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1.
Sci Rep ; 10(1): 10895, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616763

RESUMO

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Vacinas Virais/metabolismo
2.
Mol Carcinog ; 59(7): 862-870, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32386086

RESUMO

The strength of the interaction between T-cell receptors (TCRs) and their ligands, peptide/major histocompatibility complex complexes (pMHCs), is one of the most frequently discussed and investigated features of T cells in immuno-oncology today. Although there are many molecules on the surface of T cells that interact with ligands on other cells, the TCR/pMHC is the only receptor-ligand pair that offers antigen specificity and dictates the functional response of the T cell. The strength of the TCR/pMHC interaction, along with the environment in which this interaction takes place, is key to how the T cell will respond. The TCR repertoire of T cells that interact with tumor-associated antigens is vast, although typically of low affinity. Here, we focus on the low-affinity interactions between TCRs from CD8+ T cells and different models used in immuno-oncology.


Assuntos
Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Imunoterapia/métodos
3.
Scand J Immunol ; 91(6): e12888, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32281665

RESUMO

We propose a framework to explain how T cells achieve specificity and sensitivity, how the affinity of the TcR peptide/MHC interaction controls positive and negative thymic selection and mature T cell survival, and whether antigen-dependent activation and inactivation takes place. Two distinct types of signalling can lead to mature T cell multiplication. One requires the TcR to recognize with a certain affinity an antigen-derived peptide, an agonist peptide, bound to an MHC molecule. The other, the tonic signal, leads to naïve T cell survival and modest proliferation if the T cell successfully competes for endogenous, self-peptide/MHC ligands, involving lower affinity TCR/ligand interactions. Many suggest lymphopenia contributes to autoimmunity by increasing the strength of TcR-tonic signalling, and so activation of anti-self T cells. We suggest T cell activation requires antigen-mediated cooperation between T cells. Increased tonic signalling under lymphopenic conditions facilitates T cell proliferation and so antigen-dependent cooperation and activation of anti-self T cells.


Assuntos
Linfopenia/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/imunologia , Animais , Apresentação do Antígeno , Autoantígenos/imunologia , Autoantígenos/metabolismo , Autoimunidade , Comunicação Celular , Diferenciação Celular , Sobrevivência Celular , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ativação Linfocitária , Modelos Imunológicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
4.
Int J Mol Sci ; 21(2)2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31940843

RESUMO

Contact hypersensitivity (CHS) is an established animal model for allergic contact dermatitis. Dendritic cells (DCs) play an important role in the sensitization phase of CHS by initiating T cell responses to topically applied haptens. The cannabinoid receptors 1 (CB1) and 2 (CB2) modulate DC functions and inflammatory skin responses, but their influence on the capacity of haptenized DCs to induce CHS is still unknown. We found lower CHS responses to 2,4-dinitro-1-fluorobenzene (DNFB) in wild type (WT) mice after adoptive transfer of haptenized Cnr2-/- and Cnr1-/-/Cnr2-/- bone marrow (BM) DCs as compared to transfer of WT DCs. In contrast, induction of CHS was not affected in WT recipients after transfer of Cnr1-/- DCs. In vitro stimulated Cnr2-/- DCs showed lower CCR7 and CXCR4 expression when compared to WT cells, while in vitro migration towards the chemokine ligands was not affected by CB2. Upregulation of MHC class II and co-stimulatory molecules was also reduced in Cnr2-/- DCs. This study demonstrates that CB2 modulates the maturation phenotype of DCs but not their chemotactic capacities in vitro. These findings and the fact that CHS responses mediated by Cnr2-/- DCs are reduced suggest that CB2 is a promising target for the treatment of inflammatory skin conditions.


Assuntos
Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Quimiotaxia , Células Dendríticas/citologia , Dermatite Alérgica de Contato/genética , Dinitrofluorbenzeno/toxicidade , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptores CCR4/genética , Receptores CCR4/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo
5.
Biochimie ; 168: 220-230, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31756401

RESUMO

G9a (also known as EHMT2 - Euchromatin histone methyltransferase 2) is a protein lysine methyltransferase which introduces methylation modification in variety of proteins including histones. G9a catalyzes the dimethylation of lysine 9 on histone 3 (H3K9me2) which is a repressive epigenetic modification. H3K9me2 is associated with the silencing of several genes including tumor suppressor genes in many cancers and hence G9a is a well characterized drug target for cancer therapy. Here, we report the discovery of CSV0C018875 as a novel quinoline based G9a inhibitor through virtual screening strategy from a HTS database. Sub-structure querying based on the known G9a inhibitors, followed by docking based virtual screening, led to the identification of CSV0C018875 as G9a inhibitor. We found that CSV0C018875 inhibits the activity of G9a in both enzyme and cell based assays. Importantly, the toxicity of CSV0C018875 is much lesser than that of the well-studied G9a inhibitor, BIX-01294. Molecular dynamics simulations shows that CSV0C018875 binds deeper inside the active site cavity of G9a, which facilitates the tight binding and also increases the compounds residence time, which in turn reflects better G9a inhibition. The novel quinoline CSV0C018875 could be further optimized to improve the ADME as well pharmacodynamic property.


Assuntos
Inibidores Enzimáticos , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Quinolinas , Azepinas/química , Domínio Catalítico , Bases de Dados de Compostos Químicos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Ligação Proteica , Quinazolinas/química , Quinolinas/química , Quinolinas/metabolismo
6.
Nucleic Acids Res ; 48(D1): D948-D955, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31667505

RESUMO

The IPD-IMGT/HLA Database, http://www.ebi.ac.uk/ipd/imgt/hla/, currently contains over 25 000 allele sequence for 45 genes, which are located within the Major Histocompatibility Complex (MHC) of the human genome. This region is the most polymorphic region of the human genome, and the levels of polymorphism seen exceed most other genes. Some of the genes have several thousand variants and are now termed hyperpolymorphic, rather than just simply polymorphic. The IPD-IMGT/HLA Database has provided a stable, highly accessible, user-friendly repository for this information, providing the scientific and medical community access to the many variant sequences of this gene system, that are critical for the successful outcome of transplantation. The number of currently known variants, and dramatic increase in the number of new variants being identified has necessitated a dedicated resource with custom tools for curation and publication. The challenge for the database is to continue to provide a highly curated database of sequence variants, while supporting the increased number of submissions and complexity of sequences. In order to do this, traditional methods of accessing and presenting data will be challenged, and new methods will need to be utilized to keep pace with new discoveries.


Assuntos
Alelos , Biologia Computacional , Bases de Dados Genéticas , Antígenos de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/genética , Software , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Navegador
7.
Biomed Res ; 40(6): 243-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839668

RESUMO

Recently, the first series of small molecule inhibitors of PD-1/PD-L1 were reported by Bristol-Myers Squibb (BMS), which were developed using a homogeneous time-resolved fluorescence (HTRF)-based screening investigation of the PD-1/PD-L1 interaction. Additional crystallographic and biophysical studies showed that these compounds inhibited the interaction of PD-1/PD-L1 by inducing the dimerization of PD-L1, in which each dimer binds one molecule of the stabilizer at its interface. However, the immunological mechanism of the antitumor effect of these compounds remains to be elucidated. In the present study, we focused on BMS-202 (a representative of the BMS compounds) and investigated its antitumor activity using in vitro and in vivo experiments. BMS-202 inhibited the proliferation of strongly PD-L1-positive SCC-3 cells (IC50 15 µM) and anti-CD3 antibody-activated Jurkat cells (IC50 10 µM) in vitro. Additionally, BMS-202 had no regulatory effect on the PD-1 or PD-L1 expression level on the cell surface of these cells. In an in vivo study using humanized MHC-double knockout (dKO) NOG mice, BMS-202 showed a clear antitumor effect compared with the controls; however, a direct cytotoxic effect was revealed to be involved in the antitumor mechanism, as there was no lymphocyte accumulation in the tumor site. These results suggest that the antitumor effect of BMS-202 might be partly mediated by a direct off-target cytotoxic effect in addition to the immune response-based mechanism. Also, the humanized dKO NOG mouse model used in this study was shown to be a useful tool for the screening of small molecule inhibitors of PD-1/PD-L1 binding that can inhibit tumor growth via an immune-response-mediated mechanism.


Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Animais , Antineoplásicos/química , Antígeno B7-H1/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade/genética , Humanos , Camundongos , Camundongos Knockout , Estrutura Molecular , Receptor de Morte Celular Programada 1/genética , Ligação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Medicine (Baltimore) ; 98(48): e18212, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31770281

RESUMO

As one of the most serious cancers, gastric cancer (GC) represents the third leading cause of malignancy-related deaths. G9a is a histone lysine methyltransferase and has been reported to be involved in the progression of some human cancers. In the present study, we aimed to explore the expression patterns and clinical value of G9a in GC patients.The expression of G9a in 142 paired GC tissues and adjacent non-cancerous tissues (no less than 5 cm from tumor edge) was examined with quantitative real-time polymerase chain reaction (qRT-PCR). To estimate the association of G9a expression with clinical characteristics of GC patients, Chi-square test and t test were conducted. Kaplan-Meier survival and multivariate Cox regression analyses were performed to explore the prognostic value of G9a in GC.Upregulated expression of G9a was found in GC tissues compared with noncancerous tissues (P < .001). Elevated G9a expression was significantly correlated with patients' lymph node metastasis (P = .007) and TNM stage (P < .001). Kaplan-Meier survival curves demonstrated that patients with high G9a expression had shorter survival time than those with low expression (log-rank test, P < .05), reaching a median OS of 24 months. According to the results of Cox regression, G9a could be considered as an independent prognostic biomarker in patients with GC (HR = 3.912, 95% CI = 2.213-6.915, P < .001). Additionally, the diagnosis cut-off value of G9a in GC patients was 1.515.Taken together, G9a expression was upregulated in GC tissues and could be an effective prognostic biomarker for GC.


Assuntos
Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias Gástricas , Biomarcadores Tumorais/genética , China/epidemiologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Regulação para Cima
9.
PLoS One ; 14(11): e0225161, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31747418

RESUMO

BACKGROUND: As the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors. METHODS: The bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs. FINDINGS: There were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18. CONCLUSIONS: Universal blood donor- type Standardbred MSCs from passages 2-4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.


Assuntos
Células da Medula Óssea/metabolismo , Antígenos de Histocompatibilidade/genética , Cavalos/genética , Células-Tronco Mesenquimais/metabolismo , Animais , Biomarcadores/sangue , Células Cultivadas , Antígenos de Histocompatibilidade/metabolismo , Cavalos/sangue , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Medicina Veterinária/métodos
10.
ACS Appl Mater Interfaces ; 11(49): 45427-45441, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31718136

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high mortality but limited therapeutic options. Epigenetic regulations including DNA methylation and histone modification control gene expressions and play a crucial role during tumorigenesis. G9a, also known as EHMT2 (euchromatic histone-lysine N-methyltransferase 2), is a histone methyltransferase predominantly responsible for dimethylation of histone H3 lysine 9 (H3K9). G9a has been shown to play a key role in promoting tumor progression. Recent studies have identified that G9a is a critical mediator of HCC pathogenesis. UNC0646 is a G9a inhibitor that has shown potent in vitro efficacy. However, due to its water insolubility, the in vivo efficacy of UNC0646 is not satisfactory. In this study, nanodiamonds (NDs) were utilized as a drug delivery platform to improve in vivo delivery of this small-molecule inhibitor. Our results showed that ND-UNC0646 complexes could be rapidly synthesized by physical adsorption, meanwhile possessing favorable drug delivery properties and was able to improve the dispersibility of UNC0646 in water, therefore making it amenable for intravenous administration. The release profile of UNC0646 from ND-UNC0646 was demonstrated to be pH-responsive. Moreover, ND-UNC0646 maintained the biological functionality of UNC0646, with higher efficacy in reducing H3K9 methylation as well as enhanced invasion suppressive effects. Most importantly, increased in vivo efficacy was demonstrated using an orthotopic HCC mouse model, which paves the way of translating this small-molecule inhibitor toward HCC treatment. Our work demonstrates the potential of NDs in the clinical application for HCC treatment.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Neoplasias Hepáticas/tratamento farmacológico , Nanodiamantes/química , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade/química , Código das Histonas/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/química , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Nanodiamantes/uso terapêutico , Quinazolinas/química , Quinazolinas/farmacologia
11.
Genetica ; 147(5-6): 337-350, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31782071

