Bilateral Increase of Matrix Metalloproteinase-9 Expression in Tears After the Unilateral Decrease of Corneal Sensitivity.

PURPOSE: The purpose of this study is to analyze bilateral changes of matrix metalloproteinase-9 (MMP-9) expression in the tear film in patients with unilateral neurotrophic keratitis. METHODS: We included 24 eyes from 24 subjects with normal corneal sensitivity according to Cochet-Bonnet corneal esthesiometry in the control group and 25 subjects with the unilateral neurotrophic keratitis in the unilateral decrease of corneal sensitivity (UDCS) group. The expression of MMP-9 in tears was assessed using point-of-care immunoassay. The clinical parameters including meibomian gland plugging, quality of the secreted meibum, tear secretion using Schirmer I without anesthesia, and corneal erosions were evaluated among the control, affected, and contralateral unaffected eyes in the UDCS group. RESULTS: Tear MMP-9 grades in affected (2.5 ± 1.0) and contralateral eyes (2.6 ± 0.9) in the UDCS group were higher than those in control eyes (1.5 ± 0.7, P = 0.0003 and P = 0.0006, respectively). The lacrimal secretion decreased in the affected eyes but preserved in the contralateral eyes. There was no difference in corneal erosion scores between control eyes and contralateral eyes in the UDCS group. Tear MMP-9 grades in the contralateral eyes were significantly correlated with the meibum quality grades in the contralateral eyes (r = 0.525 and P = 0.025). CONCLUSIONS: Patients with the unilateral neurotrophic keratitis demonstrated a significant bilateral increase of the MMP-9 expression in the tear film as compared with controls. Despite the tearing reflex and corneal surface barrier being preserved in the unaffected contralateral eyes, it is necessary to pay attention to the possible attenuation of meibomian gland function in the opposite eyes as well.

Association of tear matrix metalloproteinase 9 immunoassay with signs and symptoms of dry eye disease: A cross-sectional study using qualitative, semiquantitative, and quantitative strategies.

PURPOSE: This study aimed to analyze the association of tear matrix metalloproteinase 9 (MMP-9) immunoassay with the severity of dry eye (DE) signs and symptoms through qualitative, semiquantitative, and quantitative evaluations of immunoassay band. MATERIALS AND METHODS: This cross-sectional study enrolled 320 eyes of 320 patients. The clinical signs of DE were assessed using the Ocular Surface Disorder Index (OSDI) score, visual analogue scale (VAS), tear breakup time (tBUT), tear volume evaluation by tear meniscometry, and staining scores of the cornea and conjunctiva by the Oxford grading scheme. The tear MMP-9 immunoassay results were interpreted using qualitative (positive or negative), semi-quantitative (reagent band density on a four-point scale: 0 = negative; 1 = weakly positive; 2 = moderately positive; 3 = strongly positive), and quantitative (ratio of reagent band density to control band density) indicators. RESULTS: Positive MMP-9 immunoassay results were significantly related to shorter tBUT, tBUT ≤3 seconds, higher corneal staining score, corneal staining score ≥2, and conjunctival staining score ≥2. The semi-quantitative results of the MMP-9 immunoassay were positively correlated with higher corneal staining score (r = 0.122, p = 0.029) and negatively correlated with tBUT (r = -0.125, p = 0.025). However, in the quantitative analysis, none of the DE signs or symptoms were correlated to the band density of the MMP-9 immunoassay. CONCLUSIONS: The positive MMP-9 immunoassay results were related to the severity of ocular signs of DE. However, using quantitative measures of the MMP-9 immunoassay to assess the clinical severity of DE requires further investigation.

In vitro validation of the tear matrix metalloproteinase 9 in-situ immunoassay.

We aimed to validate a tear MMP-9 in-situ immunoassay (InflammaDry) and to identify factors that could affect results or interpretation. Three factors were examined: sample concentration, volume, and time. Recombinant human (rh) MMP-9 (10 or 20 µl; 0, 12.5, 25, 50, 100, 200, 500, and 1,000 ng/ml) was applied to the kit and the detection limit and assay reproducibility were examined. At a rhMMP-9 volume of 10 µl (≥ 50 ng/ml), all positive results were identified by densitometry at 10 and 20 min; however, after 20 min, more than half of the nine ophthalmologists interpreted a positive result. At a rhMMP-9 volume of 20 µl (≥ 25 ng/ml), ophthalmologists and densitometry identified almost all test lines at 10 and 20 min. At 10 µl, densitometry showed a linear dose-response pattern. At 20 µl, densitometry showed a linear dose-response pattern at concentrations up to 500 ng/ml; however, full saturation was achieved at concentrations ≥ 500 ng/ml. When the same amount of rhMMP-9 was applied, the density result increased significantly upon doubling of the solvent volume (i.e., by adding the same volume of PBS to a sample). InflammaDry showed a high inter- and intra-assay coefficient of variation at 10 min (28.4% and 24.7%, respectively). The results of the MMP-9 in-situ immunoassay varied significantly depending on sample volume. Therefore, when interpreting the results, careful attention must be paid to tear volume.

Tear Osmolarity and Matrix Metallopeptidase-9 in Dry Eye Associated with Sjögren's Syndrome.

PURPOSE: To evaluate the correlations between tear osmolarity and matrix metallopeptidase-9 (MMP-9) and dry eye (DE) indices in patients with DE associated with Sjögren's syndrome (SS). METHODS: Sixty-three patients with DE associated with SS who underwent tear analysis were included. DE tests performed were ocular surface disease index, tear break-up time, Schirmer's test, ocular staining score, and tear osmolarity and MMP-9 tests. Correlations between tear osmolarity and DE indices, differences between patients with abnormal and normal tear osmolarity, and those between positive and negative MMP-9 patients were analyzed. Patients were classified into four groups according to tear osmolarity and MMP-9 results, and between-group differences were analyzed (group 1: abnormal tear osmolarity, MMP-9 positive; group 2: abnormal tear osmolarity, MMP-9 negative; group 3: normal tear osmolarity, MMP-9 positive; group 4: normal tear osmolarity, MMP-9 negative). RESULTS: Mean age of patients was 54.2 ± 13.9 years, and 96.2% were female. Thirty-five patients had abnormal tear osmolarity and 40 patients were MMP-9 positive. DE indices differed between groups with abnormal and normal tear osmolarity (p < 0.01), but not between positive and negative MMP-9 groups. There were 22 patients in group 1, 13 in group 2, 18 in group 3, and 10 in group 4. Compared to group 4, tear break-up time was shorter in groups 1 (p < 0.01) and 2 (p = 0.02). Schirmer's test values in group 1 were lower than those in group 4 (p = 0.03). Ocular staining score was higher in groups 1 (p < 0.01) and 2 (p < 0.05) than in group 4. CONCLUSIONS: Tear osmolarity was correlated with ocular surface indices in DE associated with SS. Combination of tear osmolarity and MMP-9 test results may be helpful to determine the severity of DE associated with SS.

Changes in the Matrix Metalloproteinase 9 Point-of-Care Test Positivity According to MMP-9 Concentration and Loading Volume.

PURPOSE: To measure changes in the matrix metalloproteinase 9 (MMP-9) point-of-care test, InflammaDry (Rapid Pathogen Screening, Inc, Sarasota, FL) positivity, based on ocular surface MMP-9 concentrations and loading volume. METHODS: Two different MMP-9 products, preform and active, were analyzed using the InflammaDry test, detecting MMP-9 levels of more than 40 ng/mL of both preform and active MMP-9. Preform MMP-9 (Natural human MMP-9 protein; Abcam, Cambridge, UK) was analyzed at different concentrations (50, 100, 500, 1000, and 1500 ng/mL) and loading volumes (5, 10, and 20 µL). Active MMP-9 (Human MMP-9 protein; Novus Biologicals, Littleton, CO) was also analyzed using the InflammaDry test at different concentrations (50 and 100 ng/mL) and loading volumes (10, 20, and 40 µL). RESULTS: Natural human MMP-9 protein (preform) of 50, 100, and 500 ng/mL exhibited negative results for every loading volume. At 1000 ng/mL, the 20 µL volume was positive, whereas the 5 and 10 µL volumes were negative. At 1500 ng/mL, all loading volumes were positive, but the density of positive bands varied depending on the loading volume; larger loading volumes had higher band density. Human MMP-9 protein (active) of 50 ng/mL was negative for every loading volume. In 100 ng/mL, the 20 and 40 µL volumes showed positive results with similar positive band densities. CONCLUSIONS: The InflammaDry test had a different detection range depending on MMP-9 formulas; higher concentrations of preform MMP-9 protein were needed to yield positive results. In addition, InflammaDry positivity varied based on the loading volumes. Clinicians should be aware of the possibility of false negatives with low tear volumes despite elevated MMP-9 concentrations.

