RESUMEN
Psoriasis is a chronic, immune-mediated inflammatory skin disease with a high negative impact on patient's quality of life. Secukinumab, the first interleukin 17A inhibitor, has been used for the systemic treatment of psoriasis, but its long-term, real-world retention rates in Japan have not been fully investigated. In this multicenter, noninterventional, retrospective chart review study, the retention rate of secukinumab and its effectiveness among patients with psoriasis in Japan was evaluated up to 5 years. Data of patients who received secukinumab after December 26, 2014, were collected from medical charts obtained from seven sites, all certified for biologics use by the Japanese Dermatological Association. Patient characteristics, secukinumab retention, factors affecting secukinumab retention, reason for drug discontinuation, and effectiveness data were collected. The primary end point was secukinumab retention rate at week 52. A total of 123 patients were included in the analysis. Of these, 27 patients discontinued secukinumab by week 52, yielding a 78.0% (95% confidence interval, 69.6-84.4) retention rate at week 52. For patients whose Psoriasis Area and Severity Index (PASI) score was available, mean ± standard deviation PASI at baseline and at week 52 were 9.21 ± 7.37 and 1.4 ± 2.6, respectively. During the entire study period, "insufficient response" was the most common reason for discontinuation, and "history of biologics use" was a factor significantly associated with secukinumab discontinuation (hazard ratio, 1.72; p = 0.018). This study demonstrates the real-world retention rate and effectiveness of secukinumab in patients with psoriasis in Japan for up to 5 years and provides clinical insights into psoriasis treatment strategies.
Asunto(s)
Productos Biológicos , Psoriasis , Humanos , Anticuerpos Monoclonales/uso terapéutico , Estudios Retrospectivos , Calidad de Vida , Resultado del Tratamiento , Índice de Severidad de la Enfermedad , Psoriasis/tratamiento farmacológico , Productos Biológicos/uso terapéuticoRESUMEN
Understanding patient preferences concerning the use of biologics for psoriasis treatment can support proper treatment selection to satisfy their needs. In Japan, limited studies have reported psoriasis patients' preferences for the use of biologics, and many of those focused on the improvement of skin symptoms. The present study was conducted as a web-based questionnaire survey using the discrete choice experiment approach to investigate the preferences of psoriasis patients for the use of biologics, as well as to describe social and clinical factors that influence these preferences. The following attributes were selected for the discrete choice experiment: efficacy at 1 year, risk of serious infections requiring hospitalization, incidence of injection site reactions, administration route and visits, co-payment, indications, and efficacy on skin symptoms and other manifestations (the last two have not been evaluated in previous studies). Data were collected from October 4 to October 8, 2021. An analysis of data from 357 psoriasis patients indicated that the most preferred attributes for biologics selection were administration route and visits, followed by the risk of serious infections requiring hospitalization. Some differences were observed among specific subgroups. This study demonstrated that patients with psoriasis prefer biologics with a less frequent administration route and visit schedule and a lower risk of serious infections requiring hospitalization, which contrasts with the results obtained in previous studies where the highest importance was placed on drug effectiveness. These results may reflect the personal and social impact of the coronavirus disease outbreak at the time of the survey. The results of this study might help physicians properly select biologics that satisfy psoriasis patients' needs, leading to better treatment adherence.
Asunto(s)
Productos Biológicos , Coronavirus , Psoriasis , Humanos , Prioridad del Paciente , Productos Biológicos/efectos adversos , Psoriasis/tratamiento farmacológico , Encuestas y CuestionariosRESUMEN
Little attention has been given to the burden of chronic urticaria (CU) in Japan compared with other skin diseases, such as atopic dermatitis (AD) and psoriasis. The primary objective of the RELEASE study was to evaluate the real-life quality-of-life impairment in CU patients in Japan. Data were collected from 1443 urticaria, 1668 AD and 435 psoriatic patients; 552 urticaria patients who presented urticaria symptoms for over 6 weeks were defined as CU. The mean Dermatology Life Quality Index (DLQI) total score was 4.8, 6.1 and 4.8 in CU, AD and psoriatic patients, respectively. Disease control of urticaria evaluated by the Urticaria Control Test (UCT) and DLQI exhibited a strong correlation with a Spearman's rank correlation coefficient of -0.7158. CU and AD patients had relatively higher scores in all Work Productivity and Activity Impairment - General Health subscales except for absenteeism. At the time of the survey, approximately 64% of CU patients reported UCT scores of <12 and demonstrated higher work productivity loss and activity impairment versus patients with UCT scores of ≥12. Patients with lower UCT scores also displayed a higher percentage of dissatisfaction with their health state and the treatment they received. Approximately 85% of patients with CU had visited dermatology clinics, and less than 20% had visited hospital, indicating existence of a highly burdened population outside specialized centers. These results highlight the unmet medical needs of CU patients, suggesting the need to increase awareness of CU burden among both physicians and patients and to pursue improved real-life patient care.
Asunto(s)
Costo de Enfermedad , Estado de Salud , Satisfacción del Paciente/estadística & datos numéricos , Calidad de Vida , Urticaria/complicaciones , Absentismo , Adulto , Enfermedad Crónica , Dermatitis Atópica/complicaciones , Dermatitis Atópica/epidemiología , Dermatitis Atópica/fisiopatología , Dermatitis Atópica/terapia , Eficiencia , Femenino , Necesidades y Demandas de Servicios de Salud/estadística & datos numéricos , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Medición de Resultados Informados por el Paciente , Prevalencia , Psoriasis/complicaciones , Psoriasis/epidemiología , Psoriasis/fisiopatología , Psoriasis/terapia , Urticaria/epidemiología , Urticaria/fisiopatología , Urticaria/terapiaRESUMEN
BACKGROUND: Neutrophils, the most populous innate immune cell type, are the first responders to sites of infection and inflammation. Neutrophils can release their DNA to form extracellular traps (NETs), webs of DNA and granular proteases that contribute to pathogen clearance and promote thrombus formation. At present, the study of NETs is in part limited to the qualitative analysis of fluorescence microscopy-based images, thus quantification of the interactions between NETs and coagulation factors remains ill-defined. AIM: Develop a quantitative method to measure the spatial distribution of DNA and colocalization of coagulation factor binding to neutrophils and NETs utilizing fluorescence-based microscopy. APPROACH: Human neutrophils were purified from peripheral blood, bound to fibronectin and treated with the PKC-activator phorbol myristate acetate (PMA) to induce neutrophil activation and NETs formation. Samples were incubated with purified coagulation factors or plasma before staining with a DNA-binding dye and coagulation factor-specific antibodies. The spatial distribution of DNA and coagulation factors was imaged via fluorescence microscopy and quantified via a custom-built MATLAB-based image analysis algorithm. The algorithm first established global thresholding parameters on a training set of fluorescence image data and then systematically quantified intensity profiles across treatment conditions. Quantitative comparison of treatment conditions was enabled through the normalization of fluorescent intensities using the number of cells per image to determine the percent and area of DNA and coagulation factor binding per cell. RESULTS: Upon stimulation with PMA, NETs formation resulted in an increase in the area of DNA per cell. The coagulation factor fibrinogen bound to both the neutrophil cell body as well as NETs, while prothrombin, FX and FVIIa binding was restricted to the neutrophil cell body. The Gla domain of FX was required to mediate FX-neutrophil binding. Activated protein C (APC), but not Gla-less APC, bound to neutrophil cell bodies and NETs in a punctate manner. Neither FXIIa nor FXIa were found to bind either neutrophil cell bodies or NETs. Fibrinogen binding was dependent on extracellular DNA, while FX and APC required phosphatidylserine exposure for binding to activated neutrophils. CONCLUSIONS: We have developed a quantitative measurement platform to define the spatial localization of fluorescently-labeled coagulation factor binding to neutrophils and extracellular DNA during NETosis.
