Complement Cascade 8 - Alpha and Calpain-2 in Extracellular Vesicles of Human Vitreous as Biomarkers of Infectious Endophthalmitis.

Purpose: Extracellular vesicles (EVs) are messenger pigeons of the cells that communicate about cellular microenvironment. In this study, we evaluated the expression of C8α and calpain-2 in EVs from vitreous of patients with bacterial endophthalmitis to assess its utility as a diagnostic marker. Methods: EVs were isolated from vitreous of patients with bacterial endophthalmitis (culture positive and culture negative) and noninfectious control by exosome isolation reagent and characterized, and the levels of C8α and calpain-2 was assessed by enzyme-linked immunosorbent assay in isolated EVs and direct vitreous. The receiver operating characteristic curve was generated to assess the diagnostic performance. Results: Scanning electron microscopy (SEM) and dynamic light scattering (DLS) confirmed the presence of EVs having a diameter (nm) of 275.2 ± 93, 92 ± 22, and 77.28 ± 12 in culture-positive (CP), culture-negative (CN), and control respectively. The expression level (ng/mL) of C8α in the EVs obtained from CP was 144 ± 22 and CN was 31.2 ± 9.8, which was significantly higher (P < 0.01) than control 3.7 ± 2.4. Interestingly, C8α is not expressed directly in the vitreous of CN and controls. Calpain-2 was significantly downregulated (P ≤ 0.0001) in CP (0.94 ± 0.16) and CN (0.70 ± 0.14) than control. The sensitivity and specificity of 1 for C8α and calpain-2 in the EVs implied that its diagnostic accuracy was significant. Conclusions: This study showed that the EV proteins C8α and calpain-2 could be suitable diagnostic markers for endophthalmitis. However, the presence of C8α in the EVs of CN samples but not in direct vitreous promises EVs as the future of diagnostics. Translational Relevance: Expression levels of EV-calpain-2 and EV-C8α could diagnose CN bacterial endophthalmitis.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

An X-ray inactivated vaccine against Pseudomonas aeruginosa Keratitis in mice.

Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent pathogens of bacterial keratitis. Bacterial keratitis is a major cause of blindness worldwide. The rising incidence of multidrug resistance of P. aeruginosa precludes treatment with conventional antibiotics. Herein, we evaluated the protective efficiency and explored the possible underlying mechanism of an X-ray inactivated vaccine (XPa) using a murine P. aeruginosa keratitis model. Mice immunized with XPa exhibit reduced corneal bacterial loads and pathology scores. XPa vaccination induced corneal macrophage polarization toward M2, averting an excessive inflammatory reaction. Furthermore, histological observations indicated that XPa vaccination suppressed corneal fibroblast activation and prevented irreversible visual impairment. The potency of XPa against keratitis highlights its potential utility as an effective and promising vaccine candidate for P. aeruginosa.

Assessment of Keratitis Severity Using Quantitative Image Analysis in an In Vivo Murine Model of Staphylococcus aureus Bacterial Keratitis.

Purpose: Bacterial keratitis (BK) severity in murine models has traditionally been measured by subjective clinical grading or quantification of ocular bacterial burden. This investigation explores an objective and repeatable quantification of slit lamp photography (SLP) images to measure BK severity. Methods: BALB/c strain mice underwent three parallel scratches of the right cornea with subsequent inoculation of 107Staphylococcus aureus cells. SLP imaging and clinical severity grading were performed at 48 hours post-infection. Stromal infiltrate (SI) area on SLP images were quantified. Bacterial burden was determined after enucleation and homogenization. Spearman rank correlations (rs) were used to estimate associations between SI area, clinical severity grades, and bacterial burden. Results: BALB/c strain mice (n = 14) were evaluated with an average SI area of 0.92 mm2 (standard deviation, SD = 0.65) and average bacterial burden of 3.16 × 105 colony forming units per milliliter (CFU/mL) (SD = 8.3 × 105). Clinical severity grade positively correlated with SI area (rs = 0.59, p = 0.0276) and bacterial burden (rs = 0.66, p = 0.0106). There was a trend towards positive association between SI area and bacterial burden (rs = 0.51, p = 0.0543). Conclusions: SLP annotation of SI area is correlated with clinical severity and may provide an objective, quantitative, and repeatable assessment of BK disease severity. Translational Relevance: SLP annotation of SI area is a novel quantitative method to evaluate bacterial keratitis severity longitudinally in mouse models which may be a powerful tool to better understand BK pathogenesis and response to treatments.

