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Background: Ocular allergy (OA) is a localized subset of allergy characterized by ocular surface itchiness, redness and inflammation. Inflammation and eye-rubbing, due to allergy-associated itch, are common in OA sufferers and may trigger changes to the ocular surface biochemistry. The primary aim of this study is to assess the differences in the human tear proteome between OA sufferers and Healthy Controls (HCs) across peak allergy season and off-peak season in Victoria, Australia., Methods: 19 participants (14 OA sufferers, 5 HCs) aged 18-45 were recruited for this study. Participants were grouped based on allergy symptom assessment questionnaire scoring. Proteins were extracted from human tear samples and were run on an Orbitrap Mass Spectrometer. Peaks were matched to a DIA library. Data was analyzed using the software MaxQuant, Perseus and IBM SPSS., Results: 1267 proteins were identified in tear samples of OA sufferers and HCs. 23 proteins were differentially expressed between peak allergy season OA suffers vs HCs, and 21 were differentially expressed in off-peak season. Decreased proteins in OA sufferers related to cell structure regulation, inflammatory regulation and antimicrobial regulation. In both seasons, OA sufferers were shown to have increased expression of proteins relating to inflammation, immune responses and cellular development., Conclusion: Tear protein identification showed dysregulation of proteins involved in inflammation, immunity and cellular structures. Proteins relating to cellular structure may suggest a possible link between OA-associated itch and the subsequent ocular surface damage via eye-rubbing, while inflammatory and immune protein changes highlight potential diagnostic and therapeutic biomarkers of OA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Aydin, Nie, Azizoglu, Chong, Gokhale and Suphioglu.)

Objective: Aim: to determine the state of local immunity in DED on the background of hormonal dysfunction., Patients and Methods: Materials and Methods: Of 32 women, 17 patients with diagnosed SM and 15 women of the control group were examined. The Ocular Surface Disease Index and the state of local immunity were defined by determining Ig As in lacrimal fluid (LF) by radial immunodiffusion in Mancini agar., Results: Results: During the OSDI questionnaire, a mild degree of DED was detected in 21 (65.6%) women, and an average degree was observed in 11 (34.4%) patients with SM. On average, OSDI was 34.54 ±2.01. As a result of studies of the state of local immunity in patients with SM, a tendency to increase Ig As was noted, compared with the control group. An increase in Ig As in the lacrimal fluid in patients with SM to 0.34 ±0.09 g/l was found, compared with the control group (0.24 ±0.03 g/l)., Conclusion: Conclusions: Using the OSDI questionnaire, the presence of DED was detected in women with SM, mainly mild and moderate degree. The obtained results of the state of local immunity indicate in favor of a nonspecific inflammatory process, accompanied by a decrease in local immune protection and leading to further changes in the ocular surface.

Purpose: To determine the effect of discontinuing chronic topical immune modulating (IM) treatment on Schirmer tear test (STT) values in dogs with dry eye disease (DED). Methods: Serial measurements of STTs from 14 dogs (16 eyes) previously diagnosed with DED were obtained before and after discontinuation of topical IM agents. Dogs with moderate to severe DED that had been well controlled with a topical IM treatment were included. After initial assessment topical IM treatment was discontinued, but topical lubricant was continued, and STT values were obtained sequentially. A mixed-effects regression model was used to evaluate the effects of age, gender, breed, clinical score, frequency of treatment, baseline STT value, and drug type on final STT values after IM withdrawal. P  < 0.05 was considered statistically significant. Results: During the follow-up period after the IM treatment had been discontinued (136 ± 29 days), 50% of the eyes ( n  = 8) exhibited STT values that never decreased to <10 mm/min. In the other 50% ( n  = 8), STT values decreased from 15.9 ± 4.7 mm/min to 6.1 ± 0.9 mm/min. In this group, the time it took to decrease the STT to <10 mm/min was 21.1 ± 9.5 days. Severe clinical signs of DED and low baseline STT pre-IM treatment significantly affected STT post-IM treatment withdrawal ( P  < 0.05). Conclusions: The duration that a residual effect of topical IM treatment persists needs to be taken into consideration when studies are designed utilizing dogs with previous IM treatment for DED.

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.

Purpose: To demonstrate the tear IgE (measured/exuded) ratio (R) as a useful biological marker of ocular allergy in order to distinguish severe from less severe inflammatory status., Methods: Tear samples and sera from 78 ocular allergy patients and 19 control subjects were analyzed. Total IgE and albumin were measured for calculating the tear IgE-R defining two subgroups (SG) of samples: R ≥ 4-SG and R < 4-SG. Eosinophil cationic protein, Th1 and Th2 cytokines (IFN-γ, IL-4, -5, -6, -8 and -10) and protein electrophoretic profiles were also investigated in tears., Results: The R < 4-SG compared to the R ≥ 4-SG shows higher levels of tear albumin, eosinophil cationic protein, and Th1 and Th2 cytokines. Moreover, each subgroup presents a specific protein profile., Conclusion: This study showed that an IgE-R lower than four must be carefully interpreted as a warning sign of a severe inflammatory context and should be also associated with an exploration of immunological profile.

Homeostasis of the lacrimal functional unit is needed to ensure a well-regulated ocular immune response comprising innate and adaptive phases. When the ocular immune system is excessively stimulated and/or immunoregulatory mechanisms are disrupted, the balance between innate and adaptive phases is dysregulated and chronic ocular surface inflammation can result, leading to chronic dry eye disease (DED). According to the Tear Film and Ocular Surface Society Dry Eye Workshop II definition, DED is a multifactorial disorder of the ocular surface characterized by impairment and loss of tear homeostasis (hyperosmolarity), ocular discomfort or pain, and neurosensory abnormalities. Dysregulated ocular immune responses result in ocular surface damage, which is a further contributing factor to DED pathology. Several therapeutics are available to break the vicious circle of DED and prevent chronic disease and progression, including immunosuppressive agents (steroids) and immunomodulators (cyclosporine and lifitegrast). Given the chronic inflammatory nature of DED, each of these agents is commonly used in clinical practice. In this study, we review the immunopathology of DED and the molecular and cellular actions of current topical DED therapeutics to inform clinical decision making.

Alterations in serum cytokine levels and profiles have been reported in association with a variety of disease conditions (e.g., allergic, immune-mediated, etc.) in both humans and animals. In comparison to serum cytokine measurements, tear cytokine measurements might be expected to more accurately reflect the inflammatory milieu associated with periocular disease. The purpose of this study was to use a multiplexed assay to compare the cytokine profile of tears in healthy dogs to those with inflammatory skin and periocular disease. We were able to detect IL-2, IL-6, IL-8, and TNF-α in >47 % of tear samples from both healthy canine patients and those with inflammatory dermatologic disease (with or without concurrent periocular involvement). In contrast, IL-7, IL-10 and IFN-γ were rarely detected. Dogs with both dermatologic and periocular disease (but not dermatologic disease alone) had higher levels of IL-8 (P < 0.001, P > 0.05, respectively) relative to healthy dogs. Patients with concurrent dermatologic and periocular disease also demonstrated significantly greater variability in IL-8 concentrations between eyes than did healthy dogs (P < 0.0001). Our findings suggest that tear cytokine analysis may prove to be a useful tool to investigate the role and interactions of the local ocular immune response in patients with inflammatory periocular disease., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose of Review: During allergic reaction, nervous and immune systems mutually interact through release of mediators, including neurotrophic factors and nerve growth factor (NGF). These mediators modulate allergic reaction through binding their receptors expressed by immune and structural cells and by stimulating neuropeptide release by nerves. The role of neuropeptides and NGF has been demonstrated in allergic asthma and rhinitis, and, to a lesser extent, in allergic conjunctivitis. The aim of this review are to elucidate the evidence of the role of NGF and neuropeptides in the pathogenesis of allergic conjunctivitis., Recent Findings: NGF modulates allergic reaction by stimulating release of cytokines, inflammatory mediators and neuropeptides by immune and structural cells and nerve endings at the site of inflammation. Evidence showed that local and systemic NGF levels increase in patients with allergic conjunctivitis, including allergic rhinoconjuncivitis, vernal keratoconjunctivitis and atopic keratoconjunctivitis. We recently described an increase of conjunctival p75NTR expression in patients with allergic rhinoconjuncivitis, and an increase of tear levels of NGF after conjunctival provocation test with allergen., Summary: NGF modulates ocular allergic reaction. Increasing understanding of the role of neuropeptides in allergic conjunctivitis may pave the way to the development of novel therapeutic approaches and improvement of patients' management.

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 H 3 N 2 , 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-1β, 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.

Objective: The objective of this study was to study the levels of interleukin (IL)-15 and IL-17 in tears and orbital tissues of the patients with the Graves ophthalmopathy (GO) (active and inactive stages) and in controls., Methods: Twenty-four active GO patients (CAS ≥ 3/7) were collected in our research. All patients were treated with corticosteroids during the therapy process (4-15 months) and developed into the inactive phase (CAS ≤ 2/7). The tear samples were gathered from inactive and active stages of patients with GO and sex-matched and age-matched controls (volunteers, n = 24) for the enzyme-linked immunosorbent assay measurement of IL-15 and IL-17. The orbital tissues were collected from inactive stage of patients with GO and healthy volunteers for the analysis of IL-15 and IL-17 positive cells using the immunohistochemical analysis., Results: In comparison with the volunteers, significant upregulation of IL-15 and IL-17 were identified in tears of active (P < 0.05) and inactive patients with GO ( P < 0.01). Compared with inactive patients with GO, the levels of IL-15 and IL-17 in the tears were obviously higher than those in the active patients with GO. In addition, higher expression of IL-15 and IL-17 positive cells were found in the orbital organization of inactive patients with GO compared with volunteers., Conclusion: The differences in IL-15 and IL-17 expression between GO patients (active and inactive stages) and controls suggested both IL-15 and IL-17 expression were significantly correlated with thyroid-associated ophthalmopathy pathogenesis and development., (© 2018 Wiley Periodicals, Inc.)

We report a case of xeroderma pigmentosum (XP) with endothelial dysfunction where the analysis of tears revealed elevated levels of proinflammatory cytokines, even in the absence of active inflammation and neovascularisation of the ocular surface. Although the role of ultraviolet (UV) radiation-induced inflammation in the occurrence of ocular manifestations of XP is known, little is published on the molecular mechanisms and there are no reports quantifying the presence of inflammatory cytokines in the tears of patients with ocular involvement of XP. Tear analysis demonstrated an increase in inflammatory cytokines and chemokines, especially interleukin-8 (2.38 ng/µg), tumour necrosis factor alpha (0.87 ng/µg) and granulocyte monocyte colony stimulating factor (0.44 ng/µg) as compared with the control eye. Effective management of the underlying UV-induced inflammation and promoting DNA repair may play a vital role in managing ocular manifestations and its sequelae in patients of XP., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: The purpose of this pilot study was to use a multiplexed assay to measure cytokines in normal stimulated canine tears., Methods: 25 healthy dogs were included in the study. Stimulated tears were collected in capillary tubes from the right (OD) and left (OS) eyes and stored at -80 °C until batch sample analysis was performed. The samples were analyzed utilizing Luminex ® canine-validated multiplex beads on a Bio-Rad multiplex analyzer for IL-2, IL-6, IL-7, IL-8, IL-10, TNF-α, and IFN-γ. Based upon previous human studies, tears were initially evaluated at a 1:10 dilution. Eight random samples were later re-analyzed without dilution., Results: Diluting the samples 1:10 rendered all analytes undetectable except IL-8. A repeat analysis of eight randomly selected undiluted samples still demonstrated very low cytokine levels except for IL-8 (16/16 eyes; 2254 ± 1677 pg/ml OD, 1095 ± 786.8 pg/ml OS); and IFN-γ (15/16 eyes; 13.37 ± 13.08 pg/ml OD,16.08 ± 19.4 pg/ml OS)., Conclusion: This pilot study is the first to analyze cytokines in canine tears. This study demonstrated that IL-8 is consistently detected in both diluted and undiluted samples, but undiluted samples may be superior to 1:10 diluted samples for evaluation of other cytokines in canine tears., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense., (Published by Elsevier Inc.)

Objective: To determine the expression profile of immune response and inflammation (IRI) mediator molecules in tears from patients with dry eye (DE), and those suspected of having or have primary open-angle glaucoma (POAG) under treatment and compare them with healthy controls., Methods: A prospective observational cohort study including 107 participants sub-divided into: healthy controls (CG; n=30), patients with DE (DEG; n=41) and patients suspected of having or have POAG and on hypotensive treatment (POAG-G; n=36). Tear samples were collected by capillary to be processed using a multi-immunoassay system based on flow cytometry (Luminex R-200 ®), in order to determine the interleukins (IL): 1β, 2, 4, 5, 6, and 10, and the growth factors: Tumour necrosis alpha (TNF-α), vascular endothelial (VEGF), and granulocyte-macrophage colony stimulating- (GM-CSF). Data were processed using the SPSS 20.0 program., Results: Molecules that significantly increased in tears from DEG vs. POAG-G patients were: IL-1 (P=.01), IL-6 (P=.004), IL-10 (P=.04), whereas VEGF significantly decreased in the DEG. The POAG-G showed significantly higher IL-6 values (P<.0001) as compared to the CG. When comparing both the DEG and POAG-G, significant differences were observed in tear expression of IL-4 (P=.004), IL-6 (P=.002), TNF-α (P=.03), GM-CSF (P=.03), and VEGF (P=.002)., Conclusions: The increased expression of IRI mediators in tears from patients with DE or POAG strongly demonstrated the importance of immune response in both pathologies. However, the different molecules involved also suggest distinct signalling pathways for these processes that still require further research., (Copyright © 2017 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Purpose of Review: Conventional diagnostic tests in allergy are insufficient to clarify the cause of vernal conjunctivitis. Component-resolved diagnostic (CRD) by microarray allergen assay may be useful in detecting allergens that might be involved in the inflammatory process., Recent Findings: In a recent trial in patients suffered from eosinophilic esophagitis, after 2 years of the CRD-guided exclusion diet and specific immunotherapy, significant clinical improvement was observed, and 68% of patients were discharged (cure based on negative biopsy, no symptoms, and no medication intake). Our new objective was to evaluate IgE-mediated hypersensitivity by CRD in tears and serum from patients with vernal conjunctivitis and treat patients with identified triggering allergens by specific immunotherapy. Twenty-five patients with vernal conjunctivitis were evaluated. The identified triggering allergens were n Lol p 1 (11 cases), n Cyn d 1 (eight cases), group 4 and 6 grasses (six cases) and group 5 of grasses (five cases). Prick test and pollen IgE were positive in one case. Clinical improvement was observed in 13/25 vernal conjunctivitis patients after 1-year specific immunotherapy., Summary: CRD seems to be a more sensitive diagnostic tool compared with prick test and IgE detection. Specific CRD-led immunotherapy may achieve clinical improvements in vernal conjunctivitis patients.

