Detection of pro-inflammatory cytokines in healthy canine tears using Canine Cytokine SpikeMix™ mass spectrometry via multiple reaction monitoring.

PURPOSE: To evaluate the efficacy of the Canine Cytokine SpikeMix™ and MRM-MS for detecting pro-inflammatory cytokines in canine tears from healthy research Beagles. METHODS: A complete ophthalmic examination was performed on 15 healthy research Beagles to verify no ophthalmic diseases. Tears were collected OU by placing a Weck-Cel® cellulose spear in the ventral conjunctival fornix for 1 min. The Weck-Cel® spear was placed in a 2.0 mL tube with a centrifuge filter forcing tears to flow through the filter into the bottom of the tube. The tears were analyzed using the Canine Cytokine SpikeMix™ and MRM-MS. Descriptive data from this study was reported as the normalized total peak area (nTPA) and median (range) using data imported from the online MRM-MS Skyline program. RESULTS: The level of 16 pro-inflammatory cytokines was successfully detected in all 15 dogs. The four cytokines with the highest median amounts in the samples were IL-2 = 0.1243 (0.019-6.7289), IL-6 = 0.964 (0.0036-16.9365), TNFα = 0.1644 (0.0096-0.7138), and CSF-2 = 0.4022 (0.1475-2.6208). CONCLUSIONS: This study revealed that 16 pro-inflammatory cytokines in canine tears from healthy dogs can be detected with Canine Cytokine SpikeMix™ and MRM-MS.

Detecting SARS-CoV-2 RNA in conjunctival secretions: Is it a valuable diagnostic method of COVID-19?

The main purpose of this study is to evaluate the presence of viral RNA of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in conjunctival swab specimen of coronavirus disease 2019 (COVID-19) patients with and without conjunctivitis to establish the diagnostic value of reverse transcription-polymerase chain reaction (RT-PCR) in each case and to describe its clinical characteristics. A cross-sectional study was conducted at the Hospital Clinico San Carlos of Madrid, Spain. Thirty-six subjects from the COVID admission unit with laboratory-confirmed SARS-CoV-2 infection were included. Conjunctival swabs were collected from 18 patients with conjunctivitis and 18 patients without conjunctivitis and RT-PCR was performed. Conjunctival swab was collected from both eyes of 36 patients (72 eyes), detecting SARS-CoV-2 RNA in conjunctival swab of two patients (5.5%). Among the 18 patients with conjunctivitis, only one of them (5.5%) showed positive results. Likewise, SARS-CoV-2 RNA was detected in one patient without conjunctivitis (5.5%). The mean age of the 36 patients was 67.9 years (range, 28-92 years) and the male-to-female ratio was 0.44 (16:20). The mean days since the onset of COVID-19 symptoms until conjunctivitis manifestation was 8 (range, 1-24 days). The mean duration of the conjunctivitis was 3 days (range, 1-7 days). SARS-CoV-2 RNA may be detected in conjunctival swabs of both patients with and without conjunctivitis. This study revealed the same rate of positive results amongst the group with and without conjunctivitis, suggesting that detecting SARS-CoV-2 in ocular fluids is not conditioned on the presence of conjunctivitis. The presence of SARS-CoV-2 RNA in ocular samples highlights the role of the eye as a possible route of transmission of the disease.

Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection.

OBJECTIVE: This study aimed to assess the presence of novel coronavirus in tears and conjunctival secretions of SARS-CoV-2-infected patients. METHODS: A prospective interventional case series study was performed, and 30 confirmed novel coronavirus pneumonia (NCP) patients were selected at the First Affiliated Hospital of Zhejiang University from 26 January 2020 to 9 February 2020. At an interval of 2 to 3 days, tear and conjunctival secretions were collected twice with disposable sampling swabs for reverse-transcription polymerase chain reaction (RT-PCR) assay. RESULTS: Twenty-one common-type and nine severe-type NCP patients were enrolled. Two samples of tear and conjunctival secretions were obtained from the only one patient with conjunctivitis yielded positive RT-PCR results. Fifty-eight samples from other patents were all negative. CONCLUSION: We speculate that SARS-CoV-2 may be detected in the tears and conjunctival secretions in NCP patients with conjunctivitis.

