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Purpose of Review: Ocular surface disorders (OSDs) are a prevalent and often debilitating condition encountered in clinical practice, particularly by allergists and clinical immunologists., Recent Findings: A comprehensive guide to office procedures for evaluating and managing OSDs, with a specific focus on ocular allergies, would assist in the evaluation process that begins with an initial patient assessment utilizing standardized forms to systematically gather detailed medical history, symptomatology, and environmental exposure data. This structured approach ensures a thorough understanding of the patient's condition and facilitates targeted interventions. In addition to allergy testing, the assessment of the tear film is essential for a comprehensive evaluation of OSDs. The Schirmer test is employed to quantify tear production, providing objective data on tear film adequacy and guiding interventions for tear film deficiencies., Summary: This multifaceted diagnostic approach ensures that all contributing factors to OSDs are identified and appropriately managed. By integrating these office procedures, allergists and clinical immunologists can enhance their diagnostic accuracy and therapeutic efficacy, ultimately improving patient outcomes. This manuscript provides a practical resource, outlining some of the methodologies and clinical applications of each procedure, and highlighting their role in the holistic management of OSDs in allergic patients., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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.)

The literature is filled with citations reporting an increased incidence of chronic dry eye disease, also known as keratoconjunctivitis sicca, in patients with systemic autoimmune diseases such as rheumatoid arthritis, Sjögren's Syndrome, systemic sclerosis and lupus. As the most environmentally exposed mucosal surface of the body, the conjunctiva constantly responds to environmental challenges which are typically self limited, but when persistent and unresolved may provoke pathogenic innate and adaptive immune reactions. Our understanding of the pathophysiological mechanisms by which systemic autoimmune diseases cause dry eye inducing ocular surface inflammation continues to evolve. Conjunctival immune tone responds to self or foreign danger signals (including desiccating stress) on the ocular surface with an initial non-specific innate inflammatory response. If unchecked, this can lead to activation of dendritic cells that present antigen and prime T and B cells resulting in an adaptive immune reaction. These reactions generally resolve, but dysfunctional, hyper-responsive immune cells found in systemic autoimmune diseases that are recruited to the ocular surface can amplify inflammatory stress responses in the ocular surface and glandular tissues and result in autoimmune reactions that disrupt tear stability and lead to chronic dry eye disease. We here propose that unique features of the ocular surface immune system and the impact of systemic immune dysregulation in autoimmune diseases, can predispose to development of dry eye disease, and exacerbate severity of existing dry eye., Competing Interests: Declaration of competing interest M. Stern – Consultantships - Kala. A. Theofilopoulos - None. P. Steven- Bausch & Lomb, URSA-PHARM, Marinomed, Bitop J. Niederkorn - None. R. Fox - None. M. Calonge - None. C. Scheid - None. S. Pflugfelder - None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

This study evaluated human papillomavirus's (HPV) role in pterygium pathogenesis, its autoinoculation from genitalia to ocular surface, potential cytokines involved, and crosstalk cytokines between pterygium and dry eye (DE). This cross-sectional study enrolled 25 healthy controls (HCs) and 116 pterygium patients. Four subgroups of pterygium and DE were used in cytokine evaluations. Conjunctival and pterygium swabs and first-void urine samples (i.e., genitalia samples) were collected for HPV DNA detection using real-time polymerase chain reaction. Tear cytokines interleukin (IL)-6, IL-18, and vascular endothelial growth factor (VEGF) in tears were evaluated. No HPV DNA was detected in conjunctival or pterygium swabs. No association was found between HPV DNA in urine samples and that from conjunctival or pterygium swabs. Tear VEGF levels were significantly higher in pterygium patients than in HCs, with no markedly different levels between primary and recurrent pterygia. Tear IL-6, IL-18, and tear VEGF were significantly higher in participants with DE, regardless of pterygium status. In conclusion, HPV infection was not a pathogenic factor of pterygia. The hypothesis of HPV transmitting from the genitals to ocular surfaces was nullified. Tear VEGF was involved in both pterygia and DE, whereas tear IL-6 and IL-18 played roles only in DE.

Background: Whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody tests are increasingly being used to estimate the prevalence of SARS-CoV-2 infection, the determinants of these antibody responses remain unclear., Objectives: Our aim was to evaluate systemic and mucosal antibody responses toward SARS-CoV-2 in mild versus severe coronavirus disease 2019 (COVID-19) cases., Methods: Using immunoassays specific for SARS-CoV-2 spike proteins, we determined SARS-CoV-2-specific IgA and IgG in sera and mucosal fluids of 2 cohorts, including SARS-CoV-2 PCR-positive patients (n = 64) and PCR-positive and PCR-negtive health care workers (n = 109)., Results: SARS-CoV-2-specific serum IgA titers in patients with mild COVID-19 were often transiently positive, whereas serum IgG titers remained negative or became positive 12 to 14 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers after symptom onset. Very high titers of SARS-CoV-2-specific serum IgA were correlated with severe acute respiratory distress syndrome. Interestingly, some health care workers with negative SARS-CoV-2-specific serum antibody titers showed SARS-CoV-2-specific IgA in mucosal fluids with virus-neutralizing capacity in some cases. SARS-CoV-2-specific IgA titers in nasal fluids were inversely correlated with age., Conclusions: Systemic antibody production against SARS-CoV-2 develops mainly in patients with severe COVID-19, with very high IgA titers seen in patients with severe acute respiratory distress syndrome, whereas mild disease may be associated with transient production of SARS-CoV-2-specific antibodies but may stimulate mucosal SARS-CoV-2-specific IgA secretion., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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 of Review: Chemokines are a large group of low molecular weight cytokines that attract and activate leukocytes throughout the body and therefore have a key role in the framework of late-phase allergic responses. The purpose of this article is to provide an overview of the main chemokines involved in allergic conjunctivitis, their primary functions and their physiological roles, and therapies targeted at chemokines and their receptors for ocular allergic diseases., Recent Findings: In recent years, there have been considerable advances in the understanding of ocular pathophysiology of ocular surface inflammatory diseases including both allergic eye diseases and dry eye syndrome. Several therapies being developed for dry eye inflammation are recognized as possible therapies for ocular allergic diseases as there are often common chemokines involved in both disease spectra., Summary: Chemokines represent an integral part of the late-phase cascade of ocular allergic inflammation. A deep understanding of specific chemokines and their interactions will help in targeting therapies to effectively manage ocular clinical findings and symptoms of allergic eye disease.

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.

Background-It is recognized that inflammation is an underlying cause of dry eye disease (DED), with cytokine release involved. We systematically reviewed literature with meta-analyses to quantitatively summarize the levels of tear cytokines in DED. Methods-The PubMed, Embase, Web of Science, Ovid, Cochrane, and Scopus databases were reviewed until September 2019, and original articles investigating tear cytokines in DED patients were included. Differences of cytokines levels of DED patients and controls were summarized by standardized mean differences (SMD) using a random effects model. Study quality was assessed by applying Newcastle-Ottawa-Scale and the GRADE quality score. Methods of analytical procedures were included as covariate. Results-Thirteen articles investigating 342 DED patients and 205 healthy controls were included in the meta-analysis. The overall methodological quality of these studies was moderate. Systematic review of the selected articles revealed that DED patients had higher tear levels of interleukin (IL)-1β, IL-6, chemokine IL-8, IL-10, interferon-γ, IFN-γ, and tumor necrosis factor-α, TNF-α as compared to controls. Evidence was less strong for IL-2 and IL-17A. Conclusions-Data show that levels of tear cytokines in DED and control display a great variability, and further studies of higher quality enrolling a higher number of subjects are needed, to define a cut-off value.

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.

Background: Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms., Objective: We developed a new strategy to treat Fel d 1-induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1., Methods: A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin-derived universal T-cell epitope tt830-843 (CuMV TT ) was used to immunize cats. A first tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMV TT vaccine alone or in combination with an adjuvant., Results: The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti-Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both in vitro and in vivo. A reduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed., Conclusion: Vaccination of cats with Fel-CuMV TT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Investigating cytokines in tear fluid and saliva may offer valuable information for understanding the pathogenesis of primary Sjögren's syndrome (pSS). Cytokine profiles in both tear fluid and saliva of pSS patients, non-Sjögren's syndrome (non-SS) subjects with sicca symptoms, and healthy controls without sicca complaints were analysed. Furthermore, relationships associating the severity of clinical ocular and oral manifestations with the upregulated cytokines were assessed. In tear fluid, pSS patients showed elevated levels of IL-1ra, IL-2, IL-4, IL-8, IL-12p70, IL-17A, IFN-γ, IP-10, MIP-1b, and Rantes compared to non-SS subjects and healthy controls. The increased cytokine levels (except IP-10) correlated significantly with reduced tear production, less stable tear film, and greater ocular surface damage. In saliva, pSS patients had a higher IP-10 level, which correlated with higher candida score; and an elevated MIP-1a level, which correlated significantly with lower unstimulated and stimulated whole saliva secretion rates. The upregulated cytokines identified in tear fluid and saliva of pSS patients show a clear interplay between innate and adaptive immune responses that may contribute to disease pathogenesis. The increase of IP-10 and MIP in both tears and saliva further emphasises the essential role of macrophages and innate immunity 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.)

Background: Dry eye disease (DED) affects one third of the population worldwide. In prior studies, experimental autoimmune lacrimal keratoconjunctivitis (EALK) induced by desiccating stress in mice has been used as a model of DED. This model is complicated by a requirement for exogenous epithelial cell injury and administration of anticholinergic agents with broad immunologic effects., Objective: We sought to develop a novel mouse model of EALK and to demonstrate the responsible pathogenic mechanisms., Methods: CD4 + CD45RB high naive T cells with and without CD4 + CD45RB low regulatory T cells were adoptively transferred to C57BL/10 recombination-activating gene 2 (Rag2) -/- mice. The eyes, draining lymph nodes, lacrimal glands, and surrounding tissues of mice with and without spontaneous keratoconjunctivitis were evaluated for histopathologic changes, cellular infiltration, and cytokine production in tissues and isolated cells. Furthermore, the integrity of the corneal nerves was evaluated using whole-tissue immunofluorescence imaging. Gene-deficient naive T cells or RAG2-deficient hosts were evaluated to assess the roles of IFN-γ, IL-17A, and IL-23 in disease pathogenesis. Finally, cytokine levels were determined in the tears of patients with DED., Results: EALK developed spontaneously in C57BL/10 Rag2 -/- mice after adoptive transfer of CD4 + CD45RB high naive T cells and was characterized by infiltration of CD4 + T cells, macrophages, and neutrophils. In addition to lacrimal keratoconjunctivitis, mice had damage to the corneal nerve, which connects components of the lacrimal functional unit. Pathogenic T-cell differentiation was dependent on IL-23p40 and controlled by cotransferred CD4 + CD45RB low regulatory T cells. T H 17 rather than T H 1 CD4 + cells were primarily responsible for EALK, even though levels of both IL-17 and IFN-γ were increased in inflammatory tissues, likely because of their ability to drive expression of CXC chemokines within the cornea and the subsequent influx of myeloid cells. Consistent with the findings of this model, the tears of patients with DED had increased levels of inflammatory cytokines, including IL-17A and TNF-α., Conclusion: We describe a novel model of spontaneous EALK that supports a role for T H 17 cells in disease pathogenesis and that will contribute to our understanding of autoimmune lacrimal keratoconjunctivitis in many human eye diseases, including DED., (Copyright © 2017. Published by Elsevier Inc.)