Outcomes of amoebic, fungal, and bacterial keratitis: A retrospective cohort study.

BACKGROUND: Acanthamoeba keratitis is challenging to treat and thought to result in poor outcomes, but very few comparative studies exist to assess whether ulcers caused by Acanthamoeba are worse than those caused by bacteria or fungus. METHODS: In a retrospective cohort study, all cases of smear- or culture-proven Acanthamoeba keratitis diagnosed from January 2006 to June 2011 at an eye hospital in South India were identified from the microbiology database. Random samples of the same number of cases of bacterial and fungal keratitis, matched by year, were identified from the same database in order to compare outcomes between the three types of organism. The main outcomes were the time until the following events: re-epithelialization, discontinuation of antimicrobials, perforation/keratoplasty, elevated intraocular pressure, and new cataract. RESULTS: The median time until re-epithelialization was 113 days for Acanthamoeba keratitis, 30 days for fungal keratitis, and 25 days for bacterial keratitis, and the median time until discontinuation of antimicrobial therapy was 100 days for Acanthamoeba keratitis, 49 days for fungal keratitis, and 40 days for bacterial keratitis. Compared to the other two organisms, Acanthamoeba ulcers took significantly longer to re-epithelialize (adjusted HR 0.4, 95% CI 0.3 to 0.6 relative to bacterial ulcers and HR 0.3, 95% CI 0.2 to 0.5 relative to fungal ulcers; overall p<0.001) and had significantly longer courses of antimicrobials (adjusted HR 0.3, 95% CI 0.2 to 0.6 relative to bacterial ulcers and HR 0.5, 95%CI 0.3 to 0.8 relative to fungal ulcers; overall p<0.001). No statistically significant difference was observed between the three organisms for the other time-to-event outcomes. CONCLUSIONS: Acanthamoeba keratitis was more difficult to treat and had worse clinical outcomes than bacterial or fungal ulcers, highlighting the lack of adequate treatment regimens for this infection.

Hyperkeratinization and Proinflammatory Cytokine Expression in Meibomian Glands Induced by Staphylococcus aureus.

Purpose: This exploratory study aimed to investigate the morphological and pathological alterations of the meibomian gland (MG) with the Staphylococcus aureus crude extracts (SACEs) treatment. Methods: Mouse MG explants were cultured and differentiated with or without SACEs for 48 hours. Explant's viability and cell death were determined by thiazolyl blue tetrazolium bromide (MTT) assay and TUNEL assay. MG morphology was observed by Hematoxylin and Eosin staining. Lipid droplet production was detected by Nile Red staining and LipidTox immunostaining. The pro-inflammatory cytokines were detected by ELISA. The relative gene and protein expression in MG explants was determined via quantitative RT-PCR, immunostaining, and immunoblotting. The components of the SACEs were analyzed by immunoblotting and silver staining. Results: Our findings demonstrated that the SACEs treatment induced overexpression of keratin 1 (Krt1) in the ducts and acini of MG explants, accompanied by a decrease in viability and an increase in cell death in explants. Furthermore, the SACEs treatment dose-dependently increased the levels of TNF-α, IL-1ß, and IL-6 in MG explants. The SACEs treatment induced activation of the nuclear factor kappa B (NF-κB) and AIM2 (absent in melanoma 2)/ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) inflammasome signaling pathway in explants. Further investigation showed expression of the key adipogenesis-related molecule peroxisome proliferator-activated receptor γ was decreased after SACEs treatment. However, no change was found in the lipid synthesis of MG explants after treatment with the SACEs. Staphylococcal enterotoxins B (SEB) was detected in the SACEs. SEB induced the overexpression of Krt1 and IL-1ß in ducts and acini of MG explants. Conclusions: Our findings confirm that Staphylococcus aureus induced hyperkeratinization and pro-inflammatory cytokines expression in MG explants ducts and acini. These effects might be mediated by SEB. Activation of the NF-κB and AIM2/ASC signaling pathway is involved in this process.

