-2 makes requisite contributions to pathology in CIA, indicating that targeting Ang-2 may be of therapeutic benefit in the treatment of RA.
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Angiopoyetina 2/farmacología , Artritis Experimental/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 1/farmacología , Angiopoyetina 2/inmunología , Angiopoyetina 2/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Psoriásica/metabolismo , Artritis Psoriásica/patología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Quimiocina CCL3/biosíntesis , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Trombospondinas/biosíntesisRESUMEN
The targeted delivery of therapies to diseased tissues offers a safe opportunity to achieve optimal efficacy while limiting systemic exposure. These considerations apply to many disease indications but are especially relevant for rheumatoid arthritis (RA), as RA is a systemic autoimmune disease which affects multiple joints. We have identified an antibody that is specific to damaged arthritic cartilage (anti-ROS-CII) that can be used to deliver treatments specifically to arthritic joints, yielding augmented efficacy in experimental arthritis. In the current study, we demonstrate that scaffolds enriched with bioactive payloads can be delivered precisely to an inflamed joint and achieve superior efficacy outcomes consistent with the pharmacological properties of these payloads. As a scaffold, we have used extracellular vesicles (EVs) prepared from human neutrophils (PMNs), which possess intrinsic anti-inflammatory properties and the ability to penetrate inflamed arthritic cartilage. EV fortified with anti-ROS-CII (EV/anti-ROS-CII) retained anti-ROS-CII specificity and bound exclusively to the damaged cartilage. Following systemic administration, EV/anti-ROS-CII (a) exhibited the ability to localize specifically in the arthritic joint in vivo and (b) was able to specifically target single (viral IL-10 or anti-TNF) or combined (viral IL-10 and anti-TNF) anti-inflammatory treatments to the arthritic joint, which accelerated attenuation of clinical and synovial inflammation. Overall, this study demonstrates the attainability of targeting a pro-resolving biological scaffold to the arthritic joint. The potential of targeting scaffolds such as EV, nanoparticles, or a combination thereof alongside combined therapeutics is paramount for designing systemically administered broad-spectrum of anti-inflammatory treatments.
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Antiinflamatorios/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Cartílago/inmunología , Cartílago/patología , Sistemas de Liberación de Medicamentos/métodos , Vesículas Extracelulares , Animales , Femenino , Voluntarios Sanos , Humanos , Interleucina-10/administración & dosificación , Articulación de la Rodilla/efectos de los fármacos , Leucocitos/citología , Ratones , Ratones Endogámicos C57BL , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/inmunología , Proteínas Virales/administración & dosificaciónRESUMEN
BACKGROUND: Continuous exposure to tobacco smoke (TS) is a key cause of chronic obstructive pulmonary disease (COPD), a complex multifactorial disease that is difficult to model in rodents. The spontaneously hypertensive (SH) rat exhibits several COPD-associated co-morbidities such as hypertension and increased coagulation. We have investigated whether SH rats are a more appropriate animal paradigm of COPD. METHODS: SH rats were exposed to TS for 6 hours/day, 3 days/week for 14 weeks, and the lung tissues examined by immunohistochemistry. RESULTS: TS induced a CK13-positive squamous metaplasia in proximal airways, which also stained for Ki67 and p63. We hypothesise that this lesion arises by basal cell proliferation, which differentiates to a squamous cell phenotype. Differences in staining profiles for the functional markers CC10 and surfactant D, but not phospho-p38, indicated loss of ability to function appropriately as secretory cells. Within the parenchyma, there were also differences in the staining profiles for CC10 and surfactant D, indicating a possible attempt to compensate for losses in proximal airways. In human COPD sections, areas of CK13-positive squamous metaplasia showed sporadic p63 staining, suggesting that unlike the rat, this is not a basal cell-driven lesion. CONCLUSION: This study demonstrates that although proximal airway metaplasia in rat and human are both CK13+ and therefore squamous, they potentially arise by different mechanisms.
