RESUMEN
Antisynthetase syndrome (ASSD) is an autoimmune disease characterized by circulating autoantibodies against one of eight aminoacyl-tRNA synthetases (aaRSs). Although these autoantibodies are believed to play critical roles in ASSD pathogenesis, the nature of their roles remains unclear. Here we describe ASSD pathogenesis and discuss ASSD-linked aaRSs - from the WHEP domain that may impart immunogenicity to the role of tRNA in eliciting the innate immune response and the secretion of aaRSs from cells. Through these explorations, we propose that ASSD pathogenesis involves the tissue-specific secretion of aaRSs and that extracellular tRNAs or tRNA fragments and their ability to engage Toll-like receptor signaling may be important disease factors.
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Aminoacil-ARNt Sintetasas , Miositis , Humanos , Aminoacil-ARNt Sintetasas/genética , ARN de Transferencia/genética , AutoanticuerposRESUMEN
Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.
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Ataxias Espinocerebelosas , Trombocitopenia , Trombosis , Tirosina-ARNt Ligasa , Virosis , Ratones , Animales , Trombopoyesis , Ratones TransgénicosRESUMEN
Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody-induced ITP.
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Anticuerpos Monoclonales/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/toxicidad , Reacciones Antígeno-Anticuerpo , Plaquetas/inmunología , Modelos Animales de Enfermedad , Inyecciones Intravenosas , Inyecciones Subcutáneas , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Opsoninas/inmunología , Agregación Plaquetaria/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/antagonistas & inhibidores , Púrpura Trombocitopénica Idiopática/etiología , ARN Mensajero/biosíntesis , Ratas , Bazo/patología , Trombopoyetina/biosíntesis , Trombopoyetina/genética , Regulación hacia ArribaRESUMEN
Type 2B von Willebrand disease (VWD) is caused by gain-of-function mutations in von Willebrand factor (VWF). Increased VWF affinity for GPIba results in loss of high molecular weight multimers and enhanced platelet clearance, both contributing to the bleeding phenotype. Severity of the symptoms vary among type 2B VWD patients, with some developing thrombocytopenia only under stress conditions. Efforts have been made to study underlying pathophysiology for platelet abnormalities, but animal studies have been limited because of species specificity in the VWF-GPIba interaction. Here, we generated a severe form of type 2B VWD (p.V1316M) knockin mice in the context of human VWF exon 28 (encoding A1 and A2 domains) and crossed them with human GPIba transgenic strain. Heterozygous mutant mice recapitulated the phenotype of type 2B VWD in autosomal dominant manner and presented severe macrothrombocytopenia. Of note, platelets remaining in the circulation had extracytoplasmic GPIba shed-off from the cell surface. Reciprocal bone marrow transplantation determined mutant VWF produced from endothelial cells as the major cause of the platelet phenotype in type 2B VWD mice. Moreover, altered megakaryocyte maturation in the bone marrow and enhanced extramedullary megakaryopoiesis in the spleen were observed. Interestingly, injection of anti-VWF A1 blocking antibody (NMC-4) not only ameliorated platelet count and GPIba expression, but also reversed MK ploidy shift. In conclusion, we present a type 2B VWD mouse model with humanized VWF-GPIba interaction which demonstrated direct influence of aberrant VWF-GPIba binding on megakaryocytes.
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Trombocitopenia , Enfermedad de von Willebrand Tipo 2 , Enfermedades de von Willebrand , Animales , Plaquetas/metabolismo , Células Endoteliales/metabolismo , Humanos , Ratones , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Trombocitopenia/metabolismo , Enfermedad de von Willebrand Tipo 2/genética , Enfermedades de von Willebrand/metabolismo , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.