Oxidative stress markers dynamics in keratoconus patients' tears before and after corneal collagen crosslinking procedure.

Keratoconus (KC) is a controversial ophthalmological disease, often considered both multifactorial and multigenic with poor or not entirely understood etiopathogenesis. Corneal collagen crosslinking (CXL) procedure is the most common surgical therapy for KC which both slows corneal thinning and halts disease progression. While extensive studies provide consistent evidence on systemic oxidative stress in KC patients and animal models, little is known on the tear fluid oxidative stress markers such as antioxidant enzymes activity or lipid peroxidation markers. Also, little is known considering the oxidative status dynamics following CXL. In this way, we aimed to evaluate three oxidative stress markers in the tears of KC patients before and after CXL procedure. Total superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymatic activity and malondiladehyde (MDA) levels were assessed from the tears of 20 kC patients who received the recommendation for CXL procedure. Significantly decreased SOD activity (p = 0.0014) was observed in KC patients tears, as compared to age and sex-matched controls which could lead to significant lipid peroxidation boost (p < 0.001). Significantly higher GPx enzyme activity was observed in KC patients, as compared to control (p < 0.001), suggesting a compensatory response to intense lipid peroxidation. Following CXL, SOD activity significantly decreases and GPx activity extensively increases, as compared to baseline KC levels and controls (p < 0.001). This work provides additional evidence on oxidative stress status in the tears of KC considering general oxidative stress markers dynamics both before and after the CXL procedure. We also demonstrated that the CXL procedure could have further relevance in the management of this disorder.

Differential effects of specific cathepsin S inhibition in biocompartments from patients with primary Sjögren syndrome.

OBJECTIVE: Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. METHODS: Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1ß, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. RESULTS: CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. CONCLUSION: CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS.

The Protective Effect of a Topical Mucin Secretagogue on Ocular Surface Damage Induced by Airborne Carbon Black Exposure.

Purpose: Exposure to airborne particulate matter can induce ocular surface damage and inflammation. We evaluated the effects of a topical mucin secretagogue on the mitigation of ocular surface damage induced by exposure to airborne carbon black (CB). Methods: Sprague-Dawley rats were exposed to ambient CB for 2 hours twice daily for 5 days. Corneal staining score and tear lactic dehydrogenase (LDH) activity were measured to evaluate ocular surface damage. Serum immunoglobulin (Ig) G and IgE levels and the sizes of cervical lymph nodes were also measured. The expressions of interleukin (IL)-4, IL-17, and interferon (IFN)-γ were measured by Western blot analysis. Diquafosol tetrasodium was instilled six times a day for 5 days, and the extent of ocular surface damage was evaluated. Results: After exposure to airborne CB, the median corneal staining score and LDH activity were significantly increased. Serum IgG and IgE levels and the sizes of cervical lymph nodes were also significantly increased. Additionally, the expression of IL-4 and IFN-γ was elevated in the anterior segment of the eyeball. Furthermore, the expression of IL-4, IL-17, and IFN-γ was elevated in the cervical lymph nodes. When exposed to airborne black carbon, topical diquafosol tetrasodium significantly increased tear MUC5AC concentration and decreased tear LDH activity. Conclusions: Exposure to airborne CB induced ocular surface damage and increased proinflammatory cytokines in the eyes and cervical lymph nodes. Topical mucin secretagogues seem to have a protective effect on the ocular surface against exposure to airborne particulate matters.

Tear Martix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 in Post-Lasik Ectasia.

