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1.
Leukemia ; 35(1): 75-89, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32205861

RESUMO

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.


Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva , Leucemia de Células B/imunologia , Leucemia de Células B/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD19/química , Antígenos de Neoplasias , Biomarcadores , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imunoterapia Adotiva/métodos , Leucemia de Células B/genética , Leucemia de Células B/terapia , Camundongos Transgênicos , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Relação Estrutura-Atividade , Transdução Genética , Transgenes , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Scand J Immunol ; 93(1): e12993, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33151595

RESUMO

The discovery that NK cells are able to specifically recognize cells lacking the expression of self-MHC class I molecules provided the first insight into NK cell recognition of tumour cells. It started a flourishing field of NK cell research aimed at exploring the molecular nature of NK cell receptors involved in tumour cell recognition. While much of the important early work was conducted in murine experimental model systems, studies of human NK cells rapidly followed. Over the years, human NK cell research has swiftly progressed, aided by new detailed molecular information on human NK cell development, differentiation, molecular specificity, tissue heterogeneity and functional capacity. NK cells have also been studied in many different diseases aside from cancer, including viral diseases, autoimmunity, allergy and primary immunodeficiencies. These fields of research have all, indirectly or directly, provided further insights into NK cell-mediated recognition of target cells and paved the way for the development of NK cell-based immunotherapies for human cancer. Excitingly, NK cell-based immunotherapy now opens up for novel strategies aimed towards treating malignant diseases, either alone or in combination with other drugs. Reviewed here are some personal reflections of select contributions leading up to the current state-of-the-art in the field, with a particular emphasis on contributions from our own laboratory. This review is part of a series of articles on immunology in Scandinavia, published in conjunction with the 50th anniversary of the Scandinavian Society for Immunology.


Assuntos
Imunoterapia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Animais , Antígenos de Neoplasias/imunologia , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Neoplasias/patologia , Especificidade de Órgãos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Resultado do Tratamento
3.
Gene ; 766: 145151, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32950635

RESUMO

Smoking tobacco is the major hazard for lung cancer in Indian subcontinent especially men, compare to woman where, other important risk factors such as air pollutions are responsible. So, the aim of the study is to compare chronic smokers (CS) and non-smokers living in areas with air quality categorized as poor (AQI 201-300) or moderate (AQI 101-200). We measured the expression of non-small cell lung cancer (NSCLC) biomarkers;. IDH1, CEA, Cyfra21-1, and TPA through quantitative Real-Time PCR (qRT-PCR) and compared the levels of upregulation of the transcripts in stage IIIa NSCLC over control benign tissues among the smoking and AQI settings. Though the all biomarkers were significantly up-regulated in tumor tissues compared to control benign tissues, the fold change increase of IDH1 and CEA was highest in CS-poor/moderate AQI, followed by non-smokers-poor AQI and non-smokers moderate AQI. This indicates the aggressiveness and poor prognosis in CS living in either poor or moderate AQI areas. The level of Cyfra21-1 was lower in in the CS groups in comparison to non-smokers in the poor AQI area. This suggest higher Lung Squamous cell carcinoma histology in non-smokers living areas with poor AQI. Hence, we conclude that poor air quality can be as injurious for lung cancers as chronic smoking.


Assuntos
Antígenos de Neoplasias/genética , Antígeno Carcinoembrionário/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Isocitrato Desidrogenase/genética , Queratina-19/genética , Neoplasias Pulmonares/genética , Fumar/genética , Poluição do Ar , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/genética , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , não Fumantes , Prognóstico , Fumantes , Transcriptoma/genética
4.
Nat Commun ; 11(1): 6305, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33298915

RESUMO

The features of peptide antigens that contribute to their immunogenicity are not well understood. Although the stability of peptide-MHC (pMHC) is known to be important, current assays assess this interaction only for peptides in isolation and not in the context of natural antigen processing and presentation. Here, we present a method that provides a comprehensive and unbiased measure of pMHC stability for thousands of individual ligands detected simultaneously by mass spectrometry (MS). The method allows rapid assessment of intra-allelic and inter-allelic differences in pMHC stability and reveals profiles of stability that are broader than previously appreciated. The additional dimensionality of the data facilitated the training of a model which improves the prediction of peptide immunogenicity, specifically of cancer neoepitopes. This assay can be applied to any cells bearing MHC or MHC-like molecules, offering insight into not only the endogenous immunopeptidome, but also that of neoepitopes and pathogen-derived sequences.


