Conjunctival epitheliopathy induced by topical exposure to bacterial peptidoglycan, muramyl dipeptide.

Noninfectious exudative conjunctivitis can be experimentally produced in rabbits by application of the apoptogenic bacterial cell wall peptidoglycan, muramyl dipeptide (MDP) to the ocular surface. The purpose of this study was to investigate the acute conjunctival cytopathology induced by unilateral ocular surface exposure to MDP. Hematoxylin and eosin staining assessed bilateral tear cytopathology and conjunctival histopathology. The caspases levels in conjunctival tissue and tears were measured in standard assays utilizing p-nitroanaline tagged caspase-specific substrates. Immunofluorescent antibody identified intracellular caspase-3, nuclear factor-κß (NF-κß), and oxidative DNA damage (8-OHdG; 8-oxo-2'-deoxyguanosine) in tear and conjunctiva cells. DNA extracted from conjunctival tissues and pooled tear fluids were visualized by ethydium bromide agarose gel electrophoresis. Onset of ipsilateral conjunctivitis was due to an epitheliopathy characterized by loss of conjunctival epithelial cell adherence, exuviation of conjunctival epithelial cells, and neutrophil infiltration. Caspase-3 levels were significantly higher in exuviated cells in ipsilateral than contralateral tear (p's ≤ 0.001) collected at 3-5 h post MDP. Significantly higher caspase-2, -3, -6, -8 and -9 (p's ≤ 0.03) levels were detected in ipsilateral than contralateral conjunctival tissue at 5 h. Polymeric DNA was detected in ipsilateral but not contralateral conjunctival tissue and tears. Caspase-3, NF-κß, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κß epithelial cells and polymeric DNA in conjunctival tissue and shedding of caspase positive cells and polymeric DNA into ipsilateral tears support MDP induction of acute programmed cell death in vivo. The results suggest that ipsilateral exudative conjunctivitis is due to acute caspase-mediated conjunctival epitheliopathy induced by topical exposure to the bacterial peptidoglycan MDP.

Occupational effect of sugarcane biomass burning on the conjunctival mucin profile of harvest workers and residents of an adjacent town - A Brazilian panel study.

Pre-harvest burning of sugarcane fields produces large amounts of air pollutants which are known to cause health problems, including ocular surface abnormalities. In this study, we evaluated the effect of biomass burning on mucus quality and mucin gene expression (MUC1, MUC5AC, MUC16) in the conjunctiva of sugarcane workers (SWs) and residents of an adjacent town (RTs). Impression cytology samples of the inferior tarsal and bulbar conjunctiva of 78 SWs and 32 RTs were collected before (T1) and immediately after (T2) a 6-month harvest period. The neutral, acid and total mucus content of goblet cells was determined by PAS and AB staining. The levels of MUC5AC, MUC1 and MUC16 mRNA in the conjunctiva were measured by real-time PCR. Compared to RTs, SWs had higher levels of bulbar acid mucus and MUC16 mRNA and tarsal MUC5AC mRNA at T2 and lower levels of neutral mucus at T1 and T2. In the SW group, MUC1 mRNA levels were higher at T2 than at T1, but the levels of neutral and acid mucus were similar. In the RT group, acid mucus decreased and neutral mucus increased in the bulbar and tarsal conjunctiva at T2. In conclusion, our findings show that sugarcane harvesting is associated with abnormalities in mucus quality and content and changes in mucin mRNA levels on the ocular surface. This may help explain the ocular inflammatory signs and symptoms observed in subjects exposed to air pollutants and high temperatures from sugarcane biomass burning.

Isoliquiritigenin protects against angiotensin II-induced fibrogenesis by inhibiting NF-κB/PPARγ inflammatory pathway in human Tenon's capsule fibroblasts.

