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1.
Rinsho Ketsueki ; 60(9): 1166-1175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31597840

RESUMO

The classical myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by clonal myeloproliferation without features of myelodysplasia. The diagnostic approach proposed by the World Health Organization (WHO) uses clinical features, peripheral blood counts and smear analysis, bone marrow (BM) morphology, karyotype and molecular genetic tests to classify MPN subtypes. The detection of characteristic driver mutations like JAK2V617F, JAK2 exon 12, MPL, and calrecticulin (CALR) is a major diagnostic feature. JAK2 mutations are detected in more than 90% of patients with PV and are therefore used as highly sensitive clonal marker in this subtype. However, JAK2 mutations may also occur in ET and PMF, while CALR is virtually not seen in PV. Therefore, BM remains the central diagnostic platform and is essential for distinguishing ET from pre-fibrotic PMF and diagnosing cases which do not express JAK2, MPL or CALR ('wild-type' or 'triple-negative' MPN). The standardization of relevant BM features is mandatory to recognize characteristic and easy to assess patterns that enable an accurate discrimination between the MPN subtypes. Key parameters include cellularity, erythropoiesis and neutrophil granulopoiesis in context with specific features of megakaryocytes as well as the BM fiber content, especially in early stage MPN that present with thrombocytosis and clinically mimic essential thrombocythemia.


Assuntos
Transtornos Mieloproliferativos/diagnóstico , Medula Óssea/patologia , Calreticulina/genética , Diagnóstico Diferencial , Eritropoese , Humanos , Janus Quinase 2/genética , Megacariócitos/citologia , Mutação , Neutrófilos/citologia , Policitemia Vera , Trombocitemia Essencial , Organização Mundial da Saúde
2.
Rinsho Ketsueki ; 60(8): 915-919, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31484889

RESUMO

A 83-year-old female patient was admitted to our hospital due to hematological manifestation of juvenile granulocytes and macrocytic anemia. Bone marrow (BM) examination revealed erythroid dysplasia and cytoplasmic blasts, and hence the patient was diagnosed with myelodysplastic syndrome with ring sideroblasts and with single lineage dysplasia (MDS-RS-SLD). Erythrocyte transfusion was performed as a supportive therapy, and there was a gradual increase in the number of blood cells. Therefore, BM re-examination was performed and it was confirmed that the number of megakaryocytes increased, so the patient's condition was determined as myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T). Incidentally, gene mutation analysis showed CALR gene mutation. Thereafter, administration of hydroxycarbamide and anagrelide did not show adverse events and complications, and a good blood count control was obtained. Furthermore, it was also confirmed that an SF3B1 gene mutation is highly positive in MDS-RS. There was no report on CALR-mutant MDS/MPN in Japan, and it is a rare disease overseas.


Assuntos
Calreticulina/genética , Neoplasias Hematológicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Trombocitose , Idoso de 80 Anos ou mais , Feminino , Humanos , Japão , Mutação , Trombocitose/genética
3.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura Ambiente , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
4.
Cancer Immunol Immunother ; 68(9): 1479-1492, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31463653

RESUMO

RIG-I is a cytosolic RNA sensor that recognizes short 5' triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)-calreticulin, HMGB1 and ATP-and high levels of ICD-related cytokines CXCL10, IFNß, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.


Assuntos
Antineoplásicos/uso terapêutico , Células Dendríticas/imunologia , Melanoma/tratamento farmacológico , Nelfinavir/análogos & derivados , Alarminas/imunologia , Apresentação do Antígeno/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Calreticulina/metabolismo , Caspase 3/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proteína DEAD-box 58/antagonistas & inibidores , Proteína HMGB1/metabolismo , Humanos , Imunização , Interferons/metabolismo , Terapia de Alvo Molecular , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
5.
Hematol Oncol ; 37(4): 434-437, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31465530

