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1.
Mol Ther Nucleic Acids ; 29: 657-671, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36090760

RESUMO

The use of messenger RNA (mRNA) enables the transient production of therapeutic proteins with stable and predictable translational kinetics and without the risk of insertional mutagenesis. Recent findings highlight the enormous potential of mRNA-based therapeutics. Here, we describe the synthesis of chemically modified thrombopoietin (TPO) mRNA through in vitro transcription and in vivo delivery via lipid nanoparticles (LNPs). After delivery of TPO mRNA in mice, compared with normal physiological values, plasma TPO protein levels increased over 1000-fold in a dose-dependent manner. Moreover, through a single intravenous dose of TPO mRNA-loaded LNPs, both reticulated and total platelet count increased significantly in mice, demonstrating that TPO protein derived from the exogenous mRNA was able to maintain normal activity. Submicrogram quantity of N1-methylpseudouridine-modified TPO mRNA showed a similar effect in promoting thrombopoiesis as that by the TPO receptor agonist romiplostim. In addition, a therapeutic value was established in anti-GPIbα (CD42b) antibody-induced thrombocytopenia mouse models that showed a fast recovery of platelet count. Our study demonstrated chemically modified in-vitro-transcribed TPO mRNA as a potentially safe therapeutic intervention to stimulate thrombopoiesis.

2.
Adv Sci (Weinh) ; 9(7): e2103228, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35023301

RESUMO

Conventional antiplatelet agents indiscriminately inhibit both thrombosis and hemostasis, and the increased bleeding risk thus hampers their use at more aggressive dosages to achieve adequate effect. Blocking integrin αIIbß3 outside-in signaling by separating the ß3/Src interaction, yet to be proven in vivo, may nonetheless resolve this dilemma. Identification of a specific druggable target for this strategy remains a fundamental challenge as Src SH3 is known to be responsible for binding to not only integrin ß3 but also the proteins containing the PXXP motif. In vitro and in vivo mutational analyses show that the residues, especially E97, in the RT loop of Src SH3 are critical for interacting with ß3. DCDBS84, a small molecule resulting from structure-based virtual screening, is structurally validated to be directed toward the projected target. It specifically disrupts ß3/Src interaction without affecting canonical PXXP binding and thus inhibits the outside-in signaling-regulated platelet functions. Treatment of mice with DCDBS84 causes a profound inhibition of thrombosis, equivalent to that induced by extremely high doses of αIIbß3 antagonist, but does not compromise primary hemostasis. Specific targets are revealed for a preferential inhibition of thrombosis that may lead to new classes of potent antithrombotics without hemorrhagic side effects.


Assuntos
Plaquetas , Trombose , Animais , Plaquetas/metabolismo , Hemostasia/fisiologia , Integrina beta3/química , Integrina beta3/metabolismo , Camundongos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/metabolismo , Trombose/prevenção & controle
3.
Front Med ; 16(4): 584-595, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35038106

RESUMO

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC + rLC protein had a higher activity than the hHC + hLC protein at comparable expression levels. The specific activity of hHC + rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC + rLC treatment exhibited a visibly higher activity than those treated with hHC + hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC + rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.


Assuntos
Hemofilia A , Animais , Dependovirus/genética , Fator VIII/genética , Fator VIII/metabolismo , Terapia Genética/métodos , Hemofilia A/genética , Hemofilia A/terapia , Humanos , Camundongos , Ratos
4.
Thromb Haemost ; 122(5): 755-766, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34587639

RESUMO

Platelet-targeted FVIII gene therapy can efficiently recover bleeding phenotype for hemophilia A (HA), yet characteristics of thrombus formation with this ectopic expression of factor VIII (FVIII) in platelets remain unclear. Here, we generated 2bF8trans mice restrictively expressing human B-domain-deleted FVIII (hBDD FVIII) in platelets on a hemophilic (FVIIInull) mice background. The results showed no statistical difference in clot strength and stability between wild-type (WT) and 2bF8trans mice, but with a prolonged reaction time (R-time), by thromboelastography. Fluid dynamics analysis showed that at the shear rates of 500 to 1,500 s-1, where physiological hemostasis often develops, the thrombi formed in 2bF8trans mice were more stable than those in FVIIInull mice, while at high pathological shear rates (2,500 s-1), mimicking atherosclerosis, thrombus size and fibrin deposition in 2bF8trans mice were less than those in WT mice. Thrombus morphology analysis showed that there was a locally concentrated deposition of fibrin in thrombus at the injured site and fibrin co-localized with activated platelets in 2bF8trans mice. Moreover, a higher ratio of fibrin to platelets was found in thrombus from 2bF8trans mice following laser-induced injury in cremaster arterioles, which might be the underlying mechanism of thrombus stability in 2bF8trans mice at physiological arterial circumstance. These observations suggest that specific morphological features of the thrombi might contribute to the efficacy and safety of platelet-targeted FVIII gene therapy for HA.


Assuntos
Hemofilia A , Hemostáticos , Trombose , Animais , Plaquetas/metabolismo , Fator VIII/genética , Fator VIII/metabolismo , Fibrina/metabolismo , Hemofilia A/terapia , Hemostáticos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Trombose/genética , Trombose/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(42)2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34649996

RESUMO

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset-dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.


Assuntos
Alelos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dependovirus/genética , Fator IX/genética , Hepatócitos/metabolismo , Elementos Facilitadores Genéticos , Edição de Genes/métodos , Hemofilia B/genética , Mutação , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
6.
Clin Transl Med ; 11(3): e375, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33783994

RESUMO

BACKGROUND: Treatment of hemophiliacs with inhibitors remains challenging, and new treatments are in urgent need. Coagulation factor X plays a critical role in the downstream of blood coagulation cascade, which could serve as a bypassing agent for hemophilia therapy. Base on platelet-targeted gene therapy for hemophilia by our and other groups, we hypothesized that activated factor X (FXa) targeted stored in platelets might be effective in treating hemophilia A (HA) and B (HB) with or without inhibitors. METHODS: To achieve the storage of FXa in platelets, we constructed a FXa precursor and used the integrin αIIb promoter to control the targeted expression of FXa precursor in platelets. The expression cassette (2bFXa) was carried by lentivirus and introduced into mouse hematopoietic stem and progenitor cells (HSPCs), which were then transplanted into HA and HB mice. FXa expression and storage in platelets was examined in vitro and in vivo. We evaluated the therapeutic efficacy of platelet-stored FXa by tail bleeding assays and the thrombelastography. In addition, thrombotic risk was assessed in the recipient mice and the lipopolysaccharide induced inflammation mice. RESULTS: By transplanting 2bFXa lentivirus-transduced HSPCs into HA and HB mice, FXa was observed stably stored in platelet α-granules, the stored FXa is releasable and functional upon platelet activation. The platelet-stored FXa can significantly ameliorate bleeding phenotype in HA and HB mice as well as the mice with inhibitors. Meanwhile, no FXa leakage in plasma and no signs of increased risk of hypercoagulability were found in transplantation recipients and lipopolysaccharide induced septicemia recipients. CONCLUSIONS: Our proof-of-principle data indicated that target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α-granules, the platelet-stored FXa is effective in treating HA and HB with inhibitors, suggesting that this could be a novel choice for hemophilia patients with inhibitors.


Assuntos
Plaquetas , Fator Xa/metabolismo , Terapia Genética/métodos , Hemofilia A/terapia , Hemofilia B/terapia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
Medicine (Baltimore) ; 99(44): e22931, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33126356

