Wei Chen

Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients.

In studies on dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and inflammation severity elicited through receptor-induced increases in proinflammatory cytokine and chemokine release. These immune reactions might be mediated by inflammasomes, macromolecular complexes mounted around the NLRP3 protein and can be activated by reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3 inflammasomes mediate hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1β axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor, N-acetyl-l-cysteine (NAC). HCECs transfected with NLRP3 siRNA or a negative control (NC) siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2,7-dichlorofluorescin diacetate (DCFH-DA). Real-time PCR evaluated NLRP3, ASC, pro-caspase-1 and pro-IL-1β mRNA levels. Western blot analysis assessed NLRP3 protein expression whereas caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1β release was assessed by ELISA. ROS production, NLRP3 inflammasome and pro-IL-1β gene expression as well as IL-1β secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3 inflammasome formation and activation, caspase-1 activity and IL-1β release. On the other hand, NLRP3 siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC, pro-caspase-1 and pro-IL-1β mRNA down-regulation followed by suppression of associated caspase-1 activity and IL-1β secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC, pro-caspase-1 and pro-IL-1β gene expression as well as IL-1β secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE development.

Sequence Analysis of pKF3-70 in Klebsiella pneumoniae: Probable Origin from R100-Like Plasmid of Escherichia coli

Background Klebsiella pneumoniae is a clinically significant species of bacterium which causes a variety of diseases. Clinical treatment of this bacterial infection is greatly hindered by the emergence of multidrug-resistant strains. The resistance is largely due to the acquisition of plasmids carrying drug-resistant as well as pathogenic genes, and its conjugal transfer facilitates the spread of resistant phenotypes. Methodology/Principal Findings The 70,057 bp plasmid pKF3-70, commonly found in Klebsiella pneumoniae, is composed of five main functional modules, including regions involved in replication, partition, conjugation, transfer leading, and variable regions. This plasmid is more similar to several Escherichia coli plasmids than any previously reported K. pneumoniae plasmids and pKF3-70 like plasmids share a common and conserved backbone sequence. The replication system of the pKF3-70 is 100% identical to that of RepFII plasmid R100 from E. coli. A beta-lactamase gene ctx-m-14 with its surrounding insertion elements (ISEcp1, truncated IS903 and a 20 bp inverted repeat sequence) may compose an active transposon which is directly bordered by two putative target repeats “ATTAC.” Conclusions/Significance The K. pneumoniae plasmid pKF3-70 carries an extended-spectrum beta-lactamase gene, ctx-m-14. The conjugative characteristic makes it a widespread plasmid among genetically relevant genera which poses significant threat to public health.

Dynamic Ocular Surface and Lacrimal Gland Changes Induced in Experimental Murine Dry Eye

Dry eye disease can be a consequence of lacrimal gland insufficiency in Sjögren’s Syndrome or increased tear film evaporation despite normal lacrimal gland function. To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period. Dry eye was induced in 6-week-old female C57BL/6 mice by exposing them to an Intelligently Controlled Environmental System (ICES). Sixty mice were housed in ICES for 1, 2, 4 and 6 weeks respectively. Twelve were raised in normal environment and received subcutaneous injections of scopolamine hydrobromide (SCOP) 3 times daily for 5 days. Another sixty mice were housed in a normal environment and received no treatment. Corneal fluorescein staining along with corneal MMP-9 and caspase-3 level measurements were performed in parallel with the TUNEL assay. Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs). Immunohistochemistry of excised LGs along with flow cytometry in cervical lymph nodes evaluated immune cell infiltration. Light and transmission electron microscopy studies evaluated LGs cytoarchitectural changes. ICES induced corneal epithelial destruction and apoptosis peaked at 2 weeks and kept stable in the following 4 weeks. In the ICES group, lacrimal gland proinflammatory cytokine level increases were much lower than those in the SCOP group. In accord with the lower proinflammatory cytokine levels, in the ICES group, lacrimal gland cytosolic vesicular density and size exceeded that in the SCOP group. ICES and SCOP induced murine dry eye effects became progressively more severe over a two week period. Subsequently, the disease process stabilized for the next four weeks. ICES induced local effects in the ocular surface, but failed to elicit lacrimal gland inflammation and cytoarchitectural changes, which accounts for less dry eye severity in the ICES model than that in the SCOP model.

