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.

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.

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.

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.

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.

Neutrophil collagenase, gelatinase, and myeloperoxidase in tears of patients with stevens-johnson syndrome and ocular cicatricial pemphigoid.

OBJECTIVE: To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP). DESIGN: Prospective, noninterventional cohort study. PARTICIPANTS: Four SJS patients (7 eyes), 19 OCP patients (37 eyes), and 20 healthy controls who underwent phacoemulsification (40 eyes). METHODS: Tear washes were collected from all patients and were analyzed for levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 using multianalyte bead-based enzyme-linked immunosorbent assays. Total MMP activity was determined using a fluorometric assay. Correlation studies were performed between the various analytes within study groups. MAIN OUTCOME MEASURES: Levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 (in nanograms per microgram of protein) and total MMP activity (in relative fluorescent units per minute per microgram of protein) in tears; MMP-8-to-TIMP-1 ratio; MMP-9-to-TIMP-1 ratio; and the correlations between MMP-8 and MMP-9 and both MMP and MPO. RESULTS: MMP-8, MMP-9, and MPO levels were elevated significantly in SJS and OCP tears (SJS>OCP) when compared with controls. The MMP activity was highest in SJS patients, whereas OCP patients and controls showed lower and similar activities. The TIMP-1 levels were decreased in SJS and OCP patients when compared with those in controls, with levels in OCP patients reaching significance. The MMP-8-to-TIMP-1 and MMP-9-to-TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS>OCP) when compared with those of controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels, and MMP-8 correlated with MPO, but it did not reach significance in SJS patients. There was no relationship between MMP-7 and MPO. CONCLUSIONS: Because MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes, including MMP-9, in SJS and OCP tears. Elevated MMP-to-TIMP ratios and MMP activity suggest an imbalance in tear MMP regulation that may explain the predisposition of these patients to demonstrate corneal melting and chronic complications associated with persistent inflammation. Myeloperoxidase in tears may be a sensitive and specific marker for the quantification of ocular inflammation.

Class specific peptide inhibitors for secretory phospholipases A2.

Phospholipases A2 (PLA2) catalyze the hydrolytic cleavage of free fatty acids from the sn-2 OH-moiety of glycerophospholipids. These enzymes have a number of functions, from digestion to signaling and toxicity of several venoms. They have also been implicated in inflammation and are connected to diverse diseases, such as cancer, ischemia, atherosclerosis, and schizophrenia. Accordingly, there is a keen interest to develop selective inhibitors for therapeutic use. We recently proposed a novel mechanism for the control of PLA2 activity with highly active protofibrils of PLA2 existing transiently before conversion to inactive amyloid fibrils [19]. In keeping with the above mechanism several algorithms identified (85)KMYFNLI(91) and (17)AALSYGFYG(25) in bee venom (bv) and human lacrimal fluid (Lf) PLA2, respectively, as a regions potentially forming amyloid type aggregates. Interestingly, in keeping with the proposed role of these sequences in the control of the activity of these enzymes, preincubation of 2nM bvPLA2 with (85)KMYFNLI(91) caused complete inhibition of PLA2 activity while the scrambled control peptide YNFLIMK had no effect. Approximately 36% attenuation of the hydrolytic activity of LfPLA2 present in human lacrimal fluid was observed in the presence of 80nM (17)AALSYGFYG(25).

Matrix metalloproteinase (MMP-2, -9) and tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric type 1 diabetic patients: MMPs in tear samples from type 1 diabetes.