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Plaquetas/metabolismo , Hematopoyesis , Megacariocitos/metabolismo , Poliploidía , Trombocitopenia/metabolismo , Tirosina-ARNt Ligasa/metabolismo , Plaquetas/patología , Técnicas de Cultivo de Célula , Células Cultivadas , Femenino , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Megacariocitos/patología , Transducción de Señal , Trombocitopenia/patología , Trombopoyetina/metabolismoRESUMEN
Sitosterolemia is a rare, autosomal recessive disease caused by mutations in the adenosine triphosphate-binding cassette transporter genes ABCG5 or ABCG8 that result in accumulation of xenosterols in the body. Clinical manifestations include tendon xanthomas, premature coronary artery disease, hemolytic anemia, macrothrombocytopenia, and bleeding. Although the effect of sterol accumulation on the predisposition for atherosclerosis is evident, how xenosterol accumulation leads to defects in platelet physiology is unknown. Sitosterolemia induced in Abcg5- and Abcg8-deficient mice fed a high plant sterol diet resulted in accumulation of free sterols in platelet plasma membranes, leading to hyperactivatable platelets characterized by constitutive binding of fibrinogen to its αIIbß3 integrin receptor, internalization of the αIIbß3 complex, generation of platelet-derived microparticles, and changes in the quantity and subcellular localization of filamin. The latter was associated with macrothrombocytopenia, shedding of GPIbα, impaired platelet adhesion to von Willebrand factor, and inability to form stable thrombi. Plasma levels of soluble GPIbα were strongly correlated with plasma sitosterol levels in samples from human sitosterolemic patients, implicating a similar mechanism of sterol-induced platelet passivation in the human disease. Intercalation of plant sterols into the plasma membrane therefore results in dysregulation of multiple platelet activation pathways, leading to macrothrombocytopenia and bleeding.
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Plaquetas/fisiología , Hemorragia/sangre , Hipercolesterolemia/sangre , Enfermedades Intestinales/sangre , Errores Innatos del Metabolismo Lipídico/sangre , Fitosteroles/efectos adversos , Activación Plaquetaria/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Modelos Animales de Enfermedad , Fibrinógeno/metabolismo , Filaminas/metabolismo , Hemorragia/genética , Humanos , Hipercolesterolemia/genética , Enfermedades Intestinales/genética , Errores Innatos del Metabolismo Lipídico/genética , Lipoproteínas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Fitosteroles/sangre , Fitosteroles/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genéticaRESUMEN
Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib-IX-V complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of alloimmunization. We have previously reported successful expression of human GPIbα (hGPIbα) in human megakaryocytes using a lentiviral vector (LV) encoding human GP1BA under control of the platelet-specific integrin αIIb promoter (2bIbα). In this study, we examined the efficacy of this strategy for the gene therapy of BSS using GPIbα(null) as a murine model of BSS. GPIbα(null) hematopoietic stem cells (HSC) transduced with 2bIbα LV were transplanted into lethally irradiated GPIbα(null) littermates. Therapeutic levels of hGPIbα expression were achieved that corrected the tail bleeding time and improved the macrothrombocytopenia. Sequential bone marrow (BM) transplants showed sustained expression of hGPIbα with similar phenotypic correction. Antibody response to hGPIbα was documented in 1 of 17 total recipient mice but was tolerated without any further treatment. These results demonstrate that lentivirus-mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a promising strategy for gene therapy of BSS patients.
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Síndrome de Bernard-Soulier/terapia , Terapia Genética , Vectores Genéticos/genética , Lentivirus/genética , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Síndrome de Bernard-Soulier/genética , Síndrome de Bernard-Soulier/inmunología , Plaquetas/metabolismo , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Hemorragia/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria , Unión Proteica , Trombocitopenia/inmunologíaRESUMEN
Various stress conditions are signaled through phosphorylation of translation initiation factor eukaryotic initiation factor 2α (eIF2α) to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. Here, we report that tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family that responds to diverse stress conditions through cytosol-nucleus translocation to activate stress-response genes, also inhibits global translation. However, it occurs at a later stage than eIF2α/ATF4 and mammalian target of rapamycin (mTOR) responses. Excluding TyrRS from the nucleus over-activates translation and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. We propose that TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and strategically located nuclear localization signal and integrate them by nucleus translocation to effect protective responses against chronic stress.
