Intraocular DHODH-inhibitor PP-001 suppresses relapsing experimental uveitis and cytokine production of human lymphocytes, but not of RPE cells.

BACKGROUND: Uveitis is a potentially blinding inflammatory disease of the inner eye with a high unmet need for new therapeutic interventions. Here, we wanted to investigate the suppressive effect of the intraocular application of the small molecule dihydroorotate dehydrogenase (DHODH)-inhibitor PP-001 on experimental relapsing rat uveitis and furthermore determine its effect on proliferation and cytokine secretion of human peripheral blood lymphocytes (PBL) and human retinal pigment epithelial (RPE) cells in vitro. METHODS: Spontaneously relapsing uveitis was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide R14. PP-001 was injected intravitreally after resolution of the primary disease to investigate further relapses. Proliferation and metabolic activity of phytohemagglutinin (PHA)-stimulated human peripheral lymphocytes with and without PP-001 and cytokine secretion were determined by XTT assay and bioplex bead assay. The RPE cell line ARPE-19 as well as primary human RPE cells treated with PP-001 or anti-vascular endothelial growth factor (VEGF) antibody bevacizumab were also investigated for metabolic activity and cytokine/chemokine secretion. RESULTS: Injection of PP-001 into rat eyes reduced the number of relapses by 70%, from 20 relapses (57% of the rats affected) in the control group to 6 relapses (33% of the rats) in the treatment group. In human PBL cultures, PP-001 reduced the proliferation in a dose-dependent manner. The secretion of several cytokines such as IL-17, IFN-γ, and VEGF was suppressed by PP-001, as previously observed with rat T cells in the experimental autoimmune uveitis (EAU) model. In contrast, human RPE cells were not affected by PP-001, while the anti-VEGF antibody bevacizumab severely impaired the secretion of various cytokines including VEGF. CONCLUSIONS: For the first time, intravitreal injection of PP-001 demonstrated an effective, but transient reduction of relapses in the rat EAU model. In vitro PP-001 suppressed proliferation and cytokine/chemokine secretion of human lymphocytes, while neither human RPE cell line ARPE-19 nor primary RPE cells were affected.

Sirolimus for the treatment of noninfectious uveitis.

INTRODUCTION: Noninfectious posterior uveitis (NIPU) remains a significant burden of legal blindness. Because of its immune mediated and chronic recurrent nature, common therapy includes (systemic) corticosteroids and immune modulatory agents. Most treatments bear the risk of significant adverse effects. Therefore efforts are made to administer therapeutic agents directly into the vitreous cavity. The purpose of this article is to identify the role of intravitreally applied sirolimus as a recently approved therapeutic option in NIPU. AREAS COVERED: A MEDLINE database search was conducted through August 2015 using the terms: intravitreal injection, pharmacology, sirolimus, treatment and uveitis. To provide ongoing and future perspectives in treatment options, also clinical trials as registered at ClinicalTrials.gov were included. Sirolimus (Opsiria) was in licensed from SANTEN in 2015 and approved in Phase III registration trials in the US, Europe and other countries for NIPU. Current information results mainly from registration and Phase III trials. EXPERT OPINION: Intravitreal sirolimus appears to be an interesting option in the treatment algorithms of NIPU because of its highly targeted molecular effects, nonsteroidal nature and good safety profile. It has the advantage to avoid systemic side effects, but this has to be balanced against the fact that treatment covers one eye only and bears the risks of any intraocular procedure. Nevertheless a careful evaluation of this agent has to be made, as current experience is almost exclusively based on registration trials and long-term effects still have to be explored.

Fast and Successful Management of Intraocular Inflammation with a Single Intravitreal Dexamethasone Implant.

Purpose: To investigate the efficacy and safety of a single dexamethasone intravitreal implant (Ozurdex®, 700 µg). Methods: In this prospective noncomparative case series, 84 patients (54 females) received a dexamethasone intravitreal implant. At weeks 4, 12 and 24 after the injection, vitreous haze, macular thickness and best corrected visual acuity (BCVA) were assessed and adverse events reported. Results: Clearance of vitreous haze could be achieved after 4 weeks in 61% of all eyes (p < 0.001) and remained significant until week 24 (p < 0.001). This was paralleled by a reduction of central retinal thickness after 4 (p < 0.001), 12 (p < 0.001) and 24 weeks (p < 0.006). Significant and fast improvement of BCVA was already achieved after 4 weeks (p < 0.001) but vanished by week 24. Intraocular pressure reached ≥35 mm Hg in 3 eyes and was significantly more frequent in intermediate uveitis compared to posterior uveitis (p < 0.016). Conclusions: The dexamethasone implant is effective in controlling intraocular posterior segment inflammation and reduces central retinal thickness fast and effectively. © 2014 S. Karger AG, Basel.

