Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

Parental Uveitis Influences Offspring With an Increased Susceptibility to the Experimental Autoimmune Uveitis.

Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring. Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161-180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 µg IRBP161-180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization. Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis. Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.

Matairesinol ameliorates experimental autoimmune uveitis by suppression of IRBP-specific Th17 cells.

We investigated the effects of matairesinol (MAT) in the experimental autoimmune uveitis (EAU), a classical animal model of uveitis. We found that treatment with MAT could alleviate intraocular inflammation of EAU. Notably, Th17 cells in eyes of EAU mice could be predominantly restrained by MAT. Furthermore, MAT could inhibit Th17 differentiation in vitro. In addition, MAT inhibited the signaling of MAPK and ROR-γt, a pivotal transcription factor for Th17 cell differentiation in vitro and in vivo. Taken together, these results suggested that MAT had immune-suppressive effects on autoimmune inflammation through Th17 cells.

Effect of berberine on spleen transcriptome and gut microbiota composition in experimental autoimmune uveitis.

BACKGROUND: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? METHODS: EAU was induced in B10R.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. RESULTS: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. CONCLUSIONS: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

Vorinostat Modulates the Imbalance of T Cell Subsets, Suppresses Macrophage Activity, and Ameliorates Experimental Autoimmune Uveoretinitis.

The purpose of the study was to investigate the anti-inflammatory efficiency of vorinostat, a histone deacetylase inhibitor, in experimental autoimmune uveitis (EAU). EAU was induced in female C57BL/6J mice immunized with interphotoreceptor retinoid-binding protein peptide. Vorinostat or the control treatment, phosphate-buffered saline, was administrated orally from 3 days before immunization until euthanasia at day 21 after immunization. The clinical and histopathological scores of mice were graded, and the integrity of the blood-retinal barrier was examined by Evans blue staining. T helper cell subsets were measured by flow cytometry, and the macrophage functions were evaluated with immunohistochemistry staining and immunofluorescence assays. The mRNA levels of tight junction proteins were measured by qRT-PCR. The expression levels of intraocular cytokines and transcription factors were examined by western blotting. Vorinostat relieved both clinical and histopathological manifestations of EAU in our mouse model, and the BRB integrity was maintained in vorinostat-treated mice, which had less vasculature leakage and higher mRNA and protein expressions of tight junction proteins than controls. Moreover, vorinostat repressed Th1 and Th17 cells and increased Th0 and Treg cells. Additionally, the INF-γ and IL-17A expression levels were significantly decreased, while the IL-10 level was increased by vorinostat treatment. Furthermore, due to the reduced TNF-α level, the macrophage activity was considerably inhibited in EAU mice. Finally, transcription factors, including STAT1, STAT3, and p65, were greatly suppressed by vorinostat treatment. Our data suggest that vorinostat might be a potential anti-inflammatory agent in the management of uveitis and other autoimmune inflammatory diseases.

Amelioration of experimental autoimmune uveoretinitis by inhibition of glyceraldehyde-derived advanced glycation end-product formation.

AGEs are permanently modified macromolecule derivatives that form through nonenzymatic glycation of amino groups of proteins. Glycer-AGEs are highly toxic and play an important role in the pathogenesis of chronic inflammatory diseases. However, the contribution of glycer-AGEs to the pathogenesis of uveitis is unclear. In this study, we measured serum levels of glycer-AGEs in 100 patients with endogenous uveitis (22 with HLA-B27-associated uveitis, 20 with VKH disease, 14 with Behçet's disease, and 44 with sarcoidosis) and 33 healthy volunteers. We then examined the effect of the AGE inhibitor in a mouse model of human endogenous uveitis (EAU) by continuous oral administration of pyridoxamine at 200 or 400 mg/kg/day. Regardless of the etiology, serum glycer-AGE levels were significantly higher in patients with uveitis than in healthy subjects. Treatment with 400 mg/kg pyridoxamine significantly reduced the clinical and histological severity of EAU and was accompanied by a significant decrease in serum and retinal glycer-AGE levels and suppression of translocation of NF-κB p65 into the nucleus of retinal cells. Serum glycer-AGE levels may therefore serve as a biomarker of human uveitis, as well as systemic inflammation, and may contribute to the progression of uveitis, including diabetic iritis, via the activation of NF-κB.

