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ABSTRACT: A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by â¼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an â¼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was â¼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 µg/kg) was equal to that of the WT (2 µg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.
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Coagulación Sanguínea , Factor VIII , Hemofilia A , Mutación , Animales , Factor VIII/genética , Factor VIII/metabolismo , Ratones , Humanos , Hemofilia A/genética , Hemofilia A/sangre , Cricetinae , Trombina/metabolismo , Sustitución de Aminoácidos , Línea Celular , Modelos Animales de EnfermedadAsunto(s)
Anticuerpos Biespecíficos , Anticuerpos Monoclonales Humanizados , Factor IXa , Factor X , Humanos , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Factor IXa/metabolismo , Factor X/metabolismo , Factor X/química , Transferencia Resonante de Energía de Fluorescencia/métodosRESUMEN
ABSTRACT: Introduction: Acute respiratory distress syndrome (ARDS) is a severe hypoxemic respiratory failure with a high in-hospital mortality. However, the molecular mechanisms underlying ARDS remain unclear. Recent findings have indicated that the onset of severe inflammatory diseases, such as sepsis, is regulated by epigenetic changes. We investigated the role of epigenetic changes in ARDS pathogenesis using mouse models and human samples. Methods: Acute respiratory distress syndrome was induced in a mouse model (C57BL/6 mice, myeloid cell or vascular endothelial cell [VEC]-specific SET domain bifurcated 2 [Setdb2]-deficient mice [Setdb2 ff Lyz2 Cre+ or Setdb2 ff Tie2 Cre+ ], and Cre - littermates) by intratracheal administration of lipopolysaccharide (LPS). Analyses were performed at 6 and 72 h after LPS administration. Sera and lung autopsy specimens from ARDS patients were examined. Results: In the murine ARDS model, we observed high expression of the histone modification enzyme SET domain bifurcated 2 ( Setdb2 ) in the lungs. In situ hybridization examination of the lungs revealed Setdb2 expression in macrophages and VECs. The histological score and albumin level of bronchoalveolar lavage fluid were significantly increased in Setdb2 ff Tie2 Cre+ mice following LPS administration compared with Setdb2 ff Tie2 Cre- mice, whereas there was no significant difference between the control and Setdb2 ff Lyz2 Cre+ mice. Apoptosis of VECs was enhanced in Setdb2 ff Tie2 Cre+ mice. Among the 84 apoptosis-related genes, the expression of TNF receptor superfamily member 10b ( Tnfrsf10b ) was significantly higher in Setdb2 ff Tie2 Cre+ mice than in control mice. Acute respiratory distress syndrome patients' serum showed higher SETDB2 levels than those of healthy volunteers. SETDB2 levels were negatively correlated with the partial pressure of oxygen in arterial blood/fraction of inspiratory oxygen concentration ratio. Conclusion: Acute respiratory distress syndrome elevates Setdb2 , apoptosis of VECs, and vascular permeability. Elevation of histone methyltransferase Setdb2 suggests the possibility to histone change and epigenetic modification. Thus, Setdb2 may be a novel therapeutic target for controlling the pathogenesis of ARDS.
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Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible interstitial pneumonia caused by the excessive production and deposition of extracellular matrix components, including type I collagen. Activated fibroblasts, called α-SMA (α-smooth muscle actin)-expressing myofibroblasts, are the major source of type I collagen in pulmonary fibrosis (PF), but the mechanisms underlying disease progression have not been fully elucidated. Here, we obtained lung fibroblasts from patients with IPF from both nonfibrotic and fibrotic areas as determined by a lung computed tomography scan and compared gene expression between these areas by DNA microarray. We found that ANGPTL4 (angiopoietin-like 4) was highly expressed only in fibroblasts from the fibrotic area. ANGPTL4 was selectively expressed in the fibroblastic area of IPF lungs, where the myofibroblast marker α-SMA was also expressed. ANGPTL4 also regulates the gene expression of fibrosis-related markers, cell migration, and proliferation. In addition, ANGPTL4 expression in a murine model of PF induced by treatment with bleomycin was significantly induced in the lungs from the acute to the chronic phase. Single-cell transcriptome analysis during the course of bleomycin-induced PF revealed that Angptl4 was predominantly expressed in the activated fibroblasts and myofibroblasts. Moreover, the administration of recombinant ANGPTL4 to the bleomycin-induced fibrosis model significantly increased collagen deposition and exacerbated the PF. In contrast, the pathogenesis of PF in Angptl4-deficient mice was improved. These results indicate that ANGPTL4 is critical for the progression of PF and might be an early diagnostic marker and therapeutic target for IPF.
