技术资料

Glycyrrhetinic acid (GA) is one of the main components of the traditional Chinese medicine of licorice,which can coordinate and promote the effects of other medicines in the traditional prescription. We found that GA could promote the proliferation,decrease the apoptotic rate,and attenuate DFMO-elicited growth arrest and delay in restitution after wounding in IEC-6 cells via HuR. GA failed to promote proliferation and to suppress apoptosis after silencing HuR by siRNA in IEC-6 cells. Furthermore,with the model of small intestinal organoids developed from intestinal crypt stem cells,we found that GA could increase HuR and its downstream ki67 levels to promote intestinal organoid development. In the in vivo assay,GA was shown to maintain the integrity of the intestinal epithelium under the circumstance of 48 h-fasting in rats via raising HuR and its downstream genes such as EGF,EGFR,and MEK. These results suggested that via HuR modulation,GA could promote intestinal epithelium homeostasis,and therefore contribute to the absorption of constituents from other medicines co-existing in the traditional prescription with licorice in the small intestine. Our results provide a new perspective for understanding the effect of licorice on enhancing the therapeutic effect of traditional prescriptions according to the traditional Chinese medicine theory. View Publication

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood,initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however,the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome,RNA-seq),Colony forming assay and xenograft studies,we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones,both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells,JMML-PCs are not always restricted to this compartment,highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML,and the identity of JMML-PCs,and provides robust models to monitor the disease and test novel therapeutic approaches. View Publication

Acute megakaryoblastic leukemia (AMKL) represents ∼10{\%} of pediatric acute myeloid leukemia cases and typically affects young children ({\textless}3 years of age). It remains plagued with extremely poor treatment outcomes ({\textless}40{\%} cure rates),mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60{\%} to 70{\%} of cases and include nucleoporin 98 (NUP98) gene rearrangements,most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking,and patient samples are rare,further limiting biomarkers and drug discovery. To overcome these impediments,we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest,sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia,including AMKL,that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP,MPIG6B,and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib,a clinically approved JAK2 inhibitor. Overall,synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia,which lack effective and rationally designed treatments. View Publication

Current guidelines encourage administering pneumococcal vaccine Prevnar-13 to patients with lupus,but whether such vaccinations affect disease severity is unclear. To address this issue,we treated 3-month-old MRL-lpr mice,that spontaneously develop a lupus-like syndrome,with Prevnar-13 or vehicle control. After 3 months,we quantified circulating anti-Pneumococcal polysaccharide capsule (PPS) antibodies and signs of disease severity,including albuminuria,renal histology and skin severity score. We also compared immunophenotypes and function of T and B cells from treated and untreated animals. Prevnar-13 elicited the formation of anti-pneumococcal IgM and IgG. Prevnar-13 treated animals showed reduced albuminuria,renal histological lesions,and milder dermatitis compared to vehicle-treated controls. Mitigated disease severity was associated with reduced and increased T follicular helper cells (TFH) and T follicular regulatory cells (TFR),respectively,in Prevnar-treated animals. T cells from Prevnar-13 vaccinated mice showed differential cytokine production after aCD3/aCD28 stimulation,with significantly decreased IL-17 and IL-4,and increased IL-10 production compared to non-vaccinated mice. In conclusion,pneumococcal vaccination elicits anti-pneumococcal antibody response and ameliorates disease severity in MRL-lpr mice,which associates with fewer TFH and increased TFR. Together,the data support use of Prevnar vaccination in individuals with SLE. View Publication

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS),such as hydrogen peroxide (H2O2),toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS,however,has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here,we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2,a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-$\gamma$ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings,we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation,as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2,chronic inflammation caused IECs to release higher levels of H2O2. Furthermore,colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively,these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa. View Publication

The macrophages from Crohn's Disease (CD) patients are defective to control the replication of CD-associated adherent-invasive E. coli (AIEC). We aimed to identify the host factors associated with AIEC replication focusing on polymorphisms related to autophagy. Peripheral blood monocyte-derived macrophages (MDM),obtained from 95 CD patient,30 ulcerative colitis (UC) patients and 15 healthy subjects,were genotyped for several CD-associated polymorphisms. AIEC bacteria survival increased within MDM from CD patients compared to UC (p = 0.0019). AIEC bacteria survival increased in patients with CD-associated polymorphism IRGM (p = 0.05) and reduced in those with CD-associated polymorphisms XBP-1 (p = 0.026) and ULK-1 (p = 0.033). AIEC infection led to an increase of pro-inflammatory cytokines IL-1$\beta$ (p {\textless} 0.0001) and TNF-$\alpha$ (p {\textless} 0.0001) in CD macrophages. ULK-1 expression increased in AIEC-infected MDM from CD patients compared to MDM from UC patients or healthy subjects (p = 0.0056) and correlated with AIEC survival (p = 0.0013). Moreover,the expression of ULK-1 phosphorylation on Serine 757 decreased following to AIEC infection (p {\textless} 0.0001). Short-term silencing of ULK-1 and IRGM genes restricted and promote,respectively,AIEC survival within MDM (p = 0.0018 and p = 0.0291). In conclusion,the macrophage defect to mediate AIEC clearance in CD patients is linked to polymorphisms related to autophagy such as IRGM and ULK-1. View Publication