Evaluation of a Novel Artificial Tear in the Prevention and Treatment of Dry Eye in an Animal Model

Abstract Purpose: To evaluate effects of a novel multi-ingredient artificial tear formulation containing carboxymethylcellulose (CMC) and hyaluronic acid (HA) in a murine dry eye model. Methods: Dry eye was induced in mice (C57BL/6) using an intelligently controlled environmental system (ICES). CMC+HA (Optive Fusion™), CMC-only (Refresh Tears®), and HA-only (Hycosan®) artificial tears and control phosphate-buffered saline (PBS) were administered 4 times daily and compared with no treatment (nu2009=u200964 eyes per group). During regimen 1 (prevention regimen), mice were administered artificial tears or PBS for 14 days (starting day 0) while they were exposed to ICES, and assessed on days 0 and 14. During regimen 2 (treatment regimen), mice exposed to ICES for 14 days with no intervention were administered artificial tears or PBS for 14 days (starting day 14) while continuing exposure to ICES, and assessed on days 0, 14, and 28. Corneal fluorescein staining and conjunctival goblet cell density were measured. Results: Artificial tear-treated mice had significantly better outcomes than control groups on corneal staining and goblet cell density (Pu2009<u20090.01). Mice administered CMC+HA also showed significantly lower corneal fluorescein staining and higher goblet cell density, compared with CMC (Pu2009<u20090.01) and HA (Pu2009<u20090.05) in both regimens 1 and 2. Conclusions: The artificial tear formulation containing CMC and HA was effective in preventing and treating environmentally induced dry eye. Improvements observed for corneal fluorescein staining and conjunctival goblet cell retention suggest that this combination may be a viable treatment option for dry eye disease.

Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells

Tear film hyperosmolarity and anterior ocular inflammation are two clinical signs that may be indicative of dry eye disease (DED). This condition can cause pathological and functional changes to the anterior ocular surface tissues. A contributing factor may be dysfunctional aquaporin 5 (AQP5) water channels as they are the AQP subtype that expressed in the corneal epithelium and contribute to fluid efflux needed for corneal function. We determined if described hyperosmolarity-induced increases in proinflammatory cytokine expression and cell death are mediated through AQP5 upregulation and JNK1/2 MAPK signaling activation in both primary human corneal epithelial cells (HCECs), and in a HCEC line. Real time RT-PCR identified rises in IL-1β, IL-6, IL-8, TNF-α, caspase-1, and AQP5 mRNA levels upon step increases in osmolarity up to 550u2009mOsm. Western blot analysis and the TUNEL assay identified corresponding rises in AQP5 and p-JNK1/2 protein expression and cell death respectively. JNK1/2 inhibition with SP600125, or siRNA AQP5 gene silencing reduced hypertonic-induced rises in proinflammatory cytokine expression and cell death. Taken together, hypertonicity-induced AQP5 upregulation leads to increases in proinflammatory cytokine expression and cell death through JNK1/2 MAPK activation. These results suggest that drug targeting AQP5 upregulation may be a therapeutic option in DED management.

Characteristic of entire corneal topography and tomography for the detection of sub-clinical keratoconus with Zernike polynomials using Pentacam

The study aimed to characterize the entire corneal topography and tomography for the detection of sub-clinical keratoconus (KC) with a Zernike application method. Normal subjects (nu2009=u2009147; 147 eyes), sub-clinical KC patients (nu2009=u200977; 77 eyes), and KC patients (nu2009=u2009139; 139 eyes) were imaged with the Pentacam HR system. The entire corneal data of pachymetry and elevation of both the anterior and posterior surfaces were exported from the Pentacam HR software. Zernike polynomials fitting was used to quantify the 3D distribution of the corneal thickness and surface elevation. The root mean square (RMS) values for each order and the total high-order irregularity were calculated. Multimeric discriminant functions combined with individual indices were built using linear step discriminant analysis. Receiver operating characteristic curves determined the diagnostic accuracy (area under the curve, AUC). The 3rd-order RMS of the posterior surface (AUC: 0.928) obtained the highest discriminating capability in sub-clinical KC eyes. The multimeric function, which consisted of the Zernike fitting indices of corneal posterior elevation, showed the highest discriminant ability (AUC: 0.951). Indices generated from the elevation of posterior surface and thickness measurements over the entire cornea using the Zernike method based on the Pentacam HR system were able to identify very early KC.

