技术资料

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells,through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However,the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes,that are hyperproliferative,yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0-/- enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1,but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity. View Publication

Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However,the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity,we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids,we found increased excitatory bursting activity,which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability. View Publication

The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) is a major complication of hemophilia A treatment. The sole clinical therapy to restore FVIII tolerance in patients with inhibitors remains immune tolerance induction (ITI) which is expensive,difficult to administer and not always successful. Although not fully understood,the mechanism of ITI is thought to rely on inhibition of FVIII-specific B cells (1). Its efficacy might therefore be improved through more aggressive B cell suppression. Fc$\gamma$RIIB is an inhibitory Fc receptor that down-regulates B cell signaling when cross-linked with the B cell receptor (BCR). We sought to investigate if recombinant FVIII Fc (rFVIIIFc),an Fc fusion molecule composed of FVIII and the Fc region of immunoglobulin G1 (IgG1) (2),is able to inhibit B cell activation more readily than FVIII. rFVIIIFc was able to bind FVIII-exposed and na{\{i}}ve B cells from hemophilia A mice as well as a FVIII-specific murine B cell hybridoma line (413 cells). An anti-Fc$\gamma$RIIB antibody and FVIII inhibited binding suggesting that rFVIIIFc is able to interact with both Fc$\gamma$RIIB and the BCR. Furthermore incubation of B cells from FVIII-exposed mice and 413 cells with rFVIIIFc resulted in increased phosphorylation of SH-2 containing inositol 5-phosphatase (SHIP) when compared to FVIII. B cells from FVIII-exposed hemophilia A mice also exhibited decreased extracellular signal-regulated kinase (ERK) phosphorylation when exposed to rFVIIIFc. These differences were absent in B cells from na{\"{i}}ve non-FVIII exposed hemophilic mice suggesting an antigen-dependent effect. Finally rFVIIIFc was able to inhibit B cell calcium flux induced by anti-Ig F(ab)2. Our results therefore indicate that rFVIIIFc is able to crosslink Fc$\gamma$RIIB and the BCR of FVIII-specific B cells causing inhibitory signaling in these cells.""" View Publication

Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research,particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y),termed conditionally reprogrammed cell (CRC) technology,enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings,we then created six CF cell CRC populations,three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted,and cells were subcultured,without irradiated feeders plus Y,into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y,dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage,but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus,CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers. View Publication

Approaches to deplete persistent HIV infection are needed. We investigated the combined impact of the latency reversing agent vorinostat (VOR) and AGS-004,an autologous dendritic cell immunotherapeutic,on the HIV reservoir. HIV+,stably treated participants in whom resting CD4+ T cell-associated HIV RNA (rca-RNA) increased after VOR exposure ex vivo and in vivo received 4 doses of AGS-004 every 3 weeks,followed by VOR every 72 hours for 30 days,and then the cycle repeated. Change in VOR-responsive host gene expression,HIV-specific T cell responses,low-level HIV viremia,rca-RNA,and the frequency of resting CD4+ T-cell infection (RCI) was measured at baseline and after each cycle. No serious treatment-related adverse events were observed among five participants. As predicted,VOR-responsive host genes responded uniformly to VOR dosing. Following cycles of AGS-004 and VOR,rca-RNA decreased significantly in only two participants,with a significant decrease in SCA observed in one of these participants. However,unlike other cohorts dosed with AGS-004,no uniform increase in HIV-specific immune responses following vaccination was observed. Finally,no reproducible decline of RCI,defined as a decrease of {\textgreater}50{\%},was observed. AGS-004 and VOR were safe and well-tolerated,but no substantial impact on RCI was measured. In contrast to previous clinical data,AGS-004 did not induce HIV-specific immune responses greater than those measured at baseline. More efficacious antiviral immune interventions,perhaps paired with more effective latency reversal,must be developed to clear persistent HIV infection. View Publication

Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However,the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1),YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP),a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction,in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally,advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly,gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together,our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy. View Publication

Transcriptional status determines the HIV replicative state in infected patients. However,the transcriptional mechanisms for proviral replication control remain unclear. In this study,we show that,apart from its function in HIV integration,LEDGF/p75 differentially regulates HIV transcription in latency and proviral reactivation. During latency,LEDGF/p75 suppresses proviral transcription via promoter-proximal pausing of RNA polymerase II (Pol II) by recruiting PAF1 complex to the provirus. Following latency reversal,MLL1 complex competitively displaces PAF1 from the provirus through casein kinase II (CKII)-dependent association with LEDGF/p75. Depleting or pharmacologically inhibiting CKII prevents PAF1 dissociation and abrogates the recruitment of both MLL1 and Super Elongation Complex (SEC) to the provirus,thereby impairing transcriptional reactivation for latency reversal. These findings,therefore,provide a mechanistic understanding of how LEDGF/p75 coordinates its distinct regulatory functions at different stages of the post-integrated HIV life cycles. Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection. View Publication

Glioblastoma (GBM) is the most common and aggressive malignant tumor in adult brain. Even with the current standard therapy including surgical resection followed by postoperative radiotherapy and chemotherapy with temozolomide (Temo),GBM patients still have a poor median survival. Reprogramming of tumor cells into non-malignant cells might be a promising therapeutic strategy for malignant tumors,including GBM. Based on previous studies using small molecules to reprogram astrocytes into neuronal cells,here we further identified a FTT cocktail of three commonly used drugs (Fasudil,Tranilast,and Temo) to reprogram patient-derived GBM cells,either cultured in serum containing or serum-free medium,into neuronal like cells. FTT-treated GBM cells displayed a neuronal like morphology,expressed neuronal genes,exhibited neuronal electrophysiological properties,and showed attenuated malignancy. More importantly,FTT cocktail more significantly suppressed tumor growth and prolonged survival in GBM patient derived xenograft than Temo alone. Our study provided preclinical evidence that the neuronal reprogramming drug cocktail might be a promising strategy to improve the existing treatment for GBM. View Publication

