Expression of TNF Inhibitor Gene in the Lacrimal Gland Promotes Recovery of Tear Production and Tear Stability and Reduced Immunopathology in Rabbits with induced Autoimmune Dacryoadenitis

TEAR FILM & OCULAR SURFACE EXPRESSION OF TNF INHIBITOR GENE IN THE LACRIMAL GLAND PROMOTES RECOVERY OF TEAR PRODUCTION AND TEAR STABILITY AND REDUCED IMMUNOPATHOLOGY IN RABBITS WITH INDUCED AUTOIMMUNE DACRYOADENITIS. Melvin D. Trousdale,1 Zejin. Zhu,1 Douglas Stevenson,1 Moonseok Na,1 Joel E. Schechter,2 Thomas Ritter,4 and Austin K. Mircheff.3 1Doheny Eye Institute, and Departments of 2 Cell & Neurobiology, 3Physiology & Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, CA. and 4Institute of Medical Immunology, CharitéUniversity Medicine Berlin. Purpose. To evaluate the effect of adenovirus-mediated transfer of tumor necrosis factor (TNF)-D inhibitor gene on induced autoimmune dacryoadenitis in a rabbit model. Methods. Autoimmune dacryoadenitis was induced in adult NZW rabbits by injecting the lacrimal glands with PBL activated by 5 days of co-culture with autologous lacrimal gland acinar cells in a mixed cell reaction. Two weeks later, after clinical assessments verified severe dry eye, the dysfunctional gland was inoculated with 1x108 pfu in0.2 ml of AdTNFRp55-Ig. Two weeks later, frozen sections of the glands were immunostained for expression of CD4, CD8, RTLA and CD18 markers. Results. Two weeks after induction of disease, tear production was reduced by about 40%, while tear film stability declined by 43%. Two weeks after gene therapy, tear production returned to normal levels, tear BUT improved and rose bengal scores declined. Glands from animals with induced dacryoadenitis (with and without treatment) contained immune cell infiltrates that stained positive for CD4, CD8, RTLA, and CD18 antigens. In the lacrimal gland, the CD4 to CD8 T cell ratio was 4:1 in the disease group compared to 1:2 in the treated group. Infiltration of T cells and CD18+ cells was reduced approximately 50% after gene therapy. Conclusions. Therapeutic levels of soluble TNF inhibitor were achieved in the lacrimal gland and on the corneal surface. Anti-inflammatory cytokine gene expression might offer a potential therapeutic modality for the treatment of autoimmune dacryoadenitis, once suitable vectors become available. Commercial Relationship(s): None; Support: NIH grants EY12689, EY05801, EY10550, EY03040 and grants from DFG Ri 764/10-1, Research to Prevent Blindness, Inc., and Allergan Pharmaceutical Company

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PROTEOMIC EVALUATION OF HUMAN MEIBOMIAN GLAND SECRETIONS. Patricia S. Tsai,1,2,3 James E. Evans,5 Karin M. Green,5 Rose M. Sullivan,1 Debra A. Schaumberg,1,2,3,4 Stephen M. Richards,1,3 M. Reza Dana,1,2,3 David A. Sullivan.1,3 Schepens Eye Research Institute,1 Massachusetts Eye and Ear Infirmary,2 Harvard Medical School3 and Brigham and Women’s Hospital,4 Boston, MA; University of Massachusetts Medical School,5 Worcester, MA, USA. Purpose. Human tears contain hundreds of proteins that may exert a significant influence on tear film stability, ocular surface integrity and visual function. However, the origin of many of these proteins is unknown. We hypothesize that a major source is the meibomian gland. Our rationale is that the meibomian gland is a large sebaceous gland, and sebaceous glands are known to secrete a variety of proteins (e.g. IgA, IL-1D, TNF-D) and lipids in non-ocular sites. To begin to test our hypothesis, we sought to develop an optimal methodology for the identification of proteins in human meibomian gland secretions. Methods. Meibomian gland secretions were collected from the left and right lower eyelids of adult male and female volunteers and processed for proteomic analysis by one of 3 different methods: [a] secretions were partitioned in a chloroform-methanol-water biphasic system. Non-lipid phase materials were dissolved in 8 M urea, reduced with dithiothreitol, alkylated by iodoacetamide, and digested in trypsin to generate peptides for microcapillary (μ) HPLC/mass spectrometry (MS)/MS protein identification. This peptide mixture was separated by μcapillary reverse phase chromatography and the effluent examined by nanoelectrospray mass spectrometry and data dependant MS/MS. SEQUEST software was used to identify proteins from the MS/MS spectra by searching a human protein database; [b] samples were processed and digested as in the first method, peptides were separated by 2-dimensional liquid chromatography and the resulting effluent was analyzed by Multidimensional Protein Identification Technology (MudPIT); and [c] secretions were transferred onto immobilized pH gradient (IPG) strips, isoelectrically focused, reduced, alkylated and then analyzed by 2dimensional (2D) gel electrophoresis and SYPRO Ruby staining. Results. Our results to date indicate that μHPLC/ MS/MS is the most sensitive technique for the analysis of proteins in human meibomian gland secretions. This procedure has led to the identification of over 80 proteins, including D2-macroglobuin receptor (may function in lipoprotein metabolism, proteinase regulation, and growth factor regulation), farnesoid-X receptor (a regulator of cholesterol homeostasis) and Tro alpha 1 H (an IgA heavy chain variant). In contrast, the MudPIT approach has not yet yielded sufficient information to identify any tryptic peptides. Studies with 2D gel electrophoresis have revealed approximately 20 spots, that are in the process of being isolated, digested and analyzed by Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Conclusions. Our findings suggest that the human meibomian gland secretes numerous proteins into the tear film. In addition, our results indicate that μHPLC/ MS/MS, and possibly 2D gel electrophoresis, may be appropriate for the analysis of these proteins. It is quite possible that these meibomian gland proteins contribute to the dynamics of the tear film in both health and disease. Support: Allergan, NIH (EY05612) and the Schepens Eye Research Institute/ Massachusetts Eye and Ear Infirmary (MEEI) Joint Clinical Research Center, as well as funds from the MEEI Cornea Service

THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT