Browsing by Subject "Autoimmune Disease"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Biomarker Discovery in Autoimmune Diseases: A Proteomics Approach(2009-01-14) Tan, Chiang "Niclas"; Kodadek, Thomas J.Proteins constitute the functional machinery of cells and are prime candidates for disease marker discovery. Mass spectrometry-based proteomics biomarker discovery holds the ability to interrogate a constellation of proteins simultaneously in a high-throughput manner to uncover a panel of markers that are specific to the presence of a disease. However, the rate of introduction of novel biomarkers with clinical currency has declined in the past few years due to challenges faced by both the discovery and validation stages. Surface retentate chemistry-mass spectrometry is a powerful platform that allows on-chip simplification of complex biological samples to better match the current dynamic range of mass spectrometers. Initial reports on differential protein profiling using this approach produced profiles with high sensitivities and specificities for disease classification. As with any maturing technology, issues that were overlooked during its introduction are now the main barriers to its clinical utility. The improved workflow described here aims to address some of these pending issues. Specifically, the experimental design incorporated knowledge of the disease pathway into sample selection, elected sample sources that are rich in diagnostic markers, and adopted biological and technical replicates to minimize variance. To ensure reproducibility, complete automation of the process from sample preparation to data acquisition was incorporated along with the adoption of a high performance mass spectrometer with minimal mass drift. A robust data analysis approach was implemented to overcome the issue of overfitting and to effectively trim down the list of candidate biomarkers to the selected few with true discriminatory power to facilitate downstream validation. As a demonstration of the robustness and utility of the workflow, profiling studies were performed on two autoimmune diseases. Protein profiles with high mass peak fidelity were obtained with high discriminatory power. Selective differential peaks were further investigated and confirmed to display differential levels in clinical samples. Validation in a larger sample set should determine the diagnostic potential of these markers for clinical application. Finally, a high-throughput study is reported showing that peptoids are, in general, a relatively more cell permeable class of molecules than peptides, rendering them ideal for drug development to target disease biomarkers.Item Enhancement of the An1 Population by BAFF: Potential Role in Autoimmune Disease(2010-01-12) Sadat, Eva Lasmin; Scheuermann, Richard H.The cytokine BAFF is a TNF family member found to be essential for the homeostasis of B cells. Numerous studies have shown BAFF to be involved in the pathogenesis of autoimmune diseases, including Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis. In an effort to understand the role of BAFF in the pathogenesis of autoimmune diseases, extensive in vitro studies on primary B cells have been performed. A recent study has described the existence of an anergic or silent autoreactive B cell population called An1, present within the wild-type repertoire of B cells in the periphery. Our differentiation studies using primary murine splenic B cells indicate an increase in this An1 population in response to BAFF stimulation. In order to ascertain whether this population is truly anergic, calcium flux assays have been performed. Interestingly, these assays show a decrease in response to BCR signaling in BAFF-treated cultures compared to untreated controls. Additional studies with sorted populations of cells indicate that these An1 cells are emerging mainly from the mature and the T2 populations. Survival studies using Annexin V as an apoptosis marker show that BAFF increases the survival of B cells in each of their different stages affirming the role of BAFF in promoting survival of the An1 cells. Microarray gene expression studies done on splenic B cells treated with or without BAFF show higher expression of a set of genes that have been reported to be upregulated in anergic B cells. Purified B cells from mice injected with BAFF also showed an increase in AA4.1 hi cells, which include the An1 cells. B cells from these mice show a lowered calcium flux upon BCR stimulation indicating anergic properties. These data suggest that BAFF stimulation results in the induction of B cell anergy, both in vitro and in vivo. The induction of anergy in B cells in response to stimulation through the antigen receptor may provide a mechanism by which autoreactive cells can evade deletion. Ultimately, the presence of these anergic B cells in the periphery poses a risk of activation and reversion to autoreactivity thus leading to automimmune disease.