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1.
Am J Transplant ; 2022 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-35434896

RESUMO

Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99 days, whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors, A2-CAR Tregs slightly increased median graft survival from 11 to 14 days, which was further extended to >100 days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients.

2.
Signal Transduct Target Ther ; 6(1): 375, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34728602

RESUMO

The scope and variety of the metabolic intermediates from the mitochondrial tricarboxylic acid (TCA) cycle that are engaged in epigenetic regulation of the chromatin function in the nucleus raise an outstanding question about how timely and precise supply/consumption of these metabolites is achieved in the nucleus. We report here the identification of a nonclassical TCA cycle in the nucleus (nTCA cycle). We found that all the TCA cycle-associated enzymes including citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 3 (IDH3), oxoglutarate dehydrogenase (OGDH), succinyl-CoA synthetase (SCS), fumarate hydratase (FH), and malate dehydrogenase 2 (MDH2), except for succinate dehydrogenase (SDH), a component of electron transport chain for generating ATP, exist in the nucleus. We showed that these nuclear enzymes catalyze an incomplete TCA cycle similar to that found in cyanobacteria. We propose that the nTCA cycle is implemented mainly to generate/consume metabolic intermediates, not for energy production. We demonstrated that the nTCA cycle is intrinsically linked to chromatin dynamics and transcription regulation. Together, our study uncovers the existence of a nonclassical TCA cycle in the nucleus that links the metabolic pathway to epigenetic regulation.


Assuntos
Núcleo Celular/genética , Cromatina/genética , Ciclo do Ácido Cítrico/genética , Epigênese Genética/genética , Aconitato Hidratase/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Citrato (si)-Sintase/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Metabolismo Energético/genética , Fumarato Hidratase/genética , Humanos , Isocitrato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/genética , Malato Desidrogenase/genética , Transcrição Genética , Ácidos Tricarboxílicos/metabolismo
3.
Front Vet Sci ; 8: 740472, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34746280

RESUMO

Wild aquatic birds are the primary natural reservoir of influenza A viruses (IAVs), although a small number of viruses can spill over to mammals and circulate. The focus of IAV infection in mammals was largely limited to humans and swine variants, until the emergence of H3N2 canine influenza viruses (CIVs), which provides new perspective for interspecies transmission of the virus. In this study, we captured 54 canine-adaptive signatures in H3N2 CIVs through entropy computation, which were largely concentrated in the interaction region of polymerase proteins on ribonucleoprotein complex. The receiver operating characteristic curves of these sites showed >95% accuracy in distinguishing between the hosts. Nine of the 54 canine-adaptive signatures were shared in avian-human/equine or equine-canine (PB2-82; PB1-361; PA-277; HA-81, 111, 172, 196, 222, 489), suggesting their involvement in canine adaptation. Furthermore, we found that IAVs can establish persistent transmission in lower mammals with greater ease compared to higher mammals, and 25 common adaptation signatures of H3 IAVs were observed in diverse avian-mammals comparison. There were few human-like residues in H3N2 CIVs, which suggested a low risk of human infection. Our study highlights the necessity of identifying and monitoring the emerging adaptive mutations in companion animals by enhanced surveillance and provides a basis for mammal adaptation of avian influenza viruses.

4.
J Exp Med ; 218(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34115115

RESUMO

Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/genética , Mutação/genética , Fator de Transcrição STAT3/genética , Animais , Autoimunidade , Proliferação de Células , Quimiotaxia/genética , Apresentação Cruzada/imunologia , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Epigênese Genética , Mutação com Ganho de Função , Heterozigoto , Humanos , Camundongos , Fenótipo , Regulação para Cima
5.
Mol Cell ; 81(14): 2960-2974.e7, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111398

RESUMO

The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that fails to rescue Uhrf1-/- phenotype, is physically and functionally associated with BER complex. We show that UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates XRCC1's interaction with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that Uhrf2-fostered TDG-RAD23B-BER complex is functionally linked to the completion of DNA demethylation at active promoters and that Uhrf2 ablation impedes DNA demethylation on latent enhancers that undergo poised-to-active transition during neuronal commitment. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.