Assuntos
Genes MHC Classe I/genética , Ensaios de Triagem em Larga Escala/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia/métodos , Peptídeos/imunologia , Alelos , Apresentação do Antígeno , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular , Conjuntos de Dados como Assunto , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Temperatura Alta/efeitos adversos , Humanos , Ligantes , Neoplasias/imunologia , Neoplasias/terapia , Redes Neurais de Computação , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Estabilidade Proteica , Proteômica/métodos , Espectrometria de Massas em Tandem
5.
Gan To Kagaku Ryoho ; 47(11): 1621-1623, 2020 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-33268740

RESUMO

We treated 3,164 patients with advanced cancer with dendritic cell therapy between July 2005 and March 2020. The effective rate in patients treated with dendritic cell therapy more than 3 times was 19.0%. Among them, we treated 133 cancer patients with a combination of immune checkpoint inhibitors and dendritic cell therapy between June 2015 and March 2020. The effective rate in these patients was 54.1%. We treated 98 cancer patients with dendritic cell therapy with neoantigens between March 2018 and March 2020. The effective rate in these patients treated with neoantigens was 38.7%. The effective rate in patients treated without neoantigens was 18.3%. Dendritic cell therapy with neoantigens enhanced the effective rate. The effective rate of dendritic cell therapy with both immune checkpoint inhibitors and neoantigens was 60.7%.


Assuntos
Antígenos de Neoplasias , Neoplasias , Células Dendríticas , Humanos , Imunoterapia , Neoplasias/terapia
6.
Medicine (Baltimore) ; 99(46): e22773, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33181649

RESUMO

Nasopharyngeal carcinoma (NPC) has a distinctive geographical distribution in China, especially southern China. There are several risk factors for NPC, such as Epstein-Barr virus, genetics, and environmental exposures. Although the incidence of eye metastasis (EM) is lower than metastasis in other body parts, it often indicates poor prognosis.We assessed several serum biomarkers for their ability to predict EM in NPC. Patients with NPC were selected (n = 963), and were separated into two groups, EM and no eye metastasis. Ten factors were analyzed in both groups including triglyceride (TG), high-density lipoprotein, low-density lipoprotein, alkaline phosphatase, alpha fetoprotein, carbohydrate antigen-199, cancer antigen-153, apolipoproteins AI, apolipoprotein B, and cytokeratin fragment 19 (CYFRA21-1). Independent t tests, binary logistic regression, and receiver operating characteristic curves were used to assess the data.The EM group had significantly higher CYFRA21-1 and lower TG compared with the no eye metastasis group. Areas under the curve for CYFRA21-1, TG and CYFRA21-1/TG were 0.966, 0.771, and 0.976, respectively. The corresponding cut-off values were 12.12 ng/ml, 0.41 mmol/L, and 13.5. The sensitivity and specificity of CYFRA21-1/TG were 100% and 92.2%, respectively.The increased ratio of CYFRA21-1 to TG can be an accurate method to detect EM in patients with NPC.


Assuntos
Antígenos de Neoplasias/análise , Neoplasias Oculares/etiologia , Queratina-19/análise , Neoplasias Nasofaríngeas/genética , Metástase Neoplásica/diagnóstico , Tireoglobulina/análise , Adulto , Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/análise , China/epidemiologia , Neoplasias Oculares/epidemiologia , Neoplasias Oculares/genética , Feminino , Humanos , Queratina-19/sangue , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/epidemiologia , Metástase Neoplásica/diagnóstico por imagem , Metástase Neoplásica/fisiopatologia , Fatores de Risco , Sensibilidade e Especificidade , Tireoglobulina/sangue
7.
Rinsho Ketsueki ; 61(9): 1433-1439, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33162546

RESUMO

Cancer cells harboring somatic mutations give rise to neoantigens, which are immunologically foreign in nature to be distinguished from itself, showing high immunogenicity and, thus, induce specific T-cell responses against cancer. Therefore, neoantigens are expected to be promising targets for anti-cancer immunotherapy. The general methods used to identify candidate neoantigens are as follows: (1) non-synonymous mutations are identified by whole exome and RNA sequencing; (2) neoantigens from the mutations are predicted based on in silico MHC ligand prediction algorithm; (3) specific T-cell responses toward the candidate neoantigens are verified using tumor infiltrating T cells or peripheral blood mononuclear cells. In hematological malignancy, several neoantigens have been identified as an important treatment target. In contrast with solid malignancies, the occurrence of frameshift mutations and fusion genes producing neoantigens are high. A shared neoantigen derived from frameshift mutation of nucleophosmin I, which is often observed in acute myeloid leukemia, was reported to induce specific immune responses in vitro and in vivo. We should examine neoantigens as possible target of novel immunotherapy despite several issues to be addressed for clinical application.


