Role of Integrin Expression in Adenovirus-Mediated Gene Delivery to the Intestinal Epithelium

Overview summary Previous studies of adenovirus-mediated gene transfer of the interleukin-1 receptor antagonist protein to rat jejunum have produced limited gene expression. In the present study, we have shown that integrins play a significant role in efficient adenoviral infection of the intestinal epithelium. As enterocytes differentiate, integrin expression decreases. This coincides with significant reduction in adenoviral transduction efficiency. Results from two in vitro models of the intestinal epithelium (Caco-2 and IEC-18 cells) show that αvβ3 integrins have the most influence on adenoviral internalization. Results from screening of rat intestinal segments for expression of the αvβ5 integrin suggest that, based on integrin expression, the ileum is a prime target for efficient adenovirus-mediated gene transfer. We have also found that administration of immunomodulating agents and inflammatory disease states provide a favorable environment for efficient internalization of adenoviral vectors due to up-regulation of integrin receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63160/1/hum.1998.9.4-561.pd

Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant

Abstract We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.http://deepblue.lib.umich.edu/bitstream/2027.42/109454/1/13075_2000_Article_409.pd

Imaging of joints with laser‐based photoacoustic tomography: An animal study

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134964/1/mp4166.pd

Inhibition of Interleukin-1-Induced Effects in Synoviocytes Transduced with the Human IL-1 Receptor Antagonist cDNA Using an Adenoviral Vector

Overview summary Adenovirus-mediated gene transfer into the cells of the synovial membrane may provide a means to deliver therapeutically active proteins for the local modification of the immune response in inflammatory arthropathies. In this study, we infected type B human synoviocytes in vitro and rabbit synovial lining membrane in vivo with a recombinant human adenovirus containing the cDNA for the human interleukin-1 receptor antagonist protein (IL-1ra). Expression of human IL-1ra was observed both in the transduced synoviocytes in vitro and in the microenvironment of the transduced rabbit synovial membrane in vivo, and the functional activity of the transgenic IL-1ra was suggested by in vitro inhibition of interleukin-1 (IL-1)-induced prostaglandin E2 (PGE2) production and by in vivo inhibition of IL-1-induced glycosaminoglycan (GAG) degradation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63124/1/hum.1995.6.3-307.pd

Pulmonary Inflammation Induced by Incomplete or Inactivated Adenoviral Particles

Overview summary The amount of pulmonary inflammation induced in mice by intratracheal administration of high doses of adenoviral vectors was compared to that induced by viral particles that lack the ability to express the genes that they contain. The number of inflammatory cells infiltrating the lung 6 days after particle administration was similar between animals receiving normal versus defective adenoviral particles.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63169/1/hum.1995.6.12-1553.pd

Perifollicular transgenic expression of human interleukin-1 receptor antagonist protein following topical application of novel liposome-plasmid dna formulations in vivo

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34491/1/8_ftp.pd

Synthesis of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli

Naturally occurring mutations in hypoxanthine-guanine phosphoribosyltransferase (HPRT) have been identified by amino acid sequencing, cDNA cloning, and direct nucleotide sequencing of PCR-amplified transcripts. To determine the effect these mutations have on the catalytic properties of the molecule, knowledge of the three-dimensional structure of HPRT is required. A prerequisite for this, however, is the availability of a large amount of purified product for crystallization and x-ray diffraction analysis. For these reasons we have developed an effective means of producing high levels of human HPRT in Escherichia coli using the expression cassette PCR. By taking advantage of a T7 polymerase/promoter system, we have expressed both normal and variant human hprt sequences in E. coli. The proteins synthesized from these sequences are immunologically and enzymatically active, and are physically indistinguishable from the HPRT in B-lymphoblasts derived from normal and three HPRT-deficient subjects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31002/1/0000677.pd

Direct plasmid mediated transfection of adult murine brain cells in vivo using cationic liposomes

Gene transfer to the central nervous system (CNS) is complicated by the anatomic and physiologic isolation of the brain. Direct injection techniques circumvent this, and allow delivery of transgenes to specific areas of the CNS. Previously, direct transfection of cellular components of the CNS has been achieved using plasmid DNA. We report the use of cationic liposomes as a means of transfecting plasmids into adult mammalian brain. Using the gene for E. coli [beta]-galactosidase or the cDNA or human [beta]-glucuronidase as reporters, we demonstrate plasmid mediated gene transfer into the caudate putamen of adult mice with expression of the transgene for at least 21 days post-transfection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31777/1/0000718.pd