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

Recent compelling evidence indicates that Th17 confer host immunity against a variety of microbes,including extracellular and intracellular pathogens. Therefore,understanding mechanisms for the induction and activation of Ag-specific Th17 is important for the rational design of vaccines against pathogens. To study this,we employed an in vitro system in which influenza hemagglutinin (HA) 1 was delivered to dendritic cells (DCs) via Dectin-1 using anti-human Dectin-1 (hDectin-1)-HA1 recombinant fusion proteins. We found that healthy individuals maintained broad ranges of HA1-specific memory Th17 that were efficiently activated by DCs targeted with anti-hDectin-1-HA1. Nonetheless,these DCs were not able to induce a significant level of HA1-specific Th17 responses even in the presence of the Th17-promoting cytokines IL-1? and IL-6. We further found that the induction of surface IL-1R1 expression by signals via TCRs and common ?-chain receptors was essential for naive CD4(+) T cell differentiation into HA1-specific Th17. This process was dependent on MyD88,but not IL-1R-associated kinase 1/4. Thus,interruptions in STAT3 or MyD88 signaling led to substantially diminished HA1-specific Th17 induction. Taken together,the de novo generation of pathogen-specific human Th17 requires complex,but complementary,actions of multiple signals. Data from this study will help us design a new and effective vaccine strategy that can promote Th17-mediated immunity against microbial pathogens. View Publication

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor,HSV-1 can infect certain dendritic cells,which becomes an event in the viral strategy to interfere with the host's immune system. We previously generated the HSV-1 mutant strain M6,which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype,but varied transcriptional profiles were observed,implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity,probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells,leading to a series of deviations in immune responses,ultimately generating the deficient immune phenotype observed in infected individuals in the clinical. View Publication

BACKGROUND Two opposing B cell subsets have been defined based on their cytokine profile: IL-6 producing effector B cells (B-effs) versus IL-10 producing regulatory B cells (B-regs) that respectively positively or negatively regulate immune responses. B-regs are decreased and/or impaired in many autoimmune diseases and inflammatory conditions. Since there is increasing evidence that links B cells and B cell-rich lymphoid follicles to the pathogenesis of COPD,the aim of this study was to investigate the presence and function of B-regs in COPD. METHODS First,presence of IL-10 producing regulatory B cells in human lung tissue was determined by immunohistochemistry. Secondly,quantification of IL-10??+??B-regs and IL-6??+??B-effs in peripheral blood mononuclear cells (PBMCs) from healthy controls,smokers without airflow limitation,and COPD patients (GOLD stage I-IV) was performed by flow cytometry. Thirdly,we exposed blood-derived B cells from COPD patients in vitro to cigarette smoke extract (CSE) and quantified IL-10??+??B-regs and IL-6??+??B-effs. Furthermore,we aimed at restoring the perturbed IL10 production by blocking BAFF. Fourthly,we determined mRNA expression of transcription factors involved in IL-10 production in FACS sorted memory- and naive B cells upon exposure to medium or CSE. RESULTS The presence of IL-10 producing regulatory B cells in parenchyma and lymphoid follicles in lungs was confirmed by immunohistochemistry. The percentage of IL-10??+??B-regs was significantly decreased in blood-derived memory B cell subsets from smokers without airflow limitation and patients with COPD,compared to never smokers. Furthermore,the capacity of B cells to produce IL-10 was reduced upon in vitro exposure to CSE and this could not be restored by BAFF-blockade. Finally,upon CSE exposure,mRNA levels of the transcription factors IRF4 and HIF-1$\alpha$,were decreased in memory B cells. CONCLUSION Decreased numbers and impaired function of B-regs in smokers and patients with COPD might contribute to the initiation and progression of the disease. View Publication

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here,we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both,Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells,but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly,two principle stem cell regulatory pathways WNT/{\ss}-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore,both treatments strikingly eliminated CD133+ CSC population,accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear {\ss}-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of {\ss}-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU,both,Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together,combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells. View Publication

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection,specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein,we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model,we found that Treg cells play a role during the initial stages after T. cruzi infection,restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived,effector T cell subsets,without affecting memory precursor cell formation or the expression of activation,exhaustion and functional markers. In addition,Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially,the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses,preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model. Author summaryChagas disease,caused by Trypanosoma cruzi,can result in severe health complications. While the exact mechanisms underlying the disease’s pathogenesis remain incompletely understood,the host’s inflammatory immune response is believed to play a critical role. To shed light on disease mechanisms and potential treatments,we investigated the impact of regulatory T (Treg) cells on the development of effector immune responses against T. cruzi. Our findings reveal that Treg cells dampen parasite-specific CD8+ T cells,a crucial arm of the immune response in counteracting the parasite. Notably,this regulatory influence occurs primarily during the early stages of T. cruzi infection. Furthermore,we observed that while Treg cells have minimal effects on antigen-presenting cells,they modulate the magnitude and phenotype of conventional CD4+ T cells. Importantly,we identified CD39,a molecule involved in the purinergic pathway,as essential for the suppressive functions of Treg cells during T. cruzi infection. Our findings enhance the understanding of the regulatory response during the acute phase of T. cruzi infection and may have implications for the development of novel therapeutic strategies. View Publication

