Tear cytokine profiling in patients with superior limbic keratoconjunctivitis who underwent medical treatment or in conjunction with surgical management.

PURPOSE: To determine the correlation between superior limbic keratoconjunctivitis (SLK) and selected tear cytokines and to evaluate the efficacy of these cytokines in monitoring the response of patients with SLK to either medical treatment alone or in combination with conjunctival resection. DESIGN: A cohort study. METHODS: Twenty-five eyes of 13 patients with SLK were assigned to either medically responsive or surgical treatment groups depending on their responses 1 month after initial medical treatment. Treatment efficacy was assessed by improvements in clinical grading and decreases in the levels of tear cytokines. RESULTS: Fourteen eyes were improved by medical treatment alone, whereas satisfactory outcomes were achieved for the remaining 11 eyes after surgical management. The overall grading prior to medical treatment was lower in medically responsive group compared with surgical treatment group (p=0.0139). Among the examined tear cytokines, monocyte chemoattractant protein (MCP)-1 was positively associated with the severity of clinical grading (p=0.0251). While both treatments significantly decreased the levels of MCP-1 and interleukin-6, surgical treatment also decreased the levels of interferon-γ and tumour necrosis factor-α. Notably, overall cytokine levels after surgical treatment were lower than those after medical treatment alone. CONCLUSIONS: Since an association of tear MCP-1 level with the clinical grading and treatment response of SLK was observed in this study, tear MCP-1 may be a potential indicator of SLK disease severity. According to the degree to which the tear cytokine levels were decreased, surgical treatment appears to be an effective treatment modality for patients with SLK who are refractory to medical treatment alone.

Human adenovirus type 26 uses sialic acid-bearing glycans as a primary cell entry receptor.

Adenoviruses are clinically important agents. They cause respiratory distress, gastroenteritis, and epidemic keratoconjunctivitis. As non-enveloped, double-stranded DNA viruses, they are easily manipulated, making them popular vectors for therapeutic applications, including vaccines. Species D adenovirus type 26 (HAdV-D26) is both a cause of EKC and other diseases and a promising vaccine vector. HAdV-D26-derived vaccines are under investigation as protective platforms against HIV, Zika, and respiratory syncytial virus infections and are in phase 3 clinical trials for Ebola. We recently demonstrated that HAdV-D26 does not use CD46 or Desmoglein-2 as entry receptors, while the putative interaction with coxsackie and adenovirus receptor is low affinity and unlikely to represent the primary cell receptor. Here, we establish sialic acid as a primary entry receptor used by HAdV-D26. We demonstrate that removal of cell surface sialic acid inhibits HAdV-D26 infection, and provide a high-resolution crystal structure of HAdV-D26 fiber-knob in complex with sialic acid.

Sialic Acid-Containing Glycans as Cellular Receptors for Ocular Human Adenoviruses: Implications for Tropism and Treatment.

Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.

High Inflammatory Infiltrate Correlates With Poor Symptomatic Improvement After Surgical Treatment for Superior Limbic Keratoconjunctivitis.

PURPOSE: Superior limbic keratoconjunctivitis (SLK) is a chronic and recurrent condition of unknown etiology. It is often managed conservatively, but there is a high rate of success with surgical management for severe or recalcitrant cases. The purpose of this article is to describe and analyze clinicopathological features of patients with SLK who underwent surgical treatment and their association with the clinical outcome. METHODS: A total of 22 eyes from 18 patients who underwent surgical SLK management were retrospectively analyzed. Clinicopathological data were collected including details of follow-up and patient satisfaction (n = 15). Moreover, 12 cases had specimens available for review of histopathologic findings and COX-2 expression analysis by immunohistochemistry. RESULTS: From a clinical perspective, 66.7% of the SLK eyes had nonmechanical factors contributing to SLK, and 66.7% of eyes demonstrated significant symptomatic improvement after surgery. Histopathological analysis of all the lesions showed acanthosis and goblet cell loss. Unexpectedly, in 93% of the eyes, dilated lymphatic vessels were found. Furthermore, a high inflammatory infiltrate correlated with minimal symptomatic improvements (P = 0.013). Moreover, COX-2 expression was higher in patients with SLK than in a normal conjunctiva (P = 0.001). CONCLUSIONS: In this study, the most common systemic association with SLK was the patient's autoimmune status. Histopathological evaluation revealed that high inflammatory infiltration in the biopsy might be predictive of minimal symptomatic improvement with surgical management. Finally, the higher COX-2 expression in patients with SLK compared with that in individuals with a normal conjunctiva supports the use of anti-COX-2 drugs as a possible therapeutic target.

Stem Cell Factor and Thymic Stromal Lymphopoietin Overexpression With Correlation to Mast Cells in Superior Limbic Keratoconjunctivitis.

PURPOSE: To investigate the differential expression of stem cell factor (SCF) and thymic stromal lymphopoietin (TSLP) and their correlation to mast cells, between patients diagnosed with superior limbic keratoconjunctivitis (SLK) and normal subjects. METHODS: A laboratory investigation included 22 surgical specimens of the superior bulbar conjunctiva from 17 patients with medically refractory SLK and 5 control subjects who underwent cataract or retinal surgery. Protein expression of tryptase, SCF, and TSLP in conjunctival specimens was detected by immunohistochemistry. The number of mast cells was correlated with immunohistochemistry intensity of SCF and TSLP. RESULTS: In patients with SLK, higher immunostaining intensity of SCF and TSLP was found in the conjunctival epithelium than that in the conjunctival subepithelial stroma. SCF and TSLP staining in the conjunctival epithelium was significantly more intense in patients with SLK than in normal subjects. In addition, there was a significant correlation between the number of tryptase (+) mast cells in conjunctival subepithelial stroma and TSLP immunointensity in the conjunctival epithelium and subepithelial stroma. CONCLUSIONS: Overexpression of SCF and TSLP was found in the conjunctival epithelium of patients with SLK. Significant correlation between TSLP grading and the number of mast cells was also found. SCF and TSLP may be involved in promoting mast cell migration and activation contributing to the pathogenesis of SLK.

Involvement of corneal lymphangiogenesis in a mouse model of allergic eye disease.

PURPOSE: The contribution of lymphangiogenesis (LA) to allergy has received considerable attention and therapeutic inhibition of this process via targeting VEGF has been considered. Likewise, certain inflammatory settings affecting the ocular mucosa can trigger pathogenic LA in the naturally avascular cornea. Chronic inflammation in allergic eye disease (AED) impacts the conjunctiva and cornea, leading to sight threatening conditions. However, whether corneal LA is involved is completely unknown. We addressed this using a validated mouse model of AED. METHODS: Allergic eye disease was induced by ovalbumin (OVA) immunization and chronic OVA exposure. Confocal microscopy of LYVE-1-stained cornea allowed evaluation of corneal LA, and qRT-PCR was used to evaluate expression of VEGF-C, -D, and -R3 in these mice. Administration of VEGF receptor (R) inhibitor was incorporated to inhibit corneal LA in AED. Immune responses were evaluated by in vitro OVA recall responses of T cells, and IgE levels in the serum. RESULTS: Confocal microscopy of LYVE-1-stained cornea revealed the distinct presence of corneal LA in AED, and corroborated by increased corneal expression of VEGF-C, -D, and -R3. Importantly, prevention of corneal LA in AED via VEGFR inhibition was associated with decreased T helper two responses and IgE production. Furthermore, VEGFR inhibition led a significant reduction in clinical signs of AED. CONCLUSIONS: Collectively, these data reveal that there is a distinct involvement of corneal LA in AED. Furthermore, VEGFR inhibition prevents corneal LA and consequent immune responses in AED.

A comprehensive review on vernal keratoconjunctivitis with emphasis on proteomics.

Allergic conjunctivitis presents as a spectrum of different clinical entities, such as perennial allergic conjunctivitis, seasonal allergic conjunctivitis, atopic keratoconjunctivitis and vernal keratoconjunctivitis. Vernal keratoconjunctivitis (VKC) is a disorder that is often associated with allergens and is seen during the spring season. Herein, we focused on vernal keratoconjunctivitis (VKC) and reviewed its epidemiology, clinical presentations, ocular associations, available treatment options, and the progressive understanding of its histopathological features; we have also systematically elaborated on the various studies on proteomics. Initial theories of a solely IgE-mediated mechanism have been replaced by those considering IgE and non-IgE mechanisms. Developments in basic and clinical research will open novel diagnostic approaches for the early detection and cure of the disease.

Highly expressed cytoplasmic FcεRIß in human mast cells functions as a negative regulator of the FcRγ-mediated cell activation signal.

BACKGROUND: We recently reported that the interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell (MC) activation and that FcεRIß functions as an amplifier of FcεRI-mediated activation signal. Some of FcεRIß in cytoplasm appeared not to be co-localized with FcεRIα. The function of FcεRIß in the cytoplasm remains unknown. METHODS: The localization of FcεRIß and FcεRIα in giant papillae specimens from patients with allergic keratoconjunctivitis and of FcεRIß, FcεRIα, and Lyn in cultured human MCs was examined using confocal microscopy. FcεRIß was overexpressed using an adenovirus vector system. Mediators were measured by enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS: In the subepithelial region, FcεRIß was mainly localized in the cell membrane of MCs. In the perivascular region, FcεRIß expression was scattered throughout the cytoplasm and in the cell membrane of MCs. Overexpression of FcεRIß in MCs mainly increased its cytoplasmic expression and slightly up-regulated cell surface FcεRI expression. However, overexpression of FcεRIß in MCs resulted in down-regulation of the tyrosine phosphorylation levels of FcεRIß and Syk and down-regulation of the Ca(2+) influx soon after FcεRI aggregation and then resulted in down-regulation of degranulation, PGD2 synthesis, and production of a set of cytokines. This negative regulatory effect may be due to inhibition of the redistribution of Lyn to small patches within the plasma membrane. CONCLUSION: Cytoplasmic FcεRIß, which is not co-localized with FcεRIα, may function as a negative regulator, as it can capture important signalling molecules such as Lyn.

Alarmins from corneal epithelial cells upregulate CCL11 and VCAM-1 in corneal fibroblasts.

PURPOSE: Severe ocular allergic diseases are characterized by pronounced conjunctival inflammation triggered by T helper 2 (Th2) cells and corneal epithelial damage induced by eosinophils. To examine the role of alarmins released by damaged corneal epithelial cells in tissue eosinophilia, we investigated the effects of a supernatant derived from necrotic human corneal epithelial (HCE) cells on expression of the chemokine CCL11 (eotaxin) and the adhesion molecule VCAM-1 in human corneal fibroblasts. METHODS: An alarmin preparation was obtained as the material released from HCE cells after three cycles of freezing and thawing. CCL11 released into culture medium and cell surface expression of VCAM-1 were measured with enzyme-linked immunosorbent assays, and the amounts of CCL11 and VCAM-1 mRNAs were quantitated by reverse transcription and real-time polymerase chain reaction analysis. Signaling by the transcription factor NF-κB was evaluated by immunoblot and immunofluorescence analyses. RESULTS: The combination of the necrotic HCE cell supernatant and either interleukin (IL)-4 or IL-13 induced synergistic increases in CCL11 release, VCAM-1 expression, and the abundance of CCL11 and VCAM-1 mRNAs in corneal fibroblasts. The necrotic HCE cell supernatant also induced NF-κB activation in corneal fibroblasts, whereas an inhibitor of NF-κB and IL-1 receptor antagonist each attenuated CCL11 release induced by the alarmin preparation and either IL-4 or IL-13. CONCLUSIONS: Alarmins including IL-1 released from necrotic corneal epithelial cells cooperate with Th2 cytokines to induce CCL11 production and VCAM-1 expression in corneal fibroblasts, and may thereby play an important role in tissue eosinophilia associated with ocular allergic diseases.

Chemotactic responses of peripheral blood eosinophils to prostaglandin D2 in atopic keratoconjunctivitis.

BACKGROUND: Eosinophils appear to be key cells in the pathogenesis of conjunctival inflammation in atopic keratoconjunctivitis (AKC). Chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2) mediates prostaglandin D2 (PGD2)-dependent migration of eosinophils. However, it is unclear whether CRTH2/PGD2-dependent eosinophil migration is upregulated in allergic diseases. OBJECTIVE: To compare the chemotactic responses of peripheral blood eosinophils to prostaglandin D2 in patients with severe AKC and healthy individuals. METHODS: We used an enzyme immunoassay system to measure PGD2 levels in tears and blood samples from healthy individuals and patients with AKC. CRTH2 expression on peripheral blood eosinophils was determined using reverse-transcriptase polymerase chain reaction (RT-PCR), flow cytometry, and an oligonucleotide array system. Chemotaxis experiments were performed using a modified Boyden chamber technique and an optical assay system. RESULTS: The PGD2 concentrations were higher in tears from patients with severe AKC compared with healthy individuals. RT-PCR (severe and mild cases), flow cytometry (mild cases), and GeneChip analyses revealed a significantly higher expression of CRTH2 on peripheral blood eosinophils in patients with AKC than in healthy individuals. PGD2 and its stable metabolite 13,14-dihydro-15-keto-PGD2, a CRTH2 agonist, induced chemotaxis of eosinophils from patients with AKC; chemotaxis was significantly enhanced in eosinophils from patients with severe AKC compared with those from healthy individuals. CONCLUSION: CRTH2 is more abundantly expressed on eosinophils from patients with AKC and promoted PGD2-dependent migration to a greater extent than in healthy individuals.

IgE and eosinophil cationic protein (ECP) as markers of severity in the diagnosis of atopic keratoconjunctivitis.

AIMS: To evaluate tear and serum IgE and eosinophil cationic protein (ECP) as severity markers for atopic keratoconjunctivitis (AKC). METHODS: Thirty eyes of 30 patients with AKC and 10 eyes of 10 healthy control subjects were examined in this prospective study. All subjects underwent fluorescein staining, conjunctival injection, conjunctival oedema and papillary formation grading. Tear and serum IgE and ECP levels were measured, and correlations between them investigated with reference to the ocular surface clinical parameters. RESULTS: The mean fluorescein scores, conjunctival injection, oedema scores and papillary formation were significantly higher in AKC patients compared to controls (p<0.05). Higher total IgE and ECP levels were detected in AKC tears compared with the control group. Tear ECP levels showed a significant correlation with fluorescein staining, conjunctival injection and oedema scores (r=0.70, 0.62 and 0.62, respectively). Tear IgE had no correlation with clinical signs. Serum IgE and ECP levels were elevated in AKC patients but did not show any correlation with clinical signs. CONCLUSION: This study suggests the presence of an eosinophilic response in AKC disease independent of IgE sensitisation. Tear ECP was a useful marker delineating the severity of ocular surface disease in AKC.

Cellular and tissue architecture of conjunctival membranes in epidemic keratoconjunctivitis.

PURPOSE: Conjunctival membranes are differentiated from pseudomembranes by bleeding on removal. The authors sought to study the cellular constituents and extracellular structure of formed conjunctival exudates in epidemic keratoconjunctivitis (EKC), and to determine whether these represent true conjunctival membranes. METHODS: Formed conjunctival exudates harvested from 2 patients with EKC were studied by immunohistochemistry. Adenoviral infection was evaluated by PCR. RESULTS: Each patient's tears were PCR positive for adenovirus, but not HSV-I. Both conjunctival specimens contained a fibrinous stroma richly populated with cells positive for CD4, CD8, CD20, CD68, myeloperoxidase, and fascin. Both specimens also contained cells positive for CD31 and Tie-2, and demonstrated expression of transforming growth factor-ß and vascular endothelial growth factor. CONCLUSIONS: Conjunctival exudates in EKC contain a range of leukocytes representing both innate and acquired immune responses, along with angiogenic factors and proliferating endothelial cells. Therefore, true conjunctival membranes can and do form in EKC.

Mathematical modelling of epidemics under specific regard of adenoviral keratoconjunctivitis.

At first ADV is presented as a typical pandemic. The contagiosity of adenovirus is high because of the viability of the virus on inorganic surfaces in medical offices up to 35 days. Outbreaks and epidemics occur 3-30 days after infection, which is mainly contracted from medical facilities. EKC is considered a notifiable condition in most countries, and outbreaks, suspects and infections must be reported. Symptoms like "pink eye", foreign body sensations, photophobia, pain, signs such as follicles, hemorrhages and corneal infiltrates, and vision decrease associated with malaise are frequently observed first in one eye, later involving the fellow eye. Unilateral disease has a high rate of misdiagnosis. Currently no vaccine or virustatic is available, which is effective, cost-efficient and tolerable. Treatment is symptomatic and antiinflammatory. Late scarring may be amenable to phototherapeutic keratectomy. Infection control measures focus on the disinfection of equipment and hands of staff, the handling of infected patients with gloves, spatial separation of infected individuals resp. cohorting of infected patients, use of unit-dose eye solutions, and the chlorination of pools by approved and registered disinfectants and germicides. In connection with this it is shown how to handle the dynamics of infections by mathematical models like cellular automation, systems of differential equations and to visualize periodic effects by Fourier Analysis and to calculate costs by mathematical programming. Using mathematical analysis the percentage of a population needing vaccination to prevent spreading of pandemic can be calculated. It is shown here that especially the method of cellular automation is a simple way to simulate complex epidemiological situations without completely knowing the mathematical details.

Alterations of the ocular surface epithelial MUC16 and goblet cell MUC5AC in patients with atopic keratoconjunctivitis.

BACKGROUND: An increased understanding of the ocular surface at cellular level in the conjunctiva and the cornea may help explain the pathogenesis and the subsequent clinical appearance of atopic ocular allergies, which may be potentially blinding. PURPOSE: To investigate the MUC16 and MUC5AC alterations, tear function and the ocular surface disorder in patients with atopic keratoconjunctivitis (AKC). METHODS: Thirty-six eyes of 18 AKC patients as well as 28 eyes of 14 age- and sex-matched normal subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT), fluorescein and Rose-Bengal staining of the ocular surface, conjunctival impression cytology and brush cytology. Impression cytology samples underwent periodic acid schiff and immunohistochemical staining with MUC16 and MUC5AC antibodies. Brush cytology specimens underwent evaluation for inflammatory cell numbers and quantitative real-time polymerase chain reaction (PCR) for MUC16 and MUC5AC mRNA expression. RESULTS: The mean corneal sensitivity and BUT values were significantly lower in patients with AKC, compared with controls (P < 0.001). Brush cytology specimens from AKC patients revealed significantly higher numbers of inflammatory cells (P < 0.001). Specimens from patient eyes showed positive staining for MUC5AC and MUC16. MUC16 mRNA expression was significantly upregulated with significant downregulation of MUC5AC mRNA expression in eyes with AKC compared with the eyes of control subjects. CONCLUSION: Ocular surface inflammation, decline in corneal sensitivity, tear film instability, changes in conjunctival epithelial MUC5AC and MUC16 mRNA expressions were thought to be important in the pathogenesis of atopic ocular surface disease.

Immunopathogenesis of ocular allergy: a schematic approach to different clinical entities.

PURPOSE OF REVIEW: The immunopathogenesis of ocular allergic disorders is generally related to the specific immunoglobulin E-mediated mast cell activation and the following cascade of inflammatory mediators. Seasonal and perennial allergic conjunctivitis, however, are the only ocular diseases to involve solely type I hypersensitivity. The other main forms, vernal and atopic keratoconjunctivitis, have a more complex immunological basis and a chronic inflammatory component. Involvement of inflammatory cells, particularly eosinophils and T cells, cytokines and proteases can lead to more serious corneal damage with vision-threatening potential. RECENT FINDINGS: Experimental allergic conjunctival models and clinical research studies have shown that T helper type 2-related mechanisms are definitely involved in the sensitization phase of ocular allergy, however, both T helper type 1 and type 2 cytokines are overexpressed in the active disease, contributing to the development of ocular inflammation. SUMMARY: A review of the recent literature allows us to better understand the mechanisms involved in the development of ocular allergy and to guide us toward a more schematic approach, which could possibly be useful in forming a new classification, standardizing clinical phases and individuating new treatment targets.

Another way to think of tears: blood, sweat, and... "dacruon".

Eugene Wolff's 1946 concept of an interpalpebral, trilaminar, preocular tear film does not sit comfortably with more recent scientific observations. A film so defined could exist only in the interblink phase and would exclude unshed retropalpebral fluid. A modern, evidence-based, clinically relevant model is needed, one that includes retropalpebral and meniscal fluids and is applicable throughout the blink cycle. The "ocular surface" (OS) concept combines adjacent discrete epithelia into a single continuous mucosal sheet. This "surface" constitutes an integrated organ. The OS concept has revolutionized the understanding and management of ocular pathology and therapeutics. Further practical advances can be expected once Wolff's trilaminar hypothesis is replaced by a new concept that recognizes a duality: a voluminous muco-aqueous pool extending retropalpebrally, separated always from the atmosphere by its accessory lipid sealant. The neologism "dacruon" (pronounced dacroo-on) is introduced for this composite fluid body. The respective differences between the two components--in their origins, structures, thicknesses, chemistries, motilities, secretion rates, turnovers, functions and manners of disposal--support the duality of the dacruon concept. Adoption of this alternative view of tear structure requires new descriptors to encourage precision and consensus in terminology.

Nuevos paradigmas en la patología ocular infecciosa: queratoconjuntivitis por adenovirus / New paradigms in infectious eye disease: adenoviral keratoconjunctivitis

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Ocular allergy in pediatric practice.

Allergies occur frequently in all pediatric age groups, affecting up to 40% of children. Allergic conjunctivitis is the most common ocular allergy syndrome among children, with atopic keratoconjunctivitis and vernal keratoconjunctivitis comprising less common, but potentially more severe, forms of ocular allergy. In this article, we review the impact, diagnosis, potential complications, and treatment of these ocular allergic pediatric conditions. Early detection is necessary to prevent potentially serious consequences of pediatric ocular allergy. Involvement of pediatric ophthalmologists may be necessary to avoid preventable vision loss in severe cases.