Structural characteristics and drivers of greenhouse gas emissions at county-level and long-time scales: A case study of the Anji County, China.

To achieve carbon neutrality, the Chinese government needs to gain a comprehensive understanding of the sources and drivers of greenhouse gas (GHG) emissions, particularly at the county level. Anji County in eastern China is a typical example of an industrial transformation from quarrying to a low-carbon economy. This study analyzed the decoupling types and structural characteristics of GHG emissions and the driving factors of carbon dioxide (CO2) emissions in the Anji from 2006 to 2019, and explored the differences between county-level and provincial-level or city-level results. It was observed that energy-related activities are the main source of GHG emissions in Anji and that economic development is the driving factor behind the increasing CO2 emissions. However, industrial transformation and upgradation coupled with the alternative use of clean energy limit the growth of GHG emissions. This study details the GHG emissions of county during the industrial transformation stage and provides corresponding policy recommendations for county governments.

Role of Inhibiting Inflammation of LC3-Associated Phagocytosis in Dry Eye Disease.

PURPOSE: To confirm the expression and investigate the role of LC3-associated phagocytosis (LAP) in dry eye disease (DED). METHODS: The DED model of mice was established by scopolamine subcutaneous injection in a low-humidity environment chamber. Tear secretion test and corneal fluorescein sodium staining were used to evaluate the severity of DED. Expression levels of Rubicon, microtubule-associated protein light chain 3-II (LC3-II), Beclin-1 and autophagy-related gene-7 (Atg-7) in corneas of mice with DED were tested by western blot. Cell Counting Kit-8 (CCK-8) assay was used to detect the effects of different concentrations of hypertonic solutions on the proliferation activity of human corneal epithelial cells (HCECs). The expression levels of Dectin-1, IL-6 and IL-1ß in HCECs after stimulation with different concentrations of hypertonic solutions were tested. The expressions of Rubicon, LC3-II, Beclin-1 and ATG-7 in HCECs were detected by reverse transcription polymerase chain reaction (RT-PCR). After being pretreated with 10 µM si-Rubicon, the severity of the disease was documented by corneal fluorescein sodium staining. And the expression levels of IL-6 and IL-1ß were also tested by RT-PCR. RESULTS: Compared with the normal control group, the corneal fluorescein sodium staining scores and tear secretion were significantly reduced. Rubicon, LC3-II, Beclin-1 and ATG-7 were significantly elevated. CCK-8 showed that the 400 and 450 mOsM hypertonic solutions did not affect the proliferation activity of HCECs. The expression of Dectin-1, IL-1ß and IL-6 were elevated after stimulation with 450 mOsM solution. LC3-II, Rubicon, ATG-7 and Beclin-1 increased after stimulation with 450 mOsM hyperosmolar solution in HCECs. Corneal fluorescein staining showed that si-Rubicon increased the severity of DED in mice. Moreover, the mRNA expressions of inflammatory factors IL-1ß and IL-6 in the cornea of mice were significantly increased. CONCLUSION: DED increased the expression of proteins associated with LAP. LAP could play an anti-inflammatory effect in DED.

Inhibition of AIF-1 alleviates laser-induced macular neovascularization by inhibiting endothelial cell proliferation via restrained p44/42 MAPK signaling pathway.

Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.

Biomimetic, Injectable, and Self-Healing Hydrogels with Sustained Release of Ranibizumab to Treat Retinal Neovascularization.

Retinal neovascularization (RNV) is a typical feature of ischemic retinal diseases that can lead to traction retinal detachment and even blindness in patients, in which the vascular endothelial cell growth factor (VEGF) plays a pivotal role. However, most anti-VEGF drugs currently used for treating RNV, such as ranibizumab, need frequent and repeated intravitreal injections due to their short intravitreal half-life, which increases the incidence of complications. Herein, a hydrogel intravitreal drug delivery system (DDS) is prepared by a dynamic Schiff base reaction between aminated hyaluronic acid and aldehyde-functionalized Pluronic 127 for sustained release of ranibizumab. The prepared hydrogel system named HP@Ran exhibits excellent injectability, self-healing ability, structural stability, cytocompatibility, and blood compatibility. According to an in vitro drug release study, the hydrogel system continuously releases the model drug bovine serum albumin for more than 56 days. Importantly, in an in vivo rabbit persistent RNV model, the HP@Ran hydrogel system continuously releases pharmacologically active ranibizumab for more than 7 weeks and also exhibits superior anti-angiogenic efficacy over ranibizumab treatment by decreasing vascular leakage and neovascularization at 12 weeks. Thus, the developed HP@Ran hydrogel system possesses great potential for intravitreal DDS for the treatment of RNV.