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.)

Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by severe inflammation of exocrine glands such as the salivary and lacrimal glands. When it affects the lacrimal glands, many patients experience keratoconjunctivitis due to severely dry eyes. This study investigated the pathological and immunological characteristics of ocular lesions in a mouse model of SS. Corneal epithelial injury and hyperplasia were confirmed pathologically. The number of conjunctival mucin-producing goblet cells was significantly decreased in the SS model mice compared with control mice. Expression levels of transforming growth factor (TGF)-β, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C-X-C motif chemokine (CXCL) 12 were significantly higher in the corneal epithelium of the SS model mice than in control mice. Inflammatory lesions were observed in the Harderian, intraorbital, and extraorbital lacrimal glands in the SS model mice, suggesting that the ocular glands were targeted by an autoimmune response. The lacrimal glands of the SS model mice were infiltrated by cluster of differentiation (CD)4⁺ T cells. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed significantly increased mRNA expression of TNF-α, TGF-β, CXCL9, and lysozyme in the extraorbital lacrimal glands of the SS model mice compared with control mice. These results add to the understanding of the complex pathogenesis of SS and may facilitate development of new therapeutic strategies., Competing Interests: The authors declare no conflicts of interest.

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.)

Non-recirculating resident memory (T RM ) and recirculating T cells mount vigorous immune responses to both self and foreign antigens in barrier tissues like the skin, lung and gastrointestinal tract. Using impression cytology followed by flow cytometry we identified two T RM subsets and four recirculating T-subsets in the healthy human ocular surface. In dry eye disease, principal component analysis (PCA) revealed two clusters of patients with distinct T-cell signatures. Increased conjunctival central memory and naïve T cells characterized Cluster-1 patients, and increased CD8 + T RM s and CD4 + recirculating memory T cells characterized Cluster-2 patients. Interestingly these T-cell signatures are associated with different clinical features: the first signature correlated with increased ocular redness, and the second with reduced tear break up times. These findings open the door to immune-based characterization of dry eye disease and T-subset specific immunotherapies to suppress T-subsets involved in disease. They may also help with patient stratification during clinical trials of immunomodulators.

Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvβ3 and α4β1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.

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.

Eye-derived fluids, including tears, aqueous humour and vitreous humour often contain molecular signatures of ocular disease states. These signatures can be composed of cytokines, chemokines, growth factors, proteases and soluble receptors. However, the small quantities (<10 µl) of these fluids severely limit the detection of these proteins by traditional enzyme-linked immunosorbent assay or Western blot. To maximise the amount of information generated from the analysis of these specimens, many researchers have employed multiplex immunoassay technologies for profiling the expression or modification of multiple proteins from minute sample volumes.

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.

