技术资料

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons,the presence and functions of SOCE are still in question. Here,we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase,which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs),being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions,but the underlying mechanism and,specifically,the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons,we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD,pointing out a key function of calcium and SOCE in synaptic plasticity. View Publication

Several somatic ribosome defects have recently been discovered in cancer,yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10 R98S) found in T-cell acute lymphoblastic leukemia (T-ALL). The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10 R98S mouse lymphoid cells,which we confirmed in hematopoietic cells from transgenic Rpl10 R98S mice and T-ALL xenograft samples. RPL10 R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation,as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10 R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10 R98S functionally mimics JAK-STAT activation. Mechanistically,besides transcriptional changes,RPL10 R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes,as well as decreased Jak1 degradation. Of further medical interest,RPL10 R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively,we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation,and expand the relevance of this cascade in leukemia. View Publication

Transitioning CD19-directed chimeric antigen receptor (CAR) T cells from early-phase trials in relapsed patients to a viable therapeutic approach with predictable efficacy and low toxicity for broad application among patients with high unmet need is currently complicated by product heterogeneity resulting from transduction of undefined T-cell mixtures,variability of transgene expression,and terminal differentiation of cells at the end of culture. A phase 1 trial of 45 children and young adults with relapsed or refractory B-lineage acute lymphoblastic leukemia was conducted using a CD19 CAR product of defined CD4/CD8 composition,uniform CAR expression,and limited effector differentiation. Products meeting all defined specifications occurred in 93{\%} of enrolled patients. The maximum tolerated dose was 106 CAR T cells per kg,and there were no deaths or instances of cerebral edema attributable to product toxicity. The overall intent-to-treat minimal residual disease-negative (MRD-) remission rate for this phase 1 study was 89{\%}. The MRD- remission rate was 93{\%} in patients who received a CAR T-cell product and 100{\%} in the subset of patients who received fludarabine and cyclophosphamide lymphodepletion. Twenty-three percent of patients developed reversible severe cytokine release syndrome and/or reversible severe neurotoxicity. These data demonstrate that manufacturing a defined-composition CD19 CAR T cell identifies an optimal cell dose with highly potent antitumor activity and a tolerable adverse effect profile in a cohort of patients with an otherwise poor prognosis. This trial was registered at www.clinicaltrials.gov as {\#}NCT02028455. View Publication

The use of an interleukin beta$ antibody is currently being investigated in the clinic for the treatment of acne,a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1beta$ into active IL-1beta$,caspase-1,could be an alternative small molecule approach. This report describes the discovery of uracil 20,a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration,topical administration challenges such as phototoxicity,organic and aqueous solubility,chemical stability in solution,and skin metabolic stability are discussed and successfully resolved. View Publication

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However,methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately,these methods entail numerous challenges,including poor efficiency,variable cell type heterogeneity,and lengthy,expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol,these lines can be inducibly differentiated into either cortical (i3 Neurons) or lower motor neurons (i3 LMN) in a rapid,efficient,and scalable manner (Wang et al.,2017). In this manuscript,we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3 Neurons or i3 LMNs,and we present neuronal culture conditions for various experimental applications. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

BACKGROUND Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and also that insulators can improve FV vector safety. However,in a previous analysis of insulator effects on FV vector safety,strong viral promoters were used to elicit genotoxic events. In the present study,we developed and analyzed the efficacy and safety of a high-titer,clinically relevant FV vector driven by the housekeeping promoter elongation factor-1alpha$ and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). METHODS Human CD34+ cord blood cells were exposed to an enhanced green fluorescent protein expressing vector,FV-EGW-A1,at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. RESULTS FV-EGW-A1 resulted in high-marking,multilineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. CONCLUSIONS An FV vector with an elongation factor-1alpha$ promoter and an A1 insulator is a promising vector design for use in the clinic. View Publication

The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile,easily constructed,and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types,delivery by this method is not efficient in many primary cell types,including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction,allowing the CRISPR-Cas9 system to be integrated into the genome of the cell,resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel,advancements have been made in the culture,expansion,selection,and differentiation of AECs,which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods,we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery,(2) efficiently transduce AECs,(3) culture and select for a bulk edited AEC population,(4) molecularly screen AECs for Cas9 cutting and specific sequence edits,and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease. View Publication

The sympathetic (SNS) and parasympathetic (PNS) branches of the autonomic nervous system have been implicated in the modulation of the renewal of many tissues,including the intestinal epithelium. However,it is not known whether these mechanisms are direct,requiring an interaction between autonomic neurotransmitters and receptors on proliferating epithelial cells. To evaluate the existence of a molecular framework for a direct effect of the SNS or PNS on intestinal epithelial renewal,we measured gene expression for the main autonomic neurotransmitter receptors in this tissue. We separately evaluated intestinal epithelial regions comprised of the stem,progenitor,and mature cells,which allowed us to investigate the distinct contributions of each cell population to this proposed autonomic effect. Notably,we found that the stem cells expressed the receptors for the SNS-associated alpha2A adrenoreceptor and the PNS-associated muscarinic acetylcholine receptors (M1 and M3). In a separate experiment,we found that the application of norepinephrine or acetylcholine decreases the expression of cyclin D1,a gene necessary for cell cycle progression,in intestinal epithelial organoids compared with controls (P {\textless} 0.05). Together,these results provide evidence of a direct mechanism for the autonomic nervous system influence on intestinal epithelial stem cell proliferation. View Publication

