Abstract: By using a rhinosvirus/poliovirus type 1 chimera, PV1(RIPO), with the cognate internal ribosome entry site (IRES) of human rhinovirus type 2 (HRV2), we set out to shed light on the mechanism by which this variant expresses its attenuated phenotype in poliovirus-sensitive, CD155 transgenic (tg) mice and cynomolgus monkeys. Here we report that replication of PV1(RIPO) is restricted not only in human cells of neuronal origin, as was reported previously, but also in cells of murine origin at physiological temperature. This block in replication was enhanced at 39.5°C but, remarkably, it was absent at 33°C. PV1(RIPO) variants that overcame the replication block were derived by serial passage under restrictive conditions in either mouse cells or human neuronal cells. All adapting mutations mapped to the 5'-nontranslated region of PV1(RIPO). Variants selected in mouse cells, but not in human neuronal cells, exhibited increased mouse neurovirulence in vivo. The observed strong mouse-specific defect of PV1(RIPO) at nonpermissive temperature correlated with the translational activity of the HRV2 IRES in this chimeric virus. These unexpected results must be kept in mind when poliovirus variants are tested in CD155 tg mice for their neurovirulent potential, particularly in assays of live attenuated oral poliovirus vaccine lots. Virulence may be masked by adverse species-specific conditions in mouse cells that may not allow accurate prediction of neurovirulence in the human host. Thus, novel poliovirus variants in line for possible development of human vaccines must be tested in nonhuman primates.

Abstract: Increased expression levels of CD44 (Hyaluronic acid/lymphocyte homing receptor) and CD155 (Poliovirus receptor) have been reported on human glioma cells. While CD44 has long been associated with tumour invasion, CD155 has more recently attracted interests in a similar context.

Abstract: Tumour invasion is the key element in the high rate of mortality and morbidity in glioma patients. The increased levels of expression of the cell surface adhesion molecules CD44/CD155 on neoplastic cells have been highlighted in several studies; both playing a role in glioma invasion. CD44; originally described as the lymphocyte homing receptor, is a cell adhesion molecule with two isoforms with respective molecular weights of 80-90 kDa and 150kDa. CD44 mediates glioma cell adhesion and invasion through its interaction with hyaluronic acid (HA). CD155, also known as the poliovirus receptor, is a transmembrane glycoprotein, the ectodomain of which mediates cell attachment to the extracellular matrix component, vitronectin. Within the scope of this thesis, an upregulation of CD44 and CD155 has been demonstrated on established cell line (SNB-19) and early passage cultures of biopsy-derived glioma (UPAB and UPMC) using immunocytochemistry and flow cytometry. TIRF microscopy has revealed that CD44 and CD155 are located in close proximity in all the GBM cells studied. CD44 antibody blocking and gene silencing resulted in a higher level of inhibition of invasion than that for CD155 when assessed using the Transwell assay. Interference with combined CD44/CD155 resulted in 86% inhibition of invasion in post-transfected cells. Live cell imaging showed reduced speed of motility and distance travelled in knocked-down cells over their controls. Both siRNA CD44 and siRNA CD155 cells were devoid of filopodia and were rounder in morphology compared to wild type cells. The ECM cell adhesion array demonstrated wild type cells adhered most efficiently to laminin whereas siRNAtreated cells showed decreased adhesive potential on most of the ECMs used. The BrdU cell proliferation assay showed a higher proliferative rate of siRNA CD44 and siRNA CD155 treated cells was achieved and this was inversely correlated with the reduced invasion of these cells. Confocal microscopy showed distinct overlapping of CD155 and the integrins (β1, αvβ1 and αvβ3) on extending processes of the GBM cells whereas siRNAtransfected cells showed consequent reduction in expression level of the integrins with no specific staining patterns. RHO GTPases assay showed reduced expression levels of Cdc42, Rac1/2/3 and RhoA in siRNA transfected CD44 and CD155 cells. No change in expression level of RhoC was observed in siRNA CD155 cells compared to the control cells and the expression levels of RhoB were unchanged in both transfected and control cells. Joint CD44/CD155 approaches may merit further study in targeting infiltrating glioma cells in therapeutic protocols.

Abstract: Objective—CD155 is a cell surface protein that has recently been described to exert immune regulatory functions. We have characterized the expression of CD155 on human vascular endothelial cells (E...

Abstract: Human effector memory (EM) CD4+ T cells can rapidly transmigrate across an endothelial cell (EC) monolayer in response either to chemokine or to TCR-activating signals displayed by human dermal microvascular EC under conditions of venular shear stress. We previously reported that the TCR-stimulated transendothelial migration (TEM) depends on fractalkine (CX3CL1), PECAM-1 (CD31), and ICAM-1 (CD54) expression by the EC, whereas chemokine-stimulated TEM does not. In this study, we further analyze these responses using blocking mAb and small interfering RNA knockdown to show that TCR-stimulated TEM depends on CD99 on EC as well as on PECAM-1 and depends on nectin-2 (CD112) and poliovirus receptor (CD155) as well as EC ICAM-1. ICAM-1 is engaged by EM CD4+ T cell LFA-1 (CD11a/CD18) but not Mac-1 (CD11b/CD18); nectin-2 and poliovirus receptor are engaged by both DNAX accessory molecule-1 (CD226) and Tactile (CD96). EC junctional adhesion molecule-1 (JAM-1), an alternative ligand for LFA-1, contributes exclusively to chemokine-stimulated TEM and ICAM-2 appears to be uninvolved in either pathway. These data further define and further highlight the differences in the two pathways of EM CD4+ T cell recruitment into sites of peripheral inflammation.

Abstract: Patients afflicted with glioblastoma (GBM) have poor survival due to dispersive invasion throughout the brain. Necl-5, a cell surface receptor for vitronectin, is expressed in GBM but not normal brain. In several GBM cell lines Necl-5 promotes migration and invasion but the mechanism is poorly understood. In this study, we show that knockdown of Necl-5 by RNAi results in markedly decreased invasion of A172 GBM cells in a 3-dimensional matrix. There is a concomitant decrease in the expression and activity of matrix metalloproteinase-2 (MMP-2), a known factor in GBM invasion and disease severity. Knockdown of Necl-5 diminishes basal activation of Akt, an established mediator of MMP-2 expression in gliomas. Knockdown of Necl-5 also limits the maximal Akt activation in response to vitronectin, which requires the activity of Integrin-linked kinase (ILK). During migration, Necl-5, Akt and ILK co-localize at focal contacts at the leading edge of the plasma membrane, suggesting that these molecules may act to integrate Akt signaling at the leading edge to induce MMP-2 expression. By virtue of its restricted expression in GBM and its role in invasion, Necl-5 may be an attractive target for limiting MMP-2 production in glioblastoma, and therefore limiting dispersal.