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

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans,and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein,a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore,the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here,we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs,one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies,like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs,avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore,binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies. View Publication

ARF GTPases are activated by guanine nucleotide exchange factors (GEFs) of the Sec7 family that promote the exchange of GDP for GTP. Brefeldin A (BFA) is a fungal metabolite that binds to the ARF1*GDP*Sec7 complex and blocks GEF activity at an early stage of the reaction,prior to guanine nucleotide release. The crystal structure of the ARF1*GDP*Sec7*BFA complex shows that BFA binds at the protein-protein interface to inhibit conformational changes in ARF1 required for Sec7 to dislodge the GDP molecule. Based on a comparative analysis of the inhibited complex,nucleotide-free ARF1*Sec7 and ARF1*GDP,we suggest that,in addition to forcing nucleotide release,the ARF1-Sec7 binding energy is used to open a cavity on ARF1 to facilitate the rearrangement of hydrophobic core residues between the GDP and GTP conformations. Thus,the Sec7 domain may act as a dual catalyst,facilitating both nucleotide release and conformational switching on ARF proteins. View Publication

Following haematopoietic stem cell transplantation,monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood,bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood,a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid,inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit,Accumol,Calgary,AB,Canada) (P = 0.59). Of the 303 samples tested by flow cytometry,290 (95.7%) exceeded 90% purity,and 215 (70.95%) were over 99% pure. There were some outlying samples,showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols,allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature,assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results. View Publication

Over 45,000 new cases of oral and pharyngeal cancers are diagnosed and account for over 8,000 deaths a year in the United States. An environmental chemical receptor,the aryl hydrocarbon receptor (AHR),has previously been implicated in oral squamous cell carcinoma (OSCC) initiation as well as in normal tissue-specific stem cell self-renewal. These previous studies inspired the hypothesis that the AHR plays a role in both the acquisition and progression of OSCC,as well as in the formation and maintenance of cancer stem-like cells. To test this hypothesis,AHR activity in two oral squamous cell lines was modulated with AHR prototypic,environmental and bacterial AHR ligands,AHR-specific inhibitors,and phenotypic,genomic and functional characteristics were evaluated. The data demonstrate that: 1) primary OSCC tissue expresses elevated levels of nuclear AHR as compared to normal tissue,2) Ahr mRNA expression is up-regulated in 320 primary OSCC,3) AHR hyper-activation with several ligands,including environmental and bacterial ligands,significantly increases AHR activity,ALDH1 activity,and accelerates cell migration,4) AHR inhibition blocks the rapid migration of OSCC cells and reduces cell chemoresistance,5) AHR knockdown inhibits tumorsphere formation in low adherence conditions,and 6) AHR knockdown inhibits tumor growth and increases overall survival in vivo. These data demonstrate that the AHR plays an important role in development and progression of OSCC,and specifically cancer stem-like cells. Prototypic,environmental and bacterial AHR ligands may exacerbate OSCC by enhancing expression of these properties. IMPLICATIONS This study,for the first time,demonstrates the ability of diverse AHR ligands to regulate AHR activity in oral squamous cell carcinoma cells,as well as regulate several important characteristics of oral cancer stem cells,in vivo and in vitro. View Publication

Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However,whether G9a plays a similar role in adult cells is still unclear. We identify a critical role for G9a in CD4(+) T helper (Th) cell differentiation and function. G9a-deficient Th cells are specifically impaired in their induction of Th2 lineage-specific cytokines IL-4,IL-5,and IL-13 and fail to protect against infection with the intestinal helminth Trichuris muris. Furthermore,G9a-deficient Th cells are characterised by the increased expression of IL-17A,which is associated with a loss of H3K9me2 at the Il17a locus. Collectively,our results establish unpredicted and complex roles for G9a in regulating gene expression during lineage commitment in adult CD4(+) T cells. View Publication

Both targeted therapy and immunotherapy have been used successfully to treat melanoma,but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles,but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein,we describe the development and characterization of a novel,immunogenic variant of the BrafV600ECdkn2a-/-Pten-/- YUMM1.1 tumor model that expresses the immunogen,ovalbumin (YOVAL1.1). We demonstrate that,unlike parental tumors,YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly,YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK,responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable,immunogenic and sensitive to clinical therapies,making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma. View Publication

AbstractUnsupervised clustering is a powerful machine-learning technique widely used to analyze high-dimensional biological data. It plays a crucial role in uncovering patterns,structures,and inherent relationships within complex datasets without relying on predefined labels. In the context of biology,high-dimensional data may include transcriptomics,proteomics,and a variety of single-cell omics data. Most existing clustering algorithms operate directly in the high-dimensional space,and their performance may be negatively affected by the phenomenon known as the curse of dimensionality. Here,we show an alternative clustering approach that alleviates the curse by sequentially projecting high-dimensional data into a low-dimensional representation. We validated the effectiveness of our approach,named automated projection pursuit (APP),across various biological data modalities,including flow and mass cytometry data,scRNA-seq,multiplex imaging data,and T-cell receptor repertoire data. APP efficiently recapitulated experimentally validated cell-type definitions and revealed new biologically meaningful patterns. View Publication

