ABSTRACT: Comparasion of DNA methylomes of human CD11b+ cells from healthy donors (HD) peripheral blood and CD11b+ cells isolated from ascitic fluid (a) or peripheral blood (b) from ovarian cancer patients
Project description:The methylation profiles of bisulfite-modified DNA of human CD11b+ cells from healthy donors (HD) were compared with CD11b+ cells isolated from ovarian cancer patients (blood or ascitic fluid) using the Infinium HumanMethylation850 BeadChips (Illumina, Inc., San Diego, CA,). This platform allows the interrogation of >850,000 methylation sites per sample at single-nucleotide resolution, and comprises an average of 17 CpG sites per gene in the 99% of RefSeq genes. 96% of CpG islands are covered, with additional coverage in CpG island shores and the regions flanking them. The samples were hybridized in the array following the manufacturer’s instructions.
Project description:Analysis of the role of LXR on the transcriptional signature of monocyte-derived macrophages generated in the presence of tumor ascitic fluids or rheumatoid arthritis synovial fluids. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes from independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were treated with DMSO (vehicle), 1 micromolar GW3965 or 1 micromolar GSK2033 for 1 hour, and then cultured in RPMI 1640 10% FBS supplemented with either 50% Tumor-derived Ascitic Fluid (TAF) or 20% Synovial Fluid from patients with active Rheumatoid Arthritis (RASF), at 37°C in a humidified atmosphere with 5% CO2 and 21% O2. After 72 hours, cells were lysed and RNA isolated for transcriptional analysis.
Project description:This single-cell RNA sequencing (scRNA-seq) dataset profiles human myeloid cells from healthy donors and patients with advanced oral cavity squamous cell carcinoma (OCSCC). scRNA-seq was performed on isolated CD11b⁺ cells from high- and low-density peripheral blood fractions and on isolated CD45⁺ tumor-infiltrating cells from OCSCC patients (n=8), as well as on CD11b⁺ high- and low-density peripheral blood cells from healthy donors (n=5). The dataset enables the characterization of compartment-specific transcriptional states of human myeloid cells and supports investigations into genes and pathways associated with pathologic activation and immune suppression.
Project description:A phenotypically and functinoally distinct subset of human blood dendritic cells expressing CD11b is specific of the neonatal environment. We have employed whole genome microarray expression profiling to identify the specific gene signature of CD11b+ cord blood dendritic cells as compared to their adult peripheral blood counterparts. Peripheral blood adult cDC2 (CD20- CD11c+ CD14- BDCA1+ CD11b- ), neonatal cord blood cDC2 (CD20- CD11c+ CD14- BDCA1+ CD11b-) and neonatal cord blood cDC2b (CD20- CD11c+ CD14- BDCA1+ CD11b+) were FACS purified from BDCA1+ magnetically. Neonatal monocytes (CD11c+ CD14+) and neonatal naive T cells (CD3+ CD4+ CD56- CD25- CD45RO-) were used as controls.
Project description:RNA-sequencing analysis was carried out on ascitic fluid-isolated mesothelial cells from ovarian cancer patients compared to control human peritoneal mesothelial cells.
Project description:Characterizations of ascites proteome from ovarian PC and gastric PC have demonstrated that ascites contains elevated pro-tumorigenic factors. Reasoning that the composition of ascitic fluid might offer insight into the memory of key biological events occurring intra-abdominally, we hypothesized that paracrine factors essential for survival and growth of peritoneal deposits are secreted into and circulate within ascitic fluid. Our data suggest that ascites contains biologically active ligands capable of supporting cellular functions of cancer cells.
Project description:Single-cell RNAseq (10x Genomics) analysis of human CD4+ T cells in IPEX patients, healthy donors and heterozygous mothers (blood). Human CD4+T cells from IPEX, HD and mothers were isolated from frozen peripheral blood mononuclear cells by flow cytometry as DAPI–CD3+CD4+ cells. In cohort 1, cells from separate donor were encapsulated in separate channel following 10x Genomics Single Cell 3′ Reagent Kit (V2 chemistry). In cohort 2, samples were tagged using a different Hashtags, pooled and encapsulared following 10x Genomics Single Cell 3′ Reagent Kit (V3 chemistry).
Project description:The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. Since these circulating atypical B cells in the blood of CVID patients could not be assigned to any certain B cell differentiation stage in the periphery, they were designated as CD21low B cells. Although, CD21low B cells are polyclonal and unmutated IgM+IgD+ B cells like naive B cells in the peripheral blood, they reveal several distinct phenotypic and functional features. In order to uncover the the global programme of gene expression underlying these differences and changes in CD21low B cells we have performed microarray hybridization experiments and identified differentially expressed genes which define a distinct gene expression profile in CD21low B cells compared to naive B cells. Leucocytes were separated by Ficoll gradient from peripheral blood of healthy donors (HD) and CVID patients. CD19+CD27-CD38+CD21+ naive B cells of HD as well as naive and CD19hiCD27-CD38lowCD21low (CD21low) B cells of CVID patients were sorted using a MoFlow cell sorter. RNA was extracted from sorted ex vivo B cell subpopulations and hybridized on Affymetrix microarrays. Gene expression profiles were compared between CD21low B cells and naive B cells of CVID patients as well as HD naive B cells.
Project description:To examine patterns of gene expression in ankle synovial fluid cells and peripheral blood leukocytes during serum transferred arthritis. A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle tissue, synovial fluid, and peripheral blood leukocytes.
