The Translational immunology Section (TIS) core is equipped with instrumentation to measure cellular responses at multiple levels and it supports primarily (but not solely) the research NIAMS clinical investigators. Besides having the capability to detect and quantify cell-secreted proteins and analyzing signalling events using classical techniques such as ELISA and Western blotting the facility is capable to measure the same events with powerful high-sensitivity, high-throughput instrumentation such as: Multi-mode analyzers (Bio-Plex 200;Synergy4), magnetic cell sorter (Auto MACS), nucleic acid analyzers (Illumina Gene Analyzer;Agilent 2100 Bioanalyzer;ABI 7500 real-time PCR). A major project in which involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. NIAMS clinical investigators have been interested in the development and optimization of predefined sets of assays that would allow a better categorization of the autoinflammatory patients recruited weekly in several clinical protocols. The diverse pathological features of the patients enrolled in NIAMS trials have highlighted the need of standardized of assays which will help in the discrimination between different autoinflammatory syndromes. To this end we have started a multi-pronged approach which couples a Luminex-based screening for immunoregulatory factors with a flow-cytometry-based phenotyping. We initiated studies aimed at establishing bio-signatures of human cells treated with Toll Like Receptor (TLR) ligands as well as endogenous triggers of inflammation. Recently, NIAMS clinical investigators reported the discovery and characterization of a new syndrome resulting from mutations in the gene which encodes IL-1Ra the soluble antagonist for IL-1. The TIS was involved in the project and worked extensively to accurately measure the levels of expression for IL-1Ra in the patients with this new syndrome and confirmed that two of the three patients had significantly decreased gene expression for IL-1Ra assessed by measuring IL-1Ra mRNA using qPCR. As expected, these patients responded very well to treatment with Anakinra (recombinant IL-Ra), nonetheless their clinical features are somewhat different from NOMID patients. These findings were recently reported in The New England Journal of Medicine. As mentioned above, the discovery of protein patterns as biomarkers of disease or of therapeutic efficacy is a major objective of the TIS. The laboratory of John OShea has been investigating novel therapeutic approaches for the treatment of autoimmune diseases and the TIS is currently involved in a collaboration with the OShea group and Pfizer (a CRADA is in place) aimed at defining the efficacy of the tyrosine kinase inhibitor CP690,550 on human T cells activation and differentiation. Analysis of the effects of CP-690,550 on the signaling events downstream of the IL-2 receptor has shown that this compound is capable to reduce the phosphorylation of the transcription factor STAT5 upon IL-2 stimulation of human CD4+ T cells at concentration as low as 50 nM. Notably, the concentration required to block STAT5 phosphorylation in murine cells appears to be much higher. Interestingly CP-690,550 appears to be even more effective in blocking IL-2 dependent STAT3 phosphorylation whereas for STAT1 some residual activation remains even at a high dose of compound. The availability of next generation sequencing platforms, with remarkably low costs and high-throughputs has made the comprehensive understanding of the functional elements in the human genome a possible achievement. The TIS, is providing support several projects carried out at NIAMS aimed at better understanding the genetic determinats of autoimmune, autoinflammatory and musculoskeletal using the Illumina Gene Analyzer (GA) for ultra high-throughput sequencing. In particular three major projects have been supported. The first one, in collaboration with Dr. Vittorio Sartorelli and the Laboratory of Muscle Stem Cells &Gene Regulation. Dr. Sartorellis has obtained a high-quality definition of ChIP-Seq MyoD binding events in undifferentiated and differentiated C2C12 skeletal muscle cells. The second project is on collaboration with Dr. John OShea and is aimed at defining the role of epigenetic modification in the differentiation of T helper cells. To compare the epigenetic profile of chromatin in wild type and STAT knock out mice. By using the ChIP-seq technique, the group obtained a comprehensive view of gene activation program operating in different subsets of T helper cells. Initial attempts to profile histone 3 K4me3 (Lysine 4 trimethylation) vs. K27me3 have confirmed the presence of active epigenome (K4me3) only at the corresponding cytokine loci that defines the T helper phenotype, i.e. IFN-gamma locus in Th1, IL-4 locus in Th2 and IL-17 locus in Th17. Those signature cytokine loci in opposing T helper lineages harbored repressive K27me3 mark. In contrast, both a K4me3 and K27me3 (bivalent) signatures were found at the transcription start site of some key transcription factors. Both T-bet and GATA3, critical for Th1 phenotype and Th2 phenotype respectively, were bivalently marked in opposing lineages, suggesting a potential to be quickly expressed, if triggered appropriately. The bivalent condition may be a way to define plasticity of T helper phenotype and may allow the potential to switch from one type to the other. The third project involves a collaboration with Dr. Rafael Casellas which has been utilizing the GA deep sequencing capabilites to catalogue and quantify the expression levels of all known miRNAs during mouse and human B cell ontogeny. Preliminary analysis indicates deep sequencing to be exquisitely sensitive at quantifying absolute miRNA cellular abundance.
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