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1.
Chem Commun (Camb) ; 55(88): 13311-13314, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31631199

RESUMO

Herein, we report a strategy for generating conformationally restricted α-helix mimetic small molecules by introducing covalent bridges that limit rotation about the central axis of α-helix mimetics. We demonstrate that the bridged α-helix mimetics have enhanced binding affinity and specificity to the target protein due to the restricted conformation as well as extra interaction of the bridge with the protein surface.


Assuntos
Compostos Heterocíclicos de Anel em Ponte/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Bibliotecas de Moléculas Pequenas/química , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Humanos , Células Jurkat , Modelos Moleculares , Conformação Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia
2.
Yonsei Med J ; 60(10): 924-934, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31538427

RESUMO

PURPOSE: Acute leukemia (AL) is classified as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This study aimed to investigate the effect of miR-146a on childhood AL and its underlying molecular mechanisms. MATERIALS AND METHODS: Bone marrow samples were obtained from 39 AL children and 10 non-cancer controls. The expressions of miR-146a and ciliary neurotrophic factor receptor (CNTFR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in ALL and AML pediatric patients, as well as ALL (Jurkat) and AML (HL-60) cells. Correlations between miR-146a and clinical indicators were explored. A targeting relationship between miR-146a and CNTFR was detected by dual luciferase reporter gene assay. Cell proliferation, apoptosis, migration, and invasion of Jurkat and HL-60 cells were measured by MTT assay, flow cytometry, and transwell assay, respectively. LIF expression was detected by qRT-PCR in Jurkat and HL-60 cells. The expression of p-JAK2, JAK2, p-STAT3, and STAT3 in HL-60 cells was measured by Western blot. RESULTS: miR-146a was increased in ALL and AML pediatric patients, while CNTFR was decreased. miR-146a expression was associated with immunophenotype, karyotype, fusion gene, and SIL-TAL1. CNTFR was a target gene of miR-146a. miR-146a could promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis in Jurkat and HL-60 cells by downregulating CNTFR. Meanwhile, miR-146a inhibited the expression of LIF and activated JAK2/STAT3 pathway by downregulating CNTFR. CONCLUSION: miR-146a could promote the proliferation, migration, and invasion and inhibit the apoptosis of AL Jurkat and HL-60 cells by downregulating CNTFR and activating the JAK2/STAT3 pathway.


Assuntos
Regulação para Baixo/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , MicroRNAs/metabolismo , Receptor do Fator Neutrófico Ciliar/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Apoptose/genética , Sequência de Bases , Movimento Celular/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Regulação Leucêmica da Expressão Gênica , Células HL-60 , Humanos , Lactente , Células Jurkat , Fator Inibidor de Leucemia/metabolismo , MicroRNAs/genética , Invasividade Neoplásica , Receptor do Fator Neutrófico Ciliar/metabolismo , Resultado do Tratamento , Regulação para Cima/genética
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1111-1117, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418365

RESUMO

OBJECTIVE: To investigate the apoptosis-inducing effect of Ginsenoside (Rh2) on human acute T lymphoblastic leukemia Jurkat cells and it mechamism. METHODS: The effects of different concentration of Rh2 (0, 10 , 20, 40 and 80 µg/ml) on the proliferation activity of Jurkat cells were detected by methyl thiazolyl tetrazolium (MTT) method, and the semi-inhibitory concentration (IC50) of Rh2 on Jurkat cells at 48 h was calculated. Microscopy and Hoechst 33258 fluorescence staining were used to observe the apoptosis of Jurkat cells treated with IC50 Rh2 for 48 h. And then, the cell experiment was divided into 4 groups: control, Rh2 (IC50), PI3K inhibitor LY294002 (50 µmol/l) and Rh2 (IC50) + LY294002 (50 µmol/l). After synchronous culture for 48 h, the apoptosis and cycle changes of Jurkat cells were detected by using PI single staining and Annexin V-FITC/PI double staining, respectively. Western blot was used to detect the expression level of apoptosis-related protein BAX, BCL-2, Cleaved-Caspasase 3, cell cycle-related protein Cyclin D1 and PI3K/AKT signaling pathway-related protein AKT and p-AKT. RESULTS: Rh2 (10-80 µg/ml) inhibited the Jurkat cell proliferation in a dose-time dependent manner (r48h = 0.999, P<0.01; r80 µg/ml = 0.991; P>0.05), accompanied by obvious morphological changes of apoptosis cells. Flow cytometry showed that compared with the control group, the cell apoptosis rate in Rh2 or LY294002 group significantly increased, and the cell cycle was mostly blocked in G0/G1 phase. However, the cell apoptosis and cell cycle block in Rh2+LY294002 group were more significant than that in Rh2 and LY294002 group. Western blot showed that compared with the control group, Rh2 significantly promoted the expression of BAX and Cleaved-Caspasase 3, inhibited the expression of BCL-2, Cyclin D1 and p-AKT, furthermore LY294002 significantly promoted this effect. CONCLUSION: Rh2 can induce the apoptosis of Jurkat cells in time-dose dependent manner, moreover, Rh2 also can result in an obvious block of Jurkat cells at G0/G1, that may be closely related to a series of apoptotic signaling cascades mediated by Rh2 inhibiting PI3K/AKT pathway.


Assuntos
Ginsenosídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptose , Proliferação de Células , Humanos , Células Jurkat , Fosfatidilinositol 3-Quinases
4.
Nat Immunol ; 20(9): 1129-1137, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31358998

RESUMO

Natural killer (NK) cells can recognize virus-infected and stressed cells1 using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity2,3, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations4. Using NKp44ζ+ reporter cells and primary human NKp44+ NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection5-7, graft-versus-host disease8 and inflammatory bowel disease9,10.


Assuntos
Antígenos HLA-DP/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Linhagem Celular , Doença Enxerto-Hospedeiro/imunologia , Hepatite B/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Células Jurkat
5.
Chem Biol Interact ; 310: 108726, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31255635

RESUMO

Tetrandrine (TET) and cepharanthine (CEP) are two bisbenzylisoquinoline alkaloids isolated from the traditional herbs. Recent molecular investigations firmly supported that TET or CEP would be a potential candidate for cancer chemotherapy. Prognosis of patients with glucocorticoid resistant T cell acute lymphoblastic leukemia (T-ALL) remains poor; here we examined the anti-T-ALL effects of TET and CEP and the underlying mechanism by using the glucocorticoid resistant human leukemia Jurkat T cell line in vitro. TET and CEP significantly inhibited cell viabilities and induced apoptosis in dose- and time-dependent manner. Further investigations showed that TET or CEP not only upregulated the expression of initiator caspases such as caspase-8 and 9, but also increased the expression of effector caspases such as caspase-3 and 6. As the important markers of apoptosis, p53 and Bax were both upregulated by the treatment of TET and CEP. However, TET and CEP paradoxically increased the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, and activated the survival protein NF-κB, leading to high expression of p-NF-κB. Cell cycle arrest at S phase accompanied by increase in the amounts of cyclin A2 and cyclin B1, and decrease in cylcin D1 amount in cells treated with TET or CEP will be another possible mechanism. During the process of apoptosis in Jurkat T cells, treatment with TET or CEP also increased the phosphorylation of JNK and p38. The PI3K/Akt/mTOR signaling pathway modification appears to play significant role in the Jurkat T cell apoptosis induced by TET or CEP. Moreover, TET and CEP seemed to downregulate the expressions of p-PI3K and mTOR in an independent way from Akt, since these two drugs strongly stimulated the p-Akt expression. These results provide fundamental insights into the clinical application of TET or CEP for the treatment of patients with relapsed T-ALL.