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Células Epiteliales/efectos de los fármacos , Hipertensión/complicaciones , Pulmón/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/etiología , Fumar/efectos adversos , Animales , Diferenciación Celular , Proliferación Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Hipertensión/metabolismo , Hipertensión/patología , Inmunohistoquímica , Queratina-13/metabolismo , Antígeno Ki-67/metabolismo , Pulmón/metabolismo , Pulmón/patología , Metaplasia , Fenotipo , Fosforilación , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Ratas , Ratas Endogámicas SHR , Especificidad de la Especie , Factores de Tiempo , Uteroglobina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Mobilization of hemopoietic progenitor cells from the bone marrow (BM) is a feature of inflammatory asthmatic responses. Understanding the mechanisms regulating progenitor cell mobilization and trafficking to the peripheral circulation might be important for the development of effective asthma therapies. OBJECTIVE: We investigated the role of adhesion molecules in the mobilization of hemopoietic progenitor cells from the BM during an allergen-induced asthmatic response. METHODS: BM and peripheral blood samples were obtained from dual-responders with mild asthma before and at several time points after allergen challenge. Fluctuations in expression and adhesive properties of beta1- and beta2-integrins on CD34(+)CD45(+) progenitor cells were assessed by using flow cytometry and adhesion to protein-coated wells, respectively. RESULTS: On BM-derived CD34(+)CD45(+) cells, expression of very late antigen (VLA) 4, but not VLA-5 or Mac-1, decreased significantly 24 hours after allergen challenge and had begun to recover by 48 hours after challenge. In peripheral blood allergen challenge induced a significant decrease in VLA-4 levels after 6 hours, which had not recovered by 96 hours after challenge. Similarly, VLA-5 expression decreased, most prominently at 72 to 96 hours after allergen challenge. In contrast, Mac-1 levels did not change. Chemokine-stimulated adhesion of BM-derived CD34(+)CD45(+) cells to fibronectin was significantly attenuated 24 hours after challenge. Furthermore, adhesion to fibronectin and vascular cell adhesion molecule 1 was greatly reduced by anti-VLA-4 or anti-VLA-5 antibodies. CONCLUSIONS: Preferential downregulation of beta1-integrin expression on progenitor cells can reduce the tethering forces to BM components, thus facilitating their egress to the peripheral circulation during an allergic inflammatory response.
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Alérgenos/farmacología , Asma/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Integrina beta1/biosíntesis , Adolescente , Adulto , Alérgenos/inmunología , Antígenos CD34/inmunología , Antígenos CD34/metabolismo , Asma/inmunología , Médula Ósea/inmunología , Médula Ósea/metabolismo , Antígenos CD18/biosíntesis , Antígenos CD18/inmunología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Células Cultivadas , Quimiocinas/inmunología , Quimiocinas/metabolismo , Quimiocinas/farmacología , Femenino , Fibronectinas/inmunología , Fibronectinas/metabolismo , Regulación de la Expresión Génica/inmunología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/inmunología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Integrina alfa4beta1/inmunología , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/inmunología , Integrina alfa5beta1/metabolismo , Integrina beta1/inmunología , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Antígeno de Macrófago-1/inmunología , Antígeno de Macrófago-1/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Purpose: The purpose of this study is to determine the potential of narrow spectrum kinase inhibitors (NSKIs) to treat inflammatory eye disorders. Methods: Human conjunctival epithelial (HCE) cells were retrieved from subjects via impression cytology. Real-time quantitative PCR (qPCR) was performed on HCE cells to determine gene expression of NSKI kinase targets and proinflammatory cytokines in dry eye disease (DED) patients versus healthy controls. qPCR also assessed p38α expression in hyperosmolar-treated Chang conjunctival epithelial cells. Interaction of NSKI TOP1362 with the kinases was evaluated in ATP-dependent Z-LYTE and competition binding assays. Anti-inflammatory activity was assessed in human peripheral blood mononuclear cells and primary macrophages. In an endotoxin-induced uveitis (EIU) study, lipopolysaccharide (LPS) was administered intravitreally to Lewis rats. TOP1362, dexamethasone, or vehicle was administered topically, and inflammatory cytokine levels were measured 6 hours after LPS injection. Results: HCE cells from DED patients showed significantly increased expression of p38α, spleen tyrosine kinase (Syk), Src, lymphocyte-specific protein tyrosine kinase (Lck), interleukin one beta (IL-1ß), interleukin eight (IL-8), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9). TOP1362 strongly inhibited the kinase targets p38α, Syk, Src, and Lck, blocked the rise in p38α expression in hyperosmolar Chang cells, and potently reduced inflammatory cytokine release in cellular models of innate and adaptive immunities. In the EIU model, TOP1362 dose-dependently attenuated the LPS-induced rise in inflammatory cell infiltration and ocular cytokine levels with efficacy comparable to that of dexamethasone. Conclusions: TOP1362 is a potent inhibitor of kinases upregulated in DED and markedly attenuates proinflammatory cytokine release in vitro and in vivo, highlighting the therapeutic potential of NSKIs for treating ocular inflammation, such as that observed in DED.
