CD3ε of a pan T cell marker involved in mouse Aspergillus fumigatus keratitis.

AIM: To explore whether CD3ε is involved in the adaptive immunity of Aspergillus fumigatus (A. fumigatus) keratitis in mice and the role of innate and adaptive immunity in it. METHODS: Mice models of A. fumigatus keratitis were established by intra-stromal injection and corneal epithelial scratching. Subconjunctival injections of natamycin, wedelolactone, LOX-1 inhibitor (poly I) or Dectin-1 inhibitor (laminarin) were used to treat mice with A. fumigatus keratitis. Mice were pretreated by intraperitoneal injection of anti-mouse CD3ε. We observed the corneal infection of mice under the slit lamp microscope and made a clinical score. The protein expression of CD3ε and interleukin-10 (IL-10) was determined by Western blotting. RESULTS: With the disease progresses, the degree of corneal opacity and edema augmented. In the intra-stromal injection models, CD3ε protein expression began to increase significantly on the 2nd day. However, in the scraping epithelial method models, CD3ε only began to increase on the 3rd day. After natamycin treatment, the degree of corneal inflammation in mice was significantly attenuated on the 3rd day. After wedelolactone treatment, the severity of keratitis worsened. And the amount of CD3ε protein was also reduced, compared with the control group. By inhibiting LOX-1 and Dectin-1, there was no significant difference in CD3ε production compared with the control group. After inhibiting CD3ε, corneal ulcer area and clinical score increased, and IL-10 expression was downregulated. CONCLUSION: As a pan T cell marker, CD3ε participate in the adaptive immunity of A. fumigatus keratitis in mice. In our mice models, the corneas will enter the adaptive immune stage faster. By regulating IL-10, CD3ε exerts anti-inflammatory and repairs effects in the adaptive immune stage.

CCPG1 involved in corneal Aspergillus fumigatus infection.

AIM: To investigate whether non-canonical autophagy transport receptor cell cycle progression 1 (CCPG1) is involved in the corneal antifungal immune response. METHODS: Human corneal epithelial cells (HCECs) and human myeloid leukemia mononuclear cells (THP-1) macrophages stimulated by Aspergillus fumigatus (A. fumigatus) were used as cell models. The expression of CCPG1 mRNA was detected by qRT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1ß (IL-1ß). The dectin-1 neutralizing antibody was used to detect the association between dectin-1 and CCPG1. Immunofluorescence was used to observe the colocalization of CCPG1 and C-type lectin-like receptor-1 (CLEC-1) in THP-1 macrophages. RESULTS: The expression of CCPG1 started to increase at 4h after infection and increased in a time-dependent manner in HCECs and THP-1 macrophages. With dectin-1 neutralizing antibody pretreatment, the expression of IL-1ß was down-regulated. CCPG1 up-regulation in response to A. fumigatus infection was independent of dectin-1. Immunofluorescence showed the colocalization of CCPG1 and CLEC-1 in THP-1 macrophages. CONCLUSION: As a specific autophagy protein of non-canonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.

Thymol Protects against Aspergillus Fumigatus Keratitis by Inhibiting the LOX-1/IL-1ß Signaling Pathway.

OBJECTIVE: To explore the anti-inflammatory effects and mechanisms of action of thymol in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration of thymol against A. fumigatus was detected. To characterize the anti-inflammatory effects of thymol, mouse corneas and human corneal epithelial cells were pretreated with thymol or dimethyl sulfoxide (DMSO) before infection with A. fumigatus spores. Slit-lamp microscopy, immunohistochemistry, myeloperoxidase detection, quantitative real-time polymerase chain reaction, and Western blotting were used to assess infection. Neutrophil and macrophage recruitment, in addition to the secretion of LOX-1 and IL-1ß, were quantified to evaluate the relative contribution of thymol to the inflammatory response. RESULTS: We confirmed that the growth of A. fumigatus was directly inhibited by thymol. In contrast with the DMSO group, there was a lower degree of inflammation in the mouse corneas of the thymol-pretreated group. This was characterized by significantly lower clinical scores, less inflammatory cell infiltration, and lower expression of LOX-1 and IL-1ß. Similarly, in vitro experiments indicated that the production of LOX-1 and IL-1ß was significantly inhibited after thymol treatment, in contrast with the DMSO-pretreated group. CONCLUSION: Our findings demonstrate that thymol exerted a direct fungistatic activity on A. fumigatus. Furthermore, thymol played a protective role in fungal keratitis by inhibiting LOX-1/IL-1ß signaling pathway and reducing the recruitment of neutrophils and macrophages.

