The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases

AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P 【0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.

Co-regulation of Dectin-1 and TLR2 in inflammatory response of human corneal epithelial cells induced by Aspergillus fumigates

AIM： To investigate the co-regulation of dendritic cell- associated C-type lectin-1 （Dectin-1）, Toll-like receptor 2 （TLR2）, and relative chemotactic factors in the Telomease- immortalized human corneal epithelial （THCE） cells after exposure to ,Aspergillus fumigatus （Af） hyphae. METHODS： The normal THCE cells were investigated as control. After cultured in vitro with Af hyphae, with or without laminarin and anti-TLR2 antibody for 4, 8, 16 and 24h, THCE cells were harvested. The expression of Dectin-1, TLR2, CXCL1 and CXCL8 mRNA were measured by real-time quantitative polymerase chain reaction at the stimulation of 4, 8 and 16h separately. The protein expression of Dectin-1 and TLR2 were analyzed at 8, 16, and 24h by Western blot. ~ RESULTS： The mRNA CXCL8 increased in THCE expression of CXCL1 and cells after stimulated by Af hyphae. The stimulatory effects on these inflammatory chemokines were shown in a dose-dependent manner and reached the peak at 8h. Af hyphae significantly stimulated the production of Dectin-1 and TLR2 in THCE cells at both mRNA and protein levels. The protein of Dectin-1 and TLR2 gradually increased till 16h. While pretreated with laminarin （a Dectin-1 inhibitor）, the expression of TLR2, CXCL1 and CXCL8 all decreased dramatically at the peak point, interestingly, when pretreated with TLR2 neutralizing antibody, the expression of Dectin -1, CXCL1 and CXCL8 also decreased dramatically at the peak point. CONCLUSION： These findings suggest that Dectin-1 and TLR2 co-regulated with each other after treated with inactive Af hyphae in the THCE cells, and they contribute together to the inflammatory responses by induction of chemokines CXCL1 and CXCL8.

The role of Dectin-1/Raf-1 signal cascade in innate immune of human corneal epithelial cells against Aspergillus fumigatus infection

AIM： To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1（Raf-1） and its role in the innate immune response of human corneal epithelial cells（HCECs） infected by Aspergillus fumigatus.METHODS： HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074（an inhibitor of Raf-1） group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1（Dectin-1）] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS： In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION： Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.

Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

· AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P 【0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P 【0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection

AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR.METHODSImmunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels.RESULTSWe found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01).CONCLUSIONThe VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.

Human β-NGF gene transferred to cat corneal endothelial cells

AIM： To transfect the cat corneal endothelial cells （CECs） with recombinant human β-nerve growth factor gene adeno-associated virus （AAV-β-NGF） and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS： The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco＇s modified Eagle＇s medium （DMEM） to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following： normal CEC control group, CEC-AAV control group and recombinant CEC- AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method （FCM）; cell morphology was observed under inverted phase contrast microscope. RESULTS： The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group （P〉0.05）; there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group （P〈0.05）. MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group （P〉0.05）. FCM result showed that the percentage of Glcells in CEC- AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%. CONCLUSION： Recombinant AAV-β-NGF promotes proliferation in cat CECs by expressing bioactive β-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.

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 m RNA 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 24 h.RESULTS: MBL2 m RNA and protein are expressed in normal HCECs. The expression of MBL2 m RNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 m RNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of24 h.· CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.

Epidemiological characteristics and risk factors of diabetic retinopathy in type 2 diabetes mellitus in Shandong Peninsula of China