Age-related macular degeneration (AMD) is the leading cause of blindness among senior citizens of developed countries, with currently unknown etiology. Despite the close associations between AMD development and inhibitory complement factor H mutations, the first step of complement activation, which is the antibody response in AMD patients, has not been studied. Here, we obtained blood and tear samples from AMD patients and Non-AMD controls. We found that compared to Non-AMD controls, AMD subjects had increased IgA titers in serum and tear, and had elevated levels of circulating antibody-secreting plasmablasts. The increase in antibody titer was limited to the IgA isotype, since no significant differences were observed in IgM and IgG isotypes between AMD patients and Non-AMD controls. Interestingly, this increased antibody response in AMD patients was correlated with disease severity, as late AMD patients had increased IgA titers in serum and tear, as well as elevated plasmablast frequency after staphylococcal enterotoxin B stimulation, compared to early AMD patients. Together, our results implicated a role of overreactive IgA responses in AMD pathogenesis., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Different nasal challenges induce neural and immune response leading to nasal and ocular symptoms in patients with seasonal allergic rhinitis (SAR). The release of neural mediators from nasal mucosa and conjunctiva after no-specific challenges in patients with SAR remains unknown., Objectives: To compare the release of mediators from the nose and conjunctiva with symptoms after different nasal challenges in patients with SAR., Methods: Three types of consecutive nasal challenges were performed outside the pollen season in 25 patients with SAR. Challenges consisted of 500 biological units (BU) of allergen, 80 μg of histamine, and 1 mL of 2% hypertonic saline per nostril, within 24-hour and 72-hour intervals, respectively. Before and 15 minutes after challenges, evaluation of symptoms was performed with a visual analog scale. Concentrations of tryptase, eosinophil cationic protein in nasal lavages after 15 minutes, and substance P in tears after 5 minutes were measured with enzyme immunoassays., Results: Concentrations of substance P in tears were significantly higher after nonspecific challenges. Substance P concentration in tears significantly correlated with eye itchiness after histamine and hypertonic saline and with tearing after allergen. Ocular symptoms correlated significantly with tryptase concentration in nasal lavage collected 15 minutes after allergen challenge. There is a significant correlation in tear volume comparing different nasal challenges., Conclusions: Nasal challenges with allergen, histamine, or irritants outside the pollen season induce a significant increase in nasal and ocular symptoms in patients with SAR. Interaction of the early-phase response and neurogenic inflammation define the pattern and severity of eye symptoms., (Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Ocular surface inflammation is one of the primary mechanisms associated with dysfunctional tear syndrome (DTS), also known as dry eye disease. DTS, more prevalent in older populations, causes ocular discomfort and visual disturbance due to dryness on the surface layer in the eye. We used human conjunctival fibroblast cultures (HCJVF) to investigate the effects of inflammatory cytokines IFN-γ, TNF-α and IL-1β (ITI) on the secretions of VEGF and chemokines. Our results demonstrate the elevated secretion of angiogenic VEGF molecules by ITI without affecting anti-angiogenic molecules, PEDF, endostatin, thrombospondin and sVEGF-R1. The secretion of interferon-γ inducible chemokines, CXCL9, -10, -11 by HCJVF were significantly enhanced by ITI. Our in vitro study supports previously reported observations of elevated VEGF and chemokines in tear fluids of DTS patients, reiterating the role of inflammatory reactions in DTS., (Published by Elsevier Ltd.)

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: Air pollution is one of the most environmental health concerns in the world and has serious impact on human health, particularly in the mucous membranes of the respiratory tract and eyes. However, ocular hazardous effects to air pollutants are scarcely found in the literature., Design: Panel study to evaluate the effect of different levels of ambient air pollution on lacrimal film cytokine levels of outdoor workers from a large metropolitan area., Methods: Thirty healthy male workers, among them nineteen professionals who work on streets (taxi drivers and traffic controllers, high pollutants exposure, Group 1) and eleven workers of a Forest Institute (Group 2, lower pollutants exposure compared to group 1) were evaluated twice, 15 days apart. Exposure to ambient PM2.5 (particulate matter equal or smaller than 2.5 μm) was 24 hour individually collected and the collection of tears was performed to measure interleukins (IL) 2, 4, 5 and 10 and interferon gamma (IFN-γ) levels. Data from both groups were compared using Student's t test or Mann- Whitney test for cytokines. Individual PM2.5 levels were categorized in tertiles (lower, middle and upper) and compared using one-way ANOVA. Relationship between PM2.5 and cytokine levels was evaluated using generalized estimating equations (GEE)., Results: PM2.5 levels in the three categories differed significantly (lower: ≤22 μg/m3; middle: 23-37.5 μg/m3; upper: >37.5 μg/m3; p<0.001). The subjects from the two groups were distributed unevenly in the lower category (Group 1 = 8%; Group 2 = 92%), the middle category (Group 1 = 89%; Group 2 = 11%) and the upper category (Group 1 = 100%). A significant relationship was found between IL-5 and IL-10 and PM2.5 levels of the group 1, with an average decrease of 1.65 pg/mL of IL-5 level and of 0.78 pg/mL of IL-10 level in tear samples for each increment of 50 μg/m3 of PM2.5 (p = 0.01 and p = 0.003, respectively)., Conclusion: High levels of PM2.5 exposure is associated with decrease of IL-5 and IL-10 levels suggesting a possible modulatory action of ambient air pollution on ocular surface immune response.

