The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

High Level of Inflammatory Cytokines in the Tears: A Bridge of Patients with Concomitant Exotropia and Dry Eye.

Concomitant exotropia have obvious symptoms of eye discomfort in adults, and the presence of ocular surface inflammation in patients may be important mediators between concomitant exotropia and dry eye. Oculus Keratograph eye comprehensive analyzer was performed to detect noninvasive tear break time, noninvasive tear height, and eye red index, while the ocular surface disease index and schirmer I testing were made. The levels of IL-6, IL-10, IL-17A, IL-12P70, INF-γ, and TNF-α were detected in tears in patients with concomitant exotropia and healthy controls matched by age and gender through the Simoa technology. IL-6 was significantly higher in patients with concomitant exotropia (4.683 ± 1.329) pg/mL than that in normal group (1.455 ± 0.391) pg/mL, p = 0.0304. TNF-α was also significantly higher in patients (0.2095 ± 0.0703) pg/mL than normal group (0.0513 ± 0.0149) pg/mL, p = 0.0397. The levels of inflammatory factors in strabismic patients vs. normal controls were as follows: IL-17A (0.1551 pg/mL︰0.0793 pg/mL), IL-10 (0.3358 pg/mL︰0.0513 pg/mL), IL-12p70 (0.0253 pg/mL︰0.0099 pg/mL), and INF-γ (0.0284 pg/mL︰0.009 pg/mL) were detected, and the median of them in concomitant strabismus was 1.96-6.55-fold as much as the control group. High levels of inflammatory cytokines in tears of patients with concomitant exotropia, which may be a potentially factor promoted the occurrence of dry eye in the patients with concomitant exotropia.

Corneal sub-basal nerve plexus assessment and its association with phenotypic features and lymphocyte subsets in Sjögren's Syndrome.

PURPOSE: To assess and compare corneal sub-basal nerve plexus morphology with circulating lymphocyte subsets, immunologic status and disease activity in Sjögren syndrome (SjS) patients. METHODS: Fifty-five SjS patients, 63 Sicca patients (not fulfilling SjS criteria), 18 rheumatoid arthritis (RA) patients and 20 healthy controls (HC) were included. Systemic disease activity in SjS was assessed with the ESSDAI score. Lymphocyte subpopulations were studied with flow cytometry. Corneal confocal microscopy and ImageJ software were used to characterize corneal sub-basal nerve plexus in terms of nerve density (CNFD), length (CNFL) and tortuosity (CNFT). Conventional dry eye tests were also performed. RESULTS: CNFL and CNFD were lower in SjS, Sicca and RA groups, compared to HC (p < 0.001 for both SjS and Sicca); CNFL p = 0.005, CNFD p = 0.018 in RA). CNFT was higher in SjS, followed by Sicca, RA and HC. A negative correlation was found between ESSDAI score and CNFL (r=-0.735, p = 0.012). CNFL correlated negatively with IL21+ CD8+ T cells (r=-0.279, p = 0.039) and a positively with total memory (r = 0.299, p = 0.027), unswitched memory (r = 0.281, p = 0.038) and CD24Hi CD27+ (r = 0.278, p = 0.040) B cells. CNFD showed a tendency to significance in its negative correlation with ESSDAI (r=-0.592, p = 0.071) and in its positive correlation with switched memory B cells (r = 0.644, p = 0.068). CONCLUSIONS: This is the first study aiming to correlate ocular findings with lymphocyte subsets in SjS. The associations founded between CNFL and CNFD and disease activity, IL21+ follicular T cells and some B-cell subsets suggest that corneal nerve damage may parallel systemic disease activity and inflammatory cells' dynamics.

Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation.

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Tear film analysis and evaluation of optical quality: A review of the literature.

Dry eye is a complex multifactorial disease of the ocular surface and tears. It is associated with ocular surface symptoms and is one of the most common causes for ophthalmologic consultation. Despite their frequent use in clinical practice, the usual tests to evaluate dry eye and ocular surface disease-history of symptoms, tear break-up time (TBUT), Meibomian gland evaluation, corneal fluorescein staining, Schirmer test-have shown low reproducibility and reliability. In addition, subjective symptoms are often weakly or poorly correlated with objective signs. Since the tear film is the first system through which light must pass, the optical quality of the eye is highly dependent on the homogeneity of the tear film. Various investigative methods have been developed to evaluate both the structural and functional quality of the tear film, such as corneal topography, interferometry, tear meniscus measurement, evaporation rate, tear osmolarity and even aberrometry. Some are easily accessible to clinicians, while others remain in the field of clinical research. All of these tests provide a better understanding of the pathophysiology of the tear film. This review hopes to provide an overview of the existing tests and their role in evaluating the significance of the tear film in visual function.

