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.

Determination of probability of causative pathogen in infectious keratitis using deep learning algorithm of slit-lamp images.

Corneal opacities are important causes of blindness, and their major etiology is infectious keratitis. Slit-lamp examinations are commonly used to determine the causative pathogen; however, their diagnostic accuracy is low even for experienced ophthalmologists. To characterize the "face" of an infected cornea, we have adapted a deep learning architecture used for facial recognition and applied it to determine a probability score for a specific pathogen causing keratitis. To record the diverse features and mitigate the uncertainty, batches of probability scores of 4 serial images taken from many angles or fluorescence staining were learned for score and decision level fusion using a gradient boosting decision tree. A total of 4306 slit-lamp images including 312 images obtained by internet publications on keratitis by bacteria, fungi, acanthamoeba, and herpes simplex virus (HSV) were studied. The created algorithm had a high overall accuracy of diagnosis, e.g., the accuracy/area under the curve for acanthamoeba was 97.9%/0.995, bacteria was 90.7%/0.963, fungi was 95.0%/0.975, and HSV was 92.3%/0.946, by group K-fold validation, and it was robust to even the low resolution web images. We suggest that our hybrid deep learning-based algorithm be used as a simple and accurate method for computer-assisted diagnosis of infectious keratitis.

Efficacy of compartmentalization in controlling an adenovirus type 54 keratoconjunctivitis outbreak on Oki Island, Japan.

PURPOSE: To analyze the epidemiologic characteristics of an outbreak of human adenovirus type 54 (HAdV-54) on Oki Island, Shimane Prefecture, Japan, in 2017 and to assess the effectiveness of a compartmentalization method in controlling the incidence and spread. STUDY DESIGN: Retrospective cohort study. METHODS: The infection was diagnosed in 136 individuals, and typing was confirmed by PCR and direct sequencing. The epidemiologic characteristics of the disease including the infection rate, incubation period, and basic reproductive number (R0), ie, number of cases directly infected by an infectious patient during the course of the disease, were investigated. The effectiveness of compartmentalization for infection control was determined by simulating the outbreak using the Susceptible-Exposed-Infectious-Recovered (SEIR) model. RESULTS: The majority of the HAdV-54-infected individuals were the children of 3 nursery schools (A, B, and C) and their parents on Oki Island. The infection rates in the 3 schools were 13.2%, 16.9%, and 17.2%, respectively. The one class of school B without the index case was initially compartmentalized, and the infection rate in this compartment was 0%. The incubation period was calculated to be 9.3 ± 3.5 days, and the disease duration, 13.0 ± 5.4 days. The R0 was 1.43. Using these parameters, a SEIR model was constructed. The SEIR model well predicted the daily incidence of infection and indicated that the compartmentalization method provides effective reduction in the incidence of the infection, with much earlier control. CONCLUSIONS: The compartmentalization method is effective to control HAdV-54 outbreaks.

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.

Conjunctival bacterial flora and antimicrobial susceptibility in bacterial pathogens isolated prior to cataract surgery.

PURPOSE: To optimize prophylactic antibiotic administration, antibiotic susceptibility before cataract surgery was investigated using ocular bacteria isolated preoperatively. DESIGN: Retrospective cross-sectional study. METHODS: In 204 eyes of 102 patients who underwent routine bilateral cataract surgery, conjunctival sac scrapings were collected 1-2 weeks before surgery. A total of 192 major pathogens among the 470 isolated bacteria were subjected to susceptibility testing. The major pathogens included Staphylococcus aureus, Staphylococcus epidermidis, coagulase-negative staphylococci (CNS) other than S. epidermidis, Enterococcus faecalis, and Streptococcus spp. The following antibiotics were tested: cefmenoxime (CMX), ceftazidime (CAZ), tobramycin (TOB), vancomycin (VAN), erythromycin (EM), moxifloxacin (MFLX), gatifloxacin (GFLX), levofloxacin (LVFX), chloramphenicol (CP), and imipenem (IP). RESULTS: The proportions of isolates with minimum inhibitory concentration (MIC) of S. epidermidis (N = 82), exceeding 4 µg/ml were high for CAZ (95.1%), EM (32.9%), LVFX (39.0%), and CP (82.9%). Susceptible (S) proportion was high for CMX (98.8%), VAN (100%), CP (93.9%), and IP (97.6%) but relatively low for MFLX (59.8%), GFLX (54.9%), and LVFX (54.9%). The MIC90 values were high for CMX (16 µg/ml), CAZ (64 µg/ml), TOB (32 µg/ml), EM (128 µg/ml), LVFX (16 µg/ml), and CP (8 µg/ml). The MIC of quinolonesof pathogenic bacteria other than S.epidermidis (N = 108), exceeded 4 µg/ml for 11 isolates, including two Methicillin-resistant Staphylococcus aureus. CONCLUSIONS: The increase in resistance of resident bacteria present in the conjunctival sac to antibiotics indicates that systemic and topical antibiotics are no longer effective, especially against external organisms affecting the eye.