RESUMO

The major histocompatibility complex (MHC) of the adaptive immune system and the toll-like receptor (TLR) family of the innate immune system are involved in the detection of foreign invaders, and thus are subject to parasite-driven molecular evolution. Herein, we tested for macroevolutionary signatures of selection in these gene families within and among all three major clades of birds (Paleognathae, Galloanserae, and Neoaves). We characterized evolutionary relationships of representative immune genes (Mhc1 and Tlr2b) and a control gene (ubiquitin, Ubb), using a relatively large and phylogenetically diverse set of species with complete coding sequences (34 orthologous loci for Mhc1, 29 for Tlr2b, and 37 for Ubb). Episodic positive diversifying selection was found in the gene-wide phylogenies of the two immune genes, as well as at specific sites within each gene (8.5% of codon sites in Mhc1 and 2.7% in Tlr2b), but not in the control gene (Ubb). We found 20% of lineages under episodic diversifying selection in Mhc1 versus 9.1% in Tlr2b. For Mhc1, selection was relaxed in the Galloanserae and intensified in the Neoaves relative to the other clades, but no differences were detected among clades in the Tlr2b gene. In summary, we provide evidence of episodic positive diversifying selection in key immune genes and demonstrate differential strengths of selection within Class Aves, with the adaptive gene showing an increased divergence and evolutionary rate over the innate gene, contributing to the growing understanding of vertebrate immune gene evolution.


Assuntos
Proteínas Aviárias/genética , Aves/genética , Antígenos de Histocompatibilidade/genética , Seleção Genética , Receptores Toll-Like/genética , Animais , Aves/imunologia , Taxa de Mutação , Ubiquitina/genética
12.
BMC Cancer ; 19(1): 959, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619200

RESUMO

BACKGROUND: HER3 mediates drug resistance against epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we demonstrated that EGFR induces HER3 overexpression, which facilitates the formation of cancer stem-like tumorspheres. However, the cellular mechanism through which EGFR regulates HER3 expression remains unclear. We hypothesized that EGFR downstream of STAT3 participates in HER3 expression because STAT3 contributes to cancer stemness and survival of EGFR-TKI resistant cancers. METHODS: First, RNAseq was used to uncover potential genes involved in the formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines, including HCC827, A549, and H1975, were individually treated with a panel containing 172 therapeutic agents targeting stem cell-associated genes to search for potential agents that could be applied against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used to investigate molecular mechanisms through which STAT3 regulates tumor progression and the survival in lung cancer. RESULTS: BBI608, a STAT3 inhibitor, was a potential therapeutic agent that reduced the cell viability of EGFR-positive lung cancer cell lines. Notably, the inhibitory effects of BBI608 were similar with those associated with YM155, an ILF3 inhibitor. Both compounds reduced G9a-mediated HER3 expression. We also demonstrated that STAT3 upregulated G9a to silence miR-145-5p, which exacerbated HER3 expression in this study. CONCLUSIONS: The present study revealed that BBI608 could eradicate EGFR-positive lung cancers and demonstrated that STAT3 enhanced the expression of HER3 through miR-145-5p repression by G9a, indicating that STAT3 is a reliable therapeutic target against EGFR-TKI-resistant lung cancers.


Assuntos
Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-3/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Animais , Benzofuranos/farmacologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Naftoquinonas/farmacologia , Proteínas do Fator Nuclear 90/antagonistas & inibidores , Proteínas do Fator Nuclear 90/genética , Inibidores de Proteínas Quinases/efeitos adversos , Receptor ErbB-3/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Proc Natl Acad Sci U S A ; 116(44): 22252-22261, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31570608

RESUMO

The repertoire of αß T cell antigen receptors (TCRs) on mature T cells is selected in the thymus where it is rendered both self-tolerant and restricted to the recognition of major histocompatibility complex molecules presenting peptide antigens (pMHC). It remains unclear whether germline TCR sequences exhibit an inherent bias to interact with pMHC prior to selection. Here, we isolated TCR libraries from unselected thymocytes and upon reexpression of these random TCR repertoires in recipient T cell hybridomas, interrogated their reactivities to antigen-presenting cell lines. While these random TCR combinations could potentially have reacted with any surface molecule on the cell lines, the hybridomas were stimulated most frequently by pMHC ligands. The nature and CDR3 loop composition of the TCRß chain played a dominant role in determining pMHC-reactivity. Replacing the germline regions of mouse TCRß chains with those of other jawed vertebrates preserved reactivity to mouse pMHC. Finally, introducing the CD4 coreceptor into the hybridomas increased the proportion of cells that could respond to pMHC ligands. Thus, αß TCRs display an intrinsic and evolutionary conserved bias for pMHC molecules in the absence of any selective pressure, which is further strengthened in the presence of coreceptors.


