技术资料

Aim To analyse the ability of mesenchymal stem cells (MSCs) to regulate interleukin 6 (IL-6) and transforming growth factor (TGF-$\beta$) expression in vitro under co-culture conditions in human systemic lupus erythematosus (SLE). Method This study used a post-test group design that used peripheral blood mononuclear cells (PBMCs) from SLE patients at Kariadi Hospital,Semarang,Indonesia,and MSCs from a human umbilical cord. The cells were divided into two groups. The control group of PBMCs was treated with a standard medium,and the treatment group was co-cultured with the MSCs at a 1:40 ratio. Following 24 h incubation,the levels of IL-6 and TGF-$\beta$ released in the culture medium were measured using a specific ELISA assay. Results This study showed a significant decrease in IL-6 level (p{\textless}0.05) and a significant increase in TGF-$\beta$ level (p{\textless}0.001) following 24 h of co-culture incubation of human SLE PBMCs cells and MSCs. Conclusion The PBMCs-to-MSCs ratio of 1:40 can regulate the IL-6 and TGF-$\beta$ levels in human SLE PBMCs. View Publication

COVID-19 is currently a global pandemic,but human immune responses to the virus remain poorly understood. We analyzed 125 COVID-19 patients,and compared recovered to healthy individuals using high dimensional cytometry. Integrated analysis of {\~{}}200 immune and {\~{}}50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching {\textgreater}30{\%} of circulating B cells. However,another subgroup had lymphocyte activation comparable to uninfected subjects. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. These analyses identified three immunotypes" associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19." View Publication

BACKGROUND: Despite increasing demand,current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time,labor,and cost intensive. Additionally,absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. METHODS: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained,cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy,polarized VEGF expression,establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes,RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. RESULTS: Using our 2D cytokine scarce protocol,hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless,at this stage,most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured,DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. CONCLUSIONS: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable,and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications. View Publication

Periodontitis is an irreversible,bacteria-induced,chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria,neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However,periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation,which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod,Filifactor alocis. With the discovery of these oral bacterial species,there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently,few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus,this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis,cytokine and chemokine signaling pathways,and apoptosis. Moreover,F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient,dampened p38 MAPK activation by secondary stimuli TNF$\alpha$ but not by fMLF. Functionally,the F. alocis-mediated inhibition of p38 activation by TNF$\alpha$ resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNF$\alpha$. Since the modulatory effect was characteristic of viable F. alocis only,we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses. View Publication

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here,we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C. albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors,ceramides. Mutations in the regulators render the fungus susceptible to myriocin,a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks,and the regulators are activated upon engulfment of C. albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels,one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings,therefore,indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids. View Publication

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation,here,we compared,in multiple cell and in vivo models,mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1m$\Psi$),synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1m$\Psi$ made by the modified process,while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA. View Publication

The network topology of a protein interactome is shaped by the function of each protein,making it a resource of functional knowledge in tissues and in single cells. Today,this resource is underused,as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell,thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins,each forming topological motifs called bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties display multifunctional capabilities are enriched for essential proteins and are widely expressed in other cells and tissues. Collectively the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates." View Publication

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis,regeneration and cancer,little is known about the post-translational regulation of LGR5. Here,we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation,increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically,we find that both NEDD4 and NEDD4L negatively regulate Wnt/$\beta$-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers,which subsequently enhances tumour predisposition and progression. View Publication

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells,via single-cell RNA-Seq and protein mass spectrometry,demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro,recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells,with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells,S1P ablation results in an increased production of granulocyte-macrophage progenitors,the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche. View Publication

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore,the objective was to study the effects of SCFAs on biomarkers of ISC activity,differentiation,barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET),propionate (PROP),or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments,but increased with ACET. As a result of BUT and PROP treatments,there was an increase in differentiation markers for enterocyte,Paneth,goblet,and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3$\beta$,Reg3$\gamma$,and Defb1 were stimulated by BUT and PROP,but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures,although tight junctions were influenced by BUT and PROP,but not ACET in monolayer format. Furthermore,BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall,individual SCFAs are potent stimulators of cellular gene expression,however,PROP and especially BUT show great efficacy for driving cell differentiation and gene expression. View Publication