Cyclosporine A (CsA) is a powerful immunosuppressant,but it is an ineffective stand-alone treatment for systemic lupus erythematosus (SLE) due to poor target tissue distribution and renal toxicity. We hypothesized that CD71 (transferrin receptor 1)-directed delivery of CsA to the lymphatic system would improve SLE outcomes in a murine model. We synthesized biodegradable,ligand-conjugated nanoparticles [P2Ns-gambogic acid (GA)] targeting CD71. GA conjugation substantially increased nanoparticle association with CD3+ or CD20+ lymphocytes and with intestinal lymphoid tissues. In orally dosed MRL-lpr mice,P2Ns-GA-encapsulated CsA increased lymphatic drug delivery 4- to 18-fold over the ligand-free formulation and a commercial CsA capsule,respectively. Improved lymphatic bioavailability of CsA was paralleled by normalization of anti-double-stranded DNA immunoglobulin G titer,plasma cytokines,and glomerulonephritis. Thus,this study demonstrates the translational potential of nanoparticles that enhance the targeting of lymphatic tissues,transforming CsA into a potent single therapeutic for SLE. View Publication

Activation-induced cytidine deaminase (AID) mediates immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM),critical processes for maturation of the antibody response. Epigenetic factors,such as histone deacetylases (HDACs),would underpin B cell differentiation stage-specific AID expression. Here,we showed that NAD+-dependent class III HDAC sirtuin 1 (Sirt1) is highly expressed in resting B cells and down-regulated by stimuli inducing AID. B cell Sirt1 down-regulation,deprivation of NAD+ cofactor,or genetic Sirt1 deletion reduced deacetylation of Aicda promoter histones,Dnmt1,and nuclear factor-$\kappa$B (NF-$\kappa$B) p65 and increased AID expression. This promoted class-switched and hypermutated T-dependent and T-independent antibody responses or led to generation of autoantibodies. Genetic Sirt1 overexpression,Sirt1 boost by NAD+,or allosteric Sirt1 enhancement by SRT1720 repressed AID expression and CSR/SHM. By deacetylating histone and nonhistone proteins (Dnmt1 and NF-$\kappa$B p65),Sirt1 transduces metabolic cues into epigenetic changes to play an important B cell-intrinsic role in modulating antibody and autoantibody responses. View Publication

The tumor immune microenvironment (TIME) plays a pivotal role in tumor development,progression,and prognosis. However,the characteristics of the TIME in diffuse astrocytoma (DA) are still unclear. Leveraging mass cytometry with a panel of 33 markers,we analyzed the infiltrating immune cells from 10 DA and 4 oligodendroglioma (OG) tissues and provided a single cell-resolution landscape of the intricate immune microenvironment. Our study profiled the composition of the TIME in DA and confirmed the presence of immune cells,such as glioma-associated microglia/macrophages (GAMs),CD8+ T cells,CD4+ T cells,regulatory T cells (Tregs),and natural killer cells. Increased percentages of PD-1+ CD8+ T cells,TIM-3+ CD4+ T cell subpopulations,Tregs and pro-tumor phenotype GAMs substantially contribute to the local immunosuppressive microenvironment in DA. DAs and OGs share similar compositions in terms of immune cells,while GAMs in DA exhibit more inhibitory characteristics than those in OG. View Publication

The Glioblastoma (GBM) immune microenvironment plays a critical role in tumor development,progression,and prognosis. A comprehensive understanding of the intricate milieu and its interactions remains unclear,and single-cell analysis is crucially needed. Leveraging mass cytometry (CyTOF),we analyzed immunocytes from 13 initial and three recurrent GBM samples and their matched peripheral blood mononuclear cells (pPBMCs). Using a panel of 30 markers,we provide a high-dimensional view of the complex GBM immune microenvironment. Hematoxylin and eosin staining and polychromatic immunofluorescence were used for verification of the key findings. In the initial and recurrent GBMs,glioma-associated microglia/macrophages (GAMs) constituted 59.05 and 27.87{\%} of the immunocytes,respectively; programmed cell death-ligand 1 (PD-L1),T cell immunoglobulin domain and mucin domain-3 (TIM-3),lymphocyte activation gene-3 (LAG-3),interleukin-10 (IL-10) and transforming growth factor-$\beta$ (TGF$\beta$) demonstrated different expression levels in the GAMs among the patients. GAMs could be subdivided into different subgroups with different phenotypes. Both the exhausted T cell and regulatory T (Treg) cell percentages were significantly higher in tumors than in pPBMCs. The natural killer (NK) cells that infiltrated into the tumor lesions expressed higher levels of CXC chemokine receptor 3 (CXCR3),as these cells expressed lower levels of interferon-$\gamma$ (IFN$\gamma$). The immune microenvironment in the initial and recurrent GBMs displayed similar suppressive changes. Our study confirmed that GAMs,as the dominant infiltrating immunocytes,present great inter- and intra-tumoral heterogeneity and that GAMs,increased exhausted T cells,infiltrating Tregs,and nonfunctional NK cells contribute to local immune suppressive characteristics. Recurrent GBMs share similar immune signatures with the initial GBMs except the proportion of GAMs decreases. View Publication