Assuntos
Antígenos de Neoplasias , Imunoterapia , Leucócitos Mononucleares , Neoplasias , Exoma , Humanos , Mutação , Neoplasias/genética , Neoplasias/terapia , Linfócitos T
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(10): 884-889, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33148382

RESUMO

Objective To analyze the physicochemical properties, structure and function of melanoma-associated antigen D4 (MAGE-D4) protein, and then construct the eukaryotic expression vector of MAGE-D4. Methods The physicochemical properties, structure and function of MAGE-D4 protein were analyzed by bioinformatics. Using MAGE-D4/pMAL-C2 prokaryotic recombinant plasmid as the template, PCR product digested by restriction enzyme was connected with pEGFP-C1 eukaryotic expression plasmid and transformed into E. coli. Ligation products were identified by antibiotic screening, enzyme digestion and sequencing. Then the recombinant plasmid was transfected into A549 lung cancer cells by liposome. Results MAGE-D4 protein was an unstable hydrophilic protein without transmembrane structure and signal peptide. Its secondary structure was mainly α-helix. MAGE-D4 contained multiple functional modification sites and was mainly located in the nucleus. SLLLVILGV might be a restricted T cell epitope of HLA-A*0201 derived from MAGE-D4. The first three proteins to potentially interact with MAGE-D4 were NSMCE4A, MLANA/MART-1 and BAGE5. DNA sequencing showed that the recombinant plasmid contained full-length coding sequence (CDS) of MAGE-D4 and it could be successfully transfected into A549 lung cancer cells. Conclusion MAGE-D4 protein is an unstable nuclear protein, which may play functions by interacting with a variety of melanoma-related proteins. The peptide derived from MAGE-D4 may have strong immunogenicity. The eukaryotic expression vector of MAGE-D4 has been successfully constructed.


Assuntos
Antígenos de Neoplasias/genética , Biologia Computacional , Vetores Genéticos , Proteínas de Neoplasias/genética , Células A549 , Epitopos de Linfócito T , Escherichia coli , Eucariotos , Vetores Genéticos/genética , Humanos , Plasmídeos/genética , Transfecção
9.
Nat Commun ; 11(1): 5696, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173046

RESUMO

Poorly immunogenic tumors, including triple negative breast cancers (TNBCs), remain resistant to current immunotherapies, due in part to the difficulty of reprogramming the highly immunosuppressive tumor microenvironment (TME). Here we show that peritumorally injected, macroporous alginate gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) for concentrating dendritic cells (DCs), CpG oligonucleotides, and a doxorubicin-iRGD conjugate enhance the immunogenic death of tumor cells, increase systemic tumor-specific CD8 + T cells, repolarize tumor-associated macrophages towards an inflammatory M1-like phenotype, and significantly improve antitumor efficacy against poorly immunogenic TNBCs. This system also prevents tumor recurrence after surgical resection and results in 100% metastasis-free survival upon re-challenge. This chemo-immunotherapy that concentrates DCs to present endogenous tumor antigens generated in situ may broadly serve as a facile platform to modulate the suppressive TME, and enable in situ personalized cancer vaccination.