Wide-field fundus autofluorescence corresponds to visual fields in chorioretinitis patients.

BACKGROUND AND OBJECTIVES: Detection of peripheral fundus autofluorescence (FAF) using conventional scanning laser ophthalmoscopes (SLOs) is difficult and requires pupil dilation. Here we evaluated the diagnostic properties of wide-field FAF detected by a two-laser wavelength wide-field SLO in uveitis patients. STUDY DESIGN/MATERIALS AND METHODS: Observational case series of four patients suffering from different types of posterior uveitis/chorioretinitis. Wide-field FAF images were compared to visual fields. Panretinal FAF was detected by a newly developed SLO, which allows FAF imaging of up to 200° of the retina in one scan without the need for pupil dilation. Visual fields were obtained by Goldmann manual perimetry. RESULTS: Findings from wide-field FAF imaging showed correspondence to visual field defects in all cases. CONCLUSION: Wide-field FAF allowed the detection of visual field defect-related alterations of the retinal pigment epithelium in all four uveitis cases.

Uveitis in a patient treated with Bacille-Calmette-Guérin: possible antigenic mimicry of mycobacterial and retinal antigens.

PURPOSE: To investigate the cellular immune response in uveitis developing after intravesical Bacille-Calmette-Guérin (BCG) applications. DESIGN: Experimental study. PARTICIPANTS: A 72-year-old HLA-B27-negative patient with bilateral granulomatous anterior uveitis that developed during the third cycle of intravesical BCG applications she was receiving for treatment of bladder carcinoma. METHODS: The patient's peripheral T cell reactivity to ocular autoantigens was compared with the response to purified protein derivative (PPD) from Mycobacterium tuberculosis. T-cell proliferation and cytokine and chemokine secretion were measured in vitro. MAIN OUTCOME MEASURES: Anterior uveitis was treated successfully with topical corticosteroids and cycloplegics. RESULTS: The following were demonstrated: proliferation to PPD, interphotoreceptor retinoid-binding protein (IRBP), and IRBP-peptide R16, as well as secretion of proinflammatory cytokines in response to PPD, retinal soluble antigen (S-Ag), IRBP, cellular retinal-binding protein (CRALBP), and some S-Ag and IRBP peptides. CONCLUSIONS: These data indicate the generation of a polyclonal autoimmune reaction elicited by BCG. Amino acid sequence alignments revealed homologies between proteins from M. tuberculosis, BCG, and retinal antigens, suggesting antigenic mimicry as a potential cause of uveitis in this patient.

Ocular manifestation of primary VZV infection in a splenectomized patient.

PURPOSE: To report a rare case of primary varicella zoster virus (VZV)-associated retinal vasculitis in a splenectomized patient. DESIGN: Case report. RESULTS: After manifestation of VZV-associated retinal vasculitis, a splenectomized patient experienced binocular loss of vision. CONCLUSIONS: For the development of VZV-associated uveitis, the presence of specific T cells are necessary. Here, the authors present a rare case of VZV-associated retinal vasculitis in a splenectomized patient.

Rat models of autoimmune uveitis.

Experimental autoimmune uveitis (EAU) in Lewis rats is a well-established model for human uveitis. During the last years we used this model to demonstrate extraocular induction of uveitis by antigenic mimicry of environmental antigens with retinal autoantigen and investigated the migration and intraocular reactivation of autoreactive green fluorescent protein (GFP)+ T cells. We could also elaborate several differences between EAU induced with S-antigen peptide PDSAg or R14, a peptide derived from interphotoreceptor retinoid-binding protein, suggesting two differently regulated diseases in the same rat strain. R14-mediated EAU in Lewis rats has been shown to relapse, thus we have a new model to test therapeutic approaches in an ongoing immune response instead of just preventing disease. Finally, we show antigenic mimicry of PDSAg and an HLA-B peptide for oral tolerance induction. After the successful first therapeutic trial this approach will now proceed with international multicenter clinical trials.

