Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

Modulating of ocular inflammation with macrophage migration inhibitory factor is associated with notch signalling in experimental autoimmune uveitis.

The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno-associated virus (AAV8) (Y733F)-chicken ß-actin (CBA)-MIF or AAV8 (Y733F)-CBA-enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP-injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta-like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF-injected EAU mice. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.

Protective effect of pristane on experimental autoimmune uveitis.

This study evaluates the effects of pristane and phytol, two mineral oils with pro-oxidative effects, on the course of experimental autoimmune uveitis. C57BL6 mice were immunized with IRBP1-20 peptide emulsified in CFA and treated five days prior to immunization with phytol or with pristane or with PBS as control. Administration of pristane reduces the incidence and severity of IRBP-induced uveitis as demonstrated by the decrease in vasculitis and inflammatory foci in fundus and by a reduction in histological damages and leukocyte infiltration compared to untreated or phytol-treated mice. The protective effect observed is associated with a decreased activation of peripheral CD4+ and CD8+ T lymphocytes and a decrease in the intensity of the Th1 and Th17 autoimmune response to IRBP in pristane-treated mice compared to control mice, as evidenced by the decreased production of IFNγ and IL17 by IRBP-specific lymphocytes from lymph nodes draining the site of immunization and by the increased production of anti-IRBP IgG1 over IgG2a. In addition, HUVEC and ARPE-19 cells incubated with the sera of mice treated with pristane presented a reduced production of H(2)O(2). The benefit of lowering the systemic oxidative stress by pristane in the course of EAU was confirmed by injecting the antioxidant NAC in IRBP-immunized mice. As pristane, NAC decreased clinical and histological inflammation of the retina and preserved the integrity of the hemato-retinal barrier. Finally, the protective effect of pristane on the development of EAU suggests that some mineral oils may represent a new therapeutic strategy in human uveitis.

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.

Fas and Fas ligand expressed on cells of the immune system, not on the target tissue, control induction of experimental autoimmune uveitis.

The Fas-Fas ligand (FasL) interaction is important for maintaining lymphocyte homeostasis by signaling for activation-induced cell death. Mice homozygous for the lpr or gld mutations do not express functional Fas or FasL, respectively, and spontaneously develop progressive autoimmune symptoms. Recent studies implicated expression of FasL on immunologically privileged tissues in protection from immune-mediated damage. Conversely, tissue expression of Fas may facilitate damage. We evaluated the susceptibility of lpr and gld mice to induction of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease induced with retinal Ags, which targets the neural retina. gld as well as lpr mice immunized with a retinal Ag developed disease of lower incidence and severity than wild-type controls. Delayed hypersensitivity responses were not significantly different among immunized gld, lpr, or wild-type mice, although in vitro Ag-specific lymphocyte responses of the mutant mice were lower. To evaluate whether the diminished ability of gld and lpr mice to develop EAU was due to a defect at the level of the tissue or the immune system, radiation bone marrow chimeras constructed between wild-type and mutant mice were immunized to induce EAU. Mutant recipients of wild-type bone marrow, but not wild-type recipients of mutant bone marrow, developed normal disease scores. These results indicate that normal expression of Fas and of FasL on cells of the immune system is important for EAU expression. Unexpectedly, neither lack of Fas nor lack of FasL on the ocular tissues affected expression of EAU.

Amelioration of experimental autoimmune uveoretinitis by pretreatment with a pathogenic peptide in liposome and anti-CD40 ligand monoclonal antibody.