Asunto(s)
ADN/análisis , Trampas Extracelulares , Microscopía Fluorescente/métodos , Neutrófilos/inmunología , Fenómenos Biofísicos , Factores de Coagulación Sanguínea , ADN/metabolismo , Trampas Extracelulares/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Microscopía Fluorescente/instrumentación , Activación Neutrófila , Neutrófilos/ultraestructura , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
OBJECTIVE: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. APPROACH AND RESULTS: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization, and fibrin formation on immobilized collagen and tissue factor under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pmol/L tissue factor, platelet CD62P expression, microaggregate formation, and progressive platelet consumption were significantly reduced in the presence of FXI function-blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. CONCLUSIONS: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlight FXI as a novel therapeutic target for inhibiting distal platelet consumption without affecting proximal platelet adhesion.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Factor XI/metabolismo , Mecanotransducción Celular , Activación Plaquetaria , Trombosis/sangre , Animales , Colágeno/sangre , Modelos Animales de Enfermedad , Factor XIa/metabolismo , Fibrina/metabolismo , Humanos , Masculino , Selectina-P/sangre , Papio anubis , Agregación Plaquetaria , Flujo Sanguíneo Regional , Estrés Mecánico , Trombina/metabolismo , Tromboplastina/metabolismo , Trombosis/fisiopatología , Trombosis/prevención & control , Factores de TiempoRESUMEN
Thrombus growth at the site of vascular injury is mediated by the sequential events of platelet recruitment, activation and aggregation concomitant with the initiation of the coagulation cascade, resulting in local thrombin generation and fibrin formation. While the biorheology of a localized thrombus formation has been well studied, it is unclear whether local sites of thrombin generation propagate platelet activation within the bloodstream. In order to study the physical biology of platelet activation downstream of sites of thrombus formation, we developed a platform to measure platelet activation and microaggregate formation in the bloodstream. Our results show that thrombi formed on collagen and tissue factor promote activation and aggregation of platelets in the bloodstream in a convection-dependent manner. Pharmacological inhibition of the coagulation factors (F) X, XI or thrombin dramatically reduced the degree of distal platelet activation and microaggregate formation in the bloodstream without affecting the degree of local platelet deposition and aggregation on a surface of immobilized collagen. Herein we describe the development and an example of the utility of a platform to study platelet activation and microaggregate formation in the bloodstream (convection-limited regime) relative to the local site of thrombus formation.
RESUMEN
Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration.
RESUMEN
The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.
Asunto(s)
Plaquetas/fisiología , Citoesqueleto/fisiología , Histona Desacetilasas/metabolismo , Activación Plaquetaria/fisiología , Transducción de Señal/fisiología , Tubulina (Proteína)/metabolismo , Plaquetas/citología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Histona Desacetilasa 6 , Histona Desacetilasas/genética , Humanos , Péptidos/genética , Péptidos/metabolismoRESUMEN
Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+) signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.
Asunto(s)
Quimiotaxis de Leucocito , Citoesqueleto/metabolismo , Neutrófilos/metabolismo , Quinasas p21 Activadas/metabolismo , Western Blotting , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Fluorescente , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Transporte de ProteínasRESUMEN
Colorectal cancer metastases can appear on the peritoneum and in lymph nodes, liver, and lungs, suggesting both hematogenous and lymphatic spreading of the primary tumor. While antithrombotic agents have been shown to reduce both long-term incidence and metastasis, the role of coagulation in facilitating metastasis is ill defined. We investigated the kinetics and molecular mechanisms of metastatic colon adenocarcinoma cell recruitment to thrombi under shear flow, ex vivo. Platelet aggregates were formed by perfusing citrated anticoagulated whole blood over immobilized fibrinogen or fibrillar collagen. Thrombi were formed by perfusing recalcified whole blood over fibrinogen or fibrillar collagen in the presence of coagulation. Cultured colon adenocarcinoma cells (SW620) were perfused either during or following platelet aggregate or thrombus formation. The degree of transient tumor cell interactions (recruitment, rolling, and release) and the number of firmly adhered tumor cells were quantified using fluorescence microscopy. Platelet aggregates and thrombi formed on either fibrinogen- or fibrillar-collagen supported SW620 cell interactions and adhesion under shear. Thrombi or fibrin supported a greater degree of SW620 cell interactions and adhesion compared with platelet aggregates or fibrinogen, respectively, demonstrating that coagulation promoted SW620 cell recruitment under shear. Interestingly, in the absence of anticoagulation, we observed SW620 preferentially binding to thrombus-bound polymorphonuclear leukocytes (PMNs). The addition of purified PMNs to thrombi resulted in a doubling of the number of interacting and bound SW620 cells. Since thrombi often accumulate and activate leukocytes, our findings suggest that leukocytes may play a role in localizing metastases to sites of thrombogenesis.
Asunto(s)
Adenocarcinoma/metabolismo , Coagulación Sanguínea/fisiología , Neoplasias del Colon/metabolismo , Trombosis/metabolismo , Línea Celular Tumoral , Humanos , Unión Proteica/fisiología , Resistencia al Corte/fisiologíaRESUMEN
Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, ßPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.