Assuntos
Apoptose/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Benzilisoquinolinas/uso terapêutico , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Células Jurkat , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
6.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 777-784, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204931

RESUMO

OBJECTIVE: To investigate the effect of ß-arrestin1 gene on senescence of T-ALL cells and its possible mechanism. METHODS: The bone marrow specimens of T-ALL patients and controls were collected, the expression of ß-arrestin1 and ß-arrestin1 in the T-ALL patients was detected by RT-PCR and Western blot, respectively, and the relation of ß-arrestin1 expression with the clinical pathologic characteristics and the prognosis of T-ALL patients was analyzed statistically. The stable Jurkat cell line with knocked down or overexpressed ß-arrestin1 was constructed, the CCK method was used to detect the Jurkat cell number, the ß-gal staining was used to analyze the effect of ß-arrestin1 on senescence of Jurkat cells, the cross analysis of RNA-Seg data and KEGG data was performed for screening the possible signaling pathway, and Western blot was performed for varifying the key sites of signaling pathway. RESULTS: The ß-arrestin1 expression in specimens of T-ALL patients decreased (P<0.01), moreover the ß-arrestin1 expression negatively related with peripheral blood cell number (r=-0.601), the blasts in peripheral blood (r=-0.516) and extramedullary infiltration (r=-0.359), while positively related with the response to chemotherapy (r=0.393). The detection of stable Jurkat cell line with knocked-down and overexpressed ß-arrestin1 found that the ß-arrestin 1 could decrease the Jurkat cell number and accelarate the senescence of Jurkat cells (P<0.05). The cross analysis of RNA-Seg data and KEGG data showed that the senescence of T-ALL cells may be regulated via RAS-P16-PRb-E2F1 by ß-arrestin 1. Western bolt confirmed that ß-arrestin1 promoted the expression of Ras and p16, and decreased the expression of pRB and E2F1 (P<0.05). CONCLUSIONS: ß-arrestin1 accelerates the senescence of Jurkat cells via Ras-p16-pRb-E2F1, and delays the progression in T-ALL, which may provide a new hypothesis for the pathogenesis of T-ALL.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , beta-Arrestina 1/genética , Senescência Celular , Humanos , Células Jurkat , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico
7.
J Immunoassay Immunochem ; 40(5): 459-472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31204615

RESUMO

Several plants of Satureja genus have shown anti-tumor activity. We investigated the antileukemia effects of different fractions of Satureja hortensis (Summer savory). The growth inhibitory effect of S. hortensis fractions on K562 and Jurkat leukemia cells were determined by MTT assay. The most effective fractions were analyzed by flow cytometry and colorimetric assay for apoptosis induction and cell cycle changes. Various fractions from S. hortensis showed growth inhibitory effects on leukemia cells, among them two hexane and dichloromethane fractions with IC50 values of 32.1-47.8 µg/ml (K562) and 44.3-45.7 µg/ml (Jurkat) were the most effective. According to annexin V staining, both of these fractions significantly induced apoptosis at 50µg/ml in K562 (hexane; 73.06 ± 5.11% and dichloromethane; 96.14 ± 2.33%) and Jurkat cells (hexane; 78.85 ± 11.9% and dichloromethane; 94.05 ± 2.47%) 48 h after treatment. They increased cell accumulation in sub-G1 phase (>50%, p < .001) and decreased number of cells in G0-G1, S and G2M phases. The fractions significantly increased the caspase-3 activity in both cell lines (≈2.5-3.5 fold of untreated cells). Hexane and dichloromethane fractions of S. hortensis had the capacity to induce death and change the cell cycle distribution in leukemia cells; therefore they might be good candidates for more studies in regard to their possible therapeutic usefulness in leukemia.


Assuntos
Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Hexanos/farmacologia , Leucemia de Células T/patologia , Cloreto de Metileno/farmacologia , Extratos Vegetais/farmacologia , Satureja/química , Proliferação de Células/efeitos dos fármacos , Colorimetria , Relação Dose-Resposta a Droga , Hexanos/química , Hexanos/isolamento & purificação , Humanos , Células Jurkat , Células K562 , Leucemia de Células T/tratamento farmacológico , Cloreto de Metileno/química , Cloreto de Metileno/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação
8.
Food Chem Toxicol ; 131: 110537, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150782

RESUMO

Programmed death ligand-1 (PD-L1) is an important immune checkpoint for cancer immunotherapy in clinic. In this study, we reported that platycodin D, a natural product isolated from an edible and medicinal plant Platycodon grandiflorus (Jacq.) A. DC., down-regulated the protein level of PD-L1 in lung cancer cells. Flow cytometry and immunofluorescence assay showed a weaker surface PD-L1 signal in NCI-H1975 cells after the incubation with platycodin D (10 µM) for 15 min compared to the control group. Jurkat T cells showed enhancive interleukin-2 secretion when co-cultured with platycodin D-treated NCI-H1975 cells, suggesting that platycodin D-induced PD-L1 reduction increases the activation of Jurkat T cells. An augmentation of PD-L1 protein was detected in the cell culture medium from platycodin D treatment group. Chlorpromazine (60 µM) almost abolished the platycodin D-mediated PD-L1 extracellular release and restored the membrane PD-L1. Finally, hemolysis assay exhibited that platycodin D-triggered PD-L1 extracellular release was independent of the hemolytic mechanism. Taken together, our study demonstrates that platycodin D reduces the protein level of PD-L1 in lung cancer cells via triggering its release into the cell culture medium, which sheds new light for the application of natural products in cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Humanos , Interleucina-2/metabolismo , Células Jurkat , Transporte Proteico/efeitos dos fármacos
9.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180045

RESUMO

Recent research has shown that cell-free chromatin (cfCh) particles that are released from the billions of cells that die in the body everyday can enter into healthy cells, integrate into their genomes and induce dsDNA breaks and apoptotic responses. Genomic integration of cfCh activates NF κ B suggesting a novel mechanism of induction of systemic inflammation. Since DNA damage and inflammation are underlying pathologies in multiple devastating acute and chronic disease conditions, the discovery of agents that can inactivate cfCh may provide therapeutic possibilities.


Assuntos
Anti-Inflamatórios/farmacologia , Cromatina/patologia , Histonas/genética , NF-kappa B/genética , Animais , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Transporte Biológico , Cromatina/química , Cromatina/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Desoxirribonuclease I/farmacologia , Espaço Extracelular/química , Regulação da Expressão Gênica , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Inflamação/prevenção & controle , Células Jurkat/transplante , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Transdução de Sinais
10.
Eur J Pharm Biopharm ; 142: 49-60, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31201855

RESUMO

Antibody drug conjugates (ADCs), which are obtained by coupling a potent cytotoxic agent to a monoclonal antibody (mAb), are traditionally bound in a random way to lysine or cysteine residues, with the final product's heterogeneity having an important impact on their activity, characterization, and manufacturing. A new antibody drug delivery system (ADS) based on a non-covalent linkage between a Fc-binding protein, in this case Protein A or Protein G, and a mAb was investigated in the effort to achieve greater homogeneity and to create a versatile and adaptable drug delivery system. Recombinant staphylococcal Protein A and streptococcal Protein G were chemically PEGylated at the N-terminus with a 5 kDa and a 20 kDa PEG, respectively, yielding two monoconjugates with a mass of ≈50 and ≈45 kDa. Circular dichroism studies showed that both conjugates preserved secondary structures of the protein, and isothermal titration calorimetry experiments demonstrated that their affinity for mAb was approximately 107 M-1. Upon complexation with a mAb (Trastuzumab or Rituximab), in vitro flow-cytometry analysis of the new ADSs showed high selectivity for the specific antigen expressing cells. In addition, the ADS complex based on Trastuzumab and Protein G, conjugated with a heterobifunctional 20 kDa PEG carrying the toxin Tubulysin A, had a marked cytotoxic effect on the cancer cell line overexpressing the HER2/neu receptor, thus supporting its application in cancer therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Humanos , Células Jurkat , Receptor ErbB-2/metabolismo , Rituximab/farmacologia , Trastuzumab/farmacologia
11.
Immunology ; 157(4): 296-303, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31162836

