Tear Fluid Of Patients Research Articles

Dynamics of VEGF and TGF-β indices in lacrimal fluid as a predictor of corneal transplant rejection

Graft rejection is the most common cause of corneal transplant failure. Despite the fact that the cornea is an immunoprivileged organ, corneal transplant rejection is still a pressing problem. Vascularization plays one of the key roles in triggering corneal transplant rejection. Depending on the condition of the recipient’s tissue bed, keratoplasty may be classified into “high-risk” and “low-risk” rejection. In the first case, the mechanisms of immune privilege and tolerance are disturbed. In the case of “low-risk” keratoplasty, transplantation occurs at avascular and non-inflamed bed, which is a more favorable prognostic option. The aim of our study was to evaluate the levels of vascular endothelial growth factor (VEGF) and transforming growth factor â (TGF- â) in the tear fluid of patients before and after penetrating keratoplasty. 42 patients (84 eyes) participated in the study, including 28 women (61.54%) and 14 men (38.46%) aged from 31 to 65 years, the average age was 53.1±4.38 years. Patients were divided into “high-risk” and “low-risk” groups depending on their medical history and objective clinical pattern. The levels of cytokines in the tear fluid were determined using a multiplex analysis on a Luminex Magpix 100 immunoanalyzer (USA) using a Bio-Rad multiplex analysis test system (USA) over time before surgical treatment and after 1 and 6 months of the postoperative period. The study showed an increased concentration of vascular endothelial growth factor and, conversely, a decrease in the concentration of transforming growth factor â in patients at high risk. The opposite picture, if compared to the indices of healthy controls, was observed in patients from the “low-risk” group, where low background concentrations of vascular endothelial growth factor and high levels of transforming growth factor â were determined. This finding suggests preservation of immune tolerance at the internal media of the eye, maintaining a balance of neovascularization, thus being associated with low risk of graft rejection. The risks of more frequent corneal transplant rejection as the concentration of immunosuppressive factors (e.g., TGF) decreases, and, vice versa, the risks increase with changing levels of vasoform cytokines that promote corneal neovascularization.

A study of monocyte chemoattractant protein-1 in tear fluid in patients with retinal vessel occlusion

A study of monocyte chemoattractant protein-1 in tear fluid in patients with retinal vessel occlusion

Multiplatform tear proteomic profiling reveals novel non-invasive biomarkers for diabetic retinopathy.

To investigate a comprehensive proteomic profile of the tear fluid in patients with diabetic retinopathy (DR) and further define non-invasive biomarkers. A cross-sectional, multicentre study that includes 46 patients with DR, 28 patients with diabetes mellitus (DM), and 30 healthy controls (HC). Tear samples were collected with Schirmer strips. As for the discovery set, data-independent acquisition mass spectrometry was used to characterize the tear proteomic profile. Differentially expressed proteins between groups were identified, with gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis further developed. Classifying performance of biomarkers for distinguishing DR from DM was compared by the combination of three machine-learning algorithms. The selected biomarker panel was tested in the validation cohort using parallel reaction monitoring mass spectrometry. Among 3364 proteins quantified, 235 and 88 differentially expressed proteins were identified for DR when compared to HC and DM, respectively, which were fundamentally related to retina homeostasis, inflammation and immunity, oxidative stress, angiogenesis and coagulation, metabolism, and cellular adhesion processes. The biomarker panel consisting of NAD-dependent protein deacetylase sirtuin-2 (SIR2), amine oxidase [flavin-containing] B (AOFB), and U8 snoRNA-decapping enzyme (NUD16) exhibited the best diagnostic performance in discriminating DR from DM, with AUCs of 0.933 and 0.881 in the discovery and validation set, respectively. Tear protein dysregulation is comprehensively revealed to be associated with DR onset. The combination of tear SIR2, AOFB, and NUD16 can be a novel potential approach for non-invasive detection or pre-screening of DR. Chinese Clinical Trial Registry Identifier: ChiCTR2100054263. https://www.chictr.org.cn/showproj.html?proj=143177 . Date of registration: 2021/12/12.

Dupilumab-associated ocular surface disease is characterized by a shift from Th2/Th17 toward Th1/Th17 inflammation.

Dupilumab is used for the treatment of atopic dermatitis (AD). Approximately one third of AD patients develop a dupilumab-associated ocular surface disease (DAOSD), of which the pathomechanism is poorly understood. This study aimed at investigating inflammatory markers in tear fluids of patients on dupilumab therapy. Tear fluids were collected from AD patients with DAOSD (ADwDAOSD), AD patients without DAOSD (ADw/oDAOSD), and non-AD patients before and during dupilumab therapy, and analyzed using a specialized proteomic approach quantifying inflammatory markers. The ocular surface microbiome was determined by next generation sequencing technology. Upon dupilumab therapy, an upregulation of 31 inflammatory markers was observed in DAOSD tear fluids compared to baseline in AD patients. While IL-12B was upregulated in both ADwDAOSD and ADw/oDAOSD groups, the pattern of inflammatory markers significantly differed between groups and over time. In the ADwDAOSD group, a shift from a mixed Th2/Th17 pattern at baseline toward a Th1/Th17 profile under dupilumab was observed. Furthermore, an upregulation of remodeling and fibrosis markers was seen in DAOSD. Semantic map and hierarchical cluster analyses of baseline marker expression revealed four clusters distinguishing between AD and non-AD as well as ADwDAOSD and ADw/oDAOSD patient groups. In a pilot study, dupilumab therapy was associated with a decrease in richness of the ocular surface microbiome. DAOSD is characterized by a Th1/Th17 cytokine profile and an upregulation of markers known to promote remodeling and fibrosis. The expression pattern of inflammatory markers in tear fluids at baseline might serve as a prognostic factor for DAOSD.

The detection of phosphorylated tau in tear fluid

AbstractBackgroundThe recent development of ultrasensitive immunological methods has allowed for the detection of biomarkers related to neurodegenerative disorders in blood, e.g., phosphorylated tau (p‐tau). This allows for a cost‐effective and scalable assessment of the causes behind cognitive decline. Nevertheless, other biofluids, e.g., tear fluid, have the potential to surpass these metrics. Previously, tear fluid from patients with neurodegenerative conditions has been explored both with mass‐spectrometry and immunoassays. Hereby, we established a method for the detection of p‐tau181, p‐tau217 and p‐tau231 in tear fluid from patients.MethodA total of 40 participants with neurodegenerative diseases (amyotrophic lateral sclerosis [n = 11], dementia [n = 9], Parkinson’s disease [n = 20]) and control subjects without clinical signs of neurodegeneration [n = 20], were recruited at the Department of Neurology at the Klinikum rechts der Isar, München, Germany. Tear fluid from both eyes was collected with Schirmer strips. The strips from both eyes were combined and eluted by centrifugation (16,000g) in sample buffer. The eluate was then analysed for p‐tau181, p‐tau217 and p‐tau231 by Single molecule array (Simoa). Results were normalised to tear fluid running length.ResultAll tear fluid samples were above the lower limit of detection (LLOD) for all p‐tau assays ([mean] p‐tau181, 92.1pg/ml; p‐tau217, 0.398pg/ml; p‐tau231, 52.9pg/ml) improving on previous attempts found in literature. There was a highly significant correlation between all p‐tau biomarkers in tear fluid (r = 0.90–0.98; P<0.001). The normalised concentration of tear fluid p‐tau had a significant positive correlation with age (R p‐tau181 = 0.28, R p‐tau217 = 0.27, R p‐tau231 = 0.39; P<0.05) but there was no association with the corresponding p‐tau in serum. No significant group differences in tear fluid p‐tau were observed but a trend towards increased serum p‐tau181 was found in the neurodegenerative group (6.37pg/ml) compared to controls (4.37pg/ml).ConclusionIn this pilot study, we describe quantifiable levels of p‐tau181, p‐tau217 and p‐tau231 in tear fluid samples. However, the detectable levels of p‐tau in tear fluid do not seem to correlate to p‐tau levels in serum. Further studies will show p‐tau in tear fluid of patients with suspected AD, stratified by CSF biomarkers, and thus establish the relevance of tear fluid as a diagnostic biofluid in AD.

Potential Biomarkers for Noninfectious Scleritis Identified by Serum and Tear Fluid Proteomics

