TiArA: A Virtual Appliance for the Analysis of Tiling Array Data

Genomic tiling arrays have been described in the scientific literature since 2003, yet there is a shortage of user-friendly applications available for their analysis.Tiling Array Analyzer (TiArA) is a software program that provides a user-friendly graphical interface for the background subtraction, normalization, and summarization of data acquired through the Affymetrix tiling array platform. The background signal is empirically measured using a group of nonspecific probes with varying levels of GC content and normalization is performed to enforce a common dynamic range.TiArA is implemented as a standalone program for Linux systems and is available as a cross-platform virtual machine that will run under most modern operating systems using virtualization software such as Sun VirtualBox or VMware. The software is available as a Debian package or a virtual appliance at http://purl.org/NET/tiara

Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

Convenient and well-performing protein detection methods for a wide range of targets are in great demand for biomedical research and future diagnostics. Assays without the need for washing steps while still unaffected when analyzing complex biological samples are difficult to develop. Herein, we report a well-characterized nucleic acid proximity-based assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples. Target-specific antibody pairs are linked to DNA strands that upon simultaneous binding to the target analyte create a real-time PCR amplicon in a proximity-dependent manner enabled by the action of a DNA polymerase. 3′Exonuclease-capable polymerases were found to be clearly superior in sensitivity over non-3′exonuclease ones. A PEA was set up for IL-8 and GDNF in a user-friendly, homogenous assay displaying femtomolar detection sensitivity, good recovery in human plasma, high specificity and up to 5-log dynamic range in 1 μL samples. Furthermore, we have illustrated the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-affinity antibodies. Assay performance was also confirmed for a multiplex version of PEA

Acquisition and function of NK cell-associated molecules on T cells

One challenge for multicellular organisms, including humans, is to cope with a broad variety of microorganisms and their rapid replication and alterations. To do this, both fast and specific defense mechanisms are needed that can control threatening infections. Our immune system consists of two major parts: innate immunity, which is rapid and rather non-specific, and adaptive immunity, which is highly specific but requires more time for its development. Both innate (e.g. phagocytic cells and NK cells) and adaptive immune cells (B cells and T cells) are regulated by cellular interactions as well as by soluble factors. Each cell expresses a number of receptors, which can facilitate adhesion with other cells and/or mediate downstream signalling events that determine the outcome of immune reactions. In this thesis, I have focused on the expression of different NK cell-associated molecules on CD8+ T cells and the functional consequences of this. NKT cells have been classically defined as T cells expressing NK 1.1. These NKT cells are restricted to the MHC class I-like molecule CD1d and express an invariant T cell receptor, Jalpha281 -Valpha 14. My initial finding was that IL-2-activated spleen cell cultures derived from mice deficient in classical NKT cells contained NK 1.1+ T cells (the classical definition of NKT cells). We found that these cells were derived from conventional IL-2 receptor-beta+CD8+ T cells that had acquired NK1.1 upon activation. Subsequently, we observed that similar cells also appeared in the lungs of influenza A virus-infected mice. During the peak of infection, up to 10% of the CD8+ T cells in the lungs coexpressed NK 1.1. Around one third of the NK1.1+CD8+ T cells were specific for a single influenza A virus-derived epitope (ASNENMDAM) as determined by tetramer stainings and antigen-induced IFNgamma production. Interestingly, these NK1.1+CD8+ T cells exhibited a memory phenotype and coexpressed other NK cell-associated molecules including inhibitory Ly49 receptors and 2B4. This showed that all NK1.1+ T cells do not belong to a certain lineage and that the expression of NK cell-associated molecules may represent a state of activation for T cells. We found that, similar to NK 1.1, the NK cell receptor 2B4 was expressed on a subset of memory- like CD8+ T cells and induced upon IL-2 stimulation and during influenza A virus infection. Therefore, we decided to address the function of 2B4 when expressed on CD8+ T cells. To our surprise, we found that 2B4 acted as a ligand, rather than a receptor, for CD48 expressed on neighboring T cells and augmented both antigen-driven and IL-2-induced proliferation. These results prompted us to study whether other 2B4-expressing cells could costimulate T cells through 2B4/CD48 interactions. It was observed that NK cells as well as 2B4-expressing tumor cells enhanced both activation and proliferation of the T cells in response to IL-2 or CD3-crosslinking. NK cells can regulate adaptive immune responses by shaping the cytokine milieu during infection or autoimmune conditions. These data suggest that in addition to cytokine production, direct physical interactions between NK cells and T cells influence both activation and proliferation of T cells. We speculate that 2B4/CD48 interactions between immune cells may facilitate maintenance and reactivation of CD8+ memory T cells and regulate adaptive immune responses