Toll-like receptor 9 (TLR9) recognizes bacterial,viral and self DNA and play an important role in immunity and inflammation. However,the role of TLR9 in obesity is less well-studied. Here,we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-,KO) B6 mice and model obesity using a high-fat diet. Compared with control mice,B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation,weight gain,and impaired glucose and insulin tolerance. Furthermore,the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced,and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition,gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing,we identify altered gut microbial communities including reduced Lachnospiraceae,which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9,Irf4 and Il-10 interconnecting metabolic homeostasis,with the function of B and T cells,and gut microbiota in obesity. Although the function of Toll-like receptor 9 (TLR9) in immunity and inflammation is well-established,its role in obesity is less well-studied. In this study,the authors demonstrate that TLR9 deficiency in B cells is associated with obesity in mice and results in altered frequencies of T and B lymphocyte subsets and gut microbiome dysbiosis. View Publication

PRDX2 is significantly expressed in various cancers and is associated with the proliferation of tumor cells. Nonetheless,the precise mechanism of PRDX2 in tumor immunity remains incompletely understood. This study aims to investigate the impact of PRDX2,which is highly expressed in lung adenocarcinoma,on T cells in the tumor immune microenvironment,and its immune action target to promote the immune escape of lung cancer cells,to provide a theoretical basis for lung adenocarcinoma treatment with PRDX2 as the target. Mouse animal models to verify the effect of Conoidin A treatment on tumor growth and T cell infiltration. Flow cytometry and Western blot verified tumor cell apoptosis in the in vitro co-culture system as well as granzyme B and perforin expression in T cells. RNA-Seq was used to obtain the downstream immune molecule. si-RNA knockdown of Galectin-9 was co-cultured with T cells in vitro. Immunofluorescence and Western blot verified that PRDX2 regulates Galectin-9 expression through HDAC3. PRDX2 expression was negatively correlated with CD8 + T cell expression in LUAD patients. Inhibition of PRDX2 significantly enhanced T-cell killing of LUAD cells and reduced tumor load in both in vitro and in vivo models. Mechanistically,Conoidin A or shRNA_PRDX2 decreased Galectin-9 expression by down-regulating the phosphorylation of HDAC3,consequently enhancing the infiltration and function of CD8 + T cells. This study reveals the role of the PRDX2/HDAC3/Galectin-9 axis in LUAD immune escape and indicates Galectin-9 as a promising target for immunotherapy. The online version contains supplementary material available at 10.1186/s12967-024-05888-z. View Publication

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However,little is known about the biological characteristics of recurrent HCC cells. A single patient's primary and recurrent HCC-derived cell lines,Hep-11 and Hep-12,respectively,were established by primary culture. These two cell lines have the same hepatitis B virus integration site and share many common amplifications and deletions,which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10 000 cells,injection of only 100 Hep-12 cells was sufficient to initiate tumor growth,and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11,Hep-12 cells expressed the oval cell markers AFP,NCAM/CD56,c-kit/CD117,as well as multiple stem cell markers such as Nanog,OCT4 and SOX2. In addition,textgreater90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel,but more resistant to doxorubicin,cisplatin and hydroxycamptothecin (HCPT),which had been administrated to the patient. Furthermore,Hep-12 cells expressed higher levels of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) than Hep-11,and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 cells but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells and that elevated expression of PARP-1 was related to their resistance to HCPT. View Publication

Comprehensive in vitro and in vivo studies comparing EpCAM-based methods with other cytometric CTC enrichment technologies in metastatic colorectal cancer (mCRC) patients are lacking. We compare four manual cytometric methods to detect CTCs in vitro and in mCRC patients. The EpCAM-based technology,MACS HEA MicroBeads((R)),showed a significant better tumor cell recovery rate compared to other cytometric methods (p-valuetextless0.0001). CTCs of 38 mCRC patients were enriched with MACS HEA MicroBeads(R). Progression-free survival did significantly differ between mCRC patients without detectable and with textgreateror= 1 CTCs (p=0.007). CTC enrichment with EpCAM coupled antibodies is superior to other cytometric methods and is a feasible method for CTC detection in mCRC patients. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. View Publication