Scleritis is an extremely painful and potentially blinding inflammation of the sclera with unknown pathogenesis and unpredictable course. To gain insight in its disease process and identify biomarker candidates, we performed extensive proteomics in serum and tear fluid. Prospective multicenter cohort study. A total of 121 patients with noninfectious scleritis (of which 39 active cases), 30 healthy controls, and 23 disease controls (uveitis and rheumatoid arthritis) were enrolled in the Netherlands from 2020 to2022. Serum, tear fluid of both eyes, and clinical data were gathered. The level of 368 inflammatory proteins was measured using proximity extension assays. Results were validated in an independent cohort of 15 patients with scleritis, and using addressable laser bead immunoassay, or enzyme-linked immunoassays. In addition, we studied an extended panel of matrix metalloproteinases in tear fluid of necrotizing scleritis with addressable laser bead immunoassay. Statistically significant differences in the level of inflammatory proteins between patients with scleritis and control groups. Proteomics revealed 18 significantly upregulated or downregulated serum proteins in active scleritis cases compared with all control groups in both the discovery cohort and the validation cohort. The most upregulated protein was nuclear migration protein nudC (NudC; P= 0.0032), a protein involved in neurogenesis. The other significant hits included proteins involved in T-cell activation, apoptosis, epithelial barrier maintenance, and angiogenesis. Our tear fluid analysis showed matrix metalloproteinase 9 (MMP9) to be upregulated in the tear fluid of patients with scleral necrosis. The results of our proteomics analysis suggest a role for neurogenesis, T-cell activation, disruption of epithelial barrier, and angiogenesis in the pathogenesis of scleritis, and highlight MMP9 and NudC as biomarkers with potential clinical relevance. The authors have no proprietary or commercial interest in any materials discussed in this article.

Биохимические маркеры в слезной жидкости пациентов при кератоконусе: результаты ROC-анализа

Цель. Установить прогностическое значение биохимических показателей слезной жидкости у пациентов с кератоконусом I–IV стадии. Материалы и методы. В исследование вошли 34 пациента (56 глаз) с кератоконусом и 28 здоровых добровольцев. Сформированы следующие группы: 1-я группа 28 глаз с кератоконусом I–III стадии и 2-я группа – 28 глаз с кератоконусом IV стадии, при которой заболевание выражено максимально и его лечение предполагает планирование и выполнение пересадки роговицы. Группу 3 (контрольная) составили здоровые добровольцы. Забор слезной жидкости для исследования концентраций биологически активных протеинов выполняли при помощи фильтровальных полосок. Проведено определение концентрации в слезной жидкости следующих биохимических показателей: ИЛ-1α, ИЛ-1β, ИЛ-6, ИЛ-17, коллаген IV типа, ММП-1, ММП-3 и ММП-9, ФНО-α и ФНО-β, альфа-2 макроглобулин, TФР-β, СРБ, общая антиоксидантная активность. Результаты. Установлены диагностически значимые уровни ММП-9, ИЛ-1β и ИЛ-17, α2-МГ в слезной жидкости, превышение которых может использоваться в комплексе диагностических критериев при скрининге пациентов в группе риска, при подозрении на кератоконус или при монолатеральном кератоконусе. Результаты ROC-анализа ФНО-α, ИЛ-1β, ИЛ-6, ИЛ-17, СРБ в слезной жидкости пациентов с кератоконусом I–III стадии позволяют рассматривать их в качестве предикторов патологических изменений глазной поверхности, ассоциированных с прогрессированием кератоконуса до терминальной стадии. Определены пороговые уровни биологически активных протеинов в слезе, характеризующие терминальную стадию кератоконуса. Заключение. Разработка новых диагностических инструментов повысит эффективность скрининга, диагностики прогрессирования болезни, выбора метода лечения пациентов, а также приблизит нас к пониманию патогенетических механизмов развития кератоконуса. Purpose. To establish the prognostic value of the biochemical markers of the tear fluid in patients with stage I–IV keratoconus. Materials and methods. The study included 34 patients (56 eyes) with keratoconus and 28 healthy volunteers. Group 1 consisted 28 eyes with stage I–III keratoconus, group 2 – 28 eyes with stage IV keratoconus, in which the symptoms of the disease were most pronounced and treatment required planning and performing a cornea transplant. Group 3 (control) were presented with healthy volunteers. Tear fluid was collected using filter strips to study the concentrations of biologically active proteins. The following biochemical markers were determined in the lacrimal fluid: IL-1α, IL-1β, IL-6, IL-17, type IV collagen, MMP-1, MMP-3 and MMP-9, TNF-α and TNF-β, alpha-2 macroglobulin, TGF-β, CRP, total antioxidant activity. Results. Diagnostically significant levels of MMP-9, IL-1β and IL-17, α2-MG in the tear fluid were established and can be used in the complex of diagnostic criteria for screening patients at risk, with suspected keratoconus or with monolateral keratoconus. The results of ROC analysis of TNF-α, IL-1β, IL-6, IL-17, CRP in the tear of patients with stage I–III keratoconus allow to consider them as predictors of pathological changes on the ocular surface associated with the progression of keratoconus to the terminal stage. The cut-off levels of biologically active proteins in the tear, which characterize the terminal stage of keratoconus, were determined. Conclusion. New diagnostic tools will increase the effectiveness of screening, diagnosis of the disease progression, choice of treatment method and will also bring us closer to understanding the pathogenetic mechanisms of keratoconus development.

