Interphotoreceptor Retinoid-binding Protein Research Articles

Macrophage-like iPS-derived Suppressor Cells Reduce Th1-mediated Immune Response to a Retinal Antigen

ABSTRACT Purpose To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). Methods The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. Results The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-β1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. Conclusions Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.

Induction of antigen-specific Treg cells in treating autoimmune uveitis via bystander suppressive pathways without compromising anti-tumor immunity.

BackgroundInduction of autoantigen-specific Treg cells that suppress tissue-specific autoimmunity without compromising beneficial immune responses is the holy-grail for immunotherapy to autoimmune diseases.MethodsIn a model of experimental autoimmune uveitis (EAU) that mimics human uveitis, ocular inflammation was induced by immunization with retinal antigen interphotoreceptor retinoid-binding protein (IRBP). Mice were given intraperitoneal injection of αCD4 antibody (Ab) after the onset of disease, followed by administration of IRBP. EAU was evaluated clinically and functionally. Splenocytes, CD4+CD25− and CD4+CD25+ T cells were sorted and cultured with IRBP or αCD3 Ab. T cell proliferation and cytokine production were assessed.FindingsThe experimental approach resulted in remission of ocular inflammation and rescue of visual function in mice with established EAU. Mechanistically, the therapeutic effect was mediated by induction of antigen-specific Treg cells that inhibited IRBP-driven Th17 response in TGF-β and IL-10 dependent fashion. Importantly, the Ab-mediated immune tolerance could be achieved in EAU mice by administration of retinal autoantigens, arrestin but not limited to IRBP only, in an antigen-nonspecific bystander manner. Further, these EAU-suppressed tolerized mice did not compromise their anti-tumor T immunity in melanoma model.InterpretationWe successfully addressed a specific immunotherapy of EAU by in vivo induction of autoantigen-specific Treg cells without compromising host overall T cell immunity, which should have potential implication for patients with autoimmune uveitis.FundingThis study was supported by the Natural Science Foundation of Guangdong Province and the Fundamental Research Fund of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center.

Across the great divide: revision of the genusEupetaurus(Sciuridae: Pteromyini), the woolly flying squirrels of the Himalayan region, with the description of two new species

AbstractThe woolly flying squirrel, Eupetaurus cinereus, is among the rarest and least studied mammals in the world. For much of the 20th century it was thought to be extinct, until it was rediscovered in 1994 in northern Pakistan. This study outlines the first taxonomic and biogeographical review of the genus Eupetaurus, which until now has contained only a single species. Careful review of museum specimens and published records of Eupetaurus demonstrates that the genus occurs in three widely disjunct areas situated on the western (northern Pakistan and north-western India), north-central (south-central Tibet, northern Sikkim and western Bhutan) and south-eastern margins (north-western Yunnan, China) of the Himalayas. Taxonomic differentiation between these apparently allopatric populations of Eupetaurus was assessed with an integrative approach involving both morphological examinations and molecular phylogenetic analyses. Phylogenetic reconstruction was implemented using sequences of three mitochondrial [cytochrome b (Cytb), mitochondrially encoded 12S and 16S ribosomal RNA (12S, 16S)] and one nuclear [interphotoreceptor retinoid-binding protein (IRBP)] gene fragment. Morphological assessments involved qualitative examinations of features preserved on museum skins and skulls, supplemented with principal components analysis of craniometric data. Based on genetic and morphological comparisons, we suggest that the three widely disjunct populations of Eupetaurus are each sufficiently differentiated genetically and morphologically to be recognized as distinct species, two of which are described here as new.