Comparison of clinical outcomes after femtosecond laser in situ keratomileusis in eyes with low or high myopia.

AIM: To compare the clinical results of femtosecond (FS) laser in situ keratomileusis (LASIK) in high myopic patients and low myopic patients. METHODS: This study included 212 myopic eyes undergoing LASIK using a VisuMax 500kHz FS laser. All treated eyes were assigned to one of two groups according to preoperative manifest spherical refraction: low myopia group (A, >-4.0 D) and high myopia group (B, ≤-4.0 D). Uncorrected and corrected distance visual acuity (UDVA, CDVA), refractive errors, and higher-order aberrations (HOAs) were measured preoperatively and 1wk, 1, 3 and 6mo postoperatively. RESULTS: At 6mo of follow-up, 92% and 76% had a UDVA of 20/20 or better in group A and B, respectively (P=0.037) and UDVA was significantly different between two groups (P=0.042). Six and seven percentage lost one line of CDVA in group A and B, respectively (P=0.572) and no eyes in both groups lost more than two lines. Each group had 87% and 76% of treated eyes within ±0.5 D of the intended correction (P=0.186), and 13% and 43% with a change of >0.50 D in spherical equivalent from 1wk to 6mo postoperatively (P=0.005). In terms of postoperative astigmatism, each group had 89.1% and 76.6% within ±0.50 D, respectively and there was significant difference (P=0.006). Group A tends to induce smaller HOAs than group B. CONCLUSION: FS LASIK is effective and safe for correcting high myopia as well as low myopia. However, high myopic eyes showed more postoperative astigmatism and HOAs which affect visual acuity.

High-mobility group box1 as an amplifier of immune response and target for treatment in Aspergillus fumigatus keratitis.

AIM: To determine the roles of high-mobility group box1 (HMGB1) in pro-inflammation, host immune response and its potential target for treatment in Aspergillus fumigatus (A.fumigatus) keratitis. METHODS: Expression of HMGB1 was tested in C57BL/6 normal and infected corneas. Dual immunostaining tested co-expression of HMGB1 with TLR4 or LOX-1. C57BL/6 mice were pretreated with Box A or PBS and then infected. Clinical scores, polymerase chain reaction, ELISA, and MPO assay were used to assess the disease response. Flow cytometry were used to test the effect of Box A on reactive oxygen species (ROS) expression after A.fumigatus stimulation in polymorphonuclear neutrophilic leukocytes (PMN). C57BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation, and MIP-2, IL-1ß, TNF-α, HMGB1 and LOX-1 were measured. Macrophages were pretreated with Box B or Box B combined with Poly(I) (an inhibitor of LOX-1) before stimulating with A.fumigatus, and MIP-2, IL-1ß, TNF-α, LOX-1, p38-MAPK, p-p38-MAPK were measured. RESULTS: HMGB1 levels were elevated in C57BL/6 mice after infection. HMGB1 co-expressed with TLR4, and LOX-1 in infiltrated cells. Box A vs PBS treated C57BL/6 mice had lower clinical scores and down-regulated corneal HMGB1, MIP-2, IL-1ß expression and neutrophil influx. Box B treatment amplified expression of MIP-2, IL-1ß, TNF-α, HMGB1 and LOX-1 that induced by A.fumigatus in macrophage. Compared to the treatment of Box B only, the protein expression of IL-1ß, TNF-α showed inhibition of Box B combined with Poly(I), which also reduced the A.fumigatus-evoked protein level of LOX-1 and phosphorylation level of p38-MAPK. The production of A.fumigatus-stimulated ROS was significantly declined after Box A pretreatment in PMN. CONCLUSION: Blocking HMGB1 reduces the disease response in C57BL/6 mice. HMGB1 can amplify the host immune response through p38-MAPK, and is a target for treatment of A.fumigatus keratitis.

The expression of lacrimal androgen-binding proteins in mice Pseudomonas aeruginosa keratitis.

AIM: To investigate the expression of lacrimal androgen-binding proteins (ABPs) in mice Pseudomonas aeruginosa (P. aeruginosa) keratitis. METHODS: P. aeruginosa mice model from different gender was developed by intra-stromal injection. The expression of lacrimal ABPs in lacrimal gland specimens from P. aeruginosa keratitis mice was detected by the quantitative polymerase chain reaction (qRT-PCR). Corneal virulence was evaluated based on clinical scores. To study the mechanism of lacrimal ABPs' expression, experimental subjects were pre-treated with 4E-BP1 inhibitor, and were used to evaluate the expression levels by qRT-PCR. RESULTS: Compared with control groups, the expression of ABPα, ABPη and ABPζ in lacrimal gland from P. aeruginosa keratitis mice had no meaningful changes, while ABPε and ABPδ were significantly higher at 1d after infection. The expression of ABPδ in lacrimal gland of male mice was higher than female mice, regardless of whether or not P. aeruginosa keratitis occurred. After 4E-BP1 inhibitor subconjunctival injection or lacrimal injection, the expression of ABPδ and ABPε has no significant change compared with the control group. CONCLUSION: ABPδ and ABPε secreted by mice lacrimal gland may involve in the progress of alleviating the severity of corneal damage in P. aeruginosa keratitis. The expression of ABPδ and ABPε upon P. aeruginosa infection is independent of cap-dependent mRNA translation activated by 4E-BP1.

Regulation of lipoxygenase-1 and Dectin-1 on interleukin-10 in mouse Aspergillus fumigatus keratitis.

AIM: To investigate the regulation of lipoxygenase (LOX)-1 and Dectin-1 on interleukin-10 (IL-10) production in mice with Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The corneas of C57BL/6 mice were pretreated with LOX-1 inhibitor Poly(I) or Dectin-1 siRNA separately before the infection of A. fumigatus. Polymerase chain reaction (PCR) and Western blot were used to detect the expression of IL-10. RESULTS: The mRNA and protein expressions of IL-10 were significantly increased in mice with A. fumigatus keratitis. Compared with the group pretreated with sterile water before infection, Poly(I) pretreatment suppressed IL-10 expression significantly. Compared with the group pretreated with scrambled siRNA before infection, Dectin-1 siRNA pretreatment significantly reduced IL-10 expression in response to A. fumigatus infection. CONCLUSION: LOX-1 and Dectin-1 regulate IL-10 production in mouse A. fumigatus keratitis.

Early expression of PTX3 in Aspergillus fumigatus infected rat cornea.

AIM: To investigate the expression of pentraxin 3 (PTX3) in rat corneal epithelium at the early stage of Aspergillus fumigatus (A. fumigatus) infection. METHODS: A total of 50 Wistar rats were randomly divided into control group, Sham group and experimental group (fungal keratitis group, FK group). The right eye was chosen as the experiment one and infected by A. fumigatus. Rats were executed at 8, 16 and 24h after the experimental models being established. Corneal epithelia were collected to assess the expression of PTX3 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. RESULTS: Corneal inflammation scores increased as infection prolonged (P<0.05, P<0.001). PTX3 mRNA expression was low in normal and Sham group rats' corneas. Level of PTX3 mRNA in infected rat cornea was elevated at 8h and peaked at 16h. The difference was significant compared with control group (P<0.001). Western blot analysis also showed a significant increase of PTX3 protein in experimental group at 8h and peaked at 16h (P<0.001). The synchronous expression of control group and experimental group were also in significant difference (P<0.001). CONCLUSION: PTX3 exists in cornea epithelium and is significantly increased after A. fumigatus infection. PTX3 plays an important role in the early stage of cornea innate immunity against A. fumigatus.