Content of mediators of innate immunity in the tears of patients with vascular and neurodegenerative manifestations of diabetic retinopathy

According to the results of recent studies, diabetic retinopathy can be considered not only as a vascular disease, but also as a neurodegenerative process. Study of the composition of the tear fluid is used to assess the state of local immunity in the development of eye diseases. However, studies examining the effect of tear composition in diabetic retinopathy are few. The aim of the study is to determine the levels of IL-1β, IL-10, TGF-β3, MMP-7, TIMP-2, protein S100b, BDNF and NGF in the tear fluid ofpatients with vascular and neurodegenerative manifestations of diabetic retinopathy. The study included 80 patients diagnosed with type 2 diabetes which were divided into 2 groups: the 1st group included 40 patients who had no clinical signs of diabetic retinopathy on the fundus; the 2nd group included 40 patients with initial signs of non-proliferative diabetic retinopathy. All those included in the study were examined on an optical coherent tomograph RTVue-100 (USA); the volume of focal losses of retinal ganglion cells (FLV) was determined. An increase in FLV above the normative base of the device was regarded as an OCT-sign of retinal neurodegeneration. According to the results of OCT, the participants of the first and second groups were additionally divided into 4 subgroups: 1A — patients without vascular changes in the fundus and without OCT signs of retinal neurodegeneration (n = 12); 1B — patients without vascular changes in the fundus and with the presence of OCT signs of retinal neurodegeneration (n = 28); 2A — patients with initial non-proliferative DR and without OCT signs of retinal neurodegeneration (n = 10); and 2B — patients with initial non-proliferative DR and with OCT signs of retinal neurodegeneration (n = 30). The levels of IL-1β, IL-10, TGF-β3, MMP-7, TIMP-2, protein S100 b, BDNF, and NGF in tear fluid were determined by enzyme-linked immunosorbent assay. Levels of IL-1β and IL-10 in tear fluid in all subgroups were comparable to controls throughout the study. TGF-β3 content in the tear fluid of patients in the group with initial signs of non-proliferative DR (group 2) was significantly (p = 0.001) lower compared with control and group 2. However, there was no significant difference (p > 0.05) between subgroups A and B within groups. The concentration of MMP-7 in the tear fluid in all subgroups was significantly lower than in the control (p < 0.05). However, in the subgroups with OCT signs of retinal neurodegeneration (1B and 2B), the deficiency of this metalloproteinase was more pronounced (p = 0.0001). The levels of the neuropeptides under study NGF, BDNF and S100 B in tear fluid did not differ from controls in all subgroups.

Aim2 Deficiency Ameliorates Lacrimal Gland Destruction and Corneal Epithelium Defects in an Experimental Dry Eye Model.

Dry eye disease (DED) is a multifactorial disease that is associated with inflammation. Excessive DNA is present in the tear fluid of patients with DED. Absent in melanoma 2 (AIM2) is a key DNA sensor. This study aimed to investigate the role of AIM2 in the pathogenesis of DED. DED was induced by injection of scopolamine (SCOP). Aberrant DNA was detected by cell-free DNA (cfDNA) ELISA and immunostaining. Corneal epithelial defects were assessed by corneal fluorescein staining, zonula occludens-1 immunostaining and TUNEL. Tear production was analyzed by phenol red thread test. Lacrimal gland (LG) histology was evaluated by hematoxylin and eosin staining, and transmission electron microscopy examination. Macrophage infiltration in LG was detected by immunohistochemistry for the macrophage marker F4/80. Gene expression was analyzed by RT-qPCR. Protein production was examined by immunoblot analysis or ELISA. Aim2-/- mice displayed a normal structure and function of LG and cornea under normal conditions. In SCOP-induced DED, wild type (WT) mice showed increased cfDNA in tear fluid, and aberrant accumulations of dsDNA accompanied by increased AIM2 expression in the LG. In SCOP-induced DED, WT mice displayed damaged structures of LG, reduced tear production, and severe corneal epithelium defects, whereas Aim2-/- mice had a better preserved LG structure, less decreased tear production, and improved clinical signs of dry eye. Furthermore, genetic deletion of Aim2 suppressed the increased infiltration of macrophages and inhibited N-GSDMD and IL18 production in the LG of SCOP-induced DED. Aim2 deficiency alleviates ocular surface damage and LG inflammation in SCOP-induced DED.