We have defined a peptide K2 (ADKDVVVLTSSRTGGV) that corresponds to residues 201-216 of bovine interphotoreceptor retinoid-binding protein and induces experimental autoimmune uveoretinitis (EAU)4 in H-2Ak-carrying mice (H-2Ak mice). In this study, we attempted to ameliorate EAU in the H-2Ak mice without nonspecific suppression of T cell responses. Preceding s.c. administration of liposomes including K2 (liposomal K2) specifically inhibited subsequent generation of T cell response to K2. The same result was obtained with a combination of OVA323-339 peptide and the OVA-specific TCR-transgenic T cells. It was suggested that the inhibition was mainly attributed to peripheral anergy induction of T cells specific for the peptide Ag, although specific cell death might also be involved in the inhibition. Pretreatment with liposomal K2 also considerably abolished IFN-gamma production but not IL-4 production. The specific inhibitory effect of the pretreatment with liposomal peptide was augmented by a simultaneous administration of anti-CD40 ligand (anti-CD40L) mAb. Moreover, it was shown that the pretreatment with liposomal K2 reduced both the incidence and severity of the subsequent K2-induced EAU, and the simultaneous administration of anti-CD40L mAb augmented this preventive effect by liposomal K2. Our findings demonstrate that the s.c. administration of liposomal pathogenic peptide and anti-CD40L mAb can be applied to preventing autoimmune diseases without detrimental nonspecific suppression of T cell responses.

Envolvimento renal na infecçäo do trato urinário recorrente: importância da prote1na transportadore de retinol como marcador de disfunçäo tubular / Renal envolvemen in urinary tract infection recurring: retinol binding protein importance how marker of tubular disfuncion

Com o objetivo de pesquisar o envolvimento renal na infecçäo do trato urinário (ITU), realizaram-se a ultra-sonografia e a cintilografia renal em 66 crianças (média de idade = 30 anos). Em 20 crianças e em 23 mulheres comrecorrência da ITU (ITUr), realizou-se, também, a dosagem da proteína transportadora de retinol (RBP) na urina. Como controles, foram estudadas41 crianças (média de idade =7,2 anos) e 24 mulheres (média de idade = 32 anos). Havia cicatriz pielonefrítica em 25 porcento das crianças e em 26 porcento das mulheres com ITUr. A excreçäo urinária de RBP foi significantemente maior nas crianças e nas mulheres com ITU que nas normais. Nossos resultados demonstrtam disfunçäo tubular renal na infecçäo do trato urinário recorrente.(au)

IL-10 has a protective role in experimental autoimmune uveoretinitis.

The role of IL-10 in the regulation of ocular autoimmune disease was studied in experimental autoimmune uveoretinitis (EAU) elicited in mice by immunization with the retinal antigen interphotoreceptor retinoid binding protein. IL-10-deficient mice were susceptible to EAU, indicating that pathogenesis can occur without presence of IL-10. Treatment of normal mice with IL-10 for 5 days after uveitogenic immunization ameliorated subsequent EAU scores, and down-regulated antigen-specific production of tumor necrosis factor-alpha and IFN-gamma. A concomitant treatment with IL-4 further reduced disease, and resulted in emergence of antigen-specific IL-4 and IL-10 production, as well as in enhancement of the IgG1 antibody isotype. IL-4 by itself was not protective. Only IL-10, but not IL-4, was able to inhibit the function of differentiated uveitogenic T cells in culture. Expression of mRNA for Th1 and Th2 cytokines in the eye during the course of EAU showed that while a Th1 pattern predominated early, IL-10 mRNA expression coincided with down-regulation of the Th1 response and resolution of EAU. Systemic neutralization of IL-10 during the expression phase of EAU resulted in elevated disease scores. Our results suggest that endogenous IL-10 limits expression of EAU and may play a role in the natural resolution of disease. The data further suggest that exogenous IL-10 may be useful in therapeutic control of autoimmune uveitis. While IL-10 by itself is sufficient to suppress Th1 effector development and function, a concomitant administration of IL-4 is required to shift the autoimmune response towards a non-pathogenic Th2 pathway.

The existence of two completely distinct antigenic sites within a decapeptide.