Production of interleukin-1ß related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea.

AIM: To elucidate the effect of rapamycin on regulating the production of interleukin (IL)-1ß in Aspergillus fumigatus (A. fumigatus)-induced keratitis and to verify whether the expression of IL-1ß in A. fumigatus keratitis is associated with the mammalian target of rapamycin (mTOR)/Toll-like receptor 4 (TLR4) signaling pathway. METHODS: Fungal keratitis mouse models of susceptible C57BL/6 mice were established using A. fumigatus. The mice were subsequently treated with rapamycin. The protein levels of p-mTOR, TLR4, and IL-1ß in normal and infected corneal tissue were measured by Western blot. The TLR4 and IL-1ß mRNA levels were determined by real-time polymerase chain reaction (PCR). RESULTS: In C57BL/6 mice, rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine, IL-1ß. The expression of TLR4, stimulated by A. fumigatus, was reduced as well when the mTOR signaling pathway was suppressed by rapamycin. CONCLUSION: Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1ß. The inhibitory effect on IL-1ß expression can be associated with the mTOR/TLR4 signaling pathway in A. fumigatus infection in mice.

Mincle in the innate immune response of mice fungal keratitis.

AIM: To investigate how macrophage inducible C-type lectin (Mincle) influences inflammation in mice fungal keratitis induced by Aspergillus fumigatus (A. fumigatus). METHODS: C57BL/6 mice were infected with A. fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody (MincleAb), taking DMSO or IgG as control group respectively. The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score. Mincle expression was assessed using reverse transcription-ploymerase chain reaction (RT-PCR) and immunostaining. The expression of cytokines (IL-1ß, TNF-α and IL-6) chemokines (CXCL-1 and MIP-2) was determined by RT-PCR and ELISA. Neutrophil infiltration was observed by immunostaining. The levels of nitric oxide (NO) generated by corneas were tested by Griess reaction. RESULTS: Mincle mRNA and protein levels were higher in infected corneas than normal corneas of C57BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1ß, TNF-α and IL-6 in infected corneas were significantly increased compared with the control group (P<0.01). Results of the counterpart in corneas pretreated with Mincle neutralizing antibody was decreased consistently (P<0.01). Expression of CXCL1 and MIP-2 mRNA levels were up-regulated in TDB group and down-regulated in MincleAb group (P<0.01), coincide with neutrophil aggregation degree in corneas showed by immunostaining. As for the concentration of NO, it was promoted in TDB group compared with DMSO control group, and decreased in MincleAb group compared with IgG control group. CONCLUSION: Mincle plays a dual role in mice fungal keratitis. It participates in the innate immune system by enhancing inflammation. What's more, Mincle can mediate cytotoxic effects by regulating the formation of NO.

Boxb mediate BALB/c mice corneal inflammation through a TLR4/MyD88-dependent signaling pathway in Aspergillus fumigatus keratitis.

AIM: To investigate whether high-mobility group box 1 (HMGB1) Boxb exacerbates BALB/c mice corneal immune responses and inflammatory through the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)-dependent signaling pathway in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The mice corneas were pretreated with phosphate buffer saline (PBS), Boxb before A. fumigatus infection. The abdominal cavity extracted macrophages were pretreated with PBS, Boxb, TLR4 inhibitor (CLI-095), Dimethyl sulfoxide (DMSO) separately before A. fumigatus hyphae stimulation. HMGB1 was detected in normal and infected mice corneas and macrophages by real-time reverse transcriptase polymerase chain reaction (RT-PCR), the TLR4, MyD88, interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) were detected by Western blot and PCR. RESULTS: In BALB/c mice corneas, the expressions of TLR4, HMGB1, IL-1ß, TNF-α were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, MyD88, IL-1ß, TNF-α compared with PBS control after infection. In BALB/c mice abdominal cavity extracted macrophages, pretreatment with Boxb increased the expressions of TLR4, HMGB1, MyD88, IL-1ß, TNF-α, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, MyD88, IL-1ß, TNF-α. CONCLUSION: In A. fumigatus keratitis, Boxb play a pro-inflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-MyD88 signal pathway.