Uric acid is independently associated with interleukin-1β levels in tear fluid of hyperuricemia and gout patients.

To explore pro-inflammatory cytokines status in the tear fluid of patients with hyperuricemia and gout and its association with uric acid level. A total of 58 control subjects, 58 hyperuricemia patients including 40 asymptomatic hyperuricemia and 18 gout participants were recruited in this study. For tear analysis, each patient's tears were collected using capillary action microcaps after stimulation. Tear uric acid levels were measured using chemiluminescence. Tear and serum interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were measured using enzyme-linked immunosorbent assay. The correlation of serum and tear uric acid levels with IL-1β and TNF-α were determined. Tear uric acid levels were significantly higher in hyperuricemia group (98.2 ± 51.5vs. 42.7 ± 24.0 µmol/L, p < .001) than in controls group. IL-1β concentrations were significantly higher in hyperuricemia eyes compared to control eyes (210.2 ± 113.9vs. 142.6 ± 29.8 pg/mL, p < .001). Multiple linear regression analysis showed that tear uric acid levels were independently positively associated with tear IL-1β concentrations (B = 0.192, p < .001). However, no significant correlations were found between serum or tear uric acid and TNF-α level. Moreover, there were no statistically differences of tear IL-1β and TNF-α levels between the asymptomatic hyperuricemia and gout groups. Tear uric acid levels were higher in patients with hyperuricemia and gout than in controls. There was a significant positive correlation between tear uric acid value and tear IL-1β level, implying an interaction between hyperuricemia and ocular inflammation responses.

The levels of hypoxia- and angiogenesis-related regulators and matrix metalloproteinase 9 activity in tear fluid of patients with non-penetrating ocular traumas

This article was focused on the evaluation of protein biomarkers related to thrombosis, hypoxia, angiogenesis, and tissue remodeling in tear fluid of patients with non-penetrating corneal trauma. 32 patients with non-penetrating corneal injures were enrolled in the study, the control group consisted of 15 healthy patients. Samples of tear fluid were collected from the patients and control volunteers with the use of a disposable end micropipette. Protein levels of D-dimer, hypoxia-inducible factor 1α (HIF-1α), angiostatins, and matrix metalloproteinase 9 (MMP-9) in tear fluids were determined by western blot analysis. Proteolytic activity values of MMP-9 were measured by gelatin zymography. Results of western blot and zymography assay were calculated by densitometry analysis and expressed as arbitrary units. Significant increase of D-dimer and HIF-1α levels in tear fluid of patients with injured cornea by 7.3 (p&lt;0.05) and 56 (p&lt;0.001) folds, respectively was shown, compared with control, indicating thrombotic events and hypoxia condition to be involved in pathogenesis of ocular trauma. Dramatically elevated levels/activity of MMP-9 enzyme (by 105 folds vs. control, p&lt;0.001) suggests intense tissue remodeling and degradation of extracellular matrix in the damaged cornea. Up-regulation of angiostatin level, products of proteolytical cleavage of plasminogen, in tear fluid collected from patients with traumatic eye in comparison with healthy volunteers (by 7.3 folds, p&lt;0.05), could represent an adaptive mechanism, which counteracts excessive hypoxia-induced neovascularization of injured cornea. It is summarized that there was a strong association between elevation of D-dimer, HIF-1α, angiostatins, and MMP-9 levels suggesting thrombosis- and hypoxia-mediated mechanisms triggering wound healing of injured cornea. The findings of this study are novel and provide a basis for further investigations of the reparation mechanisms during non-penetrating ocular trauma. Studied proteins of tear fluid can serve as relevant biomarkers of corneal wound healing and are appropriate for diagnostic and prognostic purposes.

PRODUCTION OF ANTI-LACTOFERRIN ANTIBODIES AND THEIR APPLICATION IN ANALYSIS OF THE TEAR FLUID IN HEALTH AND CORNEAL INJURIES

Lactoferrin is a ubiquitous and multifunctional protein, which has antimicrobial and immunomodulatory activities. Lactoferrin plays an important role in the maintenance of ocular health. The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury. Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P&lt;0.05. Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons (P&lt;0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation. Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way.

Change in Ocular Surface Staining during Eyelid Warming Is Related to Tear Cytokine Levels.

