Kei Nakai

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.

Choroidal thickness in central serous chorioretinopathy.

Purpose: To study the choroidal thickness profile using high-penetration optical coherence tomography in central serous chorioretinopathy (CSC). Methods: Thirty-five eyes of 27 subjects with CSC and 35 healthy, age-matched control eyes were included. We observed the choroid using the prototype high-penetration optical coherence tomography. Fluorescein angiography and indocyanine green angiography were performed to identify the CSC location and activity. The choroidal thicknesses was measured manually in various conditions or locations, and the choroidal thickness maps were obtained from cube scans and calculating software and composed of nine sectors in the Early Diabetic Retinopathy Study chart. Results: The subfoveal choroidal thicknesses in all eyes with CSC were significantly (P < 0.01) greater than that in the control eyes. The choroidal thickness at the fovea and the fluorescein points of leakage were significantly (P < 0.01 for both comparisons) greater in eyes with CSC than the corresponding locations in the fellow eyes in patients with unilateral disease. Dilatation of the choroidal large vessels was significantly (P < 0.01) more common in CSC. The foveal choroidal thickness was significantly greater in eyes with venous dilatation (P < 0.01) than those without. The mean choroidal thickness was significantly (P < 0.05) greater in all sectors of the Early Diabetic Retinopathy Study chart except for the inner (P = 0.087) and outer (P = 0.053) inferior sectors. The percent mean choroidal thicknesses compared with the normal controls in the nasal sector were significantly (P < 0.05 and P < 0.01, respectively) greater in the inner and outer circles than in the superior, temporal, and inferior sectors. Conclusion: The choroid is diffusely thickened in CSC likely because of the choroidal vascular dilatation. The nasal macula undergoes the greatest alterations in choroidal thickness compared with the other areas.

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 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.

Broad spectrum antiangiogenic treatment for ocular neovascular diseases.

Pathological neovascularization is a hallmark of late stage neovascular (wet) age-related macular degeneration (AMD) and the leading cause of blindness in people over the age of 50 in the western world. The treatments focus on suppression of choroidal neovascularization (CNV), while current approved therapies are limited to inhibiting vascular endothelial growth factor (VEGF) exclusively. However, this treatment does not address the underlying cause of AMD, and the loss of VEGFs neuroprotective can be a potential side effect. Therapy which targets the key processes in AMD, the pathological neovascularization, vessel leakage and inflammation could bring a major shift in the approach to disease treatment and prevention. In this study we have demonstrated the efficacy of such broad spectrum antiangiogenic therapy on mouse model of AMD. Methods and Findings Lodamin, a polymeric formulation of TNP-470, is a potent broad-spectrum antiangiogenic drug. Lodamin significantly reduced key processes involved in AMD progression as demonstrated in mice and rats. Its suppressive effects on angiogenesis, vascular leakage and inflammation were studied in a wide array of assays including; a Matrigel, delayed-type hypersensitivity (DTH), Miles assay, laser-induced CNV and corneal micropocket assay. Lodamin significantly suppressed the secretion of various pro-inflammatory cytokines in the CNV lesion including monocyte chemotactic protein-1 (MCP-1/Ccl2). Importantly, Lodamin was found to regress established CNV lesions, unlike soluble fms-like tyrosine kinase-1 (sFlk-1). The drug was found to be safe in mice and have little toxicity as demonstrated by electroretinography (ERG) assessing retinal and by histology. Conclusions Lodamin, a polymer formulation of TNP-470, was identified as a first in its class, broad-spectrum antiangiogenic drug that can be administered orally or locally to treat corneal and retinal neovascularization. Several unique properties make Lodamin especially beneficial for ophthalmic use. Our results support the concept that broad spectrum antiangiogenic drugs are promising agents for AMD treatment and prevention.

Dendritic Cells Augment Choroidal Neovascularization

PURPOSE Dendritic cells (DCs) are innate immune cells that have recently been shown to support angiogenesis in tumors, endometriosis, and lymph nodes. A major cause of legal blindness is wet age-related macular degeneration (wet ARMD), wherein abnormal blood vessels grow under the retina, an abnormality also referred to as choroidal neovascularization (CNV). The purpose of the present study was to investigate the role of DCs in the development of CNV. METHODS Laser photocoagulation was used to induce CNV in C57BL/6J mice. The authors analyzed CNV lesions for the presence of DCs using flow cytometry and immunostaining at designated times. They also analyzed the effects of intravenous DC transplantation on CNV development by measuring the lesion area using confocal microscopy 1 week after laser injury. RESULTS The authors analyzed CNV lesions for the presence of DCs by flow cytometry and observed that CD11c(+) major histocompatibility complex (MHC) class II(+) DCs transiently infiltrated the CNV lesions, reaching a peak at 2 to 4 days after laser injury. These DCs were mostly immature (CD11c(+) MHCII(low)) and expressed vascular endothelial growth factor receptor 2. Immunostaining of laser-induced CNV lesions confirmed that DCs are located at the sites of newly formed blood vessels. Intravenously injected DCs incorporated into the CNV lesions. However, only immature DCs enhanced CNV size. CONCLUSIONS These results suggest a role for DCs in promoting angiogenesis and lesion growth in laser-induced CNV. The present data suggest that DCs may represent potential cellular targets for therapeutic intervention in wet ARMD.

