Abnormal platelet parameters in inflammatory bowel disease: a systematic review and meta-analysis.

BACKGROUND: Platelet dysfunction plays a critical role in the pathogenesis of inflammatory bowel disease (IBD). Despite clinical observations indicating abnormalities in platelet parameters among IBD patients, inconsistencies persist, and these parameters lack standardization for diagnosis or clinical assessment. METHODS: A comprehensive search was conducted in the PubMed, Embase, Web of Science, and Cochrane Library databases for relevant articles published up to December 16th, 2023. A random-effects model was employed to pool the weighted mean difference (WMD) and 95% confidence interval (95% CI) of platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT) between IBD patients and healthy controls, and subgroup analyses were performed. RESULTS: The meta-analysis included 79 articles with 8,350 IBD patients and 13,181 healthy individuals. The results revealed significantly increased PLT and PCT levels (WMD: 69.910, 95% CI: 62.177, 77.643 109/L; WMD: 0.046%, 95% CI: 0.031%, 0.061%), and decreased MPV levels (WMD: -0.912, 95% CI: -1.086, -0.739 fL) in IBD patients compared to healthy individuals. No significant difference was found in PDW between the IBD and control groups (WMD: -0.207%, 95% CI: -0.655%, 0.241%). Subgroup analysis by disease type and disease activity showed no change in the differences for PLT, PCT, and MPV in the ulcerative colitis and Crohn's disease groups, as well as the active and inactive groups. Notably, the active group exhibited significantly lower PDW levels than the control group (WMD: -1.138%, 95% CI: -1.535%, -0.741%). CONCLUSIONS: Compared with healthy individuals, IBD patients display significantly higher PLT and PCT and significantly lower MPV. Monitoring the clinical manifestations of platelet abnormalities serves as a valuable means to obtain diagnostic and prognostic information. Conversely, proactive measures should be taken to prevent the consequences of platelet abnormalities in individuals with IBD. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42023493848.

Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells.

PURPOSE: To determine whether protein tyrosine phosphatase 1B (PTP1B) is expressed in rat retinal pigment epithelium (RPE) cells, to evaluate whether inhibition of PTP1B contributes to initiation of RPE cells into an active state, and to investigate the signaling pathways involved in this process. METHODS: Rat retinas were detached by trans-scleral injection of 1.4% sodium hyaluronate into the subretinal space. Immunocytochemistry evaluated the expression of PTP1B in RPE cells located at normal and detached retinas. From the cultured RPE cells treated with TCS-401, cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetracolium bromide assay, and the protein expression levels of cyclin A and cyclin D1 were determined. The effect of TCS-401 on cell differentiation was confirmed by immunostaining for α-smooth muscle actin and by western blot. Cell migration activity and PTP1B signaling mechanism were determined. Migration Assay was used to evaluate cell migration activity. PTP1B signaling mechanism was determined by use of PD98059 and LY294002. RESULTS: PTP1B was expressed in the RPE layer of the normal retina. After retinal detachment, weak immunolabeling of PTP1B was seen in the RPE cells. TCS-401 promoted the proliferation and expression of cyclin A and cyclin D1 in RPE cells. TCS-401 induced RPE cells to differentiate toward better contractility and motility. A migration assay proved that inhibiting PTP1B improved the migratory activity of RPE cells. TCS-401 activated extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) phosphorylation. Pretreatment with PD98059 and LY294002 abolished TCS-401-induced activation of Erk, Akt, cell proliferation, and cell migration. CONCLUSIONS: PTP1B may be involved in regulating the active state of RPE cells. The inhibition of PTP1B promoted the proliferation, myofibroblast differentiation, and migration of RPE cells, and MEK/Erk and PI3K/Akt signaling pathways played important roles in the proliferation and migration process.

NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation.

AIM: To explore whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease. METHODS: NOX2 expression was detected in C57BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride (DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species (ROS) and the release of cytokines [NF-κB, interleukin-17A (IL-17A), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), Nrf2, IL-10, and TGF-ß] were compared. A one-way ANOVA and an unpaired, two-tailed Student's t-test was used to determine the statistical significance. RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-ß compared to their expression levels without DPI treatment. CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.