RESUMO

The conventional thrombotic risk stratification in essential thrombocythemia (ET) distinguishes patients in two risk groups based on previous thrombosis and age (< or >60). The IPSET-thrombosis takes into account four risk factors: age greater than 60 years and the presence of CV risk factors, thrombosis history and JAK2 V617F presence. The revised IPSET-thrombosis uses three adverse variables to delineate four risk categories: age greater than 60, thrombosis history, and JAK2 V617F presence. We compared different risk models in the estimation of thrombotic risk in 191 patients with ET and the role of specific driver mutations affecting overall survival, according to thrombotic risk. We also evaluated the mutational status of patients showing history of thrombosis or cardiovascular events versus patients who did not. Finally, we verified whether the thrombotic risk had a significant impact on survival in our ET patients. The data analysis has been performed through the conventional statistics and overall survival estimated by using the Kaplan-Meyer method. Interestingly, either using the traditional system for thrombotic risk or the IPSET-t prognostic score or the current stratification for the thrombotic risk, high-risk patients are always highly represented. This evidence is of note, being the high-risk category indicated for cytoreduction, affecting quality of life, despite the good overall prognosis of patients with ET diagnosis in general. The analysis of overall survival in our patients, according to different models for thrombotic risk, highlighted the poor prognosis of high-risk patients compared with those with a lower thrombotic risk, in particular when using traditional stratification and current stratification. In conclusion, the occurrence of thrombotic or cardiovascular events represents one of the most severe complications at diagnosis or during follow-up of ET despite current recommendations, having a significant impact on morbidity and survival.


Assuntos
Índice de Gravidade de Doença , Trombocitemia Essencial/complicações , Trombofilia/etiologia , Trombose/etiologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Calreticulina/genética , Feminino , Seguimentos , Humanos , Incidência , Janus Quinase 2/genética , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Mutação de Sentido Incorreto , Prognóstico , Receptores de Trombopoetina/genética , Recidiva , Estudos Retrospectivos , Medição de Risco/métodos , Fatores de Risco , Trombocitemia Essencial/genética , Trombocitemia Essencial/mortalidade , Trombofilia/genética , Trombose/epidemiologia , Adulto Jovem
6.
Nature ; 571(7765): 355-360, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270458

RESUMO

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.


Assuntos
Genótipo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Neoplasias/genética , Neoplasias/patologia , Transcriptoma/genética , Animais , Antígenos CD34/metabolismo , Calreticulina/genética , Linhagem Celular , Proliferação de Células , Células Clonais/classificação , Células Clonais/metabolismo , Células Clonais/patologia , Endorribonucleases/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Modelos Moleculares , Transtornos Mieloproliferativos/classificação , NF-kappa B/metabolismo , Neoplasias/classificação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Resposta a Proteínas não Dobradas/genética
7.
Rinsho Ketsueki ; 60(6): 610-618, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31281152

RESUMO

Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders, including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, characterized by constitutive activation of JAK/STAT signaling. JAK2, MPL, and CALR mutations are considered "driver mutations" and are directly implicated in the disease pathogenesis by activation of JAK2/STAT signaling. In addition to these driver mutations, several other mutations in epigenome regulatory and RNA splicing molecules have been found. This genetic information, especially regarding driver mutations, is essential for the diagnosis of MPN. Furthermore, assessment of non-driver mutations is also becoming increasingly important for disease risk assessment and treatment strategy definition.


Assuntos
Mutação , Transtornos Mieloproliferativos/genética , Calreticulina/genética , Humanos , Janus Quinase 2/genética , Mielofibrose Primária , Receptores de Trombopoetina/genética , Transdução de Sinais , Trombocitemia Essencial
8.
Gene ; 714: 144004, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351124

RESUMO

Calreticulin (CRT) is calcium binding protein of endoplasmic reticulum (ER) which performs plethora of functions besides it's role as molecular chaperone. Among the three different isoforms of this protein, CRT3 is most closely related to primitive CRT gene of higher plants. Based on their distinct structural and functional organisation, the plant CRTs have been known to contain three different domains: N, P and the C domain. The domain organisation and various biochemical characterstics of plant and animal CRTs are common with the exception of some differences. In plant calreticulin, the important N-glycosylation site(s) are replaced by the glycan chain(s) and several consensus sequences for in vitro phosphorylation by protein kinase CK2 (casein kinase-2), are also present unlike the animal calreticulin. Biotic and abiotic stresses play a significant role in bringing down the crop production. The role of various phytohormones in defense against fungal pathogens is well documented. CRT3 has been reported to play important role in protecting the plants against fungal and bacterial pathogens and in maintaining plant innate immunity. There is remarkable crosstalk between CRT mediated signalling and biotic, abiotic stress, and phytohormone mediated signalling pathways The role of CRT mediated pathway in mitigating biotic and abiotic stress can be further explored in plants so as to strategically modify it for development of stress tolerant plants.