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.)

Background: Conventional diagnostic tests in allergy are insufficient to clarify the origin of vernal conjunctivitis (VC)., Objectives: To evaluate IgE-mediated hypersensitivity by component-resolved diagnosis (CRD) in tears and serum from patients with VC and to evaluate how to treat patients with identified triggering allergens by specific immunotherapy., Methods: Patients were divided into 3 groups: (1) patients with VC (25 patients), (2) patients allergic to grass pollen with seasonal allergic conjunctivitis (AC) (50 patients), and (3) healthy blood donors (50 patients). If triggering allergens were detected, specific conventional immunotherapy was administered for 1 year., Results: Twenty-five patients with VC were evaluated. The identified triggering allergens were n Lol p 1 (11 patients), n Cyn d 1 (8 patients), group 4 and 6 grasses (6 patients), and group 5 grasses (5 patients). Prick test and pollen IgE test results were positive in one patient. Clinical improvement was observed in 13 of the 25 patients with VC after 1 year of specific immunotherapy., Conclusion: 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 patients with VC., (Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Immunoglobulin G (IgG) antibodies are involved in type II and type III hypersensitivity. We evaluated the relation between perennial allergic conjunctivitis and serum levels of specific IgG for cat allergens. A prospective study was conducted in patients with seasonal allergic conjunctivitis (seasonal group, n = 10), patients with perennial allergic conjunctivitis (perennial group, n = 10), and healthy control subjects (control group, n = 10). Serum levels of specific IgE and IgG for cat allergens and total tear IgE were measured, and a skin prick test was also performed. In addition, a severity score associated with allergic conjunctivitis was calculated (0-30). The positive rates and scores of for total tear IgE, serum cat-specific IgE, and serum cat-specific IgG were all higher in the seasonal and perennial groups than in the control group (all p < 0.05). Serum cat-specific IgG levels were higher in the perennial group than in the seasonal group (p = 0.0156), but there was no significant difference in the grade of cat-specific IgE between the two groups (p = 0.3008). On multivariate analysis, the mean wheal diameter for cat allergen was associated with the serum level of cat-specific IgG (not IgE) in all patients [odds ratio (OR) = 31.979, p < 0.0001]. Multivariate analysis revealed that the total objective score was strongly associated with serum cat-specific IgG (OR = 23.015, p < 0.0001). These findings suggest that specific IgG antibodies may be involved in perennial allergic symptoms caused by indoor allergens such as cat allergens.

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.

Purpose: To investigate the efficacy of hypotonic 0.18% sodium hyaluronate (SH) eyedrops in a mouse model of experimental dry eye (EDE)., Methods: EDE was induced in C57BL/6 mice by a subcutaneous scopolamine injection and an air draft. The mice were divided into 4 groups according to topical treatment regimens: EDE control, isotonic 0.5% carboxymethycellulose (CMC), isotonic 0.1% SH, and hypotonic 0.18% SH. Tear volume, corneal smoothness, and corneal staining scores were measured at 5 and 10 days of EDE. Multiplex immunobead assay, immunohistochemistry, and flow cytometry for proinflammatory cytokines, chemokines, and inflammatory molecules were performed at 10 days of EDE., Results: The 0.18% SH group had a significantly lower corneal smoothness and staining scores than the 0.5% CMC and 0.1% SH groups at 10 days of EDE (P<0.05). The 0.18% SH group showed significantly low levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, monokine induced by interferon-γ, and interferon-γ-inducible protein 10 compared with the other groups (P<0.05). The mean percentages of CD4(+)CXCR3(+), CD40(+), and CD44(+) cells in the conjunctiva were significantly lower in the 0.18% SH group than in the other groups (P<0.05). In addition, the 0.1% SH group showed lower levels of TNF-α and IL-1β and percentages of CD40(+) and CD44(+) cells than the EDE and 0.5% CMC groups., Conclusions: Hypotonic 0.18% SH eyedrops are more effective in improving ocular surface irregularity and staining and decreasing inflammatory cytokines, chemokines, and cells on the ocular surface compared with isotonic 0.5% CMC or 0.1% SH eyedrops in the treatment of EDE.

