ovalbumin and Conjunctivitis--Allergic

Allergic conjunctivitis (AC) is a common allergic condition worldwide that requires accurate screening and early diagnosis. We found that gp130 is essential for AC, as gp130 levels are elevated in AC. Therefore, this study aimed to elucidate the functions and the possible underlying mechanisms of gp130 in AC.. To compare mRNA expression profiles, the conjunctival tissues of BALB/c mice with ovalbumin (OVA)-induced AC were subjected to RNA-sequencing (RNA-seq) analysis followed by bioinformatic analysis. A nonrandomized study involving 57 patients with AC and 24 sex- and age-matched healthy individuals was conducted. A protein chip was used to detect cytokine levels in patient tears. Differentially expressed proteins in patient serum were detected using label-free quantitative mass spectrometry. Histamine-stimulated conjunctival epithelial cells (HConEpiCs) were used to construct a cell model. LMT-28 which can inhibit gp130 phosphorylation was dropped onto the murine ocular surface, and the resulting symptoms were observed.. Gp130 is upregulated in the conjunctival tissues of OVA-induced mice, the serum and tears of patients, and the histamine-stimulated HConEpiCs. Signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) were upregulated in the conjunctival tissues of mice with OVA-induced AC and in HConEpiCs. Ocular surface inflammation was significantly relieved in LMT-28-treated mice. The expression of IgE, IL-4, IL-5, and IL-13 in serum of LMT-28-treated mice decreased. The number of mast cells in conjunctival tissue was decreased compared with OVA-induced mice.. Gp130 may play an important role in AC via the gp130/JAK2/STAT3 pathway. Inhibiting gp130 phosphorylation alleviates ocular surface inflammation in mice, presenting a potential treatment approach for AC.

Topics: Animals; Conjunctivitis, Allergic; Cytokine Receptor gp130; Histamine; Inflammation; Janus Kinase 2; Mice; Ovalbumin; STAT3 Transcription Factor

To explore the role of Th2 signaling pathway in allergic conjunctivitis (AC).. Serum Th2 cytokines IL-4 or IL-13 of patients with AC were detected using the Meso scale discovery assay to verify the correlation of Th2 immunity and AC pathogenesis. Wistar Han rats were intraperitoneally and subcutaneously injected with ovalbumin (OVA) to establish an experimental AC model and the Th2 signaling pathway was blocked by an investigational neutralizing antibody (CM310). Serum IgE and OVA-specific IgE were detected by ELISA. Conjunctivitis inflammation, infiltration of eosinophils, and mast cell degranulation were detected by histological examination. Immortalized human conjunctival epithelial cells, a conjunctival epithelial cell line, and peripheral blood mononuclear cells of patients with AC were used as the target cells to study the impact of IL-4 or IL-13 on AC progression. Finally, a STAT6 reporter gene system was constructed using immortalized human conjunctival epithelial cells to confirm whether the downstream signaling pathway activated by IL-4 or IL-13.. Serum IL-4 or IL-13 were increased in patients with AC versus healthy individuals. In an OVA-induced rat experimental AC model, blocking the Th2 signaling pathway with CM310, an investigational neutralizing antibody, alleviated the conjunctival symptoms, and decreased serum IgE, suppressed infiltration of eosinophils and mast cell degranulation. Further, an in vitro model showed CM310 suppressed the secretion of inflammatory cytokine from both immune cells and epithelial cells in both patients peripheral blood mononuclear cells and cell line.. Blocking Th2 signaling pathway alleviates the clinical symptoms and inflammation in AC.

Topics: Animals; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Humans; Immunoglobulin E; Inflammation; Interleukin-13; Interleukin-4; Leukocytes, Mononuclear; Mice; Mice, Inbred BALB C; Ovalbumin; Rats; Rats, Wistar; Signal Transduction; Th2 Cells

Allergic conjunctivitis (AC) resulting from conjunctival reactive inflammation is a common ocular surface disease. Quercetin is known for its anti-allergic properties but its effects on conjunctivitis are less well understood.. In this study, we evaluated the anti-allergic effects of quercetin in animal models of conjunctivitis, and explored its molecular mechanism(s) of action in cultured human mast cells (MCs).. Quercetin inhibited the ovalbumin (OVA) induced expression of IgE, HA, IL-4, TNF-α and substance-P in the peripheral blood of AC mouse models. Quercetin also attenuated OVA induced MC degranulation, eosinophil number, substance P concentrations, and mRNA IL-4/TNF-α expression in the conjunctival tissue of AC models. In vitro analysis showed that quercetin reduced DNP-HSA/IgE induced calcium (Ca2+) influx, and suppressed degranulation and chemokine release in LAD2 cells (human primary mast cell). Quercetin also inhibited DNP-HSA/IgE induced Lyn/PLCγ/IP3R-Ca2+ activation, Lyn/ERK1/2 signaling, and Lyn/NF-κB activation in LAD2 cells, all of which promote inflammation. When added alone, quercetin had no effect on PLCγ1 phosphorylation or expression, but potently inhibited Lyn and phosphorylation-Lyn. Quercetin (200 μM) and Lyn inhibitors (Bafetinib, 10 μM) inhibit the activity of Lyn kinase, and quercetin can reduce the activation of Lyn kinase by Lyn agonist (Tolimidone, 10 μM). These data can be preliminarily determined that quercetin can inhibit allergic conjunctivitis as a Lyn kinase inhibitor.