Activation of Conjunctiva-Associated Lymphoid Tissue in Patients With Infectious Keratitis Using In Vivo Confocal Microscopy.

Purpose: We aimed to evaluate activation of conjunctiva-associated lymphoid tissue (CALT) in patients with keratitis using in vivo confocal microscopy (IVCM) and conjunctival impression cytology (CIC). Methods: In addition to anterior segment photography and corneal fluorescein staining, IVCM revealed the palpebral conjunctiva in all subjects, and CIC and immunofluorescence staining were performed. Results: Diffuse lymphoid tissue cell density in the eyes of patients with keratitis was significantly greater compared with healthy volunteers (P < 0.001). Similar trends were found in perifollicular lymphocyte density (P < 0.001), follicular density (P = 0.029), follicular center reflection intensity (P = 0.011), and follicular area (P < 0.001). Immunofluorescence staining showed that the proportions of CD4+ (61.7% ± 8.0% vs. 17.3% ± 10.2%, respectively, P < 0.001) and CD8+ (46.9% ± 10.0% vs. 19.6% ± 11.5%, respectively, P < 0.001) cells in patients with keratitis was greater compared with healthy volunteers. Interestingly, we also observed changes in the contralateral eye in subjects with keratitis. Conclusions: Our research suggests that CALT, as an ocular immune structure, is activated and plays an important role in the pathogenesis of keratitis. This has been overlooked previously. CALT is also active in the contralateral eye of subjects with keratitis.

Oxygen Self-Supplying Nanotherapeutic for Mitigation of Tissue Hypoxia and Enhanced Photodynamic Therapy of Bacterial Keratitis.

Hypoxia, a common characteristic of bacterial infections, is known to be closely associated with the emergence of multidrug-resistant bacteria, which hastens the need to develop advanced microbicides and antibacterial techniques. Photodynamic therapy is a promising strategy to reduce bacterial antibiotic resistance and employs photosensitizers, excitation light sources, and sufficient oxygen to generate toxic reactive oxygen species (ROS). The inherent limitation of PDT is that the generation of ROS is restricted by the hypoxic microenvironment in infection sites. Here, an oxygen self-supplying nanotherapeutic is developed to enhance antibacterial activity against multidrug-resistant bacteria on the basis of fluorinated boron dipyrromethene (BODIPY)-based glycomimetics. The nanotherapeutic not only could capture the bacteria efficiently but also was able to act as an oxygen carrier to relieve the hypoxic microenvironment of bacterial infections, thus achieving enhanced PDT efficacy. In a Pseudomonas aeruginosa infection of a rat cornea, typical administration of the nanotherapeutic decreased the infiltrate and showed a faster healing capacity in comparison with BODIPY-based glycomimetics. Self-supplying oxygen nanotherapeutics that relieve the hypoxic microenvironment and interfere with bacterial colonization have been shown to be a promising candidate for the management of drug-resistant microbial keratitis.

The Role of Elastase in Corneal Epithelial Barrier Dysfunction Caused by Pseudomonas aeruginosa Exoproteins.

Purpose: To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa. Methods: Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1ß levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro. Results: Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ± 2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor. Conclusions: Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.

Intravitreal injection of povidone-iodine for the treatment of vancomycin-resistant Enterococcus faecalis endophthalmitis in rabbit eyes.