Effect of overnight orthokeratology on conjunctival goblet cells.

OBJECTIVE: To evaluate the differences between goblet cell density (GCD) and symptomatology after one month of orthokeratology lens wear. METHODS: A pilot, short-term study was conducted. Twenty-two subjects (29.7±7.0 years old) participated voluntarily in the study. Subjects were divided into two groups: habitual silicone hydrogel contact lens wearers (SiHCLW) and new contact lens wearers (NCLW). Schirmer test, tear break up time (TBUT), Ocular Surface Disease Index (OSDI) questionnaire and conjunctival impression cytology. GCD, mucin cloud height (MCH) and cell layer thickness (CLT) were measured. All measurements were performed before orthokeratology fitting and one month after fitting to assess the evolution of the changes throughout this time. RESULTS: No differences in tear volume and TBUT between groups were found (p>0.05). However, the OSDI score was statistically better after one month of orthokeratology lens wear than the baseline for the SiHCLW group (p=0.03). Regarding the goblet cell analysis, no differences were found in CLT and MCH from the baseline visit to the one month visit for the SiHCLW compared with NCLW groups (p>0.05). At baseline, the GCD in the SiHCLW group were statistically lower than NCLW group (p<0.001). There was a significant increase in GCD after orthokeratology fitting from 121±140cell/mm(2) to 254±130cell/mm(2) (p<0.001) in the SiHCLW group. CONCLUSION: Orthokeratology improves the dry eye subject symptoms and GCD after one month of wearing in SiHCLW. These results suggest that orthokeratology could be considered a good alternative for silicone hydrogel contact lens discomfort and dryness.

Interobserver variability of an open-source software for tear meniscus height measurement.

PURPOSE: Different values of the lower tear meniscus height (TMH) can be obtained depending on the method and technique of measurement employed. The aim of this study was to analyse the interobserver variability of a method for measuring TMH by using an open source software. MATERIAL AND METHODS: On a group of 176 subjects, two videos of the central lower tear meniscus, first under slit-lamp illumination and ten minutes later under Tearscope illumination, were generated by a digital camera attached to a slit-lamp. Images were extracted from each video by a masked observer. Two further observers measured in a masked and randomized order the TMH in each illumination method by using an open source software based on Java (NIH ImageJ). TMH was measured from the lower lid to the upper limit of the tear meniscus for both slit-lamp (TMH-SL) and Tearscope (TMH-Tc) illumination methods. Subsequently, in different order, observers assigned a four-grading and a healthy/abnormal subjective classification to each central meniscus. RESULTS: No significant differences were found between the TMH measurements obtained by both investigators in slit-lamp or Tearscope image datasets (t-test; both p≥0.136). When comparing TMH measurements stratified by grade, only interobserver significant differences were observed for grades 3 and 4 with silt-lamp (t-test; both p≤0.009). Significant differences on TMH results between subjective subgroups were observed for both illumination techniques (ANOVA, all p≤0.045). CONCLUSION: This study showed a useful tool to objectively measure TMH by photography.

Non invasive assessment of the human tear film dynamics.

Dry eye disease, or keratoconjunctivitis sicca, is a multifactorial syndrome with altered tear film homeostasis leading to ocular irritations. These alterations cause discomfort and stress for the patient, but only a few objective parameters allow for proper differential diagnosis into different subtypes of this condition. The mostly invasively performed standard assessment procedures for tear film diagnosis are manifold, but often correlate quite poorly with the subjectively reported symptoms. Due to the inherent limitations, e.g. the subjectivity of the commonly performed invasive tests, a number of devices have been developed to assess the human tear film non-invasively. Since the production, delivery, distribution and drainage of the tear film is a dynamic process, we have focused our review on non-invasive methods which are capable of continuous or repetitive observations of the tear film during an inter-blink interval. These dynamic methods include (1) Interferometry, (2) Pattern Projection, (3) Aberrometry, (4) Thermography; and (5) Evaporimetry. These techniques are discussed with respect to their diagnostic value, both for screening and differential diagnostic of Dry Eye Disease. Many of the parameters obtained from these tests have been shown to have the potential to reliably discriminate patients from healthy subjects, especially when the tests are performed automatically and objectively. The differentiation into subtypes based solely on a single, dynamic parameter may not be feasible, but the combination of non-invasively performed procedures may provide good discrimination results.

The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression.