BACKGROUND: The presence of matrix metalloproteinase (MMP-2, -9) and tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric type 1 diabetes mellitus (DM) patients and potential correlations with clinical parameters (Schirmer testing, glycosylated hemoglobin-HB(A1C)) were investigated. METHODS: Tear samples from the right eyes of 27 type 1 DM patients and 17 healthy control subjects were included in this study. The MMP gelatinolytic activity was determined by gelatin zymography analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while MMP and TIMP concentrations (in ng/ml) were quantified in tears of type 1 diabetic patients and healthy controls, with the use of enzyme-linked immunosorbent assay (ELISA). RESULTS: MMP-9, TIMP-1, -2 levels, MMP-9/TIMP-1, and MMP-9/TIMP-2 ratios in the patient group were significantly elevated. There was a significant correlation between TIMP-2 and HB(A1C) values, as well as between MMP-2 and MMP-9. CONCLUSIONS: Significant correlations between TIMP-2 and HB(A1C) and between Schirmer test results and HB(A1C) were revealed. Significant increase in tear MMP and TIMP levels in pediatric type 1 diabetic patients may be suggestive of disease progression and localized pathologic remodelling. Further studies are required in order to ascertain whether MMPs and TIMPs could be employed as indicators of early disease progression.

Effect of contact lens wear on the diurnal profile of matrix metalloproteinase 9 in tears.

PURPOSE: This study set out to determine how contact lens wear affects the profile of matrix metalloproteinase 9 (MMP-9) in the tear film. METHODS: Flush tears were collected from 47 healthy neophytes before lens wear, during the first day of lens wear, and after 1 month adaptation. Participants were randomized to either Acuvue Oasys or O2OPTIX with the choice of extended (EW) or daily wear (DW). Each time, tears were collected at midday, before sleep, and on waking and analyzed for concentrations of total protein, MMP-9, and its regulators TIMP-1 (tissue inhibitor of metalloproteinases 1) and NGAL (neutrophil gelatinase-associated lipocalin). RESULTS: Initial extended contact lens wear resulted in significantly elevated MMP-9 levels on waking (3598.7 ± 3229.1 ng/mL) compared with the same time point at baseline (2123.3 ± 1762.8; p = 0.02), whereas DW remained unchanged (2373.0 ± 2091.6 ng/mL; p = 0.61). After 1 month of EW, the levels on awakening were no longer different to those of baseline (2408.2 ± 1376.1 ng/mL; p = 0.63). The MMP:TIMP ratio during EW was greater after the first night (18.6 ± 19.9) than both no wear (13.2 ± 13.4) and 1 month (10.4 ± 7.7), but only the latter was significant (p = 0.048). The profile of NGAL did not differ from baseline (p = 0.055). CONCLUSIONS: In the neophyte, the initial period of overnight lens wear seems to disturb the tear film homeostasis, as indicated by a significant increase in MMP-9 on awakening. The return to baseline by 1 month suggests that an adaptive process takes place. No comparable changes are seen in DW.

Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, ß-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

[The estimation of systemic chemotherapy treatment administered in breast cancer on lysozyme activity in tears--preliminary report]. / Ocena wplywu systemowej chemioterapii stosowanej w leczeniu raka sutka na aktywnosc lizozymu zawartego we lzach--doniesienie wstepne.

PURPOSE: Estimation of cytostatics influence used in breast cancer treatment on lysozyme activity in human tears depend on time of treatment. MATERIAL AND METHODS: 8 women were treated at the base of chemotherapy schema: docetaxel with doxorubicin and 4 women treated with schema CMF: cyclophosphamide, methotrexate, 5-fluorouracil. Lysozyme activity in tears was assessed by measurement of diameter zone of Micrococcus lysodeicticus growth inhibition. RESULTS: It was revealed that both chemotherapy schema caused statistically significant reduction of diameter zone of M. lysodeicticus growth inhibition, after first and second course of chemotherapy treatment. After second chemotherapy course CMF schema induced loss of lysozyme activity in patient's tears (zero mm of M. lysodeicticus diameter zone growth inhibition). CONCLUSIONS: Systemic chemotherapy administered in breast cancer induce reduction of lysozyme activity in tears, that may cause higher morbidity of ocular surface infections caused by Gram-positive bacteria.

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.

