Potential Regulatory Function of Dendritic. Cells Overexpression Indoleamine 2,3-. Dioxygenase. Naoko Funeshima,1 Masayuki Fujino,1 Yusuke Kitazawa,1.
GENE REGULATION: TARGETS expression in conjunctival cells suggests that AAV is a useful delivery vector for a potent antiangiogenic factor. The administration route of subconjunctival injection can prevent the risk of endophthalmitits and cataract formation from the intracameral injection. Only one injection for gene delivery, endostatin is secreted from the conjunctival tissue around limbal vessels which can successfully suppress the corneal neovascularization during the wound healing process. This report showed the potential gene therapy of AAV carrying endostatin gene for use as a therapeutic agent to treat corneal neovascularization.
656. Potential Regulatory Function of Dendritic Cells Overexpression Indoleamine 2,3Dioxygenase Naoko Funeshima,1 Masayuki Fujino,1 Yusuke Kitazawa,1 Torayuki Okuyama,1 Xiao-kang Li.1 1 Department of Innovative Surgery, Natl. Res.Inst. for Child Health & Development, Tokyo, Japan. Indoleamine 2,3-dioxygenase (IDO) is an enzyme, involved in the catabolism of tryptophan and has been show to prevent rejection of the fetus during pregnancy, probably by inhibiting alloreactive T cells. In the present study, we investigated whether DCs that are transfected with IDO cDNA in inhibition of T cell proliferation after antigen-specific interaction. XS106 DCs, derived from A/J mice (H-2k) were transduced IDO with a gene delivery system using a recombinant adenoviral vector. Western blot and immune staining revealed IDO expression in XS106 DCs trasduced IDO (XS106-IDO DCs) and its catabolic effect was confirmed by increase the kynurenine concentration. Fluorescence activated cell sorter revealed that XS106-IDO DCs were no changeable for Ia, CD80 and CD86 expression. After co-cultured XS106-IDO DCs with C57BL/ 6 allogeneic splenic T cell, the proliferation of the T cell was inhibited significantly. The co-cultured T cells with XS106-IDO DCs were showed cell cycle arrest. Furthermore, injection of XS160-IDO DCs to the footpad of the C57BL/6 (H-2b) mice demonstrated that reduction of T cell response against allo-antigen. These results suggested that over expression of the IDO in the DC was effective to inhibit T cell proliferation, and may expand a new immunomodulatory strategy to prevent the allo-rejection of the organ transplantation.
657. Immune Responses to Transgene Expression Targeted to Keratinocytes Soosan Ghazizadeh,1,2 Lorne B. Taichman,2 Richard S. Kalish.3 Dermatology, Columbia University, New York, NY; 2Oral Biology and Pathology, Stony Brook University, Stony Brook, NY; 3 Dermatology, Stony Brook University, Stony Brook, NY. 1
Transgene-specific immune responses impose a major hurdle to effective implementation of clinical gene therapy, especially in situations where neoantigen is expressed. Previously, in our studies of cutaneous gene therapy, we showed that neoantigen expression leads to a potent immune response and rejection of the retroviraltransduced epidermal cells in normal mice. As cutaneous gene therapy will often require prolonged transgene expression, we seek a strategy to avoid this host response. One such strategy utilizes the fact that antigen presentation by non-professional antigen presenting cells (APCs), including keratinocytes, may result in T cell unresponsiveness. To explore this concept, the dorsal skin of immunocompetent mice was transduced in vivo with 2X10 7 transducing units of either standard retroviral vector encoding GFP or retroviral vectors in which GFP expression is targeted to the suprabasal layers of epidermis. Initial GFP expression in both groups of animals was confirmed at 1-week post transduction by fluorescent stereo microscopy. Examination at 3 weeks however, indicated loss S248
of GFP expression in both groups of transduced mice coinciding with the generation of significant levels of anti-GFP antibody. Although expression was directed to epidermal keratinocytes, the acute loss of transduced cells may have resulted from uptake of GFP entrapped in the viral particle by professional APCs, which are abundant in skin. This possibility is currently being examined with an ex vivo model of cutaneous gene transfer in which transgene expression will be confined solely to keratinocytes and transplanted to immunocompetent mice.
658. Different Regulation of Atrial and Ventricular HSP70 mRNA Expression during Warm Blood Cardioplegic Arrest in Rats Ernö Remsey-Semmelweis,1 John G. Kral,2 Richard B. Wait,3 M. A. Q. Siddiqui.4 1 Department of Cardiac Surgery, Philipps _University of Marburg, Marburg, Hessen, Germany; 2Department of Surgery, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York, NY; 3Department of Surgery, Baystate Medical Center, Springfield, MA; 4Department of Anatomy & Cell Biology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York, NY. Objectives: Normothermic blood cardioplegia (CWBCP) is considered as non-ischemic cardioprotection. Heat shock protein (HSP70) and creatine kinase (MCK) are markers of myocardial integrity and ischemia. We hypothesized that changes occur in atrial(A) and in ventricular(V) mRNA expression during CWBCP arrest and that A and V respond differently. Methods: mRNA expression was measured by RT-PCR in A and V tissue. Hemodynamic performance parameters were measured in blood perfused rat hearts (EXP.:n=10; Sham;n=6) in Langendorff mode before and after 1 hour CWBCP arrest. Protocol. EXP.: Baseline(B): 0 min., Equilibrium(EQ): 30 min., CWBCP:90 min., Reperfusion(R): 120 min., Sham (beating hearts): 90 min., and 120 min. Results: From B to EQ both A (5451±125 dpm vs 2933±295 dpm; Mean±SEM; p