AIM: To determine the epidemiological characteristics and estimate the risk factors of diabetic retinopathy (DR) in patients with type 2 diabetes mellitus (T2DM) in Shandong Peninsula of China. METHODS: The cases of T2DM admitted to Affiliated Hospital of Medical College of Qingdao University, Shandong Province, China, from January 2006 to December 2010 were retrospectively reviewed. The epidemiological characteristics of DR were estimated. The cases were divided into two groups according to degrees of retinopathy: non-DR group and DR group. Logistic regression analysis was used to study the related risk factors of DR. RESULTS: The prevalence of DR in patients with T2DM was 25.08% (834/3326). There was significant difference between the average age for men (59.08 +/- 15.43 years) and for women (62.92 +/- 18.19 years, P=0.0021). The majority of DR occurred in women (female: male ratio=1.76:1, P<0.0001). The incidence rate of DR in urban (489/834) was higher than that in rural area (345/834, P<0.0001). In 834 DR patients, the mean duration of T2DM was 8.90 +/- 4.15 years (range: 0-16 years); 440 people (52.76%) had received varying degrees of health education about prevention and primary care of DM; and 473 people (56.71%) suffered from other DM complications confirmed at the same time. In addition, the incidence rate of monocular (551/3326) and binocular retinopathy (283/3326) were statistically different (P<0.0001). Factors associated (p<0.05) with the presence of DR included old age, lower health educational level, intraocular surgery history, longer duration of T2DM, accompanying with other DM complications, no standard treatment procedure, lower body mass index (BMI) and higher fasting plasma glucose (FPG), glycated hemoglobin A(1)C (HbA(1)C), urine albumin (UA), total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C). The risk factors (P<0.05) independently associated with the presence of DR were: longer duration of T2DM, lower health educational level, higher FPG, higher UA, lower BMI and higher IC. CONCLUSION: DR is highly prevalent in the patients with T2DM in Shandong Peninsula of China. Besides blood glucose, many factors are associated with the present and development of DR.

Effects of COX-2 inhibitor NS-398 on IL-10 expression in rat fungal keratitis

AIM: To investigate the expression of interleukin-10 (IL-10) and the effect of NS-398 (COX-2 inhibitor) on the expression of IL-10 in fungal keratitis in rats, and analyze its effects on anti-fungus immunity. METHODS: Ninety Wister rats were randomly divided into 3 groups. Group A was blank control group (10 eyes). Group B was fungal keratitis group (40 eyes). Group C was fungal keratitis group treated with NS-398 (40 eyes). PAS staining, 100g/L potassium hydroxide (KOH) smear and fungal culture confirmed the successful establishment of fungal keratitis model. After the central epithelium was scraped, Fusarium solani colonies were applied and contact lens was put on the right cornea of group B and C, and plane contact lens was put on the left cornea of control eyes. Phosphate buffered saline (PBS) eyedrops were given for group B and NS-398 eyedrops for group C. The expression of IL-10 on corneas of group B and C on the 1(st) day, 3(rd) days, 75(th) days, and 14(th) days were detected by immunohistochemistry and semi- quantitative reverse transcription- polymerase chain reaction (RT-PCR). RESULTS: Histopathologic examination showed neutrophil infiltration and severe tissue necrosis in ulcer cornea. PAS staining confirmed the existence of hyphae and spores in the superficial layer of stroma. In the blank and control groups almost no expression of IL-10 was detected at any observing points. In group B the expression of IL-10 increased at first and decreased thereafter. Its expression also showed significant difference at any observing points (P < 0.01). Compared with group B, the expression of IL-10 in group C showed no difference on the 1(st)day, decrease on the 3(rd) day, but a significant increase on the 7(th) day and 14(th) day. CONCLUSION: IL-10 takes part in the occurrence and development of fungal keratitis. NS-398 can upgrade the expression of IL-10 in fungal keratitis in the later period of the ulcer. Meanwhile, pathologic observation showed a slightly corneal opacity. IL-10 may play an important role in the process of cornea anti-damage repair.

