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 in vitro with AF antige...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 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-κB and relative downstream cytokines such as TNF-α, IL-1β, 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-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, 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-α and IL-1β 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-κB pathway. SP-D possibly mediates the recognition to AF mycelium.展开更多
·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.·...·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 1st day,3rd days,7th days,and 14th 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 1stday,decrease on the 3rd day,but a significant increase on the 7th day and 14th 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.·展开更多
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...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.展开更多
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 ...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 clear 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.展开更多
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(My...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(My D88)-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, My D88, interleukin-1β(IL-1β), 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-1β, TNF-α were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, My D88, IL-1β, 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, My D88, IL-1β, TNF-α, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, My D88, IL-1β, TNF-α. CONCLUSION: In A. fumigatus keratitis, Boxb play a proinflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-My D88 signal pathway.展开更多
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 keratit...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.展开更多
AIM:To elucidate the effect of rapamycin on regulating the production of interleukin(IL)-1β in Aspergillus fumigatus(A.fumigatus)-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus kerati...AIM:To elucidate the effect of rapamycin on regulating the production of interleukin(IL)-1β in Aspergillus fumigatus(A.fumigatus)-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin(mT OR)/Toll-like receptor 4(TLR4) signaling pathway.METHODS:Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction(PCR).RESULTS:In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR 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-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.展开更多
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 M...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 vascularchanges. These features have important clinical significance in early detection of the gout and prevention of eye injury.展开更多
·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 Asp...·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-κB and proinflammatory cytokines such as TNF-α, 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-κB, TNF-α, 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-κB, TNF-α, 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 Af conidia by induction of proinflammatory cytokines such as TNF-α and IL-8 through the NF-κB pathway.展开更多
AIM: To explore the expression of S100 B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro.·METHODS: Immortalized human corneal epithelial cells(HCECs) were exposed to inactive A...AIM: To explore the expression of S100 B 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 24 h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48 h and the normal rat corneas were used for normal control. The m RNA level of S100 B was evaluated by real time quantitative reverse transcription-polymerase chain reaction(q RT-PCR).S100 B 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 S100 B at baseline. A. fumigatus significantly induced S100 B m RNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the m RNA began to rise significantly at8 h in vitro(P <0.05) and continue to rise as time prolonged(P <0.01). In vivo, S100 B m RNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12 h after infection(P <0.05) and reached to a peak at 24h(P <0.001). Immunochemistryrevealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100 B protein.·CONCLUSION: S100 B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation.S100 B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.展开更多
AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat's corneal epithelium. ·METHODS:A total of 72 Wistar rats...AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat's corneal epithelium. ·METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry. ·RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats'. There was no significantly difference of dectin-1 mRNA expression in group A and B (P >0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P <0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat's corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P<0.01). · CONCLUSION:Dectin-1 exists in rat's cornealepithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.展开更多
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...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.展开更多
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 ...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.展开更多
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 C57 BL/6 mice were pretrea...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 C57 BL/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.展开更多
● 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...● 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 24 h after the experimental models being established. Corneal epithelia were collected to assess the expression of PTX3 by quantitative reverse transcription polymerase chain reaction(q RT-PCR) and Western blot analysis.● RESULTS: Corneal inflammation scores increased as infection prolonged(P<0.05, P<0.001). PTX3 m RNA expression was low in normal and Sham group rats' corneas. Level of PTX3 m RNA in infected rat cornea was elevated at 8 h and peaked at 16 h. 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 8 h and peaked at 16 h(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.展开更多
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 wer...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 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(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 24 h 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 weredemonstrated 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.展开更多
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 detec...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-1β,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-1β.Similarly,in vitro experiments indicated that the production of LOX-1 and IL-1βwas 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-1βsignaling pathway and reducing the recruitment of neutrophils and macrophages.展开更多