Objective Dendritic cells (DCs) are key orchestrators of immune function. To date,rheumatoid arthritis (RA) researchers have predominantly focused on a potential pathogenic role for CD1c+ DCs. In contrast,CD141+ DCs and plasmacytoid DCs (pDCs) have not been systematically examined,at least in early RA. In established RA,the role of pDCs is ambiguous and,since disease duration and treatment both impact RA pathophysiology,we examined pDCs,and CD1c+ and CD141+ conventional DCs (cDCs),in early,drug-na{\{i}}ve RA (eRA) patients. Methods We analyzed the frequency and phenotype of pDCs View Publication

Asthma is a complex and heterogeneous respiratory disorder characterized by chronic airway inflammation. It has generally been associated with allergic mechanisms related to type 2 airway inflammation. Nevertheless,between 10 and 33{\%} of asthmatic individuals have nonallergic asthma (NA). Several targeted treatments are in clinical development for patients with Th2 immune response,but few biomarkers are been defined for low or non-Th2-mediated inflammation asthma. We have recently defined by gene expression a set of genes as potential biomarkers of NA,mainly associated with disease severity: IL10,MSR1,PHLDA1,SERPINB2,CHI3L1,IL8,and PI3. Here,we analyzed their protein expression and specificity using sera and isolated peripheral blood mononuclear cells (PBMCs). First,protein quantification was carried out using ELISA (in sera) or Western blot (proteins extracted from PBMCs by Trizol procedure),depending on the biomarker in 30 healthy controls (C) subjects and 30 NA patients. A receiver operating characteristic curve analysis was performed by using the R program to study the specificity and sensitivity of the candidate biomarkers at a gene- and protein expression level. Four kinds of comparisons were performed: total NA group vs C group,severe NA patients vs C,moderate-mild NA patients vs C,and severe NA patients vs moderate-mild NA patients. We found that all the single genes showed good sensitivity vs specificity for some phenotypic discrimination,with CHI3L1 and PI3 exhibiting the best results for C vs NA: CHI3L1 area under the curve (AUC) (CI 95{\%}): 0.95 (0.84-1.00) and PI3 AUC: 0.99 (0.98-1.00); C vs severe NA: PI3 AUC: 1 (0.99-1.00); and C vs moderate-mild NA: CHI3L1 AUC: 1 (0.99-1.00) and PI3 AUC: 0.99 (0.96-1.00). However,the results for discriminating asthma disease and severity with protein expression were better when two or three biomarkers were combined. In conclusion,individual genes and combinations of proteins have been evaluated as reliable biomarkers for classifying NA subjects and their severity. These new panels could be good diagnostic tests. View Publication

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However,there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein,we provide a detailed protocol for the production,enrichment,and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol,researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines,researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks,and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis,as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease,$\beta$-thalassemia,and primary immunodeficiencies. View Publication

We have developed models of HIV latency using microglia derived from adult human patient brain cortex and transformed with the SV40 T large and hTERT antigens. Latent clones infected by HIV reporter viruses display high levels of spontaneous HIV reactivation in culture. BrainPhys,a medium highly representative of the CNS extracellular environment,containing low glucose and 1{\%} FBS,reduced,but did not prevent,HIV reactivation. We hypothesized that spontaneous HIV reactivation in culture was due to the expression of pro-inflammatory genes,such as TNF-alpha$,taking place in the absence of the natural inhibitory signals from astrocytes and neurons. Indeed,expression and secretion of TNF-alpha$ is strongly reduced in HIV-latently infected microglia compared to the subset of cells that have undergone spontaneous HIV reactivation. Whereas inhibitors of NF-kappa$B or of macrophage activation only had a short-term silencing effect,addition of dexamethasone (DEXA),a glucocorticoid receptor (GR) agonist and mediator of anti-inflammation,silenced the HIV provirus in a long-term,and shRNA-mediated knock-down of GR activated HIV. DEXA also decreased secretion of a number of cytokines,including TNF-alpha$. Chromatin immunoprecipitation analysis revealed that DEXA strongly increased GR occupancy at the HIV promoter,and reduced histone 3 acetylated levels. Moreover,TNF-alpha$ expression inhibitors in combination with DEXA induced further HIV silencing and increased the histone 3 lysine 27 tri-methylated epigenetic mark of repression at the HIV promoter region. We conclude that GR is a critical repressor of HIV transcription in microglia,and a novel potential pharmacological target to restrict HIV expression in the CNS. View Publication