PURPOSE: In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear film, and the phenotype of these cells has yet to be characterized. This study was conducted to investigate the response of tear-film neutrophils to inflammatory stimuli. METHODS: Immediately upon awakening, cells from healthy participants (n = 12) were collected using a gentle eye-wash with PBS. Tear-film neutrophils were counted and cell viability was determined. Neutrophils were also isolated from blood by density-gradient centrifugation. Tear-film and blood-isolated neutrophils were stimulated with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Changes in the expression of macrophage-1 antigen, intercellular adhesion molecule-1 (ICAM-1), CD66b (a degranulation membrane marker), C3aR (complement C3a receptor), CD45 (leukocyte common antigen) as well as reactive oxygen species (using dichlorodihydro-fluorescein diacetate) were characterized by flow cytometry. RESULTS: Hundreds of thousands of leukocytes were collected upon awakening. Tear-film neutrophils were alive as shown by trypan blue and propidium iodide (PI) exclusion. While tear-film neutrophils were able to mount an oxidative response, stimulation with LPS, PMA, or fMLP did not induce receptor upregulation. This lack of response to stimulus with tear-film neutrophils was significantly different from that of blood-isolated neutrophils. Incubation in the presence of tear film proteins did not affect the tear-film neutrophil response to stimuli. CONCLUSIONS: Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood-isolated neutrophils. This refractory phenotype may be due to exposure to anti-inflammatory factors present in the tear film.

Repeatability of tear meniscus evaluation using spectral-domain Cirrus® HD-OCT and time-domain Visante® OCT.

PURPOSE: To investigate the intra-rater, inter-rater and inter-device repeatability of a spectral-domain OCT (Cirrus) and a time-domain OCT (Visante) for tear meniscus height (TMH) and area (TMA) measurements. METHODS: 20 participants with no known eye disease were recruited. Both eyes of participants were imaged with both OCTs under the similar conditions. The inferior tear meniscus was imaged at 6 o'clock position whereas the superior meniscus was imaged at 12 o'clock position. Data from the right eyes was analyzed. Two raters independently measured TMH and TMA using the OCT images, and one rater repeated the measurements. Intra-rater, inter-rater and inter-device repeatability of measurements were assessed using Bland-Altman plots and pooled standard deviation. RESULTS: For intra-rater repeatability, TMH and TMA measurements were more repeatable in Cirrus than Visante (95% limits of agreement (LOA): TMH (µm), -22 to 66 (Cirrus), -125 to 45 (Visante); TMA (µm(2)), -1632 to 5331 (Cirrus), -38,050 to 21,874 (Visante)). For inter-rater agreement, TMH and TMA were also more repeatable in Cirrus than Visante (95%LOA: TMH (µm), -29 to 107 (Cirrus), -215 to 252 (Visante); TMA (µm(2)), -6650 to 9567 (Cirrus), -33,119 to 39,272 (Visante)). Inter-device agreement was poor (95%LOA: TMH (µm), -158 to 150; TMA (µm(2)), -32,903 to 14,076). There was no significant difference in inferior TMH between Cirrus and Visante (p>0.05). Inferior TMA was significantly lower in Cirrus by a mean difference of 10,223µm(2) (95% confidence interval, 5479, 14,966) (p=0.0002). CONCLUSION: Spectral-domain OCT is superior to time-domain OCT for intra-rater and inter-rater repeatability of TMH and TMA measurements.

Goblet cell density association with tear function and ocular surface physiology.

PURPOSE: To determine the relationship of goblet cell density (GCD) with tear function and ocular surface physiology. METHODS: This was a cross-sectional study conducted in 35 asymptomatic subjects with mean age 23.8±3.6 years. Tear film assessment, conjunctiva and cornea examination were done in each subject. Conjunctival impression cytology was performed by applying Nitrocellulose Millipore MF™-Membrane filter over the superior bulbar conjunctiva. The filter paper was than fixed with 96% ethanol and stained with Periodic Acid Schiff, Hematoxylin and Eosin. GCD was determined by optical microscopy. Relation between GCD and Schirmer score, tear break-up time (TBUT), bulbar redness, limbal redness and corneal staining was determined. RESULTS: The mean GCD was 151±122 cells/mm(2). GCD was found higher in eyes with higher Schirmer score but it was not significant (p=0.75). There was a significant relationship of GCD with TBUT (p=0.042). GCD was not correlated with bulbar redness (p=0.126), and limbal redness (p=0.054) as well as corneal staining (p=0.079). No relationship of GCD with age and gender of the subjects (p>0.05) was observed. CONCLUSION: GCD was found correlated with TBUT but no significant correlation was found with the aqueous portion of the tear, limbal as well as bulbar redness and corneal staining.