Assuntos
5-Metilcitosina/análogos & derivados , Regulação Alostérica/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Desmetilação do DNA , Metilação de DNA/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética
6.
Front Immunol ; 12: 783282, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35003100

RESUMO

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.


Assuntos
Doença Enxerto-Hospedeiro/terapia , Imunoterapia Adotiva/métodos , Interleucina-6/metabolismo , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Etanercepte/farmacologia , Feminino , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Voluntários Saudáveis , Humanos , Fator de Transcrição Ikaros/análise , Fator de Transcrição Ikaros/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Cultura Primária de Células , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante , Transplante Heterólogo/efeitos adversos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto Jovem
7.
Entropy (Basel) ; 22(6)2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-33286457

RESUMO

The equipment condition monitoring based on computer hearing is a new pattern recognition approach, and the system formed by it has the advantages of noncontact and strong early warning abilities. Extracting effective features from the sound data of the running power equipment help to improve the equipment monitoring accuracy. However, the sound of running equipment often has the characteristics of serious noise, non-linearity and instationary, which makes it difficult to extract features. To solve this problem, a feature extraction method based on the improved complementary ensemble empirical mode decomposition with adaptive noise (ICEEMDAN) and multiscale improved permutation entropy (MIPE) is proposed. Firstly, the ICEEMDAN is utilized to obtain a group of intrinsic mode functions (IMFs) from the sound of running power equipment. The noise IMFs are then identified and eliminated through mutual information (MI) and mean mutual information (meanMI) of IMFs. Next, the normalized mutual information (norMI) and MIPE are calculated respectively, and norMI is utilized to weigh the corresponding MIPE result. Finally, based on the separability criterion, the weighted MIPE results are feature-dimensionally reduced to obtain the multiscale entropy feature of the sound. The experimental results show that the classification accuracies of the method under the conditions of no noise and 5 dB reach 96.7% and 89.9%, respectively. In practice, the proposed method has higher reliability and stability for the sound feature extraction of the running power equipment.

8.
Mol Biol Rep ; 47(10): 7557-7566, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32929654

RESUMO

The main pathogenesis of type 1 diabetes mellitus (T1DM) is autoimmune-mediated apoptosis of pancreatic islet ß cells. We sought to characterize the function of microRNA-203a (miR-203a) on pancreatic islet ß cell proliferation and apoptosis. In situ hybridization was used to detect the expression of miR-203a in islet ß cells in normal and hyperglycaemic non-obese diabetic (NOD) mice. Cell proliferation was measured by cell counting kit eight and cell apoptosis was detected using flow cytometry. Insulin receptor substrate 2 (IRS2/Irs2) was determined to be a direct target of miR-203a by Luciferase reporter assay. We detected the effects of miR-203a overexpression or inhibition on proliferation and apoptosis of IRS2-overexpressing or IRS2-knockdown MIN6 cells respectively, and preliminarily explored the downstream targets of the IRS2 pathway. NOD mice model was used to detect miR-203a inhibitor treatment for diabetes. Our experiment showed miR-203a was upregulated in pancreatic ß cells of hyperglycaemic NOD mice. Elevated miR-203a expression inhibited the proliferation and promoted the apoptosis of MIN6 cells. IRS2/Irs2 is a novel target gene directly regulated by miR-203a and miR-203a overexpression downregulated the expression of IRS2. Irs2 silencing reduced cell proliferation and increased apoptosis. Irs2 overexpression could abolish the pro-apoptotic and anti-proliferative effects of miR-203a on MIN6 cells. Hyperglycemia in newly hyperglycemic NOD mice was under control after treatment with miR-203a inhibitor. Our study suggests that miR-203a regulates pancreatic ß cell proliferation and apoptosis by targeting IRS2, treatment with miR-203a inhibitors and IRS2 might provide a new therapeutic strategy for T1DM.