RESUMO

RATIONALE: Synchronous development of both anaplastic large cell lymphoma (ALCL) and multiple myeloma (MM) in a patient is rare. To our knowledge, until now only one case has been reported. Treatment needs to cover both and is a challenge. Here we reported another case and discussed the diagnosis and treatment. PATIENT CONCERNS: This is a 63-year old woman who presented with a mass in upper abdominal skin. Positron emission tomography/computed tomography (PET/CT) showed the high metabolism in left abdominal skin and left axillary lymph nodes. Histopathologic and immunohistochemical evaluation identified the cutaneous mass as an ALK-negative ALCL. Bone marrow smear showed increased plasma cells which expressed CD38, CD138, and cLambda concomitantly. The increased monoclonal immunoglobulin IgD λ was detected by immunofixation electrophoresis. DIAGNOSES: Diagnosis of both ALCL and MM was confirmed. INTERVENTIONS: The patient successively received 6 cycles of B-CHOD regimen, one cycle of ID regimen, 2 cycles of DHAX regimen, one cycle of L-DA-EPOCH and autologous stem cell transplantation (ASCT). Then lenalidomide was performed as a maintenance therapy. OUTCOMES: Both ALCL and MM achieved complete remission. LESSONS: We reported a very rare case with synchronous development of ALCL and MM, in whom a good therapeutic response to chemotherapies followed by ASCT has been observed.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Lenalidomida/administração & dosagem , Linfoma Anaplásico de Células Grandes , Mieloma Múltiplo , Neoplasias Cutâneas , Parede Abdominal/patologia , Bleomicina/administração & dosagem , Exame de Medula Óssea/métodos , Ciclofosfamida/administração & dosagem , Dexametasona/administração & dosagem , Doxorrubicina/administração & dosagem , Etoposídeo/administração & dosagem , Feminino , Humanos , Imuno-Histoquímica , Linfoma Anaplásico de Células Grandes/imunologia , Linfoma Anaplásico de Células Grandes/patologia , Linfoma Anaplásico de Células Grandes/terapia , Quimioterapia de Manutenção/métodos , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Neoplasias Primárias Múltiplas/patologia , Neoplasias Primárias Múltiplas/terapia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Prednisona/administração & dosagem , Indução de Remissão , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Transplante Autólogo/métodos , Vincristina/administração & dosagem
9.
J Cell Mol Med ; 24(14): 7928-7938, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32459397

RESUMO

About 5 per cent of follicular lymphoma (FL) cases are double-hit (DH) lymphomas. Double-hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL-SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL-SJC cells demonstrated CD19++ , CD20+ , CD22++ , HLA-DR+ , CD10+ , CD38+ , Lambda+ CD23- , CD5- and Kappa- . The chromosome karyotypic analysis confirmed the co-existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL-SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL-SJC cells, the SCID mice developed solid tumour masses within 6-8 weeks. FL-SJC cells were proven to be free of Epstein-Barr (EB) virus infection and be multidrug-resistant. In a conclusion, the FL-SJC cell line has been identified as a novel MYC/BCL2 double-hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.


Assuntos
Biomarcadores Tumorais , Linhagem Celular Tumoral , Linfoma Folicular/genética , Mutação , Animais , Biópsia , Deleção Cromossômica , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Xenoenxertos , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Cariotipagem , Linfoma Folicular/diagnóstico , Linfoma Folicular/terapia , Masculino , Camundongos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Translocação Genética
10.
Arterioscler Thromb Vasc Biol ; 40(5): 1296-1310, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32237906

RESUMO

OBJECTIVE: Integrin ß3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether ß3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with ß3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The ß3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that ß3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine)720-722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/ß3/LPL complex that is required for ß3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in ß3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH720-722-mutated ß3 genes. CONCLUSIONS: This study reveals a hypertriglyceridemia in both ß3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of ß3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with ß3-deficient Glanzmann thrombasthenia.


Assuntos
Hipertrigliceridemia/etiologia , Cadeias beta de Integrinas/metabolismo , Integrina beta3/metabolismo , Lipase Lipoproteica/sangue , Trombastenia/complicações , Triglicerídeos/sangue , Adolescente , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , China , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/diagnóstico , Hipertrigliceridemia/enzimologia , Cadeias beta de Integrinas/genética , Integrina beta3/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Fatores de Risco , Trombastenia/sangue , Trombastenia/diagnóstico , Trombastenia/genética
11.
Int J Clin Exp Pathol ; 13(1): 21-25, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32055268