Genomic analysis of antibiotic resistance for Acinetobacter baumannii in a critical care center.

AIM: Acinetobacter baumannii is commonly associated with outbreaks and antibiotic-resistant nosocomial infection. This study aimed to determine the relationship between antibiotic resistance and genotypes of A. baumannii. METHODS: A study was undertaken in the critical care center (CCC) of Juntendo University Urayasu Hospital (Urayasu, Japan) between January 2012 and September 2015. Antimicrobial susceptibility tests were carried out according to the Clinical and Laboratory Standards Institute guidelines. All A. baumannii isolates were verified to carry carbapenemase genes and the ISA ba1 element using polymerase chain reaction. The genetic relationship of all A. baumannii isolates was determined by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: During the study period, 1634 patients were admitted to the CCC. Acinetobacter baumannii was detected in 43 patients (average age, 58 ± 19 years; 67.4% men). Six patients were determined to be extensively drug-resistant A. baumannii and 21 patients determined to be multidrug-resistant A. baumannii. Antimicrobial susceptibility linked genotypes of A. baumannii. Molecular characterization by pulsed-field gel electrophoresis and multilocus sequence typing showed that closely related clones of A. baumannii had spread in the CCC. CONCLUSION: Resistance to antimicrobial drugs was significantly associated with certain A. baumannii genotypic types and molecular types. Thus, we might be able to predict whether the genotype has spread in the CCC or not when the susceptibility is examined, facilitating the appropriate isolation of patients.

Effectiveness of intraoperative iodine in cataract surgery: cleanliness of the surgical field without preoperative topical antibiotics.

PURPOSE: To verify the possibility that preoperative topical antibiotics are not essential as long as iodine disinfection is performed during surgery. STUDY DESIGN: Crossover equivalence trial. PATIENTS AND METHODS: In 204 eyes of 102 patients who underwent routine bilateral cataract surgery, 1 eye was treated with intraoperative iodine, and the other, with preoperative topical antibiotics. For the intraoperative iodine eyes, 5 mL of 0.25% povidone-iodine was applied at 2 stages: (1) just after the placement of the speculum and (2) before intraocular lens (IOL) insertion. For the contralateral eyes, preoperative topical antibiotics were administered 3 days before surgery without intraoperative iodine. Conjunctival samples for culture were obtained at 3 time points: (a) presurgery, (b) beginning of surgery, and (c) postsurgery. Real-time polymerase chain reaction (PCR) samples were obtained at the beginning of surgery and before IOL insertion. Intracameral moxifloxacin was applied in all the cases. RESULTS: The respective positive bacterial culture rates for intraoperative iodine eyes and preoperative topical antibiotics eyes were 95.1% and 98.0% at (a), 7.8% and 5.9% at (b), and 60.8% and 62.7% at (c). A significant difference in the positive bacterial culture rate was not found at any time point. For the intraoperative iodine eyes, the bacterial DNA copy number at (b) was significantly lower than that for the preoperative topical antibiotics eyes. CONCLUSIONS: The cleanliness of the operative field without using topical antibiotics was revealed to be equivalent to that of the conventional method (using preoperative antibiotics without intraoperative iodine) as long as intraoperative iodine was used.