Purpose: This study investigated changes in selected tear cytokine concentrations (IL-1β, IL-1Ra, IL-6, IL-10, IL-12 (p70), and TNF-α) after a 1-week washout from soft contact lens wear (CLW), and the repeatability of cytokine measurements using custom multiplex assays., Methods: A total of 10 subjects completed this 6-visit (immediately following contact lens removal, and after 1, 2, 3, 4, and 7 [±1] days without CLW) pilot study. Approximately 20 to 30 μL of pooled basal tears were collected from both eyes at each visit. Two custom multiplex assays were used by two operators to quantify the concentration of tear cytokines. Tear samples from subjects 1 to 6 were analyzed using the first kit by operator 1. Tear samples from subject 7 to 10 plus additional tear samples from subjects 1 to 5, which were used to determine the between-kit per operator repeatability, were analyzed using the second kit by operator 2. Linear mixed models were used to determine changes in tear cytokine concentrations over time. Between-kit per operator and within-kit per operator repeatabilities were assessed using the Bland and Altman analysis., Results: There were no significant changes in tear cytokine concentrations over a 1-week washout of CLW. More than 99% of the tear samples had detectable levels of cytokines using custom multiplex assays. Within-kit per operator repeatability was good, but between-kit per operator repeatability was poor; likely due to protein degradation, differences in operator experience, and operating procedures., Conclusion: A washout period may not be necessary when evaluating changes in tear cytokines with new contact lenses or lens care products. A well-trained operator using standardized operating procedures can produce repeatable measurements using custom multiplex assays.

Approximately a quarter of the world's population at some point in life is at risk of developing shingles (Herpes Zoster). In 10-20% of cases the first branch of the trigeminal nerve gets involved (Herpes Zoster Ophthalmicus, HZO). Ophthalmic complications of HZO are able to cause a significant reduction in visual function., Aim: To study and summarize clinical features of HZO (including the rate of complications and their nature) and to determine the relationship between clinical and laboratory data from these patients., Material and Methods: The study included 133 patients with ophthalmic and neurological complications of HZO (group 1 (n=28) - retrospective analysis of outpatient records for the period 1995-2005; group 2 (n=95) - a prospective study for the period 2005-2015), who received a course of conservative treatment in either the Botkin City Hospital, branch № 1, or in the ophthalmic department of the Moscow herpes centre (Gerpeticheskiy Tsentr Ltd.). Laboratory tests were performed only in patients from group 2 and included: examination of biological fluids for six types of herpes viruses by polymerase chain reaction, examination of tears and urine for DNA of Chlamydia, Mycoplasma, and Ureaplasma, and serological blood testing for markers of herpes virus infection. Patients from group 1 were prescribed topical antiviral, antibacterial, and anti-inflammatory therapy, in rare cases - acyclovir per os. In group 2, the treatment included systemic antiviral medications and immune correction therapy. Anti-inflammatory therapy consisted of local and systemic non-steroidal agents (NSAIDs)., Results: The most common ophthalmic complications of HZO in both groups were stromal keratitis and keratoiridocyclitis, neurological - III and VI cranial nerves palsies. The duration of the disease in the first group ranged from 2 months to 3 years; in the second group, patients were divided into two subgroups: subgroup A with the disease duration of no more than one month (n=81) and subgroup B with the disease duration from 1.5 to 9 months (n=14). Varicella-zoster virus (VZV) DNA was present in tears and/or other biological fluids of patients from group 2 in more than 70% of cases (n=67). Particularly, in 27.4% of cases the virus was isolated in two fluids and in 7.4% of cases - in three fluids. The duration of virus production in tears and other biological fluids (saliva, blood, and urine) ranged from 10 days to 4 months., Conclusion: Topical non-steroidal anti-inflammatory drugs and systemic etiological treatment in case of intraocular inflammation in HZO patients may reduce the risk of severe consequences of VZV reactivation and help avoid recurrences later in life.

Background: A specific allergen sensitization can be demonstrated in approximately half of the vernal keratoconjunctivitis (VKC) patients by conventional allergic tests. The measurement of specific IgE in tears using a multiplex allergen microarray may offer advantages to identify local sensitization to a specific allergen., Methods: In spring-summer 2011, serum and tears samples were collected from 10 active VKC patients (three females, seven males) and 10 age-matched normal subjects. Skin prick test, symptoms score and full ophthalmological examination were performed. Specific serum and tear IgE were assayed using ImmunoCAP ISAC, a microarray containing 103 components derived from 47 allergens., Results: Normal subjects resulted negative for the presence of specific IgE both in serum and in tears. Of the 10 VKC patients, six resulted positive to specific IgE in serum and/or tears. In three of these six patients, specific IgE was found positive only in tears. Cross-reactivity between specific markers was found in three patients. Grass, tree, mites, animal but also food allergen-specific IgE were found in tears. Conjunctival provocation test performed out of season confirmed the specific local conjunctival reactivity., Conclusions: Multiple specific IgE measurements with single protein allergens using a microarray technique in tear samples are a useful, simple and non-invasive diagnostic tool. ImmunoCAP ISAC detects allergen sensitization at component level and adds important information by defining both cross- and co-sensitization to a large variety of allergen molecules. The presence of specific IgE only in tears of VKC patients reinforces the concept of possible local sensitization., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye., Methods: We recruited 54 patients with Sjögren's syndrome dry eye (SSDE), 50 patients with non-Sjögren's syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting., Results: NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups., Conclusions: In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye.

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4+, CD8+, CD19+ and CD3-CD56+ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3-CD56+ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.

Context: Allergy to hydrolyzed wheat protein in facial soap has become a major social issue in Japan. It has been reported that the most frequent early symptoms of allergy to hydrolyzed wheat protein in soap are allergic conjunctivitis and rhinitis, while wheat-dependent exercise-induced anaphylaxis can be induced by long-term use., Objective: We evaluated the relation between tear fluid levels of specific IgE for wheat and the features of allergic conjunctivitis., Methods: A prospective, non-randomized, cross-sectional study was conducted in 103 patients with moderate to severe allergic conjunctivitis (allergic group) and 20 age- and sex-matched healthy control subjects (control group). Specific IgE for wheat was measured in tear fluid with an immunochromatography assay, and a skin prick test (SPT) was also performed. Symptoms (sneezing, rhinorrhea, nasal obstruction, ocular itching, and lacrimation) were assessed in each subject along with the activities of daily living (ADL) score and the total ocular symptom score for allergic conjunctivitis. A severity score (0, 1, 2, or 3) was assigned for various changes of the palpebral and bulbar conjunctiva, as well as for limbal and corneal lesions associated with allergic conjunctivitis., Results: The IgE positive rate and specific IgE score were both higher in the allergic group than in the control group (71.8% versus 40.0% and 1.9 ± 0.7 versus 1.4 ± 0.5). A positive SPT for wheat was also more frequent in the allergic group than in the control group (6.8% versus 0.0%). Within the allergic group, patients with a positive SPT had higher specific IgE scores than patients with a negative SPT (3.3 ± 0.5 versus 1.8 ± 0.6, p < 0.001). In the allergic group, the wheat IgE level in tear fluid was correlated with the severity of allergic conjunctivitis symptoms, including ocular itching (r = 0.665), tearing (r = 0.672), and the total ocular symptom score (r = 0.204). Wheat IgE in tear fluid was also correlated with the severity of rhinitis symptoms, including sneezing (r = 0.610), nose blowing (r = 0.640), and nasal obstruction (r = 0.677). Furthermore, the tear fluid wheat IgE score was correlated with five objective features of allergic conjunctivitis (p < 0.05)., Conclusions: These results suggest that wheat allergy may be involved in the development of allergic conjunctivitis.

Monitoring serum antibodies against natural infections or after immunizations has been a standard clinical diagnostic procedure. However, collecting blood samples requires trained personnel, and may cause discomfort and increase the risk of complications. In this study, we investigated whether tear samples could serve as a surrogate for serum samples to measure specific antibodies. A widely used preclinical cottontail rabbit papillomavirus (CRPV)/rabbit model has been a surrogate model for high-risk human papillomavirus (HPV) infections. New Zealand white rabbits, either naturally infected with CRPV or immunized with two clinically available HPV vaccines (Gardasil and Cervarix), were examined for antibody generation in both tear and serum samples. We demonstrated that antibodies were detectable in tears from both naturally infected as well as vaccinated animals. Overall, the antibody levels in tears were ~10-fold lower than those from the corresponding serum samples, but background noise was lower in tear samples. The isotypes of antibodies in tears were predominantly IgA and IgG. These findings showed clearly that tears could be a surrogate for serum samples for monitoring antibody responses. As collecting tears causes no discomfort and poses no risk to patients, it represents a novel and promising method for monitoring future HPV epidemiological studies as well as for use in clinical practice.

Antimicrobial peptides are important as the first line of innate defense, through their tendency to disrupt bacterial membranes or intracellular pathways and potentially as the next generation of antibiotics. How they protect wet epithelia is not entirely clear, with most individually inactive under physiological conditions and many preferentially targeting Gram-positive bacteria. Tears covering the surface of the eye are bactericidal for Gram-positive and -negative bacteria. Here we narrow much of the bactericidal activity to a latent C-terminal fragment in the prosecretory mitogen lacritin and report that the mechanism combines membrane permeabilization with rapid metabolic changes, including reduced levels of dephosphocoenzyme A, spermidine, putrescine, and phosphatidylethanolamines and elevated alanine, leucine, phenylalanine, tryptophan, proline, glycine, lysine, serine, glutamate, cadaverine, and pyrophosphate. Thus, death by metabolic stress parallels cellular attempts to survive. Cleavage-dependent appearance of the C-terminal cationic amphipathic α-helix is inducible within hours by Staphylococcus epidermidis and slowly by another mechanism, in a chymotrypsin- or leupeptin protease-inhibitable manner. Although bactericidal at low micromolar levels, within a biphasic 1-10 nM dose optimum, the same domain is mitogenic and cytoprotective for epithelia via a syndecan-1 targeting mechanism dependent on heparanase. Thus, the C terminus of lacritin is multifunctional by dose and proteolytic processing and appears to play a key role in the innate protection of the eye, with wider potential benefit elsewhere as lacritin flows from exocrine secretory cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: The purpose of the study is to determine the role of certain clinical and immunological factors of the onset and development of age-related macular degeneration (AMD)., Materials and Methods: Retrospective analysis of patients records; determination of IFNalpha IFNgamma, IL-1beta, and TNFalpha cytokines levels as well as extracellular peroxidase activity (EPA) in blood serum and tear; measuring frequency of occurrence of intraocular infectious antigens with predominantly intracellular localization, such as Herpesviridae spp. (Herpes simplex, Cytomegalovirus, Epstein-Barr virus) and Chlamydiaceae spp. (Chlamydia pneumoniae, Chlamydia trachomatis) in lens matter and aqueous humor by means of polymerase chain reaction (PCR). The new evidence strongly suggests a relationship between AMD development and hypertension (p < 0.001; r = +0.30, P < 95%) as well as between AMD progression and hypertension accompanied by chronic inflammation with predominantly intracellular localization of the infectious agent (p < 0.05 for the predisciform stage, p < 0.001 for the disciform and cicatrical stages; r = +0.30, P < 95%). "Dry" AMD is characterized by hypertension-associated systemic and then local increase of EPA. In "wet" AMD it is the interferon response that is impaired: IFNalpha and IFNgamma are systemically decreased, while local level of IFNalpha is increased. Intraocular Herpes simplex infection is pathogenically significant for AMD development (18.8% in the study group vs 0% in the control group)., Conclusion: AMD progression is associated with hypertension accompanied by chronic inflammation with predominantly intracellular localization of the infectious agent as well as impairment of the interferon response (systemic decrease of IFNalpha and IFNgamma with local increase of IFNalpha). Moreover, the presence of intraocular Herpes simplex infection leads to activation of the first line antiviral immunity (IFNgalpha) with decompensation of the local interferon response (IFNgamma).

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).

Background: Corneal inflammation has long been associated with contact lens wear and the use of extended-wear lenses enhances the risk of corneal injury. Elucidation of the molecular mediators of contact lens-associated inflammation has the potential to provide injury-identifying markers early in the inflammatory process, as well as determine potential therapeutic targets., Methods: This cross-over study investigated a potential correlation between overnight contact lens wear and the concentrations of two markers of inflammation, α1-antitrypsin and C-reactive protein, in tear fluid. To obtain baseline measurements, 17 subjects adapted to wearing silicone hydrogel contact lenses wore their prescribed eye glasses for one week, after which tears were collected and ocular health assessed by a licensed optometrist. Subjects then returned to wearing their prescribed silicone hydrogel lenses continuously for one week. A second tear sample was collected and ocular inflammation was again assessed. Enzyme-linked immunosorbent assays were performed on all tear samples for both α1-antitrypsin and C-reactive protein., Results: α1-antitrypsin was significantly (p = 0.01) elevated after continuous contact lens wear, with increases above baseline concentrations averaging 2.48-fold. Optometric assessment of inflammation loosely correlated with levels of this inflammatory marker. C-reactive protein was detected in the tears of subjects at both times and levels were also slightly elevated after extended lens wear, but not significantly (p > 0.5) and not consistently in all subjects., Conclusion: The results of this study suggest that α1-antitrypsin in tear fluid may be useful as an early marker of contact lens-associated ocular irritation and inflammation. The presence of C-reactive protein in the tears of contact lens wearers is a novel finding which, while not correlative with either α1-antitrypsin concentrations or clinically observable inflammation, may warrant further study., (© 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.)