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.

IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB.

Purpose: To explore the role of IL-36α in corneas infected by Aspergillus fumigatus. Methods: The experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1ß, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry. Results: IL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1ß, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra. Conclusions: IL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1ß, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.

Evaluation of vitrectomy combined preoperative intravitreal ranibizumab and postoperative intravitreal triamcinolone acetonide for proliferative diabetic retinopathy.

BACKGROUND: To explore the treatment efficacy of the combination of preoperative intravitreal ranibizumab (IVR) and postoperative intravitreal triamcinolone acetonide (IVTA) in patients undergoing pars plana vitrectomy (PPV) for proliferative diabetic retinopathy (PDR). METHODS: A retrospective comparative study was performed on 128 eyes of 128 patients who had PDR and underwent PPV. Patients who received a single PPV were assigned to Group A. Those who received PPV with preoperative IVR were assigned to Group B. Patients in Group C underwent PPV combined preoperative IVR and postoperative IVTA. Intraoperative findings, changes in mean best-corrected visual acuity (BCVA) and postoperative adverse events, were retrospectively evaluated at 6-month follow-up. RESULTS: The incidences of iatrogenic breaks, severe intraoperative bleeding, using long-term internal tamponade agents, recurrent vitreous hemorrhage (VH), and duration of surgery were statistically significantly less in Group B and Group C than in Group A. The postoperative BCVA was statistically significantly better in Groups B and Group C than in Group A, respectively, at 1 month after surgery. The mean 3-month postoperative visual acuity was better in Group C. The incidence of high intraocular pressure (IOP) was significantly higher in Group C at the first postoperative week. There were no statistically significant differences in the incidence of exudative retinal detachment and choroidal detachment among the three groups. CONCLUSION: In patients undergoing PPV for PDR, preoperative IVR significantly reduced the occurrence of intraoperative and postoperative complications, and the combination of preoperative IVR and postoperative IVTA can better improve the postoperative visual outcome.

Indoleamine 2,3-Dioxygenase Regulates Macrophage Recruitment, Polarization and Phagocytosis in Aspergillus Fumigatus Keratitis.

Purpose: To explore the influence of indoleamine 2,3-dioxygenase (IDO) on macrophage recruitment, polarization and phagocytosis in Aspergillus fumigatus keratitis. Methods: A murine model of A. fumigatus keratitis and peritoneal macrophages incubated with the hyphae of A. fumigatus were used. Macrophage recruitment in corneas was evaluated using immunofluorescence staining. The polarization of macrophages, which was stimulated by A. fumigatus and pretreatment with or without 1-methyltryptophan (1-MT), interferon gamma (IFNG), extracellular regulated protein kinases (ERK) antagonist, and p38 antagonist, was determined using reverse-transcription polymerase chain reaction and flow cytometry. P38 and ERK levels were determined using Western blotting. Macrophage phagocytosis was examined using colony-forming units. Results: Compared with the A.F. group, recruitment of macrophages increased, tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression decreased, whereas arginase-1 (Arg-1) and interleukin-10 (IL-10) expression increased in the mouse corneas of the 1-MT+A.F. group. The ratio of CD206+/CD86+ macrophages in the corneas and spleens of 1-MT+A.F. group increased. Furthermore, in peritoneal macrophages stimulated by A. fumigatus, 1-MT promoted Arg-1 and IL-10 expression while upregulating the ratio of CD206+/CD86+ macrophages. Conversely, IDO agonist IFNG promoted TNF-α and iNOS expression, inhibited Arg-1 and IL-10 expression and downregulated the ratio of CD206+/CD86+ macrophages. The role of IFNG was reversed by the antagonist of P38 or ERK. P38 and ERK levels were downregulated in corneas of 1-MT+A.F. group. Besides, IFNG inhibited macrophage phagocytosis. Conclusion: IDO inhibited macrophage recruitment and phagocytosis in A. fumigatus keratitis. Mechanistically, IDO is involved in M1 macrophage polarization in A. fumigatus keratitis through a MAPK/ERK-dependent pathway.

