Profiling miRNAs in tear extracellular vesicles: a pilot study with implications for diagnosis of ocular diseases.

PURPOSE: To estimate the roles of extracellular vesicles (EVs) in tears and to determine whether their profiles are associated with the type of ocular disease. STUDY DESIGN: Cross-sectional study. METHODS: Tear EVs were extracted from 14 healthy participants and from 21 patients with retinal diseases (age-related macular degeneration [AMD] or diabetic macular edema [DME]). The surface marker expression of tear EVs was examined, and microRNAs (miRNAs) were extracted and profiled by use of real-time PCR array. The stability of the expression of the miRNAs was determined, and their functions were assessed by network analyses. Classification accuracy was evaluated by use of a random forest classifier and k-fold cross-validation. RESULTS: The miRNAs that were highly expressed in tear EVs were miR-323-3p, miR-548a-3p, and miR-516a-5p. The most stably expressed miRNAs independent of diseases were miR-520h and miR-146b-3p. The primary networks of the highly stably expressed endogenous miRNAs were annotated as regulation of organismal injury and abnormalities. The highly expressed miRNAs for severe retinal disease were miR-151-5p for AMD and miR-422a for DME, suggesting potential roles of tear EVs in liquid biopsy. Nine miRNAs (miR-25, miR-30d, miR-125b, miR-132, miR-150, miR-184, miR-342-3p, miR-378, and miR-518b) were identified as distinguishing individuals with AMD from healthy individuals with a classification accuracy of 91.9%. CONCLUSIONS: The finding that tear EVs contain characteristic miRNA species indicates that they may help in maintaining homeostasis and serve as a potential tool for disease diagnosis.

Association of IL-4 with pachychoroid neovasculopathy.

The purpose of this study was to identify the inflammatory cytokines that were associated with pachychoroid neovasculopathy (PNV). Seventy-five eyes of 75 patients with PNV, 145 eyes of 145 patients with neovascular age-related macular degeneration without pachyvessels, and 150 eyes of 150 normal subjects were examined for the levels of intraocular cytokines. In eyes with PNV, the levels of IL-1α, IL-1ß, IL-2, IL-4, IL-10, and VEGF were significantly higher than that of the controls. Logistic regression analysis showed that the highest association with the pachyvessels was found for IL-4, IL-2, and IL-1α. In eyes with PNV, the levels of IL-4, IL-2, IL-5, IL-13, IL-1α, and IL-1ß were significantly higher in eyes with both increased choroidal thickness and choroidal vessel diameter. The strongest correlation with the choroidal thickness and vessel diameter was observed for IL-4. In PNV eyes with polypoidal lesions, the levels of IL-4, IL-17, and TNFß were significantly correlated with the number of polypoidal lesions. Of these cytokines, IL-4 was especially associated with the thickness of the choroidal vessels and the formation of polypoidal lesions. We conclude that IL-4 is most likely involved in establishing the clinical characteristics of PNV and polypoidal vascular remodeling.

Antiviral cytotoxic T lymphocyte responses for long term prognosis of corneal infection by cytomegalovirus in immunocompetent subjects.

Ocular cytomegalovirus (CMV) infections in immunocompetent individuals are rare, but its activation can cause chronic and relapsing inflammation in anterior segment of the eye resulting in loss of corneal clarity and glaucoma. Fifty five patients with anterior segment CMV infection were assessed for their clinical characteristics, and CMV corneal endotheliitis was found to cause significant loss of corneal endothelial cells. The disease duration with recurrences was significantly correlated with the maximum intraocular level of CMV DNA. To examine why CMV is activated in healthy immunocompetent individuals and causing corneal endothelial cell damage, assays of cytotoxic T cells (CTLs) which directly target infected corneal endothelial cells were performed for 9 HLA-matched CMV corneal endotheliitis patients (HLA-A*2402). When the cell loss was analyzed for associations with CTL responses, CMV-induced endothelial cell damage was mitigated by pp65-specific CTL induction. The recurrence-free time was also prolonged by pp65-specific CTL induction (hazard ratio (HR): 0.93, P = 0.01). In contrast, IE1-specific CTL was associated with endothelial cell damage and reduced the time for corneal transplantation (HR: 1.6, P = 0.003) and glaucoma surgery (HR: 1.5, P = 0.001). Collectively, induction of pp65-specific CTL was associated with improved visual prognosis. However, IE1-specific CTL without proper induction of pp65-specific CTL can cause pathological damage leading to the need of surgical interventions.

Bilateral diffuse anterior scleritis after unilateral cataract surgery: A case report.

Surgically induced scleritis (SIS) is a rare complication after a variety of ocular surgeries. Majority of the patients had had two or more surgical procedures before the onset of SIS. We report a patient who developed bilateral diffuse scleritis after unilateral cataract surgery. A 64-year-old man presented with red eye and tenderness in the right eye. An uneventful cataract surgery had been conducted in the right eye 5 months earlier. He had lost vision in the left eye 25 years ago, and there had been no ocular symptoms in both the eyes for the previous 20 years. Scleritis of the right eye appeared 5 months after cataract surgery, and scleritis of the left eye developed three months later. No previous reports referred to the possibility that the right eye surgery could induce scleritis in the left eye. Our case implies that surgical trauma in one eye can induce scleritis in both the eyes with a history of severe inflammation.

