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

Phosphoinositide 3-kinase (PI3K) p110$\delta$ signalling negatively regulates the production of mouse IgE. However,there are disparities between the mouse and human IgE biology,and the role of PI3K p110$\delta$ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110$\delta$ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114,a small molecule inhibitor of PI3K p110$\delta$. Using FACS,RT-PCR and ELISA we examined the effect of PI3K p110$\delta$ inhibition on IgE production and determined the mechanisms involved. Unlike in mice,we observed that PI3K p110$\delta$ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However,the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110$\delta$ inhibition. The expression levels of AID,$\epsilon$ and $\gamma$1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However,we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40,specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition,PI3K p110$\delta$ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion,PI3K p110$\delta$ signalling is required for the production of human IgE,which makes it a pharmacological target for the treatment of allergic disease. View Publication

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins,the Traf and Nck interacting kinase (TNIK),is a postsynaptic density (PSD) signaling hub,with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment,the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here,using hiPSC-derived excitatory neurons,we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover,the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses,suggesting a differential role of TNIK in immature synapsis in NDD. View Publication

Human respiratory syncytial virus (RSV) is a major cause of serious respiratory tract infections in infants and the elderly. Recently it was shown that the RSV G glycoprotein mediates attachment to cells using CX3CR1 as a receptor,and that G-specific neutralizing antibodies can be detected using human airway epithelial (HAE) cell cultures. To investigate the contributions of G-specific antibodies to RSV neutralization,we performed HAE neutralization assays on sera from RSV G-immunized mice or RSV-infected infants. We confirmed that G-specific neutralization using serum from mice or humans could only be detected on HAE cultures. We also found that RSV G-specific antibodies in infants were either subgroup specific or cross-neutralizing. Altogether,our results suggest that G is an important target for generating neutralizing antibodies and would be beneficial to include in an RSV vaccine. Further,inclusion of G antigens from both RSV subgroups may enhance the vaccine cross protection potency. View Publication

Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand,stem cell factor activates several distinct early signaling pathways,including phospholipase C gamma,phosphatidylinositol 3-kinase,Src kinase,Grb2,and Grb7. The role of these pathways in Kit-induced growth,proliferation,or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However,inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and,to a lesser extent,with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably,restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth,proliferation,and cooperation with Epo-R downstream from Kit. View Publication

A series of hydroxamate based HDAC inhibitors containing a phenylisoxazole as the CAP group has been synthesized using nitrile oxide cycloaddition chemistry. An HDAC6 selective inhibitor having a potency of approximately 2 picomolar was identified. Some of the compounds were examined for their ability to block pancreatic cancer cell growth and found to be about 10-fold more potent than SAHA. This research provides valuable,new molecular probes for use in exploring HDAC biology. View Publication

BACKGROUND While lipid emulsions in modern formulations for total parenteral nutrition (TPN) provide essential fatty acids and dense calories,they also promote inflammation and immunometabolic disruptions. OBJECTIVES We aimed to develop a novel lipid emulsion for TPN use with superior immunometabolic actions compared with available standard lipid emulsions. METHODS A novel lipid emulsion [Vegaven (VV)] containing 30% of 18-carbon n-3 fatty acids ($\alpha$-linolenic acid and stearidonic acid) was developed for TPN (VV-TPN) and compared with TPN containing soybean oil-based lipid emulsion (IL-TPN) and fish-oil-based lipid emulsion (OV-TPN). In vivo studies were performed in instrumented male C57BL/6 mice subjected to 7-d TPN prior to analysis of cytokines,indices of whole-body and hepatic glucose metabolism,immune cells,lipid mediators,and mucosal bowel microbiome. RESULTS IL-6 to IL-10 ratios were significantly lower in liver and skeletal muscle of VV-TPN mice when compared with IL-TPN or OV-TPN mice. VV-TPN and OV-TPN each increased hepatic insulin receptor abundance and resulted in similar HOMA-IR values,whereas only VV-TPN increased hepatic insulin receptor substrate 2 and maintained normal hepatic glycogen content,effects that were IL-10-dependent and mediated by glucokinase activation. The percentages of IFN-$\gamma$- and IL-17-expressing CD4+ T cells were increased in livers of VV-TPN mice,and liver macrophages exhibited primed phenotypes when compared with IL-TPN. This immunomodulation was associated with successful elimination of the microinvasive bacterium Akkermansia muciniphila from the bowel mucosa by VV-TPN as opposed to standard lipid emulsions. Assay of hepatic lipid mediators revealed a distinct profile with VV-TPN,including increases in 9(S)-hydroxy-octadecatrienoic acid. When co-administered with IL-TPN,hydroxy-octadecatrienoic acids mimicked the VV-TPN immunometabolic phenotype. CONCLUSIONS We here report the unique anti-inflammatory,insulin-sensitizing,and immunity-enhancing properties of a newly developed lipid emulsion designed for TPN use based on 18-carbon n-3 fatty acids. View Publication