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BACKGROUND: Hemophilia A (HA) is a hereditary bleeding disorder caused by defects in endogenous factor (F)VIII. Approximately 30 % of patients with severe HA treated with FVIII develop neutralizing antibodies (inhibitors) against FVIII, which render the therapy ineffective. The managements of HA patients with high-titter inhibitors are especially challenging. Therefore, it is important to understand the mechanism(s) of high-titer inhibitor development and dynamics of FVIII-specific plasma cells (FVIII-PCs). AIMS: To identify the dynamics of FVIII-PCs and the lymphoid organs in which FVIII-PCs are localized during high-titer inhibitor formation. METHODS AND RESULTS: When FVIII-KO mice were intravenously injected with recombinant (r)FVIII in combination with lipopolysaccharide (LPS), a marked enhancement of anti-FVIII antibody induction was observed with increasing FVIII-PCs, especially in the spleen. When splenectomized or congenitally asplenic FVIII-KO mice were treated with LPS + rFVIII, the serum inhibitor levels decreased by approximately 80 %. Furthermore, when splenocytes or bone marrow (BM) cells from inhibitor+ FVIII-KO mice treated with LPS + rFVIII were grafted into immune-deficient mice, anti-FVIII IgG was detected only in the serum of splenocyte-administered mice and FVIII-PCs were detected in the spleen but not in the BM. In addition, when splenocytes from inhibitor+ FVIII-KO mice were grafted into splenectomized immuno-deficient mice, inhibitor levels were significantly reduced in the serum. CONCLUSION: The spleen is the major site responsible for the expansion and retention of FVIII-PCs in the presence of high-titer inhibitors.
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Hemofilia A , Humanos , Animales , Ratones , Hemofilia A/tratamiento farmacológico , Bazo , Lipopolisacáridos , Factor VIII/farmacología , Anticuerpos NeutralizantesRESUMEN
Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was â¼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was â¼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was â¼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.
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Hemofilia A , Hemostáticos , Animales , Ratones , Factor VIII/metabolismo , Factor IXa/química , Trombina/metabolismo , Estructura Terciaria de Proteína , Hemofilia A/genéticaRESUMEN
When patients with hemophilia A develop factor VIII (FVIII) inhibitors, FVIII replacement therapy becomes ineffective. Although immune-tolerance induction (ITI) therapy has been used to eradicate inhibitors, treatment is unsuccessful in approximately 30% of cases. However, the mechanism behind treatment failure remains unclarified. We retrospectively examined the longitudinal profiles of immunoglobulin G (IgG) subclasses and/or the inhibitory activities of FVIII in plasma samples from 14 Japanese patients with congenital hemophilia A during hemostatic, FVIII replacement, and/or ITI therapies. In five patients, an increase in IgG4 was observed simultaneously with a decrease in IgG1 when the patient had a history of relatively high FVIII inhibitor titers, reflecting an apparent change in humoral immunity. In addition, we examined the reactivity and specificity of the patients' anti-FVIII IgG1 and IgG4 to FVIII domains by immunoblotting. Under our experimental conditions, plasma from three patients with historically higher inhibitor titers appeared to have high titers of antibodies against the A2-a2 domain, which did not necessarily correlate with ITI failure. These observations may improve scientific understanding of the immune response to infused FVIII in patients with hemophilia A.
