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

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis,with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN,NK cells and ILC3 were the prominent subpopulations. Among the ILC3s,we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients,NK cells were prominent,and proportions of ILC3 subsets were low. In tumors and TD-LN,NK cells display reduced levels of NCR (Natural cytotoxicity receptors),despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN,all cytokine activated ILC populations produced TNF$\alpha$ in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR,but cytokine stimulation restored their functionality. View Publication

As the prognosis of invasive aspergillosis remains unacceptably poor in patients undergoing hematopoietic stem cell transplantation (HSCT),there is a growing interest in the adoptive transfer of antifungal effector cells,such as Natural Killer (NK) cells. Because immunosuppressive agents are required in most HSCT recipients,knowledge of the impact of these compounds on the antifungal activity of NK cells is a prerequisite for clinical trials. We,therefore,assessed the effect of methylprednisolone (mPRED),cyclosporin A (CsA) and mycophenolic acid (MPA) at different concentrations on proliferation,apoptosis/necrosis,and the direct and indirect anti-Aspergillus activity of human NK cells. Methylprednisolone decreased proliferation and increased apoptosis of NK cells in a significant manner. After seven days,a reduction of viable NK cells was seen for all three immunosuppressants,which was significant for MPA only. Cyclosporin A significantly inhibited the direct hyphal damage by NK cells in a dose-dependent manner. None of the immunosuppressive compounds had a major impact on the measured levels of interferon-$\gamma$,granulocyte-macrophage colony-stimulating factor and RANTES (regulated on activation,normal T cell expressed and secreted; CCL5). Our data demonstrate that commonly used immunosuppressive compounds have distinct effects on proliferation,viability and antifungal activity of human NK cells,which should be considered in designing studies on the use of NK cells for adoptive antifungal immunotherapy. View Publication

Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However,whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here,we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua,where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy,we used Homeobox a11 (Hoxa11)-deficient mice,having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11-/- mice results in stromal expansion,gland formation,and marked decidualization otherwise absent in Hoxa11-/- mice. Moreover,in Hoxa11+/- mice,which have increased pregnancy losses,BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively,these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma,thereby playing important roles in decidualization and pregnancy. View Publication

PIEZO1 is critical to numerous physiological processes,transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1,we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging,our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore,the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca2+-sensitive HaloTag ligands,achieving temporal resolution approaching that of patch clamp electrophysiology. Finally,we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems,with potential for elucidating disease mechanisms and targeted drug screening. PIEZO1 is critical in numerous physiological processes,but monitoring its activity and localization in cells can be challenging. Here,the authors present a chemogenetic platform to visualize endogenous human PIEZO1 localization and activity in native cellular conditions,expanding the knowledge on mechanotransduction across single cells and tissue organoids. View Publication

Tissue-repair regulatory T cells (trTregs) comprise a specialized cell subset essential for tissue homeostasis and repair. While well-studied in sterile injury models,their role in infection-induced tissue damage and antimicrobial immunity is less understood. We investigated trTreg dynamics during acute Trypanosoma cruzi infection,marked by extensive tissue damage and strong CD8+ immunity. Unlike sterile injury models,trTregs significantly declined in secondary lymphoid organs and non-lymphoid target tissues during infection,correlating with systemic and local tissue damage,and downregulation of function-associated genes in skeletal muscle. This decline was linked to decreased systemic IL-33 levels,a key trTreg growth factor,and promoted by the Th1 cytokine IFN-γ. Early recombinant IL-33 treatment increased trTregs,type 2 innate lymphoid cells,and parasite-specific CD8+ cells at specific time points after infection,leading to reduced tissue damage,lower parasite burden,and improved disease outcome. Our findings not only provide novel insights into trTregs during infection but also highlight the potential of optimizing immune balance by modulating trTreg responses to promote tissue repair while maintaining effective pathogen control during infection-induced injury. Author summaryDuring Chagas’ disease,caused by the protozoan Trypanosoma cruzi,severe organ damage is generated by the interplay between the parasite and the immune response. In our investigation,we examined the role of tissue-repair regulatory T cells (trTregs) during the acute phase of T. cruzi infection in mice. Surprisingly,we observed a reduction in trTregs at the peak of tissue damage,contrary to their usual accumulation after injury in other contexts. This decline aligned with decreased levels of interleukin-33,a critical factor for trTreg survival,and was promoted by the effector cytokine IFN-γ. Administering interleukin-33 at early infection times not only boosted trTregs but also expanded other reparative and antiparasitic immune cells. Consequently,these treated mice exhibited reduced damage and lower parasite levels in tissues. Our findings provide new insights into how trTreg function during infection-related injury,paving the way for strategies that balance the immune response to support tissue repair without weakening the body’s ability to fight the infection. This approach could have broader implications for treating infectious diseases and conditions involving tissue damage. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis,basophils accumulate in secondary lymphoid organs (SLO),amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here,we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis,both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation,maintenance,and function in SLO were dependent on PD-L1 and IL-4 in basophils,which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis. Basophils have been implicated in systemic lupus erythematosus (SLE),as evidenced by the fact that basophil-deficient mice do not develop the disease. Here,the authors demonstrate that PD-L1 and IL-4 expression in basophils promotes the pathogenic accumulation of follicular helper T cells in patients with SLE and murine models. View Publication

AbstractImmunodeficient mouse models are widely used for the assessment of human normal and leukemic stem cells. Despite the advancements over the years,reproducibility,as well as the differences in the engraftment of human cells in recipient mice remains to be fully resolved. Here,we used various immunodeficient mouse models to characterize the effect of donor–recipient sex on the engraftment of the human leukemic and healthy cells. Donor human cells and recipient immunodeficient mice demonstrate sex‐specific engraftment levels with significant differences observed in the lineage output of normal CD34+ hematopoietic stem and progenitor cells upon xenotransplantation. Intriguingly,human female donor cells display heightened sensitivity to the recipient mice's gender,influencing their proliferation and resulting in significantly increased engraftment in female recipient mice. Our study underscores the intricate interplay taking place between donor and recipient characteristics,shedding light on important considerations for future studies,particularly in the context of pre‐clinical research. View Publication

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

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed,at least in part,to the presence of oleuropein and hydroxytyrosol. In this study,oleuropein and hydroxytyrosol,major phenolic compound of olive oil,was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay),cell viability (Guava ViaCount assay),cell apoptosis,cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability,inhibited cell proliferation,and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 mug/mL of oleuropein or 50 mug/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G(1) to S phase transition manifested by the increase of cell number in G(0)/G(1) phase. 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

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

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture,it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition,we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted,aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis,aligned collagen substrates choreographed ordered matrix mineralization. Likewise,myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures. View Publication