The tear film coats the cornea and conjunctiva and serves several important functions. It provides lubrication, prevents drying of the ocular surface epithelia, helps provide a smooth surface for refracting light, supplies oxygen and is an important component of the innate defense system of the eye providing protection against a range of potential pathogens. This review describes both classic antimicrobial compounds found in tears such as lysozyme and some more recently identified such as members of the cationic antimicrobial peptide family and surfactant protein-D as well as potential new candidate molecules that may contribute to antimicrobial protection. As is readily evident from the literature review herein, tears, like all mucosal fluids, contain a plethora of molecules with known antimicrobial effects. That all of these are active in vivo is debatable as many are present in low concentrations, may be influenced by other tear components such as the ionic environment, and antimicrobial action may be only one of several activities ascribed to the molecule. However, there are many studies showing synergistic/additive interactions between several of the tear antimicrobials and it is highly likely that cooperativity between molecules is the primary way tears are able to afford significant antimicrobial protection to the ocular surface in vivo. In addition to effects on pathogen growth and survival some tear components prevent epithelial cell invasion and promote the epithelial expression of innate defense molecules. Given the protective role of tears a number of scenarios can be envisaged that may affect the amount and/or activity of tear antimicrobials and hence compromise tear immunity. Two such situations, dry eye disease and contact lens wear, are discussed here., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The identification of inflammatory mediators in the tear fluid have been extensively used in ocular allergy to find either a 'disease marker', to better understand the immune mechanisms involved in the ocular surface inflammation, or to identify potential targets for therapeutic interventions. While the clinical characteristics allow a relatively convincing diagnosis of ocular allergic diseases, in the initial, non active phases, or in the chronic stages, the diagnosis may not be clear. Although not highly specific, total tear IgE can be measured with local tests by inserting a paper strip in the lower meniscus. The measurement of tear specific inflammatory markers, such as histamine, tryptase, ECP, IL-4, IL-5 and eotaxin, may be useful for the diagnosis or monitoring ocular allergy. New technologies such as multiplex bead assays, membrane-bound antibody array and proteomic techniques can characterize the distribution of a wide range of bioactive trace proteins in tears. Dozens of mediators, cytokines, chemokines, growth factors, angiogenic modulators, enzymes and inhibitors were thus identified in small tear samples using these techniques, providing the possible identification of specific biomarker for either specific disease or disease activity. However, to date, there is no a single specific laboratory test suitable for the diagnosis and monitoring of allergic conjunctivitis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To investigate the role of specific secretary IgA antibodies on lipopolysaccharide (LPS-sIgA) and exotoxin A (ETA-sIgA) in tear fluid produced by immunoresponse to P. aeruginosa., Subjects and Methods: Subjects were divided into 3 groups; 41 eyes of 41 normal volunteers without a history of using contact lenses (CL) as controls, 32 eyes of 32 CL users without adverse events as a healthy CL wearer group (CL group), and 12 eyes of 12 patients with CL-related corneal ulcers from which P. aeruginosa was isolated (ulcer group). Tear fluid was obtained using filter paper, and the concentration of LPS-sIgA and ETA-sIgA in samples was determined using enzyme-linked immunosorbent assay (ELISA)., Results: The lower limit of concentration for LPS-sIgA and ETA-sIgA was 1.24 and 1.24 (U/mL), respectively. The positive eyes which exceeded this lower limit in LPS-sIgA were 17 of 22 eyes in the control group (median concentration 2.00U/ml, range 1.28 to 7.20), 15 of 25 eyes in the CL group (median 1.76 U/ml, range 1.24 to 6.92) and 1 of 12 eyes in the ulcer group (1.80 U/ml). The number of eyes which exceeded the lower limit in ETA-sIgA was 36 of 41 eyes in the control group (median concentration 6.56 U/ml, range 1.36 to 182), 28 of 32 eyes in the CL group (median 5.40 U/ml, range 1.56 to 29.20) and 5 of 12 eyes in the ulcer group (median 1.72 U/ml, range 1.40 to 2.16). There was no significant difference in LPS-sIgA and ETA-sIgA between the control group and the CL group, but they were significantly lower in the ulcer group than in the control group (p < 0.01, Steel's test)., Conclusion: Tear fluid in normal, healthy CL wearers contains LPS-sIgA and ETA-sIgA which acts as a barrier to P. aeruginosa infectious keratitis.

Developments in the field of tear film protein profiling are reviewed, including the advantages and disadvantages of the multiplex bead array technique and its potential for identifying biomarkers of ocular surface disease. Commercial kits, which specifically employ 96-well plates with polystyrene microspheres and magnetic beads, and sensitivity variations between them are discussed. Modifications to protocols of these kits (which are designed primarily for larger sample volumes, such as blood and cell culture supernatants) may be necessary for tear fluid samples. Multiplex bead array is compared to the "gold standard," ELISA. The challenges of diagnosing and monitoring dry eye disease, due to the conflicting and variable signs and symptoms presented, are illustrated by the authors' own study results. The development of a biomarker profile for the disease would be a useful approach to the ongoing problems of diagnosis. The multiplex bead array technique has important potential applications in this regard., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: The level of specific class E immunoglobulins (IgE) in tear fluid is a useful diagnostic indicator for allergic conjunctivitis, but it is still unclear whether the measurement of tear fluid IgE is helpful for assessing the severity of allergic conjunctivitis. In this study, we evaluated the relation between tear fluid levels of specific IgE and features of allergic conjunctivitis., Methods: A prospective, nonrandomized, cross-sectional study was conducted in patients with allergic conjunctivitis (n = 55, allergic group) and age- and sex-matched healthy control subjects (n = 50, control group). Levels of specific IgE for cedar pollen, cat epithelium/dander and Dermatophagoides pteronyssinus were measured in tear fluid with the Immfast Check J1®. A severity score (0, 1, 2 or 3) was assigned for various changes of the palpebral and bulbar conjunctiva, as well as for limbal and corneal lesions., Results: The levels of specific IgE for both cedar pollen, and D. pteronyssinus were significantly higher in the allergic group compared with the control group (p < 0.0001), while the level of specific IgE for cat epithelium/dander showed no significant difference between the two groups (p = 0.0777). When IgE levels were divided into four classes, the classes for both D. Pteronyssinus and cat epithelium/dander IgE were correlated with four features of allergic conjunctivitis. On the other hand, no correlation was found between the class of cedar pollen IgE and any of the features of allergic conjunctivitis., Conclusions: This study demonstrated that measurement of specific IgE in tear fluid may be useful for determining the severity of allergic conjunctivitis induced by indoor allergens. Although measurement of IgE in tear fluid is only a supplemental tool for evaluating the clinical activity of allergic conjunctivitis, the test can be useful for detecting specific IgE antibodies responsible for this condition.