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.

[Effects of exogenous interleukin-2, interleukin-23 on differentiation of IRBP 1-20-specific T cells toward Th1 and Th17 and comparison of the pathogenicity of IRBP 1-20-specific T cells].

OBJECTIVES: To explore the effects of exogenous interleukin (IL)-2 and IL-23 on differentiation of IRBP 1-20-specific T cells originated from C57BL/6 mouse with experimental autoimmune uveitis (EAU) and to investigate the difference in pathogenicity. METHODS: Experimental study. Thirty C57BL/6 mice were immunized subcutaneously with 200 µl emulsion containing 200 µg interphotoreceptor retinoid-binding protein (IRBP) 1-20 and 0.8 mg mycobacterium tuberculosis in complete Freund's adjuvant, distributed over six spots at the tail base and on the flank. On postimmunization Day 13, spleens and draining lymph nodes were removed from mice, and a part of spleens, as the control group, was reserved for examining the expression of IFN-γ mRNA and IL-17 mRNA by RT-PCR. T cells were isolated from the rest of spleens and lymph nodes by passage through a nylon wool column, and then T cells were stimulated for 48 hours with IRBP 1-20 in the presence of antigen-presenting cell and mouse recombinant cytokine IL-2 or IL-23. The IRBP 1-20-specific T cells were separated by Ficoll gradient centrifugation, the expressions of IFN-γ mRNA and IL-17 mRNA were assessed by RT-PCR, and IFN-γ and IL-17 in T cells supernatant were detected using a commercial ELISA kit. The T cells were cultured for another 48 hours, and then the proportions of IL-17(+)γδ(+)T cells were analyzed by flow cytometry. EAU models were built in 30 C57BL/6 mice, T cells from which were randomly divided into two groups according to the methods mentioned above: IL-2 group and IL-23 group. Transfer EAU models were built in other 6 mice and divided into two groups, IL-2 group and IL-23 group, by intraperitoneal injection of 3.5×10(6) IRBP-special-T cells from IL-2 group or IL-23 group respectively. Clinical changes were observed by indirect ophthalmoscopy on postimmunization day 3, 7, 14. For histopathological evaluation, whole eyes were collected on postimmunization day 21. Rank sum test, one-way ANOVA and paired t test were used to analyze the results. A comparison of pathogenicity was made between Th1 and Th17 through clinical observation and histopathological evaluation of B6 mouse. RESULTS: Rosette formation was found among T cells on poststimulation day 2. There was a statistical difference in the expression of IFN-γ mRNA between the IL-2 group and normal group or IL-23 group (0.26 ± 0.02 vs. 0.12 ± 0.05 or 0.10 ± 0.00) (F = 80.51, P = 0.003); however, the expression of IFN-γ in the IL-2 group [(13 124.67 ± 107.73) µg/L] was higher than that of the IL-23 group [(3953.67 ± 117.34) µg/L] (t = 169.61, P = 0.000); and the expression of IL-17 mRNA in the IL-2 group [(588.67 ± 77.43) µg/L] was lower than that of the IL-23 group [(5038.33 ± 88.00) µg/L] (t = -361.71, P = 0.000). Flow cytometer showed that the concentration of IL-17 in the IL-2 group [(3.23 ± 0.28)%] was significantly lower than that in the IL-23 group [(9.93 ± 0.55)%] (t = -33.18, P = 0.001). There was a significant difference in the proportion of IL-17(+)γδ(+)T cells between the IL-23 group and IL-2 group (9.93 ± 0.55 vs. 3.23 ± 0.28) (t = -33.18, P = 0.001). Inflammatory response could not be detected in either group three days after injection of IRBP 1-20-specific T cells. Both groups of mice had the most severe inflammatory response on the 14 th day, of which that of the IL-23 group was significantly more severe. CONCLUSIONS: IL-23 facilitates IRBP 1-20-specific T cells to differentiate into Th17, whereas IL-2 inhibits this process and induces these cells to differentiate towards Th1. Further studies showed that the pathogenicity of Th17 cells was significantly higher than that of Th1 cells.

Posttranslational modification of differentially expressed mitochondrial proteins in the retina during early experimental autoimmune uveitis.

PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.

The role of TLR2, TRL3, TRL4, and TRL9 signaling in the pathogenesis of autoimmune disease in a retinal autoimmunity model.

PURPOSE: Induction of tissue-specific experimental autoimmune diseases involves the use of complete Freund adjuvant containing Mycobacterium tuberculosis, whose recognition by the innate immune system depends on Toll-like receptors (TLRs) that signal through the adaptor molecule MyD88. The authors' previous study showed that MyD88(-/-) mice, but not TLR2(-/-), TLR4(-/-), or TLR9(-/-) mice, were resistant to experimental autoimmune uveitis (EAU). METHODS: The EAU induction in mice deficient in TLR3 or mice double deficient in TLR2+4, TLR2+9, and TLR4+9 was examined and the role of the TLR agonists in the adjuvant effect involved in the induction of EAU was assessed. RESULTS: TLR3-deficient and TLR2+4, TLR2+9, and TLR4+9 double-deficient mice were as susceptible to EAU as their control littermates. However, in mice immunized with a low-dose EAU regimen, TLR4 agonist lipopolysaccharide (LPS) enhanced EAU scores, delayed-type hypersensitivity responses, and antigen-specific T-cell proliferation. Antigen-specific IL-17 and IFN-gamma production by T lymphocytes was markedly increased in the LPS-treated group. The effects of LPS on EAU were abolished by treatment with an LPS deactivator polymyxin B. Inclusion of agonists for TLR2, TRL3, or TRL9 in immunization also enhanced EAU scores. CONCLUSIONS: These results suggest that signaling of TLR2, TRL3, TRL4, and TRL9 is highly redundant in the adjuvant effect needed to induce EAU and that diverse microbial infections may contribute to the pathogenesis of diseases such as uveitis.

Injection of an alpha-melanocyte stimulating hormone expression plasmid is effective in suppressing experimental autoimmune uveitis.

PURPOSE: The neuropeptide, alpha-melanocyte stimulating hormone (alpha-MSH), is an endogenous antagonist of inflammation. Injections of alpha-MSH peptide into inflamed tissues have been found to be very effective in suppressing autoimmune and endotoxin mediated diseases. We evaluated the potential to suppress ocular autoimmune disease (uveitis) by augmenting the expression of alpha-MSH through subconjunctival injections of naked adrenocorticotropic hormone amino acids 1-17 (ACTH1-17) plasmid. METHODS: We clinically scored the uveitis over time in B10.RIII, C57BL/6, and melanocortin 5 receptor knock-out (MC5r((-/-))) mice with experimental autoimmune uveitis (EAU) that were conjunctively injected with a naked DNA plasmid encoding ACTH1-17 at the time of EAU onset and three days later. The post-EAU retina histology of plasmid injected eyes was examined, and post-EAU concentrations of alpha-MSH in aqueous humor was assayed by ELISA. RESULTS: The subconjunctival injection of ACTH1-17 plasmid augmented the concentration of alpha-MSH in the aqueous humor of all post-EAU mice. The injection of ACTH1-17 suppressed the severity of EAU in the B10.RIII and C57BL/6 mice but the MC5r((-/-)) mice. In all the models of EAU, the ACTH1-17 injection helped to preserve the structural integrity of the retina; however, post-EAU aqueous humor was not immunosuppressive. CONCLUSIONS: The subconjunctival injection of the alpha-MSH expression vector ACTH1-17 plasmid is effective in suppressing EAU. The suppressive activity is dependent on MC5r expression, and possibly works though alpha-MSH antagonism of inflammation than on alpha-MSH directly modulating immune cells. The results suggest that an effective therapy for uveitis could include a gene therapy approach based on delivering alpha-MSH.

Regulation of interphotoreceptor retinoid-binding protein (IRBP)-specific Th1 and Th17 cells in anterior chamber-associated immune deviation (ACAID).

