搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

The persistence of HIV-1 latency reservoirs in CD4+ T cells is a significant obstacle for curing HIV-1. Shock-and-kill strategies,which aim to reactivate latent HIV-1 followed by cytotoxic clearance,have shown limited success in vivo due to insufficient efficacy of latency reversal agents (LRAs) and off-target effects. Natural killer (NK) cells,with their ability to mediate cytotoxicity independent of antigen specificity,offer a promising avenue for enhancing the shock-and-kill approach. Previously,we observed that pan-caspase inhibitors induce NK cells to secrete an LRA in vitro. Here,we aimed to identify this LRA using a targeted proteomic approach. We identified lymphotoxin-α (LTα) as the key LRA secreted by NK cells following pan-caspase inhibitor treatment. LTα was shown to significantly induce HIV-1 LTR promoter activity,a hallmark of viral reactivation. Neutralization of LTα effectively abolished the observed LRA activity,confirming its central role. Moreover,cytokine-primed but not resting human primary NK cells exhibited LRA activity that could be neutralized with LTα neutralizing antibodies. Finally,pan-caspase inhibitor treatment did not decrease the ability of the cytokine-primed NK cells to kill target cells. These findings demonstrate that cytokine-primed NK cells,through LTα secretion,can effectively reactivate latent HIV-1 following pan-caspase inhibitor treatment,without compromising NK cell cytotoxicity. This highlights a potential enhancement strategy utilizing NK cells for shock-and-kill approaches in HIV-1 cure research. View Publication

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs),only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients,the antitumor immune response is defective,conferring resistance and promoting tumor progression. Therefore,the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods: Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs),at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate,progression-free survival (PFS),overall survival (OS) and performance status (PS)) were performed. Results: Univariate analysis showed that before therapy,higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs,identified patients with significantly worse survival. Furthermore,CD137⁺Tregs resulted also positively correlated with worse PS,while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab,with a significant increase in non-responsive patients during therapy. Interestingly,both CD137⁺Tregs as well as LOX-1⁺PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137−Tregs and LOX-1⁻PMN-MDSCs,respectively. Multivariate analysis revealed that the circulating LOX-1⁺PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis,as confirmed in an independent validation cohort. Conclusions: The levels of blood circulating LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy,improving patient selection and suggesting novel personalized therapies. View Publication

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration,mobilized peripheral blood,or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems,we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary,the type of medium influenced in a significant way the ICs determination of some drugs. View Publication

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses with different coreceptor usage profiles were isolated from 29 South African patients with advanced AIDS. All 24 R5 isolates were inhibited by the CCR5-specific agents,PRO 140 and RANTES,while the two X4 viruses and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor,AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells that did not express CCR5. When tested using coreceptor-transfected cell lines,one R5 virus was also able to use CXCR6,and another R5X4 virus could use CCR3,BOB/GPR15,and CXCR6. The R5X4 and X4 viruses contained more-diverse V3 loop sequences,with a higher overall positive charge,than the R5 viruses. Hence,some HIV-1 subtype C viruses are able to use CCR5,CXCR4,or both CXCR4 and CCR5 for entry,and they are sensitive to specific inhibitors of entry via these coreceptors. These observations are relevant to understanding the rapid spread of HIV-1 subtype C in the developing world and to the design of intervention and treatment strategies. View Publication

We report the systematic use of large-scale cDNA microarrays to study the gene expression profiles of primary human peripheral blood monocytes (MONO) in comparison with in vitro-differentiated,M-CSF-induced MONO-derived macrophages (MAC) and primary human alveolar MAC (AM),obtained by bronchoalveolar lavage from the lungs of normal volunteers. These studies revealed large-scale differences in the gene expression profile between both MAC types (MAC and AM) and MONO. In addition,large differences were observed in the gene expression profiles of the two MAC types. Specifically,21% of genes on the array (2904 out of 13,582) were differentially expressed between AM and MONO,and 2229 out of 13,583 probes were differentially expressed between MAC and AM. Our expression data show remarkable differences in gene expression between different MAC subpopulations and emphasize the heterogeneity of different MAC populations. This study underscores the need to scrutinize models of MAC biology for relevance to specific disease processes. View Publication

BACKGROUND: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However,the involvement of bronchial epithelial cells in this process should also be investigated. METHODS: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) beta1 as an inducer of tissue remodeling for 21 days,and measured gel size,histological changes,and expression of factors related to extracellular matrix homeostasis. RESULTS: TGF-beta1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-beta1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium,suggesting the induction of epithelial-mesenchymal transition. In addition,the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. CONCLUSION: Our findings indicate that TGF-beta1 can affect both epithelial and mesenchymal cells,and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells,fibroblasts,and their interactions in the airway remodeling process. View Publication

BACKGROUND Granulocyte colony-stimulating factor (G-CSF) is a pro-inflammatory cytokine that stimulates myeloid stem cell maturation,proliferation,and migration into circulation. Despite being a known growth factor,the impact of G-CSF on solid tumours has not been well examined. G-CSF receptor (G-CSFR) is expressed by some tumours,and thus the aim of this study was to examine the expression and impact of G-CSF and G-CSFR on gastrointestinal tumours. METHODS In this study,G-CSF expression was examined in human gastric and colon tumours and by tumour-derived stromal myofibroblasts and carcinoma cells. G-CSFR expression was examined on carcinoma cells isolated from human tissues. The effects of G-CSF on gastric and colon carcinoma cell proliferation,migration,and signalling were examined. RESULTS G-CSFR was highly expressed in 90% of human gastric and colon carcinomas. G-CSF was also found to be highly produced by stromal myofibroblasts and carcinoma cells. Exposure of carcinoma cells to G-CSF led to increased proliferation and migration,and expansion of a sub-population of carcinoma cells expressing stem-like markers. These processes were dependent on ERK1/2 and RSK1 phosphorylation. CONCLUSIONS These data suggest that the G-CSF/R axis promotes gastric and colorectal cancer development and suggest they are potential tumour targets. View Publication

ABSTRACTBacteriophages (phages) are emerging as a viable adjunct to antibiotics for the treatment of multidrug‐resistant (MDR) bacterial infections. While intravenous phage therapy has proven successful in many cases,clinical outcomes remain uncertain due to a limited understanding of host response to phages. In this study,we conducted a comprehensive examination of the interaction between clinical‐grade phages used to treat MDR Escherichia coli and Klebsiella pneumoniae infections,and human peripheral blood immune cells. Using whole transcriptome as well as proteomic approaches,we identified a strong inflammatory response to E. coli phage vB_EcoM‐JIPh_Ec70 (herein,JIPh_Ec70) that was absent upon exposure to K. pneumoniae phage JIPh_Kp127. We confirmed that JIPh_Ec70's DNA recognition by the STING pathway was principally responsible for the activation of NF‐kB and the subsequent inflammatory response. We further show that monocytes and neutrophils play a dominant role in phage uptake,primarily through complement‐mediated phagocytosis. Significant differences in complement‐mediated phagocytosis of JIPh_Kp127 and JIPh_Ec70 were observed,suggesting that reduced recognition,phagocytosis,and immunogenicity all contribute to the significantly decreased response to JIPh_Kp127. Our findings contribute to the progress of our understanding of the innate immune response to therapeutic phages and offer potential insights into how to improve the safety and effectiveness of phage therapy. Clinical grade JIPh_Ec70 phages but not JIPh_Kp127 phages elicit a potent inflammatory response in peripheral immune cells. JIPh_Ec70 phagocytic engulfment is facilitated by complement opsonization,resulting in STING activation by phage DNA,driving an inflammatory signaling cascade. Understanding phage immunogenicity will be a key factor in developing effective phage therapies in the coming years. View Publication

GABA B receptor (GABA B R) activation is known to alleviate pain by reducing neuronal excitability,primarily through inhibition of high voltage‐activated (HVA) calcium (Ca V 2.2) channels and potentiating G protein–coupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons,emerging strategies to develop,study,and characterise human pluripotent stem cell (hPSC)‐derived sensory neurons present a promising alternative. In this study,hPSCs were efficiently differentiated into peripheral DRG‐induced sensory neurons (iSNs) using a combined chemical and transcription factor‐driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A,ISLET1,and PRPH,in addition to GABA B R and ion channels including Ca V 2.2 and GIRK1 in iSNs. Functional characterisation of GABA B R was conducted using whole‐cell patch clamp electrophysiology,assessing neuronal excitability under current‐clamp conditions in the absence and presence of GABA B R agonists baclofen and α‐conotoxin Vc1.1. Both baclofen (100 μM) and Vc1.1 (1 μM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage‐clamp mode,baclofen and Vc1.1 inhibited HVA Ca 2+ channel currents,which were attenuated by the selective GABA B R antagonist CGP 55845. However,modulation of GIRK channels by GABA B Rs was not observed in the presence of baclofen or Vc1.1,suggesting that functional GIRK1/2 channels were not coupled to GABA B Rs in hPSC‐derived iSNs. This study is the first to report GABA B R modulation of membrane excitability in iSNs by baclofen and Vc1.1,highlighting their potential as a future model for studying analgesic compounds. View Publication

Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair,their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells,derived CPECs (dCPECs) display canonical properties and dynamic motile multiciliated phenotypes that interact with Aβ uptake. Single dCPEC transcriptomes over time correlate well with human organoid and fetal CPECs,while pseudotemporal and cell cycle analyses highlight the direct CPEC origin from neuroepithelial cells. In addition,time series analyses define metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints,followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contract. These temporal patterns are then confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs,these findings suggest dynamic models of ChP support for the developing human brain. Subject terms: Differentiation,Neural stem cells,Functional clustering,Cell fate and cell lineage View Publication

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover,CaP metastasizes to the bone marrow,which harbors a rich source of lipids stored within adipocytes. Here,we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique,we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition,larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction,the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes,indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction. View Publication