Effect of Synthesized Onion Lachrymatory Factor on Tear Dynamics on the Soft Contact Lens

TEAR FILM & OCULAR SURFACE INTRACELLULAR TRAFFIC OF NOVEL CANDIDATE AUTOIMMUNE DACRYOADENITIS ANTIGENS. R. M. Hawk,1 R. Duncan,2 C. M. Rose,1 C. T. Chiu,1 L. Qian,1 A. K. Mircheff.1 Dept of Physiology & Biophysics, Dept of Molecular Pharmacology & Toxicology,2 Univ of Southern California, Los Angeles, CA. Purpose. The dysfunctional tear syndrome of Sjögren’s syndrome (SjS) is associated with focal CD4+ cell infiltration of the lacrimal glands and serum autoantibodies to intracellular proteins. To learn how lacrimal acinar cells (AC) expose or present autoantigen epitopes, we wish to identify autoantigens in amenable experimental models. Methods. Subcellular fractions from rat lacrimal glands were analyzed by SDS-PAGE. Western blots were probed with sera from NOD mice with diagnosed SjS-like disease, and IgG1 heavy chain was detected with isotype specific secondary antibody. Labeled bands were analyzed using matrix-associated laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry. Tryptic peptides were identified by Mascot Matrix Science search software. Compartmental distributions of candidate B cell autoantigens were determined by Western blotting of isolated membrane fractions from rabbit AC cultured with and without chronic carbachol (CCh) stimulation. Results. Subcellular fractionation analysis indicated that the candidate autoantigens were primarily associated with the membrane phase; most were primarily in compartments of the biosynthetic and lysosomal pathways. Some also were in secretory vesicle-related compartments of control cells but re-distributed to the endosomes after chronic CCh stimulation. The proteomic analysis identified Grp 78, rab3D, and ribosomal protein L7 as candidate autoantigens. Western blotting with monoclonal antibodies confirmed the presence and compartmental distributions of Grp 78 and rab3D. Protein sequence analysis with TEPITOPE confirmed that an identified B cell epitope at the N-terminal end of L7 also is a likely T cell epitope. Conclusion Grp78 and L7 are known to be antigens in other autoimmune diseases but have not previously been known to be associated with SjS. Rab3D has not previously been recognized as a disease-associated autoantigen. These and other AC autoantigens are present in intracellular compartments where they may be subject to aberrant proteolytic processing that we theorize generates otherwise cryptic epitopes. Commercial Relationship(s): None; Support: EY13720, EY05801, DK48522 MECHANISMS OF INNATE AND ADAPTIVE IMMUNE RESPONSIVENESS TO BACTERIAL INFECTION IN CORNEA. Linda D. Hazlett. Wayne State University School of Medicine, Detroit, MI. Purpose. Pseudomonas aeruginosa is a common organism associated with bacterial keratitis, especially in extended wear contact lens users. Advances in our understanding of host innate and adaptive immune responses to experimental infection have been made using inbred murine models that are classed as resistant (cornea heals) vs. susceptible (cornea perforates). Methods. A variety of immunological, microbiological, molecular and statistical analyses and approaches were used. Results. Data provide evidence that sustained IL-12 driven IFN-J production in dominant Th1 responder mouse strains such as C57BL/6 contributes to corneal destruction and perforation, while IL-18 driven production of IFN-J(by NK cells) in the absence of IL-12 p40, is associated with bacterial killing and less corneal destruction in dominant Th2 responder strains such as BALB/c. Data also suggest that IFN-J contributes to bacterial killing indirectly by regulating macrophage nitric oxide levels in cornea. The critical role of PMN in the innate response to bacterial infection also will be discussed, as mice of either strain depleted of PMN and then infected, die within 48 hours. Regulation of PMN also will be discussed and evidence from antibody neutralization experiments provided to show that persistence of PMN in B6 cornea is regulated by CD4+ T cells, while macrophages (and NK cells) regulate PMN number in the cornea of BALB/c mice. Conclusions. These studies continue to provide better understanding of the inflammatory mechanisms that are operative in the cornea after P. aeruginosa challenge and are consistent with our long-term goals of providing targets for alternative or adjunctive treatment for this disease. Commercial Relationship(s): None; Support: NIH R01EY02986 and P30EY04068

EFFECT OF SYNTHESIZED ONION LACHRYMATORY FACTOR ON TEAR DYNAMICS ON THE SOFT CONTACT LENS. Hisayo Higashihara, Norihiko Yokoi, Kunio Maruyama, Shigeru Kinoshita,1 Nobuaki Tsuge, Shinsuke Imai and Nobuo Shiomi.2 Department of ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan;1 House Foods Corporation, Chiba, Japan.2 Purpose. Tear film on the soft contact lens (SCL) often becomes thinner and unstable during SCL wear. This study was performed to investigate the effect of synthesized onion lachrymatory factor (SOLF: Propanethial SOxide) on tear dynamics on the SCL. Methods. Enrolled were all daily SCL wearer, aged 21.4 ± 1.6 (mean ± SD, n=8). Subjects wore 58% high water content hydrogel SCLs on both eyes for more than 10 hours. Their unilateral eyes, selected in a masked fashion, were exposed to 3 ȝl SOLF saturated in the original eyecup up until their limit of endurance to irritation. Before and after exposure, examinations were performed as follows: 1) the tear volume was evaluated by a video meniscometer (Yokoi, Cornea, 2000) by measuring the tear meniscus radius (TMR), 2). Using a vide-interferometer, the tear interference patterns on the contact lens (TIPCL) were classified into one of 5 grades (Maruyama, IOVS, 2004; increased grades for thinner film), and the non-invasive tear film breakup time (NIBUT) was measured. Results. TMR (mm) before and after the exposure of SOLF were 0.24 ± 0.07 (mean±SD) and 0.54 ± 0.14, respectively. The grades of TIPCL after exposure were significantly lower than those before exposure (before: 3.4 ± 1.3; after: 1.4 ± 0.5, p<0.05), NIBUT (seconds) after exposure were significantly longer than those before exposure (before: 2.5 ± 3.4; after: 9.1 ± 2.1, p<0.05). Conclusions. From this study, it was found that tear dynamics on the SCL became improved; and with the increase of tear volume, tear film on the SCL became thicker and more stable together with the smooth expansion of the tear film lipid layer. EFFECTS OF DESICCATION ON INFLAMMATORY CYTOKINE PRODUCTION IN CORNEAL EPITHELIUM. Akihiro Higuchi,1 Ayako Tagami,1 Keiji Kido,2 Junji Tomura,2 Shigeru Nakamura,2 Kazuo Tsubota.3 6N9 Research Park, Keio University School of Medicine, Tokyo, Japan;1 OPHTECS Corporation, Toyooka, Japan;2 Department of Ophthalmology, Keio University School of Medicine, Tokyo Japan.3 Purpose. Cornea epithelial cells are always affected by various physical factors, such as, temperature, humidity, ultraviolet irradiation, and airflow. Desiccation is significantly affected on these cellular conditions. We investigated the influence of desiccation on inflammatory cytokine production in corneal cells using human corneal epithelial (CEPI) cell line and dry eye model rats. Methods. CEPI was grown in keratinocyte growth medium 2 (KGM2) to approximately 80% confluence in dishes. The medium is discarded by aspiration and plates were left for 0 to 30 minutes with opening the cover to dry the cells (short term desiccation). KGM2 was poured into the dishes. Fifteen minutes later, the medium was collected to measure the concentration of the cytokines in medium by EIA. Viability of the cells was estimated with alamer Blue. To study the effect of long term desiccation, we use transwell (Corning Inc., corning, NY). CEPI was grown on membrane of transwell. KGM2 in upper-layer was discarded to dry CEPI and viability of the cells and concentration of the cytokines were measured. The expression of cytokines in cornea of dry eye model rat was measured by real time PCR. Results. In short term desiccation, CEPI started to die after 20 minute of desiccation. Secretion of IL-6 from CEPI increased by 15-20 minutes desiccation but that of TNFD did not change. In long term desiccation, secretion of IL-6 and IL-8 from CEPI increased but that of TNFD did not change. In dry eye model rat, the mRNA of IL-6 obtained from cornea also increased significantly, but that of TNFD did not change. Conclusions. The cell death induced by desiccation is suggested to related IL-6 and IL-8 secretion but not TNFD.

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