Assuntos
Materiais Biocompatíveis/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Imunoterapia/métodos , Neoplasias de Mama Triplo Negativas/terapia , Animais , Antígenos de Neoplasias/metabolismo , Biotecnologia/métodos , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos/métodos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Fatores Imunológicos/metabolismo , Fatores Imunológicos/uso terapêutico , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias de Mama Triplo Negativas/imunologia , Microambiente Tumoral/imunologia
10.
PLoS One ; 15(11): e0241551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33227008

RESUMO

Perineural invasion is a pathologic process of neoplastic dissemination along and invading into the nerves. Perineural invasion is associated with aggressive disease and a greater likelihood of poor outcomes. In this study, 3 of 9 patients with cutaneous squamous cell carcinoma and perineural invasion exhibited poor clinical outcomes. Tumors from these patients expressed high levels of MAGE-A3, a cancer testis antigen that may contribute to key processes of tumor development. In addition to perineural invasion, the tumors exhibited poor differentiation and deep invasion and were subsequently classified as Brigham and Women's Hospital tumor stage 3. Cyclin E, A and B mRNA levels were increased in these tumors compared with normal skin tissues (102.93±15.03 vs. 27.15±4.59, 36.83±19.41 vs. 11.59±5.83, 343.77±86.49 vs. 95.65±29.25, respectively; p<0.05). A431 cutaneous squamous cell carcinoma cells pretreated with MAGE-A3 antibody exhibited a decreased percentage S-phase cells (14.13±2.8% vs. 33.97±1.1%; p<0.05) and reduced closure in scratch assays (43.88±5.49% vs. 61.17±3.97%; p = 0.0058). In a syngeneic animal model of squamous cell carcinoma, immunoblots revealed overexpression of MAGE-A3 and cyclin E, A, and B protein in tumors at 6 weeks. However, knockout of MAGE-A3 expression caused a reduction in tumor growth (mean tumor volume 155.3 mm3 vs. 3.2 mm3) compared with parental cells. These results suggest that MAGE-A3 is a key mediator in cancer progression. Moreover, elevated collagen XI and matrix metalloproteases 3, 10, 11, and 13 mRNA levels were observed in poorly differentiated cutaneous squamous cell carcinoma with perineural invasion compared with normal skin tissue (1132.56±882.7 vs. 107.62±183.62, 1118.15±1109.49 vs. 9.5±5, 2603.87±2385.26 vs. 5.29±3, 957.95±627.14 vs. 400.42±967.66, 1149.13±832.18 vs. 19.41±35.62, respectively; p<0.05). In summary, this study highlights the potential prognostic value of MAGE-A3 in clinical outcomes of cutaneous squamous cell carcinoma patients.


Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Neoplasias/metabolismo , Nervos Periféricos/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Animais , Anticorpos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , Ciclinas/metabolismo , Humanos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos
11.
BMC Bioinformatics ; 21(1): 532, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208106

RESUMO

BACKGROUND: Neoantigen-based personal vaccines and adoptive T cell immunotherapy have shown high efficacy as a cancer treatment in clinical trials. Algorithms for the accurate prediction of neoantigens have played a pivotal role in such studies. Some existing bioinformatics methods, such as MHCflurry and NetMHCpan, identify neoantigens mainly through the prediction of peptide-MHC binding affinity. However, the predictive accuracy of immunogenicity of these methods has been shown to be low. Thus, a ranking algorithm to select highly immunogenic neoantigens of patients is needed urgently in research and clinical practice. RESULTS: We develop TruNeo, an integrated computational pipeline to identify and select highly immunogenic neoantigens based on multiple biological processes. The performance of TruNeo and other algorithms were compared based on data from published literature as well as raw data from a lung cancer patient. Recall rate of immunogenic ones among the top 10-ranked neoantigens were compared based on the published combined data set. Recall rate of TruNeo was 52.63%, which was 2.5 times higher than that predicted by MHCflurry (21.05%), and 2 times higher than NetMHCpan 4 (26.32%). Furthermore, the positive rate of top 10-ranked neoantigens for the lung cancer patient were compared, showing a 50% positive rate identified by TruNeo, which was 2.5 times higher than that predicted by MHCflurry (20%). CONCLUSIONS: TruNeo, which considers multiple biological processes rather than peptide-MHC binding affinity prediction only, provides prioritization of candidate neoantigens with high immunogenicity for neoantigen-targeting personalized immunotherapies.


Assuntos
Antígenos de Neoplasias/metabolismo , Medicina de Precisão , Software , Algoritmos , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Carcinoma de Células Pequenas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo
12.
J Biol Regul Homeost Agents ; 34(5): 1637-1646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33108861

RESUMO

This study aims to investigate the value of the combined detection of carcinoembryonic antigen (CEA), Neuron-specific enolase (NSE) and the level of Interleukin-18 (IL-18) in the serum in the diagnosis of lung cancer. The correlation between these parameters and the expression levels of B-cell lymphoma-2 (Bcl-2) protein were also studied. Eighty patients with lung cancer were included in the lung cancer group. These patients underwent surgery in the Department of Oncology of Huai'an Second People's Hospital between February 2016 and February 2018. During the same period, another 80 patients with benign lung lesions were registered in the benign lesion group and 80 healthy people were enrolled in the control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of CEA, NSE and IL-18. The diagnostic critical value of these factors was used as positive indicator. When CEA, NSE and IL-18 levels were positive at the same time, the combined detection was considered to be positive. WB was used to detect Bcl-2 expression level. We also analyzed the possible correlation between CEA, NSE, IL-18 levels and the Bcl-2 expression levels. The CEA, NSE and IL-18 expression levels in the serum of the lung cancer group were significantly higher than those in the benign lesion and the control groups (p<0.05). The area under ROC curve for CEA, NSE and IL-18 respectively was 0.770 (0.697-0.843), 0.829 (0.767-0.890), 0.721 (0.642-0.800) (p<0.001). IL-18 level was negatively correlated with the level of Bcl-2 mRNA in the tissue (r=-0.380, p<0.001). In conclusion, CEA, NSE and IL-18 have a good auxiliary diagnostic value in patients with lung cancer. The combined detection could improve the sensitivity and specificity of lung can¬cer diagnosis. There was a negative correlation between IL-18 and Bcl-2 levels which suggested a potential inhibitory role of IL-18 on the lung cancer cells apoptosis pathway.


Assuntos
Neoplasias Pulmonares , Antígenos de Neoplasias , Apoptose , Biomarcadores Tumorais/genética , Antígeno Carcinoembrionário , Humanos , Interleucina-18 , Queratina-19 , Neoplasias Pulmonares/diagnóstico , Fosfopiruvato Hidratase , Proteínas Proto-Oncogênicas c-bcl-2
14.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
15.
Proc Natl Acad Sci U S A ; 117(44): 27528-27539, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33067394

RESUMO

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses.


Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Macrófagos/imunologia , Neoplasias/terapia , Vacinação/métodos , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Gangliosídeos , Humanos , Imunogenicidade da Vacina , Leucócitos Mononucleares , Lipossomos , Macrófagos/metabolismo , Neoplasias/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Células THP-1 , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia
16.
Am J Pathol ; 190(12): 2330-2342, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33011110

RESUMO

Gelatinous drop-like corneal dystrophy (GDLD) is a severe inherited corneal dystrophy characterized by subepithelial corneal amyloid deposition. We had previously succeeded in identifying the responsible gene, TACSTD2, and subsequently found that the epithelial barrier function is significantly decreased. As with GDLD patients, the knockout mice showed severe loss of tight junction, progressive opacity, and neovascularization in the cornea. We devised an easy method to confirm the loss of the corneal barrier function even before corneal opacity is observed. Furthermore, by using knockout mice, we were able to verify clinical findings, such as the wound healing delay and light-induced acceleration of the disease. This mouse model should prove to be a highly useful tool for investigating the pathology of GDLD and for developing new therapies.


Assuntos
Amiloidose Familiar/patologia , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Distrofias Hereditárias da Córnea/patologia , Animais , Distrofias Hereditárias da Córnea/genética , Modelos Animais de Doenças , Gelatina/genética , Gelatina/metabolismo , Camundongos , Mutação/genética
17.
Sci Rep ; 10(1): 16772, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033277

RESUMO

The role of fibroblasts in tissue fibrosis has been extensively studied. Activated fibroblasts, namely myofibroblasts, produce pathological extracellular matrix. CD248, a type I transmembrane glycoprotein, is expressed in fibroblasts after birth. In human chronic kidney disease, upregulated CD248 in myofibroblasts is linked to poor renal survival. In this study, we demonstrated a novel interaction between CD248 and macrophages to be a key step in mediating tissue fibrosis. CD248 was upregulated in myofibroblasts in murine models of renal and peritoneal fibrosis. Cd248 knockout (Cd248-/-) could attenuate both renal and peritoneal fibrosis. By parabiosis of GFP reporter mice and Cd248-/- mice, we showed that attenuation of renal fibrosis was associated with a decrease of macrophage infiltration in Cd248-/- mice. Moreover, decrease of chemokine (C-C motif) ligand 17 and Ccl22 was found in macrophages isolated from the fibrotic kidneys of Cd248-/- mice. Because galectin-3-deficient macrophages showed decreased Ccl17 and Ccl22 in fibrotic kidneys, we further demonstrated that CD248 interacted specifically with galectin-3 of macrophages who then expressed CCL17 to activate collagen production in myofibroblasts. Mice with DNA vaccination targeting CD248 showed decreased fibrosis. We thus propose that CD248 targeting should be studied in the clinical tissue fibrosis setting.