MMP-9 and the perioperative management of LASIK surgery.

PURPOSE OF REVIEW: Hyperosmolarity is a central mechanism causing ocular surface inflammation and eye irritation in typical patients suffering from tear dysfunction. Tear composition in dry eyes, or dysfunctional tear syndrome, may destabilize the tear film and cause ocular surface epithelial disease. Increased activity of matrix metalloproteinases (MMPs), especially MMP-9, plays a critical role in wound healing and inflammation and is primarily responsible for the pathologic alterations to the ocular surface that leads to a dysfunctional tear state. RECENT FINDINGS: Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. The increased MMP-9 activity in dry eyes may contribute to deranged corneal epithelial barrier function, increased corneal epithelial desquamation, and corneal surface irregularity. SUMMARY: Dry eye is one of the most common complications of photorefractive keratectomy and laser in-situ keratomileusis (LASIK). LASIK has both a neurotrophic effect on the cornea and leads to a physical change in corneal shape that results in a change in tear dynamics, leading to ocular surface desiccation. The reduction in tear function after LASIK may induce an increase in osmolarity and consequently raise the concentration of proinflammatory cytokines and MMP-9 in the tear film, which results in dry eyes and insufficient attachment between the corneal flap and the corneal bed. Appropriate diagnosis and management of dysfunctional tear syndrome may lead to less postoperative LASIK complications.

MMP expression in leaking filtering blebs and tears after glaucoma filtering surgery.

OBJECTIVE: Glaucoma filtering surgery may be compromised by cystic blebs which develop more frequently when anti-metabolites are used to arrest wound healing. Matrix metalloproteinases (MMPs) and the naturally occurring tissue inhibitors of metalloproteinases (TIMPs) are essential in connective tissue remodeling and wound healing. This study aimed to determine whether filtering blebs display increased expression of MMP-2, MMP-9, TIMP-1 and TIMP-2, and whether it is reflected in tear fluid. METHODS: Tissue samples from leaking blebs (n = 5) and control conjunctiva (n = 5) were evaluated by immunohistochemistry for MMP-2, MMP-9, TIMP-1 and TIMP-2. Tear fluid was collected from 12 patients (12 eyes) with cystic blebs and ten patients (ten eyes) with flat blebs following trabeculectomy with Mitomycin C applied and 16 controls. MMP levels were evaluated by zymography and TIMP levels by Western blot analysis. RESULTS: Conjunctival tissue was obtained from five eyes with cystic leaking blebs and five control eyes undergoing cataract surgery. More extensive MMP-2 and MMP-9 expression was found in the epithelial and stromal layers of blebs than in control conjunctiva. TIMP-1and TIMP-2 were expressed in all layers of the blebs, but only in the epithelium of control conjunctiva. MMP-2 and proMMP-2 activity in tears from eyes with flat blebs was significantly higher than that of controls, while activity in tears of eyes with cystic blebs was significantly higher than in those with flat blebs. There was no difference in MMP-9 activity between tears of control and post-filtering surgery eyes. CONCLUSIONS: Increased MMPs and TIMPs expression is associated with the formation of filtering blebs, suggesting involvement of MMPs in bleb remodeling. MMP-2 and ProMMP-2 levels in tear fluid may be markers for bleb configuration.

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.

Quantification of tear proteins and sPLA2-IIa alteration in patients with allergic conjunctivitis.