Expression of macrophage migration inhibitory factor in Aspergillus fumigatus keratitis.

AIM: To investigate the expression of macrophage migration inhibitory factor (MIF) and detect its role in the innate immune response of fungal keratitis (FK). METHODS: We collected the paraffin-embedded cornea tissues from 10 FK and 6 ocular trauma patients to explore the MIF expression by immunohistochemistry. Then we cultured telomease-immortalized human corneal epithelial cells (THCEs), stimulated by the hyphae suspension of Aspergillus fumigatus (A. fumigatus) to detect the change of MIF with or without the pretreatment of MIF inhibitor [4-Iodo-6-phenylpyrimidine (4-IPP)] by real-time polymerase chain reaction (PCR). The protein level of MIF was also tested by immunohistochemistry, and the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA were compared between normal, hyphae stimulated and 4-IPP pretreated groups by real-time PCR to study the influence of MIF on the expression of TNF-α and IL-6. Corneal severity of rats' FK models was documented by clinical scores, and real-time PCR. Western blot and immunohistochemistry were used to test the expression of MIF, TNF-α and IL-6 in rats' corneas. RESULTS: In the corneas of FK patients, there was much stronger expression of MIF than that in the normal group showed by immunohistochemistry. In cultured THCEs stimulated by A. fumigatus, the expression of MIF became stronger in both immunohistochemistry and PCR at 16, 24, 32 and 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.

Role of angle κ in visual quality in patients with a trifocal diffractive intraocular lens.

PURPOSE: To evaluate the visual quality of patients with different angle κ sizes after a trifocal diffractive intraocular lens (IOL) implantation. SETTING: The Affiliated Hospital of Qingdao University, Qingdao, China. DESIGN: Prospective case series. METHODS: Patients who had phacoemulsification with the implantation of the trifocal IOL AT LISA tri 839MP were enrolled in the study. The patients were divided into 3 groups based on the size of the preoperative angle κ. Monocular far, intermediate, and near uncorrected visual acuities were measured during a 3-month follow-up. Other outcome measurements taken were the modulation transfer function (MTF) cutoff, the Strehl ratio, and objective scatter index. All the patients completed a subjective questionnaire survey. RESULTS: The study comprised 89 patients (89 eyes). The 3 groups showed statistically significant differences in the incidence of glare and halo after their surgery. There were no significant differences in the following variables: uncorrected far, intermediate, and near visual acuities, MTF cutoff, Strehl ratio, and spectacle independence. There was a significant difference in the MTF cutoff and Strehl ratio between the patients with the largest and the smallest angle κ. CONCLUSIONS: The patients' postoperative far, intermediate, and near vision was not affected by their angle κ. However, when angle κ was greater than 0.4 mm, the incidence of glare and halo increased and when it was greater than 0.5 mm, patients' visual quality decreased. In clinical work, for patients with a larger angle κ, the choice to implant a trifocal IOL should be carefully evaluated.

Effect of incision on visual outcomes after implantation of a trifocal diffractive IOL.