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 w...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 C57 BL/6 normal and infected corneas.Dual immunostaining tested coexpression of HMGB1 with TLR4 or LOX-1.C57 BL/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).C57 BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation,and MIP-2,IL-1β,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-1β,TNF-α,LOX-1,p38-MAPK,p-p38-MAPK were measured.RESULTS:HMGB1 levels were elevated in C57 BL/6 mice after infection.HMGB1 co-expressed with TLR4,and LOX-1 in infiltrated cells.Box A vs PBS treated C57 BL/6 mice had lower clinical scores and down-regulated corneal HMGB1,MIP-2,IL-1βexpression and neutrophil influx.Box B treatment amplified expression of MIP-2,IL-1β,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-1β,TNF-αshowed inhibition of Box B combined with Poly(I),which also reduced the A.fumigatusevoked 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 C57 BL/6 mice.HMGB1 can amplify the host immune response through p38-MAPK,and is a target for treatment of A.fumigatus keratitis.展开更多
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 ...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 mR NA expression in BALB/c cornea was higher than C57BL/6 cornea at 1 and 3 d post infection(p.i.). rV IP treatment of C57BL/6 mice showed alleviated disease and down-regulated expression of interleukin-1β(IL-1β) 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-1β, 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.展开更多
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 mye...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 Aspergil us fumigatus(A. fumigatus) were used as cell models. The expression of CCPG1 m RNA was detected by q RT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1β(IL-1β). 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 4 h 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-1β 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 noncanonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.展开更多
基金National Natural Science Foundation of China(No.81170825)
文摘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 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-κB and relative downstream cytokines such as TNF-α, IL-1β, 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-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, 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-α and IL-1β 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-κB pathway. SP-D possibly mediates the recognition to AF mycelium.
文摘·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 1st day,3rd days,7th days,and 14th 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 1stday,decrease on the 3rd day,but a significant increase on the 7th day and 14th 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.·
基金Supported by National Natural Science Foundation of China(No.81170825No.81300730)
文摘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.
基金National Natural Science Foundation of China (No.81170825)
文摘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 clear 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.
文摘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(My D88)-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, My D88, interleukin-1β(IL-1β), 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-1β, TNF-α were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, My D88, IL-1β, 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, My D88, IL-1β, TNF-α, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, My D88, IL-1β, TNF-α. CONCLUSION: In A. fumigatus keratitis, Boxb play a proinflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-My D88 signal pathway.
基金Supported by the National Natural Science Foundation of China(No.81170825No.81470609)+2 种基金Specialized Research Fund for the Doctoral Program of Higher Education(No.20123706110003)The Youth Natural Science Foundation of Shandong Province(No.ZR2013HQ007)The Key Project of Natural Science Foundation of Shandong Province(No.ZR2012HZ001)
文摘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.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81500695)
文摘AIM:To elucidate the effect of rapamycin on regulating the production of interleukin(IL)-1β in Aspergillus fumigatus(A.fumigatus)-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin(mT OR)/Toll-like receptor 4(TLR4) signaling pathway.METHODS:Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction(PCR).RESULTS:In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR 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-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.
文摘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 vascularchanges. These features have important clinical significance in early detection of the gout and prevention of eye injury.
基金National Natural Science Foundation of China (No.30672285)Qingdao Municipal Science and Technology Commission,China (No.10-3-3-10-NSH)
文摘·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-κB and proinflammatory cytokines such as TNF-α, 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-κB, TNF-α, 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-κB, TNF-α, 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 Af conidia by induction of proinflammatory cytokines such as TNF-α and IL-8 through the NF-κB pathway.
基金Supported by National Natural Science Foundation of China (No.81170825, No.81470609)Specialized Research Fund for the Doctoral Program of Higher Education (No. 20123706110003)+1 种基金The Youth Natural Science Foundation of Shandong Province (No. ZR2013HQ007)The Key Project of Natural Science Foundation of Shandong Province (No. ZR2012HZ001)
文摘AIM: To explore the expression of S100 B 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 24 h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48 h and the normal rat corneas were used for normal control. The m RNA level of S100 B was evaluated by real time quantitative reverse transcription-polymerase chain reaction(q RT-PCR).S100 B 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 S100 B at baseline. A. fumigatus significantly induced S100 B m RNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the m RNA began to rise significantly at8 h in vitro(P <0.05) and continue to rise as time prolonged(P <0.01). In vivo, S100 B m RNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12 h after infection(P <0.05) and reached to a peak at 24h(P <0.001). Immunochemistryrevealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100 B protein.·CONCLUSION: S100 B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation.S100 B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.