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Hemofilia A , Hemostáticos , Humanos , Tolerancia Inmunológica , Inmunoglobulina G , Japón , Estudios RetrospectivosRESUMEN
The spleen is comprised of spatially distinct compartments whose functions, such as immune responses and removal of aged red blood cells, are tightly controlled by the non-hematopoietic stromal cells that provide regionally-restricted signals to properly activate hematopoietic cells residing in each area. However, information regarding the ontogeny and relationships of the different stromal cell types remains limited. Here we have used in vivo lineage tracing analysis and in vitro mesenchymal stromal cell assays and found that Tlx1, a transcription factor essential for embryonic spleen organogenesis, marks neonatal stromal cells that are selectively localized in the spleen and retain mesenchymal progenitor potential to differentiate into mature follicular dendritic cells, fibroblastic reticular cells and marginal reticular cells. Furthermore, by establishing a novel three-dimensional cell culture system that enables maintenance of Tlx1-expressing cells in vitro, we discovered that signals from the lymphotoxin ß receptor and TNF receptor promote differentiation of these cells to express MAdCAM-1, CCL19 and CXCL13, representative functional molecules expressed by different subsets of mature stromal cells in the spleen. Taken together, these findings indicate that mesenchymal progenitor cells expressing Tlx1 are a subset of lymphoid tissue organizer-like cells selectively found in the neonatal spleen.
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Proteínas de Homeodominio/metabolismo , Tejido Linfoide/metabolismo , Células Madre Mesenquimatosas/metabolismo , Bazo/metabolismo , Células del Estroma/metabolismo , Animales , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Regulación de la Expresión Génica , RatonesRESUMEN
Extramedullary hematopoiesis (EMH) in postnatal life is a pathological process in which the differentiation of hematopoietic stem/progenitor cells (HSPCs) occurs outside the bone marrow (BM) to respond to hematopoietic emergencies. The spleen is a major site for EMH; however, the cellular and molecular nature of the stromal cell components supporting HSPC maintenance, the niche for EMH in the spleen remain poorly understood compared to the growing understanding of the BM niche at the steady-state as well as in emergency hematopoiesis. In the present study, we demonstrate that mesenchymal progenitor-like cells expressing Tlx1, an essential transcription factor for spleen organogenesis, and selectively localized in the perifollicular region of the red pulp of the spleen, are a major source of HSPC niche factors. Consistently, overexpression of Tlx1 in situ induces EMH, which is associated with mobilization of HSPC into the circulation and their recruitment into the spleen where they proliferate and differentiate. The alterations in the splenic microenvironment induced by Tlx1 overexpression in situ phenocopy lipopolysaccharide (LPS)-induced EMH, and the conditional loss of Tlx1 abolished LPS-induced splenic EMH. These findings indicate that activation of Tlx1 expression in the postnatal splenic mesenchymal cells is critical for the development of splenic EMH.
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Hematopoyesis Extramedular/fisiología , Proteínas de Homeodominio/fisiología , Bazo/citología , Nicho de Células Madre , Animales , Proliferación Celular , Hematopoyesis Extramedular/efectos de los fármacos , Proteínas de Homeodominio/genética , Lipopolisacáridos/toxicidad , Ratones , Ratones NoqueadosRESUMEN
PKnox1 (also known as Prep1) belongs to the TALE family of homeodomain transcription factors that are critical for regulating growth and differentiation during embryonic and postnatal development in vertebrates. We demonstrate here that PKnox1 is required for adult spermatogenesis in a germ cell-intrinsic manner. Tamoxifen-mediated PKnox1 loss in the adult testes, as well as its germ cell-specific ablation, causes testis hypotrophy with germ cell apoptosis and, as a consequence, compromised spermatogenesis. In PKnox1-deficient testes, spermatogenesis was arrested at the c-Kit+ spermatogonia stage, with a complete loss of the meiotic spermatocytes, and was accompanied by compromised differentiation of the c-Kit+ spermatogonia. Taken together, these results indicate that PKnox1 is a critical regulator of maintenance and subsequent differentiation of the c-Kit+ stage of spermatogonia in the adult testes.
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Proteínas de Homeodominio/fisiología , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Animales , Diferenciación Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Espermatogonias/citología , Testículo/citología , Testículo/metabolismoRESUMEN
Prep1, a TALE-family homeodomain transcription factor, has been demonstrated to play a critical role in embryonic hematopoiesis, as its insufficiency caused late embryonic lethality associated with defective hematopoiesis and angiogenesis. In the present study, we generated hematopoietic- and endothelial cell-specific Prep1-deficient mice and demonstrated that expression of Prep1 in the hematopoietic cell compartment is not essential for either embryonic or adult hematopoiesis, although its absence causes significant hematopoietic abnormalities in the adult bone marrow. Loss of Prep1 promotes cell cycling of hematopoietic stem/progenitor cells (HSPC), leading to the expansion of the HSPC pool. Prep1 deficiency also results in the accumulation of lineage-committed progenitors, increased monocyte/macrophage differentiation and arrested erythroid maturation. Maturation of T cells and B cells is also perturbed in Prep-deficient mice. These findings provide novel insight into the pleiotropic roles of Prep1 in adult hematopoiesis that were unrecognized in previous studies using germline Prep1 hypomorphic mice.