Assuntos
Apoptose , Proliferação de Células , Hiperglicemia/metabolismo , Proteínas Substratos do Receptor de Insulina/biossíntese , Células Secretoras de Insulina/metabolismo , MicroRNAs/metabolismo , Animais , Linhagem Celular , Feminino , Hiperglicemia/patologia , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos NOD
9.
Sci Adv ; 6(11): eaay4697, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32201722

RESUMO

Previously, we reported that chromodomain Y-like (CDYL) acts as a crotonyl-coenzyme A hydratase and negatively regulates histone crotonylation (Kcr). However, the global CDYL-regulated crotonylome remains unclear. Here, we report a large-scale proteomics analysis for protein Kcr. We identify 14,311 Kcr sites across 3734 proteins in HeLa cells, providing by far the largest crotonylome dataset. We show that depletion of CDYL alters crotonylome landscape affecting diverse cellular pathways. Specifically, CDYL negatively regulated Kcr of RPA1, and mutation of the Kcr sites of RPA1 impaired its interaction with single-stranded DNA and/or with components of resection machinery, supporting a key role of RPA1 Kcr in homologous recombination DNA repair. Together, our study indicates that protein crotonylation has important implication in various pathophysiological processes.


Assuntos
Proteínas Correpressoras/metabolismo , Hidroliases/metabolismo , Processamento de Proteína Pós-Traducional , Reparo de DNA por Recombinação , Proteína de Replicação A/metabolismo , Sobrevivência Celular/genética , Proteínas Correpressoras/genética , Dano ao DNA , DNA de Cadeia Simples/genética , Técnicas de Silenciamento de Genes , Células HeLa , Histonas/metabolismo , Humanos , Hidroliases/genética , Proteoma
10.
PLoS Pathog ; 14(12): e1007193, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30543715

RESUMO

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.


Assuntos
Antivirais , Peste Suína Clássica/prevenção & controle , Engenharia Genética/métodos , Animais , Animais Geneticamente Modificados , Vírus da Febre Suína Clássica , Suínos
11.
Cardiovasc Diabetol ; 17(1): 142, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30409151

RESUMO

BACKGROUND: It is well known that angiopoietin-like protein 8 (ANGPTL8) exerts its effects on lipid metabolism through the inhibition of lipoprotein lipase and subsequent elevation of plasma triglyceride. However, it is not clear whether ANGPTL8 could affect lipid metabolism via other pathways. The study was aimed to investigate the effects of ANGPTL8 on the function of high-density lipoprotein (HDL), which plays a protective role in atherosclerosis progression. METHODS: Two hundred and ten subjects were recruited. Plasma ANGPTL8 was measured by enzyme-linked immunosorbent assays. Cholesterol efflux capacity was chosen as the biomarker of HDL function and measured via H3-cholesterol loading THP-1 cell models. RESULTS: ANGPTL8 exhibited no significant difference between CAD group and nonCAD group, but ANGPTL8 in DM group was significantly higher than that in the nonDM group [568.3 (406.2-836.8) vs 458.2 (356.8-755.6), P = 0.023]. Compared to controls, subjects in CAD group and DM group exhibited significantly lower cholesterol efflux capacity [CAD: 14.58 ± 2.06 vs 12.51 ± 2.83%, P < 0.0001; DM: 13.62 ± 2.57 vs 12.34 ± 3.16%, P = 0.0099]. ANGPTL8 was inversely correlated with cholesterol efflux capacity (r = - 0.188, P < 0.01). Regression analysis revealed that plasma ANGPTL8 was an independent contributor to cholesterol efflux capacity (standardized ß = - 0.143, P = 0.023). CONCLUSION: ANGPTL8 presents a negative effect on HDL-mediated cholesterol efflux capacity.


Assuntos
Síndrome Coronariana Aguda/sangue , Proteínas Semelhantes a Angiopoietina/sangue , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Macrófagos/metabolismo , Hormônios Peptídicos/sangue , Síndrome Coronariana Aguda/diagnóstico , Idoso , Transporte Biológico , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células THP-1
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