Asunto(s)
Plaquetas/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacología , Quinasas p21 Activadas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Plaquetas/citología , Plaquetas/metabolismo , Adhesión Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Quinasas Lim/genética , Quinasas Lim/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Quinasas p21 Activadas/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Lysophosphatidylcholine (LPC) is one of the major lysophospholipids mainly generated by phospholipase A2 (PLA2)-mediated hydrolysis of phosphatidylcholine (PC). We previously found that LPC displays neurotrophin-like activity in the rat pheochromocytoma PC12 cells and in cerebellar granule neurons, but the molecular mechanism remains unclear. We report here that LPC specifically enhances nerve growth factor (NGF)-induced signals in PC12 cells. When PC12 cells were treated with NGF, MAPK was phosphorylated, but this phosphorylation was significantly elevated when LPC was added together. In accordance, NGF-induced expression of immediate early genes, c-fos and NGF-IA, was upregulated by LPC. Phosphorylation of the upstream components, MEK and NGF receptor TrkA, was also promoted by LPC, which was in line with increased phosphorylation of Akt. In contrast, LPC did not enhance epidermal growth factor (EGF)-, basic fibroblast growth factor-, or insulin-like growth factor-1-induced signals. Studies using TrkA/EGF receptor chimeras demonstrated that the extracellular domain, but not the transmembrane or intracellular domains, of TrkA is responsible for the effect of LPC. Exogenously-added secretory PLA2 (sPLA2) enhanced NGF-induced MAPK phosphorylation at a comparable level to LPC, suggesting that LPC generated in situ by sPLA2-mediated hydrolysis of membrane PC stimulated NGF-TrkA signal. Taken together, these results indicate a specific role and function of LPC on NGF-TrkA signaling pathway.
RESUMEN
Autophagy is an essential cellular mechanism for cell homeostasis and survival by which damaged cellular proteins are sequestered in autophagosomal vesicles and cleared through lysosomal machinery. The autophagy pathway also plays an important role in immunity and inflammation via pathogen clearance mechanisms mediated by immune cells, including macrophages and neutrophils. In particular, recent studies have revealed that autophagic activity is required for the release of neutrophil extracellular traps (NETs), representing a distinct form of active neutrophil death, namely NETosis. Although NET formation is beneficial during host defense against invading pathogens, the mechanisms that promote excessive NETosis under pathological conditions remain ill defined. In the present study, we aimed to characterize the role of the mammalian target of rapamycin (mTOR) in NETosis. As mTOR kinase is known as a key regulator of autophagy in many mammalian cells including neutrophils, we hypothesized that mTOR may play a regulatory role in NET release by regulating autophagic activity. Our data show that the pharmacological inhibition of the mTOR pathway accelerated the rate of NET release following neutrophil stimulation with the bacteria-derived peptide formyl-Met-Leu-Phe (fMLP), while autophagosome formation was enhanced by mTOR inhibitors. This increased mTOR-dependent NET release was sensitive to inhibition of respiratory burst or blockade of cytoskeletal dynamics. Overall, this study demonstrates a pivotal role for the mTOR pathway in coordinating intracellular signaling events downstream of neutrophil activation leading to NETosis.
Asunto(s)
Autofagia/fisiología , Activación Neutrófila , Neutrófilos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , ADN/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/inmunología , Fagocitosis , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Rho GTPase proteins play a central role in regulating the dynamics of the platelet actin cytoskeleton. Yet, little is known regarding how Rho GTPase activation coordinates platelet activation and function. In this study, we aimed to characterize the role of the Rho GTPase effector, p21 activated kinase (PAK), in platelet activation, lamellipodia formation, and aggregate formation under shear. APPROACH AND RESULTS: Stimulation of platelets with the glycoprotein receptor VI agonist, collagen-related peptide, rapidly activated PAK in a time course preceding phosphorylation of PAK substrates, LIM domain kinase LIMK1 and the MAPK/ERK kinase MEK, and the subsequent activation of MAPKs and Akt. Pharmacological inhibitors of PAK blocked signaling events downstream of PAK and prevented platelet secretion as well as platelet aggregation in response to collagen-related peptide. PAK inhibitors also prevented PAK activation and platelet spreading on collagen surfaces. PAK was also required for the formation of platelet aggregates and to maintain aggregate stability under physiological shear flow conditions. CONCLUSIONS: These results suggest that PAK serves as an orchestrator of platelet functional responses after activation downstream of the platelet collagen receptor, glycoprotein receptor VI.