PURPOSE: Intracameral (anterior chamber) injection of antigen inhibits the development of delayed-type hypersensitivity, a phenomenon known as anterior chamber-associated immune deviation (ACAID). The authors investigated the effect of intracameral injection of interphotoreceptor retinoid-binding protein (IRPB) peptides on the development of IFN-gamma(+) and IL-17(+) pathogenic T cells. METHODS: A uveitogenic (IRBP1-20) or nonuveitogenic (IRBP161-180) peptide was injected into the anterior chamber (AC) of B6 mice. Seven days later, the mice were primed with a pathogenic dose of IRBP1-20 in adjuvant. Thirteen days later, the pathogenic activity of the T cells isolated from the spleens of treated and untreated mice were compared, and the numbers of Th1 and Th17 T cells were assessed by intracellular staining. Regulatory T-cell activity was assessed by antibody staining and functional assays. The authors also compared the effect of inhibition on EAU of ocular injection to various sites, including the AC, the vitreous cavity, and the subretinal space. RESULTS: Intraocular injection of the uveitogenic peptide (IRBP1-20), but not the nonuveitogenic peptide (IRBP161-180), inhibited the generation of IFN-gamma(+) and IL-17(+) uveitogenic T cells and the development of experimental autoimmune uveitis (EAU). AC administration of IRBP1-20, but not IRBP161-180, significantly decreased the number of circulating gammadelta T cells after subsequent systemic immunization with IRBP1-20. Absence of the gammadelta T-cell population prohibited the development of ACAID. CONCLUSIONS: Injection of a uveitogenic peptide into the AC inhibited the development of EAU by regulation of Th1 and Th17 IRBP-specific T cells. The circulating gammadelta T-cell population was reduced and was associated with decreased activation of IL-17(+) uveitogenic T cells.

Autoimmunity in the immune privileged eye: pathogenic and regulatory T cells.

Experimental autoimmune uveitis (EAU) in animals serves as a model of human uveitis. EAU can be induced in mice by immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP) in complete Freund's adjuvant (CFA) or by IRBP-pulsed mature dendritic cells, and can be driven either by a Th17 or a Th1 effector response, depending on the model. The direction of the response is affected by conditions present during the exposure to antigen, including the quality/quantity of innate receptor stimulation and/or type of APC. IL-17 and IFN-gamma production by innate cells such as NKT may also affect the disease process. If exposure to antigen is via a hydrodynamic DNA vaccination with an IRBP-encoding plasmid, the response is directed to a regulatory phenotype, and disease is ameliorated or prevented. Our data shed light on effector and regulatory responses in autoimmune disease, provide balance to the Th1/Th17 paradigm and help to explain the clinical heterogeneity of human uveitis, which occurs in the face of responses to the same ocular antigen(s).

Beacon-like/ubiquitin-5-like immunoreactivity is highly expressed in human hypothalamus and increased in haloperidol-treated schizophrenics and a rat model of schizophrenia.

The beacon gene is involved in the regulation of energy metabolism, food intake, and obesity. We localized its gene product, beacon-/ubiquitin 5-like immunoreactivity in brains of normal-weight, non-psychotic individuals, adipose (BMI over 32), non-psychotic individuals, and haloperidol-treated schizophrenics. The protein was found to be highly expressed in many neurons of the paraventricular and supraoptic hypothalamic nuclei. Besides, it was detected in neurons of other hypothalamic areas (suprachiasmatic, arcuate, and ventromedial nuclei) as well as outside the hypothalamus (Nuc. basalis Meynert, thalamus, hippocampus, and some neocortical areas). A morphometric analysis of beacon-immunoreactive hypothalamic and neocortical neurons revealed that compared to normal-weight controls in haloperidol-treated schizophrenics, there was a significant increase of protein-expressing supraoptic, paraventricular, and orbitofrontal neurons. However, a significant increase in beacon-expressing supraoptic neurons was also seen in adipose, non-psychotic individuals in comparison with normal-weight controls. Haloperidol at different doses has no effect on beacon expression in SHSY5Y neuroblastoma cells, which makes the assumption unlikely that haloperidol per se is responsible for the increased neuronal expression of the peptide in schizophrenics. In rats with a neonatal lesion of the ventral hippocampus (a widely used animal model of schizophrenia), we found an increased neuronal expression of beacon in the paraventricular and supraoptic nuclei. We suppose that elevated hypothalamic expression of beacon-like protein in non-obese schizophrenics is not primarily related to metabolic alterations, but to a certain role in schizophrenia, which is possibly unrelated to aspects of weight gain and obesity. The latter assumption finds some support by data obtained in rats with ventral hippocampus lesion.