Role of IL-4 in bone marrow driven dysregulated angiogenesis and age-related macular degeneration.

Age-associated sterile inflammation can cause dysregulated choroidal neovascularization (CNV) as age-related macular degeneration (AMD). Intraocular fluid screening of 234 AMD patients identified high levels of IL-4. The purpose of this study was to determine the functional role of IL-4 in CNV formation using murine CNV model. Our results indicate that the IL-4/IL-4 receptors (IL4Rs) controlled tube formation and global proangiogenic responses of bone marrow cells. CCR2+ bone marrow cells were recruited to form very early CNV lesions. IL-4 rapidly induces CCL2, which enhances recruitment of CCR2+ bone marrow cells. This in vivo communication, like quorum-sensing, was followed by the induction of IL-4 by the bone marrow cells during the formation of mature CNVs. For CNV development, IL-4 in bone marrow cells are critically required, and IL-4 directly promotes CNV formation mainly by IL-4R. The IL-4/IL-4Rα axis contributes to pathological angiogenesis through communications with bone marrow cells leading to retinal degeneration.

Effectiveness of 16S ribosomal DNA real-time PCR and sequencing for diagnosing bacterial keratitis.

PURPOSE: To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis. METHODS: We studied 272 eyes of 272 patients with keratitis. Eyes with keratitis were classified as "definite" (N = 118), "likely" (N = 71), or "non-bacterial" (N = 83) to have bacterial keratitis. The diagnostic efficacy of real-time PCR and conventional testing was determined by receiver operating characteristic analysis. The copy numbers of bacterial DNA and clinical characteristics were retrospectively analyzed for association with concordant culture results in the "definite" cases. RESULTS: The level of bacterial DNA was significantly associated with the diagnostic probability of the three diagnostic categories. The level of bacterial DNA had comparable diagnostic efficacy with the area under the curve (AUC) at 0.67, by culture at 0.65, and by smear testing at 0.73. The efficacy was significantly improved by combining the DNA level with the conventional culture testing with an AUC of 0.81. Analysis of the "definite" cases showed culture positivity in 51.8% (58 eyes), and of these, 41 eyes (70.7%) were higher than the cutoff PCR values and 40 eyes were identified by 16S r-DNA sequencing. In the culture-negative eyes, the level of bacterial DNA was significantly lower (P = 0.0008). Eyes with higher bacterial DNA levels had significantly concordant outcomes with sequencing and culture results (P = 0.006). Previous antibiotic treatments decreased the bacterial DNA amount by 0.09-fold, and it was a significant factor for discordance (P = 0.006). CONCLUSION: Quantification of the bacterial DNA level and conventional testing improves the diagnostic efficacy of infectious bacterial keratitis.

Effectiveness of real-time PCR for diagnosis and prognosis of varicella-zoster virus keratitis.

PURPOSE: To determine the efficacy of real-time PCR for the diagnosis and prognosis of varicella-zoster virus (VZV) keratitis. STUDY DESIGN: Retrospective case series. METHODS: Patients: 545 consecutive patients with keratitis were examined to quantify copy numbers of VZV DNA by real-time PCR. Association of copy numbers of VZV DNA to clinical signs and disease course was assessed by logistic regression analysis and Cox proportional hazard model. RESULTS: Of the 545 eyes, 38 (6.9%) were diagnosed as VZV keratitis. The median copy number of the VZV DNA was 104.19 copies; this number was significantly associated with diagnosis of VZV keratitis with the highest odds ratio of 3390 (for median copy) compared to the clinical signs. The diagnostic accuracy of the VZV DNA copy indicated good diagnostic value of area under the curve (0.92) by receiver operating characteristic analysis, and detection of unrelated VZV DNA from the cornea was very rare (0.2%). When the VZV DNA copy and clinical signs were assessed for association with the disease course of herpes zoster ophthalmicus, the disease duration was significantly prolonged in VZV keratitis cases with higher numbers of VZV DNA copies, iritis, and history of recurrences. The amount of VZV DNA led to a continuous risk of prolonged disease duration until the ocular inflammation subsided (hazard ratio 0.17, 95% CI 0.07-0.42, for median copies). CONCLUSIONS: Higher VZV DNA copy numbers are associated with the refractoriness of VZV keratitis, and its evaluation may be a useful way to clinically diagnose and manage VZV keratitis.

Corneal endothelial cells activate innate and acquired arm of anti-viral responses after cytomegalovirus infection.