Purpose To investigate the changes in the tear cytokine profile of patients with meibomian gland dysfunction (MGD) treated with eyelid warming and to correlate these changes with clinical parameters for dry eye disease (DED). Methods Seventy patients with MGD were included and treated with the warming of eyelids. Of these, 61 still used the treatment three months after baseline, while 48 completed the whole treatment period of six months. The concentrations of 39 cytokines in the tear fluid were measured at baseline and after three and six months of treatment. All participants were examined with tests for DED, including tear film break-up time (TBUT), ocular surface staining (OSS), and the self-reporting Ocular Surface Disease Index (OSDI). Changes in cytokine concentrations were assessed from baseline to three months, from three to six months, and from baseline to six months. Correlation analyses were performed between changes in the cytokine concentrations and changes in TBUT, OSS, and OSDI during the same time intervals. Results No significant changes were found in the concentrations of the 39 cytokines during any of the three treatment intervals. However, several correlations were detected between changes in the level of cytokines and OSS from baseline to three months of treatment. Decreasing concentrations of granulocyte chemotactic protein 2 (GCP-2/CXCL6, mean effect 2.36, p=0.042), interleukin 10 (IL-10, mean effect 1.04, p=0.045), and IL-16 (mean effect 1.36, p=0.035) were associated with decreasing OSS. Decreasing concentrations of granulocyte macrophage colony-stimulating factor (GM-CSF, mean effect −2.98, p=0.024), IL-8 (IL-8/CXCL8, mean effect −1.35, p=0.026), and macrophage migration inhibitory factor (MIF, mean effect −2.44, p=0.033) were related to increasing OSS. Conclusions Warming of eyelids did not change the concentration of cytokines in the tear fluid of patients with MGD significantly. However, alterations in the level of several cytokines were associated with changes in the OSS. This finding indicates a close connection between tear cytokines and OSS in MGD patients treated with eyelid warming.

PROTEIN MARKERS OF HYPOXIA AND ANGIOGENESIS IN TEAR FLUID OF PATIENTS WITH TRAUMATIC CORNEAL INJURY

The aim of our study was to evaluate tear levels of some protein endpoints that can reflect intensities of hypoxia, angiogenesis and tissue remodeling in wounded cornea. Methods. We examined 21 patients (21 eyes) with nonpenetrating corneal injuries. The patients underwent standard ophthalmological examination including previous history and ocular symptoms, visual acuity test, complete anterior and posterior eye segments examination using slit lamp biomicroscopy, evaluation of corneal staining with fluorescein, ophthalmoscopy. Healthy volunteers (n = 10) served as a control. Tear fluid was collected from patients and control volunteers with the use of a disposable tip micropipette. From the lower arch of the conjunctiva without instillation of anesthetic, tears were collected in a sterile plastic Eppendorf tube and frozen at -20 oC before laboratory examination. Proteins of tear fluids were separated by SDS-PAGE (loading 50 µg total protein per track). Then, levels of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and angiostatins were measured by western blot. Active MMP-9 levels were evaluated by gelatin zymography. The results of blot and zymography assays were processed by densitometric software and then analyzed statistically with the use of Mann-Whitney U-test. Results. Elevated HIF-1α (P&lt;0.001) and angiostatins (P&lt;0.05) levels were revealed by western blot in tear fluid samples collected from patients with injured cornea in comparison with the control group. It is noteworthy that extremely low amounts of VEGF were detected in tear fluid from injured eyes, in spite of abundance of its transcription inducer HIF-1α. Dramatically increased levels of active MMP-9 were found in the tear fluids of patients with corneal wounds, while no significant collagenolytic activity was observed in tears from healthy eyes. There is a strong correlation between extent of corneal lesions and changes in markers expression. Conclusions. Tear levels of HIF-1α and angiostatin as well as MMP-9 activity could represent valuable biomarkers of corneal injury severity in traumatic eye.

Tear Proteomic Approach and Identification of Tear Film Biomarkers with Rigid Gas Permeable Scleral Contact Lens (ScCLs) Wear for Keratoconus