Assuntos
Evolução Molecular , Antígenos de Histocompatibilidade/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Seleção Genética
14.
PLoS Comput Biol ; 15(9): e1007338, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31498801

RESUMO

T cells use their T-cell receptors (TCRs) to scan other cells for antigenic peptides presented by MHC molecules (pMHC). If a TCR encounters a pMHC, it can trigger a signalling pathway that could lead to the activation of the T cell and the initiation of an immune response. It is currently not clear how the binding of pMHC to the TCR initiates signalling within the T cell. One hypothesis is that conformational changes in the TCR lead to further downstream signalling. Here we investigate four different TCRs in their free state as well as in their pMHC bound state using large scale molecular simulations totalling 26 000 ns. We find that the dynamical features within TCRs differ significantly between unbound TCR and TCR/pMHC simulations. However, apart from expected results such as reduced solvent accessibility and flexibility of the interface residues, these features are not conserved among different TCR types. The presence of a pMHC alone is not sufficient to cause cross-TCR-conserved dynamical features within a TCR. Our results argue against models of TCR triggering involving conserved allosteric conformational changes.


Assuntos
Antígenos de Histocompatibilidade , Receptores de Antígenos de Linfócitos T , Biologia Computacional , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
15.
Cytogenet Genome Res ; 158(4): 205-212, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31434093

RESUMO

EHMT2 (euchromatic histone lysine methyltransferase 2), a histone methyltransferase, has been shown to be involved in multiple human cancers. In this study, we determined mRNA and protein expression of EHMT2 in cervical cancer cells and normal cervical epithelial cells. EHMT2 was inhibited with short hairpin RNA (shEHMT2) in cervical cancer cells. Cell viability, colony proliferation, apoptosis, adhesion, and invasion assays and Western blot were performed to assess the function of EHMT2. As a result, EHMT2 was upregulated in human cervical cancer cells compared to normal cervical epithelial cells. Suppression of EHMT2 expression impairs cell proliferation and induces apoptosis. Furthermore, EHMT2 silencing inhibited cell adhesion and invasion. Finally, knockdown of EHMT2 resulted in a reduction of the expression of the tumorigenic proteins Bcl-2, Mcl-1, and Survivin and in an increase in the expression of the anti-malignant protein E-cadherin. In conclusion, our data suggest that EHMT2 plays a key role in cell proliferation and metastatic capacity in cervical cancer cells and could serve as a potential therapeutic target.


Assuntos
Inativação Gênica , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/patologia , Apoptose/genética , Caderinas/biossíntese , Adesão Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Neoplasias do Colo do Útero/genética
16.
Cancer Sci ; 110(11): 3442-3452, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31432592

RESUMO

Functional E-cadherin loss, a hallmark of epithelial-mesenchymal transition (EMT), is important for metastasis. However, the mechanism of Snail2 in hepatocellular carcinoma (HCC) EMT and metastasis remains unclear. Here, we showed that Snail2 was upregulated in primary HCC, and significantly increased during transforming growth factor-ß-induced liver cell EMT. Snail2-overexpressing and knockdown cell lines have been established to determine its function in EMT in HCC. H3K9 methylation was upregulated and H3K4 and H3K56 acetylation were downregulated at the E-cadherin promoter in Snail2-overexpressing cancer cells. Furthermore, Snail2 interacted with G9a and histone deacetylases (HDACs) to form a complex to suppress E-cadherin transcription. Snail2 overexpression enhanced migration and invasion in HCC cells, whereas G9a and HDAC inhibition significantly reversed this effect. Moreover, Snail2 overexpression in cancer cells increased tumor metastasis and shortened survival time in mice, whereas G9a and HDAC inhibitors extended survival. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also suggests novel treatment strategies for HCC.