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).

Factors responsible for the persistence of Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis vaccines in commercial flocks and the high frequency of isolation of ArkDPI-type infectious bronchitis viruses in respiratory cases are still unclear. We compared dynamics of vaccine viral subpopulations, viral loads, persistence in trachea and cloaca, and the magnitude of infectious bronchitis virus (1BV)-specific antibody induction after vaccination with two commercial ArkDPI-derived Arkansas (Ark) serotype vaccines. One of the vaccines (coded vaccine B) produced significantly higher vaccine virus heterogeneity in vaccinated chickens than the other vaccine (coded A). Chickens vaccinated with vaccine B had significantly higher viral loads in tears at 5 days postvaccination (DPV) than those vaccinated with vaccine A. Vaccine B also induced a significantly higher lachrymal immunoglobulin M response at 11 DPV, an earlier peak of IBV-specific lachrymal immunoglobulin A, and higher serum antibodies than vaccine A. In addition, a significantly higher proportion of birds vaccinated with vaccine B had vaccine virus detected in the trachea at 20 DPV than those vaccinated with vaccine A. Furthermore, the virus detected at 20 DPV in most of the chickens vaccinated with vaccine B was a single specific subpopulation (subpopulation 4) selected from multiple vaccine subpopulations detected earlier at 5 and 7 DPV in the same chickens. On the other hand, a higher proportion of chickens vaccinated with vaccine A had virus detected in cloacal swabs at 20 DPV. Thus we found differences in mucosal antibody induction and selection and persistence of vaccine viruses between two ArkDPI-derived vaccines from different manufacturers. The higher vaccine virus heterogeneity observed in chickens vaccinated with vaccine B compared with those vaccinated with vaccine A may be responsible for these differences. Thus the high frequency of Ark IBV viruses in the field may be due to the inherent ability of some ArkDPI-derived vaccine viruses to be selected and persist in vaccinated chickens. Vaccine virus persistence may offer genetic material for recombination or may undergo mutations with the potential to result in increased virulence.

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).

Infectious bovine keratoconjunctivitis (IBK) caused by Moraxella bovis is the most common eye disease of cattle. The pathogenesis of M. bovis requires the expression of pili that enable the organism to attach to the ocular surface and an RTX (repeats in the structural toxin) toxin (cytotoxin or hemolysin), which is cytotoxic to corneal epithelial cells. In this pilot study, ocular mucosal immune responses of steers were measured following intranasal (i.n.) vaccination with a recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid. Beef steers were vaccinated with either 500 μg (n = 3) or 200 μg (n = 3) of recombinant M. bovis cytotoxin plus adjuvant. Control group steers (n = 2) were vaccinated with adjuvant alone, and all steers were given a booster on day 21. Antigen-specific tear IgA and tear IgG, tear cytotoxin-neutralizing antibody responses, and serum cytotoxin-neutralizing antibody responses were determined in samples collected prevaccination and on days 14, 28, 42, and 55. Changes in tear antigen-specific IgA levels from day 0 to days 28, 42, and 55 were significantly different between groups; however, in post hoc comparisons between individual group pairs at the tested time points, the differences were not significant. Our results suggest that i.n. vaccination of cattle with recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid effects changes in ocular antigen-specific IgA concentrations. The use of intranasally administered recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid could provide an alternative to parenteral vaccination of cattle for immunoprophylaxis against IBK.

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.)