Natamycin in the treatment of fungal keratitis : a systematic review and Meta-analysis

AIM: To review published clinical studies examining the effect of natamycin in the treatment of fungal keratitis.METHODS: We selected the publications in CENTRAL,MEDLINE, EMBASE, CNKI, and CBM. This study systematically reviewed published randomized controlled trials(RCTs) that compared natamycin to other antifungal agents, and conducted feasible Meta-analysis of efficacy results using Revman 5.2 software.RESULTS: We included seven trials which were mainly carried out in developing countries of Asia, with five trials conducted in India, one each in China and Bangladesh. A total of 804 participants were randomized to following comparisons: 2% econazole versus 5%natamycin showed little difference in the effects of treatment of fungal keratitis [RR =0.99, 95% confidence interval(CI), 0.8 to 1.21]; chlorhexidine gluconate versus5% natamycin indicated that the results on healing of the ulcer at 21 d was less conclusive(RR=0.77, 95% CI, 0.55 to 1.08; I2=0%); 1% voriconazole versus 5% natamycin suggested that natamycin treatment appeared to be significantly better outcomes than voriconazole(regression coefficient =-0.18 log MAR; 95% CI,-0.30 to-0.05; P =0.006), especially in Fusarium cases(regression coefficient=-0.41 log MAR; 95% CI,-0.61 to-0.20; P 【0.001);natamycin versus fluconazole showed a significant difference in cure rate(χ2=5.048, P 【0.05) and natamycin group was more effective than fluconazole in average period of therapy(t =7.94, P 【0.01).CONCLUSION: Natamycin was a preferable choice in the treatment of fungal keratitis, especially in the early period of Fusarium cases.

Effect of corneal graft diameter on therapeutic penetrating keratoplasty for fungal keratitis

AIM: To evaluate the effect of corneal graft diameter on therapeutic penetrating keratoplasty(PKP) for fungal keratitis. METHODS: A total of 116 patients (116 eyes) suffered from fungal keratitis underwent PKP at the Affiliated Hospital of Medical College Qingdao University from May 2006 to May 2010. They were divided into two groups according to the corneal graft diameter. 64 eyes' corneal graft diameter was 8.00mm or larger and 52 eyes' graft diameter was smaller than 8.00mm. The follow-up time was 2 years. The postoperative visual acuity and complications were documented and compared. RESULTS: Sixty-two (96.88%) eyes and fifty (96.15%) eyes preserved eyeballs respectively in two groups. There was no statistical difference in postoperative visual acuity (P = 0.961), corneal graft dear rate (P=0.132) or the incidence of recurred fungal infection (P=0.770) between two groups. But there was a higher incidence of graft rejection (P=0.020) and secondary glaucoma (P=0.039) in group with corneal graft diameter 8.00mm or larger. CONCLUSION: PKP is an effective treatment approach for fungal keratitis. There is a higher incidence of complications in large-diameter PKP for fungal keratitis.Effective, preventive and therapeutic measures can improve the prognosis.

The role of LOX-1 on innate immunity against Aspergillus keratitis in mice

AIM：To explore the effects of lectin-like ox-LDL receptor（LOX-1） on innate immunity against Aspergillus fumigatus（A.fumigatus） in mice cornea.METHODS：The mRNA levels of LOX-1 were tested in normal and A.fumigatus infected corneas of C57BL/6and BALB/c mice.The expression of LOX-1,pro-inflammatory cytokines TNF-α,CXCL1 and IL-6,antiinflammatory cytokines IL-10,and matrix metalloproteinase 9（MMP9） were tested with treatment with LOX-1 neutralizing antibody or control IgG in A.fum/gatus infected corneas of C57BL/6.Macrophages and neutrophils were extracted from susceptible C57BL/6mice,and pretreated with LOX-1 neutralizing antibody or IgG,then stimulated with A.fum/gatus.The mRNA levels of LOX-1,TNF-α,CXCL1,IL-6,IL-10 and MMP9 were evaluated by polymerase chain reaction.RESULTS：The expression of LOX-1 was significantly increased in C57BL/6 mice corneas after A.fum/gatus infection compared with BABL/c mice.After treatment with LOX-1 neutralizing antibody,the expression of LOX-1,TNF-α,CXCL1,IL-6,MMP9 and IL-10 in C57BL/6corneas were significantly decreased compared with treatment with control IgG;the expression of LOX-1,CXCL1,IL-6 and IL-10 were significantly decreased in macrophages,while TNF-α and MMP9 expressions had no change;LOX-1,TNF-α,CXCL1,IL-6,MMP9 and IL-10 expressions were significantly decreased in neutrophils.CONCLUSION：The expression of LOX-1 can affect the expression of pro-inflammatory and anti-inflammatory cytokines in fungal infected corneas,macrophages and neutrophils of C57BL/6.LOX-1 inhibition rebalances the inflammatory response of fungal keratitis in mice.

Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway

AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for &#x003b1;-smooth muscle actin (&#x003b1;-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay.RESULTSThe mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. &#x003b1;-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, &#x003b1;-SMA expression and cell migration.CONCLUSIONPTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

Nucleotide oligomerization domain 2 contributes to the innate immune response in THCE cells stimulated by Aspergillus fumigatus conidia

AIM: To investigate the expression of nucleotide oligomerization domain 2 (NOD2) in the immortalized human corneal epithelial cell line (THCE), and its role in the innate immune response triggered by inactive Aspergillus fumigatus (Af) conidia. METHODS: The normal THCE cells were investigated as controls. After incubation with inactive Af conidia for 0.5, 2, 4, 6, and 8 hours, THCE cells were harvested, mRNA expression of NOD2 and receptor interacting protein 2 (RIP2) was detected by RT-PCR. Intracellular proteins including NOD2, NF-kappa B and proinflammatory cytokines such as TNF-alpha, IL-8, IL-6 in the cell supernatant were analyzed by ELISA. RESULTS: Our data indicate that NOD2 expressed in the normal THCE cells. After triggered by the inactive Af conidia, the expression of NOD2, RIP2 mRNA and the secretion of NOD2, NF-kappa B, TNF-alpha, IL-8, IL-6 both increased in a time-depended manner, and reached the peak point at 4, 6, 6, 4, 6, 6, 4 hours, respectively. And after pretreated with NOD2 neutralizing antibody, the expression of RIP2, NF-kappa B, TNF-alpha, IL-8 both decreased dramatically at the peak point, while the secretion of IL-6 changed little. CONCLUSION: The results of this study suggest that NOD2 exists and expresses in the THCE cells, and contributes to the innate immune responses triggered by inactive Afconidia by induction of proinflammatory cytokines such as TNF-alpha and IL-8 through the NF-kappa B pathway.

Expression of macrophage migration inhibitory factor in Aspergillus fumigatus keratitis

AIM: To investigate the expression of macrophage migration inhibitory factor(MIF) and detect its role in the innate immune response of fungal keratitis(FK). METHODS: We collected the paraffin-embedded cornea tissues from 10 FK and 6 ocular trauma patients to explore the MIF expression by immunohistochemistry. Then we cultured telomease-immortalized human corneal epithelial cells(THCEs), stimulated by the hyphae suspension of Aspergillus fumigatus(A. fumigatus) to detect the change of MIF with or without the pretreatment of MIF inhibitor [4-Iodo-6-phenylpyrimidine(4-IPP)] by real-time polymerase chain reaction(PCR). The protein level of MIF was also tested by immunohistochemistry, and the level of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) mRNA were compared between normal, hyphae stimulated and 4-IPP pretreated groups by real-time PCR to study the influence of MIF on the expression of TNF-α and IL-6. Corneal severity of rats' FK models was documented by clinical scores, and real-time PCR. Western blot and immunohistochemistry were used to test the expression of MIF, TNF-α and IL-6 in rats' corneas.RESULTS: In the corneas of FK patients, there was much stronger expression of MIF than that in the normal group showed by immunohistochemistry. In cultured THCEs stimulated by A. fumigatus, the expression of MIF became stronger in both immunohistochemistry and PCR at 16, 24, 32 and 48 h post infection(p.i.; P<0.01, P<0.01, P<0.01, P<0.05). After pretreated with 4-IPP, the expression of MIF reduced at 4, 8, 16 h p.i.(P<0.05, P<0.05, P<0.05) and the downstream TNF-α and IL-6 decreased obviously(P<0.05, P<0.01). In rats with A. fumigatus keratitis, the relative mRNA and protein level of MIF increased than thosein the normal group by PCR(at 1 d: P<0.01, 3 d: P<0.01, 5 d: P<0.01), Western blot and immunohistochemistry. After blocked MIF with 4-IPP, the clinical outcomes of rat keratitis showed markedly reduced inflammatory response(P<0.01), with TNF-α and IL-6 decreased in accordance with those in THCEs by PCR(P<0.05, P<0.01). CONCLUSION: The expression of MIF increased significantly in FK patients, THCEs and rats stimulated by A. fumigatus. After blocked with 4-IPP, the expression of MIF reduced, and so did its downstream cytokines: TNF-α and IL-6. The inflammation reaction of the rats' corneas lightened after pretreated with 4-IPP. MIF may play a role in the innate immune response of the corneal resistance against A. fumigatus.