Ocular Impression-Taking-Which Material Is Best?

OBJECTIVES: To assess the efficacy and effect on clinical signs of a polyvinylsiloxane (Tresident; Shütz Dental Group GmbH, Germany) compared with an irreversible hydrocolloid (Orthoprint; Zhermack SpA, Badia Polesine, Italy) for ocular impression-taking. METHODS: Twenty subjects were recruited (13 female and 7 male), with mean age 31.1±4.6 years (SD) (range 25.8-39.7). Subjects attended for 2 sessions, each of 1-hr duration, on 2 separate days. Each session was scheduled at the same time on each day. At each visit, the subject underwent an ocular impression procedure, using either Tresident or Orthoprint, in random order and to one eye only. Investigator 2 was blind to this assignment. Two experienced practitioners conducted the study, investigator 1 performed the ocular impression procedures and investigator 2 observed and assessed the clinical signs: logMAR visual acuity, ocular surface staining, tear break-up time (TBUT), and ocular hyperemia. RESULTS: Visual acuity was unaffected by either material; TBUT was marginally disrupted by both materials, but was not clinically significant according to published criteria; ocular redness increased with both materials; and corneal staining was significantly greater after Orthoprint impression. Less redness and clinically insignificant staining after impression-taking, with fewer clinical complications, was found after use of Tresident. CONCLUSIONS: Tresident offers a quicker, more effective, and clinically viable method of obtaining ocular impression topography compared with the traditional Orthoprint, and Orthoprint causes significantly more superficial punctuate staining of the corneal epithelium than Tresident.

Rebamipide suppresses TNF-α production and macrophage infiltration in the conjunctiva.

PURPOSE: To evaluate the anti-inflammatory effect of rebamipide during corneal epithelial wound healing using a mouse wound repair model. METHODS: A 2-mm circular disc of the central cornea was demarcated in the right eye of C57BL/6 mice and the epithelium removed. Rebamipide 2% eyedrop was instilled onto the wounded eye 5 times a day (n = 26). Phosphate-buffered saline (PBS) was used in the control group (n = 26). Corneal and conjunctival IL-1ß and TNF-α levels were measured at 6 h and 24 h postinjury by ELISA. The wounded area was evaluated by fluorescein staining at 24 h postinjury. Macrophage infiltration was assessed immunohistochemically, and TNF-α secretion from macrophages was examined in vitro. RESULTS: Conjunctival IL-1ß and corneal IL-1ß levels were not significantly different between PBS-treated and rebamipide-treated groups. However, conjunctival TNF-α level was significantly lower in the rebamipide-treated group compared with the PBS-treated group. Macrophage migration into the conjunctiva, but not into the cornea, was suppressed by rebamipide treatment. In addition, TNF-α secretion from cultured macrophages was suppressed by rebamipide in a concentration-dependent manner. Rebamipide treatment significantly accelerated corneal epithelial wound healing at 24 h postinjury. CONCLUSIONS: In a mouse corneal epithelial wound model, rebamipide suppressed TNF-α secretion and macrophage infiltration in the conjunctiva, which might have contributed to accelerated corneal epithelial wound healing in the first 24 h following injury.

Isolation of microRNA from conjunctival impression cytology.

Impression cytology (IC) is an easy and safe technique that has been used in the past for harvesting epithelial cells from the cornea and conjunctiva for various applications including histology, immunohistology and molecular studies. Previous investigations have shown the usage of different types of membranes for the purpose of investigating pathophysiology and staging of diseases. This contributes to a better understanding of ocular surface conditions and helps to provide information for diagnosis, therapeutic options and prognosis. Recently, there has been a shift of focus in research towards understanding the contribution of microRNAs (miRs) to ocular disease. Thus far, impression cytology has been explored for measuring gene expression but not for quantifying miR expression. This study describes how miRs and mRNA can be isolated from conjunctival epithelial cells obtained by impression cytology and determines the optimum membrane and technique for this purpose. The IC technique was optimized using Biopore, Immobilon-P(SQ) and Millicell Hanging Cell Culture Insert membranes on healthy controls. miRs and mRNAs were isolated from the conjunctival epithelial cells (CEC) obtained and measured. Biopore membrane provided the optimum yield of miRs (38.8 ng/µL ± 10.8) and mRNA (155.3 ng/µL ± 20.1) as well as subjectively found to be best tolerated with minimum discomfort. Appreciable levels of miRs and mRNAs were detected from the CEC from healthy controls, confirming that it is possible to isolate miR and mRNA from CEC. Here, we give a detailed description of the application of conjunctival impression cytology to isolate miRs and the convenience of the technique by using the best membrane available. This method can be readily adopted in both clinical and laboratory settings. This technique will facilitate the measurement of miRs to improve our understanding of the pathogenesis of ocular surface conditions as well as potentially identifying novel therapeutic targets.

Cleaning effects of eyewashes on ocular surface symptoms caused by air pollution in a single-center, two-arm, nonrandomized trial in Indonesia: first report from Jakarta study.

PURPOSE: Air pollutants, such as Asian sand and particulate matter (PM) 2.5, have become a global concern for causing ocular inflammation and allergic symptoms. This study, as part of an international investigation, examined the effects of eyewashes for ocular damage caused by air pollution in Indonesia. METHODS: This was a single-center, patient- and-evaluator-blinded, parallel two-arm, nonrandomized trial. In Jakarta, Indonesia, 30 eyes of 15 car commuters and 30 eyes of 15 motorcycle commuters were recruited from healthy volunteers. After commuting to work, both eyes were washed with a commercial eyewash. Before and after eyewashing, eight items of ocular surface symptoms and four items of rhinitis subjective symptoms were scored using a modified Japanese Allergic Conjunctival Disease Quality-of-Life Questionnaire. RESULTS: Five of the 12 subjective symptom scores before eyewashing were higher in motorcycle commuters than in car commuters (p < 0.05). Motorcycle commuters showed improvement in the five symptom scores of "itchy eyes, foreign body sensation, eye mucus, dryness, and eye strain" after eyewashing compared to before eyewashing (p < 0.05). In all patients, sootlike particles and ocular mucus were found in the solutions collected after eyewashing. CONCLUSION: These findings indicate that eyewashing for ocular symptoms caused by airborne particles may be effective in removing foreign particles from the ocular surface and relieving subjective symptoms.

Localisation and quantification of benzalkonium chloride in eye tissue by TOF-SIMS imaging and liquid chromatography mass spectrometry.

Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C12 and benzyldimethyltetradecylammonium C14 and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in the field of pharmaco-toxicology in order to localise, identify and quantify drugs or xenobiotic compounds present at biological sample surfaces.

Non-invasive hemoglobin estimation from conjunctival images using deep learning.

Hemoglobin, a crucial protein found in erythrocytes, transports oxygen throughout the body. Deviations from optimal hemoglobin levels in the blood are linked to medical conditions, serving as diagnostic markers for certain diseases. The hemoglobin level is usually measured invasively with different devices using the blood sample. In the physical interpretation, some signs are traditionally used. These signs are the palms, face, nail beds, pallor of the conjunctiva, and palmar wrinkles. Studies have shown that conjunctival pallor can yield more effective results in detecting anemia than the pallor of the palms or nail beds. This study is aimed to predict the hemoglobin level by deep learning method, non-invasive, cheap, fast, high accuracy, and without creating medical waste. In this context, conjunctival images and age, weight, height, gender, and hemoglobin values were collected from 388 people who donated blood to the Turkish Red Crescent. A dataset was generated by augmenting the gathered data with body mass index data. Within the scope of this investigation, the limits of agreement (LoA) value at a 95% confidence interval was computed to be 1.23 g/dL, while the bias was established as 0.26 g/dL. The mean absolute percentage error (MAPE) values were determined to be 3.4%, and the root mean squared error (RMSE) was calculated to be 0.68 g/dL. These findings exhibit a successful outcome compared to similar investigations, signifying that this non-invasive method can be employed for hemoglobin level estimation. Furthermore, the estimated hemoglobin levels could aid in diagnosing several hemoglobin-related ailments.

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples.

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.

Immunohistochemical profile of VEGF, TGF-ß and PGE2 in human pterygium and normal conjunctiva: experimental study and review of the literature.

Human pterygium is made up of chronic proliferative fibro-vascular tissue growing on the ocular surface. This disease exhibits both degenerative and hyperplastic properties. Some fibroangiogenic factors have recently been shown to play a potential role in fibrovascular diseases via the angiogenesis process. The aim of this study is to evaluate VEGF, TGF-ß and PGE2 expression in the epithelial, endothelial and stromal cells of human pterygium and normal conjunctiva in order to determine whether these factors participate in the development of pterygium. Ten specimens from patients with pterygium and two normal conjunctivas (cadavers) were analyzed by immunohistochemistry using specific antibodies against these growth factors. The technique used was ABC/HRP (Avidin complexed with biotinylated peroxidase). Immunoreactivity of VEGF was significantly increased in the epithelium, vascular endothelium and stromal cells in primary pterygium as compared with normal conjunctiva. A moderate expression of TGF-ß in the pterygium was observed in the epithelial and stromal layers. On the contrary, immunolabeling of this growth factor in the human normal conjunctiva was weak. PGE2 was strongly expressed in the epithelium of patients with pterygium, as in control conjunctival tissues, and the immunolabeling was moderate in the stroma from the same patients. Our results suggest that these growth factors may contribute to the progression of primary pterygium by increasing angiogenesis, thus leading to the formation of new blood vessels from the pre-existing vasculature. We conclude that VEGF, TGF-ß and PGE2 may be potential therapeutic targets in the treatment of this disease although proof of this evidence requires further studies.

Presence of SARS-CoV-2 on the conjunctival mucosa in patients hospitalized due to COVID-19: Pathophysiological considerations and therapeutic implications.

Introduction: Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a worldwide pandemic, due to its great capacity to invade the human body. Previous studies have shown that the primary route of invasion of this virus is the human respiratory tract via the co-expression of ACE2 receptor and TMPRSS2, a serine protease on the cellular surface. Interestingly, this condition is present not only on the respiratory epithelium but on the conjunctival mucosa, as well. Thus, we hypothesized that SARS-CoV-2 is present on the conjunctival mucosa. Aim: To prove that SARS-CoV-2 can be detected in the conjunctiva. Methods: Previously nasopharyngeal swab-sample based real-time polymerase chain reaction (PCR) positive COVID-19 infected patients were selected at the COVID Care Centers of Semmelweis University, Budapest, Hungary. The study was approved by the ethical committee of Semmelweis University. During their recovery, both nasopharyngeal and conjunctival swab-samples were taken and PCR method was used to detect the presence of SARS-CoV-2 RNA. Appropriate statistical analysis was performed. Results: The study population consisted of 97 patients, 49 females (50.5%) and 48 males (49.5%), with a mean age of 67.2 ± 11.9 years. During recovery, with nasopharyngeal swabs, the PCR test was positive in 55 cases (56.70%), whereas with conjunctival swabs it was positive in 8 cases (8.25%). Both tests were positive in 5 cases (5.15%). In some patients, ocular symptoms were observed as well. The rest of the patients (29 cases) had negative nasopharyngeal PCR tests during recovery. Conclusions: Although only in few cases, the data of the present study provides a proof of concept that SARS-CoV-2 can be present on the conjunctival mucosa even in nasopharyngeal negative patients, a finding, which can have clinical importance. Also, on the basis of these findings one can hypothesize that - in addition to the respiratory tract - the conjunctiva can be an entrance route for SARS-CoV-2 to the human body. Thus, in high-risk conditions, in addition to covering the mouth and nose with mask, the protection of the eyes is also strongly recommended.

A novel cell-associated protection assay demonstrates the ability of certain antibiotics to protect ocular surface cell lines from subsequent clinical Staphylococcus aureus challenge.

In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 µg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 µg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 µg/ml, whereas AZM displayed toxicity to one cell line at 512 µg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Pharmacokinetics of pirfenidone after topical administration in rabbit eye.

PURPOSE: Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a new, broad-spectrum agent that has an inhibition effect on the proliferation, migration, and collagen contraction of human Tenon's fibroblasts, and thus modulating the wound healing process of glaucoma filtering surgical site. This study investigated the pharmacokinetics of topically administered pirfenidone (0.5%) in rabbit eyes. METHODS: Pirfenidone solution (50 µl) was instilled into the rabbit's conjunctival sac. The rabbits were quickly sacrificed at 2, 5, 8, 10, 15, 20, 30, 60, 90, and 120 min after the administration and ocular tissues were obtained. The concentrations of pirfenidone in conjunctiva, sclera, cornea, aqueous humor, and vitreous were determined by high performance liquid chromatography. RESULTS: After topical administration, there was wide distribution and fast clearance of pirfenidone among the various ocular tissues. The mean maximum concentrations (C(max)) of pirfenidone in cornea, conjunctiva, sclera, aqueous humor, and vitreous were 9.64 mg/g, 9.62 mg/g, 2.13 mg/g, 34.88 mg/l and 0.52 mg/l, respectively. The half-life for these tissues was 18.26, 34.16, 15.71, 70.91, and 39.48 min, respectively. CONCLUSIONS: Measurable concentrations of pirfenidone are achieved in ocular tissues after topical application in rabbit model. Topical administration of pirfenidone may be an effective approach for modulation of wound healing responses in glaucoma filtration surgical site.

Immunohistochemical differences between normal and chronically inflamed conjunctiva: diagnostic features.

PURPOSE: There is widespread misinterpretation of normal conjunctival fibrinogen. In differentiating between normal conjunctiva and cicatrizing conjunctivitis, including ocular cicatricial pemphigoid, atopic keratoconjunctivitis, and lichen planus, it is important to properly evaluate and characterize the histologic appearance of the structures seen and not base a diagnosis on just the presence or absence of certain features. One feature of conjunctival histology prone to misinterpretation and misdiagnosis is the presence of subepithelial fibrinogen, particularly when the diagnosis of lichen planus is being considered. Although the presence of subepithelial fibrinogen in oral mucous membranes and in skin can be indicative of lichen planus, such is not the case for conjunctiva. An erroneous diagnosis of lichen planus based on the presence of conjunctival subepithelial fibrinogen can initiate prolonged treatment with topical steroids leading to avoidable, blinding, complication, and further, delay therapy for the real cause of the conjunctivitis. We conducted a cross sectional, controlled, blinded and prospective Institutional Review Board-approved study on the occurrence and pattern of fibrinogen at the epithelial basement membrane zone (BMZ) of normal and inflamed conjunctiva. METHODS: Bulbar conjunctiva was obtained from 10 cases of undiagnosed chronic conjunctivitis of at least 6 months duration and 8 patients with normal conjunctiva. Immunofluorescent staining with antifibrinogen antibodies, periodic acid-schiff stain (PAS), and Giemsa staining were performed. RESULTS: BMZ fibrinogen was found in all cases. This layer was linear, smooth, and continuous in normal conjunctiva and 7 cases of chronic conjunctivitis. It was fragmented and lumpy in 1 case of ocular cicatricial pemphigoid (OCP) and showed spikes and spurs in 2 cases of lichen planus. CONCLUSIONS: BMZ fibrinogen is a normal component of the conjunctiva and its morphological features rather than its mere presence should be assessed as a diagnostic tool.

The Middle East respiratory syndrome coronavirus (MERS-CoV) nucleic acids detected in the saliva and conjunctiva of some naturally infected dromedary camels in Saudi Arabia -2019.

Dromedary camels are playing essential roles in the evolution and transmission of MERS-CoV. MERS-CoV shedding in some dromedary camel secretions, particularly nasal swabs, were studied in more detail. However, the roles of viral shedding in saliva and ocular secretions are still required further detailed studies. We performed a longitudinal study on a farm of dromedary camel herd from 10th March until 7th April, 2019, in eastern Saudi Arabia. This is a closed management herd including a large number of colour-based breed animals and include animals of both sexes. We collected saliva and ocular swabs from 18% of the target animal population. Detection of the MERS-CoV-RNAs in these samples was conducted by the real-time PCR technique. We detected the viral RNAs in the saliva of and conjunctival swabs of some of the tested animals at 33%, 77% and 88% during the three-time points, respectively. Moreover, we also detected the viral RNAs in the conjunctival swabs at 11%, 22% and 33% at similar time intervals. Our results are suggesting the possibility of MERS-CoV shedding in the saliva and the ocular discharges of the infected dromedary camels. This explains, at least in part, the mechanism of transmission of MERS-CoV from animals to humans. More studies are needed for a better understanding of the transmission of MERS-CoV from animals to humans; thus, the risk of virus spread can be mitigated.

Transcriptome analysis identifies the differentially expressed genes related to the stemness of limbal stem cells in mice.

Limbal stem cells (LSCs) reside in the basal layer of limbal epithelial cells (LECs). They are crucial for maintenance of corneal epithelium homeostasis and corneal wound healing. Their stemness is determined by their gene expression pattern. Despite of several positive identifiers have been reported, the unique biomarker for LSCs still remain elusive. Differentially expressed genes (DEGs) between stem cells and differentiated cells affect the fate of stem cells via specific signaling pathway. In order to understand the DEGs in the LSCs, RNA-seq was firstly conducted using a mouse model. A total of 1907 up-regulated DEGs and 395 down-regulated DEGs were identified in the limbus (L) compared to central cornea (CC) and conjunctiva (Cj). Reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The expression pattern of putative biomarkers was considered to be age-related. In up-regulated DEGs GO analysis, 570 gene ontology (GO) terms were significantly enriched. Five groups of genes related with biological processes from these significantly enriched GO terms comprised ionic transport, regulation of tissue development, muscle contraction, visual perception, and cell adhesion, which were clustered as a weighted similar network. Whereas, in down-regulated DEGs GO analysis, 61 GO terms were significantly enriched and only one group of ATP biosynthesis and metabolic process were clustered. Furthermore, we identified 55 signaling pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database based on up-regulated genes and 14 KEGG pathways based on down-regulated genes. In this study, we provide a landscape of the expression of putative LSCs biomarkers and stemness-related signaling pathways in a mouse model. Our findings could aid in the identification of LSC niche factors that may be related to the stemness of the LSCs.

Contact lens deposits, adverse responses, and clinical ocular surface parameters.

PURPOSE: To correlate clinical responses during contact lens wear with the amount of protein or cholesterol extracted from lenses after wear. METHODS: Clinical parameters, including adverse response rates and corneal staining, and symptomatology rating during lens wear were collected from a series of clinical tests comprising four different silicone hydrogel lenses with four different multipurpose solutions. To test for correlates, the amount of total protein or cholesterol extracted from lenses after daily wear were compared statistically to clinical parameters. RESULTS: The amount of protein (p = 0.008) or cholesterol (p = 0.01) extracted from lenses was higher for those subjects who showed solution-induced corneal staining. Amount of protein extracted was correlated (p < 0.01) with conjunctival staining (R = -0.23), lens front surface wetting (r = 0.14), and lens fit tightness (R = -0.20). These clinical parameters accounted for 48% of lens protein deposition. The amount of cholesterol extracted from lenses was much more weakly associated with clinical variables. Amount of protein or cholesterol extracted from lenses was not associated with the production of any corneal infiltrative or mechanical adverse event during wear and was only very weakly correlated with insertion comfort of lenses. CONCLUSIONS: These results suggest that there may be no physiologically relevant consequence of cholesterol depositing on silicone hydrogel lenses. The amount of protein that deposits onto silicone hydrogel lenses during wear may have more affect on lens performance on-eye. However, the correlations were generally small and may still not indicate any causative relevant physiological response. Further work is required to determine whether there is any direct causative effect to support these correlative findings.

[Expression of the antimicrobial peptide beta-defensin-2 in the conjunctival epithelial cells in primary open-angle glaucoma and over time in the postoperative period].

The time course of changes in the postoperative expression of the antimicrobial peptide beta-defensin-2 (HBD-2) in the conjunctival epithelial cells was studied in 13 patients (13 eyes) with primary open-angle glaucoma (POAG) and 9 individuals (9 eyes) without ophthalmopathology. Scrapes from the eyelid conjunctiva and transient folds were used as a material for an immunological study. In the study group, the scrapes were taken a few hours before and at 7, 30, and 90 days after surgery. The expression of the HBD-2 gene in the study material was determined by real-time polymerase chain reaction. In the subjects without pathology of the organ of vision, the conjunctival epithelial cells exhibited HBD-2; in the patients with POAG, the baseline HBD-2 gene expression was significantly higher. The baseline HBD-2 expression did not depend on gender, the duration of local antihypertensive therapy, the stage of disease stage, and the level of ophthalmotonus. HBD-2 gene expression was by 5-10 times higher than that preoperatively in all the patients with POAG on postoperative day 7. One month after surgery, the HBD-2 gene expression considerably rose in 31% of the examined patients while it became normal in 38% of the patients.

Altered mucins and aquaporins indicate dry eye outcome in patients undergoing Vitreo-retinal surgery.

Vitreo-retinal (VR) surgeries induce conjunctival changes. However, there are no study reports regarding prevalence and severity of dry eye after these surgeries. This study evaluated dry eye outcome after VR surgery. Patients undergoing VR surgery classified as scleral buckle and microincision vitrectomy surgery (n = 44, mean age: 56.09±10.2 years) were recruited. Dry eye evaluation was done before and 8 weeks after surgery (2 weeks after omitting topical eye drops). Conjunctival imprint cytology for goblet cell count and tear Mucin 5AC (MUC5AC) protein estimation was done. Gene expressions of MUC5AC, MUC4, MUC16, Aquaporin 4 (AQP4) and AQP5 were analyzed in the conjunctival imprint cells by qPCR. None of the patients exhibited clinical signs of dry eye after VR surgery. But the conjunctival goblet cell density (GCD) was significantly lowered post-VR surgery (63% cases, **p = 0.012) with no alterations in the tear MUC5AC protein. Post-VR surgery, the conjunctival cell gene expression of MUC4, MUC16 and AQP4 were significantly increased (*p = 0.025, *p = 0.05 and *p = 0.02 respectively) and AQP5 was significantly lowered (*p = 0.037), with no change in MUC5AC expression. Tear cytokines were significantly increased post-VR surgery (anti-inflammatory: IL1RA, IL4, IL5, IL9, FGF; PDGFbb and pro-inflammatory: IL2, IL6, IL15, GMCSF and IFNg). Though clinical signs of dry eye were not observed after VR surgery, ocular surface changes in the form of reduced GCD, altered MUC5AC, MUC4, MUC16, AQP4, AQP5 and cytokines are suggestive of dry eye outcome at the molecular level especially inpatients aged above 51 years, especially female gender and those who are diabetic.