Role of vasoactive intestinal peptide in Aspergillus fumigatus-infected cornea.

AIM: To investigate the anti-inflammatory role of vasoactive intestinal peptide (VIP) in Aspergillus fumigatus (A. fumigatus) ketatitis. METHODS: Expression of VIP was tested by polymerase chain reaction (PCR) in C57BL/6 and BALB/c normal and A. fumigatus infected corneas. C57BL/6 mice were pretreated with recombinant (r) VIP, while BALB/c mice were pretreated with VIP antagonist, and then infected with A. fumigatus. Clinical score was recorded. Expression of pro- and anti-inflammatory cytokines, toll-like receptor 4 (TLR4), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and neutrophil infiltration were tested by PCR, enzyme-linked immunosorbent assay (ELISA), and myeloperoxidase (MPO) assay. RESULTS: VIP mRNA expression in BALB/c cornea was higher than C57BL/6 cornea at 1 and 3d post infection (p.i.). rVIP treatment of C57BL/6 mice showed alleviated disease and down-regulated expression of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), while IL-10 expression was up-regulated. Neutrophil infiltration and TLR4, IL-17 expression were decreased after rVIP treatment, while LOX-1 expression was up-regulated in C57BL/6. VIP antagonist pretreatment showed increased disease and higher IL-1ß, TNF-α, TLR4, IL-17 and MPO levels, while IL-10 and LOX-1 levels were down-regulated in BALB/c mice. CONCLUSION: rVIP alleviate disease response of C57BL/6 mice. VIP antagonist resulted in worsened disease of BALB/c mice. VIP proposed anti-inflammatory role in A. fumigatus keratitis.

Meta-analysis of the efficacy and safety of combined surgery in the management of eyes with coexisting cataract and open angle glaucoma.

AIM: To conduct a systematic review and quantitative Meta-analysis of the efficacy and safety of combined surgery for the eyes with coexisting cataract and open angle glaucoma. METHODS: We performed a systematic search of the related literature in the Cochrane Library, PubMed, EMBASE, Web of Science databases, CNKI, CBM and Wan Fang databases, with no limitations on language or publication date. The primary efficacy estimate was identified by weighted mean difference of the percentage of intraocular pressure reduction (IOPR%) from baseline to end-point, the percentage of number of glaucoma medications reduction from pre- to post-operation, and the secondary efficacy evaluations were performed by odds ratio (OR) and 95% confidence interval (CI) for complete and qualified success rate. Besides, ORs were applied to assess the tolerability of adverse incidents. Meta-analyses of fixed or random effect models were performed using RevMan software 5.2 to gather the consequences. Heterogeneity was evaluated by Chi2 test and the I2 measure. RESULTS: Ten studies enrolling 3108 patients were included. The combined consequences indicated that both glaucoma and combined cataract and glaucoma surgery significantly decreased IOP. For deep sclerectomy vs deep sclerectomy plus phacoemulsification and canaloplasty vs phaco-canaloplasty, the differences in IOPR% were not all statistically significant while trabeculotomy was detected to gain a quantitatively greater IOPR% compared with trabeculotomy plus phacoemulsification. Furthermore, there was no statistical significance in the complete and qualified success rate, and the rates of adverse incidents for trabeculotomy vs trabeculotomy plus phacoemulsification. CONCLUSION: Compared with trabeculotomy plus phacoemulsification, trabeculectomy alone is more effective in lowering IOP and the number of glaucoma medications, while the two surgeries can not demonstrate statistical differences in the complete success rate, qualified success rate, or incidence of adverse incidents.

Expression of indoleamine 2,3-dioxygenase in a murine model of Aspergillus fumigatus keratitis.

AIM: To observe the presence and expression of indoleamine 2,3-dioxygenase (IDO) during the corneal immunity to Aspergillus fumigatus (A. fumigatus) in the murine models. METHODS: The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A. fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO mRNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO mRNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO mRNA measured by qRT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group. CONCLUSION: IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage.

Expression of S100B during the innate immune of corneal epithelium against fungi invasion.

AIM: To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS: Immortalized human corneal epithelial cells (HCECs) were exposed to inactive Aspergillus fumigatus (A. fumigatus) conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC). RESULTS: Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn't express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05) and continue to rise as time prolonged (P<0.01). In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05) and reached to a peak at 24h (P<0.001). Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION: S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.

Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells.

AIM: To investigate the cytotoxic effect on human corneal epithelial cells (HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose (CMC) and hyaluronic acid (HA). METHODS: HCECs were exposed to 0.5% CMC (Refresh plus(®), Allergan, Irvine, California, USA) and 0.1% and 0.3% HA (Kynex(®), Alcon, Seoul, Korea, and Hyalein mini(®), Santen, Osaka, Japan) for the period of 30min, and 4, 12, and 24h. Methyl thiazolyl tetrazolium (MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase (LDH) leakage assay to assess the cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24h after confluent HCECs were scratch wounded. RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC. CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.

Early expression of mannose-binding lectin 2 during Aspergillus fumigatus infection in human corneal epithelial cells.

AIM: To evaluate the early expression of mannose-binding lectin 2 (MBL2) in human corneal epithelial cells (HCECs) infected by Aspergillus fumigatus (AF). METHODS: HCECs cultured in vitro with AF antigens and sampled at 0, 0.5, 1, 2, 4, 6 and 8h. The expression of MBL2 mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression of MBL2 protein in supernatant fluid was shown by enzyme linked immunosorbent assay (ELISA). MBL2 protein in HCECs was detected by immunocytochemistry at 0 and 24h. RESULTS: MBL2 mRNA and protein are expressed in normal HCECs. The expression of MBL2 mRNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 mRNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of 24h. CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.

Expression of dectin-1 during fungus infection in human corneal epithelial cells.

AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) in human corneal epithelial (HCE) cells infected by fungus. METHODS: A total of 20 cases of healthy donor corneas were group A, and 20 patients (20 eyes) suffered from fungal keratitis (FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus (AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0, 4, 8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h, and dectin-1 protein expression increased after stimulation at 24h. CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.

Characteristics of ocular abnormalities in gout patients.

AIM: To characterize the clinical features of ocular surface in gout patients in coastal area of Shandong Province in China. METHODS: A total of 380 consecutive gout patients were examined from January 2011 to May 2011. According to the course of gout, patients were divided into group A (<5 years), B (5-10 years) and C (>10 years). Group D (control group) was consist of 50 healthy subjects. Eyelids, lateral canthus, medial canthus, palpebral conjunctiva, sclera and cornea, anterior chamber, lens, anterior vitreous were examined by slit lamp to find whether there were deposition of uric acid crystals, ocular vascular tortuosity, redness and subconjunctival hemorrhage. The ophthalmic exams of visual acuity, intraocular pressure, fundus were used to assess any gout-related eye disease. RESULTS: Uric acid crystals were found in 3 patients and the positions of the deposite were in corneal stroma, corneal epithelium and superficial stroma, and sclera respectively. The incidence was 0.79%. Dilatated and tortuous blood vessels in conjunctiva and sclera surface were found in 38 (23.8%), 40 (44.0%), 58 (45.0%), 9 (18.0%) patients in groups A, B, C and D, respectively. The differences between group B and D, group C and D were statistically significant (P<0.01, P<0.01). Transparent vesicles with metal-like reflected light in subconjunctiva were seen in 26 (16.2 %), 29 (31.9%), 41 (31.8%), 2 (4.00%) patients in groups A, B, C and D, respectively. The differences between A and D, B and D, C and D were statistically significant (P<0.05, P<0.01, P<0.01). Subconjunctival hemorrhage was found in all groups, the difference among the four groups showed no statistically significance. CONCLUSION: Gout can cause ocular surface abnormalities, such as tophi deposition, subconjunctival transparent vesicles and hemorrhage, and vascular changes. These features have important clinical significance in early detection of the gout and prevention of eye injury.