The aim of this study was to investigate the efficacy of intravitreal povidone-iodine (PI) in the treatment of vancomycin-resistant Enterococcus faecalis (VRE) endophthalmitis. Fifty New Zealand white rabbits were divided into 5 groups (n = 10 in each group). After the induction of endophthalmitis using VRE (minimum inhibitory concentration [MIC] ≥ 40 µg/mL) in the right eye, Group A, B, C, and D received intravitreal injections of 0.1% PI, 0.3% PI, 0.05% vancomycin, and 0.5% vancomycin, respectively. Eyes in Group E were used as controls. Fundus photography, vitreous culture, electroretinography (ERG), and histologic examinations of the retina were conducted on day 14. A marked improvement in endophthalmitis was observed in Group A, B, C and D, compared to Group E. Fundus photographs showed mild vitreous opacities in Group A and B, and moderate vitreous opacity in Group C. All eyes in Group D had a clear vitreous. In vitreous culture, bacterial growth was found in 6 eyes (100, 200, 200, 400, 500, and 500 colony-forming units) in Group C, but not in Groups A, B, or D. ERG and histological examination also indicated intraocular damage in Group C. Our results show that intravitreal injection of PI, even at low concentrations, was effective for treatment of VRE endophthalmitis, although some vitreous opacity remained. Intravitreal vancomycin injection was also useful to treat resistant strains, if used at a higher concentration within the safety threshold.

Design, synthesis and antiamoebic activity of dysprosium-based nanoparticles using contact lenses as carriers against Acanthamoeba sp.

PURPOSE: Contact lenses have direct contact with the corneal surface and can induce sight-threatening infection of the cornea known as Acanthamoeba keratitis. The objective of this study was to evaluate the dysprosium-based nanoparticles (Dy-based NPs), namely Fe3 O4 -PEG-Dy2 O3 nanocomposites and Dy(OH)3 nanorods, as an active component against Acanthamoeba sp., as well as the possibility of their loading onto contact lenses as the drug administering vehicle to treat Acanthamoeba keratitis (AK). METHODS: The Dy-based NPs were synthesized, and they were loaded onto commercial contact lenses. The loading content of the NPs and their release kinetics was determined based on the absorbance of their colloidal solution before and after soaking the contact lenses. The cytotoxicity of the NPs was evaluated, and the IC50 values of their antiamoebic activity against Acanthamoeba sp. were determined by MTT colorimetric assay, followed by observation on the morphological changes by using light microscopy. The mechanism of action of the Dy-based NPs against Acanthamoeba sp. was evaluated by DNA laddering assays. RESULTS: The loading efficiencies of the Dy-based NPs onto the contact lens were in the range of 30.6-36.1% with respect to their initial concentration (0.5 mg ml-1 ). The Dy NPs were released with the flux approximately 5.5-11 µg cm-2  hr-1 , and the release was completed within 10 hr. The emission of the NPs consistently showed a peak at 575 nm due to Dy3+ ion, offering the possible monitoring and tracking of the NPs. The SEM images indicated the NPs are aggregated on the surface of the contact lenses. The DNA ladder assay suggested that the cells underwent DNA fragmentation, and the cell death was due most probably to necrosis, rather than apoptosis. The cytotoxicity assay of Acanthamoeba sp. suggested that Fe3 O4 -PEG, Fe3 O4 -PEG-Dy2 O3 , Dy(NO3 )3 .6H2 O and Dy(OH)3 NPs have an antiamoebic activity with the IC50 value being 4.5, 5.0, 9.5 and 22.5 µg ml-1 , respectively. CONCLUSIONS: Overall findings in this study suggested that the Dy-based NPs can be considered as active antiamoebic agents and possess the potential as drugs against Acanthamoeba sp. The NPs could be loaded onto the contact lenses; thus, they can be potentially utilized to treat Acanthamoeba keratitis (AK).

Preparation and in vitro/in vivo evaluations of novel ocular micelle formulations of hesperetin with glycyrrhizin as a nanocarrier.

