Recharacterization of superior limbic keratoconjunctivitis via a subdividing grading method in 236 Chinese patients.

PURPOSE: Superior limbic keratoconjunctivitis (SLK) is an uncommon and often overlooked chronic ocular surface disease. This retrospective consecutive case series study on Chinese patients aimed to characterize the features of this disease, including those undescribed in previous literature. METHODS: Two hundred thirty-six patients diagnosed with SLK were enrolled into this consecutive case study from 2016 to 2019. The demographics, symptoms, Ocular Surface Disease Index, and ocular signs were collected and analyzed. A scoring system (SLK scale index, SSI) that integrated five major sign scores was applied to evaluate SLK severity. RESULTS: Of the 236 SLK patients, dryness was the most common complaint (59.3%). Of 459 SLK eyes, superior limbus/conjunctival staining (SCS) was present in 98% eyes, followed by the superior tarsal conjunctival alterations (85.2%) and superior bulbar conjunctiva hyperemia (80.8%). Approximately 63% of eyes were accompanied by corneal staining. Superior bulbar conjunctivochalasis was a relatively rare sign (41.6%). Among the five major signs, only the prevalence of SCS gradually increased with its severity. In addition, fluorescein staining at the inferior limbus and adjacent conjunctiva (ICS) was found positive in 163 eyes of 84 patients (36%) who had significantly higher SSI than those without ICS (p = 0.013). CONCLUSIONS: We found that SCS is the most common out of the 5 typical signs of SLK. ICS, a new sign, occurred in one-third of patients. SCS, a simple marker of SLK, as well as SSI, an integrated evaluation system, had the advantage of evaluating the severity and objectively characterizing SLK in clinical practice.

Efficacy of sodium polyanethol sulfonate on herpes simplex virus-1 infection in vitro.

Objective: To investigate the effect of sodium polyanethol sulfonate (SPS) on herpes simplex virus type 1 (HSV-1) infection in vitro. Methods: Human corneal epithelial (HCE-T) cells and Vero cells were infected with HSV-1 [HSV-1 f strain, HSV-1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV-1g]. SPS was added to the culture medium at various concentrations in time-of-addition assay. Experiments including photography of fluorescence in HSV-1g or plaque formation by HSV-1f, western blot assays, real-time RT-PCR assays, cytopathic effect inhibition assays, cytotoxicity assays, and viral absorption and penetration assays were performed to explore the antiviral effect and mechanism of the compounds. Results: We identified that SPS reduced the replication of HSV-1 in HCE-T and Vero cells in a dose-dependent manner. HSV-1g fluorescence was reduced by 66.3% and 65.4% in HCE-T and Vero cells, respectively, after treatment with 0.4 µg/ml SPS. Furthermore, the viral fluorescence intensities were inhibited by SPS in a dose-dependent manner when the viruses or cells were preincubated with SPS. Relative levels of the ICP4 protein and VP16 mRNA were decreased by SPS in a dose-dependent manner. Moreover, the IC50 values of SPS for HSV-1g and HSV-1f in HCE-T cells were 0.69±0.09 µg/ml and 1.63±0.44 µg/ml, respectively. Even 10,000 µg/ml SPS had no obvious cytotoxicity toward HCE-T and Vero cells. Importantly, viral absorption and penetration assays showed that the relative fluorescence intensity of HSV-1g was significantly reduced by SPS in a dose-dependent manner in the absorption test, but no change was observed in the penetration test. Conclusions: SPS inhibits HSV-1 replication in HCE-T and Vero cells, indicating that SPS has the potential for treating HSV-1 infection, particularly HSV-1 keratitis.

Anti-viral activity of Staphylococcus aureus lysates against herpes simplex virus type-I infection: an in vitro and in vivo study.

AIM: To investigate the effect of Staphylococcus aureus (S. aures) lysates (SALs) on herpes simplex virus type-I (HSV1) infection in human corneal epithelial (HCE) cells and in a mouse model of HSV1 keratitis. METHODS: HCE, Vero, HeLa, and BV2 cells were infected with HSV1 [HSV1 f strain, HSV1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV1g]. Pre- or post-infection, SAL at various concentrations was added to the culture medium for 24h. GFP fluorescence in HSV1g or plaque formation by HSV1f were examined. The effects of heat-treated SAL, precooled acetone-precipitated SAL, and SAL subjected to ultrafiltration (100 kDa) were evaluated. The effects of other bacterial components and lysates on HSV1 infection were also tested, including lipoteichoic acid (LTA), peptidoglycan (PGN), staphylococcal protein A (SPA), and α-hemolysin from S. aureus (α-toxin) as well as lysates from a wild-type S. aureus strain, S. epidermidis, and Escherichia coli (W-SAL, SEL, and ECL, respectively). In addition, SAL eye drops were applied topically to BALB/c mice with HSV1 keratitis, followed by in vivo observations. RESULTS: The cytopathic effect, plaque formation (HSV1f), and GFP expression (HSV1g) in infected cells were inhibited by SAL in a dose-dependent manner. The active component of SAL (≥100 kDa) was heat-sensitive and retained activity after acetone precipitation. In HSV1g-infected cells, treatment with LTA-sa, α-toxin, PGN-sa, or SPA did not inhibit GFP expression. SAL, W-SAL, and SEL (but not ECL) decreased GFP expression. In mice with HSV1 keratitis, SAL reduced corneal lesions by 71%. CONCLUSION: The results of this study demonstrate that SAL can be used to inhibit HSV1 infection, particularly keratitis. Further studies are needed to determine the active components and mechanism underlying the effects of SAL.

Lipopolysaccharide enhances human herpesvirus 1 replication and IL-6 release in epithelial cells.

OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS) on human herpesvirus 1 (HHV-1) infection in epithelial cells. METHODS: Two strains of HHV-1, HHV-1 F strain (HHV-1f) and HHV-1 strain-H129 with GFP knock-in (HHV-g4), were used to infect HCE-T and VERO cells at MOIs of 0.04 and 0.02, respectively. After 1 h, 0, 10, 50, and 100 µg/ml LPS was added to serum-free medium and the cells were cultured for up to 24 h. GFP fluorescence of HHV-g4 in cells was examined under a fluorescence microscope and imaged. HHV-1f titer was determined by quantitative real-time polymerase chain reaction (qPCR) in HCE-T cells and plaque assays in VERO cells. The expression of the viral ICP4 protein of HHV-1f was detected by Western blot assay. IL-6 and IL-10 levels in culture medium were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Similar changes but at different degrees were found in HCE-T and VERO cells that were infected with HHV-1. GFP fluorescence of HHV-g4 and cell lesions increased in a dose-dependent manner. Virus titer was also enhanced by LPS stimulation in HCE-T and VERO cells. ICP4 expression was promoted at higher LPS concentrations (P = 0.04). In addition, viral infection resulted in increased expression of IL-6 in a dose-dependent manner at 12 and 24 h (P = 0.01), while IL-10 expression was unaffected by either HHV-1 infection or LPS stimulation. CONCLUSION: LPS promotes HHV-1 infection in epithelial cells, which suggests that gram-negative bacteria on ocular surfaces may aggravate HHV-1 infection.