PURPOSE To assess the potential for CUE-101,a novel therapeutic fusion protein,to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors,including head and neck squamous cell carcinoma (HNSCC),cervical,and anal cancers. EXPERIMENTAL DESIGN CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex,an HPV16 E7 peptide epitope,reduced affinity human IL2 molecules,and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101,whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS CUE-101 demonstrates selective binding,activation,and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice,and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS Consistent with its design,CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells,a favorable safety profile,and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689). View Publication

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays,cell culture systems,and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate (EGCG),the major polyphenol present therein. We have earlier shown that EGCG treatment results in apoptosis of several cancer cells,but not of normal cells (J. Natl. Cancer Inst. 89,1881-1886 (1997)). The mechanism of this differential response of EGCG is not known. In this study,we investigated the involvement of NF-kappaB during these differential responses of EGCG. EGCG treatment resulted in a dose-dependent (i) inhibition of cell growth,(ii) G0/G1-phase arrest of the cell cycle,and (iii) induction of apoptosis in human epidermoid carcinoma (A431) cells,but not in normal human epidermal keratinocytes (NHEK). Electromobility shift assay revealed that EGCG (10-80 microM) treatment results in lowering of NF-kappaB levels in both the cytoplasm and nucleus in a dose-dependent manner in both A431 cells and NHEK,albeit at different concentrations. EGCG treatment was found to result in a dose-based differential inhibition of TNF-alpha- and LPS-mediated activation of NF-kappaB in these cells. The inhibition of NF-kappaB constitutive expression and activation in NHEK was observed only at high concentrations. The immunoblot analysis also demonstrated a similar pattern of inhibition of the constitutive expression as well as activation of NF-kappaB/p65 nuclear protein. This inhibition of TNF-alpha-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha. Taken together,EGCG was found to impart differential dose-based NF-kappaB inhibitory response in cancer cells vs normal cells; i.e.,EGCG-mediated inhibition of NF-kappaB constitutive expression and activation was found to occur at much higher dose of EGCG in NHEK as compared to A431 cells. This study suggests that EGCG-caused cell cycle deregulation and apoptosis of cancer cells may be mediated through NF-kappaB inhibition. View Publication

BACKGROUND Induced pluripotent stem cells (iPSCs) hold tremendous potential,both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children,a highly relevant and easily accessible tissue for pediatric populations. METHODS We performed detailed comparative analysis on the transcriptomes and methylomes of NECs,iPSCs derived from NECs (NEC-iPSCs),and embryonic stem cells (ESCs). RESULTS NEC-iPSCs express pluripotent cell markers,can differentiate into all 3 germ layers in vivo and in vitro,and have a transcriptome and methylome remarkably similar to those of ESCs. However,residual DNA methylation marks exist,which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro,suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming,which are indicative of possible roles in airway epithelium development. CONCLUSION NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients,and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory. View Publication

Purpose: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. Experimental design: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a stem-like" phenotype (CD44+CD24low/-� View Publication

The cell surface marker CD133 has been proposed as a brain tumor stem cell marker. However,there have been substantial controversies regarding the necessity and role of CD133 in tumorigenesis. This study aimed to characterize CD133(+) cells in brain tumors. Human brain tumor specimens and whole blood were collected from the same patients (N=12). We carried out dual FACS staining for CD133/CD34 and functional tumorigenesis and angiogenesis analyses of CD133(+) cells from different origins. We also investigated the in vivo tumorigenic potential and histological characteristics of four distinct groups on the basis of expression of CD133/CD34 markers (CD133(+),CD133(+)/CD34(+),CD133(+)/CD34(-),and CD133(-)). CD133(+) brain tumor cells coexpressed significantly higher positivity for CD34 (70.7±5.2% in CD133(+) vs. 12.3±4.2% in CD133(-) cells,P<0.001). CD133(+) brain tumor cells formed neurosphere-like spheroids and differentiated into multiple nervous system lineages unlike CD133(+) blood cells. They showed biological characteristics of endothelial cells,including vWF expression,LDL uptake and tube formation in vitro,unlike CD133(-) brain tumors cells. Pathologic analysis of brains implanted with CD133(+) cells showed large,markedly hypervascular tumors with well-demarcated boundary. CD133(+)/CD34(-) cells produced smaller but highly infiltrative tumors. Notably,pure angiogenic cell fractions (CD133(+)/CD34(+)) and CD133(-) tumor cells did not generate tumors in vivo. Our data suggest the presence of a distinct subpopulation of CD133(+) cells isolated from human brain tumors,with characteristics of endothelial progenitor cells (EPCs). View Publication