PURPOSE: To estimate the concentrations of matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase-1 (TIMP-1) in the tear film of cases with post-Lasik ectasia (PLE) to spot any role of these mediators. SETTINGS: Ophthalmology department, Benha University hospitals, Egypt. METHODS: Twelve eyes of 12 patients with PLE, 30 eyes of 30 patients with KC, 25 eyes of 25 subjects with uncomplicated Lasik and finally 25 eyes of 25 healthy subjects as a control group were studied. Subjects with ocular surface diseases, previous ocular surgeries except for Lasik in PLE group and Lasik group, were excluded. All subjects had full ophthalmic examination and Pentacam imaging. The concentration of tear MMP-9 and TIMP-1 was measured by ELISA. RESULTS: Our results showed a significant elevation in the level of MMP-9 and a significant reduction in the level of TIMP-1 in tear samples from PLE cases (MMP-9 was 59.17 ± 28.15 ng/ml, and TIMP-1 was 110.3 ± 50.6 ng/ml) and also in KC cases (MMP-9 was 53.12 ± 17.35 ng/ml, and TIMP-1 was 105.8 ± 56.3 ng/ml) when compared to post-Lasik group (MMP-9 was 35.65 ± 17.32 ng/ml, and TIMP-1 was 155.2 ± 39.4 ng/ml) and control group (MMP-9 was 31.92 ± 20.78 ng/ml, and TIMP-1 was 162.5 ± 48.2 ng/ml). CONCLUSION: The results pointed to potential role of MMP-9 in the pathogenesis of PLE and also referred to a biochemical similarity between PLE and KC. More studies are needed in the future to investigate larger number of tear mediators.

Prevalence of ocular surface dysfunction in patients presenting for cataract surgery evaluation.

PURPOSE: To report the prevalence of ocular surface dysfunction in patients presenting for cataract surgery evaluation. SETTING: Duke University Eye Center and Weill Cornell Ophthalmology, single-physician practices. DESIGN: Prospective case series. METHODS: Consecutive patients presenting for cataract surgery evaluation were identified. Patient information including demographics, medical history, slitlamp findings, tear osmolarity, and tear matrix metalloproteinase-9 (MMP-9) levels were recorded. Patients were considered to have ocular surface dysfunction if any of the following outcomes were present: visually significant abnormal corneal surface examination, positive MMP-9 test, or abnormal osmolarity values (>307 mOsm/L or >7 mOsm/L intereye difference). Patient symptoms were recorded using the ocular surface disease index (OSDI) or Symptom Assessment iN Dry Eye questionnaires. RESULTS: There were 120 patients (69% women), mean age 69.5 years ± 8.4 (SD). Abnormal osmolarity was found in 68 patients (56.7%), and abnormal MMP-9 in 76 patients (63.3%). Clinical findings showed that 47 patients (39.2%) had positive corneal staining on presentation, 9 patients (7.5%) had epithelial basement membrane dystrophy, and 2 patients (1.6%) had Salzmann nodules. Questionnaire data showed 54 (54.0%) of 100 patients reported symptoms suggestive of ocular surface dysfunction. In the asymptomatic group of 46 patients, 39 (85%) had at least 1 abnormal tear test (osmolarity or MMP-9) and 22 (48%) had both tests abnormal. Overall, 96 (80%) of 120 patients had at least 1 abnormal tear test result suggestive of ocular surface dysfunction and 48 patients (40%) had 2 abnormal results. CONCLUSIONS: Objective ocular surface dysfunction findings were common among patients presenting for cataract surgery, yet many presented undiagnosed. Clinicians should be aware of this high prevalence and consider screening with tear testing before surgery.

Functional Activity of Matrix Metalloproteinases 2 and 9 in Tears of Patients With Glaucoma.

Purpose: To evaluate the differential expression of tear matrix metalloproteinases (MMP) 2 and 9 in of patients with various forms of glaucoma. Methods: Tear samples were collected with a Schirmer's strip from 148 eyes of 113 patients (medically naïve patients with primary open-angle [POAG] or angle closure glaucoma [PACG] and those with pseudoexfoliation syndrome [PXF] or glaucoma [PXG]). These were compared to patients undergoing cataract surgery (controls) for this cross-sectional study. Functional activities of tear MMP-9 and MMP-2 were analyzed by gelatin zymography. Tenon's capsules (n = 15) were harvested from the inferior quadrant in those undergoing cataract surgery and protein expression of MMP-9 was analyzed by immunohistochemistry (IHC). Hydrogen peroxide (H2O2) stress-induced effects on in vitro activities of MMP-9 in human trabecular meshwork (HTM) cells were analyzed. Results: The MMP-9 activity in tears was increased significantly in POAG, (n = 27), PACG (n = 24), and PXF (n = 40) eyes compared to controls (n = 35), and was increased significantly in eyes with glaucoma compared to moderate/severe glaucoma (P < 0.001). The MMP-9 expression was significantly lower in PXG (n = 22) eyes. Immunohistochemistry of Tenon's capsule revealed increased expression of MMP-9 in primary glaucoma eyes. Increased MMP-9 activity was seen in in vitro by gelatin zymography and was confirmed by Western and immunofluorescent assay on HTM upon 800 and 1000 µM H2O2-induced stress for 2 to 3 hours with approximately 80% cell death. Conclusions: Increased tear MMP-9 activity in early glaucoma and pseudoexfoliation syndrome suggesting activation of extracellular matrix (ECM) degradation can be used as a tear-based predictive biomarker. Decreased expression in advanced stages suggests exhaustion of the degradation response.

