Dendritic Cells as Danger-Recognizing Biosensors

Dendritic cells (DCs) are antigen presenting cells that are characterized by a potent capacity to initiate immune responses. DCs comprise several subsets with distinct phenotypes. After sensing any danger(s) to the host via their innate immune receptors such as Toll-like receptors, DCs become mature and subsequently present antigens to CD4+ T cells. Since DCs possess the intrinsic capacity to polarize CD4+ helper cells, it is critical to understand the immunological roles of DCs for clinical applications. Here, we review the different DC subsets, their danger-sensing receptors and immunological functions. Furthermore, the cytokine reporter mouse model for studying DC activation is introduced

Preparation and Evaluation of Single-Stranded DNA Aptamer-Based Immunological Adjuvant in Broiler Chickens

Mineral oils and metal salts are commonly used as adjuvants to enhance acquired immunity. Recently, monoclonal antibodies (MAbs) and recombinant peptides agonist CD40 receptor have shown remarkable promise for induction of rapid and robust immune responses. Limitations of this approach MAb production costs and multiple administrations due to anti-MAb immune responses. Here we demonstrate the development of a unique and sophisticated DNA aptamer-based alternative for CD40-directed delivery of universal antigens as an alternative in chickens, and potentially other vertebrate species. This receptor, expressed by antigen-presenting cells, acts as a costimulatory molecule for activated T helper lymphocytes. After initially selecting for high affinity aptamers of independent sequence, we utilized a polymerization process where multiple aptamers were simultaneously and sequentially arranged through rolling circle amplification products (RCA-p), potentially capable of binding multiple recognition sites and causing receptor clustering. Selected sequences were demonstrated to effectively activate chicken macrophage HD11 cell line through CD40 receptors, demonstrating proof of concept in vitro. Additionally, using limited proteolysis liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), we deduced the actual amino acid sequences targeted by these aptamer products, which are essential for activation. In chapter III, we produced a biotinylated version of an effective CD40-activating RCA-p, and streptavidin-conjugated this RCA-p to a biotinylated peptide of the highly conserved type-A influenza peptide M2e. This conjugate was administered subcutaneously at either (25μg/bird) or (50μg/bird) at age 7 and 21 days. Anti-M2e IgG responses were determined at 14, 21, 28 and 35 days of age by ELISA. The RCA-p-M2e complex, at 50μg/bird, was able to induce robust seroconversion as early as 7 days post-immunization, with high antibody titer consistent through the end of the experiment. The lower dose, however, showed delayed responses and required the second administration. Taken together, these results constitute proof of concept for RCA-p-directed antigen delivery for peptide antigens and a potential tool for rapid generation of new vaccines against animal diseases. In chapter IV we discuss PCR artifacts that develop during traditional DNA aptamers selection and proposed a repetitive thermal correction cycles [(65°C/10min, 20°C/1min)x6] of the PCR. This may improve future selection of aptamers for similar purposes

Targeting the tumor microenvironment in colorectal peritoneal metastases

Peritoneal metastasis (PM) occurs in approximately one in four colorectal cancer (CRC) patients. The pathophysiology of colorectal PM remains poorly characterized. Also, the efficacy of current treatment modalities, including surgery and intraperitoneal (IP) delivery of chemotherapy, is limited. Increasingly, therefore, efforts are being developed to unravel the PM cascade and at understanding the PM-associated tumor microenvironment (TME) and peritoneal ecosystem as potential therapeutic targets. Here, we review recent insights in the structure and components of the TME in colorectal PM, and discuss how these may translate into novel therapeutic approaches aimed at re-engineering the metastasis-promoting activity of the stroma

Developing Caspase-1 Biosensors to Monitor Inflammation in Vitro and in Vivo

Inflammation is central to the pathogenesis of numerous human diseases. in many contexts, inflammation drives immune responses which are critical to protection against many pathogens. However, uncontrolled inflammation can drive the pathology of numerous diseases, including neurodegenerative diseases, intestinal disorders, cancer, and autoimmune diseases. a primary mediator of inflammation is the inflammasome complex, which drives the release of inflammatory mediators through caspase-1 activation. Our goal was to develop a tool that would allow us to monitor caspase-1 activation in vitro and ultimately in vivo. to this end, we employed a circularly permuted form of luciferase in which the N- and C-terminal domains necessary for bioluminescence are physically separated by a linker region. Caspase-1 target sequences were cloned into this linker region, so that upon caspase-1 activation, these sequences are cleaved allowing the two domains of luciferase to come together and luminescence can be quantified. in pilot experiments, we identified putative sensors that exhibited a significant increase in bioluminescence following caspase-1 activation in vitro. We generated transgenic mice expressing our top biosensor and identified lines that exhibited biosensor expression in all tissues. to determine if the caspase-1 reporter was functioning in vivo, mice were challenged with various inflammatory stimuli and bioluminescence was quantified in living animals and tissues ex vivo. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. Our results suggest that caspase-1 biosensors may serve as an outstanding tool to monitor and quantify inflammation in living animals

Toll-Like Receptor 2 Dependent Signaling of Low Molecular Weight Hyaluronan Upregulates T-Helper 17 Differentiation and Promotes the Expression of Hyaluronidase 2

Immune cells have the capacity to differentiate and proliferate after stimulation through pattern recognition receptors (PRRs). In the case of potentially autoreactive T-­cells, signaling from self molecules through PRRs can stimulate these cells to attack otherwise healthy tissue leading to autoinflammatory diseases such as multiple sclerosis. One self molecule that may induce this effect is hyaluronan, a structural polysaccharide found in the extracellular matrix. We initially hypothesized that low molecular weight hyaluronan, indicative of injury, could signal through toll-­like receptor 2, a PRR, to upregulate Th17 cell differentiation and proliferation. Naïve CD4+ murine T-cells were isolated and exposed to low and high molecular weight hyaluronan to determine the molecule’s effect. Surprisingly, our results showed evidence for both hyaluronan and hyaluronidase II in upregulating T-­helper 17 cell differentiation and proliferation. Further research is necessary to describe the mechanism for this effect, as well as to establish its physiological relevance

Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided