Nobuyuki Ohguro

Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens

The eye lens is composed of fibre cells, which develop from the epithelial cells on the anterior surface of the lens. Differentiation into a lens fibre cell is accompanied by changes in cell shape, the expression of crystallins and the degradation of cellular organelles. The loss of organelles is believed to ensure the transparency of the lens, but the molecular mechanism behind this process is not known. Here we show that DLAD (‘DNase II-like acid DNase’, also called DNase IIβ) is expressed in human and murine lens cells, and that mice deficient in the DLAD gene are incapable of degrading DNA during lens cell differentiation—the undigested DNA accumulates in the fibre cells. The DLAD-/- mice develop cataracts of the nucleus lentis, and their response to light on electroretinograms is severely reduced. These results indicate that DLAD is responsible for the degradation of nuclear DNA during lens cell differentiation, and that if DNA is left undigested in the lens, it causes cataracts of the nucleus lentis, blocking the light path.

Involvement of Th17 cells and the effect of anti-IL-6 therapy in autoimmune uveitis

OBJECTIVES Human endogenous uveitis is one of the sight-threatening diseases associated with variety of systemic disorders, such as Behcets disease and sarcoidosis. Recently, biosynthesized antibodies against inflammatory cytokines have been recognized to be useful to control the regional inflammation. In this study, we focused on the possibility of IL-6-based biological therapies for endogenous uveitis. We initially confirmed the significant increase of several inflammatory soluble factors including IL-6 in the vitreous fluids from refractory/chronic engogenous uveitis patients. METHODS To investigate the role of IL-6 in the formation of refractory ocular inflammation, we used the mouse experimental autoimmune uveitis (EAU) model. Both IL-6 and IL-23 are required for the development of IL-17-producing helper T subset (Th17) from naïve CD4(+) T cells. Results. In the EAU model, neither IL-6-deficient mice nor IL-23-deficient mice could induce Th17 cells and the EAU score was decreased in these mice in the entire time course. We also confirmed that systemic administration of anti-il-6 receptor antibody ameliorates EAU By suppressing both systemic and regional TH17 responses. CONCLUSIONS IL-6 is responsible for causing ocular inflammation, and it is, at least partially, due to IL-6-dependent Th17 differentiation. IL-6 may be a target for therapy of refractory endogenous uveitis in humans.

iTRAQ-based proteomic identification of leucine-rich α-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases

Objective To identify a novel serum biomarker of disease activity in inflammatory autoimmune disorders. Methods Sera obtained from rheumatoid arthritis (RA) patients before and after anti-TNF therapy were analysed by iTRAQ (isobaric tags for relative and absolute quantitation) quantitative proteomic analysis and further validated by ELISA. Results Of 326 proteins identified by proteomic analysis, increased serum levels of leucine-rich α-2 glycoprotein (LRG) was identified in RA patients before therapy. Serum LRG concentrations were significantly elevated in RA patients compared with healthy controls and decreased after anti-TNF therapy. Furthermore, serum LRG concentrations correlated with disease activity in RA and Crohns disease (CD). Interestingly, in a subpopulation of patients with active CD and normal C-reactive protein levels, serum LRG concentrations were elevated. Conclusions LRG represents a novel serum biomarker for monitoring disease activity during therapy in autoimmune patients, particularly useful in patients with active disease but normal CRP levels.

Blockade of interleukin-6 signaling suppresses experimental autoimmune uveoretinitis by the inhibition of inflammatory Th17 responses.

The aim of this study was to investigate the effect of anti-mouse IL-6 receptor monoclonal antibody (MR16-1) treatment on CD4 T cell differentiation and compared it to the effect of anti-TNF mAb treatment with using a murine model of experimental autoimmune uveoretinitis (EAU). C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) to induce ocular inflammation treatment with control IgG or MR16-1 or anti-TNF mAb. Helper T cells differentiation was analyzed during the development of EAU. Immunization with IRBP increased the frequency of Th17 cells rather than Th1 cells in the early stage of EAU. Treatment with MR16-1 on the same day as immunization (day 0) or one day after (day 1) suppressed ocular inflammation in EAU mice. Treatment with MR16-1 on day 0 inhibited the induction of Th17 cells in vivo, and inhibited not only IRBP-responsive Th17 cells but also their Th1 counterparts and induced IRBP-responsive regulatory T (Treg) cells in vitro. The administration of anti-TNF mAb had no significant protective effect in EAU mice. The protective effect of anti-IL-6R mAb treatment, but not anti-TNF mAb treatment on EAU correlated with the inhibition of Th17 differentiation. This finding suggests that IL-6 blockade may have a therapeutic effect on human ocular inflammation which is mediated via mechanisms distinct from those of TNF blockade. IL-6 blockade may thus represent an alternative therapy for patients with ocular inflammation who are refractory to anti-TNF mAb therapy.

Blockade of interleukin-6 signaling suppresses not only th17 but also interphotoreceptor retinoid binding protein-specific Th1 by promoting regulatory T cells in experimental autoimmune uveoretinitis.