Purpose: To assess the herpes simplex virus-specific tear secretory IgA (sIgA) and its correlation with specific serum antibodies and to evaluate the possible value of specific tear sIgA for prediction of herpes simplex keratitis (HSK) recurrence., Methods: Tear sIgA and paired serum IgG/IgM/IgA from 41 stromal HSK patients and 15 controls were analyzed by enzyme-linked immunosorbent assay. Subsequent follow-up for monitoring recurrence was performed from the time of first cure on all patients for 4 months to 1 year., Results: Serum IgG was positive in all participants. The concentration of serum IgA increased with the levels of tear sIgA (P = 0.017). The tear sIgA-positive rate was 36.59% in stromal keratitis, whereas none of the controls were found as sIgA positive. Positivity of sIgA was the only potential factor for predicting the risk of herpes keratitis recurrence (hazard ratio = 9.04; 95% confidence interval, 1.06-77.44; P = 0.045), with a sensitivity of 83.3% and a specificity of 71.4% (P = 0.034; 95% confidence interval, 0.58-0.97). Tear sIgA-positive patients had significantly shorter relapse-free survival compared with tear sIgA-negative patients (P = 0.015)., Conclusions: This is the first preliminary study suggesting that the levels of tear sIgA could be a potential prognostic parameter for the recurrence of HSK.

Analysis of functional tear production indices in patients with Grave's disease and thyroid eye disease confirmed lacrimal gland involvement in inflammatory autoimmune process. Close direct correlation was found between tear production and main disease characteristics - severity and activity.

Purpose: To identify clinical, demographic, immunologic, and health-related quality-of-life data from a cohort of vernal keratoconjunctivitis (VKC) patients with the onset of the disease after puberty (VKC-like disease)., Design: Retrospective, observational case series., Methods: Forty-nine patients with late-onset VKC-like disease from among 600 consecutive VKC patients. History of disease, test results for allergen sensitivity, signs and symptoms, impact of disease on work productivity, health-related quality of life, and treatment satisfaction were assessed. In addition, multiplex bead analysis for Th1/Th2 cytokines were carried out in tear samples from 20 VKC patients (10 adults and 10 children) and from 10 normal subjects., Results: A family history of allergy was positive in only 28% and positive prick test results were present in 55% of the 49 VKC-like adult patients. Based on typical signs and symptoms, 48% were affected by the limbal form, 33% were affected by the tarsal form, and 19% were affected by the mixed form. Corneal ulcer complicated the disease in only 2 adult patients. Although the disease was not considered a limiting factor for work, productivity was reduced by 26% and social activities were reduced by 31% during active flare-ups. No significant differences were found in tear cytokine pattern production between VKC in children and VKC in adults., Conclusions: A late onset VKC-like disease can appear in young adults with signs and symptoms similar to those in pediatric disease, but with less corneal involvement., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

Purpose: We evaluated the levels of lipid oxidative stress markers and inflammatory cells from tears and conjunctiva of patients with Sjögren syndrome (SS) and normal subjects., Methods: We examined 31 eyes of 16 patients (16 females) with SS and 15 eyes of 10 healthy controls (2 males and 8 females) in this prospective study. All subjects underwent a Schirmer test, measurement of tear film break-up time, vital stainings, confocal microscopy of the conjunctiva, tear collection for hexanoyl-lysine (HEL), ELISA, and conjunctival brush cytology. Brush cytology samples underwent immunohistochemistry (IHC) staining with HEL and 4-hydroxy-2-nonenal (4HNE). Hematoxylin-eosin and IHC staining with HEL and 4HNE also were performed on conjunctival samples of SS patients and controls., Results: The tear stability and vital staining scores were significantly worse in eyes of SS patients compared to the controls. Conjunctival inflammatory cell density was significantly higher in SS subjects compared to controls. The numbers of conjunctival cells stained positively for HEL and 4HNE were significantly higher in SS patients compared to controls. Tear HEL concentrations correlated significantly with staining scores and inflammatory cell density in confocal microscopy. Conjunctival specimens also revealed higher numbers of cells stained positively for inflammatory markers, as well as HEL and 4HNE in the IHC stainings., Conclusions: Increase of the oxidative stress status in the conjunctiva of SS patients appears to have a role in the pathogenesis of dry eye disease. A close relationship may exist between reactive oxygen species (ROS) production, lipid peroxidation related membrane damage, and inflammatory processes in dry eye.

Purpose: A new commercial immunochromatographic test (Allerwatch) has recently been developed for determining total immunoglobulin E (IgE). We previously reported on the use of this kit for analyzing tear fluid samples collected during the spring. In this study, we examined the relationship between the total IgE level in tears and the specific serum IgE levels in patients with autumnal allergic conjunctivitis., Methods: A prospective, nonrandomized cross-sectional study was conducted in 36 patients with autumnal allergic conjunctivitis (autumnal allergic group), in 32 age-matched and sex-matched healthy control subjects (control group), and in 10 patients with epidemic keratoconjunctivitis. Total tear IgE score was determined in all subjects using the Allerwatch test (0, 1, and 2), whereas the serum levels of total IgE and specific IgE for 12 inhaled allergens were measured using the Phadezym PRIST and CAP-RAST systems, respectively., Results: Positivity rates for total tear IgE were significantly higher in the autumnal allergic group as compared with the control and epidemic keratoconjunctivitis groups (88.9% vs. 0.0% vs. 0.0%, P < 0.00001). In the autumnal allergic group, there was a significant correlation between the total tear IgE score and the log-transformed total serum IgE level (r = 0.84). Significant correlations were also seen with the IgE serum levels for Dermatophagoides pteronyssinus (r = 0.53), house dust (r = 0.51), acarus (r = 0.49), cedar pollen (r = 0.33), and mugwort (r = 0.33). Multivariate logistic regression analysis showed that the only significant predictor of the total tear IgE score was the log-transformed total serum IgE level (odds ratio = 2.76, P < 0.00001)., Conclusions: The correlation between the Allerwatch total tear IgE score and the specific IgE serum levels against house dust mites suggests that house dust mite allergens might be the primary cause of allergic conjunctivitis during autumn in Japan. The Allerwatch methodology can provide rapid measurements of total IgE in the tear fluid, which makes it possible to easily diagnose allergic conjunctivitis in an outpatient setting.