Elevated Neutrophil Elastase in Tears of Ocular Graft-Versus-Host Disease Patients.

PURPOSE: To investigate the levels of neutrophil elastase (NE), matrix metalloproteinases (MMPs), and myeloperoxidase (MPO) in tear washes of patients with ocular graft-vs-host disease (oGVHD). DESIGN: Case-control study. METHODS: Based on established criteria, oGVHD patients (n = 14; 28 eyes) and age-/sex-matched healthy controls (n = 14; 28 eyes) were enrolled. Tear washes were collected and analyzed for NE using a single-analyte enzyme-linked immunosorbent assay (ELISA). MMPs (1, 2, 3, 7, 8, 9, 12), MPO, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were analyzed using multianalyte bead-based ELISA assays. Total MMP activity was measured using a fluorimetric assay. Correlation studies were performed between NE, MMP-8, MMP-9, and MPO within study groups. RESULTS: NE, MMP-8, MMP-9, and MPO levels were elevated in oGVHD tears when compared with controls (P < .0001). NE was the most elevated analyte. MMP activity was higher and TIMP-1 levels were lower in oGVHD than in control (P < .0001). In oGVHD, NE significantly correlated with MMP-8 (r = 0.92), MMP-9 (r = 0.90), and MPO (r = 0.79) (P < .0001). MMP-8 correlated with MMP-9 (r = 0.96, P < .0001), and MPO (r = 0.60, P = .001). MMP-9 correlated with MPO (r = 0.55, P = .002). In controls, NE, MMP-9, and MPO significantly correlated with each other (P < .0001). CONCLUSIONS: The marked increase in NE in oGVHD tears that correlated strongly with elevated MMP-8, MMP-9, and MPO suggests a common neutrophilic source and provides evidence of neutrophil activity on the ocular surface of oGVHD patients.

Heparanase Overexpresses in Keratoconic Cornea and Tears Depending on the Pathologic Grade.

BACKGROUND: Keratoconus has classically been defined as a noninflammatory disorder, although recent studies show elevated levels of inflammatory markers suggesting that keratoconus could be, at least in part, an inflammatory condition. Heparanase upregulation has been described in multiple inflammatory disorders. In this article, we study the differential expression of heparanase in cornea and tears from keratoconus patients and healthy controls. METHODS: A transcriptomic approach was used employing quantitative polymerase chain reaction to analyze the expression of heparanase and heparanase 2 in stromal and epithelial corneal cells. The protein expression was analyzed by immunohistochemistry in corneal sections. Enzymatic activity in tears was measured using [3H]-labeled heparan sulfate as substrate. RESULTS: Heparanase transcription was detected in stromal and epithelial cells and appeared upregulated in keratoconus. Overexpression of the enzyme was also detected by immunohistochemistry. Corneal expression of heparanase 2 was detected in some cases. Heparanase catalytic activity was found in tears and displayed a positive correlation with the degree of keratoconus. CONCLUSIONS: Heparanase overexpresses in keratoconic corneas, possibly reinforcing the inflammatory condition of the pathology. The presence of heparanase activity in tears allows us to propose its use as a biomarker for the diagnosis of the disorder.

Matrix Metalloproteinase 9 Testing in Dry Eye Disease Using a Commercially Available Point-of-Care Immunoassay.