PURPOSE. Both Th17 and Th1 cells contribute to experimental autoimmune uveoretinitis (EAU). Interleukin-6 (IL-6) blockade inhibits Th17 differentiation in EAU and potently suppresses ocular inflammation, although its effect on Th1 cells is unknown. To clarify the mechanism of IL-6 blockade, the authors investigated T helper cells with particular focus on Th1 and regulatory T cells (Treg) in EAU of IL-6 gene knockout (KO) mice. METHODS. EAU was induced in wild-type (WT) mice and in mice lacking IL-6 (IL-6KO), IL-17 (IL-17KO), and IFN-γ (GKO) on a C57BL/6 background. Clinical scores of EAU, cytokine levels in supernatants from ocular tissue homogenates, and T helper cell differentiation in lymph nodes in each mouse were examined. To study the roles of Treg cells, EAU was induced in IL-6KO mice treated with anti-CD25 monoclonal antibody (mAb) to deplete Treg cells in vivo. RESULTS. Inflammation was comparable between WT, IL-17KO, and GKO mice but was absent in IL-6KO mice. Th17 and interphotoreceptor retinoid binding protein (IRBP)-specific Th1 cells were increased in GKO and IL-17KO mice, respectively, whereas both populations were reduced in IL-6KO mice. Th1-dominant EAU in IL-17KO mice was suppressed by anti-IL-6R mAb treatment. Treg cell depletion in vivo induced EAU in IL-6KO mice. CONCLUSIONS. After the induction of EAU, IL-6 deficiency resulted in the inhibition of the IRBP-specific Th1 response and enhanced the generation of IRBP-specific Treg cells. Furthermore, Treg was needed to inhibit Th1 responses and ocular inflammation in IL-6KO mice. Protective effects of IL-6 signaling blockade in EAU involve not only Th17 cell inhibition but also IRBP-specific Treg cell promotion.

Use of a Comprehensive Polymerase Chain Reaction System for Diagnosis of Ocular Infectious Diseases

PURPOSE To measure the genomic DNA of ocular infectious pathogens in ocular fluids and to analyze the clinical relevance of these pathogens in uveitis and endophthalmitis. DESIGN Prospective clinical case series. PARTICIPANTS A total of 500 patients with infectious uveitis and endophthalmitis were examined at Tokyo Medical and Dental University, Tokyo Medical University, Kyushu University, Osaka University, and Kyoto Prefectural University, all in Japan. METHODS Genomic DNA of bacteria, fungi, parasites, and viruses in collected intraocular samples were examined by comprehensive polymerase chain reaction (PCR). Samples were analyzed first by multiplex PCR and quantitative real-time PCR for human herpes viruses (HHVs) 1 through 8 and toxoplasma. Subsequently, samples were examined by broad-range real-time PCR for bacterial 16S and fungal 18S/28S ribosomal DNA (rDNA). MAIN OUTCOME MEASURES Infectious uveitis and endophthalmitis diagnoses were obtained when using the PCR system. Calculations of the positivity and the diagnostic parameters such as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) also were evaluated. RESULTS In all of the tested infectious uveitis and endophthalmitis patients, either herpes simplex virus type 1 (n = 18), herpes simplex virus type 2 (n = 4), varicella-zoster virus (n = 55), Epstein-Barr virus (n = 17), cytomegalovirus (n = 68), HHV type 6 (n = 2), toxoplasma (n = 6), bacterial 16S (n = 33), or fungal 18S/28S (n = 11) genome was detected. Neither HHV type 7 nor HHV type 8 DNA was detected in any of the samples. Of the 21 false-negative results found during the PCR analyses, 12 cases were negative for patients clinically suspected of having bacterial endophthalmitis. Conversely, false-positive results for the comprehensive PCR examinations occurred in only 3 cases that subsequently were found to have bacterial 16S rDNA. Diagnostic parameters for the sensitivity, specificity, PPV, and NPV of our PCR examinations were 91.3%, 98.8%, 98.6%, and 92.4%, respectively. CONCLUSIONS Use of our comprehensive PCR assay to examine ocular samples in patients with endophthalmitis and uveitis seems to be clinically useful for detecting infectious antigen DNA. Thus, this PCR method is a reliable tool for both diagnosing ocular disorders and further screening of patients for intraocular infections. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Evaluation of pulse corticosteroid therapy for vogt-koyanagi-harada disease assessed by optical coherence tomography

PURPOSE To evaluate the rapid effects of pulse corticosteroid therapy on the serous retinal detachment found at the acute phase of Vogt-Koyanagi-Harada disease. DESIGN Interventional case series. METHODS Nine Japanese patients determined to be at the acute phase of Vogt-Koyanagi-Harada disease with serous retinal detachment were treated with pulse corticosteroid therapy. Optical coherence tomography was used to follow the resolution of the retinal detachment. RESULTS Optical coherence tomography images showed a marked decrease in the retinal detachment immediately after the first intravenous injection of corticosteroid and subsequent resolution. CONCLUSION The rapid improvement of serous retinal detachment associated with Vogt-Koyanagi-Harada disease following pulse corticosteroid therapy supports an early therapeutic mechanism related to improved permeability of capillaries and permeability of the blood-retinal barrier rather than an anti-inflammatory or immunosuppressive action.