RESUMO

Lymphadenopathy is an important characteristic of POEMS syndrome, and a Castleman disease (CD)-like pathologic change in the lymph nodes is one of the major diagnostic criteria. However, the characteristics of lymphadenopathy in POEMS still have not been completely elucidated. The lymph node biopsies are available only for a small proportion of patients. A simple and safe way is needed to rule CD in or out. This study aimed to analyse the features of lymphadenopathy and estimate the role of imaging methods, including computed tomography (CT) and positron emission tomography-CT (PET/CT), in the diagnosis of lymphadenopathy in patients with POEMS syndrome. We conducted a retrospective analysis of 23 patients with confirmed POEMS syndrome. All of the patients received chest and abdominal CT scan and/or superficial ultrasound examinations. Four patients underwent PET/CT examinations, and 6 patients received lymph node biopsies. Enlarged lymph nodes (short diameter ≥ 1 cm) were found in 48% (11/23) of patients, but only 1 patient had an enlarged lymph node with a diameter ≥ 2 cm. Lymph nodes with CD-like pathologic changes from 2 patients showed increased maximum standard uptake values (SUVmax) of 18F-deoxyglucose (18FDG) on PET/CT, while lymph nodes with reactive pathologic changes from 2 other patients showed a normal metabolic PET/CT profile. The extent of lymph node enlargement in patients with POEMS was less than that in patients with CD per se. We draw the conclusion that most of the enlarged lymph nodes had diameters ≤ 2 cm, which is less than that in cases of CD per se and PET/CT may be helpful in determining whether enlarged lymph nodes are characterized by CD-like changes or not.

13.
J Hematol Oncol ; 12(1): 26, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845955

RESUMO

Integrins are a family of transmembrane glycoprotein signaling receptors that can transmit bioinformation bidirectionally across the plasma membrane. Integrin αIIbß3 is expressed at a high level in platelets and their progenitors, where it plays a central role in platelet functions, hemostasis, and arterial thrombosis. Integrin αIIbß3 also participates in cancer progression, such as tumor cell proliferation and metastasis. In resting platelets, integrin αIIbß3 adopts an inactive conformation. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbß3 to switch from a low- to high-affinity state for fibrinogen and other ligands. Ligand binding causes integrin clustering and subsequently promotes outside-in signaling, which initiates and amplifies a range of cellular events to drive essential platelet functions such as spreading, aggregation, clot retraction, and thrombus consolidation. Regulation of the bidirectional signaling of integrin αIIbß3 requires the involvement of numerous interacting proteins, which associate with the cytoplasmic tails of αIIbß3 in particular. Integrin αIIbß3 and its signaling pathways are considered promising targets for antithrombotic therapy. This review describes the bidirectional signal transduction of integrin αIIbß3 in platelets, as well as the proteins responsible for its regulation and therapeutic agents that target integrin αIIbß3 and its signaling pathways.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Abciximab/farmacologia , Sequência de Aminoácidos , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Eptifibatida/farmacologia , Humanos , Terapia de Alvo Molecular , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/agonistas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Transdução de Sinais , Tirofibana/farmacologia
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(1): 227-232, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30738475

RESUMO

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin ß3 cytoplasmic tail on αⅡbß3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length ß3 or mutant ß3, including ß3-ΔNITY (ß3 cytoplasmic tail NITY motif deleted), ß3-Δ754 (ß3 cytoplasmic tail TNITYRGT motif deleted) and ß3-Δ759 (ß3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbß3ΔNITY, 293T-αⅡbß3Δ754, 293T-αⅡbß3Δ759 and 293T-αⅡbß3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbß3 cells which expressed full ß3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbß3 cells, the 293T-αⅡbß3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbß3Δ754 cells and 293T-αⅡbß3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION: To the cell spreading function mediated by integrin ß3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different ß3 mutants provide a solid basis for a further analysis of mass spectrometry.


Assuntos
Integrina beta3/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Células CHO , Adesão Celular , Cricetinae , Cricetulus , Células HEK293 , Humanos , Integrina beta3/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética
15.
Proc Natl Acad Sci U S A ; 116(6): 2220-2225, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659143

RESUMO

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Genes myc , Mepesuccinato de Omacetaxina/farmacologia , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Biomarcadores Tumorais , Linhagem Celular Tumoral , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Mepesuccinato de Omacetaxina/química , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Repressoras/química , Fator de Transcrição RelA/metabolismo , Transcrição Genética , Translocação Genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cell Death Dis ; 9(10): 963, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237564

RESUMO

Non-small cell lung cancer (NSCLC) is characterized by hyperexpression and/or gain-of-function mutations of the epidermal growth factor receptor (EGFR), resulting in an elevated overall kinase activity. Gefitinib is remarkably effective in patients with the L858R or ΔE746-A750-mutated of EGFR. However, drug resistance tends to develop because of the emergence of T790M mutation on EGFR. New strategies other than repressing kinase activity are thus required to treat NSCLC, thereby circumventing the resistance. In this study, arsenic trioxide (ATO) at 2 µM significantly inhibited the proliferation of the gefitinib-resistant NCI-H1975 cells of the EGFR L858R/T790M mutant compared with a modest inhibition in the gefitinib-sensitive HCC827 cells of ΔE746-A750 mutant and A549 cells of wild-type EGFR. Moreover, ATO significantly inhibited the overall kinase activity of EGFR primarily through quantitatively diminishing the EGFR in NCI-H1975 cells to an extent comparable with that reached by gefitinib in HCC827 cells. Furthermore, ATO promoted autophagic degradation of EGFR in NSCLC cells by directly binding to P62, which interacted with EGFR, preferentially the L858R/T790M mutant providing a plausible explanation for a more favorable effect of ATO on NCI-H1975 cells. Accordingly, the effect of ATO was further confirmed in the NSCLC xenograft mouse models. Our results reveal a new target for ATO with a unique molecular mechanism, i.e., ATO suppresses the overall catalytic potential of EGFR, significantly those with the L858R/T790M mutant in NCI-H1975 cells, through an autophagic degradation by interacting with P62. This study potentially offers an innovative therapeutic avenue for the NSCLC with L858R/T790M-mutated EGFR.


Assuntos
Arsênio/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/metabolismo , Gefitinibe/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Ligação a RNA/metabolismo , Células A549 , Animais , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Fosforilação , Proteínas de Ligação a RNA/genética
17.
Blood Cells Mol Dis ; 73: 47-54, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30249384

RESUMO

The development of a novel coagulation factor VIII (FVIII) expression cassette with an enhanced activity for gene therapy of hemophilia A (HA) is essential. The biological properties of several non-human FVIII sequences, such as porcine and canine, have been evaluated. Here, we compared the activity level of rat FVIII (rFVIII) and human FVIII (hFVIII) by using single-chain and dual-chain strategies in 293 T cells and the HA mice. In both in vitro and hydrodynamic injection studies, the activity of rFVIII detected by the activated partial thromboplastin time assay was higher than that of hFVIII both by single-chain (~2.96-fold and ~1.72-fold, respectively) and dual-chain (~7.69-fold and ~2.35-fold, respectively). Moreover, the dual chain exerted a potentially higher delivery efficacy compared with the single chain (~4.96-fold and ~2.99-fold, respectively). The blood loss of HA mice administrated with rFVIII was less than those with hFVIII. AAV-delivered rFVIII and hFVIII also exerted long-term therapeutic effects on HA mice and caused a transient ALT elevation. These data might help to the development of novel, optimized FVIII expression cassettes based on the amino acid difference between rFVIII and hFVIII. These data indicate that the dual-chain strategy would likely enhance the delivery efficiency of the AAV-mediated FVIII gene therapy.


Assuntos
Fator VIII/administração & dosagem , Terapia Genética/métodos , Hemofilia A/tratamento farmacológico , Adenoviridae/genética , Animais , Fator VIII/genética , Vetores Genéticos , Células HEK293 , Humanos , Tempo de Tromboplastina Parcial , Ratos , Transfecção
18.
Front Med ; 12(3): 324-329, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29075958