Asunto(s)
Plaquetas/enzimología , Activación Plaquetaria , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Seudópodos/enzimología , Transducción de Señal , Quinasas p21 Activadas/sangre , Plaquetas/efectos de los fármacos , Proteínas Portadoras/farmacología , Forma de la Célula , Activación Enzimática , Humanos , Quinasas Lim/sangre , Quinasas Quinasa Quinasa PAM/sangre , Péptidos/farmacología , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/agonistas , Inhibidores de Proteínas Quinasas/farmacología , Seudópodos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Estrés Mecánico , Factores de Tiempo , Quinasas p21 Activadas/antagonistas & inhibidoresRESUMEN
Inflammatory demyelinating diseases like multiple sclerosis are characterized by mononuclear cell infiltration into the central nervous system. The glycosaminoglycan hyaluronan and its receptor, CD44, are implicated in the initiation and progression of a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Digestion of hyaluronan tethered to brain vascular endothelial cells by a hyaluronidase blocks the slow rolling of lymphocytes along activated brain vascular endothelial cells and delays the onset of EAE. These effects could be due to the elimination of hyaluronan or the generation of hyaluronan digestion products that influence lymphocytes or endothelial cells. Here, we found that hyaluronan dodecasaccharides impaired activated lymphocyte slow rolling on brain vascular endothelial cells when applied to lymphocytes but not to the endothelial cells. The effects of hyaluronan dodecasaccharides on lymphocyte rolling were independent of CD44 and a receptor for degraded hyaluronan, Toll-like receptor-4. Subcutaneous injection of hyaluronan dodecasaccharides or tetrasaccharides delayed the onset of EAE in a manner similar to subcutaneous injection of hyaluronidase. Hyaluronan oligosaccharides can therefore act directly on lymphocytes to modulate the onset of inflammatory demyelinating disease.
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Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Células Endoteliales/fisiología , Ácido Hialurónico/farmacología , Rodamiento de Leucocito/efectos de los fármacos , Linfocitos/fisiología , Oligosacáridos/farmacología , Animales , Encéfalo/irrigación sanguínea , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Expresión Génica , Receptores de Hialuranos/metabolismo , Activación de Linfocitos , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microvasos/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Human herpesviruses establish lifelong latency. Viral recrudescence can lead to the development of cancers, immunoproliferative disorders, transplantation complications, and thrombocytopenia. Although platelet-specific autoantibodies have been reported in patients infected with the Epstein-Barr virus (EBV), the mechanisms by which thrombocytopenia is induced remain unclear, as do the relative contributions of lytic viral replication and latent viral gene expression. The human gammaherpesviruses are tightly restricted in their ability to infect other mammals, so they are difficult to study in live animal models. Here we show that infection of mice with murine gammaherpesvirus-68 (γHV68), a rodent-specific pathogen closely related to EBV, induces the production of platelet-binding antibodies and causes thrombocytopenia. Infection of antibody-deficient mice does not lead to thrombocytopenia, indicating the platelet decrease is mediated by antibody. Additionally, infection with a latency-null recombinant γHV68 does not induce thrombocytopenia, suggesting factors associated with viral latency drive the infection-induced antibody-mediated thrombocytopenia. These studies describe an important animal model of gammaherpesvirus-induced autoimmune thrombocytopenia and demonstrate that this pathology is mediated by antibody and dependent on viral latency. This model will allow studies of the underlying mechanisms of disease progression and the testing of therapeutic strategies for the alleviation of virus-induced thrombocytopenia.
Asunto(s)
Anticuerpos/inmunología , Trastornos de las Plaquetas Sanguíneas/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Gammaherpesvirinae/fisiología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/inmunología , Latencia del Virus , Animales , Trastornos de las Plaquetas Sanguíneas/etiología , Plaquetas/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/complicaciones , Femenino , Infecciones por Herpesviridae/complicaciones , Humanos , Cadenas mu de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la Especie , Replicación ViralRESUMEN
The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 µm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.