BACKGROUND: To evaluate visual acuity, corneal astigmatism and corneal higher-order aberrations (HOAs) after implantation of trifocal diffractive IOLs operated with either a corneal steep-axis incision or 135° incision. METHOD: This prospective study enrolled patients randomly assigned to different groups. According to preoperative corneal astigmatism, 101 eyes of 77 patients were assigned into group A1 (0 ~ 0.50 D) or A2 (0.51 ~ 1.00 D) with a corneal steep-axis incision or group B1 (0 ~ 0.50 D) or B2 (0.51 ~ 1.00 D) with a 135° incision. Visual acuity, corneal astigmatism and corneal higher-order aberrations (HOAs) were followed-up for 3 months. RESULTS: Corneal astigmatism in group A2 significantly decreased 3 months after surgery (P < 0.01) and was significantly lower than that in group B2 1 day, 2 weeks, 1 month, and 3 months postoperatively (all values of P < 0.01). The following parameters were better in group A2 than in group B2: uncorrected intermediate visual acuity (UIVA) at 1 day, 2 weeks, 1 month, and 3 months (P = 0.00, 0.00, 0.01, 0.01, respectively);uncorrected distance visual acuity (UDVA) at 1 day and 2 weeks (P = 0.00, 0.01); and uncorrected near visual acuity (UNVA) at 1 day, 2 weeks, and 1 month postoperatively (P = 0.00, 0.01, 0.02, respectively). CONCLUSIONS: After a corneal steep-axis incision, patients with preoperative corneal astigmatism of 0.51 D to 1.00 D exhibited reduced corneal astigmatism and achieved better UIVA and early postoperative UDVA/UNVA. TRIAL REGISTRATION: Retrospectively Registered Trials ISRCTN10086721 , 23/06/2018.

Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium.

Interleukin (IL)-32, a novel cytokine, participates in a variety of inflammatory disorders. Thymic stromal lymphopoietin (TSLP) plays important roles in mucosal epithelial cells, especially in allergy-induced inflammation, through the TSLP-TSLPR (thymic stromal lymphopoietin receptor) signalling pathway. However, the association of IL-32 with TSLP on the ocular surface remains unclear. The present work aimed to assess the functional association of IL-32 with TSLP in the control of pro-inflammatory cytokine levels in the corneal epithelium. Human corneal tissue specimens and human corneal epithelial cells (HCECs) were administered different concentrations of IL-32 in the presence or absence of various inhibitors to assess TSLP levels and localization, as well as the molecular pathways that control pro-inflammatory cytokine production. TSLP mRNA levels were determined by real time RT- PCR, while protein levels were quantitated by ELISA and immunohistochemical staining. TSLP protein expression was examined in donor corneal epithelium samples. IL-32 significantly upregulated TSLP and pro-inflammatory cytokines (TNFα and IL-6) in HCECs at the gene and protein levels. The production of pro-inflammatory molecules by IL-32 was increased by recombinant TSLP. Interestingly, both NF-κB (quinazoline) and caspase-1 (VX-765) inhibitors suppressed the IL-32-related upregulation of pro-inflammatory cytokines (TNFα and IL-6). These findings demonstrate that IL-32 and IL-32-induced-TSLP are critical cytokines that participate in inflammatory responses through the caspase-1 and NF-κB signalling pathways in the corneal epithelium, suggesting new molecular targets for inflammatory diseases of the ocular surface. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assays and RT-PCR,respectively. The results demonstrated that IL-32 inhibits cells apoptosis in HCECs.

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 inhibits neutrophils and macrophages apoptosis in A. fumigatus keratitis.

PURPOSE: To determine whether macrophage inducible C-type lectin (Mincle) regulates neutrophils and macrophages apoptosis in A. fumigatus keratitis. MATERIALS AND METHODS: A murine model (C57BL/6) of fungal keratitis (AF) was established by gently scraping corneal central epithelium, smearing A. fumigatus on the epithelium surface and covering the eye with contact lenses. AF cell model was established by extracting neutrophils (PMN) and macrophages, and then infecting cells with A. fumigatus. Animals and cells were randomly divided into control and A. fumigatus keratitis group, which were treated with Mincle ligand Trehalose-6,6-dibehenate (TDB), Mincle neutralizing antibody (MincleAb) or PBS before infection. The cornea infection was monitored using a slit lamp and further analyzed using H&E assay. PCR, Western blot, immunostaining, TUNEL staining and flow cytometry were used to examine the expression of Mincle and apoptosis factors, PMN infiltration and cell apoptosis, respectively. RESULTS: Higher levels of Mincle mRNA and protein, as well as epithelial thickness and presence of inflammatory cells in the stroma, were observed in the AF group compared to control. In addition, higher Mincle mRNA levels were observed in normal and stimulated neutrophils and macrophages. Furthermore, Fas, FasL and CASP3 mRNA levels, neutrophils infiltration rate and TUNEL-positive cells were significantly increased in AF+MincleAb mice compared with the control. Similar results, as well as significantly higher neutrophils and macrophages apoptosis, were observed by treating cells with MincleAb in vitro. Most importantly, opposite results i.e. lower mRNA levels, neutrophils infiltration rate and TUNEL-positive cells, as well as lower cell apoptosis in vitro, were observed in mice and cells treated with TDB. CONCLUSION: Mincle-participated in inflammatory process which inhibits neutrophils and macrophages apoptosis induced by A. fumigatus involved in Fas-dependent apoptotic pathways.