基金National Natural Science Foundation of China (No.81170825)
文摘AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat's corneal epithelium. ·METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry. ·RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats'. There was no significantly difference of dectin-1 mRNA expression in group A and B (P >0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P <0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat's corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P<0.01). · CONCLUSION:Dectin-1 exists in rat's cornealepithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81500695)
文摘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.
基金National Natural Science Foundation of China (No. 81170825)
文摘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.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81300730+4 种基金No.81500695)China Postdoctoral Science Foundation(No.2018M630482)the National Natural Science Foundation of Shandong(No.ZR2017BH025)Key Research Project of Shandong(No.2018GSF118193)Applied Basic Research Project of Qingdao(No.16-5-1-65-jch)
文摘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 C57 BL/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.
基金Supported by National Natural Science Foundation of China (No.81170825 No.81470609)+2 种基金Specialized Research Fund for the Doctoral Program of Higher Education (No.20123706110003)the Youth Natural Science Foundation of Shandong Province (No.ZR2013HQ007)the Key Project of Natural Science Foundation of Shandong Province (No.ZR2012HZ001).
文摘● 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 24 h after the experimental models being established. Corneal epithelia were collected to assess the expression of PTX3 by quantitative reverse transcription polymerase chain reaction(q RT-PCR) and Western blot analysis.● RESULTS: Corneal inflammation scores increased as infection prolonged(P<0.05, P<0.001). PTX3 m RNA expression was low in normal and Sham group rats' corneas. Level of PTX3 m RNA in infected rat cornea was elevated at 8 h and peaked at 16 h. 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 8 h and peaked at 16 h(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.
基金Supported by Biomedical Research Institute Grant(No.2009-39)Pusan National University Hospital
文摘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 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(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 24 h 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 weredemonstrated 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.
基金the National Natural Science Foundation of China(No.82171019)Natural Science Foundation of Shandong Province(No.ZR2021MH368)+1 种基金Traditional Chinese Medicine Research Project of Qingdao(No.2020-zyy055)Shandong Qingdao Outstanding Health Professional Development Fund.
文摘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-1β,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-1β.Similarly,in vitro experiments indicated that the production of LOX-1 and IL-1βwas 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-1βsignaling pathway and reducing the recruitment of neutrophils and macrophages.
基金Supported by Natural Science Foundation of Shandong Province(No.ZR2017BH025)。
文摘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 C57 BL/6 normal and infected corneas.Dual immunostaining tested coexpression of HMGB1 with TLR4 or LOX-1.C57 BL/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).C57 BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation,and MIP-2,IL-1β,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-1β,TNF-α,LOX-1,p38-MAPK,p-p38-MAPK were measured.RESULTS:HMGB1 levels were elevated in C57 BL/6 mice after infection.HMGB1 co-expressed with TLR4,and LOX-1 in infiltrated cells.Box A vs PBS treated C57 BL/6 mice had lower clinical scores and down-regulated corneal HMGB1,MIP-2,IL-1βexpression and neutrophil influx.Box B treatment amplified expression of MIP-2,IL-1β,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-1β,TNF-αshowed inhibition of Box B combined with Poly(I),which also reduced the A.fumigatusevoked 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 C57 BL/6 mice.HMGB1 can amplify the host immune response through p38-MAPK,and is a target for treatment of A.fumigatus keratitis.
基金Supported by the National Natural Science Foundation of China(No.81470609No.81500695)+1 种基金the Natural Science Foundation of Shandong Province(No.ZR2013HQ007No.ZR2017BH025)
文摘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 mR NA expression in BALB/c cornea was higher than C57BL/6 cornea at 1 and 3 d post infection(p.i.). rV IP treatment of C57BL/6 mice showed alleviated disease and down-regulated expression of interleukin-1β(IL-1β) 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-1β, 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.
基金Supported by the National Natural Science Foundation of China(No.82171019)the Natural Science Foundation of Shandong Province(No.ZR2021MH368)+1 种基金Traditional Chinese Medicine Research Project of Qingdao(No.2020-zyy055)Shandong Qingdao Outstanding Health Professional Development Fund。
文摘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 Aspergil us fumigatus(A. fumigatus) were used as cell models. The expression of CCPG1 m RNA was detected by q RT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1β(IL-1β). 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 4 h 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-1β 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 noncanonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.