Neutrophil Chemotaxis and Local Expression of Interleukin-10 in the Tolerance of Endotoxin-Induced Uveitis

PURPOSE To study the mechanism of lipopolysaccharide (LPS) tolerance in a rat model of footpad injection endotoxin-induced uveitis (EIU). METHODS EIU was produced by footpad injection of 1 mg/kg LPS in male Sprague-Dawley rats. Four experiments were undertaken in this study. First, on days 3, 7, 14, 28, 56, and 84 after LPS injection, the iris-ciliary body (ICB) was isolated. LPS tolerance-associated gene expression in the ICB was determined by quantitative polymerase chain reaction. Second, the distribution of IL-10-producing cells in frozen sections of ocular tissues was analyzed by fluorescence and confocal microscopy. Third, peripheral blood neutrophil chemotaxis was determined using a fluorescent in vitro migration assay. Fourth, for in vivo neutrophil chemotaxis assay, neutrophils isolated from EIU-tolerant or control rats were transfused into green fluorescence protein (GFP) rats injected with LPS 18 hours earlier. Six hours after transfusion, the percentage of GFP-negative neutrophils in the aqueous humor was determined by flow cytometry. RESULTS IL-10 gene expression in ICB was significantly upregulated for at least 1 month. Immunohistochemical examination indicated that dendritic cells in the ICB produced IL-10. Peripheral blood neutrophil chemotaxis in EIU-tolerant rats was inhibited significantly in vitro and in vivo. IL-10 enhanced the reduction of neutrophil chemotaxis in EIU-tolerant rats in vitro. CONCLUSIONS The results suggest that continuous high expression of IL-10 in the eye and the reduction of peripheral blood neutrophil chemotaxis play significant roles in the mechanism of LPS tolerance in a rat model of footpad injection EIU.

Laser-Induced Choroidal Neovascularization in Mice Attenuated by Deficiency in the Apelin-APJ System

PURPOSE To investigate the role of the apelin-APJ system in the development of choroidal neovascularization (CNV). METHODS Experimental CNV was induced by laser photocoagulation in wild-type (WT), apelin-deficient (apelin-KO), and apelin receptor (APJ)-deficient (APJ-KO) mice. The gene expression levels of angiogenic or inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction. APJ expression in CNV lesions was examined by immunohistochemistry. The sizes of the CNV lesions in the three mouse models were measured and compared histologically using isolectin B4 staining. Macrophage recruitment was measured by flow cytometric analysis. Proliferation of endothelial cells was determined using the alamar Blue assay. RESULTS Laser photocoagulation significantly increased expression of apelin and APJ in the retina-retinal pigment epithelium (RPE) complex. APJ immunoreactive cells were found in the CNV lesions and colocalized with platelet endothelial cell adhesion molecule-1, an endothelial cell marker. The sizes of the CNV lesions in apelin-KO or APJ-KO mice decreased significantly compared with those in the WT mice. Macrophages in the RPE complex of the apelin-KO mice, in which gene expression of the inflammatory factors was almost equal to that in WT mice, were recruited as a result of laser photocoagulation to the same degree as in WT mice. In addition, apelin small and interfering RNA (siRNA) suppressed proliferation of endothelial cells independently of vascular endothelial growth factor (VEGF) receptor 2 signaling, while VEGF increased expression of apelin and APJ in human umbilical vein endothelial cells. CONCLUSIONS The results suggested that the apelin-APJ system contributes to CNV development partially independent of the VEGF pathway.

Invasive human pancreatic carcinoma cells adhere to endothelial tri-cellular corners and increase endothelial permeability

Although adhesive interactions between metastasizing cancer cells and vascular endothelial cells are critical in hematogenous metastasis, the early molecular events of the cancer–endothelial interaction remain largely obscure. Here we investigated the functional impact of cancer cells on endothelial permeability. We examined the binding of human pancreatic carcinoma cells MIA PaCa‐2, PANC‐1 and PSN‐1 to a human umbilical vein endothelial cell (HUVEC) monolayer and the subsequent changes in the transendothelial electronic resistance (TEER) of the HUVEC. We found that MIA PaCa‐2 and PANC‐1 cells preferentially bound to the tri‐cellular corners of HUVEC and induced a rapid and irreversible reduction of TEER. The reduction of HUVEC TEER was associated with the focal disengagement of endothelial junctional adhesion molecules VE‐cadherin and CD31. Blocking antibodies to integrin β1, CD44, or CD9 affected neither the MIA PaCa‐2 binding to HUVEC nor the reduction of TEER. Specific inhibitors for metalloproteinases, tyrosine‐kinases and lipoxigenases, and a neutralizing anti‐vascular endothelial growth factor antibody failed to affect the MIA PaCa‐2‐induced reduction of HUVEC TEER, whereas treatment of the cells with paraformaldehyde or cytochalasin B abrogated the TEER reduction. These findings indicate that the MIA PaCa‐2 cells bind selectively to endothelial tri‐cellular corners, triggering a reduction of HUVEC TEER, which requires the active metabolism and intact actin cytoskeleton of the carcinoma cells, and is apparently unrelated to previously described cell adhesion and soluble factor pathways. Our data indicate a novel cell‐contact‐dependent mechanism for the cancer cell‐mediated breakdown of endothelial barrier functions, which may be important in hematogenous cancer metastasis. (Cancer Sci 2005; 96: 766–773)

Choroidal and cutaneous metastasis from gastric adenocarcinoma

Choroidal or cutaneous metastasis of gastric cancer is rare. Gastrointestinal cancer was found in only 4% in patients with uveal metastasis. Choroidal metastasis from gastric cancer was reported in two cases in earlier literature. The frequency of gastric cancer as a primary lesion was 6% in cutaneous metastasis of men, and cutaneous metastasis occurs in 0.8% of all gastric cancers. We report a patient with gastric adenocarcinoma who presented with visual disorder in his left eye and skin pain on his head as his initial symptoms. These symptoms were diagnosed to be caused by choroidal and cutaneous metastasis of gastric adenocarcinoma. Two cycles of chemotherapy consisted of oral S-1 and intravenous cisplatin (SPIRITS regimen); this was markedly effective to reduce the primary gastric lesion and almost all the metastatic lesions.