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Conjuntiva/citología , Citocinas/metabolismo , Síndromes de Ojo Seco/metabolismo , Células Epiteliales/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Animales , Estudios de Casos y Controles , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Endogámicas Lew , TranscriptomaRESUMEN
BACKGROUND: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition. METHODS: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa. RESULTS: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays. CONCLUSIONS: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease.
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Benzamidas/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/enzimología , Citocinas/metabolismo , Dasatinib/uso terapéutico , Naftalenos/uso terapéutico , Niacinamida/análogos & derivados , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Benzamidas/farmacología , Biopsia , Colitis Ulcerosa/patología , Citocinas/efectos de los fármacos , Dasatinib/farmacología , Células HT29 , Humanos , Interleucina-8/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Miofibroblastos/metabolismo , Naftalenos/farmacología , Niacinamida/farmacología , Niacinamida/uso terapéutico , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Respiratory syncytial virus (RSV) infection causes severe lower respiratory diseases in infancy, early childhood and the elderly. RSV infections respond poorly to current therapies. Therefore, we initiated a search for novel drug targets by investigating the characteristics and identity of RSV adhesion receptors on mammalian cells. Soluble human lectins, complex polysaccharides and a low molecular selectin antagonist, TBC1269, were used to characterise and isolate the RSV receptor on a human epithelial cell line (Hep2 cells). The binding characteristics of the RSV receptor on Hep2 cells were similar to those reported for L-selectin. The carbohydrate-based selectin antagonists, fucoidan and TBC 1269, inhibit RSV infection both in vitro and in a mouse model of infection. Furthermore, we have isolated annexin II as a potential RSV receptor on Hep2 cells. The expression of annexin II was increased after RSV infection. Recombinant annexin II binds to RSV G-protein, heparin and plasminogen and the binding is inhibited by a selectin antagonist, TBC1269. These findings indicate that inhibitors of annexin II could have potential in treating RSV infection.
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Anexina A2/metabolismo , Células Epiteliales/metabolismo , Receptores Virales/aislamiento & purificación , Receptores Virales/metabolismo , Virus Sincitial Respiratorio Humano/patogenicidad , Animales , Anexina A2/antagonistas & inhibidores , Anexina A2/genética , Anexina A2/aislamiento & purificación , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Línea Celular , Humanos , Selectina L/efectos de los fármacos , Selectina L/metabolismo , Manosa/análogos & derivados , Manósidos/farmacología , Manósidos/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Receptores Virales/química , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/metabolismo , Proteínas Virales/metabolismoRESUMEN
1. The antigen-induced inflammatory response in the Brown Norway rat is a model commonly used to assess the impact of novel compounds on airway eosinophilia. A detailed functional, cellular and molecular characterization of this model has not yet been performed within a single study. This information together with the temporal changes in this phenomenon should be known before this model can be used, with confidence, to elucidate the mechanisms of action of novel anti-inflammatory drugs. 2. Antigen challenge caused an accumulation of eosinophils in lung tissue 24 h after challenge. Accumulation of CD2(+) T cells was not apparent until after 72 h. 3. Interestingly, mRNA for the Th2 type cytokines interleukin (IL)-4, IL-5 and IL-13 and eotaxin were elevated in lung tissue after challenge and the expression of IL-13 and eotaxin protein increased at around 8-12 h. The temporal changes in both the biomarker production and the functional responses are important factors to consider in protocol design prior to initiating a compound screening program. 4. A neutralising antibody (R73) against alphabeta-TCR caused a significant reduction in T cell numbers accompanied by a significant suppression of eosinophil accumulation. 5. Airway hyperreactivity (AHR) was not apparent in this specific Brown Norway model in sensitized animals after a single or multiple challenges although eosinophil influx was seen in the same animals. 6. In conclusion, this is a convenient pre-clinical model (incorporating the measurement of biomarkers and functional responses) for screening novel small molecule inhibitors and/or biotherapeutics targeted against T cell/eosinophil infiltration/activation.