Automatic assessment of tear film break-up dynamics.

Dry eye syndrome is a common disorder of the tear film which affects a remarkable percentage of the population. The Break-Up Time (BUT) is a clinical test used for the diagnosis of this disease, which computes the time the first tear film break-up appears. This work describes a fully automatic methodology to compute the BUT measurement and evaluate the break-up dynamics until the final blink. This analysis provides useful additional information for the assessment of tear film stability.

Development of a new grading scale for tear ferning.

PURPOSE: This paper reports on the development of a new tear ferning (TF) subjective grading scale, and compares it with the Rolando scale. METHOD: TF patterns obtained from tear film samples collected from normal and dry eye subjects in previous studies were collated into a large image library. From this library, 60 images were selected to represent the full range of possible TF patterns, and a further sub-set of 15 images was chosen for analysis. Twenty-five optometrists were asked to rank the images in increasing order between extreme anchors on a scale of TF patterns. Interim statistical analysis of this ranking found 7 homogeneous sub-sets, where the image rankings overlapped for a group of images. A representative image (typically the mean) from each group was then adopted as the grade standard. Using this new 7-point grading scale, 25 optometrists were asked to grade the entire 60 image library at two sessions: once using the 4-point Rolando scale and once using the new 7-point scale, applying 0.25 grade unit interpolation. RESULTS: Statistical analysis found that for the larger image set, the Rolando scale produced 3 homogeneous sub-sets, and the 7-point scale produced 5 homogeneous sub-sets. With this refinement, a new 5-point TF scale (Grades 0-4) was obtained. CONCLUSIONS: The Rolando grading scale lacks discrimination between its Type I and II grades, reducing its reliability. The new 5-point grading scale is able to differentiate between TF patterns, and may provide additional support for the use of TF for both researcher and clinician.

[Sjögren's syndrome]. / Sjögren-Syndrom.

Sjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltration of the tear and salivary glands leading to dryness of the mouth and eyes. The awareness that extraglandular manifestations, such as polyneuropathy, arthritis or recurrent airway infections may indicate Sjögren's syndrome is important. In the diagnostic procedure, the tear and saliva production and antibodies against Sjögren's syndrome A (SS-A) and SS-B should be measured. A salivary gland biopsy should be performed when the diagnosis is not still clear. The therapy of oral and ocular dryness is mainly symptomatic whereas the treatment of extraglandular manifestations is based on experience with treatment of these manifestations in systemic lupus erythematosus (SLE).

Cellular changes in tears associated with keratoconjunctival responses induced by nasal allergy.

BACKGROUND: Allergic keratoconjunctivitis occurs in a primary form, caused by an allergic reaction localized in the conjunctiva, and in a secondary form, induced by an allergic reaction originating in the nasal mucosa. Various hypersensitivity mechanisms involved in the keratoconjunctivitis forms result in different keratoconjunctival response types. PURPOSE: To investigate the cytologic changes in tears during the secondary immediate (SIKCR), late (SLKCR), and delayed (SDYKCR) keratoconjunctival responses. METHODS: In 61 patients, comprising 20 SIKCRs, 23 SLKCRs, and 18 SDYKCRs, nasal provocation tests (NPTs) with allergens and 61 phosphate-buffered control challenges were repeated and supplemented with cell counting in the tears. RESULTS: The SIKCR (P<0.01), appearing 10-120 min after the NPT, was associated with increased eosinophil and mast cell counts in tears. The SLKCR (P<0.01), appearing 5-12 h after the NPT, was accompanied by increased counts of eosinophils, neutrophils, basophils, and conjunctival epithelial and goblet cells. The SDYKCR (P<0.05), appearing 24-48 h after NPT, was associated with increased counts of lymphocytes, neutrophils, monocytes, basophils, conjunctival epithelial, corneal epithelial and goblet cells. CONCLUSIONS: The SIKCR, SLKCR, and SDYKCR, induced by nasal allergy, were associated with different cellular profiles in the tears. The cells, except mast, epithelial and goblet cells, displaying no intracellular changes, migrated probably from the conjunctival capillaries, in response to the factors released during the primary allergic reaction in the nasal mucosa and subsequently penetrating into the conjunctiva. These results demonstrate a causal role of nasal allergy and diagnostic value of NPT combined with recording of ocular features and cellular profiles in tears in some keratoconjunctivitis patients.

Effect of orbital protrusion and vertical interpalpebral distance on pterygium formation.

PURPOSE: To evaluate the risk factors in the development of pterygium in the Marmara region of Turkey as well as the efficacy of vertical interpalpebral distance, protrusion level and tear function in the development of pterygium. MATERIALS AND METHODS: The cases were grouped in two as the research group consisted of patients with pterygium and the control group consisted of healthy people. A total of 294 patients with pterygium (108 bilateral, 186 unilateral) and 200 controls were included in the study. All patients and control group underwent a thorough ophthalmic examination, including tear function analysis using tear film breakup time measurement, protrusion level and vertical interpalpebral distance. RESULTS: No statistically significant difference was found between the bilateral pterygium subgroup and control group in terms of vertical interpalpebral distance and protrusion value (p=0.733, p=0.625). When the pterygium eyes and the control group were compared in the unilateral pterygium subgroup, no significant difference was found in terms of vertical interpalpebral distance and protrusion value (p=0.533, p=0.209). CONCLUSIONS: While UV efficiency in pterygium was obvious, protrusion value and vertical interpalpebral distance were not found to be a risk factor in the formation of pterygium.

Tear fluid extracellular DNA: diagnostic and therapeutic implications in dry eye disease.

PURPOSE: To determine the abundance of extracellular DNA (eDNA) in tear fluid of patients with dry eye disease (DED) and to report clinical outcomes after DNase I eyedrops use to reduce excessive tear fluid eDNA. METHODS: Tear fluid was collected from healthy control subjects and patients with DED. The eDNA abundance was determined with the PicoGreen dye assay. The DED symptoms and clinical signs were recorded and correlated with eDNA abundance. Two patients with DED having excessive eDNA in tear fluid were treated with DNase I eyedrops. RESULTS: The PicoGreen dye assay measures tear fluid eDNA abundance after a 2-minute incubation time. With longer incubations, admixed cells also contribute to eDNA measurements. The mean (SE) eDNA abundance in healthy control subjects' tear fluid was 1.4 (0.2) µg/mL. The mean (SE) eDNA abundance in tear fluid of patients with nonautoimmune DED, autoimmune DED, and graft versus host disease was significantly higher: the values were 2.9 (0.6), 5.2 (1.2), and 9.1 (2.3) µg/mL, respectively (P < 0.05). In most of these patients, the PicoGreen dye kinetic assay of tear fluid showed an increase in fluorescence signal due to the presence of viable cells in tear fluid. Tear fluid eDNA had the best correlation with corneal Rose Bengal staining (r = 0.55). Treatment of patients having DED with DNase I eyedrops reduced eDNA abundance, abrogated signal increase, and improved comfort. CONCLUSIONS: Excessive eDNA is present in tear fluid of patients with dry eyes. A novel therapeutic approach for managing DED may be to measure eDNA abundance in tear fluid with the PicoGreen dye assay and reduce excessive amounts with DNase I eyedrops.

Cytological changes in tears during the secondary conjunctival response induced by nasal allergy.

PURPOSE: Allergic conjunctivitis (AC) can be caused by an allergic reaction localised exclusively in the conjunctivae and initiated by direct exposure of conjunctivae to an allergen (primary AC form) or it can be induced secondarily by an allergic reaction occurring primarily in the nose (secondary AC form). OBJECTIVES: To investigate the cellular profiles in tears accompanying the particular types of secondary conjunctival response (SCR). METHODS: In 53 seasonal AC or perennial AC patients developing 53 positive SCRs (17 immediate, 21 late, 15, delayed) and 31 negative responses to the nasal provocation test with allergen (NPT), the NPTs were repeated and supplemented with, cytological examination of the tears. RESULTS: The immediate SCRs (p<0.01), appearing 10-120 min after the NPT, were associated with increased eosinophil and mast cell counts. The late SCRs (p<0.01), occurring 5-12 h, were accompanied by increased eosinophil, neutrophil, basophil and epithelial cell counts. The delayed SCRs (p<0.05), appearing 24-48 h, were associated with increased lymphocyte, neutrophil, monocyte, epithelial and goblet cell counts. CONCLUSIONS: The secondary immediate, late and delayed conjunctival responses, induced by nasal allergy, were associated with different cellular profiles in the tears. The cells, except mast, epithelial and goblet cells, displaying no intracellular changes, had probably migrated from conjunctival capillaries, affected by factors released during the initial nasal allergic reaction, without participating in the allergy processes. In AC patients demonstrating insufficient therapeutic compliance, the role of nasal allergy should be examined.