The purpose of this study was to explore the potential of formulating hesperetin into an ophthalmic solution with dipotassium glycyrrhizinate (DG) as a micelle nanocarrier. A DG-based micelle ophthalmic solution encapsulating hesperetin (DG-Hes) was developed and its in vitro/in vivo characterizations were evaluated. The optimal formulation featured a DG/hesperetin (Hes) weight ratio of 12:1 and an encapsulation efficiency of 90.4 ± 1.7%; The optimized DG-Hes was characterized as small uniform spheres with an average micelle size of 70.93 ± 3.41 nm, a polydispersity index of 0.11 ± 0.02, and an electrically negative surface (-36.12 ± 2.79 mV). The DG-Hes ophthalmic solution had good tolerance in rabbit eyes. DG-Hes significantly improved the in vitro passive permeation, ex vivo corneal permeation, and in vivo ocular bioavailability of Hes. DG-Hes showed markedly increases in in vitro antioxidant activity. In vitro antibacterial activity tests revealed a lower minimum inhibitory concentration and lower minimum bactericidal concentration for DG-Hes ophthalmic solution were lower than for free Hes. DG-Hes ophthalmic solution also significantly reduced symptoms of eye infection in the rabbit bacterial keratitis model when compared to a Hes suspension. These results suggest that DG-Hes eye drops may be useful as a new ophthalmic preparation for the treatment of ocular diseases, especially bacterial ophthalmopathy.

A new ophthalmic formulation containing antiseptics and dexpanthenol: In vitro antimicrobial activity and effects on corneal and conjunctival epithelial cells.

Antibiotic resistance is increasing even in ocular pathogens, therefore the interest towards antiseptics in Ophthalmology is growing. The aim of this study was to analyze the in vitro antimicrobial efficacy and the in vitro effects of an ophthalmic formulation containing hexamidine diisethionate 0.05%, polyhexamethylene biguanide (PHMB) 0.0001% disodium edetate (EDTA) 0.01%, dexpanthenol 5% and polyvinyl alcohol 1.25% (Keratosept, Bruschettini, Genova, Italy) on cultured human corneal and conjunctival cells. The in vitro antimicrobial activity was tested on Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mitis. For each microbial strain 10 µL of a 0.5 MacFarland standardized bacterial inoculum were incubated at 25 °C with 100 µL of ophthalmic solution for up to 6 h. After different periods of time, samples were inoculated on blood agar with 5% sheep blood. Moreover, a 0.5 MacFarland bacterial inoculum was seeded in triplicate on Mueller-Hinton Agar or on Mueller-Hinton Fastidious Agar; then a cellulose disc soaked with 50 µL of ophthalmic solution was applied on the surface of agar and plates were incubated for 18 h at 37 °C, in order to evaluate the inhibition of bacterial growth around the disc. Human corneal and conjunctival epithelial cells in vitro were incubated for 5, 10 and 15 min with Keratosept or its components. The cytotoxicity was assessed through the release of cytoplasmic enzyme lactate dehydrogenase (LDH) into the medium immediately after exposure to the drugs; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the metabolic cell activity. Our results show that Keratosept ophthalmic solution gave an average logarithmic (log) reduction of bacterial load of 2.14 ± 0.35 within 6 h of exposure (p-value < 0.05 versus control saline solution). On agar plates, all microbial strains, excluding P. Aeruginosa, showed an inhibition zone of growth around the Keratosept-soaked discs. Keratosept and its components after 5 and 10 min did not show any cytotoxic effect on cultured corneal and conjunctival cells, and only after 15 min a significant reduction of cell viability and an increase of cytotoxicity compared to control (vehicle) was seen; dexpanthenol 5% and polyvinyl alcohol accelerated the wounding of corneal cells in vitro. In conclusion, Keratosept showed good antimicrobial activity on the tested strains; the ophthalmic solution and its components were safe and non-toxic for the corneal and conjunctival epithelial cells for 5 and 10 min at the concentrations analyzed, and dexpanthenol 5% and polyvinyl alcohol promoted the wounding of corneal cells.

