The FADS mouse: A novel mouse model of atopic keratoconjunctivitis.

BACKGROUND: Atopic keratoconjunctivitis (AKC) is a chronic allergic conjunctival disease. However, a mouse model of AKC to investigate the underlying mechanism of the therapeutic agents and estimate their efficacy has not been established. We recently generated mice in which Ikk2 is specifically deleted in facial skin fibroblasts and found that these mice spontaneously develop atopic dermatitis (AD)-like facial skin inflammation and scratching behaviors; thus, we named them facial AD with scratching (FADS) mice. OBJECTIVE: We sought to evaluate whether the ocular lesions that FADS mice spontaneously develop are similar to those of patients with AKC and to estimate the efficacy of topical treatments with tacrolimus and betamethasone for FADS mice by using tear periostin, a novel biomarker for allergic conjunctival disease. METHODS: FADS mice, in which Ikk2 is deleted in dermal fibroblasts, were generated by crossing female Ikk2Flox/Flox mice to male Nestincre; Ikk2Flox/+ mice. We conducted histologic analysis of the ocular lesions in FADS mice. Furthermore, we measured periostin in the tears collected from FADS mice untreated or treated with tacrolimus or betamethasone. RESULTS: The FADS mice exhibited severe blepharitis and scratch behaviors for their faces. In these mice, corneal epithelium and stroma showed hyperplasia and infiltration of eosinophils, mast cells, and TH2/TC2 cells. Periostin was significantly expressed in the lesions and tear periostin was upregulated. Betamethasone showed more suppressive effects than did tacrolimus on severe corneal lesions and increased tear periostin level. CONCLUSIONS: The FADS mouse is a novel mouse model of AKC and is useful to examine the therapeutic effects of anti-AKC agents.

Increased frequency of Demodex blepharitis in rosacea and facial demodicosis patients.

BACKGROUND: Rosacea is an inflammatory disease with 50% of ocular involvement rate. Primary demodicosis is an eruption caused by Demodex mites, and there is no data about the rate of ocular involvement in primary demodicosis. AIMS: In this cross-sectional study, it is aimed to reveal the frequency of Demodex blepharitis in rosacea and primary demodicosis patients. METHODS: In total, 58 rosacea, 33 primary demodicosis patients, and 31 healthy volunteers were included in the study. Four samples were obtained from eyelashes with a forceps and from facial skin by standardized skin surface biopsy. A positive result is described as detecting at least one Demodex mite on an eyelash or at five mites in the face. The patients were also examined by an ophthalmologist in terms of ocular involvement. RESULTS: Both rosacea and primary demodicosis patients had significantly more complaints like burning and stinging in the eyes compared to the control patients (P = .001). Primary demodicosis and papulopustular rosacea patients had the highest numbers of eyelash mites, respectively, and significantly a higher rate of blepharitis than the control group. CONCLUSION: As a result, the Demodex count was significantly higher in the primary demodicosis and rosacea patients than the control group. We think that every Demodex-positive patients should be evaluated for also eyelash mites to prevent a possible chronic blepharitis.

IgE Ratio in Tears: A Predictive Tool of Ocular Allergic Inflammation.

PURPOSE: To demonstrate the tear IgE (measured/exuded) ratio (R) as a useful biological marker of ocular allergy in order to distinguish severe from less severe inflammatory status. METHODS: Tear samples and sera from 78 ocular allergy patients and 19 control subjects were analyzed. Total IgE and albumin were measured for calculating the tear IgE-R defining two subgroups (SG) of samples: R ≥ 4-SG and R < 4-SG. Eosinophil cationic protein, Th1 and Th2 cytokines (IFN-γ, IL-4, -5, -6, -8 and -10) and protein electrophoretic profiles were also investigated in tears. RESULTS: The R < 4-SG compared to the R ≥ 4-SG shows higher levels of tear albumin, eosinophil cationic protein, and Th1 and Th2 cytokines. Moreover, each subgroup presents a specific protein profile. CONCLUSION: This study showed that an IgE-R lower than four must be carefully interpreted as a warning sign of a severe inflammatory context and should be also associated with an exploration of immunological profile.

Idiopathic inflammatory diseases of orbit and ocular adnexa: Histopathological and immunochemical analysis.

Purpose: : To present histopathological and immunohistochemical analysis of idiopathic inflammatory diseases of orbit and ocular adnexa. Methods: Design- A retrospective laboratory-based study. The study was carried out in an ocular pathology laboratory in a tertiary institute of northeast India where analysis of 93 cases was done in 5 years, during the period from 2011 to 2016. Hematoxylin--eosin and special stains were done for the diagnoses. Immunohistochemistry (IHC) panel was also carried out. For infectious pathology, Grocott's methenamine silver (GMS) stain for fungus, tissue Gram's stain for bacteria's, and acid-fast stains for tubercular bacilli were done. IHC panels were done for CD 20 (B-cells), CD-3 (T-cells), CD-45 (Leukocyte common antigen, LCA), BCL-2, CD-138 (Plasma cells), Kappa, Lambda, IgG-4 in tissue, IgG-4 in serum, etc. IHCs were done using kit methods (standardized) and adequate controls were taken for each sample. Results: 93 cases of nonspecific orbital inflammation were reported out of 1,467 specimens. Orbital pseudotumors (idiopathic orbital inflammatory disease, IOID) were seen in 27 cases (sclerosing variety-6); benign lymphoid hyperplasia in two cases; reactive lymphoid hyperplasia in 10 cases; atypical plasma lymphoproliferative reactive (polyclonal immunophenotypically, IgG4 negative) lesions in four cases; IgG-4 related disease in one case; nonspecific inflammatory reactions (conjunctiva, sclera, and lid) in 49 cases. In all the diagnoses, infections and lymphomas were excluded. Conclusion: Biopsy supported study on nonspecific orbital inflammation was important to know the pattern.

Efficacy of long-term intralesional triamcinolone in Morbihan's disease and its possible association with mast cell infiltration.

Morbihan's disease is characterized by chronic persistent facial edema of the upper half of the face, absence of typical diagnostic findings, and refractoriness to treatment. A 44-year-old man was diagnosed with Morbihan's disease based on clinical signs and histopathology, which showed dermal edema in upper dermis, discrete lymphocytic infiltrate without granulomatous reaction, and mast cell infiltration. After long-term therapy with intralesional triamcinolone a remarkable objective and subjective clinical response was observed. Reported cases of Morbihan's disease are reviewed, with respect to their treatment and histopathological findings. Mast cell infiltration has been observed on histopathology in most patients who responded to intralesional triamcinolone, suggesting a possible marker of response. The long-lasting response seen in our case indicates the efficacy of intralesional triamcinolone in this rare condition.

[Atopic keratoconjunctivitis: One allergy may mask another. A clinical observation with two types of hypersensitivity reactions: IgE-mediated and non-IgE-mediated]. / Kératoconjonctivite atopique : une allergie peut en cacher une autre. À propos d'une observation clinique avec hypersensibilité IgE médiée et hypersensibilité non IgE médiée.

Allergies are frequently implicated in ophthalmologic practice. These typically benign allergies can be potentially severe for the ocular surface and have an impact in everyday life. We relate, through a case of keratoconjunctivitis involving 2 types of hypersensitivity, the various triggers and therapeutic choices to allow a more effective treatment.

Tear cathepsin S as a candidate biomarker for Sjögren's syndrome.

OBJECTIVE: The diagnosis of Sjögren's syndrome (SS) in routine practice is largely a clinical one and requires a high index of suspicion by the treating physician. This great dependence on clinical judgment frequently leads to delayed diagnosis or misdiagnosis. Tear protein profiles have been proposed as simple and reliable biomarkers for the diagnosis of SS. Given that cathepsin S activity is increased in the lacrimal glands and tears of NOD mice (a murine model of SS), the aim of this study was to explore the clinical utility of using tear cathepsin S (CTSS) activity as a biomarker for SS. METHODS: A method to measure CTSS activity in tears eluted from Schirmer's test strips was developed and validated. Schirmer's tests were performed and CTSS activity measurements were obtained in 278 female subjects, including 73 with SS, 79 with rheumatoid arthritis, 40 with systemic lupus erythematosus, 10 with blepharitis, 31 with nonspecific dry eye disease, and 12 with other autoimmune diseases, as well as 33 healthy control subjects. RESULTS: The median tear CTSS activity in patients with SS was 4.1-fold higher than that in patients with other autoimmune diseases, 2.1-fold higher than that in patients with nonspecific dry eye disease, and 41.1-fold higher than that in healthy control subjects. Tear CTSS levels were equally elevated in patients with primary SS and those with secondary SS, independent of the Schirmer's test strip values or the levels of circulating anti-SSA or anti-SSB antibodies. CONCLUSION: Markedly high levels of tear CTSS activity are suggestive of SS. CTSS activity in tears can be measured in a simple, quick, economical, and noninvasive manner and may serve as a novel biomarker for autoimmune dacryoadenitis during the diagnostic evaluation for SS.

Diagnostics and new developments in the treatment of ocular allergies.

About 30% of people suffer from allergic symptoms, and 40% to 80% of them have eye symptoms. Atopic conjunctivitis is divided into seasonal allergic conjunctivitis and perennial allergic conjunctivitis. The treatment of seasonal allergic conjunctivitis is simple: antihistamines, anti-inflammatory agents, or cromoglycate. Perennial allergic conjunctivitis needs longer therapy with mast cell stabilizers and sometimes local steroids. Atopic keratoconjunctivitis requires long-term treatment of the lid eczema and keratoconjunctivitis. Vernal keratoconjunctivitis mainly affects children and young people. It commonly calms down after puberty. It demands intensive therapy, often for many years, to avoid serious complicating corneal ulcers. Giant papillary conjunctivitis is a foreign body reaction in contact lens users or patients with sutures following ocular surgery. Nonallergic eosinophilic conjunctivitis affects mostly middle-aged and older women with eosinophilic conjunctivitis and dry eye. Contact allergic blepharoconjunctivitis is often caused by cosmetics and eye medication. Work-related ocular allergies should be considered as a cause of resistant ocular symptoms in workplaces.

Clinical and immunological responses in ocular demodecosis.

The purpose of this study was to investigate clinical and immunological responses to Demodex on the ocular surface. Thirteen eyes in 10 patients with Demodex blepharitis and chronic ocular surface disorders were included in this study and treated by lid scrubbing with tea tree oil for the eradication of Demodex. We evaluated ocular surface manifestations and Demodex counts, and analyzed IL-1ß, IL-5, IL-7, IL-12, IL-13, IL-17, granulocyte colony-stimulating factor, and macrophage inflammatory protein-1ß in tear samples before and after the treatment. All patients exhibited ocular surface manifestations including corneal nodular opacity, peripheral corneal vascularization, refractory corneal erosion and infiltration, or chronic conjunctival inflammatory signs before treatment. After treatment, Demodex was nearly eradicated, tear concentrations of IL-1ß and IL-17 were significantly reduced and substantial clinical improvement was observed in all patients. In conclusion, we believe that Demodex plays an aggravating role in inflammatory ocular surface disorders.

Xanthogranulomatous variant of immunoglobulin G4 sclerosing disease presenting as ptosis, proptosis and eyelid skin plaques.

A 70-year-old male was referred to the oculoplastic clinic with left-sided ptosis and floppy eyelids. During follow-up, bilateral upper lid xanthelasma developed with worsening ptosis and proptosis, which was worse on the left side. A left orbital biopsy showed xanthogranulomatous inflammation of the orbit. The patient was treated with a variety of immune modulator regimes but due to a variety of side-effects, treatment was discontinued. The left orbit was surgically debulked twice and histology revealed xanthogranulomatous inflammation, with the additional features of sclerosis, lymphoid aggregates and a prominent population of plasma cells. Around 80% of the plasma cells expressed immunoglobulin G4 (IgG4). This case report reveals an association between xanthogranulomatous inflammation of the orbit and a prominent population of IgG4-positive plasma cells. We propose that the overall disease is a novel variant of IgG4 sclerosing disease of the orbit and suggest that cases of histologically proven xanthogranulomatous inflammation should be stained for IgG4 if there is an accompanying plasma cell population.

18ß-glycyrrhetic acid inhibits immune activation triggered by HMGB1, a pro-inflammatory protein found in the tear fluid during conjunctivitis and blepharitis.

PURPOSE: High-mobility group proteins are chromatin-binding factors with key roles in nuclear homeostasis. Evidence indicates that extracellularly released high-mobility group box 1 protein (HMGB1) behaves as a cytokine, promoting inflammation and disease pathogenesis. HMGB1 release occurs during endophtalmitis or uveoretinitis. METHODS: The authors investigated the presence of HMGB1 in tear fluid of patients with different inflammatory disorders of the external eye. RESULTS: Data demonstrate that HMGB1 content is close to detection limit in tears of control subjects but highly increased (about 15-fold) in patients with conjunctivitis or blepharitis. The authors also report that 18ß-glycyrrhetic acid impairs antibody recognition of HMGB1, suggesting direct binding to the protein. Accordingly, 18ß-glycyrrhetic acid prevented HMGB1-dependent COX2 expression and cluster formation in primary cultures of human macrophages. CONCLUSION: Together, these findings suggest that HMGB1 contributes to inflammatory disorders of the external eye, and 18ß-glycyrrhetic acid may scavenge the protein and inhibit its detrimental effects.

Tear cytokines and chemokines in patients with Demodex blepharitis.

PURPOSE: The purpose of the study is to evaluate the causes of inflammation in Demodex-induced blepharitis by analyzing cytokine levels in lacrimal fluid. METHODS: Fifteen Demodex blepharitis patients were selected for assessment of tear cytokine concentrations. Fifteen Demodex-free blepharitis patients and 15 subjects with no ocular symptoms were selected as control groups. Minimally stimulated tear samples (20µl) were collected from each eye and analyzed using a Luminex® 200™ Total System for detection of IL-1ß, IL-5, IL-7, IL-12, IL-13, IL-17, granulocyte colony-stimulating factor (G-CSF), and macrophage inflammatory protein-1 beta (MIP-1ß). RESULTS: The concentration of IL-17 in tears was significantly higher in the Demodex blepharitis group than in the Demodex-free blepharitis group. Tear IL-7 and IL-12 levels show serial increases for these three groups (p<0.05). There were no significant differences in the other cytokines levels between both blepharitis groups. We confirmed that elevated cytokines normalized after treatments. CONCLUSIONS: Infestation of Demodex mites induces change of tear cytokine levels, IL-17 especially, which cause inflammation of the lid margin and ocular surface. These findings might increase our understanding of the mechanism of ocular discomfort and telangiectasias frequently found in Demodex blepharitis patients.

Involvement of STAT4 in IgG subtype switching and ocular HSV-1 replication in mice.

PURPOSE: To assess the relative impact of elevated T-helper 2 (T(H)2)- and reduced T-Helper 1 (T(H)1)-dependent immune responses on ocular herpes simplex virus type 1 (HSV-1) infection. METHODS: Signal transducer and activator of transcription protein 4 knockout mice (BALB/c-STAT4(-/-)) and wild-type BALB/c control mice were immunized with avirulent HSV-1 strain KOS or were mock-immunized. Three weeks after the third immunization, neutralizing antibody titers were determined by plaque reduction assays. Following ocular infection with virulent HSV-1 strain McKrae, viral replication in the eye, blepharitis, corneal scarring (CS), survival, and immunoglobulin (Ig) isotypes in sera were determined. RESULTS: Vaccinated STAT4(-/-) and BALB/c mice contained significant and similar neutralizing antibody titers and were completely protected against HSV-1-induced death and CS. In contrast to vaccinated STAT4(-/-) mice, mock-vaccinated STAT4(-/-) mice had higher ocular HSV-1 titers than mock-vaccinated BALB/c mice on days 2-3 post-ocular infection. There were also significant differences in the levels of IgG2a, IgG2b, and IgG3 in the sera of STAT4(-/-) mice when compared to the control BALB/c mice. CONCLUSIONS: These results suggest that the absence of T(H)1 cytokine responses did alter protection against viral replication and IgG isotypes but not eye disease or survival.

Idiopathic fibroinflammatory disease of the face, eyelids, and periorbital membrane with immunoglobulin G4-positive plasma cells.

Progressive sclerosing orbital pseudotumors are a subset of usually primary and localized idiopathic fibroinflammatory disorders. We report on a 66-year-old man who developed this condition along the facial tissue planes with extension into the orbit and preferential involvement of the periorbital membrane. Fibrocollagenous tissue with scattered lymphoid aggregates without follicle formation dominated the process. There was a light dispersion of B and T lymphocytes and histiocytes in the stroma. Immunoglobulin G4 (IgG4)-positive plasma cells (>35 per high-power field) were identified mostly in the lymphoid clusters, as has been discovered in similar IgG4-related fibrosclerosing conditions of other nonorbital sites. No associated systemic disease emerged during a 20-year clinical course. Previously reported orbital cases of IgG4-positive disease have all involved the lacrimal gland, usually bilaterally, and more closely resembled hypercellular reactive lymphoid hyperplasias with moderate interlobular fibrosis, rather than representing an essentially sclerosing process from the beginning.

A new model of experimental autoimmune keratoconjunctivitis sicca (KCS) induced in Lewis rat by the autoantigen Klk1b22.

PURPOSE: This study was designed to generate an inducible autoimmune model of keratoconjunctivitis sicca (KCS) for study of pathogenesis of the disease. METHODS: Lewis rats were immunized with a mixture of lacrimal and salivary gland extract or recombinant mouse protein kallikrein 1b22 (Klk1b22) emulsified in complete Freund's adjuvant (CFA). For disease induction by adoptive transfer of primed cells, donor rats were received with T-cell blasts. KCS were observed by either clinical signs or histology. RESULTS: The autoantigen Klk1b22, isolated from the lacrimal and salivary glands, readily induced Sjögren's syndrome (SS)-like KCS in the recipients. The diseased animals presented the clinical and pathologic symptoms that resemble related human disease. Most immunized rats showed an increase, then a decrease in tear volume, together with corneal opacity and ocular lesions. Histologic examination revealed that the rats displayed the cardinal signs of primary SS-like KCS, including marked lymphocytic infiltration of the lacrimal and salivary glands and destruction of the acinar cells. Immunofluorescence studies showed that both CD8(+) and CD4(+) T cells were heavily infiltrated, with the former cells predominant in the damaged ducts. Finally, adoptive transfer of Klk1b22-reactive T cells induced more severe disease with earlier onset. CONCLUSIONS: Klk1b22 is an autoantigen for inducing an experimental SS-like KCS in Lewis rats. The availability of this new and reproducible rat model should provide a new and needed tool for studying the pathogenesis of SS.

Loss of MLL5 results in pleiotropic hematopoietic defects, reduced neutrophil immune function, and extreme sensitivity to DNA demethylation.

MLL5 is a divergent member of the Drosophila Trithorax-related (SET) domain and plant homeodomain (PHD) domain-containing chromatin regulators that are involved in the regulation of transcriptional "memory" during differentiation. Human MLL5 is located on chromosome 7q22, which frequently is deleted in myeloid leukemias, suggesting a possible role in hemopoiesis. To address this question, we generated a loss-of-function allele (Mll5(tm1Apa)) in the murine Mll5 locus. Unlike other Mll genes, Mll5(tm1Apa) homozygous mice are viable but display defects in immunity and hematopoiesis. First, Mll5(tm1Apa) homozygous mice show increased susceptibility to spontaneous eye infections, associated with a cell-autonomous impairment of neutrophil function. Second, Mll5(tm1Apa/tm1Apa) mice exhibit a mild impairment of erythropoiesis. Third, Mll5(tm1Apa/tm1Apa) hematopoietic stem cells (HSCs) have impaired competitive repopulating capacity both under normal conditions and when subjected to self-renewal stimulation by NUP98-HOXA10. Fourth, Mll5(tm1Apa) homozygous HSCs show a dramatic sensitivity to DNA demethylation-induced differentiation (5-azadeoxycytidine). Taken together, our data show that MLL5 is involved in terminal myeloid differentiation and the regulation of HSC self-renewal by a mechanism that involves DNA methylation. These data warrant investigation of MLL5 expression levels as a predictive marker of demethylating-agent response in patients with myelodysplastic syndromes and leukemias and identify MLL5 as a key regulator of normal hematopoiesis.

Corneal abnormalities in the NC/Nga mouse: an atopic dermatitis model.

PURPOSE: To study corneal abnormalities in the NC/Nga mouse, which is an animal model of atopic dermatitis. METHODS: To study histopathologic changes of the eyelid, conjunctiva, and cornea, we extracted the eyeballs together with upper and lower eyelids and fixed them for examination by light and electron microscopy or snap-froze them for immunohistochemistry. Transferase mediated-dUTP digoxigenin nick-end labeling staining was performed to detect apoptotic cells. In order to assess eye scratching behavior and the effect of tacroliums hydrate ointment, we made video recordings. RESULTS: Mice kept in a conventional room suffered from various grades of blepharoconjunctivitis and scratched their eyes furiously. Tacrolimus hydrate ointment reduced their eye-scratching behavior. Histopathologic study of the eyelid and conjunctiva showed that this blepharoconjunctivitis was caused by allergic inflammation. Mice with severe blepharoconjunctivitis showed thinning of the corneal epithelium, an irregular interface between the epithelium and stroma, subepithelial deposition of materials, and neovascularization of the stroma. Their corneas were cone shaped. Many transferase mediated-dUTP digoxigenin nick-end labeling-positive cells were recognized among superficial epithelial cells and keratocytes. CONCLUSIONS: NC/Nga mice are a useful animal model of atopic blepharoconjunctivitis. Various corneal disorders in these mice may depend on their eye-scratching behavior.