Purpose: To assess the clinical relevance of tear anti-herpes simplex virus (HSV) antibody measurement for the diagnosis of herpes simplex keratitis., Methods: Records of 364 patients clinically suspect of HSV-related keratitis who had tear anti-HSV IgG assessment (tear-quantified anti-HSV IgG/filtrated IgG ratio) in our institution between January 2000 and August 2008 were retrospectively analyzed. Patients were classified into 4 groups as follows: group 1, anti-HSV IgG negative in serum and tears; group 2, anti-HSV IgG negative in tears and positive in serum; group 3, anti-HSV IgG nonsignificantly positive in tears and positive in serum; and group 4, anti-HSV IgG significantly positive in serum and tears. Randomly selected patient charts from each group were reviewed for clinical data., Results: The prevalence of anti-HSV IgG in blood increased with age from >70% before 20 years to 95% after 70 years. The prevalence of anti-HSV IgG in tears increased with age from 20% before 20 years to >50% after 70 years. The presence (either significant or not) of anti-HSV IgG in tears was significantly associated with decreased corneal sensation, presence of stromal opacities, and with neurotrophic keratitis. Logistic regression showed no significant association between age and clinical signs except for herpetic ulcers and herpetic necrotizing keratitis., Conclusions: Tear production of anti-HSV IgG increases with age, and it is associated with sequelae of herpes simplex keratitis. Conversely, it is poorly associated with clinical signs of acute herpes simplex keratitis.

Purpose: Measurement of total tear immunoglobulin E (IgE) is useful for the diagnosis of allergic conjunctivitis, but it is still unknown whether measurement of total tear IgE is useful for assessment of the severity of allergic conjunctivitis. We evaluated the relation between the total IgE level in tears and objective signs of allergic conjunctivitis., Methods: A prospective, nonrandomized, and cross-sectional study was conducted in 84 patients with allergic conjunctivitis (allergic group) and 80 age- and sex-matched healthy control subjects (control group). The total tear IgE score was obtained with the Allerwatch(®) test (0, 1, and 2), and ten severity scores (0, 1, 2, 3) were determined for objective ocular findings of the palpebral and bulbar conjunctiva, as well as limbal and corneal lesions., Results: The scores for total tear IgE and each of the objective ocular findings were higher in the allergic group than in the control group (all p < 0.01). The total tear IgE score was correlated with the severity of each clinical feature of allergic conjunctivitis (p < 0.01), except for corneal epithelial damage in the allergic group. Multivariate analysis revealed that the severity of giant papillae was the most important indicator of the total tear IgE score (odds ratio = 1.33, p < 0.00001)., Conclusion: The total tear IgE score was correlated with the scores for objective signs of allergic conjunctivitis. This rapid test for total IgE in tear fluid is easy to perform and could be used to assess the severity of allergic conjunctivitis on an outpatient basis.

Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease of cattle caused by Moraxella bovis (Mb). Parenterally administered immunogens used to prevent the disease do not offer complete protection possibly because they stimulate a poor ocular mucosal secretory response, in which locally secreted immunoglobulin-A (sIgA) is one of the main components. The principal aim of this study was to evaluate by an indirect enzyme linked immunosorbent assay (ELISA), the local ocular mucosal sIgA response against Mb purified pili, produced after intranasal inoculation of experimental vaccines. Pili were adjuvanted by several different adjuvants (QuilA, Marcol Arlacel, Marcol Span, microencapsulated pili with PLGA polymers). Results were compared to sIgA response produced by adjuvant placebo inoculations and by IBK natural infection. Significantly higher anti-pili IgA response (p<0.05) was detected in calves vaccinated intranasally with pili QuilA and pili Marcol Span compared to control calves, although this specific immune response did not seem to be related to protection against Mb infection or typical IBK lesion development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the efficacy of subjective symptoms, objective findings, and of a total tear IgE test kit in the diagnosis of allergic conjunctival diseases (ACD)., Subjects and Methods: The subjects were 223 patients with ACD, diagnosed according to the clinical practice guidelines for allergic conjunctival diseases in 28 medical facilities in Japan. The ACD patients were divided into the following five groups: 84 patients with seasonal allergic conjunctivitis (SAC), 52 patients with perennial allergic conjunctivitis (PAC), 41 patients with atopic keratoconjunctivitis (AKC), 38 patients with vernal keratoconjunctivitis (VKC) and 8 patients with giant papillary conjunctivitis (GPC). All cases were examined for clinical ACD, subjective symptoms, objective findings by the clinical score and by a total tear IgE test. An eosinophil examination by conjunctival smear was also performed in 87 ACD patients., Results: The most prevalent subjective symptoms were itching (81.6%) and hyperemia (77.6%). The clinical scores of the objective findings in the patients with SAC, PAC, AKC, VKC and GPC were 16.3 +/- 3.8 (mean +/- SD) points, 16.2 +/- 2.8, 19.8 +/- 6.5, 23.1 +/- 5.3, and 21.4 +/- 3.9, respectively. In the total tear IgE test 72.2% of the ACD patients were positive; i.e., the ratios were 61.9% in SAC, 65.4% in PAC, 80.5% in AKC, 94.7% in VKC, and 75.0% in GPC. In the examination of eosinophils in the 87 ACD patients, 42.5% (37 eyes) were positive, i.e., the ratio was 20.0% in SAC, 36.8% in PAC, 53.3% in AKC, 75.0% in VKC and 33.3% in GPC. The rate of agreement between the total tear IgE test and the examination of eosinophils was kappa = 0.28 (Cohen kappa coefficient)., Conclusion: Because of the high positive ratio in patients quasi-definitively diagnosed with ACD, we conclude that the total tear IgE test is useful as an auxiliary method for diagnosing ACD.

Background: Seasonal allergic conjunctivitis can manifest itself through tear film instability and symptoms of eye discomfort during the pollen season. This study investigated whether seasonal allergic inflammation defines tear film instability outside the season., Methods: Twenty-three control subjects and 13 ragweed-allergic patients were involved (21 female, 15 male; mean age 26.6 ± 5.4 years). Outside the pollen season, subjective ocular symptoms, non-invasive tear film break-up time, lower tear meniscus height and the tear lipid layer's interference pattern grade were recorded. C3a complement activation level was also measured in collected tear samples., Results: Non-invasive tear film break-up time, lower tear meniscus height, C3a complement activation level and the incidence of the different grades of tear lipid pattern did not differ significantly in the two examined groups (p ≥ 0.223). The mean eye symptom score outside the season was greater in the allergic group, but the difference was not significant (p = 0.062). The C3a complement activation level showed a significant and inverse correlation with the lipid layer grade (r = -0.343, p = 0.017). Among the participants with thinner tear lipid layers, the complement activation in the tear samples was higher than among those patients with normal tear lipid layers., Conclusion: Seasonal allergic inflammation did not cause permanent tear film instability and eye symptoms were not observed outside the pollen season., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: To explore non-invasive, protein-based, membrane array technology as a means to evaluate the global immune and angiogenic profile of tear proteins in patients with active ocular cicatricial pemphigoid (OCP)., Methods: Forty-three proteins consisting of cytokines, angiogenic/growth factors, and immunoinflammatory modulators were measured by membrane array in tear samples of four control patients and four OCP patients during active disease and after treatment., Results: Signals for several distinct and consistent molecular entities were upregulated in all four active OCP tear samples relative to controls. In particular, interleukin-8 and matrix metalloproteinase-9 were elevated during active disease and decreased after systemic immunomodulatory therapy., Conclusions: Protein array analysis may provide a well-tolerated assay to monitor levels of inflammatory markers in the tears of OCP patients in response to therapy.

