参考文献

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health,with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom,phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding ({\textgreater}103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium,it is the relatively rapid and higher levels of Stx2a induction,compared to Stx2c,that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium. View Publication

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset,progression,and treatment of a broad spectrum of inflammatory conditions. In this study,we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form,as well as in its partially demannosylated and partially desialylated form,on HEK293 cells expressing TLR-8,and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells,N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent,i.e.,chloroquine (CQN),that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8,it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold,p {\textless} 0.0001),partially demannosylated (0.3-fold,p {\textless} 0.0001) and partially desialylated (0.4-fold,p {\textless} 0.0001) N-glycans. Furthermore,the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN,the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune,infections or other inflammatory disorders. View Publication

Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including increased regulatory T cells (Tregs),prevention of auto-immune disease and ability to reduce growth of human cancer cells. We have previously shown in large cohorts that elevated serum mannose levels are associated with future development of type 2 diabetes (T2D) and cardiovascular disease. However,potential direct effects of mannose on insulin sensitivity in vivo or in vitro are unknown. We here show that administration of mannose (0.1 g/kg BW twice daily) for one week in man did not elicit negative effects on meal-modified glucose tolerance,markers of inflammation or insulin levels. Tregs number and insulin signaling in human liver cells were unchanged. These data suggest that mannose is a marker,and not a mediator,of insulin resistance. To verify this,we examined serum mannose levels during long-term euglycemic hyperinsulinemic clamps in non-diabetic and T2D individuals. Mannose was reduced by insulin infusion in proportion to whole-body insulin sensitivity. Thus,mannose is a biomarker of insulin resistance which may be useful for the early identification of diabetic individuals with insulin resistance and increased risk of its complications. View Publication

The placenta and umbilical cord are pre-eminent candidate sources of mesenchymal stem cells (MSCs). However,placenta-derived MSCs (P-MSCs) showed greater proliferation capacity than umbilical cord-derived MSCs (UC-MSCs) in our study. We investigated the drivers of this proliferation difference and elucidated the mechanisms of proliferation regulation. Proteomic profiling and Gene Ontology (GO) functional enrichment were conducted to identify candidate proteins that may influence proliferation. Using lentiviral or small interfering RNA infection,we established overexpression and knockdown models and observed changes in cell proliferation to examine whether a relationship exists between the candidate proteins and proliferation capacity. Real-time quantitative polymerase chain reaction,western blot analysis,and immunofluorescence assays were conducted to elucidate the mechanisms underlying proliferation. Six candidate proteins were selected based on the results of proteomic profiling and GO functional enrichment. Through further validation,yes-associated protein 1 (YAP1) and $\beta$-catenin were confirmed to affect MSCs proliferation rates. YAP1 and $\beta$-catenin showed increased nuclear colocalization during cell expansion. YAP1 overexpression significantly enhanced proliferation capacity and upregulated the expression of both $\beta$-catenin and the transcriptional targets of Wnt signaling,CCND1,and c-MYC,whereas silencing $\beta$-catenin attenuated this influence. We found that YAP1 directly interacts with $\beta$-catenin in the nucleus to form a transcriptional YAP/$\beta$-catenin/TCF4 complex. Our study revealed that YAP1 and $\beta$-catenin caused the different proliferation capacities of P-MSCs and UC-MSCs. Mechanism analysis showed that YAP1 stabilized the nuclear $\beta$-catenin protein,and also triggered the Wnt/$\beta$-catenin pathway,promoting proliferation. View Publication

The tumor microenvironment (TME) comprises complex interactions of multiple cell types that determines cell behavior and metabolism such as nutrient competition and immune suppression. We discuss the various types of heterogeneity that exist in solid tumors,and the complications this invokes for studies of TME. As human subjects and in vivo model systems are complex and difficult to manipulate,simpler 3D model systems that are compatible with flexible experimental control are necessary for studying metabolic regulation in TME. Stable Isotope Resolved Metabolomics (SIRM) is a valuable tool for tracing metabolic networks in complex systems,but at present does not directly address heterogeneous metabolism at the individual cell level. We compare the advantages and disadvantages of different model systems for SIRM experiments,with a focus on lung cancer cells,their interactions with macrophages and T cells,and their response to modulators in the immune microenvironment. We describe the experimental set up,illustrate results from 3D cultures and co-cultures of lung cancer cells with human macrophages,and outline strategies to address the heterogeneous TME. View Publication

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation,allowing symbiotic co-existence with the intestinal microbiota. However,upon acute events such as epithelial damage and concomitant influx of microbes,these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here,we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly,these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition,Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together,our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling. View Publication

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here,we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFR$\alpha$+Sca1+ (P$\alpha$S) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition,chromatin structure and gene expression profiles,including the reduced expression of adhesion-related genes. Functionally,the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs,leading to reduced quiescence and diminished myeloid output. Most notably,HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus,genetic alterations in the BM niche lead to long-term functional changes of HSCs. View Publication

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues,large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells,the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs,fibroblasts,and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival,while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs. View Publication

Gelatin coatings are effective in increasing the retention of MSCs injected into the heart and minimizing the damage from acute myocardial infarction (AMI),but early studies suffered from low fractions of the MSCs coated with gelatin. Biotinylation of the MSC surface is a critical first step in the gelatin coating process,and in this study,we evaluated the use of biotinylated cholesterol lipid insertion" anchors as a substitute for the covalent NHS-biotin anchors to the cell surface. Streptavidin-eosin molecules where eosin is our photoinitiator can then be bound to the cell surface through biotin-streptavidin affinity. The use of cholesterol anchors increased streptavidin density on the surface of MSCs further driving polymerization and allowing for an increased fraction of MSCs coated with gelatin (83{\%}) when compared to NHS-biotin (52{\%}). Additionally the cholesterol anchors increased the uniformity of the coating on the MSC surface and supported greater numbers of coated MSCs even when the streptavidin density was slightly lower than that of an NHS-biotin anchoring strategy. Critically this improvement in gelatin coating efficiency did not impact cytokine secretion and other critical MSC functions. Proper selection of the cholesterol anchor and the biotinylation conditions supports cellular function and densities of streptavidin on the MSC surface of up to {\~{}}105 streptavidin molecules/$\mu$m2 . In all these cholesterol anchors offer an effective path towards the formation of conformal coatings on the majority of MSCs to improve the retention of MSCs in the heart following AMI." View Publication

The discovery and study of human neural stem cells has advanced our understanding of human neurogenesis,and the development of novel therapeutics based on neural cell replacement. Here,we describe methods to culture and cryopreserve human neural stem cells (hNSCs) for expansion and banking. Importantly,the protocols ensure that the multipotency of hNSCs is preserved to enable differentiation to neurons and supporting neuroglia. View Publication

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs),though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms,including platelet activation and neutrophil-extracellular trap formation; therefore,an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here,we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC),a sulfhydryl-containing compound with broad effects on glutathione replenishment,free radical scavenging,and reducing disulfide bonds,to investigate its antithrombotic effects in the context of MPN. Strikingly,NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo,we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore,NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN. View Publication

Cancer stem cells are critical for cancer initiation,development,and treatment resistance. Our understanding of these processes,and how they relate to glioblastoma heterogeneity,is limited. To overcome these limitations,we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells,and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells,and,using RNA velocity,is often the originator of the other cell types. Finally,we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development,suggests a possible origin for glioblastoma hierarchy,and helps to identify cancer stem cell-specific targets. View Publication