Evaluation of ocular involvement in patients with Hansen's disease.

Hansen's disease (HD) belongs to the group of neglected diseases and can cause physical deformities and disabilities, in addition to leading to social discrimination. Ocular involvement in HD is estimated at 70-75% worldwide. About 10-50% suffer from severe ocular symptoms and loss of vision occurs in approximately 5% of cases. Ocular changes may persist or worsen even after patients are considered cured and it is necessary to better understand these conditions in order to determine the need for additional public policies. The objective of this study was to identify the prevalence of ocular involvement in patients with HD at two specialist referral centers for treatment of the disease. A cross-sectional study was conducted with ophthalmological evaluations of patients with HD from June 2017 to June 2018. Diagnostic ocular findings, corrected visual acuity, and refractive error were described. Findings were correlated with patients' clinical and epidemiological variables. A total of 86 patients were evaluated, with a mean age of 50.1 years, predominantly males (59.3%), and with multibacillary HD (92%). The prevalence of ophthalmologic changes was 100% and the most common were dysfunction of the Meibomian glands (89.5%) and dry eye syndrome (81.4%). Cataracts were observed in 22 patients (25.6%), but best corrected visual acuity was normal or near normal in 84 patients (97.7%) and there were no cases of bilateral blindness. Patients with some degree of physical disability had more ophthalmological alterations, involving both the ocular adnexa (p = 0.03) and the ocular globe (p = 0.04). Ocular involvement is common in patients with Hansen's disease, reinforcing the importance of ophthalmologic examination in the evaluation and follow-up of these patients.

Measurement Reliability for Keratitis Morphology.

PURPOSE: To evaluate the reliability of manual annotation when quantifying cornea anatomical and microbial keratitis (MK) morphological features on slit-lamp photography (SLP) images. METHODS: Prospectively enrolled patients with MK underwent SLP at initial encounter at 2 academic eye hospitals. Patients who presented with an epithelial defect (ED) were eligible for analysis. Features, which included ED, corneal limbus (L), pupil (P), stromal infiltrate (SI), white blood cell (WBC) infiltration at the SI edge, and hypopyon (H), were annotated independently by 2 physicians on SLP images. Intraclass correlation coefficients (ICCs) were applied for reliability assessment; dice similarity coefficients (DSCs) were used to investigate the area overlap between readers. RESULTS: Seventy-five MK patients with an ED received SLP. DSCs indicate good to fair annotation overlap between graders (L = 0.97, P = 0.80, ED = 0.94, SI = 0.82, H = 0.82, WBC = 0.83) and between repeat annotations by the same grader (L = 0.97, P = 0.81, ED = 0.94, SI = 0.85, H = 0.84, WBC = 0.82). ICC scores showed good intergrader (L = 0.98, P = 0.78, ED = 1.00, SI = 0.67, H = 0.97, WBC = 0.86) and intragrader (L = 0.99, P = 0.92, ED = 0.99, SI = 0.94, H = 0.99, WBC = 0.92) reliabilities. When reliability statistics were recalculated for annotated SI area in the subset of cases where both graders agreed WBC infiltration was present/absent, intergrader ICC improved to 0.91 and DSC improved to 0.86 and intragrader ICC remained the same, whereas DSC improved to 0.87. CONCLUSIONS: Manual annotation indicates usefulness of area quantification in the evaluation of MK. However, variability is intrinsic to the task. Thus, there is a need for optimization of annotation protocols. Future directions may include using multiple annotators per image or automated annotation software.

Comparison of two methods for obtaining and transporting corneal samples in suspected infectious keratitis.

