We have focused on understanding at the molecular level the pathogenesis of human non-infectious, sight-threatening uveitis using multiple established molecular and cellular strategies to analyze tissue samples from patients with ocular inflammatory disease. Initial studies on patient specimen have provided information that has not been observed in animal and in vitro cell line studies. In addition, a great deal of effort has been made to minimize limitations and difficulties of handling patient specimen. Progress has also been made in standardizing laboratory methodology and synchronizing works between clinicians and basic researchers. I: Molecular mechanisms of non-infectious uveitis: Despite intensive studies on animal models and using in vitro culturing system, the molecular mechanisms of non-infectious uveitis in human are still an open question. In conjunction with our microarray studies described elsewhere, we have studied a recently described TNF receptor family member, GITR (glucocorticoid-induced TNF receptor family-related gene, TNFRSF18), and its expression in non-infectious uveitis patients vs. normal donors. GITR has been recently suggested a marker for CD4+CD25+ regulatory T cells which are thought critical for peripheral tolerance. GITR is also shown to be involved in T cell co-stimulation and apoptosis. We found that GITR expression correlated well with the clinical status of uveitis, hence, a marker for the disease status. In addition, flow cytometry analysis suggested that GITR expression, as well as CD25 on T cell and non-T cell surface in normal human donor is much higher than that shown in mice studies, which seems consistent with our earlier obervation that the outcome of blocking human IL-2R (CD25) may not be the same as what would be predicted based on studies in IL-2 and IL2R gene knock-out mice. If GITR and CD25 are true markers for a sub-population of regulatory T cells, our data suggested that the phenotypes of this sub-population in human may differ from that in mice. Further studies to address this question is intensively pursued in the laboratory. Intensive studies are also underway to understand the implication of GITR expression in autoimmune uveitis patients as well as in other autoimmune diseases by studying the effects of blocking the interaction between GITR and GITR ligand in vitro as well as in uveitis animal model. In addition, we have also focused on the role of GITR lignad (GITRL) in uveitis as well as the interaction of GITRL-GITR at the molecular level. An effort to generate GITRL deficient mice is under way which should provide great insight in understanding this aspect. In addition, we continue our interest in the search for antigens associated with uveitic conditions. Tropomycin has been recently reported to cause uveitis in rats. We are now testing the uveitogenic tropomycin peptides. The recall responses (proliferation, cytokine profiles) of immune cells from Behcet's disease patients and from normal donors to those tropomycin peptides, as well as to established human retinal S-antigen, are being investigated. II: Mechanistic effects of daclizumab (humanized anti-CD25, anti-Tac, HAT) treatment on uveitis patients: Daclizumab, or humanized anti-CD25, or anti-Tac, or HAT, has been successful in treating non-infectious intermediate and posterior uveitis. Understanding the mechanisms of this therapy at the molecular level can facilitate and improve clinical treatment of autoimmune uveitis. We believe that these findings will have implications for other autoimmune disorders. Currently, we are addressing several basic questions in those patients who have been successfully treated with daclizumab: 1) will daclizumab therapy change the cellular components of immune system? e.g., CD3, CD4, CD8 or CD4+CD25+ population, which has recently been described as a key peripheral regulatory sub-population. We are using multi-color FACS analysis system to address this question. Similar studies in normal donors are also being used. 2) Does therapy change the functionality of the immune system? e.g., proliferative responses to a variety of stimuli such as PHA, human retinal S-Ag and tetanus toxoid. 3) Does therapy change the TH1/TH2 balance? e.g. Th1 and/or Th2 cytokine and chemokine expression profiles in response to different stimuli. Initial results suggested that CD4+CD25+ population in either normal donor or uveitis patients is much higher than that shown in mice studies. But, we have not seen significantly higher CD4+CD25+ cells in HAT treated patients than in normal donors. Further studies to compare among HAT treated patients vs. non-HAT treated patients, as well as normal donors are underway. III: Streamline molecular and cellular methodology and maximize windows that help reveal potential mechanisms for the disease: The advantage of studying patient samples is its direct clinical relevance and potential impact on clinical diagnosis and therapy. But the challenges of clinical studies are the limitation of sample sources and quantity, inconsistency of patients' compliance, often difficulty to repeat the results, as well as influences of constantly changing environments that may alter the outcome of studies. One of the ways to minimize those factors is to streamline the methodologies that are used to examine patients' specimen so that multiple measurements will be performed simultaneously for a single specimen and mutually supportive data will be generated . We have made a lot of efforts to standardize methodology for analysis. Several molecular and cellular technologies, e.g. multi-color, whole blood flow cytometry analysis, multi-task proliferation assay (e.g., multiple comparison of stimuli, simultaneous analysis of cytokine profiles from the same proliferation assay) as well as microarray analysis have proven high through-put, highly reliable and reproducible.