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.

Blockade of the OX40 ligand prolongs corneal allograft survival.

Although corneal transplantation is one of the most common tissue transplantations and is known to have a high graft acceptance rate, occasional corneal graft rejection remains a cause of blindness. OX40, a member of the TNF receptor superfamily, is expressed on activated T cells, and transmits a costimulatory signal by binding to OX40 ligand (OX40L) expressed on several cells with antigen-presenting functions. Using a blocking monoclonal antibody (mAb) against murine OX40L, we investigated the role of OX40 in a murine model of corneal transplantation. C3H/He mouse corneas were transplanted to BALB/c mice orthotopically. Administration of anti-OX40L mAb significantly reduced allograft rejection, and increased graft survival rate to 40% at 8 weeks after transplantation, while all corneas were rejected within 5 weeks in control IgG-treated mice. Similar reduced rejection was observed when wild-type donor corneas were transplanted to OX40L-deficient recipients. In vitro study revealed that the anti-OX40L mAb treatment reduced proliferative response and IFN-gamma production of draining lymph node cells in response to stimulation with donor alloantigen. These results demonstrate that OX40L blockade is effective for prolongation of corneal allograft survival by inhibiting recipient T cell activation.

Minimally activated CD8 autoreactive T cells specific for IRBP express a high level of Foxp3 and are functionally suppressive.

PURPOSE: Results in previous reports have demonstrated that immunization of the EAU-prone B6 mouse activates both CD4 and CD8 IRBP-specific T cells. The purpose of this study was to investigate structural and functional differences between CD4 and CD8 autoreactive T cells activated by the uveitogenic peptide. METHODS: Purified CD4 and CD8 isolated from B6 mice immunized with an uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP)1-20, were stimulated in vitro with various doses of immunizing peptide. The activated T cells were determined for cytokine production, expression of Foxp3, and suppressor activity. RESULTS: CD4 autoreactive T cells underwent full activation when stimulated with high or medium concentrations of immunizing peptide, whereas a high dose of antigenic peptide resulted in only modest activation of CD8 autoreactive T cells. When stimulated by a low dose (<0.1 microg/mL) of antigen or by of a high dose of antigen and a small amount of TGF-beta1, the minimally activated CD8 T cells expressed a high level of Foxp3 and gained suppressor function. CONCLUSIONS: Minimally activated CD8 autoreactive T cells can be functionally suppressive and may neutralize the tissue-damaging effect of the CD4 autoreactive T cells.

Supplementation of CD4+CD25+ regulatory T cells suppresses experimental autoimmune uveoretinitis.

AIMS: To investigate whether supplementation of natural CD4+CD25+ regulatory T cells ameliorates mouse experimental autoimmune uveoretinitis (EAU) induced by CD4+ T cell-dependent interphotoreceptor retinoid-binding protein (IRBP). METHODS: C57BL/6 mice were immunised with human interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP(1-20)), and IRBP(1-20)-sensitised T cells were obtained. CD4+CD25+ T cells derived from naive mice were cocultured with IRBP(1-20)-sensitised T cells, and their proliferation responses and cytokine production were measured. In addition, CD4+CD25+ T cells were transferred intravenously into mice 7 or 15 days after immunisation with IRBP(1-20), and the severity of EAU and T cell proliferation responses were evaluated. RESULTS: CD4+CD25+ regulatory T cells effectively inhibited both the proliferation of, and interleukin (IL)2, IL5 and interferon (IFN)gamma production by, IRBP(1-20)-sensitised T cells. Adoptive transfer of CD4+CD25+ regulatory T cells to IRBP(1-20)-immunised mice conferred considerable protection from EAU development and inhibition of T cell proliferation responses to IRBP(1-20). CONCLUSION: These findings show that natural CD4+CD25+ regulatory T cells possess the ability to inhibit activation of IRBP-reactive T cells that have been already sensitised in vivo, and adoptive transfer of these cells ameliorates EAU even in the effector phase. Supplementation of natural CD4+CD25+ regulatory T cells may have therapeutic potential for effective treatment of uveitis.

Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins.

PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.