A decapeptide (1182-1191) derived from the bovine interphotoreceptor retinoid-binding protein (IRBP) was found to contain two completely distinct antigenic sites when tested in Lewis rats. One site, localized in sequence 1182-1191, is the core of the immunodominant and highly uveitogenic determinant of IRBP. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at position 1182 is deleted. Lymphocytes sensitized against the first, larger site recognized all longer peptides within sequence 1169-1191, as well as whole IRBP. In contrast, lymphocytes sensitized against the second, short epitope recognized only two peptides, 1184-1191 and (to a lesser degree) 1183-1191. The responses to both sites were restricted by the same major histocompatibility complex (MHC) product (I-A), as shown by monoclonal antibody blocking and by the finding that the lymphocyte response to 1184-1191 was competitively inhibited by peptide 1181-1191. The unique finding of two completely distinct antigenic sites within a decapeptide could be explained by the hypothesis that peptides of the two sites combine with the MHC molecule on antigen-presenting cells by different configurations, thus forming two distinct antigenicities.

Adoptive transfer of experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein.

The immunopathogenic mechanisms of experimental autoimmune uveoretinitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) were studied. Lymph node (LN) cells or spleen cells were collected from donor rats 14 days after the immunization with IRBP. After the 3-day pre-incubation of these cells with either IRBP or Concanavalin A (Con A), the cells were transferred to naive syngeneic rats intraperitoneally. EAU was successfully transferred by LN cells pre-cultured with IRBP, while EAU was poorly transferred by LN cells pre-cultured with Con A. On the other hand, spleen cells pre-cultured with either IRBP or Con A were quite potent to transfer EAU. In addition, the enriched helper/inducer T-cells repeatedly transferred EAU, while the enriched suppressor/cytotoxic T-cells did not. There also existed the simultaneous involvement of pinealitis. The histopathological features of EAU and pinealitis were generally similar to those in actively immunized rats. The immune responses to IRBP in recipients with EAU were mostly cellular (delayed hypersensitivity type skin response and proliferative responses of lymphocytes), while humoral responses (Arthus type skin response and antibody activities) were quite weak in most of the recipients. Thus, it was confirmed that cellular immunity plays a major role in the adoptive transfer of EAU by IRBP as in S-antigen-induced EAU.

Antigen-specific suppressor cells induced by FK506 in experimental autoimmune uveoretinitis in the rat.

The authors previously reported that FK506 effectively suppressed the induction of experimental autoimmune uveoretinitis (EAU) in rats with much lower doses than cyclosporine A. This study was aimed at analyzing the immune status of the FK506-treated and EAU-suppressed rats and examining the hypothesis whether the agent could induce antigen-specific suppressor T (Ts) cells. It was found that spleens from S-antigen-immunized and FK506-treated rats contained a population of Ts cells inhibiting the proliferative responses of S-antigen-sensitized lymphocytes to S-antigen, yet these cells did not affect the proliferative responses of interphotoreceptor retinoid-binding protein (IRBP)-sensitized lymphocytes to IRBP. The helper T (Th) cells did not exhibit such suppressor activities. Furthermore, transfer of Ts cells from S-antigen-immunized and FK506-treated rats to naive syngenic rats induced partial inhibition of EAU induction or delay of EAU onset after immunizing the recipient rats with S-antigen. Lymphocytes from the EAU-suppressed recipients showed low proliferative response to S-antigen and low levels of antibody to S-antigen. These data thus indicate that FK506 treatment after S-antigen immunization induces an activation of Ts cells specific to S-antigen and that the Ts cells might contribute, at least in part, to the uniquely prolonged and intensive immunosuppression by FK506.

Serial adoptive transfer of experimental autoimmune uveoretinitis in rats.

Experimental autoimmune uveoretinitis (EAU) and pinealitis induced by an interphotoreceptor retinoid-binding protein (IRBP)-derived peptide (R4) was serially transferred into naive recipient rats, using spleen cells from recipients of previous "orders" of transfer. The cells initiating the disease in recipients of the first order were either lymph node cells from rats immunized against peptide R4, or lymphocytes of a cell line specific toward this peptide. The serial transfer was successfully carried out through as many as four orders of sequential recipients.

