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In patients with inactivating mutations in myosin Vb (Myo5B),enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis,the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus,apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes. View Publication

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent,submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages,reduces tumor cell proliferation and angiogenesis,and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33,an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11,which is required for the growth of gastric cancers in mice. Accordingly,ablation of the cognate IL-33 receptor St2 limits tumor growth,and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2,Ccl3,and Il6. Conversely,genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore,tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer. View Publication

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result,or possibly originate,from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore,we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether,our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses. View Publication

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression,yet their role in cell fate transitions remains largely unexplored. Here,we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant,hyper-pluripotent" state which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer heterochromatin and DNA methylation landscapes of undifferentiated cell types. Collectively our data establish a link between P-body homeostasis chromatin organization and stem cell potency." View Publication

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2),a known regulator of cell volume,is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity,as well as activation of Akt and Erk,leading to activation of mTORC1 in cells,colonic organoids,and mouse colon. Moreover,NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1,therefore,suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly,by linking ion transport and cell volume regulation to mTORC1 function,NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation. View Publication

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory properties and have been used as immune regulators for the treatment of graft-versus-host disease (GVHD). Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to BM-MSCs for potential clinical applications because of their accessibility and easy preparation. The aim of this in vitro study was to compare MSCs from dental pulp (DP-MSCs),gingival tissue (G-MSCs),and periodontal ligament (PDL-MSCs) in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells to determine which MSCs would be the most appropriate for in vivo immunoregulatory applications. BM-MSCs were included as the gold standard. Our results demonstrated,in vitro,that MSCs from DP,G,and PDL showed immunoregulatory properties similar to those from BM,in terms of the cellular proliferation inhibition of both CD4+- and CD8+-activated T-cells. This reduced proliferation in cell co-cultures correlated with the production of interferon-$\gamma$ and tumor necrosis factor alpha (TNF-$\alpha$) and the upregulation of programmed death ligand 1 (PD-L1) in MSCs and cytotoxic T-cell-associated Ag-4 (CTLA-4) in T-cells and increased interleukin-10 and prostaglandin E2 production. Interestingly,we observed differences in the production of cytokines and surface and secreted molecules that may participate in T-cell immunosuppression in co-cultures in the presence of DT-MSCs compared with BM-MSCs. Importantly,MSCs from four sources favored the generation of T-cell subsets displaying the regulatory phenotypes CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+. Our results in vitro indicate that,in addition to BM-MSCs,MSCs from all of the dental sources analyzed in this study might be candidates for future therapeutic applications. View Publication

BACKGROUND The inhibition of Janus kinases (JAKs) and subsequent signal transducers and activators of transcription (STATs) by tofacitinib represents a new therapeutic strategy in inflammatory bowel diseases (IBD) as clinical trials have led to approval of tofacitinib for ulcerative colitis (UC) and hint at a possible efficacy for Crohn`s disease (CD). However,the impact of tofacitinib on cellular response of monocytes,which are key players in inflammatory responses,has not been investigated so far. We aimed to analyze JAK/STAT-inhibition by tofacitinib in monocytes of IBD patients and healthy controls. METHODS Primary monocytes of IBD patients with active disease and healthy controls (n = 18) were analyzed for cytokine expression and phenotype after granulocyte macrophage colony-stimulating factor (GM-CSF)/interferon (IFN)$\gamma$-stimulation and tofacitinib pretreatment (1-1000 nM) and capacity to induce Foxp3+-regulatory T cells (Tregs) in cocultures. In total,20 UC patients and 21 CD patients were included. Additionally,dose-dependent inhibition of JAK/STAT-phosphorylation was analyzed in controls. RESULTS Pro-inflammatory costimulation with GM-CSF/IFN$\gamma$ resulted in significant tumor necrosis factor (TNF$\alpha$) and interleukin (IL)-6 increase,whereas IL-10 expression decreased in monocytes. Tofacitinib modulated the responses of activated monocytes toward a regulatory phenotype through reduced TNF$\alpha$ and IL-6 secretion and enhanced Treg induction in cocultures. However,in monocytes from active IBD patients,higher tofacitinib dosages were needed for blockade of pro-inflammatory cytokines. Tofacitinib induced stronger regulatory phenotypes in monocytes of UC patients,including more effective inhibition of pro-inflammatory pathways and better restoration of anti-inflammatory mechanisms as compared with CD-derived monocytes. CONCLUSION Tofacitinib dose-dependently reprograms monocytes toward a more regulatory cell type. This beneficial effect possibly results from selective JAK/STAT-blockade by adequate tofacitinib dosage with inhibition of pro-inflammatory responses and permission of a balance-shift toward regulatory pathways. View Publication

INTRODUCTION Bone marrow mesenchymal stem cells (BMSCs) have been extensively investigated from a perspective on cardiac regeneration therapy. The current study aimed to investigate the protective effect conferred by BMSCs in subacute myocardial injury,and to identify an appropriate BMSC reinfusion time. METHODS BMSCs were isolated from human bone marrow blood. Daunorubicin (DNR)-induced subacute myocardial models were subsequently established. The rats with DNR-induced subacute myocardial injury were injected with dexrazoxane (DZR) and/or BMSCs at varying time points,after which cardiac function was evaluated by assessing left ventricular ejection fraction (LVEF) and fraction shortening (FS). The myocardial structural changes were analyzed,after which the levels of CD3 and human leukocyte antigen DR (HLA-DR) were examined to further validate the mechanism by which BMSCs could influence subacute myocardial injury. RESULTS BMSCs combined with DZR treatment enhanced the cardiac function of rats with DNR-induced myocardial injury,as reflected by increased LVEF and FS. DNR-induced myocardial injuries were mitigated via the application of BMSCs combined with treatment of DZR,accompanied by diminished infiltration or vacuolization. Moreover,BMSCs were observed to alleviate infiltration of T lymphocyte and antigen-presenting cells,as evidenced by reduced expression of CD3 and HLA-DR. CONCLUSION Taken together,this study demonstrates that BMSCs could protect against DNR-induced myocardial injury,especially in the first three days of DNR administration. BMSCs combined with DZR exert a better therapeutic effect,but there are individual differences. View Publication

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

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 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