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 m RNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction（q RT-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 m RNA 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 m RNA measured by q RT-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.

TREM-1 expression in rat corneal epithelium with Aspergillus fumigatus infection

AIM: To investigate the expression of triggering receptor expressed on myeloid cells-1(TREM-1) in the aberrant inflammation within the corneal epithelium at early period of fungal infection.METHODS: A total of 65 Wistar rats were randomly divided into control group, sham group and fungal keratitis(FK) group, in which the cornea was infected by Aspergillus fumigatus(A. fumigatus). After executed randomly at 8, 16, 24, 48 and 72 h after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1through quantitative reverse transcription-polymerase chain reaction(RT-PCR), immunofluorescence technique and Western blot analysis. The correlation between FK inflammation and expression of TREM-1 was also analyzed.RESULTS: Corneal inflammation scores increased with time after fungal infection(F =49.74, P =0.000). The inflammation scores in FK group were obviously higher than those in sham group on the whole(F =137.78, P =0.000). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h(P 【0.001,compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24 h and 48 h after fungal infection.Immunofluorescence technique showed that TREM-1mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1expression in FK(r =0.942, P =0.000).CONCLUSION: TREM-1 may contribute to amplify theinflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses.

The expressions of metadherin and LEF-1 in mucosa-associated lymphoid tissue lymphoma of ocular adnexal

AIM：To investigate the expressions of metadherin（astrocyte elevated gene-1, AEG-1） and lymphoid enhancerbinding factor-1（LEF-1） in ocular adnexal mucosaassociated lymphoid tissue（MALT） lymphoma.METHODS：The expressions of AEG-1 and LEF-1 were detected on specimens harvested from patients suffering from MALT lymphoma and lymphadenosis of ocular adnexal in Ophthalmology Department, Affiliated Hospital of Qingdao University from 2000 to 2015 by immunohistochemical and polymerase chain reaction（PCR） analysis.RESULTS：AEG-1 and LEF-1 expressions in MALT lymphoma was respectively higher than that in lymphadenosis, both by immunohistochemical and PCR analysis（P〈0.05）. Diversity of AEG-1 and LEF-1 expressions in different Ann Arbor clinical stages showed a statistically significant result（P〈0.05）. A positive relevance between AEG-1 and LEF-1 was observed in MALT ocular adnexal lymphoma（r=0.435, P=0.016）.CONCLUSION：The over expressions of AEG-1 and LEF-1 at the level of protein and mR NA participates in the tumorigenesis of ocular adnexal MALT lymphoma. They should act as a new biological marker for pathological diagnosis in the future.

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： C57 BL/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 transcriptionploymerase chain reaction（RT-PCR） and immunostaining. The expression of cytokines（IL-1β, TNF-α and IL-6） chemokines（CXCL-1 and MIP-2） was determined by RTPCR 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 C57 BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1β, 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 upregulated in TDB group and down-regulated in Mincle Ab 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 Mincle Ab group compared with Ig G 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.