ICAM1 expression is upregulated on activated endothelium and is important in leukocyte adhesion and transendothelial migration (TEM). ICAM1 engagement initiates several signaling events including cortactin phosphorylation and association with the ICAM1 cytoplasmic tail. We hypothesized that interactions between cortactin and a putative SH3 binding domain in the ICAM1 tail regulate leukocyte TEM. To test this we mutated 3 amino acids within the ICAM1 cytoplasmic tail such that the putative SH3 binding domain was no longer present (SH3Mut ICAM1). We used adenoviral infection to express WT ICAM1 or SH3Mut ICAM1 in human umbilical vein endothelial cells (HUVEC). Standard biochemical methods and transmigration assays were used to assess functional relevance of the SH3 mutation. ICAM1 engagement triggers cortactin association with ICAM1. Approximately 50% less cortactin associates with SH3Mut ICAM1 (n=3, p<0.05). Neutrophil TEM is also reduced by 43% in cells expressing SH3Mut ICAM1. Furthermore, ICAM1 ligation induces the formation of a large transmigratory complex which includes ICAM1, VE‐Cad, p120, pSrc, and cortactin. Formation of this complex is, in part, dependent on the SH3 binding domain. Our study identifies an SH3 binding motif in the ICAM‐1 cytoplasmic tail that is necessary for association with cortactin and provides evidence that transmigratory complex formation is important for TEM.

e14263 Background: We have previously demonstrated that SPAR modified T cells effectively detect bacterial and viral pathogens. SPAR cells are engineered to express a modified T cell receptor (TCR) capable of antibody-directed signal transduction and respond with a Ca2+-mediated production of luminescent signal upon target detection. Here we describe the ability of SPAR cells to rapidly detect the cell surface expression of melanoma biomarkers without a requirement for extensive sample preparation. Methods: SPAR cells expressing both an engineered T cell receptor and the luminescent reporter protein aequorin were developed via the transfection of Jurkat cells with the aequorin expression vector pEF1-Aeq and an engineered TCR complex composed of mouse FcγRI fused to the CD3ζ subunit. The mFcγRI-CD3ζ receptor binds to the Fc region of full-length mouse IgG2 antibodies: SPAR cells are programmed for target detection via the addition of target-specific antibody. The ability of programmed SPAR cells to accurately detect melanoma-specific cell surface biomarkers was evaluated using whole cells from cultured mouse (B16-F10) and human (SK-Mel-28) melanoma cell lines. SPAR cells were programmed by incubation with murine antibodies directed toward the melanoma biomarkers CD133 or TRP1, then mixed with melanoma cells or K562 control cells and evaluated for signal generation. Results: SPAR cells programmed with anti-CD133 or anti-TRP1 antibody produced luminescent signal within minutes when combined with human SK-Mel-28 cells or mouse B16-F10 cells, respectively, known to abundantly express the appropriate biomarker. No signal was generated when programmed SPAR cells were incubated with K562 cells. Further studies also document cytokine production following receptor engagement. Conclusions: SPAR cells can be programmed for the rapid and specific detection of known cell surface cancer biomarkers. The Ca2+-dependent production of luminescent signal and cytokine release in response to TCR engagement suggests SPAR cell activation. Thus, in addition to biomarker detection the SPAR system may ultimately provide predictive insights into the potency of antibody-directed cell therapy.

8Efficient pathogen detection is essential for the successful treatment and prevention of infectious 9 disease; however, current methods are often too time intensive to be clinically relevant in cases 10 requiring immediate intervention. We have developed a Surface Programmable Activation 11 Receptor (SPAR) diagnostic platform comprised of universal biosensor cells engineered for use 12 in combination with custom or commercial antibodies to achieve rapid and sensitive pathogen 13 detection. SPAR cells are stably transfected Jurkat T cells designed to constitutively express a 14 modified T cell mouse FcγRI receptor on the cell surface and a high level of the luminescent 15 reporter protein aequorin in the cytoplasm. The modified mFcγRI-CD3ζ receptor protein binds 16 with high affinity to the Fc region of any full-length mouse IgG2a and some IgG2 antibodies: 17 this allows customized target detection via the selection of specific antibodies. T-cell receptor 18 aggregation in response to target antigen binding results in signal transduction which, when 19 amplified via the endogenous T cell signal cascade, triggers the rapid intracellular release of 20 calcium. Increased Ca 2+ concentrations activate the expressed reporter protein aequorin resulting 21 in the immediate emission of detectable light. Testing demonstrates the accurate and specific 22 2detection of numerous targets including P. aeruginosa, E. coli O111, and E. coli O157. We 23 report that the SPAR biosensor cell platform is a reliable pathogen detection method that enables 24 the rapid identification of bacterial causative agents using standard laboratory instrumentation. 25The technology lends itself to the development of efficient point-of-care testing and may aid in 26 the implementation of effective and pathogen-specific clinical therapies. 27 Introduction 28The rapid and accurate identification of causative agents is critical to the prompt application of 29 directed, pathogen-specific antibiotic therapies. Effective and timely clinical intervention is 30 essential for the control of infectious disease as well as in the successful treatment of bacterial 31 infections. The observed increases in the frequency and severity of nosocomial infections (1) 32 and the increasing prevalence of antibiotic resistance induced by non-specific antibiotic use (2) 33 further highlight the need for informed antibiotic selection based upon precise and efficient 34 pathogen detection. 35 Current bacterial identification methods include both classic procedures and novel molecular 36 techniques. Traditional culture-based methods, while sensitive and reliable, are also labor-37 intensive and time-consuming and therefore often cannot provide definitive diagnostics within a 38 clinically relevant timeframe (3). Molecular diagnostic methods include immunological assays 39 such as ELISA (4), microarray immunoblot (5), or serological assays (6); nucleic acid-based 40 techniques including PCR (7), DNA sequencing (8), hybridization techniques (9), or DNA/RNA 41 microarrays (10);...