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

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

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

Although CD31 expression on human thymocytes has been reported,a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study,we provide a global picture of the evolution of CD31 expression from the CD34(+) hematopoietic precursor to the CD45RA(+) mature CD4(+) and CD8(+) single-positive (SP) T cells. Using nine-color flow cytometry,we show that CD31 is highly expressed on CD34(+) progenitors and stays high until the early double-positive stage (CD3(-)CD4(+)CD8α(+)β(-)). After β-selection,CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3(high)CD4(+)CD8(+) double-positive thymocytes. However,following positive selection,CD31 expression differs dramatically between CD4(+) and CD8(+) lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells,including a subset of CD45RA(+)CD31(-) mature CD4(+) thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably,there is a substantial subset of semimature (CD45RA(-)) CD4 SP thymocytes that lack CD31 expression. Moreover,FOXP3(+) and ICOS(+) cells are overrepresented in this CD31(-) subpopulation. Despite this CD31(-)CD45RA(-) subpopulation,most egress-capable mature CD45RA(+) CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: β-selection,positive selection,and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases,our results suggest a significant role for CD31 during T cell development. View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with S1647T variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model will be useful for further function studies and therapeutic screening. View Publication

Peripheral blood was collected from a clinically diagnosed 79-year old male sporadic Parkinson's disease 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 can be used to study the mechanism of sporadic Parkinson's disease and to test new drugs. Resource Table. View Publication