Assuntos
Proteínas de Arabidopsis/genética , Calreticulina/genética , Transdução de Sinais/genética , Estresse Fisiológico/genética , Animais , Regulação da Expressão Gênica de Plantas/genética , Imunidade Vegetal/genética , Isoformas de Proteínas/genética
9.
Hematol Oncol ; 37(4): 424-433, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359447

RESUMO

Currently available prognostic scoring systems in primary myelofibrosis (PMF) do not integrate clinical, histological, and molecular data, or they also required information on "other" mutations that are available in the clinical practice only in a very limited number of laboratories. In the present multicenter study, including 401 PMF patients, an integrated International Prognostic Scoring System (I-IPSS) was developed by combining IPSS, grade of bone marrow fibrosis (GBMF), and driver mutations molecular status (MS) to define PMF prognosis at diagnosis. Four prognostic categories were identified: I-IPSS-low risk (113 patients), I-IPSS-intermediate-1 risk (56 patients), I-IPSS-intermediate-2 risk (154 patients), and I-IPSS-high risk (78 patients). Median overall survival was 26.7 years in I-IPSS-intermediate-1, 10.8 in I-IPSS-intermediate-2, and 6.4 in I-IPSS-high-risk patients (log-rank test <0.0001); instead, it was not reached in the I-IPSS-low-risk cohort because of the extremely low number of registered deaths. The addition of GBMF and MS to IPSS improved the efficacy for predicting the risk of death. Indeed, the sensitivity of I-IPSS was significantly higher (P < .05) than that of IPSS, considering both total deaths and 5- and 10-year mortality. This comprehensive approach allows clinicians to evaluate mutual interactions between IPSS, GBMF, and MS and identify high-risk patients with poor prognosis who may benefit from aggressive treatments. More importantly, this integrated score can be easily applicable worldwide as it only required information that represent the good clinical practice in the management of PMF patients.


Assuntos
Mielofibrose Primária/diagnóstico , Índice de Gravidade de Doença , Adulto , Idoso , Biomarcadores , Medula Óssea/patologia , Calreticulina/genética , Feminino , Humanos , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Mielofibrose Primária/genética , Mielofibrose Primária/mortalidade , Mielofibrose Primária/patologia , Prognóstico , Receptores de Trombopoetina/genética , Reticulina/ultraestrutura , Estudos Retrospectivos , Fatores de Risco
10.
Life Sci ; 232: 116591, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228513

RESUMO

AIMS: Calreticulin (CRT) is a multifunctional protein that protects endothelial cells by alleviating actin cytoskeleton injury, but the underlying mechanism remains unclear. CRT was recently identified as a novel acyltransferase; acetylation at the N-terminus of actin monomers strengthens actin polymerization. This study was undertaken to determine whether CRT protects human microvascular endothelial cells (HMECs) against microwave radiation through actin acetylation. MATERIALS AND METHODS: We prepared a eukaryotic-derived recombinant CRT and incubated the HMECs with it prior to microwave exposure. We then assessed cell injury and endothelial function, detected actin polymerization and acetylation after HMECs exposure to S-band high-power microwaves. Coimmunoprecipitation, pull-down, and ex vitro acetylation reaction were performed to determine whether actin is a novel substrate of CRT acyltransferase. Finally, we employed the mutant experiments to demonstrate the acetylation sites contributing to CRT acetyltransferase activity. KEY FINDINGS: Microwave radiation induced severe cell injury and endothelial contact dysfunction, reduced the polymerization of actin filaments, and destroyed the actin arrangement, ultimately reducing acetylated actin expression. CRT treatment upregulated actin acetylation levels, promoted polymerization, and facilitated thicker and longer F-actin stress fibre formation. Pre-incubation with CRT rescued microwave-induced cell injury, decreased actin acetylation, and rendered the actin cytoskeleton radiation-retardant. The level of acetyl-actin was positively correlated with actin polymerization. Actin was identified as a novel substrate of CRT, being acetylated mainly through the CRT P-domain at lys-206 and -207. SIGNIFICANCE: This work provides a better understanding of the underlying mechanism of CRT-induced cytoprotection, and suggests a novel therapeutic target for microwave radiation-related diseases with endothelial dysfunction.


