Neil Taylor

Enhanced Tolerance to Autoimmune Uveitis in CD200-Deficient Mice Correlates with a Pronounced Th2 Switch in Response to Antigen Challenge

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200−/− mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class IIlow APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.

Total dose and frequency of administration critically affect success of nasal mucosal tolerance induction

AIMS Nasal tolerance induction with autoantigens can effectively protect against a variety of experimental models of autoimmune disease. The aims of this study were to characterise the dosage and kinetics of inhibition of experimental autoimmune uveoretinitis (EAU) via intranasal administration of the uveitogenic antigen interphotoreceptor retinal binding protein (IRBP) in the murine model of IRBP induced EAU. METHODS B10RIII mice were tolerised by intranasal administration of IRBP either with a long term multiple low dose or a short term/high dosing regimen before subcutaneous immunisation with IRBP in complete Freunds adjuvant (CFA). On day 15 post-immunisation, mice were killed and eyes were removed for histological examination and quantification of inflammatory cell infiltration and degree of target organ (rod outer segment, ROS) destruction. RESULTS Nasal administration of multiple low doses of IRBP (1 μg or 3 μg IRBP per mouse per day for 10 days) significantly protected mice from IRBP induced EAU. Short term/high dose regimens were only effective when given either as a single or, at most, as two consecutive doses (40 μg per dose). Multiple doses in the range of 45–120 μg over 3 days afforded no protection. CONCLUSIONS These results indicate that both dose and frequency of intranasal antigen administration are pivotal to tolerance induction and subsequent suppression of T cell mediated autoimmune disease.

Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium

AIM To investigate the efficacy of “ex vivo” adenoviral vector mediated gene transfection of human conjunctival epithelial cell as a possible route for gene therapy for the distribution of anti-inflammatory agents for the potential treatment of immune mediated ocular inflammatory disorders. METHODS Human conjunctival cells (HCs) were cultured with various concentrations of recombinant adenoviral vectors carrying a reporter gene LacZ, GFP, or an immunomodulating cytokine vIL-10. vIL-10 in culture supernatant was detected by sandwich ELISA and biological activity was assessed by suppression of ConA stimulated splenocyte proliferation. X-gal and GFP expression was assessed by histochemistry. RESULTS The extent of adenoviral vector mediated transfer of both reporter genes and vIL-10 was dose dependent. LacZ expression could be detected for at least 50 day after infection with multiple of infection (MOI) 200. Following AdCMVvIL-10 transduction, vIL-10 protein expression occurred between 4–6 days post-transduction, and was maintained at a detectable level for at least 1 month. Secreted vIL-10 showed biological activity, significantly inhibiting Con A induced splenocyte proliferation. Additionally, transfection of HCs with two Adv vectors, one carrying LacZ and the other carrying GFP, resulted in co-expression within a single cell. CONCLUSION These results confirm previous successful adenoviral vector mediated gene transfer to HCs and further show that expression can be maintained. Furthermore the data show HCs can secrete biologically active vIL-10 that could be developed as a strategy to suppress immune mediated disorders. The successful co-transduction of HCs as described for other tissues, opens avenues to develop a multiple target gene therapy locally.