Labrafil--a new adjuvant for peptide-specific oral tolerance in rat experimental autoimmune uveitis.

Application of soluble antigen via the oral route results in systemic antigen-specific tolerance, a therapeutic approach that has already been used for uveitis patients. In the Lewis rat experimental autoimmune uveitis (EAU) can be induced by active immunisation with retinal antigens such as retinal soluble antigen (S-Ag) or interphotoreceptor retinoid-binding protein (IRBP) and peptides thereof. These normally pathogenic antigens can also be used to induce oral tolerance. In order to optimize oral tolerance induction we analysed the effect of Labrafil M 2125 CS, an orally administrable composition for pharmaceutical use, consisting of fatty acid esters and glycerides and capable of forming micro emulsions. Feeding peptide emulsified in Labrafil M 2125 CS/PBS prior to immunisation significantly improved oral tolerance compared to feeding peptide in PBS only. We observed a delayed onset of disease, reduced intraocular inflammation and less retinal destruction. Application of Labrafil M 2125 CS without tolerogen had no effect. Combined feeding of peptide with Labrafil M 2125 CS even allowed 10-fold reduction of the tolerogenic peptide dose. Furthermore, the effect of Labrafil M 2125 CS upon oral tolerance was dose-dependent, a peptide emulsion containing 0.5-2% Labrafil M 2125 CS achieved a maximal enhancement of oral tolerance induction, suggesting that Labrafil M 2125 CS might be a useful adjuvant to enhance therapeutic use of oral tolerance.

CRALBP is a highly prevalent autoantigen for human autoimmune uveitis.

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.

The effect of the CC chemokine receptor antagonist Met-RANTES on experimental autoimmune uveitis and oral tolerance.

Lymphocyte trafficking is controlled in part by the actions of chemokines. In rat experimental autoimmune uveitis (EAU) we observed differential therapeutic effects of Met-RANTES, a CCR1/CCR5 receptor antagonist, depending on the retinal antigen peptides inducing the disease and the time of application during the afferent or efferent immune response. CCR1 and/or CCR5 blockade may have inhibitory effects on different phases of the autoimmune response, depending on the antigen specificity of T cells in EAU. In contrast, Met-RANTES enhanced therapeutic oral tolerance independently of orally applied antigen.

The fate of autoreactive, GFP+ T cells in rat models of uveitis analyzed by intravital fluorescence microscopy and FACS.

Experimental autoimmune uveitis (EAU) is an inflammatory disease of the immune privileged inner eye, mediated by CD4(+) Th1 cells specific for retinal autoantigens. To elucidate the fate of the T cells in the eye we adoptively transferred green fluorescent protein-positive (GFP(+)) T cells with specificity for R14, a peptide from interphotoreceptor retinoid-binding protein (IRBP) or OVA as foreign control antigen to naive Lewis rats. We also used the model of immunogenic uveitis, an inflammatory eye disease induced by intraocular injection of soluble OVA 1 day post transfer of OVA-specific GFP(+) cells. We investigated the timing of ocular T cell infiltration and their immunological activation state by intravital fluorescence microscopy (IVFM) of the iris until onset of intraocular inflammation. Within 30 min of injection, GFP(+) cells invaded the iris tissue, irrespective of their antigen specificity, whereas intraocular inflammation was only observed 3 days later, if cells recognized their respective antigen (R14-specific cells in EAU, OVA-specific cells in immunogenic uveitis). Using FACS analysis we found that activation markers were upregulated only on cells from uveitic eyes, but not from other sources, suggesting that intraocularly presented specific antigen is a prerequisite for T cell reactivation and subsequent recruitment of inflammatory cells.

Long-term follow-up of oral tolerance induction with HLA-peptide B27PD in patients with uveitis.

Oral tolerance induction with peptide B27PD had an ameliorating effect in uveitis patients, which was also observed in the five-year follow-up period. Repeated treatments with peptide B27PD were also effective. Side effects did not occur in this patient group, not even in patients receiving peptide for up to 42 weeks.

Gamma-delta T cells as orally induced suppressor cells in rats: in vitro characterization.

Gamma-delta T cells from orally tolerized rats adoptively transfer suppression of experimental autoimmune uveitis. In vivo and in vitro these regulatory cells specifically recognize retinal autoantigen peptide PDSAg and its mimotope B27PD, but not other mimicry peptides. Proliferation of gamma/delta T cells was MHC class II and CD8 dependent.