PURPOSE: To measure matrix metalloproteinase 9 (MMP-9) in the tear film of patients with dry eye disease (DED) compared with controls and to correlate clinical findings. DESIGN: In a prospective study, 101 patients and controls underwent MMP-9 testing of the tear film. Thereafter, they were evaluated for symptoms and signs of DED. PARTICIPANTS: Included patients were those who showed 3 of the following 4 dry eye criteria: ocular surface disease index (OSDI) score of more than 12, tear film break-up time (TBUT) of 10 seconds or less, Schirmer test results without anesthesia of less than 10 mm/5 minutes, and corneal staining results of 1 or more. Fifty-four healthy eyes and 47 eyes fulfilling diagnostic criteria for DED of various levels of severity were included in this study. METHODS: The tear film was analyzed for MMP-9 by a commercially available test (InflammaDry; Rapid Pathogen Screening, Inc, Sarasota, FL) detecting MMP-9 levels of more than 40 ng/ml. Symptoms and signs of DED were evaluated using the OSDI questionnaire, TBUT, conjunctival and corneal staining, Schirmer test results without anesthesia, and meibomian gland examination. These findings were correlated to results of the MMP-9 test in tears. MAIN OUTCOME MEASURES: Positive MMP-9 results in tears. RESULTS: In 19 of 47 patients confirmed with dry eye (40.4%) and in 3 of 54 controls (5.6%), the MMP-9 results were positive. This difference was statistically significant (P < 0.001). Thus, the MMP-9 results indicated a clinically significant inflammation in 40% of dry eye patients. Positive results correlated well with subjective symptoms of DED evaluated by OSDI (P = 0.001), TBUT of less than 5 seconds (P < 0.013), Schirmer test results (P < 0.001), conjunctival staining (P < 0.001), and corneal staining (P = 0.007). Moreover, MMP-9 results correlated with the number of obstructed meibomian ducts (P = 0.005) and a pathologic meibomian gland secretion (P = 0.001). The MMP-9 results were increased significantly in women (P < 0.001) and in patients with autoimmune disease (P = 0.005), especially Sjögren's syndrome (P = 0.001) and thyroid disease (P = 0.012). CONCLUSIONS: Matrix metalloproteinase 9 testing in DED is a valuable new diagnostic tool. It correlated well with other dry eye tests and identified the presence of ocular surface inflammation in 40% of confirmed dry eye patients. It may be especially helpful to identify patients with ocular surface inflammation and autoimmune disease and may facilitate the decision to institute anti-inflammatory treatment in these patients.

Protein-directed synthesis of highly monodispersed, spherical gold nanoparticles and their applications in multidimensional sensing.

An in-situ reduction method has been reported to prepare gold nanoparticles (GNPs) of 40-110 nm by using the green reducing agents of proteins, which are activated by H2O2 and the superoxide anion (). The protein of collagen turns HAuCl4 to the aqueous Au(I) ainions, which are further reduced by other proteins to be highly monodispersed and spherical GNPs of different sizes. The GNPs reduced by different proteins are found to be with the exposed {100} facets, the distinctive UV-vis absorption spectra and various colors (See Fig. 1). By means of extracting the color responses, such as red, green and blue (RGB) alterations, an in-situ reduction method-based multidimensional sensing platform is fabricated in the process of GNPs synthesis. Without further modification of GNPs, nine common proteins are found to be well detected and discriminated at different concentrations. Moreover, this sensing platform also demonstrates great potentials in qualitative and semiquantitative analysis on the individuals of these proteins with high sensitivity. Furthermore, the validation of this multidimensional sensing platform has been carried out by analysis on the spiked proteins in human urine and the target proteins in complex matrix (e.g. lysozyme in human tear).

Tear Matrix Metalloproteinases and Myeloperoxidase Levels in Patients With Boston Keratoprosthesis Type I.