Molecular characterization and phylogeography of Mediterranean picarels (Spicara flexuosa, S. maena and S. smaris) along the coasts of Turkey and the Eastern Mediterranean

In this study, molecular taxonomy associated with Spicara flexuosa, Spicara maena, and Spicara smaris was examined with a geographical focus along the coasts of the Turkey and the Mediterranean. In addition, the effects of the Turkish Straits System on the evolutionary history and phylogeography of Spicara flexuosa were investigated. The mitochondrial cytochrome c oxidase I, cytochrome b, and the nuclear Interphotoreceptor retinoid–binding protein genes were used for these purposes. Our results indicated that the distinction of the three taxa under Spicara is possible with the two different mitochondrial DNA markers. Nuclear DNA analyses indicated that reproductive isolation of the S. maena and S. flexuosa was complete. Demographic analyses support the sudden population growth of S. flexuosa after a potential bottleneck. The result of the dating analyses suggested that the three Spicara species were differentiated during the early or middle Pliocene. An absence of genetic structure between Spicara flexuosa subpopulations from Turkey indicated the connectivity, within sampling locations, suggesting that the Turkish Straits System is a corridor for gene flow for this species. As picarel populations have experienced rapid decline in their distribution areas in recent years, separate population studies are necessary to help make informed conservation and management decisions for S. maena and S. flexuosa.

Correlation of autoantigen-specific Treg frequency with development of spontaneous organ-specific autoimmunity in a mouse model of uveitis

Abstract About 50% of AireGW/+Lyn−/− mice developed autoimmune uveitis, and this was accompanied by the expansion of interphotoreceptor retinoid-binding protein (IRBP) specific CD4+T cells. Moreover, the response to IRBP was necessary for disease initiation, as deletion of IRBP prevented uveitis. Mass cytometry (CyTOF) analysis of CD4+T cells in the retina of the mice with uveitis identified activated T cell subsets characterized as Ly6Chi effectors, PD-1hi effectors, and Treg. To study cellular mechanisms that promoted development of uveitis, we did single-cell RNA sequencing (scRNA-seq) of eye-draining lymph node (LN) CD4+T cells that recognize the P2 epitope of IRBP (amino acid 271–290). Uniform manifold approximation and projection (UMAP) analysis showed that these CD4+T cells included distinct subsets that were Ly6c1hi , Pdcd1hi and Treg, with the Treg population being overrepresented in the mice without disease. Pseudotime analysis indicated that the Ly6c1hi T cell subset represented an earlier stage in activation, whereas the Pdcd1hi subset represented a later stage. Similar analysis of P2–specific CD4+T cells from the retina of mice with uveitis identified 6 subsets of P2-specific T cells, and was largely consistent with the CyTOF analysis of total CD4+ T cells from the retina. Pseudotime analysis indicated that toward the earlier time were cells characterized by being proliferative (Mki67hi), followed by a Lag3hi subset and at other end of the pseudotime axis by a Lag3hi / Pdcd1hi subset. Our results are consistent with the hypothesis that the fraction of Treg within the IRBP-specific CD4+ T cells in the draining LN determines whether these genetically susceptible mice develop eye-specific autoimmunity or are protected.

T-cell receptor repertoire of mice with organ-specific autoimmunity resulting from a partial defect in T cell negative selection and dendritic cell enhancement

Abstract Previously, we reported a new genetic model of spontaneous autoimmune disease in which mice with a hypomorphic mutation of Aire (AireGW/+) combined with deletion of Lyn in dendritic cells develop autoimmune uveitis with a 50% penetrance. Here we demonstrate that a key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+ Lyn−/− IRBP−/− mice failed to develop uveitis. The expansion of CD4+ T cells recognizing amino acids 271–290 (“P2”) of IRBP correlated strongly with autoimmune disease in mice with intact IRBP. To study the role of these T cells, we did single-cell RNA-seq with TCR sequencing for P2-specific CD4+ T cells isolated from eye-draining lymph nodes (LN) of AireGW/+ Lyn−/− mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen, with some sharing of TCR Vα or Vβ sequences. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCR clonotypes was expressed in developing thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR clonotype. Thus, deficiency of negative selection of P2 tetramer–specific TCR promotes the development of uveitis in AireGW/+ Lyn−/− mice, even if negative selection is only partly compromised.