[Expression of substance P in experimental fungal keratitis].

OBJECTIVE: To detect the change process of substance P (SP)expression and the difference between fungus and bacterial keratitis and explore effects of SP on the damage and repair process of keratitis model of Wistar rats. METHODS: Wistar rats were divided randomly as blank controlled group, fungal keratitis group and bacterial keratitis group. Inoculate fusarium and staphylococcus into cornea of Wistar rats to make animal keratitis model. Choose the right eye as experimental eye and the left eye as control. After the model was created successfully, 25 rats were executed randomly at 1, 5, 8, 10 and 14 days after the surgery. Detailed record and hematoxylin-eosin dye were done to each group to examine the process of ulcer. Expression of SP were detected through immunohistochemistry and reverse transcription polymerase chain reaction. Use variance analysis, T test and Q test to evaluate the results. RESULTS: One day after infection, the cornea of experimental group showed ulcer with disordered shallow matrix layer collagenous fibre arrangement and neutral granular cell infiltration; after 5 days, ulcer worsened and granular cells infiltrated through the whole layer; after 8 days, ulcer shrinked and fibroblast began to increase with new vessels formed; after 10 days, new vessels began to decrease; after 14 days, the matrix level textile fiber assumes scar type restructuring. The expression of SP increased in endothelium cells, inflammatory cells, fibroblast cells and endothelium of new vessels 1 d after surgery (absorbance for fusarium and staphylococcus group were 0.3313 ± 0.0133 and 0.3995 ± 0.0191 respectively; mRNA were 0.4525 ± 0.0170 and 0.5532 ± 0.0258), peaked at 8 d (for fusarium and staphylococcus group were 0.5525 ± 0.0171 and 0.7050 ± 0.0119 respectively; mRNA were 0.5975 ± 0.0221 and 0.7150 ± 0.0238), began to decrease at 10 d (for fusarium and staphylococcus group were 0.1533 ± 0.0176 and 0.3125 ± 0.0170 respectively; mRNA were 0.2416 ± 0.0082 and 0.4835 ± 0.0082) and dropped to lower than normal at 14 d (for fusarium and staphylococcus group were 0.1150 ± 0.0128 and 0.1675 ± 0.0126 respectively; mRNA were 0.1275 ± 0.0126 and 0.2325 ± 0.0171), which had significant difference (the absorbance of SP: for fusarium group, F = 832.24, q = 35.3675, 12.9044, 27.3621, 34.6506, 22.4632, 62.7296, 70.0182, 40.2664, 47.5550, 7.2886, P < 0.01;for staphylococcus group F = 636.17, q = 38.7494, 11.4245, 10.8298, 28.6082, 27.3249, 49.5791, 67.3575, 22.2543, 40.0327, 17.7784, P < 0.01. mRNA expression: for fusarium group F = 658.60, q = 18.9941, 9.5132, 27.4719, 42.0007, 9.4809, 46.4661, 60.9948, 36.9852, 51.5139, 14.5287, P < 0.01; for staphylococcus group F = 335.13, q = 16.9266, 4.1677, 7.1081, 32.4724, 12.7589, 24.0347, 49.3990, 11.2759, 36.6402, 25.3643, P < 0.01). Two experimental groups showed similar changes as time changes, the bacterial group showed more SP expression than fungal group, which had significant difference (absorbance of SP t = 6.5493, 7.3867, 16.0505, 14.5479, 6.5360, P < 0.05; mRNA expression t = 7.2878, 9.5232, 8.8149, 43.6256, 11.1269, P < 0.05). Controlled eyes showed increased SP expression at 1 d (absorbance was 0.2840 ± 0.0212; mRNA was 0.3950 ± 0.0129) and dropped to normal at 5 d (absorbance was 0.2125 ± 0.0174; mRNA was 0.3321 ± 0.0041). There was significant difference between bacterial group, fungal group and controlled eyes (absorbance of SP: compared with fusarium group t = 4.2261, 18.3314, 35.5163, 5.3609, 13.4826; compared with staphylococcus group t = 9.0508, 25.6639, 63.4924, 7.4828, 7.1301, P < 0.05. mRNA expression: compared with fusarium group t = 6.0249, 31.9158, 26.0413, 9.1550, 19.1741; compared with staphylococcus group t = 12.2636, 53.4404, 36.8727, 15.8687, 8.2939, P < 0.05). CONCLUSION: SP possibly participated in the early time damage and the later period repair process in fungus keratitis and its lower expression level possibly participated in the mechanism of lighter ache in fungus keratitis.