Ocular toxoplasmosis can result in recurrent uveitis. Studies have shown that a correlation between active ocular toxoplasmosis and the presence of anti-Toxoplasma gondii secretory IgA (SIgA) in tears. This study compares anti-T. gondii SIgA levels in patients' tears during the acute and inactive phases of toxoplasmic uveitis. Twenty-nine positive tear specific SIgA for T. gondii patients with acute toxoplasmic uveitis were selected and were followed-up for at least two years, when the anti-T. gondii SIgA tears levels were determined. Specific SIgA for T. gondii was negative in 22 patients (75.86%) and positive in seven patients (24.13%) of whom six (85.7%) were followed over three years. Average SIgA levels during the acute phase are 1.54 and decrease significantly to 0.72 (p = 0.0001) during the inactive phase of disease. Because anti-T. gondii SIgA in the tear is negative in 75.86% of patients after the acute phase of infection, T. gondii SIgA levels may be used as a complementary diagnostic marker for active ocular toxoplasmosis.

Purpose: Recently, a new commercial test for total tear immunoglobulin E (IgE), based on immunochromatography (Allerwatch), was developed. We examined the relation between the total IgE level in tears and specific serum IgE., Methods: A prospective, nonrandomized, cross-sectional study was conducted in 35 patients with allergic conjunctivitis (allergic group), 30 age- and sex-matched healthy control subjects (control group), and 8 patients with epidemic keratoconjunctivitis. In all subjects, the total tear IgE score was determined with the Allerwatch test (0, 1, and 2), and serum levels of total IgE and specific IgE for 12 inhaled allergens were measured with the Phadezym PRIST and CAP-RAST systems, respectively., Results: The total tear IgE-positive rate was significantly higher in the allergic group than in the control and epidemic keratoconjunctivitis groups (100.0% vs. 0.0% vs. 0.0%; P < 0.00001). In the allergic group, the total tear IgE score was significantly correlated with the log-transformed total serum IgE level (r = 0.61) and with the serum levels of IgE for cedar pollen (r = 0.35), house dust (r = 0.46), Dermatophagoides pteronyssinus (r = 0.49), and acarus (r = 0.36). Multivariate logistic regression analysis showed that the log-transformed total serum IgE level was the only significant predictor of the total tear IgE score (odds ratio = 1.85, P = 0.00008)., Conclusions: The total tear IgE score, determined with the Allerwatch test, was correlated with the total and specific serum IgE levels. This rapid test is easy to perform, sensitive, and highly specific for the detection of ocular allergy on an outpatient basis.

Purpose: To evaluate whether Immfast Check J1, a new commercial immunochromatographic test for the measurement of specific immunoglobulin (Ig) E, could be used as a screening test for the diagnosis of allergic conjunctivitis., Methods: A prospective, nonrandomized, cross-sectional study was conducted in 64 moderate to severe cases of allergic conjunctivitis (allergic group) and 44 age- and sex-matched healthy control subjects (control group). Specific IgE levels for cedar pollen, cat epithelium, and Dermatophagoides pteronyssinus were measured in tear fluid with the Immfast Check J1., Results: Specific IgE levels in tear fluid could be assayed within 20 minutes in all subjects. The positive rate of specific IgE was significantly higher in the allergic group than in the control group (cedar pollen, 96.9% vs. 2.3%, P < 0.00001; cat epithelium, 23.4% vs. 0.0%, P = 0.00018; and D. pteronyssinus, 53.1% vs. 2.3%, P < 0.00001). Specific IgE scores were also significantly higher in the allergic group than in the control group (cedar pollen, P < 0.00001; cat epithelium, P = 0.03907; and D. pteronyssinus, P < 0.00001)., Conclusions: These results suggest that Immfast Check J1 is a rapid and easy test for detection of allergen-specific IgE in tear fluid samples from patients with allergic conjunctivitis. The test is reliable and easy to perform on an outpatient basis.

Background: Herpes Simplex Virus (HSV) keratitis is a leading cause of corneal blindness. Definitive laboratory diagnosis is essential for timely management. Collection of corneal scrapings in patients with advanced epithelial keratitis and corneal thinning poses perforation risks; tear fluid is a feasible and convenient alternative but has not been widely evaluated for HSV detection., Methods: Tear fluid alone (229) or along with corneal scrapings (153) from patients of suspected herpetic keratitis was tested for HSV-1 antigen by indirect immunofluorescence assay, virus isolation in Hep 2 cells and PCR to amplify the 111bp region of the thymidine kinase (tk) coding gene and the 144bp region from the DNA polymerase coding gene of HSV., Results: HSV 1 antigen was detected in 31/229 (13.53%) tear specimen and 35/153 (22.87%) corneal scrapings in immunofluorescence assay; virus was isolated from 12/229 (5.2%) tear and 17/153 (11.11%) corneal scrapings, and PCR was positive for both the genes in 32/229 (13.97%) tear specimen and 56/153 (36.66%) corneal scrapings., Conclusion: Corneal scrapings yielded a significantly better HSV positivity than tears in both the PCR assay (p<0.0005) and immunofluorescence assay. PCR was much more sensitive than immunofluorescence and virus isolation. However, tears should be tested for definitive laboratory diagnosis of HSV infection whenever corneal scraping collection is not possible.

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

Background: Recently, a new commercial test for specific IgE based on immunochromatography (Immfast Check J1®) has been developed. We previously reported on the use of this kit for tear fluid samples during spring. In this study, we compared the level of specific IgE in tear fluid among patients with seasonal allergic conjunctivitis during spring or autumn and control subjects., Methods: A nonrandomized, cross-sectional study was conducted in patients with moderate-to-severe allergic conjunctivitis occurring in spring (n = 56, spring group) or autumn (n = 52, autumn group), as well as in age- and sex-matched healthy control subjects (n = 50, control group). Levels of specific IgE for cedar pollen, cat epithelium and Dermatophagoides pteronyssinus were measured in tear fluid with the Immfast Check J1., Results: Specific IgE scores for cedar pollen and D. pteronyssinus were significantly higher in the spring and autumn groups than in the control group (p < 0.0001). In the autumn group, the highest detection rate for a specific allergen was 96.2% for D. pteronyssinus, followed by cedar pollen (86.5%) and cat epithelium (25.5%). In the spring group, the highest detection rate was 98.3% for cedar pollen, followed by D. pteronyssinus (51.7%) and cat epithelium (19.0%)., Conclusions: These results suggest that both house dust mite allergen and outdoor allergens such as cedar pollen can be causes of allergic conjunctivitis during both spring and autumn in Japan. The Immfast Check J1 provides rapid measurement of specific IgE in tear fluid, which allows easy diagnosis of allergic conjunctivitis in an outpatient setting., (Copyright © 2011 S. Karger AG, Basel.)