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Modelos Animales de Enfermedad , Eosinofilia/etiología , Hipersensibilidad/etiología , Inflamación/etiología , Enfermedades Pulmonares/etiología , Animales , Biomarcadores , Hiperreactividad Bronquial , Quimiocina CCL11 , Quimiocinas CC/genética , Citocinas/genética , Eosinófilos/fisiología , Pulmón/inmunología , Pulmón/patología , Masculino , ARN Mensajero/análisis , Ratas , Ratas Endogámicas BN , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Linfocitos TRESUMEN
OBJECTIVE: To evaluate the efficacy and tolerability of the selective CRTh2 (DP2) receptor antagonist AZD1981 compared with placebo in patients with moderate to severe COPD. METHODS: In this multicentre, randomised, double-blind, parallel-group, phase IIa study (ClinicalTrials.gov identifier: NCT00690482) patients with moderate to severe COPD received either AZD1981 1000 mg twice daily or matching placebo for 4 weeks. Inhaled terbutaline was used as-needed as reliever medication throughout. The co-primary endpoints were change from baseline to end of treatment in pre-bronchodilator forced expiratory volume in 1 s [FEV1] and the Clinical COPD Questionnaire (CCQ). Additional endpoints included other lung function measures, 6-min walk test (6-MWT), COPD symptom score, reliever medication use and tolerability. RESULTS: 118 patients were randomised to treatment (AZD1981 n = 61; placebo n = 57); 83% of patients were male and the mean age was 63 years (range 43-83). There were no significant differences in the mean difference in change from baseline to end of treatment between AZD1981 and placebo for the co-primary endpoints of pre-bronchodilator FEV1 (AZD1981-placebo: -0.015, 95% CI: -0.10 to 0.070; p = 0.72) and CCQ total score (difference: 0.042, 95% CI: -0.21 to 0.30; p = 0.75). Similarly, no differences were observed between treatments for the other outcomes of lung function, COPD symptom score, 6-MWT, BODE index, and use of reliever medication. AZD1981 was well tolerated. CONCLUSION: There was no beneficial clinical effect of AZD1981, at a dose of 1000 mg twice daily for 4 weeks, in patients with moderate to severe COPD. AZD1981 was well tolerated and no safety concerns were identified.
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Acetatos/uso terapéutico , Broncodilatadores/uso terapéutico , Indoles/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Acetatos/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Broncodilatadores/efectos adversos , Método Doble Ciego , Femenino , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Indoles/efectos adversos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Capacidad Vital/efectos de los fármacosRESUMEN
The respiratory tract is the primary site of exposure to airborne compounds, with the bronchial epithelium providing one of the first lines of defence. A growing need exists for an accurate in vitro model of the bronchial epithelium. Here, normal human bronchial epithelial (NHBE) cells cultured at an air/liquid interface create a fully differentiated, in-vivo-like model of the human bronchial epithelium. Developmental characterisation includes (i) trans-epithelial electrical resistance, (ii) morphology and (iii) bronchial cell specific stains/markers. It is concluded that the basal/progenitor cells create a pseudo-stratified, mucociliary NHBE model containing basal, serous, Clara, goblet and ciliated cells, reflective of the normal human bronchial epithelium (days 24-33 ALI culture).
