参考文献

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

Here,we report on the results of a phase I/II trial (NCT00490529) for patients with mantle cell lymphoma who,having achieved remission after immunochemotherapy,were vaccinated with irradiated,CpG-activated tumor cells. Subsequently,vaccine-primed lymphocytes were collected and reinfused after a standard autologous stem cell transplantation (ASCT). The primary endpoint was detection of minimal residual disease (MRD) within 1 yr after ASCT at the previously validated threshold of ≥1 malignant cell per 10,000 leukocyte equivalents. Of 45 evaluable patients,40 (89{\%}) were found to be MRD negative,and the MRD-positive patients experienced early subsequent relapse. The vaccination induced antitumor CD8 T cell immune responses in 40{\%} of patients,and these were associated with favorable clinical outcomes. Patients with high tumor PD-L1 expression after in vitro exposure to CpG had inferior outcomes. Vaccination with CpG-stimulated autologous tumor cells followed by the adoptive transfer of vaccine-primed lymphocytes after ASCT is feasible and safe. View Publication

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