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Tirosina-ARNt Ligasa , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismo , Transporte de Proteínas , Fosforilación , Señales de Localización Nuclear , Estrés OxidativoRESUMEN
Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20 to 30%) despite modern supportive care. In a human bacteremia cohort, we found that development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used after myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thereby providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus, a "toxin-platelet-AMR" regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof of concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.
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Bacteriemia , Preparaciones Farmacéuticas , Infecciones Estafilocócicas , Animales , Bacteriemia/tratamiento farmacológico , Plaquetas , Humanos , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureusRESUMEN
BACKGROUND: Super-resolution microscopy has enabled high-resolution imaging of the actin cytoskeleton in megakaryocytes and platelets. These technologies have extended our knowledge of thrombopoiesis and platelet spreading using megakaryocytes and platelets cultured in vitro on matrix proteins. However, for better understanding of megakaryocytopoiesis and platelet production, high-resolution imaging of cells in an in vivo bone marrow microenvironment is required. Development of Kawamoto's film method greatly advanced the techniques of thin cryosectioning of hard tissues such as undecalcified bones. One obstacle that remains is the spherical aberration that occurs due to the difference in the refractive index for the light path, limiting the usage of Kawamoto's film method to lower magnification observation. OBJECTIVES: To overcome the weakness of the conventional Kawamoto's film method for higher magnification observation of undecalcified bone marrow. METHODS: We have modified the original method with a very simple method: flipping the film at the step of mounting the sections on the glass. RESULTS AND CONCLUSIONS: This new method successfully led to the adjustment of the refractive index and enabled super-resolution imaging of megakaryocytes in undecalcified mouse femurs. Our modified method will expand the application of Kawamoto's film method and enable precise analysis of megakaryocytopoiesis and platelet production in the bone marrow microenvironment under pathophysiological conditions.
RESUMEN
The von Willebrand factor ristocetin cofactor activity assay (VWF:RCo) is used for diagnosis of von Willebrand disease (VWD) because of its ability to evaluate VWF binding to platelets. VWF sequence variant p.D1472H is associated with lower VWF:RCo levels in the absence of associated bleeding symptoms, indicating the VWF:RCo may not be accurate for characterizing VWF function in individuals with this variant. Thus, this study aimed to determine the implications of the variant on VWF functioning in vivo. Mice were engineered with humanized wild-type (WT*) VWF A1/A2 and VWF with the p.D1472H (1472H) variant along with humanized platelet GPIbα and bred to homozygosity. VWF antigen and VWF binding to GPIbα were measured using enzyme-linked immunosorbent assay. Gel electrophoresis was used for VWF multimer analysis. Tail bleeding assays were performed at a 3-mm defined length. Normal VWF multimers were preserved in both WT* and 1472H mice. VWF expression was normal in the WT* and 1472H mice, and VWF binding to GPIbα did not statistically differ between the groups. Additionally, tail bleeding times were similar for WT* and 1472H mice. These results show the p.D1472H variant does not impair hemostasis in mice, and support the conclusion that p.D1472H is a normal variant in humans.
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Enfermedades de von Willebrand , Factor de von Willebrand , Animales , Tiempo de Sangría , Plaquetas , Hemorragia , Ratones , Enfermedades de von Willebrand/diagnóstico , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genéticaRESUMEN
BACKGROUND: Tyrosyl-tRNA synthetase (YRS) belongs to the family of enzymes that catalyzes the tRNA aminoacylation reaction for protein synthesis, and it has been recently shown to exert noncanonical functions. Although database results indicate extremely low levels of YRS mRNA in platelets, YRS protein is abundantly present. The source of YRS in platelets, as well as the physiological role of platelet-stored YRS, remains largely unknown. OBJECTIVES: To clarify how YRS accumulates in platelets and determine the potential role of platelet-stored YRS. METHODS: Recombinant YRS proteins with epitope tags were prepared and tested in vitro for proteolytic cleavage in human plasma. Fluorescent-labeled YRS was examined for uptake by platelets, as demonstrated by western blotting and confocal microscopy analysis. Using RAW-Dual reporter cells, Toll-like receptor and type I interferon activation pathways were analyzed after treatment with YRS. RESULTS: Full-length YRS was cleaved by both elastase and matrix metalloproteinases in the plasma. The cleaved, N-terminal YRS fragment corresponds to the endogenous YRS detected in platelet lysate by western blotting. Both full-length and cleaved forms of YRS were taken up by platelets in vitro and stored in the α-granules. The N-terminal YRS fragment generated by proteolytic cleavage had monocyte activation comparable to that of the constitutive-active mutant YRS (YRSY341A) previously reported. CONCLUSION: Platelets take up both full-length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N-terminal YRS fragment stored in α-granules may have potential to activate monocytes.