PURPOSE: To examine the protective effects of Isoliquiritigenin (ISL) in angiotensin II (ANG II)-induced inflammation and fibrosis on Human Tenon's capsule Fibroblasts (HTFs) and Mouse Peritoneal Macrophages (MPMs). This study also investigated the potential mechanism of action of ISL. METHOD: Methyl-thiazolyl tetrazolium (MTT) assay was used to test ISL toxicity. An ELISA and an RT-qPCR assay detected the inflammatory cytokines (TNF-α, IL-6, COX-2, and ICAM-1). A Western blot investigated the expression levels of inflammation-related signals [nuclear factor-κB (NF-κB), peroxisome proliferator-activated receptor γ (PPARγ)], and fibrogenesis, including fibronectin and alpha-smooth muscle actin (α-SMA)]. Protein expressions of α-SMA were measured by immunofluorescence. RESULTS: Pre-treatment with ISL (10 or 20 µM) dose-dependently decreased the mRNA levels of TNF-α, IL-6, ICAM-1, and COX-2 induced by ANG II (1 µg/ml) in both MPMs and HTFs. ANG II remarkably increased the amount of P65 in the nuclei and decreased the amount of P65 in the cytoplasm. Additionally, ANG II reduced PPARγ expression levels in a time-dependent manner. Furthermore, these effects which were induced by ISL were remarkably neutralized by ISL pre-treatment. Finally, ANG II markedly elevated the expression of fibronectin and α-SMA. CONCLUSION: ISL could alleviate ANG II-induced fibrogenesis by inhibiting the NF-κB/PPARγ inflammatory pathway. In addition, ISL may be a potential agent for the treatment of conjunctival fibrosis. Most importantly, the NF-κB/PPARγ signaling pathway could be an effective therapeutic target for the prevention and treatment of conjunctival fibrosis after glaucoma surgery.

Impact of diurnal variation, sex, tear collection method, and disease state on tear protein levels in dogs.

OBJECTIVE: To investigate the effects of various biological factors on total protein concentration (TPC) and serum albumin levels in canine tears. ANIMALS STUDIED: 10 healthy beagles (5 female, 5 male) were used. PROCEDURES: Experiments were conducted on separate days, collecting tears with either capillary tubes or Schirmer strips, as follows: (i) Tear collection at 3 hours intervals (from 6 am to 12 am); and (ii) Tear collection before and 20 minutes following topical histamine application (1, 10, 375 mg/mL) to induce mild, moderate, and severe conjunctivitis, respectively. TPC and serum albumin were measured with infrared spectroscopy and ELISA, respectively. RESULTS: Tear film TPC and serum albumin ranged from 9.7-26.1 mg/mL and 6.4-1662.6 µg/mL, respectively. Protein levels did not differ significantly among time points (P ≥ .080). Median coefficient of variation (CV%) was lower with Schirmer strips compared to capillary tubes for both TPC (12% vs 15%, P = .020) and serum albumin (57% vs 78%, P = .232). TPC (P < .001), but not serum albumin was greater in male vs. female dogs. Serum albumin, but not TPC (P ≥ .099), increased significantly with each grade of conjunctivitis severity (P < .001), with no differences between collection devices (P ≥ .322); median increase was 106%, 1389%, and 2871% in eyes with mild, moderate, and severe conjunctivitis, respectively. CONCLUSIONS: There is no apparent diurnal variation in canine tear protein levels. Blood-tear barrier breakdown with conjunctivitis allows serum albumin to leak into the tear film at high concentrations. Schirmer strips compare well with capillary tubes for bioanalytical purposes in healthy and diseased eyes, and this collection method may offer improved reproducibility for protein quantification.

Preinflammatory Signs in Established Reusable and Disposable Contact Lens Wearers.

SIGNIFICANCE: Established reusable contact lens (CL) wearers show higher tear inflammatory cytokine concentrations and greater conjunctival metaplasia in the region covered by standard soft CLs. The balance of proinflammatory to anti-inflammatory cytokines, but not individual tear cytokine concentrations, was associated with self-reported CL discomfort. PURPOSE: Daily disposable (DD) lenses are often used to improve CL discomfort, but the effect on ocular inflammatory responses has not been fully investigated. This study aimed to compare the concentrations of tear cytokines and conjunctival cell morphology in healthy habitual DD and reusable soft CL wearers. METHODS: Thirty-six established daily CL wearers, including 14 DD and 24 reusable wearers, were enrolled. Symptoms and ocular surface integrity were evaluated. The concentration of tear cytokines (interleukin 1ß [IL-1ß], IL-6, IL-10, IL-12(p70), IL-17A, and tumor necrosis factor α) were determined using Multiplex assays. The ratios of proinflammatory and anti-inflammatory cytokines were calculated. Impression cytology was performed on the conjunctiva, and goblet cell density and epithelial squamous metaplasia were quantified. Differences in variables by CL replacement schedules and the associations between variables were analyzed. RESULTS: Reusable CL wearers had higher concentrations (in pg/mL) of IL-1ß (26 ± 7 vs. 16 ± 11), IL-6 (42 ± 14 vs. 25 ± 20), IL-10 (83 ± 23 vs. 49 ± 36), IL-12(p70) (145 ± 44 vs. 91 ± 68), IL-17A (93 ± 26 vs. 54 ± 44), and tumor necrosis factor α (312 [171 to 468] vs. 189 [6 to 447]) (all P < .01) and greater conjunctival metaplasia in the region covered by CLs (0.7 [0.2 to 1.6] vs. 0.4 [0.04 to 1.2], P = .01) compared with DD wearers. There was a positive association between CL discomfort and ratios of IL-1ß to IL-10 and IL-12(p70) to IL-10 (ρ = 0.42 and ρ = 0.33, P < .05). CONCLUSIONS: Higher ocular inflammatory responses, as indicated by higher tear cytokine concentrations and higher conjunctival epithelial metaplasia, were found in reusable CL wearers than in DD CL wearers. The balance of proinflammatory and anti-inflammatory cytokines may be helpful to assess the inflammatory status of the eye.

Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro.

Tear hyperosmolarity is known to cause ocular surface inflammation in dry eye syndrome. Benzalkonium chloride (BAK), an eyedrop preservative, is known to induce dry eye in long-term-treated patients. Analyzing the modulation of the proinflammatory potential of hyperosmolarity in the presence of BAK on the conjunctiva could give new insights into the effect of this preservative on the disease. In a hyperosmolar model on a conjunctiva-derived cell line, and in the presence of BAK, we evaluated key inflammatory markers [CCL2, IL-8, IL-6, macrophage migration inhibitory factor (MIF) and intercellular adhesion molecule (ICAM)-1] as well as the osmoprotectant element nuclear factor of activated T cells (NFAT)5 using ELISA, RT-qPCR or immunofluorescence staining. Hyperosmolarity highly stimulated CCL2 and NFAT5 in these cells. BAK alone only increased IL-6 expression. The stress-combined condition stimulated CCL2, NFAT5, MIF and IL-8 secretion. ICAM-1 was not modulated by any of the conditions tested. In this model, hyperosmolarity and BAK induced the release of different proinflammatory mediators, and, when combined, they lead to the release of additional inflammatory cytokines. This in vitro study highlights the importance of avoiding long-term ophthalmic treatments containing BAK, as tear film hyperosmolarity can be a result of its detergent action.

Histopathological clues in the diagnosis of fungal infection by Scedosporium in a case of endophthalmitis starting as conjunctivitis.

Cutaneous fungal infections can result in disastrous episodes if improperly diagnosed and treated, especially in immunosuppressed patients. Although dermatopathologists are highly familiar with some filamentous fungi - such as Aspergillus and Zygomycetes - they are not so aware of other less common species. We report a case of ocular infection by Scedosporium apiospermum that started as conjunctivitis and resulted in Phthisis bulbi and subsequent exeresis of the left eye. We describe some of the main morphological features of the fungus as well as the important morphological clues for the differential diagnosis with some similar species, such as Aspergillus, Scopulariopsis, Fusarium, Paecilomyces and Zygomycetes.

Basophil expression of diamine oxidase: a novel biomarker of allergen immunotherapy response.

BACKGROUND: Immunotherapy inhibits basophil histamine release, but the assay is cumbersome, and no one has studied the effects of immunotherapy withdrawal. OBJECTIVE: Intracellular fluorochrome-labeled diamine oxidase (DAO) was used as a novel functional readout of basophil histamine release after immunotherapy. Results were compared with conventional basophil surface expression of activation markers. METHODS: Subcutaneous immunotherapy (SCIT)-treated patients (n = 14), sublingual immunotherapy (SLIT)-treated patients (n = 12), participants who completed 3 years of treatment with grass pollen sublingual immunotherapy (the SLIT-TOL group; n = 6), patients with untreated seasonal allergic rhinitis (SAR; n = 24), and nonatopic control subjects (n = 12) were studied. Intracellularly labeled DAO(+) and surface expression of CD203c(bright), CD63(+), and CD107a(+) on chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTh2)-positive basophils were measured by means of flow cytometry. Serum IgG4 levels and serum inhibitory activity for IgE-allergen complex binding to B cells (IgE-FAB) and basophil histamine release were also determined. RESULTS: Proportions of allergen-stimulated DAO(+)CRTh2(+) basophils were higher in participants in the SCIT, SLIT, and SLIT-TOL groups (all P < .0001) compared with those in patients in the SAR group. Similarly, there were lower proportions of CRTh2(+) basophils expressing surface CD203c(bright) (all P < .001), CD63 (all P < .001), and CD107a (all P < .01). Rhinitis symptoms were lower in the SCIT, SLIT, and SLIT-TOL groups (P < .001) compared with those in the SAR group. Serum inhibitory activity for IgE-FAB and basophil histamine release were also significantly greater in all immunotherapy groups (P < .05) compared with the SAR group. CONCLUSION: These results support long-term clinical and immunologic tolerance during and after grass pollen immunotherapy. Intracellularly labeled DAO expression by basophils merits further investigation as a surrogate biomarker for monitoring efficacy and tolerance after immunotherapy.

Rabbit conjunctivae edema and release of NO, TNF-α, and IL-1ß from macrophages induced by fractions and esculentosides isolated from Phytolacca americana.

CONTEXT: The roots of Phytolacca americana L. (Phytolaccaceae) may be toxic. Despite heated controversy over the toxic compounds of P. americana, especially esculentosides, relevant studies remain scarce. OBJECTIVE: The objective of this study is to screen the toxic fractions and compounds of P. americana, to determine the controlling indices, and to provide evidence for unraveling the mechanism. MATERIALS AND METHODS: Petroleum ether (PE), CH2Cl2, n-BuOH, and water fractions were isolated from 70% ethanol extract of P. americana. The n-BuOH fraction was dissolved in 50% ethanol and precipitated by adding ethyl ether. The resultant supernatants and precipitates were referred to as SUPs and SEDs fractions, respectively. SUPs fraction was separated by column chromatography into four main stimulating esculentosides that were identified by HR-ESI/MS and NMR as EsA, EsB, EsC, and EsF. The irritating effects of esculentosides on rabbit conjunctivae (500 µg/eye) was observed by pathological examination and those on macrophages (5, 25, 50 and 100 µg/mL) were evaluated by detecting changes of NO, TNF-α, and IL-1ß levels. RESULTS AND DISCUSSION: n-BuOH, SUP fractions, and EsC induced severe conjunctival edema. The four esculentosides induced dose-dependent releases of proinflammatory mediators NO, TNF-α, and IL-1ß from macrophages, and releasing amounts peaked after 2 h of treatment. EsC and EsF induced macrophages to release mediators most significantly. EsC (50 µg/mL) functioned more effectively than EsF did, and similarly n-BuOH and SUPs fractions functioned more effectively than the esculentoside mixture. Thus, the four esculentosides exerted proinflammatory effects synergistically. CONCLUSION: All extracted esculentosides, especially EsC, induced inflammatory stimulation. Phytolacca americana-induced irritation of the gastrointestinal tract may be associated with esculentosides such as EsC.

Ocular surface mucins and local inflammation--studies in genetically modified mouse lines.

Mucins locate to the apical surfaces of all wet-surfaced epithelia including ocular surface. The functions of the mucins include anti-adhesive, lubrication, water retention, allergens and pathogen barrier function. Ocular surface pathologies, i.e. dry eye syndrome or allergic conjunctivitis, are reportedly associated with alteration of expression pattern of mucin components. Recent investigations indicated anti-bacterial adhesion or anti-inflammatory effects of members of mucins in non-ocular tissues, i.e., gastrointestinal tracts or airway tissues, by using genetically modified mouse lines that lacks an expression of a mucin member. However, examination of ocular phenotypes of each of mucin gene-ablated mouse lines has not yet fully performed. Muc16-deficient mouse is associated with spontaneous subclinical inflammation in conjunctiva. The article reviews the roles of mucin members in modulation of local inflammation in mucous membrane tissues and phenotype of mouse lines with the loss of a mucin gene. Analysis of ocular surface of mucin-gene related mutant mouse lines are to be further performed.

Exhaled nitric oxide in spray painters exposed to isocyanates: effect modification by atopy and smoking.

BACKGROUND: Isocyanate asthma is one of the most frequently identified forms of occupational asthma in industrialised countries. The underlying mechanisms have not been clarified. There is only limited information about the relationship between exhaled nitric oxide (eNO) and occupational exposure to isocyanates and asthma. OBJECTIVES: To investigate the association between isocyanate exposure and eNO levels in isocyanate-exposed workers and to elucidate whether eNO acts as a marker of airway inflammation controlling for smoking and atopy in an industry-wide survey. METHODS: Information on estimated personal isocyanate exposure, measured eNO levels, health effects and sensitisation were analysed in 229 workers from a cross-sectional study. Univariate and multiple regression analyses were used to explore the exposure-response relationships between isocyanate exposure and eNO, stratified by smoking and atopy. RESULTS: A marginally significant exposure-response relationship was found between isocyanate exposure and eNO in atopic, non-smokers (p=0.054). eNO was significantly associated with atopy and smoking, bronchial hyper-responsiveness (BHR), work-related conjunctivitis and rhinitis after adjustment for age, gender, atopy and smoking (p<0.05). A borderline significant association was found between eNO and asthma-like symptoms after adjustment for age, gender, atopy and current smoking (p=0.055). In a small group of isocyanate-exposed workers with positive serum-specific immunoglobulin E (IgE) antibodies to hexamethylene diisocyanate (HDI), elevated eNO levels were clearly exposure related. eNO was associated with the positive specific IgG antibodies to HDI in non-atopic, non-smokers (p=0.03). CONCLUSIONS: Increased eNO levels may indicate increased airway inflammation in atopic, non-smokers exposed to isocyanates especially at higher levels of isocyanate exposure.

Azithromycin reduces inflammation in a rat model of acute conjunctivitis.

PURPOSE: Macrolide antibiotics are known to have various anti-inflammatory effects in addition to their antimicrobial activity, but the mechanisms are still unclear. The effect of azithromycin on inflammatory molecules in the lipopolysaccharide-induced rat conjunctivitis model was investigated. METHODS: Twenty-four Wistar rats were divided into two groups receiving topical ocular azithromycin (15 mg/g) or vehicle. In total, six doses (25 µl) were administered as one dose twice a day for three days before subconjunctival lipopolysaccharide injection (3 mg/ml). Before the rats were euthanized, mucus secretion, conjunctival and palpebral edema and redness were evaluated. Real-time polymerase chain reaction was used to determine gene expression for interleukin-6, cyclooxygenase-2, tumor necrosis factor-α, matrix metalloproteinase (MMP)-2, and MMP-9. Interleukin-6 was determined with enzyme-linked immunosorbent assay, nuclear factor-kappa B with western blot, and MMP-2 activity with gelatin zymogram. Four eyes per group were processed for histology and subsequent periodic acid-Schiff staining and CD68 for immunofluorescence. The Student t test or the Wilcoxon test for independent samples was applied (SPSS v.15.0). RESULTS: Azithromycin-treated animals showed a significant reduction in all clinical signs (p<0.05) compared to controls. Interleukin-6 (p<0.05), nuclear factor-kappa B protein expression (p<0.01), and MMP-2 activity (p<0.05) in conjunctival homogenates were significantly reduced compared with the control animals. MMP-2 gene expression showed a tendency to decrease in the azithromycin group (p=0.063). Mucus secretion by goblet cells and the macrophage count in conjunctival tissue were also decreased in the azithromycin group (p<0.05). CONCLUSIONS: These results suggest that azithromycin administration ameliorates induced inflammation effects in a rat model of acute conjunctivitis.

Bilateral acute pyogenic conjunctivitis with iritis induced by unilateral topical application of bacterial peptidoglycan muramyl dipeptide in adult rabbits.

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5-5.0 × 10(6) cells/mL) were elevated 4-8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation.

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.

Nuclear Factor-κB: central regulator in ocular surface inflammation and diseases.

The nuclear factor-κB (NF-κB) is a key transcription factor pathway that is responsible for many key biological processes, such as inflammation, apoptosis, stress response, corneal wound healing, angiogenesis, and lymphangiogenesis. Numerous recent studies have investigated NF-κB in the context of ocular surface disorders, including chemical injury, ultraviolet radiation-induced injury, microbial infections, allergic eye diseases, dry eye, pterygium, and corneal graft rejection. The purpose this article is to summarize key findings with regard to the pathways regulating NF-κB and processes governed by the NF-κB pathway. In the innate defense system, NF-κB is involved in signaling from the toll-like receptors 2, 3, 4, 5 and 7, which are expressed in conjunctival, limbal, and corneal epithelial cells. These determine the ocular responses to infections, such as those caused by Pseudomonas aeruginosa, Staphylococcus aureus, adenovirus, and herpes simplex-1 virus. Natural angiogenic inhibitors enhance NF-κB, and this may occur through the mitogen-activated protein kinases and peroxisome proliferator-activated receptor γ. In alkali injury, inhibition of NF-κB can reduce corneal angiogenesis, suggesting a possible therapeutic strategy. The evaluation of NF-κB inhibitors in diseases is also discussed, including emodin, besifloxacin, BOL-303242-X (mapracorat), thymosin-ß4, epigallocatechin gallate, Perilla frutescens leaf extract and IKKß-targeting short interfering RNA.

Should we care about the ocular surface in the anophthalmic patient?

PURPOSE: To assess clinical and biomolecular changes of the conjunctival epithelium in anophthalmic patients wearing an ocular prosthesis. METHODS: Thirty-five unilateral anophthalmic patients were enrolled. Patients with blepharitis, lid abnormalities, and topical/systemic medication affecting the ocular surface were excluded. Symptom Assessment in Dry Eye (SANDE) questionnaire and tear function test (Schirmer Test Type I) were recorded. Conjunctival inflammation and meibomian gland dysfunction (MGD) were graded in the anophthalmic side and fellow eye. Impression cytology sampling of the upper, lower tarsal, and posterior/bulbar conjunctiva from the anophthalmic socket were collected and compared to healthy controls. RESULTS: Patients had significantly higher SANDE (p < 0.001), Schirmer I test (p = 0.004), conjunctival inflammation (p < 0.001), and MGD scores (p < 0.001) on the anophthalmic side compared to the fellow eye. Mucin 5AC, inflammatory markers (MMP-9, ICAM-1) expression (p < 0.001), and response to oxidative stress (NRF2-KEAP1 signaling pathway) (p < 0.05) were significantly upregulated in the posterior conjunctival surface in the anophthalmic socket. CONCLUSIONS: Anophthalmic patients complained of more pronounced dry eye symptoms and presented more significant signs of inflammation and MGD on the anophthalmic side. The bulbar conjunctiva, behind the prosthesis, showed more significant hyperexpression of mucins, markers of inflammation, and increased response to oxidative stress compared to the tarsal conjunctiva. Patients wearing ocular prosthesis had signs of inflammation resembling dry eye disease.

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.

Efficacy of Intense Pulsed Light Treatment for Moderate to Severe Acute Blepharitis or Blepharoconjunctivitis: A Retrospective Case Series

Objectives: We aimed to evaluate the efficacy of periocular intense pulsed light (IPL) therapy in the treatment of moderate to severe acute blepharitis or blepharoconjunctivitis. Materials and Methods: This was a retrospective study performed in one institution. Eleven patients who received bilateral periocular IPL therapy using an IPL device (E>Eye, ESwin, Paris, France) were retrospectively evaluated. The following findings obtained at baseline and 10 weeks after the treatment were recorded: slit-lamp examinations; symptom scores of the Compression of the Eyelid (COTE) grading system and Ocular Surface Disease Index (OSDI); ocular surface staining with Oxford grading scale (OXFORD) scores; lipid layer thickness (LLT); and non-invasive tear meniscus test (TMH), non-invasive break up time measurement (NIBUT), and meibography performed by using I.C.P. Ocular Surface Analyzer (SBM System, Turin, Italy). Results: Significant improvements in OSDI symptom scores (p<0.0001), LLT (p<0.0001), and meibography (p<0.0001) were obtained at 10 weeks after bilateral periocular IPL therapy. COTE and ocular surface staining scores decreased by 59.72% and 57.14% respectively, while NIBUT and TMH increased by 47.34% and 22.16%, respectively. In parallel to the improvement in OSDI, LLT, and meibography, findings of acute blepharitis or blepharoconjunctivitis improved in slit-lamp examination. There were no adverse effects. Conclusion: Serial IPL therapy improves the clinical signs and symptoms of moderate to severe acute blepharitis or blepharoconjunctivitis, meibomian gland morphology, and secretion quality.

Substance P Level in Tears as a Potential Biomarker for Contact Lens Discomfort.

Purpose: To analyze the effects of contact lens (CL) wear, time of the day, and CL discomfort (CLD) on clinical signs, tear inflammatory mediators and substance P.Methods: Thirty symptomatic and 30 asymptomatic CL wearers attended two visits (morning and afternoon) on two days (non-CL and CL wearing days). Comfort, meniscus area, noninvasive breakup time (NIBUT), tear collection, hyperemia, lid parallel conjunctival folds, fluorescein staining, and sensitivity were performed. The tear levels of 23 inflammatory mediators and substance P were measured.Results: Comfort, meniscus area, NIBUT, and MMP-9 were lower while conjunctival staining and EGF higher (p ≤ 0.015) on the CL wearing day. Comfort, IL-8/CXCL8, and VEGF were lower while EGF, IP-10/CXCL10, and MCP-1/CCL2 higher (p ≤ 0.047) in the afternoon. Comfort was lower and substance P higher (p ≤ 0.006) in symptomatic wearers.Conclusion: Substance P may be implicated in CLD etiology; its role and potential application as a biomarker should be further studied.

Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca2+ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.