Keratoconus (KC) is a classical non-inflammatory disorder associated with elevated serum levels of IgE, IgG, and IgM [1,2]. Lema et al. [3] have described specific cytokines and protease profiles in patients with keratoconus; and found levels of IL-6, TNF-alpha, and MMP-9 were elevated in keratoconus subjects as compared to normal. The differential expression of tear film proteins such as MMP-1, keratins, and mammaglobin B can be found in keratoconus subjects [4]. Tear fluid has been successfully used as a source of biomarkers in several well-studied eye diseases. Studies performed on tear fluid in patients of keratoconus provided insights into the pathology of the disease and revealed probable prognostic and diagnostic biomarkers for the disease [5]. A multi-omics approach integrating data from proteomics, lipidomics and metabolomics is the need of the hour for studying tear fluid as an important source of biomarkers in keratoconus to lead to effective prognosis and treatment of the disease. Tear fluid is a highly complex chemical and biological mixture containing proteins, peptides, electrolytes, lipids, and metabolites. It is proposed to be a surrogate representative for many eye diseases. It is an integral part of the ocular surface and represents the extracellular matrix for ocular surface epithelial cells [4,6]. Nano-mass Spectrometry and Liquid Chromatography were used in tear analysis, including analysis in proteomics, metabolomics, and lipidomics. Dry eye and meibomian gland dysfunction diseases involve the disruption of the lacrimal functional unit, resulting in symptoms of discomfort, visual disturbance, and tear film instability. These illnesses may exist independently as either symptomatic or asymptomatic disorders. However, they are frequently found in the same patient. The progression of these diseases typically leads to alterations in the tear film, tear hyperosmolarity, and the secretion of inflammatory mediators into the tears, initiated by cytokine release and metalloproteinase activation. Ocular dryness disorders also promote squamous metaplasia, mainly as a consequence of inflammation, whose severity can be used for grading different diseases affecting the ocular surface. In this study, we explored the efficacy of Rigid Gas Permeable Scleral Contact Lens in keratoconus regarding the expression of inflammatory Mediators.

Metabolomic Alterations in the Tear Fluids of Patients With Superior Limbic Keratoconjunctivitis.

PurposeSuperior limbic keratoconjunctivitis (SLK) is a bilateral, chronic inflammatory disease that recurs for up to several years; however, the fundamental processes involved in its pathogenic mechanisms remain unknown. We aimed to investigate the metabolomic alterations in the tear fluids of patients with superior limbic keratoconjunctivitis (SLK) compared with those of healthy volunteers (Ctrl group).MethodsWe performed a cross-sectional study involving 42 subjects. Tear fluid was taken from one eye of 24 SLK patients (40.13 ± 14.55 years, 83.33% female) and 18 healthy volunteers (Ctrl, 39.89 ± 9.2 years, 72.22% female) using Schirmer strips. After the liquid extraction of tear metabolites, samples were infused into the QE HFX Orbitrap mass spectrometer in both positive and negative ion mode. Metabolites were quantitatively analyzed and matched with entries in the HMDB database. Metabolic differences between the SLK group and the control group were identified based on multivariate statistical analysis. Open database sources, including SMPDB and MetaboAnalyst, were used to identify metabolic pathways.ResultsAmong 179 metabolites retained for annotation, 133 metabolites were finally identified, among which 50 were found to be significantly changed in SLK patients. Of these 50 metabolites, 31 metabolites significantly increased and 19 metabolites decreased in SLK patients. The altered metabolites are mainly involved in α linolenic acid and linoleic acid metabolism, ketone body metabolism, butyrate metabolism, mitochondrial electron transport chain, carnitine synthesis, and so on. The most significantly changed pathway was linoleic acid metabolism. To explore the utility of tear biomarkers, a model combining 9 metabolites (phenol, ethyl glucuronide, eicosapentaenoic acid, 12-keto-leukotriene B4, linoleic acid, hypoxanthine, triethanolamine, 1-nitrohexane, and terephthalic acid) was selected as a candidate biomarker.ConclusionThe results reveal that SLK has a specific metabolomic profile, of which some key elements can serve as potential biomarkers of SLK for diagnostic and prognostic purposes. The findings of this study are novel and provide a basis for further investigations of the mechanism of SLK.

Cystatin C and cystatin SN as possible soluble tumor markers in malignant uveal melanoma.

BackgroundThe aim of the study was to determine the concentration of endogenous cystatin C and cystatin SN, as potential tumor biomarkers, in the serum and biological fluids of the eye in both healthy controls and patients with uveal melanoma.Patients and methodsThe concentration of both cystatins was determined in the intraocular fluid (IOF), tear fluid, and serum of patients with uveal melanoma and compared to baseline measurements in IOF, tears, serum, cerebral spinal fluid, saliva and urine of healthy controls.ResultsThe concentration of cystatin C in all the biological matrices obtained from healthy controls significantly exceeded the concentration of cystatin SN and was independent of gender. Cystatin C concentrations in the tear fluid of patients with uveal melanoma (both the eye with the malignancy, as well as the contralateral, non-affected eye), were significantly greater than cystatin C concentrations in the tear fluid of healthy controls and was independent of tumor size. The concentration of cystatin SN in IOF of patients with uveal melanoma was significantly less than the corresponding concentration of cystatin SN in healthy controls.ConclusionsThe ratio of cystatins (CysC:CysSN) in both the serum and tear fluid, as well as the concentration of cystatin SN in IOF, would appear to strongly suggest the presence of uveal melanoma. It is further suggested that multiple diagnostic criteria be utilized if a patient is suspected of having uveal melanoma, such as determination of the cystatin C and cystatin SN concentrations in serum, tears, and IOF, ocular fundus and ultrasound imaging, and biopsy with histopathological evaluation.