RESUMEN
The integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2, and the cell cycle regulation gene PELO, embedded within ITGA1 intron 1. ITGA1 contains a CArG box that is bound by serum response factor (SRF), while PELO contains two Sp1 binding elements. A comparison of mRNA levels in megakaryocytic (MK) and non-megakaryocytic (non-MK) cell lines and an analysis of the transcriptional activity of promoter-LUC reporter gene constructs in transfected cells revealed that ITGA1 is selectively suppressed in the MK lineage. Sodium bisulfite genomic sequencing established that a CpG-rich ITGA1 promoter region (-209/+115) is fully methylated at 19 CpG sites in MK cells that do not express alpha1beta1, but completely demethylated in expressing cells. In vitro methylation of ITGA1 suppresses transcription, while treatment of megakaryocytic cells with 5-aza-2'-deoxycytidine, but not Trichostatin A, resulted in de novo expression of ITGA1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, we observed rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of alpha1beta1 regulation that coincides with the initiation of megakaryocyte differentiation.
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Cromosomas Humanos Par 5/metabolismo , Metilación de ADN , Epigénesis Genética/fisiología , Integrina alfa1/biosíntesis , Integrina alfa1beta1/biosíntesis , Integrina alfa2/biosíntesis , Megacariocitos/metabolismo , Proteínas Nucleares/biosíntesis , Sitios de Carácter Cuantitativo/fisiología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Cromosomas Humanos Par 5/genética , Metilación de ADN/efectos de los fármacos , Decitabina , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Células HeLa , Humanos , Ácidos Hidroxámicos/farmacología , Integrina alfa1/genética , Integrina alfa1beta1/genética , Integrina alfa2/genética , Intrones/fisiología , Células K562 , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Megacariocitos/citología , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/fisiología , Trombopoyetina/farmacología , Transcripción GenéticaRESUMEN
Adipose tissue that consists of mature and immature adipocytes is suggested to contain mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. Here we show that three-dimensional collagen gel culture of rat sc adipose tissue fragments maintained viable mature adipocytes for a long term, producing immature adipocytes and MSC-like cells from the fragments, using immunohistochemistry, ELISA, and real time RT-PCR. Bromodeoxyuridine uptake of mature adipocytes was detected. Adiponectin and leptin, and adipocyte-specific genes of adiponectin, leptin, and PPAR-gamma were detected in culture assembly, whereas the lipogenesis factor insulin (20 mU/ml) and inflammation-related agent TNF-alpha (2 nm) increased and decreased, respectively, all of their displays. Both spindle-shaped cell types with oil red O-positive lipid droplets and those with expression of MSC markers (CD105 and CD44) developed around the fragments. The data indicate that adipose tissue-organotypic culture retains unilocular structure, proliferative ability, and some functions of mature adipocytes, generating both immature adipocytes and CD105+/CD44+ MSC-like cells. This suggests that our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome.
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Adipocitos/citología , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Células Madre Mesenquimatosas/citología , Técnicas de Cultivo de Órganos/métodos , Adipoquinas/genética , Animales , División Celular/fisiología , Supervivencia Celular/fisiología , Colágeno , Medios de Cultivo/farmacología , Endoglina , Técnica del Anticuerpo Fluorescente , Geles , Expresión Génica/fisiología , Receptores de Hialuranos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leptina/genética , Lípidos , Ratas , Ratas WistarRESUMEN
The receptor binding to interleukin (IL)-13 is composed of the IL-13 receptor alpha1 chain (IL-13Ralpha1) and the IL-4 receptor alpha chain (IL-4Ralpha). In order to investigate the interaction of IL-13 with IL-13Ralpha1 and IL-4Ralpha, the DNA fragments coding the extracellular regions of human IL-13Ralpha1 and the IL-4Ralpha (containing a cytokine receptor homologous region) were fused with mouse Fc and expressed by a silkworm-baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. After further purification with anion-exchange chromatography, these receptors were used to investigate the ligand-receptor interaction. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13Ralpha1 showed predominant affinity to IL-4Ralpha, although neither detectable affinity of IL-13 nor IL-13Ralpha1 was observed against IL-4Ralpha. Combining these data with the moderate affinity of IL-13 to IL-13Ralpha1, this indicates that IL-13 first binds to IL-13Ralpha1 and recruits consequently to IL-4R.
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Baculoviridae/genética , Bombyx/genética , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Animales , Clonación Molecular , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/aislamiento & purificación , Subunidad alfa del Receptor de Interleucina-4/genética , Subunidad alfa del Receptor de Interleucina-4/aislamiento & purificación , Estructura Terciaria de ProteínaRESUMEN
We report a 6-year-old girl with amegakaryocytic thrombocytopenia, the first case of this rare congenital disorder not to have an MPL gene mutation. Although no mutations were identified in MPL, Mpl protein was absent in the platelets and TPO induced phosphorylation of the Janus tyrosine kinase 2 (Jak2) was not detected. In addition to the defect of Mpl, the patient demonstrated markedly reduced expression of glycoprotein VI (GPVI) in contrast to normal expression of other platelet-specific proteins GPIb alpha, GPIb beta, and GPIIb. To explore the causes for the absence of Mpl, the entire coding region of Jak2 and AML1 were sequenced and no mutations were identified. To our knowledge, this is the first report that describes a case of amegakaryocytic thrombocytopenia that is not caused by a mutation in MPL and demonstrates the severe impairment of GPVI expression on platelets.
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Megacariocitos/metabolismo , Trombocitopenia/metabolismo , Regiones no Traducidas 3'/genética , Plaquetas/metabolismo , Niño , ADN Complementario/genética , Femenino , Humanos , Masculino , Sistemas de Lectura Abierta/genética , Linaje , Transducción de Señal , Trombocitopenia/genéticaRESUMEN
IL-4 and IL-13, two related Th2-type cytokines, play critical and redundant roles in the defense against gastrointestinal nematodes. These cytokines exert various immunological and physiological effects to expel these worms; however, it had not been known whether protease/protease inhibitor interaction was involved in the defense mechanism against these parasites. Many protozoan and helminth parasites generate cysteine proteases, the majority of which are orthlogues of mammalian cathepsin L, and these cysteine proteases play key roles in the life cycle of parasites as they infect and/or adapt to the hosts. We previously found that the squamous cell carcinoma antigens (SCCA1) and SCCA2, members of the ovalbumin serpin family, were secreted from epithelial cells upon stimulation by IL-4 or IL-13. SCCA1 and SCCA2 show different inhibitory profiles. We recently found that SCCA1, but not SCCA2, inhibited several parasite-derived cysteine proteases. Furthermore, SCCA molecules employed a unique inhibitory mechanism against cysteine proteases: they interacted with proteases without forming a covalent complex followed by irreversible impairment of the protease activities and they resisted cleavage by the target proteases. These results indicate that the interaction between cysteine proteases of parasites and SCCA molecules may be a novel immunodefense mechanism of Th2-type responses against parasites, particularly helminths. In this article, we summarize the roles of IL-4/IL-13 on the defense mechanism against parasites, the effects of SCCA molecules against extrinsic cysteine proteases, and the correlation between induction of SCCA molecules and allergic diseases.
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Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Parásitos/efectos de los fármacos , Parásitos/enzimología , Animales , Biomarcadores , Humanos , Hipersensibilidad/inmunología , Interleucina-13/metabolismo , Interleucina-4/metabolismoRESUMEN
The interaction of platelet glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) initiates hemostasis after vascular injury and also contributes to pathological thrombosis. GPIbα binding to the VWF A1 domain (VWFA1) is a target for antithrombotic intervention, but attempts to develop pharmacologic inhibitors have been hindered by the lack of animal models because of the species specificity of the interaction. To address this problem, we generated a knockin mouse with Vwf exon 28-encoding domains A1 and A2 replaced by the human homolog (VWFh28). VWFh28 mice (M1HA) were crossbred with a transgenic mouse strain expressing human GPIbα on platelets (mGPIbαnull;hGPIbαTg; H1MA) to generate a new strain (H1HA) with humanized GPIbα-VWFA1 binding. Plasma VWF levels in the latter 3 strains were similar to those of wild-type mice (M1MA). Compared with the strains that had homospecific GPIbα-VWF pairing (M1MA and H1HA), M1HA mice of those with heterospecific pairing had a markedly greater prolongation of tail bleeding time and attenuation of thrombogenesis after injury to the carotid artery than H1MA mice. Measurements of GPIbα-VWFA1 binding affinity by surface plasmon resonance agreed with the extent of observed functional defects. Ristocetin-induced platelet aggregation was similar in H1HA mouse and human platelet-rich plasma, and it was comparably inhibited by monoclonal antibody NMC-4, which is known to block human GPIbα-VWFA1 binding, which also inhibited FeCl3-induced mouse carotid artery thrombosis. Thus, the H1HA mouse strain is a fully humanized model of platelet GPIbα-VWFA1 binding that provides mechanistic and pharmacologic information relevant to human hemostatic and thrombotic disorders.
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Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Factor de von Willebrand/metabolismo , Animales , Biomarcadores , Plaquetas/metabolismo , Cruzamientos Genéticos , Exones , Hemostasis , Humanos , Ratones , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Trombosis/etiología , Trombosis/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/genéticaRESUMEN
The physiological significance of the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2, members of the ovalbumin serpin family, remains unresolved. In this study, we examined whether SCCA1 or SCCA2 inhibits protozoa- or helminth-derived cysteine proteases. SCCA1, but not SCCA2, potently inhibited the cysteine protease activities of CPB2.8 from Leishmania mexicana, cruzain from Trypanosoma cruzi, rhodesain from Trypanosoma brucei rhodesience, and cathepsin L2 from Fasciola hepatica. The inhibitory activities of SCCA1 were due to its resistance to cleavage by the cysteine proteases. The findings indicate that induction of cysteine protease inhibitors might be a novel defense mechanism against parasite development.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Parásitos/enzimología , Serpinas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/química , Catálisis , Línea Celular , Humanos , Queratinocitos/metabolismo , Datos de Secuencia Molecular , Serpinas/química , Relación Estructura-ActividadRESUMEN
The incidence of allergic diseases has dramatically increased in recent decades in Japan; therefore, it is important to establish ways to diagnose allergic patients based on their pathogenesis and to treat them. Allergic diseases are complicated and diverse disorders in which various cells and mediators are involved; however, it is widely accepted that they are Th2-type inflammations triggered by the invasion of allergens. It is known that either IL-4 or IL-13, particularly the latter, has an important role in the pathogenesis of bronchial asthma among Th-2 type cytokines; however, it is unclear how IL-4 or IL-13 causes asthmatic phenotypes. We have been trying to address this question by using microarray analysis. We have recently found that periostin, identified as an IL-4/IL-13-inducible gene by microarray analysis, is a novel component of subepithelial fibrosis of bronchial asthma. This finding is important to demonstrate the significance of IL-4 and/or IL-13 as a therapeutic target to inhibit fibrosis in bronchial asthma. Furthermore, it is also important to establish a way to diagnose allergic patients in which IL-4 or IL-13 is dominantly involved, and to apply the developing IL-4/IL-13 inhibitors to these patients. In this article, we show how we are addressing this issue.