Continuous-light versus pulsed-light accelerated corneal crosslinking with ultraviolet-A and riboflavin

PURPOSEnTo determine whether the pulsed-light ultraviolet-A (UVA) accelerated corneal crosslinking (CXL) procedure is more efficacious and selective than its continuous-light counterpart in rabbits.nnnSETTINGnSchool of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China.nnnDESIGNnExperimental study.nnnMETHODSnFifty-four rabbits were divided into 2 groups. Group 1 had continuous-light accelerated CXL using 9xa0mW/cm2 UVA for 10xa0minutes (5.4xa0J/cm2). Group 2 had pulsed-light accelerated CXL by exposing them to 9xa0mW/cm2 UVA for 20xa0minutes (1xa0second on/1xa0second off). Corneal stromal demarcation line depth, inxa0vivo confocal microscopic analysis, biomechanical stiffness, endothelial cell density, and keratocyte apoptosis were measured after performing these CXL procedures.nnnRESULTSnThe mean stromal demarcation line depth was 254.7xa0μmxa0±xa047.4 (SD) in Group 1 and 341.1xa0±xa036.1xa0μm in Group 2 (Pxa0<xa0.01). One day after CXL, confocal analysis and histological staining identified keratocyte apoptotic fragments in the anterior stroma in the Group 2 corneas whereas all cells were obliterated in Group1. Seven days after treatment, the thicknesses in Group 1 were significantly greater than those in Group 2 (Pxa0<xa0.05). Endothelial cell losses were reversible; however, in Group 1, some losses were still evident on day 7. Increases in both the stress-strain relationship and tangent modulus in Group 2 were greater than those in Groupxa01.nnnCONCLUSIONnThe pulsed-light accelerated CXL protocol was less injurious and more efficacious at inducing CXL than the continuous-light accelerated CXL protocol in rabbit corneas.

Hyperosmotic Stress–Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells

PurposenThe purpose of this study was to determine whether either a hyperosmotic or oxidative stress induces NLRP3 inflammasome activation and increases in bioactive IL-1β secretion through transient receptor potential melastatin 2 (TRPM2) activation in primary human corneal epithelial cells (PHCECs).nnnMethodsnReal-time PCR, Western blots, and immunofluorescent staining were used to evaluate TRPM2 and NLRP3, ASC, caspase-1, and IL-1β mRNA and protein expression levels, respectively. A CCK-8 assay evaluated cell viability. Hyperosmotic 500 mOsm and oxidative 0.5 mM H2O2 stresses were imposed. TRPM2 expression was inhibited with a TRPM2 inhibitor, 20 μM N-(p-amylcinnamoyl) anthranilic acid (ACA), or TRPM2 siRNA knockdown.nnnResultsnIn the hypertonic medium, TRPM2, NLRP3, ASC, caspase-1, and IL-1β gene and protein expression levels rose after 4 hours (P ≤ 0.043), whereas ACA preincubation suppressed these rises (P ≤ 0.044). Similarly, H2O2 upregulated TRPM2 protein expression by 80%, and induced both NLRP3 inflammasome activation and increased bioactive IL-1β secretion (P ≤ 0.036), whereas ACA pretreatment suppressed these effects (P ≤ 0.029). TRPM2 siRNA transfection reduced TRPM2 gene expression by 70% (P = 0.018) in this hyperosmotic medium and inhibited the increases in NLRP3, caspase-1, and IL-1β gene (P ≤ 0.028) and protein expression (P ≤ 0.037).nnnConclusionsnTRPM2 activation by either a hyperosmotic or oxidative stress contributes to mediating increases in NLRP3 inflammasome activity and bioactive IL-1β expression because inhibiting TRPM2 activation or its expression blunted both of these responses in PHCECs. This association points to the possibility that TRPM2 is a viable target to suppress hyperosmotic-induced corneal epithelial inflammation.

Corneal Collagen Cross-Linking With Riboflavin and UVA Regulates Hemangiogenesis and Lymphangiogenesis in Rats

PurposenThe purpose of this study was to determine whether corneal collagen crosslinking (CXL) inhibits hemangiogenesis and lymphangiogenesis during acute corneal inflammation in an in vivo rat model.nnnMethodsnInflammatory corneal neovascularization was induced by suture placement into a rat cornea. At day 3 after suture, a CXL protocol using riboflavin and UVA was administered after mechanical epithelial debridement. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically. CD45 and CD68 immunostaining evaluated corneal leucocyte and macrophage immune cell infiltration, respectively. A TUNEL assay detected stromal cell apoptosis. Quantitative RT-PCR analysis identified angiogenic and lymphangiogenic genes as well as proinflammatory cytokine expression. Western blot analysis characterized vascular endothelial cell CD31 and lymphatic vessel endothelial hyaluronan receptor (LYVE-1) protein expression.nnnResultsnCXL treatment significantly reduced corneal pathologic suture-induced hemangiogenesis and lymphangiogenesis 7 days after suture emplacement, but this procedure failed to affect hemangiogenesis and lymphangiogenesis 14 days after suture. Increased cell apoptosis and reduced CD45+ and CD68+ cell infiltration were evident in CXL-treated rats on days 7 and 14 after suture emplacement. CXL treatment significantly decreased angiogenic and lymphangiogenic mRNA expression levels and both CD31 and LYVE-1 protein expression levels, whereas it increased proinflammatory cytokine levels on day 7 after suture emplacement. However, on day 14 after corneal neovascularization, angiogenic and lymphangiogenic mRNA gene expression levels were upregulated along with hematic CD31 and lymphatic LYVE-1 protein expression.nnnConclusionsnCXL treatment only temporarily inhibits corneal inflammatory-associated hemangiogenesis and lymphangiogenesis in vivo. Such insight suggests that future studies are warranted to develop novel CXL strategies with longer-lasting effectiveness in attenuating hemantic- and lymphatic-related corneal diseases.

Short-term effect of a developed warming moist chamber goggle for video display terminal-associated dry eye

BackgroundVideo display terminal (VDT)-associated dry eye (DE) patients are the rising group worldwide, and moisture goggles are the preferable treatment since they are capable of improving tear film stability and DE discomfort. The current study aims to evaluate the short-term efficacy and safety of the developed warming moist chamber goggles (WMCGs) for VDT-associated DE patients.MethodsIn this prospective self-control study, 22 DE patients (22 eyes) working with VDTs over 4xa0h daily were enrolled and instructed to wear WMCGs for 15xa0min. Sodium hyaluronate (SH, 0.1%) eyedrops were applied as a control on another day on these same patients, however 4 subjects denied the eyedrop application. The symptomatology visual analog scale (VAS) score, tear meniscus height (TMH), noninvasive tear film break-up time (NI-BUT), tear film lipid layer thickness (LLT), and bulbar conjunctival redness were assessed with Keratographxa05xa0M at baseline, 5, 30 and 60xa0min after treatment. The WMCGs wearing comfort was also evaluated.ResultsThe ocular discomfort evaluated by VAS decreased in the WMCGs group throughout 60xa0min (P<0.001), better than the control group levels (Pxa0≤u20090.015). TMH, NI-BUT (including the first BUT and average BUT) increased than baseline level accross 60xa0min in the WMCG group (Pxa0≤u20090.012), while those in the control group only showed temporary improvements in 5xa0min. LLT also increased obviously after WMCGs wear, while the change in the control group was nearly innoticeable. No adverse responses were detected.ConclusionsTemporary use of the WMCGs is able to relieve ocular discomfort, and improves tear film stability in DE patients for at least 1xa0h, making it a promising alternative to other treatments.