PURPOSE: Allergic conjunctivitis (AC) has been reported to induce the instability of the tear film. The tear protein and the lipid layer play important roles in maintaining the tear film. The aim of this study was to quantify the alteration of the major tear protein components and a lipid related protein secretory type IIa phospholipase A2 (sPLA2-IIa) in tears of seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) patients. METHODS: Twenty-one SAC and PAC patients and thirteen normal controls completed a symptom questionnaire and underwent regular ocular examination. SAC and PAC patients were diagnosed based on the clinical presentation and elevated serum IgE levels. Schirmer test paper was used to collect tear samples from SAC and PAC patients and normal controls. Soybean trypsin inhibitor (SBTI) was used as an internal standard to analyze tear samples in 15% SDS-PAGE gel. Total tear protein and its major components from the SAC and PAC patients and normal controls were quantified by band densitometry. The major tear protein bands were determined by MALDI-TOF/TOF spectrum analysis. Western blot was used to detect the content of sPLA2-IIa in tears of allergic conjunctivitis patients and normal controls. RESULTS: Schirmer test scores were more than 10 mm in all the SAC and PAC patients and control subjects. The tear film breakup time of SAC and PAC patients was much shorter than that of the normal controls. We obtained 15 bands of tear protein by one dimensional SDS-PAGE, in which 14 bands were determined by mass-spectrum analysis. The band densitometry analysis revealed that the total tear protein concentration was much higher in SAC and PAC patients than in normal controls (p<0.05). The quantity of tear protein band 4 (serum albumin precursor), band 6 (Ig gamma-2), band 9 (leukocyte elastase inhibitor) were also significantly higher in AC patients (p<0.05). Content of sPLA2-IIa, as shown by western blot, was much higher in AC patients than in controls. CONCLUSIONS: The total tear protein concentration and some of the major tear protein components was increased in tears of SAC and PAC patients. In addition, the content of sPLA2-IIa in tears of SAC and PAC patients was elevated. The tear protein changes in SAC and PAC patients may contribute to instability of tear film.

Tear lipocalin and lysozyme concentrations in postmenopausal women.

PURPOSE: To investigate the potential relationship between subjective symptomatology, tear volume, and tear break up time with tear film lipocalin and lysozyme concentrations in a group of symptomatic dry-eyed postmenopausal (PM) women compared to age-matched controls. METHODS: Eighty-five healthy PM females (>50 years of age) were categorized as mild or moderate dry eye (DE), or asymptomatic [non-dry eye (NDE)] based on their responses to the Ocular Surface Disease Index (OSDI) questionnaire. Non invasive tear breakup time (NITBUT) and tear secretion were measured. Tears were collected via capillary tube and an eye wash method. Tear lysozyme and lipocalin concentrations were determined via Western blotting. RESULTS: Ocular Surface Disease Index responses revealed 16 mild DE, 30 moderate DE, and 39 NDE. The OSDI total score and sub scores for the DE groups were significantly greater than for the NDE group (p < 0.001). The mild and moderate DE groups exhibited significantly shorter NITBUTs compared to NDE (p < 0.004). Tear secretion using the Phenol Red Thread (PRT) test was found to be significantly lower in the moderate DE group compared to NDE (p < 0.001). No difference in tear lysozyme or lipocalin concentration was found between DE and NDE groups, irrespective of tear collection method, although method of collection significantly influenced absolute concentrations (p < 0.008). Significant correlations were not found between symptoms or signs of DE compared to either lipocalin or lysozyme concentration. CONCLUSION: Within a PM population, lipocalin and lysozyme are invariant, irrespective of the presence and severity of DE symptoms. This is the first comprehensive study of lipocalin and lysozyme in dry-eyed PM women and our results suggest that neither protein would offer utility as a biomarker of DE.

[The levels of endothelin, neuron-specific enolase, and their autoantibodies in the serum and tear fluid of patients with type 2 diabetes mellitus].

Enzyme immunoassay was used to measure the serum and tear fluid concentrations of endothelin, neuron-specific enolase (NSE), and their autoantibodies in 19 patients with nonproliferative diabetic retinopathy (NDR). The serum and tear fluid levels of endothelin were shown to increase in patients with NDR. At the same time, the content of endothelin-1 autoantibodies rose in the blood and remained in the normal range in the tear fluid. The concentration of NSE showed an 18-fold increase in the tear fluid and the content of its autoantibodies remained unchanged.

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.

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.