The roles of possible geographic barriers and geological events on the phylogeographic structure of the Eastern broad toothed field mouse (Apodemus mystacinus)

Abstract The Eastern broad toothed field mouse, Apodemus mystacinus, is a rodent species distributed in Turkey, the Middle East, and a few Aegean Islands. The aim of this study is to analyse the phylogeographic structure of A. mystacinus and possible causes of its differentiation, on the basis of mitochondrial and nuclear DNA sequences using a large number of new samples from Turkey. In this context, partial mitochondrial sequences of cytochrome b (Cytb), control region (D-loop) and a nuclear interphotoreceptor retinoid-binding protein (IRBP) gene were used to reveal the geographical differentiation among A. mystacinus populations and the validity of its subspecies. The estimated divergence times revealed that the first separation of A. mystacinus into three distinct groups (subspecies of A. mystacinus: A. m. mystacinus, A. m. smyrnensis, and A. m. euxinus) begun 0.641 Mya. The possible physical barriers in Anatolia such as high mountains and rivers could interrupt the gene flow between A. mystacinus populations. The results of the present study indicated that A. mystacinus might have used the high rocky areas along the Anatolian Diagonal as a dispersal way. Moreover, mitochondrial data in this study suggested for the first time that A. m. rhodius is synonymous with the nominative subspecies A. m. mystacinus.

High Ambient Temperature Aggravates Experimental Autoimmune Uveitis Symptoms.

Whether ambient temperature influences immune responses leading to uveitis is unknown. We thus tested whether ambient temperature affects the symptoms of experimental autoimmune uveitis (EAU) in mice and investigated possible mechanisms. C57BL/6 mice were kept at a normal (22°C) or high temperature (30°C) housing conditions for 2 weeks and were then immunized with human interphotoreceptor retinoid-binding protein (IRBP651–670) peptide to induce EAU. Histological changes were monitored to evaluate the severity of uveitis. Frequency of Th1 cells and Th17 cells was measured by flow cytometry (FCM). The expression of IFN-γ and IL-17A mRNA was measured by real-time qPCR. The generation of neutrophil extracellular traps (NETs) was quantified by enzyme-linked immunosorbent assay (ELISA). Differential metabolites in the plasma of the mice kept in the aforementioned two ambient temperatures were measured via ultra-high-performance liquid chromatography triple quadrupole mass spectrometry quadrupole time of flight mass spectrometry (UHPLC-QQQ/MS). The differential metabolites identified were used to evaluate their effects on differentiation of Th1 and Th17 cells and generation of NETs in vitro. The results showed that EAU mice kept at high temperature experienced a more severe histopathological manifestation of uveitis than mice kept at a normal temperature. A significantly increased frequency of Th1 and Th17 cells in association with an upregulated expression of IFN-γ and IL-17A mRNA was observed in the splenic lymphocytes and retinas of EAU mice in high temperature. The expression of NETs as evidenced by myeloperoxidase (MPO) and neutrophil elastase (NE), was significantly elevated in serum and supernatants of neutrophils from EAU mice kept at high temperature compared to the normal temperature group. The metabolites in the plasma from EAU mice, fumaric acid and succinic acid, were markedly increased in the high temperature group and could induce the generation of NETs via the NADPH oxidase-dependent pathway, but did not influence the frequency of Th1 and Th17 cells. Our findings suggest that an increased ambient temperature is a risk factor for the development of uveitis. This is associated with the induction of Th1 and Th17 cells as well as the generation of NETs which could be mediated by the NADPH oxidase-dependent pathway.

Detection and identification of Kinetoplastids of zoonotic interest by HRM-qPCR analysis in Canis lupus familiaris from Argentinean Mesopotamia.