Аналіз цитокінового балансу в сльозовій рідині у хворих на бактеріальний кератит і цукровий діабет залежно від тяжкості захворювання

The aim. To analyze the cytokine balance of tear fluid in patients with bacterial keratitis at presentation depending on the severity of the disease and the presence of diabetes mellitus (DM). Materials and methods. The analysis was performed through the comparison of the level of pro- and anti-inflammatory cytokines in the tear fluid of 17 patients with type 1 DM and bacterial keratitis and 15 patients with bacterial keratitis without DM at presentation. Data from 14 healthy individuals of the appropriate age were also used for comparison. The patients with bacterial keratitis were divided into subgroups according to the severity of bacterial keratitis. The levels of IL-1β, IL-6 and IL-10 in the tear fluid of the sick and the contralateral eye were determined by quantitative colorimetric enzyme-linked immunosorbent assay. Results and discussion. At presentation, patients with bacterial keratitis, both with and without DM, showed increased levels of proinflammatory cytokines, namely IL-1β and IL-6, in the tear fluid of the sick eye, which correlated with the severity of the disease, and also increased level of anti-inflammatory cytokine IL-10 in the tear fluid of the contralateral eye. In addition, the concentrations of proinflammatory cytokines IL-1β, IL-6 in the tear fluid of the contralateral eye in DM patients were increased at all degrees of severity of bacterial keratitis. Conclusions. In patients with bacterial keratitis, the cytokine balance of the tear fluid of the sick and the contralateral eye depends on the severity of the disease and the presence of DM. Keywords: diabetes mellitus, bacterial keratitis, severity of keratitis, cytokines, interleukins.

Melatonin level as a risk factor for age-related macular degeneration

Background. The current trend towards an increase in age-related macular degeneration (AMD) incidence rate in the population, including the working-age population, with a possible loss of professional activity, indicates the need for early preclinical identification of risk groups, timely prevention and treatment. In the prevention and treatment of AMD, the prospect of using melatonin is being actively discussed.Aim: to analyze serum and tear fluid levels of melatonin in patients with AMD and study their correlation with risk factors.Materials and methods. In the course of the study, two groups were formed: the main group - patients with non-exudative AMD and senile cataract (n = 40) and the reference group - conditionally healthy patients without AMD and cataract (n = 20). Patients of both groups were surveyed to identify risk factors for AMD. The content of melatonin in blood serum and lacrimal fluid was determined by enzyme-linked immunosorbent assay using the Melatonin ELISA Kit (USA).Results. In the course of the study, it was found that the concentration of melatonin in blood serum and tear fluid in patients with AMD was significantly lower than in patients of the reference group (p &lt;0.05). Serum and tear fluid melatonin levels depend on the following factors: age, body mass index (BMI), arterial hypertension, eye color, insomnia, and night work. It is possible that the local determination of melatonin in the lacrimal fluid can be a biomarker in the determination of ophthalmic pathological conditions.Conclusion. The obtained results can be used as recommendations for clarifying individual regimens for the use of melatonin, especially in the treatment of patients with AMD.

Determination of copper by AAS in tear fluid of patients with keratoconus

Keratoconus (KC) is the most common degenerative corneal disease and no single biomarker for KC has been discovered. Its causes have not yet been clarified and this work aims to be a contribution to the deepening of the knowledge of this disease and a preliminary data to the evaluation of the possibility of the use of copper (Cu) concentration in the tear fluid as a specific marker. A tear fluid sampling and Cu determination by spectrometric atomic absorption method was optimized to determine Cu levels in the tear fluid of patients with KC compared to that of healthy patients. Results demonstrate that in the KC subjects (n = 6) the concentration of Cu ions was 325.5 ± 110.7 ng/ml, while in the control group was 141.3 ± 71.1 ng/ml. A significant increase in Cu ion levels in the tear fluid was observed in the KC group compared to the control group (p value < 0.001).