Toxicities of and inflammatory responses to moxifloxacin, cefuroxime, and vancomycin on retinal vascular cells.

Prophylactic intracameral injection of antibiotics is commonly used to prevent endophthalmitis after cataract surgery. However, devastating visual complications have been reported including hemorrhagic occlusive retinal vasculitis (HORV).To determine the toxic and inflammatory effects of moxifloxacin, cefuroxime, and vancomycin on human retinal vascular cells, human retinal vascular endothelial cells (RVEC) and pericytes were exposed to three antibiotics, and the adverse effects were assessed by membrane damage, loss of intrinsic esterase activity, kinetic cell viability, and inflammatory cytokine secretion. Their retinal toxicity was examined by live/dead assays after an intravitreal injection of the three antibiotics into mice eyes. In vascular cells in culture, membrane damage and loss of esterase activity were induced after exposure to the three antibiotics. The toxic effects were most obvious after moxifloxacin (RVEC, ≥125 µg/mL; pericytes, ≥1000 µg/mL) at 24 h. Cefuroxime also reduced esterase activity and the membrane integrity of vascular cells but were less toxic than moxifloxacin. Kinetic cell viability testing showed that 500 µg/mL of moxifloxacin exposure induced significant decrease (29%) in the viability as early as 1 h. When the inflammatory effects of the antibiotics were examined, a significant induction of IL-8 was observed especially by RVECs after exposure to cefuroxime or vancomycin which was exacerbated by L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid (Tri-DAP), a NOD1 ligand. Intravitreal injections in mice showed that cefuroxime and vancomycin caused retinal and vascular toxicity extending to the inner nuclear layers. Collectively, moxifloxacin causes immediate damage to retinal vascular cells in vitro, while cefuroxime and vancomycin induced significant inflammatory effects on vascular endothelial cells and caused retinal toxicity. Surgeons need to be cautious of the toxicity when antibiotics are used prophylactically especially by intravitreal administration.

Lung metastases from cutaneous adenoid cystic carcinoma 23 years after initial treatment.

Cutaneous adenoid cystic carcinoma (CACC) is an extremely rare neoplasm of head and neck region, and is characterized by propensity for local recurrence and perineural invasion. Late distant metastases occur usually to lungs. Although patients with lung metastases from CACC cannot be cured, long-term survival may be possible due to its slow-growing malignancy. We report a case of a 69-year-old female with lung metastases from CACC 23 years after initial surgery of scalp nodule.

Effectiveness of timely intraoperative iodine irrigation during cataract surgery.

PURPOSE: To determine the antiseptic efficacy of timely intraoperative iodine irrigation during cataract surgery. METHODS: A total of 198 eyes of 99 cataract surgery patients were studied. The eyes were randomly assigned to treatment with or without timely intraoperative iodine irrigation of the surgical field with an iodine compound equivalent to 0.33 % povidone-iodine. In eyes in the timely intraoperative iodine irrigation group, the ocular surface was irrigated twice intraoperatively-before the initial incision and before insertion of the intraocular lens (IOL). The efficacy of the antiseptic treatment was evaluated by culture tests using scrapings of the surface of the sclerocornea and conjunctiva to the left of the incision and by broad-range real-time PCR for bacterial 16S ribosomal DNA using scrapings from the right side of the incision. RESULTS: Following intraoperative application of the iodine, bacteria were not detected in cultures of the samples. For the control eyes without timely iodine irrigation, cultures of samples from five and two eyes were positive before the initial incision and before IOL insertion, respectively. The bacterial DNA copy number before the initial incision was 1.7 ± 0.5 × 103, which was significantly lower than that of the control eyes (1.7 ± 0.6 × 104). For both groups of eyes, the bacterial DNA copy number was significantly lower before the IOL insertion depending on the time course. When the antiseptic effect of the iodine irrigation and time course on bacterial DNA copy number was analyzed using generalized mixed linear regression, both were found to be significantly effective. No significant intraoperative epithelial defect was observed. The postoperative corneal endothelial cell count did not differ significantly between the two groups of eyes. CONCLUSIONS: Timely iodine irrigation can serve as a simple and useful adjunctive disinfection step in cataract surgery.

