A major project in which involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. In collaboration NHGRI investigators we have shown that susceptibility to Behcet's disease is associated with genes which regulate the immune respons. We are currently investigating if the variants discovered in the IL-23R locus influence the expression levels of this receptor on T cells and monocytes of healthy individuals bymeasuring the mRNA expression levels for IL-23 in PBMCs from normal donors genotyped for their IL-23R variants. Furthermore, using a flow cytometry-based assay we are measuring the expression levels of IL-23R in specific cellular population. Moreover, we are also assessing the cytokine-secreting capabilities of patients suffering from Erdheim-Chester Disease (ECD), a rare, non-familial multisystem disorder characterized by proliferation and infiltration of non-Langerhans histocytes into multiple organs. Previous reports on the role of cytokines in ECD are few and variable, we compared the cytokine secretion levels of ECD patients with normal controls after stimulation of PBMCs. Additionally, since histiocytes are the primary cells involved in ECD lesions, we are investigating the ability of patients monocyte-derived macrophages and alveolar macrophages to ingest fluorescent particles as compared to healthy controls. These assays will allow us to better understand the mechanisms of inflammatory processes underlying these diseases and may lead to potential novel therapeutic approaches. 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 Dr. John OShea has been investigating novel therapeutic approaches for the treatment of autoimmune diseases. The TIS is currently involved in a collaboration with the O'Shea group and Pfizer (a CRADA is in place) aimed at defining the efficacy of the tyrosine kinase inhibitor Tofacitinib on the activation programs of human T cells. We examined the effects of tofacitinib in T cell metabolism. In effector T cells, tofacitinib changes T cell metabolic status from aerobic glycolysis to oxidative phosphorylation, which reflects activated T cell and quiescent T cell, respectively. Moreover, we investigated an effect of tofacitinib in CD8(+) T cells by analyzing animal model of CD8 T cell-mediated graft-versus-host like disease. We found an efficacious role in the model. Tofacitinib decreased CD8(+) T cell proliferation and activation. Also, tofacitinib directly inhibits IFNgamma-induced keratinocyte activation. Overall, Tofacitinib may improve autoimmune diseases and prevent transplant rejection. The TIS has also started a project aimed at generating inducible pluripotent stem cells (iPS). iPS are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somaticc cell, by inducing a """"""""forced"""""""" expression of specific transcription factors. Starting from a commercial source of human fibroblast we have generated iPS cells and demonstrated their pluripotency by assessing specific markers and embryoid body formation capacity. Furthermore, iPSCs were differentiated into cardiomyocytes that spontaneously began beating. Our research will now be aimed at showing that such cells are a powerful new tool for studying the pathogenesis of human diseases for pharmacological and toxicological testing as well as cell-based therapy. The TIS, is also providing support to several projects carried out at NIAMS aimed at better understanding the genetic determinants of autoimmune, autoinflammatory and musculoskeletal diseases using the Illumina HiSeq 2000 and the Genome Analyzer GAIIx for ultra high-throughput sequencing. With Dr. Kambiz Mousavi in Dr. Sartorellis group we continue studies to definie the epigenome and the transcriptional landscape during skeletal muscle formation. In particular we want to delineate the function of Polycomb group protein, Ezh1, within the myogenic system;and to characterize genome-wide occupancy of myogenic regulatory factors. we showed that the enhancement RNA Polymerase II elongation by Ezh1;and regulation of numerous short-lived intergenic non-coding RNA by MyoD during skeletal muscle formation. With Dr. Yuka Kanno our efforts are focused on analyzing genomic organization of T lymphocytes to understand gene regulatory mechanism for T helper cell fate specification. We have successfully profiled distribution of p300 to identify active enhancer landscape and extended our understanding on the intergenic regulatory region of the genome. We have performed transcriptome mapping by RNA-seq to understand phenotypic difference among T helper subsets. With Dr. Casellas we comprehensively analyzed the epigenome of B-cells. We are analyzing DNA methylation, histone modification as well as transcriptome from mature to hematopoietic/embryonic stem cells to acquire the landscape of those changes and to observe what is happening through the cell differentiation. Since B-cells undergo genome DNA editing in class switching recombination and somatic hypermutation upon encounter to the antigen. We use ChIP-seq technique to elucidate the repair pathways involved in B-cell maturation. 3. Genome organizer CTCF. CTCF is one of the critical factors to demarcate genome DNA between active and inactive loci and to organize its structure by holding genome DNA. We analyze CTCF mutants by ChIP-seq to clarify this function in details. Dr. Chao Jiang in Dr. Riveras lab is studying a subset of SLE patients that have elevated levels of circulating auto-IgE. To understand the contribution of auto-IgE to the pathogenesis of SLE, we investigated the gene expression profile associated with high levels of auto-IgE production in humans using RNA-Seq. Dr. Rosa Munoz Cano also in the Riveras lab evaluates and compares four clinical anaphylaxis phenotypes using RNA-Sequencing. With Dr. Jin-Chul Kim and Dr. Morasso we continue the study of Dlx3 function on inflammation. We want to investigate which genes are altered immediately after dlx3 deletion before we see the phenotype on the skin. Dlx3 also functions during hair development and we want to examine which genes are altered during the hair cycle as well as analyze the hair stem cell related genes expression after dlx3 deletion. With Dr. Leon Nesti and Dr. Youngmi Ji we have performed RNA-Seq studies aimed at elucidating how activation via injury affects the mesenchymal progenitor cells and to determine whether trauma may contribute to pathological differentiation of these cells and formation of heterotopic ossification. Outisde NIAMS IRP we are collaborating with Dr. Melodie Weller from NIDCR. We are sequencing of a novel RNA viral isolate identified in human serum and we are also resequencing an amplicon which carries a mutation present in a population of autoimmune patients initially identified through comparative genomic hybridization (CGH) analysis. With Dr. Nussenzweig group from NCI we are mapping replication-induced DNA damage stress in response to the drug hydroxyurea and the recruitment of DNA repair proteins to AID-dependent and in B cells.
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