RESUMO

The characterization of the architecture, structure and extracellular interactions of the CD6 glycoprotein, a transmembrane receptor expressed in medullary thymocytes and all mature T-cell populations, has been enhanced by the existence of monoclonal antibodies (mAbs) that specifically recognize the various scavenger receptor cysteine-rich (SRCR) domains of the ectodomain. Using engineered isoforms of CD6 including or excluding each of the three SRCR domains, either expressed at the membranes of cells or in soluble forms, we provide conclusive and definitive evidence that domain 2 of CD6, previously not identifiable, can be recognized by the CD6 mAbs OX125 and OX126, and that OX124 targets domain 3 and can block the interaction at the cell surface of CD6 with its major ligand CD166. Alternative splicing-dependent CD6 isoforms can now be confidently identified. We confirm that following T-cell activation there is a partial replacement of full-length CD6 by the CD6Δd3 isoform, which lacks the CD166-binding domain, and we find no evidence for the expression of other CD6 isoforms at the mRNA or protein levels.


Assuntos
Processamento Alternativo/imunologia , Anticorpos Monoclonais Murinos/química , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Anticorpos Monoclonais Murinos/imunologia , Humanos , Células Jurkat , Domínios Proteicos , Isoformas de Proteínas/imunologia , Linfócitos T/citologia
12.
Nat Commun ; 10(1): 2233, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110232

RESUMO

The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Modelos Genéticos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Células K562 , RNA Guia/genética
13.
Nat Commun ; 10(1): 2209, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101809

RESUMO

Changes in bulk transcriptional profiles of heterogeneous samples often reflect changes in proportions of individual cell types. Several robust techniques have been developed to dissect the composition of such mixed samples given transcriptional signatures of the pure components or their proportions. These approaches are insufficient, however, in situations when no information about individual mixture components is available. This problem is known as the  complete deconvolution problem, where the composition is revealed without any a priori knowledge about cell types and their proportions. Here, we identify a previously unrecognized property of tissue-specific genes - their mutual linearity - and use it to reveal the structure of the topological space of mixed transcriptional profiles and provide a noise-robust approach to the complete deconvolution problem. Furthermore, our analysis reveals systematic bias of all deconvolution techniques due to differences in cell size or RNA-content, and we demonstrate how to address this bias at the experimental design level.


Assuntos
Biologia Computacional/métodos , Modelos Genéticos , Transcriptoma , Algoritmos , Animais , Tamanho Celular , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Células Jurkat , Modelos Lineares , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de RNA/métodos
14.
PLoS Pathog ; 15(5): e1007802, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116788

RESUMO

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Provírus/imunologia , Latência Viral/imunologia , Humanos , Células Jurkat , Transdução de Sinais , Ativação Viral/imunologia , Replicação Viral/imunologia
15.
Immunogenetics ; 71(7): 465-478, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31123763

RESUMO

Invariant NKT (iNKT) cells in both humans and non-human primates are activated by the glycolipid antigen, α-galactosylceramide (α-GalCer). However, the extent to which the molecular mechanisms of antigen recognition and in vivo phenotypes of iNKT cells are conserved among primate species has not been determined. Using an evolutionary genetic approach, we found a lack of diversifying selection in CD1 genes over 45 million years of evolution, which stands in stark contrast to the history of the MHC system for presenting peptide antigens to T cells. The invariant T cell receptor (TCR)-α chain was strictly conserved across all seven primate clades. Invariant NKT cells from rhesus macaques (Macaca mulatta) bind human CD1D-α-GalCer tetramer and are activated by α-GalCer-loaded human CD1D transfectants. The dominant TCR-ß chain cloned from a rhesus-derived iNKT cell line is nearly identical to that found in the human iNKT TCR, and transduction of the rhesus iNKT TCR into human Jurkat cells show that it is sufficient for binding human CD1D-α-GalCer tetramer. Finally, we used a 20-color flow cytometry panel to probe tissue phenotypes of iNKT cells in a cohort of rhesus macaques. We discovered several tissue-resident iNKT populations that have not been previously described in non-human primates but are known in humans, such as TCR-γδ iNKTs. These data reveal a diversity of iNKT cell phenotypes despite convergent evolution of the genes required for lipid antigen presentation and recognition in humans and non-human primates.