Assuntos
Caderinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Antígenos de Histocompatibilidade/metabolismo , Histona Desacetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Acetilação , Animais , Azepinas/uso terapêutico , Carcinoma Hepatocelular/secundário , Movimento Celular , Progressão da Doença , Regulação para Baixo , Feminino , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Quinazolinas/uso terapêutico , Transcrição Genética , Fator de Crescimento Transformador beta/farmacologia
17.
Immunogenetics ; 71(8-9): 545-559, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31384962

RESUMO

Butyrophilins (BTN), specifically BTN3A, play a central role in the modulation of γδ T cells, which are mainly present in gut and mucosal tissues. BTN3A1 is known, for example, to activate Vγ9Vδ2 T cells by means of a phosphoantigen interaction. In the extended HLA region, three genes are located, designated BTN3A1, BTN3A2 and BTN3A3, which were also defined in rhesus macaques. In contrast to humans, rhesus monkeys have an additional gene, BTN3A3Like, which has the features of a pseudogene. cDNA analysis of 32 Indian rhesus and 16 cynomolgus macaques originating from multiple-generation families revealed that all three genes are oligomorphic, and the deduced amino acids display limited variation. The macaque BTN3A alleles segregated together with MHC alleles, proving their location in the extended (Major Histocompatibility Complex) MHC. BTN3A nearly full-length transcripts of macaques and humans cluster tightly together in the phylogenetic tree, suggesting that the genes represent true orthologs of each other. Despite the limited level of polymorphism, 15 Mamu- and 14 Mafa-BTN3A haplotypes were defined, and, as in humans, all three BTN3A genes are transcribed in PBMCs and colon tissues. In addition to regular full-length transcripts, a high number of various alternative splicing (AS) products were observed for all BTN3A alleles, which may result in different isoforms. The comparable function of certain subsets of γδ T cells in human and non-human primates in concert with high levels of sequence conservation observed for the BTN3A transcripts presents the opportunity to study these not yet well understood molecules in macaques as a model species.


Assuntos
Antígenos CD/genética , Butirofilinas/genética , Antígenos de Histocompatibilidade/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Sequência de Aminoácidos , Animais , Butirofilinas/metabolismo , Sequência Conservada , Feminino , Haplótipos , Humanos , Macaca mulatta , Masculino , Filogenia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Homologia de Sequência , Linfócitos T/metabolismo
18.
Cancer Sci ; 110(10): 3027-3037, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31348591

RESUMO

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS-ML. Human iPS-ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS-ML cells expressing interferon ß (iPS-ML/interferon (IFN)-ß) in xenograft cancer models. However, assessment of host immune system-mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS-ML/IFN, a mouse embryonic stem (ES) cell-derived myeloid cell line producing IFN (ES-ML/IFN). The ES-MLs producing IFN-ß (ß-ML) and IFN-γ (γ-ML) and originating from E14 ES cells derived from the 129 mouse strain (H-2b ) were generated, and the MHC (H-2Kb , Db , and I-Ab ) genes of the ES-ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES-ML/IFN to treat allogeneic BALB/c mice (H-2d ) transplanted with Colon26 cancer cells. Treatment with ß-ML but not with γ-ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES-ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES-ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen-specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC-deficient ß-ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA-deficient human iPS-ML/IFN-ß cells for therapy of HLA-mismatched allogeneic cancer patients.


Assuntos
Neoplasias do Colo/terapia , Células-Tronco Embrionárias/citologia , Antígenos de Histocompatibilidade/genética , Interferon beta/metabolismo , Células Mieloides/transplante , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Células Mieloides/metabolismo , Transplante Homólogo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Eur J Med Chem ; 179: 537-546, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276898

RESUMO

Epigenetics is the study of heritable changes in gene expression without changing the DNA sequence - a change in phenotype without a change in genotype. Epigenetic abnormalities can lead to serious diseases such as cancer in organisms. Histone methylation is one of the several manifestations of epigenetics, and requires specific enzymes to catalyze, for example, G9a, which is a histone methyl transferase. G9a catalyzes the methylation of histone 3 lysine 9 (H3K9) and histone 3 lysine 27 (H3K27). In addition, G9a also plays an essential role in DNA replication, damage and repair, and gene expression by regulating DNA methylation. Moreover, G9a has been found to be overexpressed in many tumor cells and is associated with the occurrence and development of tumors. Because of its unique characteristics, G9a has become a very promising target for anti-cancer agents. Over the last decade, dozens of G9a inhibitors have been discovered as potential anticancer therapeutic agents. In this review, we summarize and classify current G9a inhibitors, the challenges and future direction are also discussed in detail.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Antineoplásicos/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular
20.
Nat Med ; 25(7): 1073-1081, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270502

RESUMO

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.


Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Feminino , Antígenos de Histocompatibilidade , Humanos , Camundongos , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia
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