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Bronquios/citología , Células Epiteliales/citología , Mucosa Respiratoria/citología , Ingeniería de Tejidos/métodos , Autopsia , Biomarcadores/análisis , Bronquios/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Impedancia Eléctrica , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Mucosa Respiratoria/metabolismoRESUMEN
BACKGROUND: Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood. METHODOLOGY AND PRINCIPAL FINDINGS: The objective of this study was to assess IL-1 α and ß expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and ß. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and ß levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1ß- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation. CONCLUSIONS AND SIGNIFICANCE: This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.
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Proteína Antagonista del Receptor de Interleucina 1/biosíntesis , Interleucina-1alfa/biosíntesis , Neutrófilos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Fumar , Animales , Biopsia , Caspasa 1/metabolismo , Humanos , Inflamación , Interleucina-1beta/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Humo , Esputo/metabolismoRESUMEN
BACKGROUND: The costimulatory molecule OX40 and its ligand, OX40L, mediate key aspects of allergic airway inflammation in animal models of asthma, including eosinophilic airway inflammation, airway hyperresponsiveness, and T helper 2 polarization. We sought to examine OX40/OX40L and interleukin (IL)-4 expression in asthma across severities. METHODS: Bronchial biopsies were obtained from 27 subjects with asthma (mild Global Initiative for Asthma [GINA] 1 [n = 10], moderate GINA 2-3 [n = 7], and severe GINA 4-5 [n = 10]) and 13 healthy controls. The number of OX40(+), OX40L(+), IL-4(+), and IL-4 receptor alpha (IL-4Ralpha)(+) cells in the lamina propria and airway smooth muscle (ASM) bundle and the intensity of IL-4Ralpha(+) expression by the ASM were assessed. RESULTS: The number of OX40(+), OX40L(+), and IL-4(+) cells in the lamina propria and OX40(+) and IL-4(+) cells in the ASM bundle was significantly increased in subjects with mild asthma, but not in those with moderate or severe asthma, compared with healthy controls. In the subjects with asthma, OX40/OX40L expression was positively correlated with the number of eosinophils and IL-4(+) cells in the lamina propria. The number of IL-4Ralpha(+) cells in the lamina propria was significantly increased in moderate-to-severe disease, but not in mild asthma, compared with controls. IL-4Ralpha expression by the ASM bundle was not different among groups. CONCLUSIONS: OX40/OX40L expression is increased in the bronchial submucosa in mild asthma, but not in moderate-to-severe disease, and is related to the degree of tissue eosinophilia and IL-4 expression. Whether these costimulatory molecules have a role as targets for asthma requires further investigation.
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Asma/metabolismo , Bronquios/metabolismo , Interleucina-4/metabolismo , Ligando OX40/metabolismo , Receptores OX40/metabolismo , Adulto , Asma/patología , Biopsia , Bronquios/patología , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso/metabolismo , Músculo Liso/patología , Eosinofilia Pulmonar/metabolismo , Eosinofilia Pulmonar/patología , Receptores de Interleucina-4/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
CpG-containing immunostimulatory DNA sequences (ISS), which signal through TLR9, are being developed as a therapy for allergic indications and have proven to be safe and well tolerated in humans when administrated via the pulmonary route. In contrast, ISS inhalation has unexplained toxicity in rodents, which express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, the principal TLR9-expressing cells in humans. We therefore investigated the mechanisms underlying this rodent-specific toxicity and its implications for humans. Mice responded to intranasally administered 1018 ISS, a representative B class ISS, with strictly TLR9-dependent toxicity, including lung inflammation and weight loss, that was fully reversible and pDC and B cell independent. Knockout mouse experiments demonstrated that ISS-induced toxicity was critically dependent on TNF-alpha, with IFN-alpha required for TNF-alpha induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-alpha in vitro in response to ISS stimulation. Moreover, sputum of allergic humans exposed to inhaled ISS demonstrated induction of IFN-inducible genes but minimal TNF-alpha induction. These data demonstrate that ISS induce rodent-specific TNF-alpha-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.
