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

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells,resulting in reversal of Treg suppression. Importantly,TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression,which may be used as a strategy to advance tumor immunotherapy. View Publication

Mechanisms of lymphoid and myeloid lineage choice by hemopoietic stem cells remain unclear. In this study we show that the multipotent progenitor (MPP) population,which is immediately downstream of hemopoietic stem cells,is heterogeneous and can be subdivided in terms of VCAM-1 expression. VCAM-1(+) MPPs were fully capable of differentiating into both lymphoid and myeloid lineages. In contrast,VCAM-1(-) MPPs gave rise to lymphocytes predominately in vivo. T and B cell development from VCAM-1(-) MPPs was 1 wk faster than that from VCAM-1(+) MPPs. Furthermore,VCAM-1(+) MPPs gave rise to common myeloid progenitors and VCAM-1(-) MPPs in vivo,indicating that VCAM-1(-) MPPs are progenies of VCAM-1(+) MPPs. VCAM-1(-) MPPs,in turn,developed into lymphoid lineage-restricted common lymphoid progenitors. These results establish a hierarchy of developmental relationship between MPP subsets and lymphoid and myeloid progenitors. In addition,VCAM-1(+) MPPs may represent the branching point between the lymphoid and myeloid lineages. View Publication

Fetuses with homozygous alpha-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence,the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of alpha-thalassemia. These mice mimic the human alpha-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2,3,and 4 of the beta-LCR,a central polypurine tract element,and the beta-globin gene promoter directing either the EGFP or the human alpha-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery,we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human alpha-globin gene expression. In the newborn,human alpha-globin gene expression was detected in the liver,spleen,and peripheral blood. The human alpha-globin gene expression was at the peak at 3-4 months,when it reached 20% in some recipients. However,the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human alpha-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus,lentiviral vectors can be an effective vehicle for delivering the human alpha-globin gene into erythroid cells in utero,but,in the mouse model,delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression. View Publication

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied,the developmental capacity of AG ESCs is much less clear. Here,we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed,with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs,we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic,it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study,we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary,the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies. View Publication

We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration,and the level of vitamin D-binding protein (DBP) in 24 normal subjects,17 pregnant subjects,and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus,the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl,respectively,P less than 0.001),whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml,respectively). In contrast,in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%,P less than 0.001),the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml,P less than 0.001). When data from all three groups were combined,there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels,(b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels,and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions. View Publication

Carbonic anhydrases (CAs) comprise a family of zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide. CAs contribute to a myriad of physiological processes,including pH regulation,anion transport and water balance. To date,16 known members of the mammalian alpha-CA family have been identified. Given that the catalytic family members share identical reaction chemistry,their physiologic roles are influenced greatly by their tissue and sub-cellular locations. CAVI is the lone secreted CA and exists in both saliva and the gastrointestinal mucosa. An alternative,stress-inducible isoform of CAVI (CAVI-b) has been shown to be expressed from a cryptic promoter that is activated by the CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP). The CAVI-b isoform is not secreted and is currently of unknown physiological function. Here we use neuronal models,including a model derived using Car6 and CHOP gene ablations,to delineate a role for CAVI-b in ischemic protection. Our results demonstrate that CAVI-b expression,which is increased through CHOP-signaling in response to unfolded protein stress,is also increased by oxygen-glucose deprivation (OGD). While enforced expression of CAVI-b is not sufficient to protect against ischemia,CHOP regulation of CAVI-b is necessary for adaptive changes mediated by BDNF that reduce subsequent ischemic damage. These results suggest that CAVI-b comprises a necessary component of a larger adaptive signaling pathway downstream of CHOP. View Publication

The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However,this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g.,LMPPs),implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here,we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP),whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model. View Publication

During drug development,measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen,and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However,accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets,epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease,and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques,we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb,binding of two reagent antibodies,as required for a classic sandwich ELISA,was not feasible,and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites,and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method,total LTα could be accurately measured in cynomolgus macaque serum,and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody. View Publication