Associations of inflammatory cytokines with choroidal neovascularization in highly myopic eyes.

PURPOSE: To determine the relationships between the levels of intraocular inflammatory cytokines and the clinical characteristics of myopic choroidal neovascularization (mCNV) in eyes with myopic maculopathy. METHODS: One hundred eyes of 100 cases, including 51 mCNV eyes, 14 highly myopic eyes without choroidal neovascularization, and 35 normal subjects, were studied. The intraocular levels of choroidal neovascularization-related cytokines, like vascular endothelial growth factor, MCP-1, IL-8, IL-10, and IL-23, were determined. RESULTS: The levels of vascular endothelial growth factor and IL-8 were significantly higher in eyes with mCNV than in high myopia eyes without mCNV with significant odds ratio of 2.00 and 2.25 per quartile, respectively (P < 0.05). When myopic lesions of patients with mCNV were classified into 3 categories based on the severity, IL-8 and MCP-1 were significantly elevated depending on the presence of maculopathy (P < 0.05). Vascular endothelial growth factor was significantly elevated in eyes of Category 2. An advancement of the maculopathy category was significantly associated with the need for multiple treatment of intravitreal bevacizumab (P < 0.05). In 12 eyes that required multiple intravitreal bevacizumab, the MCP-1 level was significantly elevated. CONCLUSION: The significant associations of mCNV in highly myopic eyes with elevated levels of vascular endothelial growth factor or inflammatory cytokines and maculopathy lesions strongly suggest an involvement of inflammation in the etiology of mCNVs.

Safety of intracameral injection of moxifloxacin using total replacement technique (bag and chamber flushing).

PURPOSE: To assess the safety of intracameral injection of moxifloxacin (MFLX) using the total replacement technique [bag and chamber (BC) flushing]. METHODS: The anterior chamber including the area behind the intraocular lens was irrigated and replaced using BC flushing. In Group A, 36 patients received balanced salt solution irrigation in the right eye and intracameral MFLX (150 µg/mL: 33-fold dilution of 0.5% eye drops) in the left eye. In Group B, 33 patients received intracameral MFLX in the right (500 µg/mL: 10-fold dilution) and left eyes (150 µg/mL: 33-fold dilution). Visual acuity, intraocular pressure, corneal endothelial cell density, corneal thickness, and foveal thickness were recorded before and up to 3 months after surgery. RESULTS: In either group, there were no statistically significant differences between the right and left eyes with respect to visual acuity, intraocular pressure, corneal endothelial cell count, and foveal thickness before and at 3 months after surgery. There was no difference in corneal thickness before and at 3 months after surgery. In patients who received 500 µg/mL MFLX during surgery, central corneal thickness at 1 week after surgery was significantly reduced. CONCLUSIONS: In the present study, there was no adverse effect associated with intracameral injection of 150-500 µg/mL MFLX. Moreover, our results supported the safety of BC flushing. Thus, BC flushing, which can irrigate the inside of the capsular bag and attain stable antibiotic concentrations, appears to be a favorable technique for intracameral injection of MFLX.

Assessment of vitreous drug concentration in the porcine eye following intracameral injection or irrigation with moxifloxacin.