Assuntos
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Quimiocina CCL17/metabolismo , Fibroblastos/metabolismo , Nefropatias/metabolismo , Macrófagos/metabolismo , Fibrose Peritoneal/metabolismo , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Quimiocina CCL17/genética , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Nefropatias/genética , Nefropatias/patologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibrose Peritoneal/genética , Fibrose Peritoneal/patologia
18.
PLoS One ; 15(10): e0225487, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119615

RESUMO

Receptor for advanced glycation end products (RAGE) has been implicated in the pathophysiology of Alzheimers disease(AD) due to its ability to bind amyloid-beta (Aß42) and mediate inflammatory response. G82S RAGE polymorphism is associated with AD but the molecular mechanism for this association is not understood. Our previous in silico study indicated a higher binding affinity for mutated G82S RAGE, which could be caused due to changes in N linked glycosylation at residue N81. To confirm this hypothesis, in the present study molecular dynamics (MD) simulations were used to simulate the wild type (WT) and G82S glycosylated structures of RAGE to identify the global structural changes and to find the binding efficiency with Aß42 peptide. Binding pocket analysis of the MD trajectory showed that cavity/binding pocket in mutant G82S glycosylated RAGE variants is more exposed and accessible to external ligands compared to WT RAGE, which can enhance the affinity of RAGE for Aß. To validate the above concept, an in vitro binding study was carried using SHSY5Y cell line expressing recombinant WT and mutated RAGE variant individually to which HiLyte Fluor labeled Aß42 was incubated at different concentrations. Saturated binding kinetics method was adopted to determine the Kd values for Aß42 binding to RAGE. The Kd value for Aß42- WT and Aß42-mutant RAGE binding were 92±40 nM (95% CI-52 to 152nM; R2-0.92) and 45±20 nM (95% CI -29 to 64nM; R2-0.93), respectively. The Kd value of <100nM observed for both variants implicates RAGE as a high-affinity receptor for Aß42 and mutant RAGE has higher affinity compared to WT. The alteration in binding affinity is responsible for activation of the inflammatory pathway as implicated by enhanced expression of TNFα and IL6 in mutant RAGE expressing cell line which gives a mechanistic view for the G82S RAGE association with AD.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Polimorfismo de Nucleotídeo Único , Antígenos de Neoplasias/genética , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Predisposição Genética para Doença , Glicosilação , Humanos , Interleucina-6/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Fator de Necrose Tumoral alfa/genética
19.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
20.
Artigo em Alemão | MEDLINE | ID: mdl-33034694

RESUMO

The physician and scientist Paul Ehrlich put forward the thesis that the immune system not only fights infections but can also fight cancer. The possible positive effects of a simultaneous infection on the course of cancer were reported in ancient Egypt around 2600 BC. However, it was not until the 1960s that it became apparent that the immune system could specifically fight cancer cells, and it was not until the 1990s that researchers slowly clarified how this happens.Against this background, the efforts over the last 30 years to develop therapeutic vaccines against cancers are briefly summarized, and their lack of success to date is highlighted. In addition, potentially promising future developments in this context are discussed. The available scientific literature as well as our own results are taken into account.Central questions arise, such as the following: How do cancer cells differ from normal cells? How can the immune system recognize these differences? What are tumor-specific antigens? Why do they need to be selected and applied in an individualized fashion? How can an efficient immune response be induced? Which pharmaceutical formulations, adjuvants, and vaccination routes are effective?Finally, we explain why it may still be worth pursuing peptide vaccination, which has so far been completely unsuccessful (when measured in terms of already approved therapeutics).


Assuntos
Vacinas Anticâncer , Neoplasias , Antígenos de Neoplasias , Vacinas Anticâncer/uso terapêutico , Alemanha , Humanos , Imunoterapia , Neoplasias/prevenção & controle , Vacinas de Subunidades/uso terapêutico
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