Assuntos
Antígenos CD1/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Primatas/genética , Sequência de Aminoácidos , Animais , Antígenos CD1/metabolismo , Sequência Conservada , Evolução Molecular , Feminino , Humanos , Células Jurkat , Macaca mulatta/imunologia , Masculino , Fenótipo , Primatas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
16.
PLoS Pathog ; 15(5): e1007669, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31042779

RESUMO

HIV-1 is dependent on the host cell for providing the metabolic resources for completion of its viral replication cycle. Thus, HIV-1 replicates efficiently only in activated CD4+ T cells. Barriers preventing HIV-1 replication in resting CD4+ T cells include a block that limits reverse transcription and also the lack of activity of several inducible transcription factors, such as NF-κB and NFAT. Because FOXO1 is a master regulator of T cell functions, we studied the effect of its inhibition on T cell/HIV-1 interactions. By using AS1842856, a FOXO1 pharmacologic inhibitor, we observe that FOXO1 inhibition induces a metabolic activation of T cells with a G0/G1 transition in the absence of any stimulatory signal. One parallel outcome of this change is the inhibition of the activity of the HIV restriction factor SAMHD1 and the activation of the NFAT pathway. FOXO1 inhibition by AS1842856 makes resting T cells permissive to HIV-1 infection. In addition, we found that FOXO1 inhibition by either AS1842856 treatment or upon FOXO1 knockdown induces the reactivation of HIV-1 latent proviruses in T cells. We conclude that FOXO1 has a central role in the HIV-1/T cell interaction and that inhibiting FOXO1 with drugs such as AS1842856 may be a new therapeutic shock-and-kill strategy to eliminate the HIV-1 reservoir in human T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteína Forkhead Box O1/antagonistas & inibidores , Regulação da Expressão Gênica , Infecções por HIV/virologia , HIV-1/imunologia , Ativação Viral/imunologia , Replicação Viral , Animais , Linfócitos T CD4-Positivos/virologia , Ciclo Celular , Proteína Forkhead Box O1/genética , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Macaca fascicularis , Masculino , Latência Viral
17.
Georgian Med News ; (288): 158-162, 2019 Mar.
Artigo em Russo | MEDLINE | ID: mdl-31101797

RESUMO

The goal of our study was to establish the anti- proapoptotic activity of the common in Georgia crops on the Jurkat and MDCK cells. Extracts of various varieties of beans (Tirkmela, Batumi meadow, Shulavera, Udelebi, as well as green peas, Lens Culinaris lentils, soy beans) were added to the intact or incubated under oxidative stress conditions Jurkat and MDCK cells. Cell viability (apoptosis intensity) was determined by a cell proliferative activity test (MTT test). Correlation and statistical analysis of ANOVA was performed using the package (SPSS version 11.0). In the presented study the selective effectiveness of extracts with different antioxidant activity on intact and incubated under oxidative stress Jurkat and MDCK cells was revealed, related with different sensitivity of cells to the oxidative stress. In normal MDCK epithelial cells, resistant to redox-active factors (H2O2), inverse relationship between the intensity of apoptosis and the antiradical potential of the extract was found; in leukemia transformated Jurkat cells, characterized by high sensitivity to oxidants (H2O2), a violation of the redox-dependent anti-apoptotic cell protection mechanisms was revealed, which is manifested by the absence of regularity of the cytoprotective / cytotoxic effects of the extracts on intact and incubated cells under oxidative stress conditions. These results can be used in the development of schemes of anti-tumor and anti-inflammatory therapy.


Assuntos
Fabaceae , Extratos Vegetais , Animais , Apoptose , Cães , Fabaceae/química , Humanos , Peróxido de Hidrogênio , Células Jurkat , Células Madin Darby de Rim Canino/efeitos dos fármacos , Estresse Oxidativo , Extratos Vegetais/farmacologia
18.
Food Chem Toxicol ; 130: 122-129, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31100301