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Oligodesoxirribonucleótidos/toxicidad , Factor de Necrosis Tumoral alfa/metabolismo , Adyuvantes Inmunológicos/toxicidad , Administración por Inhalación , Animales , Asma/genética , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Técnicas In Vitro , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/inmunología , Especificidad de la Especie , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/deficiencia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
INTRODUCTION: Although the presence of bone marrow lesions (BMLs) on magnetic resonance images is strongly associated with osteoarthritis progression and pain, the underlying pathology is not well established. The aim of the present study was to evaluate the architecture of subchondral bone in regions with and without BMLs from the same individual using bone histomorphometry. METHODS: Postmenopausal female subjects (n = 6, age 48 to 90 years) with predominantly medial compartment osteoarthritis and on a waiting list for total knee replacement were recruited. To identify the location of the BMLs, subjects had a magnetic resonance imaging scan performed on their study knee prior to total knee replacement using a GE 1.5 T scanner with a dedicated extremity coil. An axial map of the tibial plateau was made, delineating the precise location of the BML. After surgical removal of the tibial plateau, the BML was localized using the axial map from the magnetic resonance image and the lesion excised along with a comparably sized bone specimen adjacent to the BML and from the contralateral compartment without a BML. Cores were imaged via microcomputed tomography, and the bone volume fraction and tissue mineral density were calculated for each core. In addition, the thickness of the subchondral plate was measured, and the following quantitative metrics of trabecular structure were calculated for the subchondral trabecular bone in each core: trabecular number, thickness, and spacing, structure model index, connectivity density, and degree of anisotropy. We computed the mean and standard deviation for each parameter, and the unaffected bone from the medial tibial plateau and the bone from the lateral tibial plateau were compared with the affected BML region in the medial tibial plateau. RESULTS: Cores from the lesion area displayed increased bone volume fraction but reduced tissue mineral density. The samples from the subchondral trabecular lesion area exhibited increased trabecular thickness and were also markedly more plate-like than the bone in the other three locations, as evidenced by the lower value of the structural model index. Other differences in structure that were noted were increased trabecular spacing and a trend towards decreased trabecular number in the cores from the medial location as compared with the contralateral location. CONCLUSIONS: Our preliminary data localize specific changes in bone mineralization, remodeling and defects within BMLs features that are adjacent to the subchondral plate. These BMLs appear to be sclerotic compared with unaffected regions from the same individual based on the increased bone volume fraction and increased trabecular thickness. The mineral density in these lesions, however, is reduced and may render this area to be mechanically compromised, and thus susceptible to attrition.
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Enfermedades de la Médula Ósea/patología , Huesos/patología , Calcificación Fisiológica , Osteoartritis de la Rodilla/patología , Anciano , Anciano de 80 o más Años , Densidad Ósea , Femenino , Humanos , Persona de Mediana Edad , Posmenopausia , EsclerosisRESUMEN
BACKGROUND: We modeled the expression of proteins in baseline bronchoalveolar lavage (BAL) samples from asymptomatic 60-year-old lifelong current smokers or healthy never-smokers, who were reevaluated after 6 to 7 years to record clinical outcome. METHODS: Applying a technology toolbox consisting of replicate 2-dimensional gel separations, image annotation, and mass spectrometry identification, we catalogued a global set of proteins that were differentially expressed in individuals by presence, absence, and intensity scores. RESULTS: By use of multivariate analysis, we selected a subset of proteins that accurately separated smokers from never-smokers based on composite scoring. Follow-up after 6 to 7 years identified a group of individuals who had progressed to chronic obstructive pulmonary disease (COPD), Global Initiative for Chronic Obstructive Lung Disease stage 2. The baseline BAL samples of these eventual COPD patients shared a distinct protein expression profile that could be identified using partial least-squares discriminant analysis. This pattern was not observed in BAL samples of asymptomatic smokers free of COPD at 6- to 7-year follow-up. CONCLUSIONS: Our model suggests that certain patterns of protein expression occurring in the airways of long-term smokers may be detected in smokers susceptible to a progression of COPD disease, before disease is clinically evident.