Exposure to cigarette smoke causes a multitude of pathological changes leading to tissue damage and disease. Quantifying such changes in highly differentiated in vitro human tissue models may assist in evaluating the toxicity of tobacco products. In this methods development study,well-differentiated human air-liquid-interface (ALI) in vitro airway tissue models were used to assess toxicological endpoints relevant to tobacco smoke exposure. Whole mainstream smoke solutions (WSSs) were prepared from 2 commercial cigarettes (R60 and S60) that differ in smoke constituents when machine-smoked under International Organization for Standardization conditions. The airway tissue models were exposed apically to WSSs 4-h per day for 1-5 days. Cytotoxicity,tissue barrier integrity,oxidative stress,mucin secretion,and matrix metalloproteinase (MMP) excretion were measured. The treatments were not cytotoxic and had marginal effects on tissue barrier properties; however,other endpoints responded in time- and dose-dependent manners,with the R60 resulting in higher levels of response than the S60 for many endpoints. Based on the lowest effect dose,differences in response to the WSSs were observed for mucin induction and MMP secretion. Mitigation of mucin induction by cotreatment of cultures with N-acetylcysteine suggests that oxidative stress contributes to mucus hypersecretion. Overall,these preliminary results suggest that quantifying disease-relevant endpoints using ALI airway models is a potential tool for tobacco product toxicity evaluation. Additional research using tobacco samples generated under smoking machine conditions that more closely approximate human smoking patterns will inform further methods development. View Publication

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro,and become more adult-like in their structure,cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al.,2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment,long-term survival,and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue,we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue,resulting in airway-like structures that were remarkably similar to the native adult human lung. View Publication

AIM: The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.backslashnbackslashnMETHODS: Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression,flow cytometry,teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.backslashnbackslashnRESULTS: Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival,motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein,mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette,driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4,NANOG and SOX2) and on the cell surface (SSEA4,TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and,importantly,maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging,rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces,quantification of cell attachment to nanoparticles and rapid estimation of cell survival.backslashnbackslashnCONCLUSION: The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation,making it a useful tool for bioengineering and in vivo tracking experiments. View Publication

BACKGROUND Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However,our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities,gene expression profiles,and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS In this study,we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First,we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition,we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry,we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing,we show significant TCR overlap between matched dura and meningioma samples. Finally,we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura. View Publication

Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation,the precise means through which they contribute to disease severity and chronicity remains incompletely understood,posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay in vitro even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work,by using air-liquid-interface (ALI) human airway in vitro models,we aimed to recreate COPD-related persistent bacterial infections. In particular,we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression,allowing one to monitor host-pathogen interactions for up to three weeks. Notably,the use of these models,coupled with confocal and transmission electron microscopy,revealed unique features associated with NTHi and Mcat infection,highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall,this study demonstrates the possibility to perform long term host-pathogen investigations in vitro with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets. View Publication