An HLA-peptide mimics organ-specific antigen in autoimmune uveitis: its role in pathogenesis and therapeutic induction of oral tolerance.

Autoimmune uveitis is a sight threatening disease, which is conventionally treated with immunosuppressive medication. New treatment strategies include immunological approaches and aim at antigen specificity like oral tolerance. A peptide from the sequence of certain HLA-class I molecules plays a central role in the pathogenesis. When T cells recognize the HLA-peptide and are activated they are enabled to pass the blood-retina barrier. In the eye they recognize a cross-reactive organ-specific peptide and cause inflammation, which presents as uveitis. Here, we used the HLA-peptide as oral tolerogen to treat uveitis patients in an open study. All patients showed a positive therapeutic response and could reduce their long-lasting conventional immunosuppressive treatment. We did not observe any side effects. Moreover, side effects from conventional therapy could be reduced significantly.

Oral tolerance for treating uveitis - new hope for an old immunological mechanism.

Oral tolerance induction has evolved as an attractive approach for the treatment of autoimmune uveitis. This approach is effective and generally void of the side effects associated with conventional immunosuppression. Following uptake of soluble antigen via the gut mucosa a specific systemic tolerance is generated. Experimental autoimmune diseases such as uveitis can efficiently be treated when autoantigens are fed to animals. The immunological mechanisms of oral tolerance are not well understood but are thought to involve the recognition of tolerogenic epitopes, generation of suppressor T cells and altered regulation of selected cytokines. The dose, purity of the antigen (tissue extract vs. single peptide) and concomitant treatment with cytokines were evaluated with the aim to enhance oral tolerance. Immunomodulatory drugs can abrogate oral tolerance. This requires careful evaluation with respect to therapeutic approaches in patients. The first clinical trials for treatment of uveitis with oral retinal autoantigen or an HLA-peptide crossreactive with S-Antigen show a promising therapeutic effect and confirmed the safety of this approach.

Uveitis in horses induced by interphotoreceptor retinoid-binding protein is similar to the spontaneous disease.

Equine recurrent uveitis (ERU) is an inflammatory eye disease with high similarity to uveitis in man. It is the only spontaneous animal model for uveitis and the most frequent eye disease in horses affecting up to 10% of the population. To further investigate the pathophysiology of ERU we now report the establishment of an inducible uveitis model in horses. An ERU-like disease was elicited in seven out of seven horses by injection of interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. Control horses did not develop uveitis. The disease model is characterized by a highly reproducible disease course and recurrent episodes with an identical time course elicited in all horses by repeated IRBP injections. The histology revealed the formation of lymphoid follicle-like structures in the eyes and an intraocular infiltration dominated by CD3(+) lymphocytes, morphological patterns typical for the spontaneous disease. Antigen-specific T cell proliferation of PBL was monitored prior to clinical uveitis and during disease episodes. An initial T cell response to IRBP-derived peptides was followed by epitope spreading to S-antigen-derived peptides in response to subsequent immunizations. Thus, horse experimental uveitis represents a valuable disease model for comparative studies with the spontaneous disease and the investigation of immunomodulatory therapeutic approaches after onset of the disease.

Induction of arthritis and uveitis in Lewis rats by antigenic mimicry of peptides from HLA-B27 and cytokeratin.

Ankylosing spondylitis (AS) is highly associated with HLA-B27. We have previously shown that peripheral blood lymphocytes from AS patients respond to stimulation with a peptide from the sequence of HLA-B27. Here we report on molecular mimicry of peptides from HLA-B27 and cytokeratin, the latter being specifically expressed in synovial membranes and eyes, the main targets of the autoaggressive immune response in AS patients. Immunization of rats with these peptides induced an inflammatory response in joints, spine and eyes, resembling the symptoms in AS. Furthermore, both HLA-B27- and cytokeratin-derived peptides, are effective oral tolerogens: feeding these peptides ameliorated arthritis and uveitis induced with the cytokeratin peptide. Our model might elucidate the role of peptides from the sequence of HLA-B27 as an antigen of the immune response in AS, introducing a new aspect of antigenic mimicry between HLA-B27 and tissue-specific antigens. We propose this as a mechanism directing a systemic autoimmune response to specific target organs by antigenic mimicry of T cell epitopes.