Assuntos
Actinas/metabolismo , Calreticulina/farmacologia , Microvasos/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Linhagem Celular , Citoproteção , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Microvasos/efeitos da radiação , Micro-Ondas , Substâncias Protetoras , Processamento de Proteína Pós-Traducional , Transdução de Sinais
11.
Artif Cells Nanomed Biotechnol ; 47(1): 1978-1983, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31174435

RESUMO

The aim of this study is to investigate the effect of miR-148b on cell proliferation and migration of Schwann cells and explore its mechanism. The miR-148b group, miR-con group and the anti-miR-148b group, anti-miR-con group, si-con group, si-CALR group, Ctrl group, CALR group were transfected into Schwann cells by liposome method; the expression of miR-148b was detected by qRT-PCR; the cell viability was detected by MTT assay; the migration of cells was detected by Transwell method; WB assay was used to detect the protein expression of CALR. Firstly, we found that compared with miR-con group and si-con group, the proliferation and migration of miR-148b group and si-CALR group were significantly down-regulated (P < .05). Moreover, compared with anti-miR-con group and Ctrl group, anti-miR-148b group and CALR group cells proliferation and migration were significantly up-regulated (P < .05). In addition, miR-148b was targeted to CALR, and silencing CALR could reverse the inhibitory effect of miR-148b on Schwann cell proliferation and migration. In conclusion, miR-148b can regulate the proliferation and migration of Schwann cells. The mechanism may be related to the targeted negative regulation of CALR, which will provide a basis for targeted therapy of peripheral nerve injury.


Assuntos
Calreticulina/genética , Movimento Celular/genética , MicroRNAs/genética , Células de Schwann/citologia , Proliferação de Células/genética , Humanos
12.
Ann Hematol ; 98(10): 2339-2346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250082

RESUMO

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Medula Óssea , Calreticulina , Mutação , Trombocitemia Essencial , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea/metabolismo , Medula Óssea/patologia , Calreticulina/genética , Calreticulina/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/patologia
13.
Anticancer Res ; 39(5): 2405-2413, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092433

RESUMO

BACKGROUND/AIM: Plasma medicine is a new field that provides great potential for the treatment of human diseases including cancer in addition to sterilizing the surface of skin and facilitating wound healing. Recently, non-thermal atmospheric plasma (or cold atmospheric plasma, CAP) was introduced, not only for denaturing cells and tissues, but also for operating under the threshold of thermal damage and for chemically inducing a specific response or modification. MATERIALS AND METHODS: Microwave-mediated CAP was used in this study. RESULTS: CAP increased high-mobility group box 1 protein (HMGB1) expression, thereby increasing HMGB-1 secretion. In addition, we observed that the calreticulin (CRT) protein was concentrated at the cellular membrane when plasma was treated, representing immunogenic cell death. CONCLUSION: Overall, plasma treatment induces apoptosis via immunogenic cell death in cancer cells, implying a potential application to human cancer therapy and for the treatment of other human diseases.


Assuntos
Calreticulina/genética , Proteína HMGB1/genética , Neoplasias/terapia , Gases em Plasma/uso terapêutico , Apoptose/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HCT116 , Humanos , Fenômenos Imunogenéticos/efeitos da radiação , Micro-Ondas/uso terapêutico , Neoplasias/genética , Neoplasias/patologia
14.
Inflamm Res ; 68(7): 597-611, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31119302