This work aimed to conduct a first PCR-based approach for differential diagnosis of kinetoplastidean infections in dogs. Diagnosis of Kinetoplastid infections in domestic animals is difficult, since parasitemia is intermittent and signs are nonspecific; it is mainly based on parasitological smears or concentration techniques, which lack sensitivity and depend on operator` expertise. Dogs are relevant reservoirs in transmission of Kinetoplastids; they function as sentinels to detect active transmission cycles before they involve humans. Trypanosoma cruzi, Trypanosoma evansi, and various species of Leishmania genus are multi-host parasites, capable of parasitizing dogs among a vast number of reservoirs. An algorithm based on sequential Real-Time PCR-High Resolution Melting (HRM) (qPCR-HRM) assays directed at 24S alpha ribosomal DNA, ITS1 and Hsp70 designed to distinguish among T. cruzi, T. rangeli, T. evansi and Leishmania spp. was tested in fourteen dogs with suspicion of kinetoplastid diseases. A qPCR control of DNA integrity in the tested sample, targeted to the mammalian interphotoreceptor retinoid-binding protein (IRBP) gene fragment was incorporated to the algorithm. T. evansi was detected in four dogs and L. infantum in one. Two of five qPCR positive cases were smear negative. Smear and T. evansi qPCR positive cases corresponded to animals that died despite being treated, indicating the association of parasitemia with disease severity. This laboratory tool increases the possibility of confirming outbreaks of kinetoplastid diseases with zoonotic potential and identify the etiological agents involved.

First detection of Leishmania of the subgenus viannia in Alipiopsitta xanthops, endemic bird of South America

We report the first molecular detection of Leishmania infection (subgenus Viannia) in the yellow-faced parrot (Alipiopsitta xanthops), at a wildlife rehabilitation center located in the city of Campo Grande, Brazil, an endemic area for leishmaniasis. PCRs targeting kinetoplast DNA (kDNA) and the small subunit of ribosomal RNA of Leishmania spp. were performed, both positive, followed by the sequencing of the amplified region of the SSU rDNA gene, which confirmed the identity of the parasite. This is the first report of success obtained in the use of PCR targeting the IRBP (Interphotoreceptor retinoid-binding protein) gene as an internal control in the molecular diagnosis of pathogens in bird species.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis

BackgroundThe integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets.MethodsMice (female; B10.RIII or C57Bl/6; aged 6–8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry.ResultsThere was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers.ConclusionsThis α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Regulated Tristetraprolin Overexpression Dampens the Development and Pathogenesis of Experimental Autoimmune Uveitis.

Non-infectious uveitis, a common cause of blindness in man, is often mediated by autoimmunity, a process in which cytokines play major roles. The biosynthesis and secretion of pro-inflammatory cytokines are regulated in part by tristetraprolin (TTP), an endogenous anti-inflammatory protein that acts by binding directly to specific sequence motifs in the 3’-untranslated regions of target mRNAs, promoting their turnover, and inhibiting synthesis of their encoded proteins. We recently developed a TTP-overexpressing mouse (TTPΔARE) by deleting an AU-rich element (ARE) instability motif from the TTP mRNA, resulting in increased accumulation of TTP mRNA and protein throughout the animal. Here, we show that homozygous TTPΔARE mice are resistant to the induction of experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP), an established model for human autoimmune (noninfectious) uveitis. Lymphocytes from TTPΔARE mice produced lower levels of the pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and TNFα than wild type (WT) mice. TTPΔARE mice also produced lower titers of antibodies against the uveitogenic protein. In contrast, TTPΔARE mice produced higher levels of the anti-inflammatory cytokine IL-10, and had higher frequencies of regulatory T-cells, which, moreover, displayed a moderately higher per-cell regulatory ability. Heterozygous mice developed EAU and associated immunological responses at levels intermediate between homozygous TTPΔARE mice and WT controls. TTPΔARE mice were able, however, to develop EAU following adoptive transfer of activated WT T-cells specific to IRBP peptide 651–670, and naïve T-cells from TTPΔARE mice could be activated by antibodies to CD3/CD28. Importantly, TTPΔARE antigen presenting cells were significantly less efficient compared to WT in priming naïve T cells, suggesting that this feature plays a major role in the dampened immune responses of the TTPΔARE mice. Our observations demonstrate that elevated systemic levels of TTP can inhibit the pathogenic processes involved in EAU, and suggest the possible use of TTP-based treatments in humans with uveitis and other autoimmune conditions.