Trans-Tenon’s retrobulbar triamcinolone infusion for diffuse diabetic macular edema

Diabetic macular edema may be divided into two types, focal and diffuse. The Early Treatment Diabetic Retinopathy Study demonstrated a significant benefit of focal or grid laser photocoagulation for the treatment of focal macular edema [2], but much less efficacy for diffuse macular edema [2, 4]. Vitrectomy has been reported to be somewhat effective for diabetic macular edema with [3] or without [7] a thickened and taut vitreous membrane on the macula. Yet, there still remain cases of diffuse diabetic macular edema resistant to grid laser photocoagulation and vitrectomy, and thus other treatment modalities are needed. Periocular and intravitreal injections of triamcinolone acetonide have been used to treat cystoid macular edema [1, 6], and recently transTenon’s retrobulbar infusion of the same has been shown to be effective in treating posterior inflammation associated with uveitis [5]. Here we describe the results of trans-Tenon’s retrobulbar infusion of triamcinolone acetonide (triamcinolone infusion) for the treatment of diffuse diabetic macular edema that remained unresolved after vitrectomy. Graefe’s Arch Clin Exp Ophthalmol (2004) 242:444–445

Evaluation of the Long-Term Efficacy and Safety of Infliximab Treatment for Uveitis in Behçet's Disease: A Multicenter Study

PURPOSE To evaluate the long-term efficacy and safety of infliximab for the treatment of uveitis in Behçets disease (BD). DESIGN Retrospective multicenter study using a questionnaire. PARTICIPANTS A total of 164 consecutive patients with BD treated with infliximab for more than 1 year were studied. The mean age at initiation of infliximab treatment was 42.6±11.7 years, and the mean treatment duration was 32.9±14.4 months. METHODS Data before and at the last visit during infliximab treatment were analyzed in 4 groups divided by duration of treatment: group A (n = 43, 12-<24 months), group B (n = 62, 24-<36 months), group C (n = 42, 36-<48 months), and group D (n = 17, ≥48 months). MAIN OUTCOME MEASURES Best-corrected visual acuity (BCVA), relapse of ocular inflammation, numbers of ocular inflammatory attacks per year, and adverse effects of infliximab therapy. RESULTS The frequency of ocular attacks decreased in all groups (from 5.3±3.0 to 1.0±0.3 in group A, 4.8±4.6 to 1.4±0.3 in group B, 4.1±2.9 to 0.9±0.3 in group C, and 9.5±5.8 to 1.6±0.5 in group D; all P < 0.05). The BCVA was improved in approximately 55% of the eyes after treatment. Mean BCVA converted to logarithm of the minimum angle of resolution was improved after treatment with infliximab in groups A to C (from 0.79±1.04 to 0.59±0.94 in group A, 0.59±1.07 to 0.41±1.04 in group B, and 1.15±1.77 to 0.92±1.73 in group C; all P < 0.05) but not in group D. Uveitis relapsed in 59.1% of all patients after infliximab treatment, and no difference in duration until relapse was observed between individual groups. Approximately 80% of relapses occurred within 1 year after the initiation of infliximab treatment in all groups, 90% of which were controlled by increasing doses of topical corticosteroids and shortening the interval of infliximab infusion. Adverse effects were observed in 65 cases or 35% of all subjects. Infliximab treatment was continued in 85% of the patients, but 15% of the patients discontinued infliximab treatment because of adverse effects or insufficient efficacy. CONCLUSIONS Infliximab reduced the frequency of ocular attacks and improved visual acuity in patients with BD-related uveitis and was generally well tolerated with few serious adverse events.

Concentration dependent effects of hydrogen peroxide on lens epithelial cells

AIMS To evaluate the effects of hydrogen peroxide exposure on the survival and proliferation of cultured lens epithelial cells. METHODS TOTL-86 cells, a line of rabbit lens epithelial cells, were used. The survival and proliferation of TOTL-86 cells were quantified by a rapid colorimetric assay (MTT assay). To determine the effects of hydrogen peroxide, TOTL-86 cells were exposed to different concentrations of hydrogen peroxide. To determine the effect of cell numbers on the survival and proliferation of TOTL-86 cells at a fixed concentration of hydrogen peroxide, different numbers of cells were plated and exposed to hydrogen peroxide. To determine whether there is a synergistic effect between hydrogen peroxide and EGF, bFGF, PDGF-AA, and insulin, TOTL-86 cells were exposed to hydrogen peroxide combined with one of these growth factors. RESULTS High levels (1 mM) of hydrogen peroxide killed TOTL-86 cells and sublethal levels (100 μM) suppressed their proliferation. From 1 nM to 1 μM of hydrogen peroxide, there was a dose dependent increase in the cell numbers. The initial seeded cell number dramatically affected the response to hydrogen peroxide. Although growth factors showed no synergistic effects with hydrogen peroxide on proliferation, both EGF and insulin, but not bFGF or PDGF, rescued TOTL-86 cells from the sublethal effect. CONCLUSION Hydrogen peroxide in cooperation with some growth factors plays an important role in the proliferation of lens epithelial cell.