RESUMO

OBJECTIVE: The present study was undertaken to validate whether TNF-α and calreticulin (CRT) serve as dual signaling to activate nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HUVECs. The effect of human antigen R (HuR) in NLRP3 inflammasome activation was also explored in RA FLS. METHODS: Immunofluorescence was used to determine the expression of NLRP3 and adaptor protein apoptosis associated speck-like protein containing a CARD (ASC) in RA synovial tissue and HuR location in RA FLS. Western blot and quantitative real-time PCR were employed to measure the priming effect of NLRP3 inflammasome in cells and HuR expression in synovial tissue. The concentrations of IL-1ß and IL-18 were detected by enzyme linked immunosorbent assay. Immunohistochemistry was used to visualize the expression of HuR in synovial tissue. HuR knockdown in RA FLS was achieved by siRNA-mediated gene silencing. RESULTS: Higher expression of NLRP3 and ASC in RA synovial tissue than those in osteoarthritis was detected. The staining of NLRP3, ASC and cleaved IL-1ß were observed in FLS and vascular endothelial cells in RA synovium. Expression of NLRP3 and pro-IL-1ß in RA FLS and HUVECs treated with TNF-α was increased. The pro-IL-18 expression was also enhanced in HUVECs, but not in RA FLS. TNF-α/CRT dual stimulation of cells gave rise to caspase-1 p20 expression and the secretion of IL-1ß. The secreted IL-18 was also elevated in HUVECs but not in RA FLS. HuR expression was significantly elevated in RA synovial tissue. TNF-α initiated the nucleocytoplasmic shuttling of HuR in both FLS and HUVECs. The knockdown of HuR in FLS incubated with TNF-α led to reduced caspase-1 p20 protein expression and further resulted in decreased secretion of IL-1ß in the presence of CRT. CONCLUSIONS: TNF-α/CRT dual signaling induced NLRP3 inflammasome activation, which could be suppressed by HuR knockdown presumably due to the block of HuR translocating from nucleus to cytoplasma.


Assuntos
Artrite Reumatoide/imunologia , Calreticulina/imunologia , Proteína Semelhante a ELAV 1/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Fator de Necrose Tumoral alfa/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Transdução de Sinais , Membrana Sinovial/imunologia , Sinoviócitos/imunologia
15.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(2): 433-438, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-30998150

RESUMO

OBJECTIVE: To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism. METHODS: The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot. RESULTS: The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced. CONCLUSION: The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.


Assuntos
Calreticulina/metabolismo , Calreticulina/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Vetores Genéticos , Humanos , Lentivirus , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Interferência de RNA , RNA Interferente Pequeno , Transfecção , Fator A de Crescimento do Endotélio Vascular
16.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(2): 520-526, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-30998164

RESUMO

OBJECTIVE: To explore the symptomatic burden of patients with essential thrombocythemia (ET) and its relation with clinical characteristics including the mutation status, therapeutic protocols and sex. METHODS: Total of 173 Chinese ET patients were selected and grouped on the basis of disease characteristics (mutation status, therapeutic pro to- cols, and sex). RESULTS: All the groups showed low-to-high symptom burden, with the highest in the Hu (hydroxyurea)-group (total symptom score [TSS], 14.7; range, 7.6-14.7). In the JAK2V617F-positive, Hu-treated, and female groups TSS and independent symptom scores were higher than those in the control group. The CALR-positive and IFN-α-treated groups had lower overall and individual scores as compared with groups lacking the corresponding characteristics. As the number of characteristics (JAK2V617F-positive, Hu-treated, and female) increases, the severity of symptoms gradually increased. CONCLUSION: The different characteristics have various effects on symptom burden in ET patients. The accumulation of certain characteristics will lead to more severe symptom burden, thus the patient's symptom burden should be considered comprehensively when making up the treatment schemes and prognosis.


Assuntos
Trombocitemia Essencial , Grupo com Ancestrais do Continente Asiático , Calreticulina , Feminino , Humanos , Hidroxiureia , Janus Quinase 2 , Mutação
18.
Rev. cuba. hematol. inmunol. hemoter ; 35(1): e911, ene.-mar. 2019. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-1003885

RESUMO

Las neoplasias mieloproliferativas crónicas (NMPC) son enfermedades clonales caracterizadas por un aumento en el número de células maduras circulantes; estas incluyen: policitemia vera (PV), trombocitemia esencial (TE), mielofibrosis primaria (MFP), entre otras. Una de las principales características moleculares de estas tres entidades es la ausencia del gen de fusión BCR/ABL. La primera mutación relacionada directamente con estas neoplasias fue detectada en el gen JAK2; a partir de su descubrimiento, otras mutaciones en los genes del receptor de trombopoyetina (MPL) y calreticulina (CALR) han sido fuertemente relacionadas con la presentación de la enfermedad. La calreticulina es una proteína del retículo endoplásmico con diversas funciones a nivel celular como la homeostasis del calcio y la actividad de chaperona. Hasta la fecha se ha identificado un gran número de mutaciones en el gen CALR. La mayoría de ellas son inserciones y deleciones que generan cambios a nivel proteico con implicaciones importantes en el curso clínico y pronóstico de las neoplasias. Debido a su alta frecuencia y fuerte asociación con las NMPC, las mutaciones de CALR se incluyen como criterio mayor para el diagnóstico de estas entidades. Por este motivo, se han desarrollado varias técnicas encaminadas a la detección rápida, eficiente, sensible y especifica de esta mutación como: la secuenciación, el análisis de fragmentos y el análisis de fusión de alta resolución. El conocimiento e implementación de estas técnicas en los laboratorios clínicos constituye un avance importante para el diagnóstico y la evolución de los pacientes(AU)


Chronic myeloproliferative neoplasms (NMPC) are clonal diseases characterized by an increase in the number of mature circulating cells; these diseases include: polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (MFP) among others. One of the main molecular characteristics of these three entities is the absence of the BCR/ABL fusion gene. The first mutation related to this group of neoplasms was detected in the JAK2 gene; since its discovery, other mutations in thrombopoietin receptor (MPL) and calreticulin (CALR) genes have been strongly related with the presentation of the disease. Calreticulin is an endoplásmic reticulum protein with different functions in the cell such as calcium homeostasis and the chaperone activity. To date, a large number of mutations have been identified in CALR gene most of them are insertions and deletions that generate changes in the protein that generate important implications in the clinical course and prognosis of neoplasms. Due to its high frequency and strong association with NMPC, CALR mutations are included as a major criteria for the diagnosis of these entities. For this reason, several techniques have been developed aimed at the rapid, efficient, sensitive and specific detection of this mutation as: sequencing, fragment analysis and high resolution fusion analysis. The knowledge and implementation of these techniques in clinical laboratories is an important advance for the diagnosis and in the evolution of patients(AU)


Assuntos
Humanos , Masculino , Feminino , Calreticulina/síntese química , Técnicas de Diagnóstico Molecular , Mutação
19.
Oncogene ; 38(10): 1651-1660, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30846848

RESUMO

Frameshift mutations in the calreticulin (CALR) gene are present in 30% of essential thrombocythemia and myelofibrosis patients. The two most frequent mutations are CALR del52 (type 1, approximately 60%) and CALR ins5 (type 2, around 30%), but many other rarer mutations exist accounting each for less than 2% of all CALR mutations. Most of them are structurally classified as type 1-like and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha-helix structure. Yet, several key questions remain unanswered, especially the reason of such low frequencies of these other mutations. In an attempt to investigate specific pathogenic differences between type 1-like and type 2-like CALR mutations and del52 and ins5, we modeled two type 1-like (del34 and del46) and one type 2-like (del19) mutations in cell lines and in mice. All CALR mutants constitutively activate JAK2 and STAT5/3/1 in a similar way in the presence of the thrombopoietin receptor (MPL) and induced cytokine-independent cell growth but to a lesser extent with rare mutants over time. This correlates with reduced expression levels of rare CALR mutants compared to del52 and ins5. Lethally irradiated mice that were engrafted with bone marrow transduced with the different CALR mutations developed thrombocytosis, but to a much lesser extent with ins5 and the type 2-like CALR mutation. In contrast to type 2-like mice, type 1-like mice developed marked myelofibrosis and splenomegaly 10 months after engraftment. Similar to del52, type 1-like CALR mutations induced an expansion at an early stage of hematopoiesis compared to ins5 and type 2-like mutation. Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble del52 and ins5 mutants, respectively.


Assuntos
Calreticulina/genética , Calreticulina/metabolismo , Mutação , Transtornos Mieloproliferativos/genética , Animais , Calreticulina/química , Linhagem Celular , Modelos Animais de Doenças , Humanos , Janus Quinase 2/genética , Camundongos , Transtornos Mieloproliferativos/metabolismo , Receptores de Trombopoetina/metabolismo , Fatores de Transcrição STAT/genética , Ativação Transcricional
20.
J Biol Chem ; 294(16): 6240-6252, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30833329

RESUMO

α1-Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT-ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT-ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.


Assuntos
Calreticulina/biossíntese , Exossomos/metabolismo , Mutação de Sentido Incorreto , Proteólise , alfa 1-Antitripsina/metabolismo , Substituição de Aminoácidos , Animais , Calreticulina/genética , Linhagem Celular , Exossomos/genética , Exossomos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/patologia
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