BACKGROUND AND PURPOSE: The purpose of this study is to compare two alternative methods of collecting and transporting media for the diagnosis of corneal ulcers, as not all clinical settings have conventional culture materials and transport media available. METHODS: In this open-label, prospective, comparative, and randomized study, patients with clinical suspicion of infectious keratitis with high risk of loss of vision had corneal specimens collected using two methods and transport media: Eswab scraping with Amies transport medium and 23-gauge needle scraping in BACTEC Peds broth. The order of each collection method was randomized. The samples were processed by standard methods, comparing the positivity frequencies for both by parametric and nonparametric tests, according to normality criteria. RESULTS: Corneal infiltrates from 40 eyes of 40 patients were analyzed. Culture positivity rate was 50% for Eswab and 35% for 23-gauge needle (P=0.258). The overall growth rate of the two methods combined was not higher than with the swab alone. The results obtained with a swab were not influenced by the collection sequence (P=0.112); however, the positivity rate was significantly higher when the sample taken with the needle was performed first (P=0.046). CONCLUSIONS: The single sample Eswab method of collection and transportation for the diagnosis of high risk corneal ulcers is a valid alternative and can be used in cases in which, for various reasons, there is no access to the full set of traditional culture materials.

Corneal epithelial injury-induced norepinephrine promotes Pseudomonas aeruginosa keratitis.

Tissue injury causes the secretion of stress hormone catecholamine and increases susceptibility to opportunistic infection. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that is a leading cause of microbial keratitis usually associated with ocular injury or contact lens wear. However, the effect of catecholamine on P. aeruginosa induced corneal infection is unknown. Here, we test if norepinephrine (NE) would promote the progression of P. aeruginosa keratitis in mice. Adult C57BL/6 mouse corneas were scarified and then inoculated with P. aeruginosa. The content of NE was elevated in corneas after scarification and inoculation with P. aeruginosa. Then, exogenous NE was applied to the infected corneas at 24 h after inoculation; control eyes were treated with sterile saline. Topical application of NE aggravated the severity of P. aeruginosa keratitis, accompanied with the increase of clinical score, bacterial load, pathological changes, neutrophils infiltration, bacterial virulence factors and proinflammatory factors levels. In order to further verify the role of NE, N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), a neurotoxin selected to deplete NE, was injected subconjunctivally 12 h before scarification. Pre-depletion of local NE by DSP-4 significantly alleviated the severity of corneal infection. Moreover, NE was also confirmed to increase the bacterial growth and the expression of virulence factors gene in vitro. Together, these data showed that increased corneal NE content facilitated the progression of P. aeruginosa keratitis in mice by amplifying host excessive inflammatory response and bacterial virulence. Therefore, targeting NE may provide a potential strategy for the treatment of P. aeruginosa keratitis.

Glycolytic inhibitor 2-deoxyglucose suppresses inflammatory response in innate immune cells and experimental staphylococcal endophthalmitis.

Previously, we have shown that Staphylococcus (S) aureus induces a glycolytic response in retinal residential (microglia) and infiltrated cells (neutrophils and macrophages) during endophthalmitis. In this study, we sought to investigate the physiological role of glycolysis in bacterial endophthalmitis using a glycolytic inhibitor, 2-deoxyglucose (2DG). Our data showed that 2DG treatment attenuated the inflammatory responses of mouse bone marrow-derived macrophages (BMDM) and neutrophils (BMDN) when challenged with either live or heat-killed S. aureus (HKSA). Among the inflammatory mediators, 2DG caused a significant reduction in levels of cytokines (TNF-α, IL-1ß, IL-6) and chemokines (CXCL1 and CXCL2). Western blot analysis of 2DG treated cells showed downregulation of bacterial-induced MEK/ERK pathways. In vivo, intravitreal administration of 2DG both pre- and post-bacterial infection resulted in a significant reduction in intraocular inflammation in C57BL/6 mouse eyes and downregulation of ERK phosphorylation in retinal tissue. Collectively, our study demonstrates that 2DG attenuates inflammatory response in bacterial endophthalmitis and cultured innate immune cells via inhibition of ERK signaling. Thus glycolytic inhibitors in combination with antibiotics could mitigate inflammation-mediated tissue damage in ocular infections.