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.

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.

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 production mechanism and immunosuppression effect of pulmonary surfactant protein D via toll like receptor 4 signaling pathway in human corneal epithelial cells during Aspergillus fumigatus infection.

OBJECTIVE: To observe the production mechanism of surfactant protein D (SP-D) in human corneal epithelial cells (HCECs) when infected by Aspergillus fumigatus (A. fumigatus) hyphae, and explore whether SP-D can inhibit the cell activations through toll-like receptor 4 signaling pathway during fungal infection. METHODS: mRNA and protein expressions of SP-D were evaluated in HCECs after stimulation by A. fumigatus, with or without pretreatment of TLR4 inhibitor (CLI-095) by real time PCR and Western blot. The expression levels of inflammatory cytokines IL-1ß and IL-8 evaluated when pretreated with SP-D antibody or recombinant human SP-D in fungi-stimulated HCECs by real time PCR and ELISA, IL-1ß and IL-8 expressions were also detected in A. fumigatus-stimulated HCECs that pretreated with CLI095 or MyD88 inhibitor (Pepinh-MYD) and recombinant human SP-D. RESULTS: mRNA and protein levels of SP-D increased after stimulation of A. fumigatus for 16h and 20h respectively. The upregulation of SP-D could be inhibited by CLI-095. mRNA and protein expressions of IL-1ß and IL-8 decreased significantly when pretreated HCECs with recombinant human SP-D for 4h before A. fumigatus stimulation, while IL-1ß and IL-8 increased when pretreated with SP-D antibody for 1h. Pretreatment of CLI095 or Pepinh-MYD can increase the expressions of IL-1ß and IL-8 mRNA and protein in HCECs induced by recombinant human SP-D and A. fumigatus. CONCLUSIONS: SP-D can be stimulated by TLR4 during A. fumigatus infection. Recombinant human SP-D can inhibit the expression of inflammatory cytokines through TLR4 signaling pathway.

Role of Dectin-1 in the innate immune response of rat corneal epithelial cells to Aspergillus fumigatus.

BACKGROUND: To observe Dectin-1 expression in fungal keratitis on rat models and to determine the role of Dectin-1 in innate immune response to Aspergillus fumigatus. METHODS: Wistar rats were randomly divided into control, fungal keratitis and pretreatment (pretreated with Laminarin) groups. Samples were used for conducting immunohistochemical staining and real-time PCR to observe expression of cytokines like CCL2, CCL3, CXCL1, CXCL2, IL-1ß, TNF-α, IL-6, IL-10. RESULTS: After fungal stimulations, all 7 inflammatory factors, except IL-10, increased with different levels. After 4 h of fungal stimulations, IL-1ß, IL-6, CCL2, CXCL1 and CXCL2 of pretreatment groups were significantly (p < 0.05) lower than fungal groups, while the other 3 cytokines had no significant changes. After 8 h of fungal stimulations, IL-6 and CXCL1 of pretreatment groups were still significantly (p < 0.05) lower than fungal groups. DISCUSSION: With progress of fungus stimulation, expression of IL-1ß,CXCL1 ,CXCL2,MCP-1 gradually increased, whilepretreated with Laminarin to block Dectin-1, these expression decreased, indicating that Dectin-1 maypromote immune reaction through them. IL-10 decreased in fungal group because of itsimmunosuppressive effect at 4h, and it began to increase at 8h to suppress Th1 inflammation response inorder to avoid excessive tissue damage. CONCLUSION: Dectin-1 in early period of innate immune responses in rat fungal keratitis might work through IL-1ß, IL-6, CCL2, CXCL1, CXCL2 to recruit neutrophils and macrophages to participate anti-fungal immunity.