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Linfocitos B/patología , Médula Ósea/patología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/patología , Proteínas de Homeodominio/fisiología , Linfocitos T/patología , Animales , Apoptosis , Linfocitos B/metabolismo , Western Blotting , Médula Ósea/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismoRESUMEN
The spleen is a lymphoid organ that serves as a unique niche for immune reactions, extramedullary hematopoiesis, and the removal of aged erythrocytes from the circulation. While much is known about the immunological functions of the spleen, the mechanisms governing the development and organization of its stromal microenvironment remain poorly understood. Here we report the generation and analysis of a Tlx1(Cre) (ER) (-Venus) knock-in mouse strain engineered to simultaneously express tamoxifen-inducible CreER(T2) and Venus fluorescent protein under the control of regulatory elements of the Tlx1 gene, which encodes a transcription factor essential for spleen development. We demonstrated that Venus as well as CreER expression recapitulates endogenous Tlx1 transcription within the spleen microenvironment. When Tlx1(Cre) (ER) (-Venus) mice were crossed with the Cre-inducible reporter strain, Tlx1-expressing cells as well as their descendants were specifically labeled following tamoxifen administration. We also showed by cell lineage tracing that asplenia caused by Tlx1 deficiency is attributable to altered contribution of mesenchymal cells in the spleen anlage to the pancreatic mesenchyme. Thus, Tlx1(Cre) (ER) (-Venus) mice represent a new tool for lineage tracing and conditional gene manipulation of spleen mesenchymal cells, essential approaches for understanding the molecular mechanisms of spleen development.
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Técnicas de Sustitución del Gen/métodos , Proteínas de Homeodominio/genética , Células Madre Mesenquimatosas/metabolismo , Modelos Animales , Morfogénesis/fisiología , Bazo/embriología , Animales , Proteínas Bacterianas/metabolismo , Linaje de la Célula/fisiología , Cruzamientos Genéticos , Cartilla de ADN/genética , Citometría de Flujo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Integrasas/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , TamoxifenoRESUMEN
Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igalpha, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8alpha-Igalpha chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Antígenos CD8/biosíntesis , Células COS , Diferenciación Celular , Proliferación Celular , Pollos , Chlorocebus aethiops , Humanos , Ratones , FosforilaciónRESUMEN
Pre-B cell receptor (pre-BCR) signals promote pre-B cell differentiation, in which the adaptor protein B-cell linker (BLNK) plays a crucial role. However, the molecular pathways downstream of BLNK are currently unclear. Utilizing pre-B leukemia cell lines (BKO84 and others) derived from BLNK-deficient mice as in vitro models of the pre-B cell differentiation, we have demonstrated that reconstitution of BLNK as well as an active form of protein kinase C (PKC)eta induces the differentiation events, such as pre-BCR down-regulation and kappa gene rearrangement. Here we show that the same events are induced by cross-linking of pre-BCR with anti-mu antibody in these pre-B cell lines, as well as in ex vivo pre-B cells from BLNK-deficient mice, suggesting a function of BLNK as an internal cross-linker of pre-BCR. Anti-mu treatment of BKO84 cells up-regulated membrane recruitment of PKC eta and the expression of IRF-4, a transcription factor known to promote light chain gene rearrangements. Anti-mu induction of surface kappa chain on BKO84 cells was blocked by reagents that inhibit phospholipase C or PKC. Enforced expression of the active PKC eta in BKO84 cells resulted in up-regulation of IRF-4 expression. Conversely, siRNA-mediated silencing of PKC eta expression strikingly attenuated the anti-mu-induced IRF-4 expression and kappa gene rearrangement, which were restored by PKC eta reconstitution. Finally, enforced expression of IRF-4, but not of BLNK, in the PKC eta-silenced BKO84 cells resulted in kappa gene rearrangement. These results indicate that PKC eta directs the induction of IRF-4 expression downstream of BLNK in the pre-BCR signaling pathway promoting kappa gene rearrangement.