技术资料

This study quantifies uptake of a fluorescent glucose analog,(2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG),in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I),which phosphorylates 2-NBDG,were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 lM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies,and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG,as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents,lonidamine (LND),and a-cyano-hydroxycinnamate (a-Cinn),respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely,when cells were treated with a-Cinn,a drug used to increase glycolysis,2-NBDG uptake was increased. Furthermore,tamoxifen (tam),a common endocrine therapy,was administered to estrogen receptor positive and negative (ER?/-) breast cells and demonstrated a decreased 2-NBDG uptake in ER? cells,reflecting a decrease in glycolysis. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development. View Publication

COVID-19 affects millions of patients worldwide,with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens,and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs),platelet factor 4,RANTES,and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19,with intubation (P {\textless} .0001) and death (P {\textless} .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360),whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19,and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally,COVID-19 neutrophils ex vivo displayed excessive NETs at baseline,and COVID-19 plasma triggered NET formation,which was blocked by nNIF. Thus,NETs triggering immunothrombosis may,in part,explain the prothrombotic clinical presentations in COVID-19,and NETs may represent targets for therapeutic intervention. View Publication

The pancreatic adenocarcinoma is an aggressive and devastating disease,which is characterized by invasiveness,rapid progression,and profound resistance to actual treatments,including chemotherapy and radiotherapy. At the moment,surgical resection provides the best possibility for long-term survival,but is feasible only in the minority of patients,when advanced disease chemotherapy is considered,although the effects are modest. Several studies have shown that thyroid hormone,3,3',5-triiodo-l-thyronine (T(3)) is able to promote or inhibit cell proliferation in a cell type-dependent manner. The aim of the present study is to investigate the ability of T(3) to reduce the cell growth of the human pancreatic duct cell lines chosen,and to increase the effect of chemotherapeutic drugs at conventional concentrations. Three human cell lines hPANC-1,Capan1,and HPAC have been used as experimental models to investigate the T(3) effects on pancreatic adenocarcinoma cell proliferation. The hPANC-1 and Capan1 cell proliferation was significantly reduced,while the hormone treatment was ineffective for HPAC cells. The T(3)-dependent cell growth inhibition was also confirmed by fluorescent activated cell sorting analysis and by cell cycle-related molecule analysis. A synergic effect of T(3) and chemotherapy was demonstrated by cell kinetic experiments performed at different times and by the traditional isobologram method. We have showed that thyroid hormone T(3) and its combination with low doses of gemcitabine (dFdCyd) and cisplatin (DDP) is able to potentiate the cytotoxic action of these chemotherapic drugs. Treatment with 5-fluorouracil was,instead,largely ineffective. In conclusion,our data support the hypothesis that T(3) and its combination with dFdCyd and DDP may act in a synergic way on adenopancreatic ductal cells. View Publication

During surgery,ATP from damaged cells induces the release of interleukin-1$\beta$,a potent pro-inflammatory cytokine that contributes to the development of postoperative systemic inflammation,sepsis and multi-organ damage. We recently demonstrated that C-reactive protein (CRP) inhibits the ATP-induced release of monocytic interleukin-1$\beta$,although high CRP levels are deemed to be a poor prognostic marker. Here,we retrospectively investigated if preoperative CRP levels correlate with postoperative CRP,leukocyte counts and fever in the context of anatomical lung resection and systematic lymph node dissection as first line lung cancer therapy. No correlation was found in the overall results. In men,however,preoperative CRP and leukocyte counts positively correlated on postoperative days one to two,and a negative correlation of CRP and fever was seen in women. These correlations were more pronounced in men taking statins and in statin-na{\{i}}ve women. Accordingly the inhibitory effect of CRP on the ATP-induced interleukin-1$\beta$ release was blunted in monocytes from coronary heart disease patients treated with atorvastatin compared to monocytes obtained before medication. Hence the common notion that elevated CRP levels predict more severe postoperative inflammation should be questioned. We rather hypothesize that in women and statin-na{\"{i}}ve patients high CRP levels attenuate trauma-induced increases in inflammatory markers.""" 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

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease,but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus,to date,our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture,or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic,but not mitochondrial,ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium,and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes. View Publication

Extremely premature infants are prone to severe respiratory infections,and the mechanisms underlying this exceptional susceptibility are largely unknown. Nasal epithelial cells (NEC) represent the first-line of defense and adult-derived ALI cell culture models show promising results in mimicking in vivo physiology. Therefore,the aim of this study was to develop a robust and reliable protocol for generating well-differentiated cell culture models from NECs of extremely premature infants. Nasal brushing was performed in 13 extremely premature infants at term corrected age and in 11 healthy adult controls to obtain NECs for differentiation at air-liquid interface (ALI). Differentiation was verified using imaging and functional analysis. Successful isolation and differentiation was achieved for 5 (38.5{\%}) preterm and 5 (45.5{\%}) adult samples. Preterm and adult ALI-cultures both showed well-differentiated morphology and ciliary function,however,preterm cultures required significantly longer cultivation times for acquiring full differentiation (44 ± 3.92 vs. 23 ± 1.83 days; p {\textless} 0.0001). Moreover,we observed that recent respiratory support may impair successful NECs isolation. Herewithin,we describe a safe,reliable and reproducible method to generate well-differentiated ALI-models from NECs of extremely premature infants. These models provide a valuable foundation for further studies regarding immunological and inflammatory responses and respiratory disorders in extremely premature infants. 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

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

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

B cell acute lymphoblastic leukemia (B-ALL) blasts hijack the bone marrow (BM) microenvironment to form chemoprotective leukemic BM niches facilitating chemoresistance and,ultimately,disease relapse. However,the ability to dissect these evolving,heterogeneous interactions among distinct B-ALL subtypes and their varying BM niches is limited with current in vivo methods. Here,we demonstrated an in vitro organotypic leukemia-on-a-chip" model to emulate the in vivo B-ALL BM pathology and comparatively studied the spatial and genetic heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. We revealed the heterogeneous chemoresistance mechanisms across various B-ALL cell lines and patient-derived samples. We showed that the leukemic perivascular endosteal and hematopoietic niche-derived factors maintain B-ALL survival and quiescence (e.g. CXCL12 cytokine signal VCAM-1/OPN adhesive signals and enhanced downstream leukemia-intrinsic NF-$\kappa$B pathway). Furthermore we demonstrated the preclinical use of our model to test niche-cotargeting regimens which may translate to patient-specific therapy screening and response prediction." 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