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

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

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

Human immunodeficiency virus type 1 (HIV-1) isolates from 20 chronically infected patients who participated in a structured treatment interruption (STI) trial were studied to determine whether viral fitness influences reestablishment of viremia. Viruses derived from individuals who spontaneously controlled viremia had significantly lower in vitro replication capacities than viruses derived from individuals that did not control viremia after interruption of antiretroviral therapy (ART),and replication capacities correlated with pre-ART and post-STI viral set points. Of note,no clinically relevant improvement of viral loads upon STI occurred. Virus isolates from controlling and noncontrolling patients were indistinguishable in terms of coreceptor usage,genetic subtype,and sensitivity to neutralizing antibodies. In contrast,viruses from controlling patients exhibited increased sensitivity to inhibition by chemokines. Sensitivity to inhibition by RANTES correlated strongly with slower replication kinetics of the virus isolates,suggesting a marked dependency of these virus isolates on high coreceptor densities on the target cells. In summary,our data indicate that viral fitness is a driving factor in determining the magnitude of viral rebound and viral set point in chronic HIV-1 infection,and thus fitness should be considered as a parameter influencing the outcome of therapeutic intervention in chronic infection. View Publication

BACKGROUND: Combining MEK inhibitors with other signalling pathway inhibitors or conventional cytotoxic drugs represents a promising new strategy against cancer. RDEA119/BAY 869766 is a highly potent and selective MEK1/2 inhibitor undergoing phase I human clinical trials. The effects of RDEA119/BAY 869766 as a single agent and in combination with rapamycin were studied in 3 early passage primary pancreatic cancer xenografts,OCIP19,21,and 23,grown orthotopically. METHODS: Anti-cancer effects were determined in separate groups following chronic drug exposure. Effects on cell cycle and downstream signalling were examined by flow cytometry and western blot,respectively. Plasma RDEA119 concentrations were measured to monitor the drug accumulation in vivo. RESULTS: RDEA119/BAY 869766 alone or in combination with rapamycin showed significant growth inhibition in all the 3 models,with a significant decrease in the percentage of cells in S-phase,accompanied by a large decrease in bromodeoxyuridine labelling and cell cycle arrest predominantly in G1. The S6 ribosomal protein was inhibited to a greater extent with combination treatment in all the three models. Blood plasma pharmacokinetic analyses indicated that RDEA119 levels achieved in vivo are similar to those that produce target inhibition and cell cycle arrest in vitro. CONCLUSIONS: Agents targeting the ERK and mTOR pathway have anticancer activity in primary xenografts,and these results support testing this combination in pancreatic cancer patients. View Publication

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation-prone proteins. Among them,Huntington's disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT),which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell-derived HD neurons exhibit some disease-relevant phenotypes and provide tools for HD research. However,they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition,the relationship between the phenotype and mHTT levels has not been elucidated. Herein,we present a human embryonic stem cell (hESC)-derived HD neuronal model expressing HTTexon1 fragments,which addresses the deficiencies enumerated above. The wild-type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ∼10% of mHTT is sufficient to prevent toxicity,whereas ∼90% reduction of wild-type HTT is safe and well-tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore,the hESC-derived neuronal models complement existing induced pluripotent stem cell-derived neuronal models and provide valuable tools for HD research.—Lu,B.,Palacino,J. A novel human embryonic stem cell-derived Huntington's disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT-dependent neurodegeneration. View Publication

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously,we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article,we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%),which leads to generation of cells/MC aggregates (400 $\$ in size) and high cell yields (2.4-3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (textgreater90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover,tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined,xeno-free,GMP-compatible,and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells. View Publication

Peripheral blood was collected from a clinically diagnosed 64-year old male multiple schwannoma patient. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma. View Publication

A growing body of clinical literature has described neurodevelopmental delays in infants with chronic prenatal opioid exposure and withdrawal. Despite this,the mechanism of how opioids impact the developing brain remains unknown. Here,we developed an in vitro model of prenatal morphine exposure and withdrawal using healthy human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in monolayer. To optimize our model,we identified that a longer neural induction and regional patterning period increases expression of canonical opioid receptors mu and kappa in midbrain neural progenitors compared to a shorter protocol (OPRM1,two-tailed t-test,p =? 0.004; OPRK1,p =? 0.0003). Next,we showed that the midbrain neural progenitors derived from a longer iPSC neural induction also have scant toll-like receptor 4 (TLR4) expression,a key player in neonatal opioid withdrawal syndrome pathophysiology. During morphine withdrawal,differentiating neural progenitors experience cyclic adenosine monophosphate overshoot compared to cell exposed to vehicle (p =? 0.0496) and morphine exposure conditions (p,=? 0.0136,1-way ANOVA). Finally,we showed that morphine exposure and withdrawal alters proportions of differentiated progenitor cell fates (2-way ANOVA,F =? 16.05,p View Publication

In fragile X syndrome (FXS),CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However,FXS siblings have been identified with more than 200 CGGs,termed unmethylated full mutation (UFM) carriers,without gene silencing and disease symptoms. Here,we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However,a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore,we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs,and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population. View Publication