[Research of factors related to invasion and metastasis in choroidal melanoma].

OBJECTIVE: To investigate the expression of factors related to invasion and metastasis in choroidal melanoma and to determine their relationships with malignant features. METHODS: The expression of Connexin43 (Cx43), epithelial cadherin (E-cadherin), phosphatidylinositol 3-kinase (PI3K) and connective tissue growth factor (CTGF) in choroidal melanoma and nevi were detected by immunohistochemistry, and the correlation between these factors and clinicopathological features were analyzed. RESULTS: Positive rates of Cx43, E-cadherin, PI3K and CTGF were 74.07% (20/27), 44.4% (12/27), 74.07% (20/27) and 66.67% (18/27) in choroidal melanoma tissues, respectively; and 33.33% (5/15), 86.67% (13/15), 33.33% (5/15) and 20.00% (3/15) in the nevi tissues, respectively. There were significant differences in the expression of these markers between the two groups (χ(2) = 5.060, P = 0.024; χ(2) = 5.490, P = 0.019; χ(2) = 5.060, P = 0.024; χ(2) = 6.637, P = 0.010). The expression rates of Cx43 protein were 40% (4/10), 88.89% (8/9) and 100% (8/8) in spindle, mixed and epithelioid cell type, respectively. The expression of these data was related to histological type (χ(2) = 9.874, P = 0.007). The expression rates of PI3K protein were 42.86% (3/7), 75% (9/12) and 100% (8/8), in small, medium and large tumors, respectively, and their expression were co-related to the tumor size (χ(2) = 6.357, P = 0.042). Positive rates of Cx43, E-cadherin, PI3K and CTGF were 50% (6/12), 83.33% (10/12), 50% (6/12) and 41.66% (5/12), respectively, in choroidal melanoma tissues without sclera invasion and were 93.33% (14/15), 40% (6/15), 93.33% (14/15) and 86.67% (13/15), respectively, in choroidal melanoma tissues with violation involved the sclera. There were significant differences of the expression of these markers between the two groups (χ(2) = 4.457, P = 0.016; χ(2) = 3.546, P = 0.028; χ(2) = 4.457, P = 0.016; χ(2) = 4.218, P = 0.019). CONCLUSION: Increased expression of Cx43, PI3K and CTGF and decreased expression of E-cadherin are involved in the processes of invasion and metastasis of choroidal melanoma.

[Spatio-temporal variation of atmospheric CH4 concentration and its driving factors in monsoon Asia.] / 亚洲季风区大气CH4æµ“åº¦æ—¶ç©ºå˜åŒ–ç‰¹å¾ä¸Žå½±å“å› ç´ .

Based on the ground-based observations from seven atmospheric background stations during 2009 to 2018 in monsoon Asia (including BKT station in Indonesia, LLN and WLG stations in China, RYO and YON stations in Japan, TAP station in Republic of Korea, and UUM station in Mongolia), we analyzed the temporal and spatial variation of atmospheric CH4 concentration and its driving factors using harmonic model and maximal information-based nonparametric exploration. The results showed that the CH4 concentration in monsoon Asia varied from 1853.04 to 1935.61 nmol·mol-1, higher than that in Mauna Loa (MLO) station (1838.33 nmol·mol-1) in Hawaii, USA. The CH4 concentration decreased from north to south, with the highest value in TAP station (1935.61 nmol·mol-1) in Republic of Korea and RYO station (1907.19 nmol·mol-1) in Japan. The average seasonal amplitude at YON station in Japan was the largest (108.20 nmol·mol-1); while that at WLG station in China was the smallest (29.48 nmol·mol-1). The seasonal amplitude of TAP station in Republic of Korea changed faster at the rate of 4.49 nmol·mol-1·a-1. Except for WLG and TAP stations, CH4 concentrations were low in summer and high in winter. From the long-term perspective, the CH4 concentration at LLN (7.68 nmol·mol-1·a-1) and WLG (7.56 nmol·mol-1·a-1) stations in China exhibited the most obvious growth trend. Compared with wind speed, temperature and precipitation had greater impact on CH4 concentration, which were negatively associated with CH4 concentration. Local CH4 emission at some stations had a significant positive effect on CH4 concentration.