Greater Bandicoot Rats (Bandicota indica) are Not Native to Sundaland Based on Deoxyribonucleic Acid (DNA) Analyses

Bandicoot rats (genus Bandicota) are distributed widely across the Indomalay biogeographic realm of tropical East Asia. One widely distributed species, the greater bandicoot rat (Bandicota indica), has a disjunct distribution including both north and south of the biogeographic break at the Isthmus of Kra. We compared genetic variation of greater bandicoot rats from north and south of the Isthmus of Kra using mitochondrial cytochrome b (cyt b, 1140 bp) and nuclear interphotoreceptor retinoid binding protein (IRBP, 801 bp) sequences. We found that the greater bandicoot rat (B. indica) is not native to Sundaland, the region south of the Isthmus of Kra. The species was introduced to the region recently as the genetic divergence with other regions is very low and phylogenies of both genes showed Malaysian greater bandicoot rat very closely related to conspecifics from Lao PDR. Haplotype data revealed all individuals from Malaysia are homogenous, which implied that the species was introduced recently. The greater bandicoot rats in Malaysia are so far only reported in the rice producing regions of Kedah and Perlis, but they may be increasing in number and distribution. A more detailed survey on the distribution and population demographics of Malaysian greater bandicoot rats are needed to support a management plan for this invasive species.

Phylogenetic and morphological significance of an overlooked flying squirrel (Pteromyini, Rodentia) from the eastern Himalayas with the description of a new genus.

The flying squirrels (Pteromyini, Rodentia) are the most diverse and widely distributed group of gliding mammals. Taxonomic boundaries and relationships within flying squirrels remain an area of active research in mammalogy. The discovery of new specimens of Pteromys (Hylopetes) leonardi Thomas, 1921, previously considered a synonym of Hylopetes alboniger, in Yunnan Province, China allowed a morphological and genetic reassessment of the status of this taxon. Phylogenetic reconstruction was implemented using sequences of two mitochondrial (12S ribosomal RNA and 16S ribosomal RNA) and one nuclear (interphotoreceptor retinoid-binding protein) gene fragments. Morphological assessments involved examinations of features preserved on skins, skulls, and penises of museum specimens, supplemented with principal component analysis of craniometric data. Together these assessments revealed that this taxon should be recognized not only as a distinct species, but should also be placed within a new genus, described here as Priapomysgen. nov.

The protective function of invariant natural killer T cells in the relapse of experimental autoimmune uveoretinitis

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

Interphotoreceptor Retinoid-Binding Protein (IRBP) in Retinal Health and Disease.

Interphotoreceptor retinoid-binding protein (IRBP), also known as retinol binding protein 3 (RBP3), is a lipophilic glycoprotein specifically secreted by photoreceptors. Enriched in the interphotoreceptor matrix (IPM) and recycled by the retinal pigment epithelium (RPE), IRBP is essential for the vision of all vertebrates as it facilitates the transfer of retinoids in the visual cycle. It also helps to transport lipids between the RPE and photoreceptors. The thiol-dependent antioxidant activity of IRBP maintains the delicate redox balance in the normal retina. Thus, its dysfunction is suspected to play a role in many retinal diseases. We have reviewed here the latest research on IRBP in both retinal health and disease, including the function and regulation of IRBP under retinal stress in both animal models and the human retina. We have also explored the therapeutic potential of targeting IRBP in retinal diseases. Although some technical barriers remain, it is possible that manipulating the expression of IRBP in the retina will rescue or prevent photoreceptor degeneration in many retinal diseases.

Chitosan Oligosaccharides Suppress Nuclear Factor-Kappa B Activation and Ameliorate Experimental Autoimmune Uveoretinitis in Mice.