Asunto(s)
Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Células Endoteliales/citología , Receptores de Hialuranos/biosíntesis , Ácido Hialurónico/química , Linfocitos/citología , Animales , Encéfalo/metabolismo , Enfermedades Desmielinizantes/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Exones , Femenino , Receptores de Hialuranos/genética , Inflamación , Rodamiento de Leucocito , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Hemostasis is dependent upon the successful recruitment and activation of blood platelets to the site of a breach in the vasculature. Platelet activation stimulates the rapid reorganization of the cortical actin cytoskeleton, resulting in the transformation of platelets from biconcave disks to fully spread cells. During this process, platelets extend filopodia and generate lamellipodia, resulting in a dramatic increase in the platelet surface area. Kohler-illuminated Nomarski Differential Interference Contrast microscopy has proved an effective tool to characterize platelet morphological changes in real time, and provides a useful tool to identify genetic and pharmacological regulators of platelet function.
Asunto(s)
Plaquetas/citología , Forma de la Célula/fisiología , Plaquetas/fisiología , Adhesión Celular/fisiología , Separación Celular , HumanosRESUMEN
PMN leukocytes are the most abundant leukocytes in the circulation and play an important role in host defense. PMN leukocyte recruitment and inflammatory responses at sites of infection are critical components in innate immunity. Although inflammation and coagulation are known to have bidirectional relationships, little is known about the interaction between PMN leukocytes and coagulation factors. Coagulation FXI participates in the intrinsic coagulation pathway upon its activation, contributing to hemostasis and thrombosis. We have shown previously that FXI-deficient mice have an increased survival and less leukocyte accumulation into the peritoneum in severe polymicrobial peritonitis. This result suggests a role for FXI in leukocyte trafficking and/or function. In this study, we characterized the functional consequences of FXIa binding to PMN leukocytes. FXIa reduced PMN leukocyte chemotaxis triggered by the chemokine, IL-8, or the bacterial-derived peptide, fMLP, perhaps as a result of the loss of directed migration. In summary, our data suggest that FXIa modulates the inflammatory response of PMN leukocytes by altering migration. These studies highlight the interplay between inflammation and coagulation and suggest that FXIa may play a role in innate immunity.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Factor XIa/metabolismo , Neutrófilos/fisiología , Coagulación Sanguínea , Movimiento Celular , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Factor XIa/farmacología , Humanos , Inflamación/metabolismo , Interleucina-8/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Peritonitis/metabolismo , Unión ProteicaRESUMEN
BACKGROUND AND PURPOSE: Infections with respiratory viruses induce exacerbations of asthma, increase acetylcholine release and potentiate vagally mediated bronchoconstriction by blocking inhibitory M2 muscarinic receptors on parasympathetic neurons. Here we test whether virus-induced M2 receptor dysfunction and airway hyperresponsiveness are tumour necrosis factor-alpha (TNF-α) dependent. EXPERIMENTAL APPROACH: Guinea pigs were pretreated with etanercept or phosphate-buffered saline 24 h before intranasal infection with parainfluenza. Four days later, pulmonary inflation pressure, heart rate and blood pressure were measured. M2 receptor function was assessed by the potentiation by gallamine (an M2 receptor antagonist) of bronchoconstriction caused by electrical stimulation of the vagus nerves and measured as increased pulmonary inflation pressure. Human airway epithelial cells were infected with influenza and TNF-α concentration in supernatant was measured before supernatant was applied to human neuroblastoma cells. M2 receptor expression in these neuroblastoma cells was measured by qRT-PCR. KEY RESULTS: Influenza-infected animals were hyperresponsive to vagal stimulation but not to intravenous ACh. Gallamine did not potentiate vagally induced bronchoconstriction in virus-infected animals, indicating M2 receptor dysfunction. Etanercept prevented virus-induced airway hyperresponsiveness and M2 receptor dysfunction, without changing lung viral titres. Etanercept caused a non-significant decrease in total cells, macrophages and neutrophils in bronchoalveolar lavage. Influenza infection significantly increased TNF-α release from isolated epithelial cells, sufficient to decrease M2 receptors in neuroblastoma cells. This ability of supernatants from infected epithelial cells to inhibit M2 receptor expression was blocked by etanercept. CONCLUSIONS AND IMPLICATIONS: TNF-α is a key mediator of virus-induced M2 muscarinic receptor dysfunction and airway hyperresponsiveness.