RESUMO

Simultaneous mycotoxins toxicity is complex and non-predictable based on their individual toxicities. Beauvericin and Enniatins are emerging mycotoxins highly co-occurrent in food and feed, and their cytotoxicity has been reported in several human cell lines. RNA-seq studies of individual exposure in Jurkat cells demonstrated human genome perturbation mainly affecting mitochondrial pathways, however, both mycotoxins showed differences between their toxic responses. This study investigates the transcriptional effects of combined exposure to Beauvericin and Enniatin B (1:1) (0.1, 0.5, 1.5 µM; 24 h) in Jurkat cells by qPCR on 30 selected target genes (10 mitochondrial, 20 nuclear). Gene expression after combined and individual exposures were compared and functional data analysis (ToxPi) on the most relevant biological processes (cycle and apoptosis regulation; cholesterol metabolism and transport; cellular signaling transduction; cellular stress responses; immune regulation; protein metabolism; retinoic acid metabolism; transcription regulation) was applied to RNA-seq data from individual exposure (1.5, 3, 5 µM; 24 h; Jurkat cells). Transcriptional changes, especially at mitochondrial level, were observed after Beauvericin-Enniatin B co-exposure including down-regulation of antioxidant activity related genes. Different expression patterns between combined and individual exposures were identified. ToxPi analysis confirmed different dose-dependent relationship profiles between these two mycotoxins after individual exposure.


Assuntos
Depsipeptídeos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Depsipeptídeos/administração & dosagem , Quimioterapia Combinada , Humanos , Células Jurkat , Transcriptoma/efeitos dos fármacos
19.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040235

RESUMO

We previously showed that Entamoeba histolytica kills human cells through a mechanism that we termed trogocytosis ("trogo-" means "nibble"), due to its resemblance to trogocytosis in other organisms. In microbial eukaryotes like E. histolytica, trogocytosis is used to kill host cells. In multicellular eukaryotes, trogocytosis is used for cell killing and cell-cell communication in a variety of contexts. Thus, nibbling is an emerging theme in cell-cell interactions both within and between species. When trogocytosis occurs between mammalian immune cells, cell membrane proteins from the nibbled cell are acquired and displayed by the recipient cell. In this study, we tested the hypothesis that through trogocytosis, amoebae acquire and display human cell membrane proteins. We demonstrate that E. histolytica acquires and displays human cell membrane proteins through trogocytosis and that this leads to protection from lysis by human serum. Protection from human serum occurs only after amoebae have undergone trogocytosis of live cells but not phagocytosis of dead cells. Likewise, mutant amoebae defective in phagocytosis, but unaltered in their capacity to perform trogocytosis, are protected from human serum. Our studies are the first to reveal that amoebae can display human cell membrane proteins and suggest that the acquisition and display of membrane proteins is a general feature of trogocytosis. These studies have major implications for interactions between E. histolytica and the immune system and also reveal a novel strategy for immune evasion by a pathogen. Since other microbial eukaryotes use trogocytosis for cell killing, our findings may apply to the pathogenesis of other infections.IMPORTANCE Entamoeba histolytica causes amoebiasis, a potentially fatal diarrheal disease. Abscesses in organs such as the liver can occur when amoebae are able to breach the intestinal wall and travel through the bloodstream to other areas of the body. Therefore, understanding how E. histolytica evades immune detection is of great interest. Here, we demonstrate for the first time that E. histolytica acquires and displays human cell membrane proteins by taking "bites" of human cell material in a process named trogocytosis ("trogo-" means "nibble"), and that this allows amoebae to survive in human serum. Display of acquired proteins through trogocytosis has been previously characterized only in mammalian immune cells. Our study suggests that this is a more general feature of trogocytosis not restricted to immune cells and broadens our knowledge of eukaryotic biology. These findings also reveal a novel strategy for immune evasion by a pathogen and may apply to the pathogenesis of other infections.


Assuntos
Entamoeba histolytica/imunologia , Entamoeba histolytica/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Proteínas de Membrana/metabolismo , Fagocitose , Humanos , Células Jurkat
20.
Infect Immun ; 87(8)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31109948

RESUMO

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitans A. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent.


Assuntos
Exotoxinas/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Linfócitos T/fisiologia , Receptor fas/fisiologia , Antígeno CD11a/fisiologia , Antígenos CD18/fisiologia , Caspases/fisiologia , Morte Celular , Humanos , Células Jurkat , Fatores de Virulência/fisiologia
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