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INTRODUCTION Complexins (CPLXs),initially identified in neuronal presynaptic terminals,are cytoplasmic proteins that interact with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis,their distribution and role in immune cells are still unclear. In this study,we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes. METHODS We examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo,we analyzed the immune phenotype of CPLX2 KO mice. Furthermore,antibodies secretion from the peritoneal cavity,spleen,and bone marrow cells of wild-type (WT) and CPLX2 KO mice were determined. RESULTS CPLX2 was detected in B cells but not in T cells,while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes,serum concentrations of immunoglobulin G (IgG),IgA,IgM,and IgE were measured in WT and CPLX2 KO mice using enzyme-linked immunosorbent assay. The level of IgM,which mainly consists of natural antibodies,was higher in KO mice than that in WT mice,while the levels of other antibodies were similar in both types of mice. Additionally,we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody-secreting cells (ASCs) of CPLX2 KO mice. CONCLUSION Our data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication

PURPOSE We investigated the effect of the racemic $\beta$-hydroxybutyrate precursor,R,S-1,3-butanediol (BD),on T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged,strenuous exercise. METHODS A repeated-measures,randomised,crossover study was conducted in nine healthy,trained male cyclists (age,26.7 ± 5.2 years; VO2peak,63.9 ± 2.5 mL kg-1 min-1). Participants ingested 0.35 g kg-1 of BD or placebo 30 min before and 60 min during 85 min of steady-state (SS) exercise,which preceded a {\~{}} 30 min time-trial (TT) (7 kJ kg-1). Blood samples were collected at pre-supplement,pre-exercise,post-SS,post-TT and 1-h post-TT. Whole blood cultures were stimulated with Staphylococcal enterotoxin B (SEB) for 24 h to determine T-cell-related interleukin (IL)-4,IL-10 and interferon (IFN)-$\gamma$ mRNA expression within isolated PBMCs in vitro. RESULTS Serum cortisol,total circulating leukocyte and lymphocyte,and T-cell subset concentrations were similar between trials during exercise and recovery (all p {\textgreater} 0.05). BD ingestion increased T-cell-related IFN-$\gamma$ mRNA expression compared with placebo throughout exercise and recovery (p = 0.011); however,IL-4 and IL-10 mRNA expression and the IFN-$\gamma$/IL-4 mRNA expression ratio were unaltered (all p {\textgreater} 0.05). CONCLUSION Acute hyperketonaemia appears to transiently amplify the initiation of the pro-inflammatory T-cell-related IFN-$\gamma$ response to an immune challenge in vitro during and following prolonged,strenuous exercise; suggesting enhanced type-1 T-cell immunity at the gene level. View Publication

Although a link between the gut microbiota and alcohol-related liver diseases (ALDs) has previously been suggested,the causative effects of specific taxa and their functions have not been fully investigated to date. Here,we analyze the gut microbiota of 410 fecal samples from 212 Korean twins by using the Alcohol Use Disorders Identification Test (AUDIT) scales to adjust for host genetics. This analysis revealed a strong association between low AUDIT scores and the abundance of the butyrate-producing genus Roseburia. When Roseburia spp. are administered to ALD murine models,both hepatic steatosis and inflammation significantly improve regardless of bacterial viability. Specifically,the flagellin of R. intestinalis,possibly through Toll-like receptor 5 (TLR5) recognition,recovers gut barrier integrity through upregulation of the tight junction protein Occludin and helps to restore the gut microbiota through elevated expression of IL-22 and REG3$\gamma$. Our study demonstrates that Roseburia spp. improve the gut ecosystem and prevent leaky gut,leading to ameliorated ALDs. View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Protein disulfide isomerase (PDI,PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies,although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4,CHOP,and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors. View Publication

Lambda interferons (IFN-$\lambda$s) are a major component of the innate immune defense to viruses,bacteria,and fungi. In human liver,IFN-$\lambda$ not only drives antiviral responses,but also promotes inflammation and fibrosis in viral and non-viral diseases. Here we demonstrate that macrophages are primary responders to IFN-$\lambda$,uniquely positioned to bridge the gap between IFN-$\lambda$ producing cells and lymphocyte populations that are not intrinsically responsive to IFN-$\lambda$. While CD14+ monocytes do not express the IFN-$\lambda$ receptor,IFNLR1,sensitivity is quickly gained upon differentiation to macrophages in vitro. IFN-$\lambda$ stimulates macrophage cytotoxicity and phagocytosis as well as the secretion of pro-inflammatory cytokines and interferon stimulated genes that mediate immune cell chemotaxis and effector functions. In particular,IFN-$\lambda$ induced CCR5 and CXCR3 chemokines,stimulating T and NK cell migration,as well as subsequent NK cell cytotoxicity. Using immunofluorescence and cell sorting techniques,we confirmed that human liver macrophages expressing CD14 and CD68 are highly responsive to IFN-$\lambda$ ex vivo. Together,these data highlight a novel role for macrophages in shaping IFN-$\lambda$ dependent immune responses both directly through pro-inflammatory activity and indirectly by recruiting and activating IFN-$\lambda$ unresponsive lymphocytes. View Publication

Hodgkin Lymphoma (HL) is a malignancy that frequently affects young adults. Although,there are effective treatments not every patient responds,necessitating the development of novel therapeutic approaches,especially for relapsed and refractory cases. The two TNF receptor family members CD30 and CD137 are expressed on Hodgkin and Reed Sternberg (HRS) cells,the malignant cells in HL. We found that this co-expression is specific for HRS cells. Based on this discovery we developed a bispecific antibody that binds preferentially to the CD30,CD137-double positive HRS cells. The CD30,CD137 bispecific antibody gets internalized into HRS cells opening up the possibility to use it as a carrier for a toxin. This antibody also induces antibody-dependent,cell-mediated cytotoxicity in CD30,CD137-double positive HRS cells. The enhances specificity of the CD30,CD137 bispecific antibody to HRS cells makes it a promising candidate for development as a novel HL treatment. View Publication

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists,RO6871304,and RO6871085,as well as a CB2R inverse agonist,RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions,binding,cell signaling ({\ss}-arrestin and cAMP) and early absorption,distribution,metabolism,excretion,and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity,in comparison to the reported CB2R ligand,HU910,using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice,and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils,respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R {\textgreater} CB1R,with both ligands acting as full agonists in cAMP and {\ss}-arrestin assays (EC50s in low nM range). When tested in EIU,topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist,RO6851228. The CB2R agonist,RO6871304,decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-,and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085,as well as HU910,decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases. View Publication

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously,iPSCs from the same patient were differentiated into ECs. On day 34,the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54,and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible. View Publication

Abdominal aortic aneurysm (AAA) is a prevalent life-threatening disease,where aortic wall degradation is mediated by accumulated immune cells. Although cytokines regulate inflammation within the aorta,their contribution to AAA via distant alterations,particularly in the control of hematopoietic stem cell (HSC) differentiation,remains poorly defined. Here we report a pathogenic role for the interleukin-27 receptor (IL-27R) in AAA,as genetic ablation of IL-27R protects mice from the disease development. Mitigation of AAA is associated with a blunted accumulation of myeloid cells in the aorta due to the attenuation of Angiotensin II (Ang II)-induced HSC expansion. IL-27R signaling is required to induce transcriptional programming to overcome HSC quiescence and increase differentiation and output of mature myeloid cells in response to stress stimuli to promote their accumulation in the diseased aorta. Overall,our studies illuminate how a prominent vascular disease can be distantly driven by a cytokine-dependent regulation of bone marrow precursors. View Publication

Microenvironmental factors such as oxygen concentration mediate key effects on the biology of mesenchymal stromal cells (MSCs). Herein,we performed an in-depth characterization of the metabolic behavior of MSCs derived from the placenta,umbilical cord,and adipose tissue (termed hPMSCs,UC-MSCs,and AD-MSCs,respectively) at physiological (hypoxic; 5{\%} oxygen [O2]) and standardized (normoxic; 21{\%} O2) O2 concentrations using chemical isotope labeling liquid chromatography-mass spectrometry. 12C- and 13C-isotope dansylation (Dns) labeling was used to analyze the amine/phenol submetabolome,and 2574 peak pairs or metabolites were detected and quantified,from which 52 metabolites were positively identified using a library of 275 Dns-metabolite standards; 2189 metabolites were putatively identified. Next,we identified six metabolites using the Dns library,as well as 14 hypoxic biomarkers from the human metabolome database out of 96 altered metabolites. Ultimately,metabolic pathway analyses were performed to evaluate the associated pathways. Based on pathways identified using the Kyoto Encyclopedia of Genes and Genomes,we identified significant changes in the metabolic profiles of MSCs in response to different O2 concentrations. These results collectively suggest that O2 concentration has the strongest influence on hPMSCs metabolic characteristics,and that 5{\%} O2 promotes arginine and proline metabolism in hPMSCs and UC-MSCs but decreases gluconeogenesis (alanine-glucose) rates in hPMSCs and AD-MSCs. These changes indicate that MSCs derived from different sources exhibit distinct metabolic profiles. View Publication