The effects of sodium orthovanadate-induced phosphatase inhibition on rat retinal pigment epithelium cell activity.

PURPOSE: The purpose of this study was to assess the contribution of sodium orthovanadate (SOV)-induced phosphatase inhibition to the activation of rat retinal pigment epithelium (RPE) cells. METHODS: Confluent cultures of rat RPE cells were treated with the general phosphatase inhibitor SOV. The effects of SOV on the cell cycle were determined by flow cytometry and protein detection of cyclin A and cyclin D1, two different cell cycle regulatory factors. The effects of SOV on cell differentiation were confirmed by immunostaining for α-smooth muscle actin (α-SMA). A migration assay was used to evaluate the effects of SOV on cell migration. RESULTS: SOV could accelerate the cell cycle of RPE cells. Western blotting showed that SOV significantly increased the expression of cyclin A and cyclin D1 in a dose-dependent fashion. The results of α-SMA staining and western blotting demonstrated that SOV induced RPE cells to differentiate toward better contractility and motility. The migration assay indicated that SOV improved the migration activity of RPE cells. CONCLUSIONS: Sodium orthovanadate can improve proliferation, differentiation, and migration of rat RPE cells and can also induce the reentry of contact-inhibited rat RPE cells into the cell cycle.

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 48h 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, 16h 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 those in the normal group by PCR (at 1d: P<0.01, 3d: P<0.01, 5d: 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.

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

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

Mincle in the innate immune response of mice fungal keratitis.

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

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 enhancer-binding factor-1 (LEF-1) in ocular adnexal mucosa-associated 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 mRNA participates in the tumorigenesis of ocular adnexal MALT lymphoma. They should act as a new biological marker for pathological diagnosis in the future.

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 expression of CXCL1 and CXCL8 increased in THCE 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.

Human ß-NGF gene transferred to cat corneal endothelial cells.

AIM: To transfect the cat corneal endothelial cells (CECs) with recombinant human ß-nerve growth factor gene adeno-associated virus (AAV-ß-NGF) and to observe the effect of the expressed ß-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-ß-NGF was constructed. The recombinant human AAV-ß-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-ß-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the ß-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 ß-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-ß-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-ß-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 G1cells 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-ß-NGF promotes proliferation in cat CECs by expressing bioactive ß-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.

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-1 murine 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-1was measured by Western blot. The expression of IL-6 and IL-8 mRNA 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 30min 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 mRNA 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.

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/6 and BALB/c mice. The expression of LOX-1, pro-inflammatory cytokines TNF-α, CXCL1 and IL-6, anti-inflammatory cytokines IL-10, and matrix metalloproteinase 9 (MMP9) were tested with treatment with LOX-1 neutralizing antibody or control IgG in A. fumigatus infected corneas of C57BL/6. Macrophages and neutrophils were extracted from susceptible C57BL/6 mice, and pretreated with LOX-1 neutralizing antibody or IgG, then stimulated with A. fumigatus. 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. fumigatus 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/6 corneas 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.

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

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

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

AIM: To 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. METHODS: Immunohistochemistry 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. RESULTS: We 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). CONCLUSION: The VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.

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

AIM: To 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. METHODS: ARPE-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 α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay. RESULTS: The mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-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, α-SMA expression and cell migration. CONCLUSION: PTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

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/mL) for various time. Then HCECs pretreated with or without laminarin (Dectin-1 blocker, 0.3 mg/mL) and curdlan were stimulated by A. fumigatus hyphae. The mRNA 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 mRNA 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 mRNA 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-1 expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1 blocker laminarin suppressed the mRNA 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 effects of curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.