The role of negative checkpoint regulators (NCRs) in human health and disease cannot be overstated. V-domain Ig-containing Suppressor of T-cell Activation (VISTA) is an Ig superfamily protein predominantly expressed within the hematopoietic compartment and has been studied for its role in the negative regulation of T cell responses. The findings presented in this study show that,unlike all other NCRs,VISTA deficiency dramatically impacts on macrophage cytokine and chemokine production,as well as the chemotactic response of VISTA-deficient macrophages. A select group of inflammatory chemokines,including CCL2,CCL3,CCL4,and CCL5,was strikingly elevated in culture supernatants from VISTA KO macrophages. VISTA deficiency also altered chemokine receptor recycling and profoundly disrupted myeloid chemotaxis. The impact of VISTA deficiency on chemotaxis in vivo was apparent with the reduced ability of both KO macrophages and MDSCs to migrate to the tumor microenvironment. This is the first demonstration of an NCR impacting on myeloid mediator production and chemotaxis,and will guide the use of anti-VISTA therapeutics to manipulate the chemotaxis of inflammatory macrophages or immunosuppressive MDSCs in inflammatory diseases and cancer. View Publication

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here,we show for the first time that G2.2,a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide,selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+,Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact,G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers,e.g.,CD133,DCMLK1,LGR5,and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly,G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly,G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions,blood coagulation profile,and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro,in vivo,and ex vivo mechanistic studies,we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38$\alpha$,uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence,G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs. View Publication

OBJECTIVE Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract,summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound {\#}5) efficiently mitigated gastrointestinal ARS. However,the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound {\#}5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS C3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound {\#}5 or solvent was administered subcutaneously 24 h later. 48 h after irradiation the mice were sacrificed,and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H{\&}E,anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts,mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS Compound {\#}5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally,assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound {\#}5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound {\#}5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24 h after drug treatment. A single dose of Compound {\#}5 significantly increased the number of CD45+ cells after 12 Gy TBI. Importantly,96 h after 12 Gy TBI Compound {\#}5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION In summary,our study provides evidence that Compound {\#}5 leads to an influx of immune cells into GALT,thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches. View Publication

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study,we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of {\textgreater}50 small molecules were developed using structure-activity-relationship studies of SB203580,a known p38-MAPK inhibitor. A particular analog,C7,resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p {\textless} .001). In depth phenotypic analysis revealed {\textgreater}600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p {\textless} .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p {\textless} .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion,a small molecule,C7,could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393. View Publication

The aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract,including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However,established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-$\gamma$ of intestinal organoids with and without epithelial cell-specific deletion of the interferon-$\gamma$-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-$\gamma$-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2,-7,and - 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of re-localization of claudins. In summary,we provide a novel methodologic approach for quantitative analyses of intestinal epithelial barrier functions in the 3D organoid model. View Publication

The purpose of these studies was to develop and characterize B-cell maturation antigen (BCMA)-specific peptide-encapsulated nanoparticle formulations to efficiently evoke BCMA-specific CD8+ cytotoxic T lymphocytes (CTL) with poly-functional immune activities against multiple myeloma (MM). Heteroclitic BCMA72-80 [YLMFLLRKI] peptide-encapsulated liposome or poly(lactic-co-glycolic acid) (PLGA) nanoparticles displayed uniform size distribution and increased peptide delivery to human dendritic cells,which enhanced induction of BCMA-specific CTL. Distinct from liposome-based nanoparticles,PLGA-based nanoparticles demonstrated a gradual increase in peptide uptake by antigen-presenting cells,and induced BCMA-specific CTL with higher anti-tumor activities (CD107a degranulation,CTL proliferation,and IFN-$\gamma$/IL-2/TNF-$\alpha$ production) against primary CD138+ tumor cells and MM cell lines. The improved functional activities were associated with increased Tetramer+/CD45RO+ memory CTL,CD28 upregulation on Tetramer+ CTL,and longer maintenance of central memory (CCR7+ CD45RO+) CTL,with the highest anti-MM activity and less differentiation into effector memory (CCR7- CD45RO+) CTL. These results provide the framework for therapeutic application of PLGA-based BCMA immunogenic peptide delivery system,rather than free peptide,to enhance the induction of BCMA-specific CTL with poly-functional Th1-specific anti-MM activities. These results demonstrate the potential clinical utility of PLGA nanotechnology-based cancer vaccine to enhance BCMA-targeted immunotherapy against myeloma. View Publication