RESUMO

Splenic lymphoma with villous lymphocytes (SLVL) or splenic marginal zone lymphoma with circulating villous lymphocytes is rare, and prolymphocytic transformation of SLVL is rarer. At present, only one case of SLVL with t(8;14)(q24;q32) translocation has been reported. In this study, we report a case of B-lymphoproliferative disorder with villous lymphocytes harboring t(8;14)(q24;q32) chromosome translocation that we inclined to SLVL with a prolymphocytic transformation. A 73-year-old female showed marked hepatosplenomegaly and high lymphocytosis (lymphocytes > 200 × 109/L). The abnormal lymphocytes had short coarse villi and round nuclei with prominent nucleoli. The immunophenotypes showed CD19+, CD20+, HLA-DR+, CD22+, CD5+, Kappa+, CD25dim, CD71dim, Lambda-, CD7-, CD10-, CD23-, CD34-, CD33-, CD13-, CD14-, CD117-, CD64-, CD103-, and CD11c-. The karyotype showed complex abnormality: 46XX,+ 3,-10, t(8;14)(q24; q32)[11]/46XX[9]. The cytoplasmic projection, immunological characteristics, and trisomy 3 chromosome abnormality supported the diagnosis of SLVL. However, the presence of prominent nucleoli and high lymphocytosis suggested prolymphocytic transformation, probably as a result of t(8,14) chromosome translocation. In this report, we described an unusual case of B-lymphoproliferative disorder with villous lymphocytes harboring t(8;14)(q24;q32) translocation, which could provide help in the diagnosis and differential diagnosis of B-lymphocytic proliferative diseases.


Assuntos
Linfócitos B/patologia , Transtornos Linfoproliferativos/genética , Translocação Genética , Idoso , Feminino , Humanos , Imunofenotipagem , Transtornos Linfoproliferativos/patologia
19.
Eur J Haematol ; 100(4): 344-350, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29266395

RESUMO

OBJECTIVES: The arsenic trioxide (ATO) plus all-trans retinoic acid (ATRA) therapy has demonstrated a tremendous success in the first-line treatment of acute promyelocytic leukemia (APL). Actually, early death (ED) is currently thought as a major challenge in APL. ATO has been reported to inhibit platelet function in vitro, and whether it increases the ED rate by exacerbating the hemorrhagic symptoms remains to be investigated. METHODS: Effects of ATO on platelet aggregation and adhesion were evaluated in vitro and in thirty-two complete remission (CR) and four newly diagnosed APL patients. Furthermore, concentrations of plasma total arsenic were monitored in APL patients via ICP-MS. RESULTS: The inhibition of platelet function, either aggregation or adhesion, did occur in vitro when the concentration of ATO reached 2 µmol/L. However, in CR APL patients receiving ATO with normal platelet count, the platelets responded normally when being activated and so did those in the newly diagnosed patients with thrombocytopenia. Our data further showed that the conventional dosage of ATO reached a plasma concentration substantially below the required concentration to inhibit platelets. CONCLUSIONS: In the first-line treatment of APL, the use of ATO is safe and effective and does not compromise the hemostatic potential that may eventually increase ED rate.


Assuntos
Antineoplásicos/administração & dosagem , Arsenicais/administração & dosagem , Hemorragia/etiologia , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/tratamento farmacológico , Óxidos/administração & dosagem , Adolescente , Adulto , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Trióxido de Arsênio , Arsenicais/efeitos adversos , Arsenicais/farmacocinética , Coagulação Sanguínea/efeitos dos fármacos , Feminino , Hemorragia/mortalidade , Humanos , Leucemia Promielocítica Aguda/sangue , Quimioterapia de Manutenção , Masculino , Pessoa de Meia-Idade , Óxidos/efeitos adversos , Óxidos/farmacocinética , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Indução de Remissão , Resultado do Tratamento , Adulto Jovem
20.
J Immunol ; 199(5): 1817-1826, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28739877

RESUMO

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all-trans-retinoic acid-induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization.


Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Dendríticas/imunologia , Leucemia Mieloide Aguda/imunologia , Células-Tronco Neoplásicas/fisiologia , Síndrome Oculocerebrorrenal/imunologia , Fagocitose , Fosfoproteínas/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Autorrenovação Celular , Proteínas do Citoesqueleto/genética , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/metabolismo , Fosfoproteínas/genética , Monoéster Fosfórico Hidrolases/metabolismo , Polimerização , Agregação de Receptores , Tretinoína/metabolismo
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