Infection of the corneal endothelial cells by human cytomegalovirus (CMV) is an important cause of corneal endotheliitis. CMV endotheliitis is difficult to completely cure and relapses are frequent. This can cause blinding corneal bullous keratopathy. However, the pathogenesis of CMV endotheliitis remains undetermined. To understand the immunopathology of endotheliitis, we examined how corneal endothelial cells prime the anti-viral immunity after CMV infection based on global transcriptional responses. To accomplish this, human corneal endothelial (HCEn) cells were infected with CMV, and the global transcriptional responses were determined by microarray analyses for primary anti-viral responses using network analysis. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and protein array analyses were used to examine whether anti-viral cytokines were induced, i.e., to determine whether innate immune responses were activated. To examine whether priming of acquired immune response was activated, CMV-infected HCEn cells were co-cultured with allogeneic CD8+ T cells from CMV seropositive donors and tested for priming activity for the CD8+ effector T cells by measuring interferon-γ secretion. The CMV-induced responses of HCEn cells were characterized by type I interferon and pattern recognition receptor pathways which represent innate immune priming. The global transcriptional activation was specifically associated with antigen presentation with the antimicrobial response functions. Protein array analyses indicated a significant increase in the secretion of anti-viral inflammatory cytokines including CXCL10 as innate immune responses. When HCEn cells were examined to determine whether CMV infection activated anti-viral acquired immunity, CMV-infected HCEn cells directly stimulated the proliferation of CD8+ T cells from CMV-seropositive donors, and pp65 viral epitope induced interferon-γ secretion from the CD8+ T cells. We conclude that CMV-infected HCEn cells induce innate immune priming along with provisions of acquired immune priming of CD8+ effector T cells. This information should help in the development of useful diagnostic procedures and efficacious therapeutic strategy to treat refractory corneal endotheliitis.

Subtenon triamcinolone injection for postoperative cystoid macular edema associated with tafluprost.

PURPOSE: We report the successful treatment of a case of cystoid macular edema (CME) associated with topical tafluprost, which was accompanied by serous retinal detachment (SRD). CASE: A 78-year-old woman underwent intraocular lens suture surgery, including anterior vitreous cutting, for crystalline lens dislocation in the right eye. Tafluprost was initiated 12 weeks after surgery. Intraocular pressure (IOP) was controlled at 10-14 mmHg. Visual acuity remained at 20/40-30/40. However, the patient complained of blurred vision (20/200) 9 months after surgery. CME accompanied by SRD was identified by optical coherence tomography (OCT) and treated with subtenon triamcinolone injection. Visual acuity rapidly increased to 20/50, and the volume of SRD decreased in a few days. Discontinuation of tafluprost and initiation of diclofenac eye drops improved visual acuity to 20/40 and resulted in improved OCT findings within a few weeks. Three months after injection, tafluprost was resumed along with diclofenac. No recurrence of CME occurred over the following 3 months, and IOP was controlled at 10-15 mmHg. CONCLUSION: SRD is considered to be a symptom of treatment-resistant CME, which may lead to poor visual acuity after recovery. In such cases, subtenon triamcinolone injection should be strongly considered at an early stage.

[Efficacy of Gram-Fungiflora Y double staining in diagnosing infectious keratitis].

PURPOSE: To evaluate the efficacy of Gram-Fungiflora Y double staining for corneal scraping samples collected by impression in diagnosing infectious keratitis. SUBJECTS AND METHODS: Two hundred and thirteen eyes of 192 patients suspected of having infectious keratitis were retrospectively studied for the sensitivity and specificity of Gram-Fungiflora Y based on final diagnosis. RESULTS: Of 165 infectious keratitis eyes, 107 had bacterial keratitis, 54 had fungal keratitis, and 15 had Acanthamoeba keratitis. Of these, 147 eyes were positive for one or other of the pathogens by the double staining method (overall sensitivity/specificity was 89.1% (95% confidence interval 83.1%-93.2%)/ 79.1% (64.6%-89.0%)). Sensitivity/specificity of the double staining for each of the pathogens was 88.8% 82.1% for bacterial keratitis, 81.4%/98.1% for fungal keratitis and 80.0%/97.0% for Acanthamoeba keratitis. CONCLUSION: The double staining method for impression specimens was effective in diagnosing infectious keratitis with high sensitivity, and was especially useful for the diagnosis of fungal or Acanthamoeba keratitis.

Successful treatment of prostaglandin-induced cystoid macular edema with subtenon triamcinolone.

PURPOSE: Cystoid macular edema (CME) is associated with the use of prostaglandin eye drops after cataract surgery. The study aim was to report on the treatment of two CME patients with subtenon triamcinolone injections. METHODS: Case one was a 70-year-old woman who underwent uneventful cataract surgery. Travoprost was administrated at 10 weeks after surgery, and CME occurred at 13 weeks after surgery. Case two was an 85-year-old man who underwent intraocular lens extraction and an intraocular lens suture operation. Latanoprost was readministered after the surgery. The patient complained of blurred vision 3 years later. RESULTS: For both cases, discontinuation of the prostaglandin eye drops, subsequent initiation of treatment with diclofenac eye drops, and subtenon injection of triamcinolone was followed by the rapid disappearance of the CME. After the CME had disappeared for 3 months, the prostaglandin eye drops were resumed. Diclofenac was added in case two. No CME recurrence was seen in either case over 2 years. CONCLUSION: Subtenon triamcinolone injection can be effective for CME associated with prostaglandin eye liquid after cataract surgery. Prostaglandin eye liquid that causes CME is not necessarily precluded for future use if administered under careful observation.