Pathology of experimental autoimmune uveoretinitis in mice.

The histopathology and immunopathology of murine experimental autoimmune uveoretinitis (EAU) following active immunization with the interphotoreceptor retinoid-binding protein (IRBP) were studied. The methods used included conventional light microscopy and immunoperoxidase staining. Lesions were located mainly in the uvea and the retina and were characteristically focal. The prominent histopathologic findings in the retina were vasculitis, granuloma, retinal fold, focal serous detachment, and loss of photoreceptors. Granulomas, formation of Dalen-Fuchs nodules, inflammatory cellular infiltration and increase in the thickness of the choroid and ciliary body were frequent findings. Subretinal neovascularization occurred in 10% of the experimental animals. Mild to moderate inflammation was also noted in the vitreous. The predominant infiltrating cells in the retinal and uveal granuloma and the Dalen-Fuchs nodules were macrophages. In contrast, the predominant infiltrating cell types in the vitreous were T helper/inducer lymphocytes. T suppressor/cytotoxic cells were rarely seen. Expression of Ia antigens on the ocular cells was confined to the immediate area of the inflammatory sites. The kinetics of histopathology showed two peaks at the 5th and 10th week after immunization, suggesting a relapsing course of the disease.

Auxiliary production of antibodies to ocular antigens in experimental autoimmune uveoretinitis.

In experimental autoimmune uveoretinitis (EAU), there are concurrent autoreactive humoral and cellular immune responses. Many studies have recently focused on T-cell reactivities in EAU, while analysis of autoantibody responses to uveitogenic epitopes has been less well characterized. In this study, a defined 16-mer uveitogenic interphotoreceptor retinoid binding protein (IRBP) peptide, designated #896, was used to induce EAU. Histological analysis of eyes at day 10 demonstrated extensive leukocyte infiltration of the anterior segment, with mononuclear and plasma cells in the posterior segment. The presence of plasma cells suggests local production of antibodies within the eye. ELISA analyses of serum, aqueous, and vitreous from rats with IRBP-induced, severe EAU revealed the presence of antibodies against peptide #896. There was little difference between the serum and intraocular antibody titers, presumably due to breakdown of the blood-ocular barriers. In addition to the anti-#896-IRBP antibodies, there were detectable antibodies reactive against separate and distinct IRBP epitopes, as well as, against epitopes on retinal-S antigen. These results indicate that in #896-peptide-induced severe EAU, humoral immune responses are induced to the primary immunogen and that there is also auxiliary production of autoreactive antibodies against other epitopes present on intraocular antigens. These auxiliary responses may contribute to the immunopathogenesis of severe EAU.

Identification of multiple associative and dissociative proliferative and uveitogenic T-cell sites in human interstitial retinoid-binding protein.

Human interstitial retinoid-binding protein (HIRBP) is a 136 kDa retinal protein capable of inducing experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis (LEW) rats. In order to identify the T-cell recognition sites of HIRBP responsible for uveitogenic and proliferative responses, 125 overlapping peptides corresponding to its entire 1262 amino acid sequence were synthesized. Individual peptides were tested for their ability to induce EAU in LEW rats and to stimulate lymphocyte proliferation in rats immunized with the native molecule. Our previous results showed the presence of nine uveitogenic peptides in HIRBP with a minimum requirement of eight amino acids needed to induce EAU in LEW rats. Our present studies show nine proliferative peptides, four of which are also responsible for uveitogenicity. Another four peptides known to actively induce EAU were unable to elicit proliferative responses. However, these peptides overlapped or were adjacent to peptides that elicited good proliferative responses. A single, highly proliferative peptide was located on the amino terminus of HIRBP. In addition, EAU was adoptively transferred with lymph node cells (LNC) of LEW rats previously immunized with two synthetic peptides known to be uveitogenic. Our study indicates that human IRBP is a complex molecule containing multiple and spatially distinct T-cell epitopes responsible for its uveitogenicity, adoptive transfer of EAU and proliferative responses.