技术资料

Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However,FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here,we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore,using single-cell RNA sequencing,we found two subsets of FRCs (CD55 hi and CD55 lo ) in the mesenteric FALC. The CD55 hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55 lo FRCs were enriched in gene expression related to immune response. Interestingly,we found that TLR9 is dominantly expressed in the CD55 lo subset. Activation of TLR9 signaling suppressed proliferation,cytokine production,and retinoid metabolism in the CD55 lo FRC,but not CD55 hi FRC. Notably,we found that adoptive transfer of Tlr9 -/– CD55 lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9 -/– CD55 hi FRC. Furthermore,we identified CD55 hi and CD55 lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55 lo subset. Therefore,inhibition of TLR9 in the CD55 lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis. View Publication

ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that are regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (approximately 180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP,of course). Cholera toxin was the first to be recognized,hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking,ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms,which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells,it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain,a sequence of approximately 200 amino acids that resembles a region in the yeast Sec7 gene product,which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains,among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity,recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4,5,or 6. View Publication

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here,we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal,proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive),neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells,as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs,an effect blocked by Notch pathway inhibition. Moreover,R-m-AEA treatment for 48 h,increased proliferation as assessed by BrdU incorporation assay,an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly,stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation),at 7 days,as shown by counting the number of NeuN-positive neurons in the cultures. Moreover,by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli,a method that allows the functional evaluation of neuronal differentiation,we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251,for 7 days,thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation,R-m-AEA also increased neurite growth,as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together,these results demonstrate that CB1R activation induces proliferation,self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures. View Publication

B cells play an important role in type 1 diabetes (T1D) development. However,the role of B cell activation-induced cytidine deaminase (AID) in diabetes development is not clear. We hypothesized that AID is important in the immunopathogenesis of T1D. To test this hypothesis,we generated AID-deficient (AID-/-) NOD mice. We found that AID-/-NOD mice developed accelerated T1D,with worse insulitis and high levels of anti-insulin autoantibody in the circulation. Interestingly,neither maternal IgG transferred through placenta,nor IgA transferred through milk affected the accelerated diabetes development. AID-/-NOD mice showed increased activation and proliferation of B and T cells. We found enhanced T-B cell interactions in AID-/-NOD mice,with increased T-bet and IFN-γ expression in CD4+ T cells in the presence of AID-/- B cells. Moreover,excessive lymphoid expansion was observed in AID-/-NOD mice. Importantly,antigen-specific BDC2.5 CD4+ T cells caused more rapid onset of diabetes when cotransferred with AID-/- B cells than when cotransferred with AID+/+ B cells. Thus,our study provides insights into the role of AID in T1D. Our data also suggest that AID is a negative regulator of immune tolerance and ablation of AID can lead to exacerbated islet autoimmunity and accelerated T1D development. View Publication

Extracellular vesicles (EVs) are phospholipid-based particles endogenously produced by cells. Their natural composition and selective cell interactions make them promising drug carriers. However,in order to harness their properties,efficient exogenous drug encapsulation methods need to be investigated. Here,EVs from various cellular origins (endothelial,cancer and stem cells) were produced and characterised for size and composition. Porphyrins of different hydrophobicities were employed as model drugs and encapsulated into EVs using various passive and active methods (electroporation,saponin,extrusion and dialysis). Hydrophobic compounds loaded very efficiently into EVs and at significantly higher amounts than into standard liposomes composed of phosphocholine and cholesterol using passive incubation. Moreover,loading into EVs significantly increased the cellular uptake by textgreater60% and the photodynamic effect of hydrophobic porphyrins in vitro compared to free or liposome encapsulated drug. The active encapsulation techniques,with the saponin-assisted method in particular,allowed an up to 11 fold higher drug loading of hydrophilic porphyrins compared to passive methods. EVs loaded with hydrophilic porphyrins induced a stronger phototoxic effect than free drug in a cancer cell model. Our findings create a firm basis for the development of EVs as smart drug carriers based on straightforward and transferable methods. View Publication

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-,atrial-,and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives,which in turn promoted cardiomyocyte differentiation. Moreover,a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation,improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation,measuring action potentials,and intracellular Ca(2+) dynamics. These findings are relevant for improving our understanding on human heart development,and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes,similar to those observed in adult cardiac myocytes. View Publication

The use of human pluripotent cell progeny for cardiac disease modeling,drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success,recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR,which in turn induces the Activin-A and Bmp pathways,is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis,and to ultimately promote myocyte maturation,we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq,induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors,followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm,whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency,even when CHIR alone was ineffective. Moreover,co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis,as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast,co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings,which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression,are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages,while Bmp levels regulate subsequent allocation into mesodermal cell types. View Publication

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1,a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling,we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A,a molecule that normally transduces TGF-β signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo,the underlying mechanisms have yet to be revealed. To this end,we developed a high-throughput screening (HTS) system using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds,we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models,an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC-based HO model mouse,revealed critical roles for mTOR signaling in vivo. Moreover,we identified ENPP2,an enzyme that generates lysophosphatidic acid,as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis. View Publication

Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model,we have recently shown that Nodal can block neuronal differentiation,suggesting that TGFbeta family members are involved in cell fate decisions of hESCs,including preservation of their pluripotency. Here,we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short,or by the Activin receptor inhibitor SB431542,precipitates hESC differentiation. Nevertheless,neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers,and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However,this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling,because it is blocked by SB431542. Finally,long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers,conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs. View Publication

PURPOSE: B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However,blocking B-cell receptor signaling with dasatinib,an inhibitor of SRC kinase,produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. EXPERIMENTAL DESIGN: Fresh CLL B cells were treated with dasatinib,and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules,as well as with molecular and cytogenetic prognostic factors. RESULTS: Among 50 CLL cases,dasatinib treatment reduced cell viability by 2% to 90%,with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly,phosphorylation of SRC family kinases was inhibited by dasatinib in good,as well as poor,responders. As opposed to SRC family kinases,activities of two downstream molecules,SYK and phospholipase Cgamma2,correlate well with the apoptotic response of CLL cells to dasatinib. CONCLUSIONS: Thus,SYK inhibition predicts cellular response to dasatinib. SYK,together with phospholipase Cgamma2,may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective,our study suggests the existence of alternative mechanisms or pathways that activate SYK,independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. View Publication

Astrocytes,a subtype of glial cells,have multiple roles in regulating neuronal development and homeostasis. In addition to the typical mammalian astrocytes,in the primate cortex,interlaminar astrocytes are located in the superficial layer and project long processes traversing multiple layers of the cerebral cortex. Previously,we described a human stem cell based chimeric mouse model where interlaminar astrocytes develop. Here,we utilized this model to study the calcium signaling properties of interlaminar astrocytes. To determine how interlaminar astrocytes could contribute to neurodevelopmental disorders,we generated a chimeric mouse model for Fragile X syndrome (FXS). We report that FXS interlaminar astrocytes exhibit hyperexcitable calcium signaling and are associated with dendritic spines with increased turnover rate. View Publication