2231st Conference Immunology Summit & Immunity and Immunotherapies 2018 10th World Congress and Expo on Immunology, Immunity, Inflammation & Immunotherapies October 19-20, 2018 | New York, USA Poster Presentations Page 45 Daniel Horowitz et al., J Clin Cell Immunol 2018, Volume: 9 DOI: 10.4172/2155-9899-C4-058 10th World Congress and Expo on Immunology, Immunity, Inflammation & Immunotherapies October 19-20, 2018 | New York, USA The development of a novel 3D printed prototype device to simplify blood cell subset enrichment at clinical sites Daniel Horowitz, George Szabo, Rudolf Cedro Jr. and Ling-Yang Hao Janssen Pharmaceutical Companies of Johnson & Johnson, USA eripheral blood collection is a relatively non-invasive way to obtain biomarkers during clinical trials. Protein and gene Pexpression changes can be used to assess drug/target engagement and disease state changes. To avoid functional changes in blood cells during storage, cells need to be stored in appropriate buffers. For gene expression analysis, whole blood can be collected in buffers that lyse the cells and protect RNA from degradation, but this obscures changes that may be found only in specific cell types. The separation of specific subsets requires instrumentation and trained personnel not readily available at many clinical sites, and shipping to a processing lab may affect the results. Therefore, a way to enrich for and stabilize blood cell subsets at the site of collection is needed. We have developed a portable 3D printed prototype device that can be operated with minimal training. Blood is collected into a tube containing commercially available polystyrene spheres (PluriSelect) coupled to an antibody specific for a blood cell surface antigen. After a 10 min incubation at room temperature, the tube is inserted into the device, followed by a single washing step. Between the inlet and outlet is a mesh with pores that are smaller than the polystyrene beads. All of the blood cells attached to the beads will remain on the mesh, while the unbound cells will pass through the filter. Using anti-CD3 antibodies, we were able to use FACS to verify that the device is capable of enriching more than 1,000,000 CD3+ cells from 2ml of whole blood with 90% purity. At a clinical site, these cells can be collected and processed at room temperature with minimal manipulation and stored in the appropriate buffer and transported. This prototype device may enable cell type-specific analysis for pharmacokinetic/pharmacodynamic assessments and biomarker discovery. Biography Daniel Horowitz has a BA from Temple University and has worked at Janssen for the past 13 years in the Immunology therapeutic area. Projects include biomarker discovery and development of techniques to help progress compounds through early clinical development. Disease areas include Pulmonary disease and Inflammatory bowel disease. [email protected] Notes: Journal of Clinical & Cellular Immunology | ISSN: 2155-9899 | Volume: 9 Page 46 Jamal Almitairi, J Clin Cell Immunol 2018, Volume: 9 DOI: 10.4172/2155-9899-C4-058 10th World Congress and Expo on Immunology, Immunity, Inflammation & Immunotherapies October 19-20, 2018 | New York, USA Activation of the classical complement pathway and auto-immune diseases Jamal Almitairi University of Leicester, United Kingdom he classical pathway of complement activation triggers lysis and opsonization of invading pathogens and stimulates Tinflammatory and adaptive immune responses resulting in auto-immune diseases and graft-rejection in organ transplantation. It is initiated via large multicomponent assembly, known as C1 (790kDa), that binds to immune complexes, protein modulators (e.g., C-reactive protein), and polyanionic structures on pathogens and apoptotic cells. It is composed of a large recognition subcomponent, C1q (460kDa), with a bouquet-like architecture consisting of six collagenous stems, each linked to a globular head, and four serine protease subcomponents, two C1r polypeptides (90kDa) and two C1s polypeptides (80kDa) that in the absence of C1q form a Ca2+-dependent heterotetramer. Binding to pathogens induces auto-activation in a stepwise fashion: C1r auto- activates and then activates C1s. C1s subsequently cleaves substrates C4 and C4b-bound C2 to form the C3 convertase (C4b2a), the next enzyme in the pathway. Here we describe the structure of the C1r–C1s interaction in the form of a complex between the CUB1-EGF-CUB2 fragments of each protease highlighting the conformational changes during activation. The fragments form Ca2+-dependent heterodimers both in solution and in the crystals. The interface is extensive and spans all three domains of each protease. Supporting the traditional arrangement in which C1r-C1s heterodimers are linked via interactions between the catalytic domains of C1r. In association with C1q, the C1r-C1r contacts would prevent auto-activation of C1r as the proteases fold up with the C1r-C1s dimers at the center. Disruption of the C1r contacts when C1 binds to an activating surface very likely triggers auto-activation of C1r and subsequent activation of C1s. Activation is likely facilitated through hyper flexibility at the C1s EGFCUB2 junction, enabling considerable movement of the catalytic domains. Biography Jamal Almitairi is a 3rd year PhD student at the University of Leicester working in structural biology and complement related diseases. Part of his work was published in the following journal: PNAS [email protected] Notes: Journal of Clinical & Cellular Immunology | ISSN: 2155-9899 | Volume: 9 Page 47 Luay AL-Kanan, J Clin Cell Immunol 2018, Volume: 9 DOI: 10.4172/2155-9899-C4-058 10th World Congress and Expo on Immunology, Immunity, Inflammation & Immunotherapies October 19-20, 2018 | New York, USA Characterisation of collections CL-Kl and CL-L1 and their role in the innate immune response Luay AL-Kanan University of Leicester, United Kingdom he immune system is a collection of proteins, cells and other biological components that protect an organism against Tpathogenic microbes. It prevents an attack through a series of processes collectively called the immune response. The complement system is an essential part of the innate immune response. Complement is activated by three pathways: the Classical (CP), the Lectin (LP) and the Alternative pathways (AP). The LP is activated by a range of different pathogen- recognition receptors including collecting kidney-1 (CL-K1 aka CL-11) and collecting liver-1 (CL-L1 aka CL-10), which bind to pathogen-associated molecular patterns (PAMPs), to activate three MBL-associated serine proteases (MASPs). CL-K1 and CL-L1 play an important role in host defense by recognizing a range of pathogens. These proteins can activate the LP individually or as a hetero-oligomeric complex. The sugar specificity of CL-K1 has been analyzed recently, it binds to high- mannose type structures and a range of different fucose-containing sugars including Lewis antigens and Blood group antigens. Binding of the former have been characterized structurally but binding to fucose-containing structures has not. In addition, the sugar specificities of CL-L1 is unknown. Here we present structures highlighting the ligand binding by CL-L1 and CL-K1. CL-K1 is known to bind to mannose and fucose-containing sugars, which are commonly found on bacterial surfaces. Biography Luay AL-Kanan PhD student working with Professor Wallis group in molecular biology and complement related disease. [email protected] Notes: Journal of Clinical & Cellular Immunology | ISSN: 2155-9899 | Volume: 9 Page 48 Xiao-Lin Tian et al., J Clin Cell Immunol 2018, Volume: 9 DOI: 10.4172/2155-9899-C4-058 10th World Congress and Expo on Immunology, Immunity, Inflammation & Immunotherapies October 19-20, 2018 | New York, USA Targeting both de novo biosynthesis and recycling of undecaprenyl phosphate as a new antimicrobial strategy against gram-positive bacteria Xiao-Lin Tian, Hasan Salim, Heather Rutherford and Yung-Hua Li Dalhousie University, Canada ntimicrobial agents that target bacterial cell wall biosynthesis are among the most successful armamentaria against bacterial infections. It is well known that undecaprenyl phosphate (C -P or Up) is an essential lipid carrier required for cell wall A 55 biosynthesis. Up is synthesized both via the de novo biosynthesis from dephosphorylation of undecaprenyl pyrophosphate (Upp) in the cytoplasm and via the recycling of released Upp after glycan is transferred to other molecules outside the cytoplasm. Both reactions are catalyzed by undecaprenyl pyrophosphate phosphatase (UppP). In addition to this pathway, Streptococcus mutans is found to have an alternative pathway to generate Up from phosphorylation of undecaprenol (C55-OH) catalyzed by an ortholog of diacylglycerol kinase (DagK). In this study, we aimed to determine whether simultaneous inactivation of uppP and dagK or blocking both the UppP- and DagK-catalyzed pathways affected the growth of S. mutans in response to cell wall-acting antibiotics. Two single-gene deletion mutants, ΔuppP and ΔdagK, and a double deletion mutant ΔdagK/uppP, were constructed for antibiotic susceptibility tests. The results revealed that deletion of uppP resulted in a mutant (ΔuppP) that was highly sensitive to bacitracin (MIC=0.25μg/mL), while deletion of dagK (ΔdagK) had much less effect (MIC≈20μg/mL) than the parent (MIC=40μg/mL). However, double deletion of both dagK and uppP nearly abolished the resistance of S. mutans to bacitracin, especially under pH 6.0. A combination of UppP inhibitor bacitracin (20μg/mL) with DgK inhibitor R59949 (25μM) almost completely inhibited the growth of S. mutans. It is concluded that antibacterial strategies that target both UppP- and DagK-catalyzed pathways could be an effective approach against Gram-positive bacteria such as S. mutans. Biography Xiao-Lin Tian received her MD degree in the Shanghai First Medical University. Since 1993, she worked as a researcher for Novopharm Biotech Inc for six years. She then worked in the Mount Sinai Hospital Lunenfeld Research Institute, Toronto, for another six years. Since 2006, Xiao-Lin has been working as a researcher at Dalhousie University, with the expertise of bacterial pathogenesis.