INTRODUCTION: Posterior capsule rupture causes instant vitreous contamination, resulting in endophthalmitis. However, transfer of intracameral moxifloxacin (MFLX) to the vitreous has not been examined in detail. We investigated vitreous antibiotic concentrations following intracameral MFLX in both ruptured and intact posterior capsular eyes. METHODS: Experiment 1: Intraocular lenses were inserted into 21 extracted porcine eyes by one of the following three methods: (1) Irrigation: Throughout surgery, 33-fold diluted MFLX irrigation solution (150 µg/mL) was used; (2) Bag and chamber flushing: After surgery, the anterior chamber and area behind the intraocular lenses were irrigated with 30-fold diluted MFLX (167 µg/mL) using a 5 mL syringe; (3) Simple injection: Tenfold diluted MFLX (50 µg in 0.1 mL) was injected intracamerally at the conclusion of surgery. The eyeballs were frozen and the anterior, central, and posterior portions of the vitreous were cubed. After defrosting, concentrations were measured using high-performance liquid chromatography. Experiment 2: The same procedure was conducted for 18 eyes in which the posterior capsule had been ruptured. RESULTS: Experiment 1: Transfer of intracameral MFLX to the anterior vitreous was approximately 1% (1.56-2.20 µg/mL) regardless of the administration method. Experiment 2: MFLX reached a high concentration in the vitreous with irrigation solution administration (maximum 30.22 µg/mL). The concentrations reached by simple injection or flushing were significantly less than those obtained by irrigation. CONCLUSION: With an intact posterior capsule, intracameral MFLX exhibited limited effects on vitreous concentration. Despite the fact that the risk of infection clearly increases in cases of ruptured capsule, no special infection prevention protocol has been proposed. It was confirmed that irrigation solution caused vitreous contamination in ruptured eyes within only a short irrigation time. In this case, intracameral administration did not necessarily achieve preventive concentrations for endophthalmitis, but it appears that an effective drug concentration can be achieved in the vitreous by the administration of irrigation solution.

Associations of IL-23 with polypoidal choroidal vasculopathy.

PURPOSE: To determine the relationship between the levels of intraocular inflammatory cytokines and polypoidal choroidal vasculopathy (PCV). METHODS: Prospective cohort study. Sixty-two patients with PCV and 36 control subjects were studied. The levels of cytokines, chemokines, and growth factors in the aqueous humor samples from PCV patients and control subjects were assessed for significant associations with PCV. Logistic regression analysis was used to compute the odds ratios (ORs) and 95% confidence intervals (CIs) after the study populations were divided into quartiles. RESULTS: In PCV patients, IL-4, IL-10, and IL-23 were significantly higher than in the controls. Logistic analyses showed a significantly high risk for IL-23 (OR for the highest quartile compared with the lowest quartile: 16.3; 95% CI: 3.5-75.2), VEGF (5.7; 1.2-26.1), and IL-4 (4.0; 1.3-12.7). IL-10 and IL-4, but not IL-23, were significantly correlated with the VEGF levels in PCV patients (IL-10: ρ = 0.477, IL-4: ρ = 0.281). The elevated levels of IL-5, IL-10, IL-4, IL-23, and IL-1α were found to be significantly associated with exudative lesion(s) in the fluorescein angiograms. CONCLUSIONS: The significant associations between elevated levels of IL-23 with PCV and its activity strongly suggest an involvement of inflammatory processes in the etiology of PCV, presumably independent of VEGF. (www.umin.ac.jp/ctr number, UMIN000003854.).

Multivariate analyses of inflammatory cytokines in eyes with branch retinal vein occlusion: relationships to bevacizumab treatment.

PURPOSE: To characterize the differential expression of intraocular inflammatory cytokines in eyes with branch retinal vein occlusion (BRVO) and to assess their roles as prognostic determinants of BRVO. METHODS: A prospective cohort study of 38 eyes with BRVO. Aqueous humor samples were collected just before the intravitreal injection of bevacizumab and were assessed for 18 cytokines, chemokines, and growth factors. For control, aqueous humor was collected from 28 eyes before cataract surgery. RESULTS: In the aqueous of eyes with BRVO, the IL-23, IL-8, IL-6, IL-15, IL-12, and IL-17 levels were significantly higher than that in control eyes. Pretreatment visual acuity was significantly correlated with the concentrations of IL-8, IL-10, IL-2, IL-1ß, IL-5, IL-6, IL-23, IL-4, MCP-1, IL-1α, IL-12, IL-13, IFN-γ, and IL-15. The pretreatment nonperfused area (NPA) was significantly correlated with the concentrations of IL-8, IL-2, MCP-1, and IL-6. Logistic regression analyses revealed significant associations between the BRVO and the concentrations of IL-8, IL-23, IL-12, IL-15, IL-10, IL-1ß, and IL-13. IL-8 had the highest odds ratio (OR) and was significantly associated with NPA, central retinal thickness (CRT), and visual acuity. Bevacizumab treatment significantly improved visual acuity and CRT after 1 month. Refractoriness to bevacizumab (defined as CRT recovery 1 month after treatment by <90%) was significantly associated with the IL-12 level. CONCLUSIONS: Of the induced cytokines in eyes with BRVO, IL-8 was the most significantly associated with the disease parameters of BRVO. IL-12 is most likely a factor that blocks the effect of bevacizumab treatment. (www.umin.ac.jp/ctr number, UMIN000003854.).

Prostatic androgen-repressed message-1 as a regulator of adipocyte differentiation in the mouse.

Adipocyte differentiation is an important aspect in energy homeostasis. Although the regulation of adipocyte differentiation is relatively well understood, the underlying molecular mechanism remains unclear. In this study, subcutaneous and epididymal adipose tissues were used to study the differential expression of associated genes. We found that the expression level of mouse homologue of rat prostatic androgen-repressed message-1 (mPARM-1) gene was higher in subcutaneous, perirenal and mesenteric adipose tissues than in epididymal adipose tissue. In mouse subcutaneous, perirenal, and mesenteric adipose tissues, the expression level of this gene was higher in adipocytes than in non-adipocyte cells, i.e. stromal-vascular cells. Furthermore, mPARM-1 mRNA expression was up-regulated in subcutaneous, mesenteric, and epididymal adipose tissues of mice fed a high-fat diet compared to those fed a normal-fat diet. Expression level of mPARM-1 mRNA increased in the early stage of the chemically induced adipocyte differentiation, preceding the increase in peroxisome proliferator-activated receptor-gamma 2 (PPAR-gamma2) mRNA. Tumor necrosis factor-alpha (TNF-alpha), an inhibitor of adipocyte differentiation, reduced the expression of mPARM-1 mRNA in differentiated 3T3-L1 cells and subsequently down-regulated the expression of adipogenic genes, including PPAR-gamma2, leptin and adipogenin. Moreover, knockdown of mPARM-1 expression with siRNA reduced lipid accumulation and the expression levels of PPAR-gamma2 and adipocyte protein 2 mRNAs, which suggest that the degree of adipocyte differentiation of 3T3-L1 cells has been reduced. These results indicate that mPARM-1 might be involved in the regulation of fat accumulation and adipocyte differentiation.

Blocking mast cell-mediated type I hypersensitivity in experimental allergic conjunctivitis by monocyte chemoattractant protein-1/CCR2.

PURPOSE: To characterize the roles played by monocyte chemoattractant protein-1 and its preferential receptor CCR2 (MCP-1/CCL2) in acute allergic inflammation. METHODS: The direct effects of MCP-1 were evaluated histologically after a subconjunctival injection of recombinant MCP-1 into naïve mice. The mice were sensitized to ragweed pollen, and allergic conjunctivitis was induced by an allergen challenge. The location of the induced MCP-1 was determined by immunohistochemistry. Anti-MCP-1 antibody and CCR2-specific antagonist, RS 504393, were used to determine whether an inhibition of MCP-1 or CCR2 signals would suppress the allergen-induced immediate hypersensitivity reaction. The effect of blocking CCR2 was tested in vitro with isolated mast cells from connective tissue, to evaluate the co-stimulatory signals mediated by CCR2 in mast cells directly. RESULTS: A subconjunctival injection of MCP-1 stimulated conjunctival mast cell degranulation and recruited monocytes/macrophages. In the allergic conjunctivitis model, the allergen-induced MCP-1 protein was located in the monocytes/macrophages in the substantia propria of the conjunctiva. Blocking MCP-1 significantly suppressed the allergen-induced clinical signs and mast cell degranulation without affecting the allergen-specific IgE, or the release of Th2 cytokine from the isolated draining lymph node cells. Inhibition of CCR2 similarly suppressed the acute inflammatory responses. Consistent with the outcome of the disease model, inhibition of CCR2 suppressed allergen-specific degranulation of IgE-primed, isolated conjunctival mast cells. CONCLUSIONS: Stimulation of the co-stimulatory axis of CCR2 by MCP-1 is essentially required for mast cell-mediated hypersensitivity reactions in mouse eyes.