技术资料

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach,intratumoral TSA-reactive CD4+,CD8+ T cells,and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs,TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype,TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle,FucoID should have the potential of accelerating the pace of personalized cancer treatment. View Publication

The Drosophila TEAD ortholog Scalloped is required for Yki-mediated overgrowth but is largely dispensable for normal tissue growth,suggesting that its mammalian counterpart may be exploited for selective inhibition of oncogenic growth driven by YAP hyperactivation. Here we test this hypothesis genetically and pharmacologically. We show that a dominant-negative TEAD molecule does not perturb normal liver growth but potently suppresses hepatomegaly/tumorigenesis resulting from YAP overexpression or Neurofibromin 2 (NF2)/Merlin inactivation. We further identify verteporfin as a small molecule that inhibits TEAD-YAP association and YAP-induced liver overgrowth. These findings provide proof of principle that inhibiting TEAD-YAP interactions is a pharmacologically viable strategy against the YAP oncoprotein. View Publication

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox),a drug used in antitumor therapy,triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore,the p53 inhibitor pifithrin-alpha (PFT-alpha) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However,Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-alpha and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally,Dox triggered Bcl-2 down-regulation,cytochrome c release from mitochondria,and the activation of caspases 9 and 3,suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox,related to its ability to induce apoptosis in proliferating endothelial cells. View Publication

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However,the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages,with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines,patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast,patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover,severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors,including interleukin-5 (IL-5),IL-13,immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures,representing growth factors (A),type-2/3 cytokines (B),mixed type-1/2/3 cytokines (C),and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A,whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus,we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome,as well as early immune signatures that correlate with divergent disease trajectories. View Publication

Protein arginine deiminase 4 (PAD4) is a transcriptional coregulator that catalyzes the calcium-dependent conversion of specific arginine residues in proteins to citrulline. Recently,we reported the synthesis and characterization of F-amidine,a potent and bioavailable irreversible inactivator of PAD4. Herein,we report our efforts to identify the steric and leaving group requirements for F-amidine-induced PAD4 inactivation,the structure of the PAD4-F-amidine x calcium complex,and in vivo studies with N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine),a PAD4 inactivator with enhanced potency. The PAD4 inactivators described herein will be useful pharmacological probes in characterizing the incompletely defined physiological role(s) of this enzyme. In addition,they represent potential lead compounds for the treatment of rheumatoid arthritis because a growing body of evidence supports a role for PAD4 in the onset and progression of this chronic autoimmune disorder. View Publication

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and,when defective,causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs,CFTR(inh)-172,reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR,CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels,multidrug resistance protein-1 (MDR-1),ATP-sensitive K(+) channels,or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90{\%} cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas. View Publication

OBJECTIVE To review the literature concerning the mechanism of action and pharmacodynamics of mifepristone (RU486),potential new uses of RU486,and its current use not only as an abortifacient but also as therapy for endometriosis,leiomyoma,breast cancer,and meningioma. DATA IDENTIFICATION AND SELECTION Studies that relate to RU486 were identified through a MEDLINE search. CONCLUSION(S) RU486 is an 11 beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. The receptor binding is not followed by transcription of P-dependent genes. Mifepristone effectively blocks P receptors in the placenta,resulting in the termination of pregnancy. In addition,it has been used in the treatment of leiomyomata,endometriosis,advanced breast cancer,and meningioma. It is a powerful tool to study the molecular action of P and in the future may be used as an estrogen-free contraceptive. Through an online search of MEDLINE,the authors reviewed the literature on the development of mifepristone (RU-486); RU-486's mechanism of action,pharmacodynamics,and distribution; the physiologic action of RU-486; potential new uses for RU-486; and its current use as both an abortifacient and therapy for endometriosis,leiomyoma,breast cancer,and meningioma. RU-486 is an 11beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. Receptor binding is not followed by the transcription of P-dependent genes. RU-486 effectively blocks P receptors in the placenta,resulting in the termination of pregnancy. It has also been used to treat leiomyomata,endometriosis,advanced breast cancer,and meningioma. The following therapeutic uses of RU-486 are discussed: the termination of early pregnancy,treatment with RU-486 in combination with prostaglandins,the termination of second-trimester pregnancy,cervical ripening,labor induction,postcoital contraception,uterine leiomyomata,endometriosis,breast cancer,and meningioma. View Publication

COVID-19 pathogenesis is associated with an exaggerated immune response. However,the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here,we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio,elevated serum IL-6,MCP-1 and IP-10,and most strikingly,modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme,COX-2,as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover,they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints. View Publication

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN,in concert with cytotoxic drugs,efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study,we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO),an inhibitor of GSH biosynthesis,leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C,a representative example of MYCN-amplified cells,has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore,the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53,bcl-2,and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches. View Publication

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control,a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control,and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy,suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2,termed Ad26.COV2.S,is currently being evaluated in clinical trials. View Publication