We investigated the therapeutic potential and mechanism of chitosan oligosaccharides (COS) for experimental autoimmune uveoretinitis (EAU) in mice. EAU was induced in C57/BL6 mice by injection of human interphotoreceptor retinoid-binding protein (IRBP) peptides. At the same time, a high or low dose (20 or 10 mg/kg) of COS or phosphate-buffered saline (PBS) was given to mice daily after EAU induction. We found that mouse EAU is ameliorated by the high-dose COS treatment when compared with PBS treatment. In the retinas of high-dose COS-treated mice, the nuclear translocation of NF-κB subunit (p65) was suppressed, and the expression of several key EAU inflammatory mediators, IFN-γ, TNF-α, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and MCP-1 was lowered. These results suggest that COS may be a potential treatment for posterior uveitis.

Alpha-taxonomy in the cricetid rodent Neomicroxus, a first assessment

Neomicroxus , a recently named genus, comprises small-bodied cricetid rodents patchily distributed in high-Andean ranges from Ecuador to Venezuela. Currently, two species of Neomicroxus are recognized, N. bogotensis , endemic to the Cordillera Oriental in Colombia and Cordillera de Merida and Paramo de Tama in Venezuela, and N. latebricola that occurs northern Andes of Ecuador. The genus is among the most poorly understood Neotropical rodents and to date no formal assessment about its alpha taxonomy was conducted. Based on DNA evidence of the first portion of the mitochondrial cytochrome b gene (cytb) and the first exon of the interphotoreceptor retinoid binding protein (IRBP), as well as craniodental measurements, we explored the divergence degree, genetic structure and phyletic relationships of the two species currently allocated under Neomicroxus . Our analyses support the monophyly of the genus as well as its uncertain tribal affiliation. Neomicroxus was retrieved as structured in two main branches, in agreement with the traditional recognition of two species. The populations referred to N. bogotensis exhibit deep divergence values (> 6 %) pointing to the existence of undescribed species under its concept. In contrast, populations of N. latebricola show a shallow genetic structure although implying recognizable geographical breaks. A moderate degree of genetic and morphological differentiation supports a new subspecies for the western populations of N. latebricola . Our contribution is the first attempt to better understanding the alpha taxonomy of Neomicroxus , highlighting the importance of the geographic complexity as a barrier to the genetic flow in N. bogotensis and the significance of the subspecies concept to formalize the geographic variation recovered in N. latebricola

Characterization of Clinical and Immune Responses in an Experimental Chronic Autoimmune Uveitis Model

Autoimmune uveitis is a sight-threatening intraocular inflammatory disease. For >30 years, the mouse model of experimental autoimmune uveitis has been employed to investigate disease mechanisms and test immunotherapeutic approaches. However, inflammation in this model is self-limited, and does not replicate the chronic, insidious nature prevalent in the human disease. Herein, a robust and reliable model of chronic autoimmune uveitis was developed and characterized in two strains of wild-type mice by modifying interphotoreceptor retinoid-binding protein dose and peptide fragments from conventional experimental autoimmune uveitis models. In both of these murine strains, immunization with our modified protocols resulted in a slowly progressive uveitis, with retinal scars and atrophy observed in the chronic stage by fundoscopy. Optical coherence tomography demonstrated decreased retinal thickness in chronic autoimmune uveitis mice, and electroretinography showed significantly reduced amplitudes of dark-adapted a- and b-waves and light-adapted b-waves. Histologic examination revealed prominent choroiditis with extensive retinal damage. Flow cytometry analysis showed substantially increased numbers of CD44hiIL-17+IFN-γ- memory T-helper 17 (Th17) cells in the retina, cervical lymph nodes, inguinal lymph nodes, and spleen. These data establish new modified protocols for inducing chronic uveitis in wild-type mice, and demonstrate a predominant memory Th17 cell response, suggesting an important role for memory Th17 cells in driving chronic inflammation in autoimmune uveitis.

Functionally distinct IFN-γ+ IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.

Immunopathogenic roles for both Th1 (CD4+ IFN-γ+ ) and Th17 (CD4+ IL-17A+ ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4+ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6±4.2%) and draining LNs (mean 8.4±3.9%; p=0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p=0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6±9.5% vs. 1.9±1.5%; p=0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.