搜索结果: 'gmp t cell activator'

BACKGROUND Neutrophils are the most abundant innate immune cells in the circulating blood,and they act as the first responder against bacterial and fungal infection. However,accumulation of activated neutrophils can cause severe inflammation and tissue damage. Recently,neutrophil trogocytosis or membrane transfer with neighboring cells was reported to modulate immune responses. Extracellular cold-inducible RNA binding protein (eCIRP) is a newly identified damage-associated molecular pattern (DAMP). eCIRP can activate neutrophils to be more pro-inflammatory. This study aimed to identify the role of eCIRP in neutrophil trogocytosis during their trans-endothelial migration. METHODS A trans-endothelial migration (TEM) assay using bone marrow neutrophils and mouse primary lung vascular endothelial cells was conducted using transwell chambers and neutrophil trogocytosis was assessed in vitro. In an in vivo mouse model of acute lung injury,neutrophil trogocytosis was assessed from bronchoalveolar lavage fluid. RESULTS In TEM assay,the trogocytosis of neutrophils occurred during trans-endothelial migration and eCIRP significantly increased the percentage of these neutrophils. The trogocytosed neutrophils acquired the endothelial membrane containing junctional adhesion molecule-C (JAM-C) and VE-cadherin,and these membrane patches were polarized by Mac-1 binding. Furthermore,eCIRP-induced JAM-C positive trogocytosed neutrophils are more pro-inflammatory than the JAM-C negative counterpart. JAM-C positive trogocytosed neutrophils were also observed in the bronchoalveolar lavage fluid of a mouse model of acute lung injury. CONCLUSION These data suggest that during the paracellular trans-endothelial migration of neutrophils in response to inflammation,eCIRP induces trogocytosis of neutrophils,and the trogocytosed neutrophils exhibit an exaggerated pro-inflammatory phenotype promoting acute lung injury. View Publication

The prostate is a gland that contributes to men's fertility. It is highly responsive to androgens and is often the site of carcinogenesis,as prostate cancer is the most frequent cancer in men in over a hundred countries. To study the normal prostate,few in vitro models exist,and most of them do not express the androgen receptor (AR). To overcome this issue,prostate epithelial cells can be grown in primary culture ex vivo in 2- and 3-dimensional culture (organoids). However,methods to purify these cells often require flow cytometry,thus necessitating specialized instruments and expertise. Herein,we present a detailed protocol for the harvest,purification,and primary culture of mouse prostate epithelial cells to grow prostate organoids ex vivo. This protocol does not require flow cytometry approaches,facilitating its implementation in most research laboratories,and organoids grown with this protocol are highly responsive to androgens. In summary,we present a new simple method that can be used to grow prostate organoids that recapitulate the androgen response of this gland in vivo. View Publication

INTRODUCTION Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently,cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping,which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS The immunomagnetic negative selection was used for the enrichment of B and T cells fractions,and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis,principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as $\beta$-carotene. In addition,the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability,the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system. View Publication

Juvenile dermatomyositis (JDM) is a pediatric autoimmune disease associated with characteristic rash and proximal muscle weakness. To gain insight into differential lymphocyte gene expression in JDM,peripheral blood mononuclear cells from 4 new-onset JDM patients and 4 healthy controls were sorted into highly enriched lymphocyte populations for RNAseq analysis. NK cells from JDM patients had substantially greater differentially expressed genes (273) than T (57) and B (33) cells. Upregulated genes were associated with the innate immune response and cell cycle,while downregulated genes were associated with decreased ribosomal RNA. Suppressed ribosomal RNA in JDM NK cells was validated by measuring transcription and phosphorylation levels. We confirmed a population of low ribosome expressing NK cells in healthy adults and children. This population of low ribosome NK cells was substantially expanded in 6 treatment-na{\{i}}ve JDM patients and was associated with decreased NK cell degranulation. The enrichment of this NK low ribosome population was completely abrogated in JDM patients with quiescent disease. Together these data suggest NK cells are highly activated in new-onset JDM patients with an increased population of low ribosome expressing NK cells which correlates with decreased NK cell function and resolved with control of active disease." View Publication

Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here,we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample,and 10?— Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein,we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development. For complete details on the use and execution of this protocol,please refer to Nozais et al. (2021). View Publication

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here,we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs,which are the most potent subsets,it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol,please refer to Liang et al. (2020) and Qiu et al. (2021). View Publication

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here,we show for the first time that G2.2,a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide,selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+,Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact,G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers,e.g.,CD133,DCMLK1,LGR5,and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly,G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly,G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions,blood coagulation profile,and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro,in vivo,and ex vivo mechanistic studies,we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38$\alpha$,uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence,G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs. View Publication

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA,but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages,which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™,iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints,and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery,mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1,2 and 4weeks]). 4 weeks after injection,mice were imaged by MRI,and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS,CD206,F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory,homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively,and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs,although some ferumoxytol label was detected in proximity to MSCs in macrophages,likely due to phagocytosis of apoptotic MSCs,increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time,we show that MSC-treated mice had increased ratios of {\%}CD206+: {\%}F4/80+ (homeostatic macrophages) (p{\textless}0.05),and decreased ratios of {\%}iNOS+: {\%}F4/80+ macrophages (p{\textless}0.01),supporting our hypothesis that MSCs may modulate synovial inflammation. View Publication

BACKGROUND Human pancreata contain many types of cells,such as endocrine islets,acinar,ductal,fat,and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore,whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5{\%} human platelet lysate (hPL). IPTD-MSCs were found to be highly pure,with {\textgreater} 95{\%} positive for CD90,CD105,and CD73,and negative for CD45,CD34,CD14,and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes,chondrocytes,and osteoblasts in vitro,underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-$\alpha$,gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased,compared to control. In contrast,treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly,a combination of TNF-$\alpha$ and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$ contained higher levels of pro-angiogenic (VEGF,IL-6,and IL-8) compared to controls,promoting angiogenesis of human endothelial cells in vitro. In contrast,levels of MCP-1,a pro-inflammatory cytokine,were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$. CONCLUSIONS The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application. View Publication

Reduced serum testosterone (T),or hypogonadism,affects millions of men and is associated with many pathologies,including infertility,cardiovascular diseases,metabolic syndrome,and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However,TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus,there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs),proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs,the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively,human induced pluripotent stem cells (hiPSCs),which are expandable in culture and have the potential to differentiate into all somatic cell types,have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis,synthesized T rather than cortisol,secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol,and displayed ultrastructural features resembling LCs. By contrast,hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration. View Publication

Spinal cord injury (SCI) is one of the most debilitating injuries,and transplantation of stem cells in a scaffold is a promising strategy for treatment. However,stem cell treatment of SCI has been severely impaired by the increased generation of reactive oxygen species in the lesion microenvironment,which can lead to a high level of stem cell death and dysfunction. Herein,a MnO2 nanoparticle (NP)-dotted hydrogel is prepared through dispersion of MnO2 NPs in a PPFLMLLKGSTR peptide modified hyaluronic acid hydrogel. The peptide-modified hydrogel enables the adhesive growth of mesenchymal stem cells (MSCs) and nerve tissue bridging. The MnO2 NPs alleviate the oxidative environment,thereby effectively improving the viability of MSCs. Transplantation of MSCs in the multifunctional gel generates a significant motor function restoration on a long-span rat spinal cord transection model and induces an in vivo integration as well as neural differentiation of the implanted MSCs,leading to a highly efficient regeneration of central nervous spinal cord tissue. Therefore,the MnO2 NP-dotted hydrogel represents a promising strategy for stem-cell-based therapies of central nervous system diseases through the comprehensive regulation of pathological microenvironment complications. View Publication

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism,some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study,we investigated the damage to BM stromal cells induced by a chemotherapy drug,daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence,generation of intracellular reactive oxygen species,production of cytokines and chemokines,and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function,and negative regulators of reactive oxygen species. Surprisingly,it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore,coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2'-deoxyguanosine in hematopoietic cells. Overall,our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells. View Publication