Project description:Here we demonstrate by the use of extensive controls and stringent statistical analysis that dendritic cells differentially regulate hundreds of different genes based upon sequence similarity of endogenously- and exogenously-loaded antigens and in a T-cell independent fashion. When endogenously and exogenously-derived antigens are identical, dendritic cells upregulate many different components of the Th-1 response, favoring the priming of CD8+ effectors and promulgating cellular immunity. Experiment Overall Design: Dendritic cells were loaded by 1 of 5 different methods, then matured for 48 hours. Total cellular RNA was then extracted from 9 unloaded DC populations, 10 mRNA-loaded populations, 11 lysate-loaded populations, 7 doubly-loaded (matched) populations, and 7 doubly-loaded (mismatched) populations, a total of 44 different experiments. Each population was derived from a different normal human donor. Matched/mismatched refers to the source of the mRNA and lysate used to load the DCs. Matched signifies that the mRNA and lysate came from the same source. Mismatched signifies that the mRNA and lysate came from disparate sources. Gene expression profiles were then determined according to the method by which the DCs had been loaded. Genes were identified as differentially expressed by DCs doubly-loaded with matched antigens, only if they were significantly different from all of the other four controls. Significance was defined as Cohenâ??s |d| > 1.0 (large effect) and q-value (Benjamini-Hochberg false discovery) < 0.01.
Project description:Purpose: analyze the transcriptomic differences in PBS, LPS and LPS + Riociguat treated whole lung lysate Methods: C57BL6 mice were given PBS, LPS or LPS + Riociguat treatment via trachea instillation. 24h later, homogenize lung and extract mRNA Results: Riociguat can restore LPS-induced lung injury by improving immune environment
Project description:Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by using P-SCAN and a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation. http://discover.nci.nih.gov/host/2003_profiling_abstract.jsp Keywords: Cancer cell line comparison.
Project description:Single cell profiling is a powerful tool for studying the molecular and cellular (dys)function of activated peripheral blood mononuclear cells (PBMCs) in the context of disease. We combined CITE-seq with two levels of multiplexing (cell hashing and individuals? genotypes) to derive a reference database of immune cell gene/protein responses to different activation conditions. PBMCs from 10 healthy adults were profiled before and after stimulating i) T cells via anti-CD3/CD28 or ii) monocytes via LPS. By using a comprehensive antibody panel (n=39) of cell type (e.g., CD16, CD14) and cell state (e.g., CD69, CD25) markers, we discovered that all lymphocytes responded to anti-CD3/CD28 stimulation, whereas LPS specifically induced inflammation in monocytes. Pseudo-temporal analyses further revealed cell- and condition-specific heterogeneity in responses to activation that were independent of individual-specific variation. Together, these data are shared within an interactive web application (https://czi-pbmc-cite-seq.jax.org/) and will serve as a resource to guide future studies of immune cell responses.
Project description:Human Glioblastoma Multiforme tumors taken before dendritic cell vaccination, the recurrent tumors taken after vaccination and control GBM tumors from non vaccinated patients. Experiment Overall Design: Six Glioblastoma Multiforme patients underwent surgery. Their brain tumors were removed and analyzed via microarray. The lysate from the tumors were cultured with the patients' dendritic cells and the DCs were injected back into the patients. The patients GBMs returned and they underwent surgery a second time and those tumors were also analyzed via microarray. Tumors from the first and second GBM surgeries of 5 patients who did not receive DC vaccines are included as controls.
Project description:Picky is an online method designer for targeted proteomics. To assess the performance of PRM methods designed by Picky we carried out a benchmark experiment. As reference samples we used different amounts of human proteins spiked into 1.4 µg yeast lysate. These sample where analyzed in PRM and DDA mode. All methods inlcuded a shotgun method (top10 for DDA and top3 for PRM) and can therefore all be analyzed by MaxQuant.
Project description:Goal: Comparison of protein and phospho-protein levels in primary tumors based on activation of different AKT paralogs Methods: RPPA performed at the MD Anderson RPPA Core Facility. Detailed procedures available in methods section. Results: Focus of study compared protein and phospho-protein levels of BRAFV600E;Cdkn2a-/-;Pten-/- and BRAFV600E;Cdkn2a-/-;Pten-/-;AKT1E17K cohorts. P-FAK and paxillin were upregulated in tumors that express AKT1E17K compared with controls Conclusion: AKT1E17K has a critical role in upregulating P-FAK and paxillin