No abstract

Systemic administration of a recombinant adenovirus encoding the human interferon-beta gene (H5.110CMVhIFN-beta) results in transduction of hepatocytes and detectable circulating levels of IFN-beta protein. In preclinical studies in mice, we noticed a distinctly nonlinear dose response, with low levels of virus (1-3 x 10(10) viral particles) yielding barely detectable levels of IFN-beta but with a higher viral dose (1 x 10(11) particles) resulting in disproportionately high IFN-beta levels. Further studies showed that transgene expression levels from low viral doses could be dramatically enhanced by coadministering an unrelated recombinant adenovirus (H5.110CMVlacZ), suggesting that there was a viral dose threshold effect for efficient viral transduction and/or IFN-beta expression. This enhancement of reporter expression by a nonreporter adenovirus, effective upon coadministration, was further enhanced by preadministration of H5.110CMVlacZ (up to 8 h), but was ineffective if the helper virus was administered as little as 5 min after the H5.110CMVhIFN-beta reporter virus. Our data suggest that the reticuloendothelial system plays a role in this threshold effect, such that low doses of virus are efficiently taken up by the RES/Kupffer cells without leading to appreciable transgene expression, whereas high doses saturate these cells and are able to productively transduce hepatocytes. A better understanding of this phenomenon could have an impact on gene therapy clinical trial safety and efficacy.

Growth factors play an important role in supraspinatus tendon-to-bone healing. The objective of this study was to evaluate the temporal expression of 8 different growth factors in tendon-to-bone healing in an animal model. We hypothesize that growth factors exhibit unique temporal profiles that correlate to specific stages in the acute process of the supraspinatus tendon. To test this hypothesis, rats underwent bilateral supraspinatus tendon detachment and repair. Animals were euthanized at 1, 2, 4, 8, and 16 weeks. Immunohistochemical staining was done using antibodies for basic fibroblast growth factor (bFGF), bone morphogenetic protein 12 (BMP-12), BMP-13, BMP-14, cartilage oligomeric matrix protein (COMP), connective tissue growth factor (CTGF), platelet-derived growth factor-B (PDGF-B), and transforming growth factor-beta1 (TGF-beta1). Immunoassays showed an increase in the expression of all growth factors at 1 week, followed by a return to control or undetectable levels by 16 weeks in both the insertion and midsubstance. Future studies will investigate the different impacts of growth factor expression in tendon to bone healing.

Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity towards B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs (ITAM)-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared to standard first and second generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors.

Fibrin polymerizes to produce branching fibers forming a three-dimensional network, which has been difficult to visualize by conventional microscopy. Three-dimensional images of whole clots at high resolution were obtained from stereo-pair intermediate-voltage electron micrographs. Computer software was developed to produce three-dimensional reconstructions of the networks in the form of a pattern of links that connect branching junctions. Network parameters were measured and analyzed to characterize the clots quantitatively. Models in which all links were moved to the origin, while preserving their orientation, allowed visualization of some network parameters and facilitated comparison of networks. Fibrin clots formed in three different conditions were analyzed and compared by these methods. Clots formed in 0.20 M saline buffer consist of fibers of uniform size, and most of the branching junctions consist of three links. Fibrin clots formed in 0.05 M saline buffer are made up of very large diameter fiber bundles with far fewer branching junctions and correspondingly longer links. Clots formed in 0.40 M saline buffer consist of very fine fibers with numerous branching junctions and very short links. In summary, the extent of lateral aggregation is directly related to the distance between branching junctions and inversely related to the total number of branching junctions. These observations must be considered in defining possible mechanisms of fibrin branching.

Fibrinogen Philadelphia, a hypodysfibrinogenemia described in a family with a history of bleeding, is characterized by prolonged thrombin time, abnormal fibrin polymerization, and increased catabolism of the abnormal fibrinogen. Turbidity studies of polymerization of purified fibrinogen under different ionic conditions reveal a reduced lag period and lower final turbidity, indicating more rapid initial polymerization and impaired lateral aggregation. Consistent with this, scanning and transmission electron microscopy show fibers with substantially lower average fiber diameters. DNA sequence analysis of the fibrinogen genes A, B, and G revealed a T>C transition in exon 9 resulting in a serine-to-proline substitution near the ␥ chain C-terminus (S378P). The S378P mutation is associated with fibrinogen Philadelphia in this kindred and was not found in 10 controls. This region of the ␥ chain is involved in fibrin polymerization, supporting this as the polymerization defect causing the mutation. Thus, this abnormal fibrinogen is characterized by 2 unique features: (1) abnormal polymerization probably due to a major defect in lateral aggregation and (2)

<p>FAP-specific KIR-CAR/Dap12 T cells induce bone marrow hypocellularity and loss of body weight.</p>

<p>CD19-specific KIR-CAR/Dap12 shows antigen-specific cytotoxicity in vitro.</p>