PURPOSE: To investigate the tear levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 in eyes after Boston keratoprosthesis type I (B-KPro) implantation and to correlate these markers with the established B-KPro prognostic categories. METHODS: Tear washes were collected from 40 patients (7 with autoimmune disease, 2 with chemical burn, and 31 with other noncicatrizing diagnoses). Tear levels of MMPs, MPO, and tissue inhibitor of metalloproteinase-1 were quantified using multianalyte bead-based enzyme-linked immunosorbent assays. The total MMP activity was determined using a fluorimetric assay. The analytes were compared to the underlying diagnosis and other clinical factors. RESULTS: The MMP-8, MMP-9, and MPO levels were markedly elevated in the eyes with B-KPro (80 ± 31, 291 ± 77, and 244 ± 33 pg/µg, respectively). Chemical burn was associated with significantly higher tear MMP-8 (474 ± 376 pg/µg) and MMP-9 levels (1300 ± 635 pg/µg) compared with noncicatrizing diseases (MMP-8: 41 ± 15 pg/µg, P = 0.02 and MMP-9: 196 ± 57 pg/µg, P = 0.02) and higher MMP-9 levels compared with autoimmune diseases (MMP-8: 96 ± 65 pg/µg, P = 0.21 and MMP-9: 306 ± 196 pg/µg, P = 0.04). Similar analyte levels were observed in the B-KPro eye and the contralateral non-B-KPro eye of patients with bilateral diseases. MMP-8, MMP-9, and total MMP activities correlated strongly with each other. CONCLUSIONS: In the eyes with B-KPro, tear MMP-8 and MMP-9 levels seem to be related to the underlying ocular surface pathology and not significantly influenced by the presence of the prosthesis.

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome.

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS.

Evaluation of point-of-care test for elevated tear matrix metalloproteinase 9 in post-LASIK dry eyes.

AIM: To evaluate the performance of a point-of-care test for detection of matrix metalloproteinase 9 (MMP-9) levels in post-laser-assisted in situ keratomileusis (LASIK) dry eyes. METHODS: A comparative study between patients with mild to moderate post-LASIK dry eyes and age-matched normal subjects was conducted. Ocular surface disease index (OSDI), tear break-up time (TBUT), and tear film MMP-9 and total protein levels were compared between the two groups. A point-of-care test device (RPS InflammaDry, Sarasota, Florida, USA) was utilised to confirm elevated MMP-9 levels in tear film. RESULTS: Fourteen post-LASIK dry eyes and 34 normal eyes were included. There was no significant difference in age and gender between both groups (p>0.175). The OSDI was significantly higher (25.5±7.7 vs 7.4±2.5; p<0.001) and TBUT levels were significantly lower (5.4±0.9 vs 13.5±2.3; p<0.001) in patients with dry eye compared with normal subjects. The tear film MMP-9 levels were 52.7±32.5 ng/mL in dry eyes and 4.1±2.1 ng/mL in normal eyes (p<0.001). MMP-9 levels were >40 ng/mL in 7/14 (50.0%) post-LASIK dry eyes. The InflammaDry was positive in 8/14 (57.1%) post-LASIK eyes. All positive cases had tear film MMP-9 levels ≥38.03 ng/mL. Agreement between InflammaDry and MMP-9 was excellent with Cohen κ value of 0.857 in post-LASIK dry eyes. CONCLUSIONS: Only half of post-LASIK dry eyes were found to have significant inflammation associated with elevated MMP-9. The OSDI is useful to non-specifically identify patients with symptomatic dry eye while the InflammaDry determined which patients with dry eye were associated with significant inflammation that may guide therapeutic management decisions.

Alterations in Tear Biochemistry Associated with Postanesthetic Chronic Dry Eye Syndrome.

Perioperative dry eye syndrome (DES) is a common ocular complication of long-term general anesthesia. Chronic DES can lead to permanent damage to the cornea and disturbance of visual function, up to total loss of vision. Here, a relationship between the duration of general anesthesia and the risk of chronic DES in patients was demonstrated. Using an experimental model of perioperative corneal abrasions in rabbits, it was found that introduction of animals to 3-h general anesthesia resulted in clinically significant chronic damage to the cornea in 50% of cases. The development of the complication was not associated with irreversible or long-term impairment of tear secretion, but it was accompanied by a decrease in tear film stability and growth of the total protein content as well as decrease in total antioxidant activity of the tear induced by low molecular weight antioxidants. In addition, anesthesia-induced changes in activity of tear antioxidant enzymes including superoxide dismutase and enzymes providing homeostasis of reduced glutathione (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) were observed. All these alterations were protracted (up to 1-2 weeks) and therefore might account for transition of the perioperative DES into the chronic form. These findings can be useful in the development of novel approaches for the prevention and treatment of chronic forms of DES in the postanesthetic period.

Role of Matrix Metalloproteinases 2 and 9 in Lacrimal Gland Disease in Animal Models of Sjögren's Syndrome.

PURPOSE: Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. METHODS: The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. RESULTS: There was a 2.5- and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. CONCLUSIONS: We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands.