搜索结果: 'gmp t cell activator'

Prime editing is a promising approach for correcting pathogenic variants,but its efficiency remains variable across genomic contexts. Here,we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region,4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first,the persistent challenge of AT-rich target sequence correction even with optimized editors,and second,the performance of 4-FEN suggests its potential value for other genomic targets. View Publication

Particulate matters such as diesel exhaust particles induce oxidative stress in cells and thereby have a negative impact on health. The aim of this study was to test whether the membrane-permeable,anti-inflammatory metabolite 4-Octyl Itaconate can counteract the oxidative stress induced by diesel exhaust particles and to analyze the downstream-regulated pathways both in human nasal epithelial cells and PBMCs. Human nasal epithelial cells were cultured from nasal swabs,and the response of the cells to diesel exhaust particles either alone or in combination with 4-Octyl Itaconatee was investigated using RNA sequencing,qPCR,and cytokine measurement. The presence of reactive oxygen species in the cells was analyzed using CellROX staining and flow cytometric DCFDA assay. Diesel exhaust particles caused an upregulation of CYP1A1 in nasal epithelial cells. The administration of 4-Octyl Itaconate reduced the reactive oxygen species and increased the expression of antioxidant genes regulated by the transcription factor NRF2,which was also confirmed in PBMCs. IL-6 secretion from NEC was elevated by diesel exhaust particles and reduced when 4-Octyl Itaconate was administered. 4-Octyl Itaconate can reduce the diesel-exhaust-particle-induced oxidative damage by the activation of NRF2-regulated antioxidative pathways. View Publication

The origin and function of blood IgM+IgD+CD27+ B cells is controversial,and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM+IgD+CD27+: IgMhi and IgMlo B cells. Here,we confirmed this finding and compared the phenotype,transcriptome,in vitro function,and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c,CD69,IL21R,CD27,MTG,CD45RB,CD5,CD184,CD23,BAFFR,and CD38) with the IgMhi phenotypically resembling previously reported marginal zone B cells and the IgMlo resembling both na{\{i}}ve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis but differed in expression of 78 genes. Moreover IgMhi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines significantly (p {\textless} 0.05) higher frequencies (62.5{\%}) of IgMhi B cells proliferated compared with IgMlo B cells (35.37{\%}) and differentiated to antibody secreting cells (14.22{\%} for IgMhi and 7.19{\%} for IgMlo). IgMhi B cells had significantly (p {\textless} 0.0007) higher frequencies of mutations in IGHV and IGKV regions IgMlo B cells had higher usage of IGHJ6 genes (p {\textless} 0.0001) and both subsets differed in their HCDR3 properties. IgMhi B cells shared most of their shared IGH clonotypes with IgM only memory B cells and IgMlo B cells with IgMhi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM+IgD+CD27+ B cells with the IgMhi B cells having similarities with previously described marginal zone B cells that passed through germinal centers and the IgMlo B cells being the least differentiated amongst the IgM+CD27+ subsets." View Publication

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly,we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2,3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized),and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. View Publication

Human-based body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers sensory neurons) and efferent (extrafusal fibers motoneurons) cells in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling." 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

Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research,particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y),termed conditionally reprogrammed cell (CRC) technology,enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings,we then created six CF cell CRC populations,three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted,and cells were subcultured,without irradiated feeders plus Y,into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y,dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage,but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus,CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers. View Publication

Overcoming the global health threat of HIV infection requires continuous pipelines of novel drug candidates. We identified the $\gamma$-pyrone polyketides Aureothin/Neoaureothin as potent hits by anti-HIV screening of an extensive natural compound collection. Total synthesis of a structurally diverse group of Aureothin-derivatives successfully identified a lead compound ({\#}7) superior to Aureothin that combines strong anti-HIV activity (IC90{\textless}45 nM),photostability and improved cell safety. Compound {\#}7 inhibited de novo virus production from integrated proviruses by blocking the accumulation of HIV RNAs that encode the structural components of virions and include viral genomic RNAs. Thus,the mode-of-action displayed by compound {\#}7 is different from those of all current clinical drugs. Proteomic analysis indicated that compound {\#}7 does not affect global protein expression in primary blood cells and may modulate cellular pathways linked to HIV infection. Compound {\#}7 inhibited multiple HIV genotypes,including HIV-type 1 and 2 and synergistically inhibited HIV in combination with clinical reverse transcriptase and integrase inhibitors. We conclude that compound {\#}7 represents a promising new class of HIV inhibitors that will facilitate the identification of new virus-host interactions exploitable for antiviral attack and holds promise for further drug development. View Publication

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study,we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly,increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD),independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated,and inhibited or enhanced by KD of CRBN or IKZF3,respectively,independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent,as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity. View Publication

Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely,how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK),which is blocked with a selective p38$\alpha$/$\beta$ MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically,we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether,these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation. View Publication