Topics: Animals; Anti-Allergic Agents; Cell Line; Chemokines; Conjunctivitis, Allergic; Eosinophils; Humans; Immunoglobulin E; Male; Mast Cells; Mice, Inbred C57BL; Ovalbumin; Protein Kinase Inhibitors; Quercetin; Signal Transduction; Skin; src-Family Kinases

Allergic conjunctivitis (AC), a common eye inflammation that affects patients' health and quality of life, is still a therapeutic challenge for ophthalmologists. Tofacitinib, a new Janus kinase (JAK) inhibitor, has been successfully used in the treatment of several disorders. Nonetheless, its effect in AC and the potential anti-allergic mechanisms are still unclear. The objective of the current study was to explore the roles of tofacitinib in preventing AC and elucidate the potential underlying mechanisms.. Tofacitinib was used topically in BALB/c mice with experimental allergic conjunctivitis (EAC). Ocular allergic symptoms and biological modifications were examined. To assess the anti-allergic mechanisms of tofacitinib, RBL-2H3 cells and HUVECs were cultured in vitro. The inhibitory effects and mechanisms of tofacitinib were studied and measured by real-time quantitative PCR, ELISA, western blot analysis, and flow cytometry.. Topical administration of tofacitinib reduced the clinical symptoms of OVA-induced EAC, with a substantial mitigation in inflammatory cell infiltration, histamine release, and TNF-α mRNA as well as IL-4 mRNA expression. In vitro, tofacitinib repressed the degranulation and cytokine production in RBL-2H3 cells and reduced histamine-induced vascular hyperpermeability. The underlying mechanism might involve the downregulation of phosphorylation of JAK3/STATs signaling molecules in RBL-2H3 cells and HUVECs.. Our findings provide evidence that tofacitinib prevented EAC by targeting the JAK3/STATs pathway. We recommend the use of tofacitinib as an innovative approach for the treatment of AC.

Topics: Allergens; Animals; Anti-Allergic Agents; Cell Degranulation; Cell Line; Conjunctivitis, Allergic; Disease Models, Animal; Female; Humans; Immunosuppression Therapy; Mast Cells; Mice; Mice, Inbred BALB C; Ovalbumin; Piperidines; Pyrimidines; Rats; Signal Transduction; Tumor Necrosis Factor-alpha

We previously reported that the nonsteroidal compound CpdX, which was initially characterized 20 y ago as a possible gestagen and, shortly afterward, as a possible drug for treatments of inflammatory diseases, selectively triggers the NFκB/AP1-mediated tethered indirect transrepression function of the glucocorticoid receptor (GR), and could therefore be a selective glucocorticoid receptor agonistic modulator (SEGRAM). We now demonstrate that, upon administration to the mouse, CpdX and one of its deuterated derivatives, CpdX-D3, repress as efficiently as a synthetic glucocorticoid (e.g., Dexamethasone) an induced skin atopic dermatitis, an induced psoriasis-like inflammation, a house dust mite (HDM)-induced asthma-like allergic lung inflammation, a collagen-induced arthritis, an induced ulcerative colitis, and an ovalbumin-induced allergic conjunctivitis. Interestingly, in the cases of an HDM-induced asthma-like allergic lung inflammation and of a collagen-induced arthritis, the CpdX antiinflammatory activity was selectively exerted by one of the two CpdX enantiomers, namely, CpdX(eA) or CpdX-D3(eA).

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Asthma; Conjunctivitis, Allergic; Dermatitis, Atopic; Dexamethasone; Disease Models, Animal; Glucocorticoids; Humans; Inflammation; Mice; NF-kappa B; Ovalbumin; Progestins; Receptors, Glucocorticoid; Skin; Transcriptional Activation

Myopia is a highly prevalent eye disease. There is limited information suggesting a relationship between myopia and inflammation. We found children with allergic conjunctivitis (AC) had the highest adjusted odds ratio (1.75, 95% confidence interval [CI], 1.72-1.77) for myopia among the four allergic diseases. A cohort study was conducted and confirmed that children with AC had a higher incidence and subsequent risk of myopia (hazard ratio 2.35, 95%CI 2.29-2.40) compared to those without AC. Lower refractive error and longer axial length were observed in an AC animal model. Myopia progression was enhanced by tumor necrosis factor (TNF)-α or interleukin (IL)-6 administration, two cytokines secreted by mast cell degranulation. The TNF-α or IL-6 weakened the tight junction formed by corneal epithelial (CEP) cells and inflammatory cytokines across the layer of CEP cells, which increased the levels of TNF-α, IL-6, and IL-8 secreted by retinal pigment epithelial cells. The expression levels of TNF-α, IL-6, IL-8, monocyte chemoattractant protein-1, and nuclear factor kappa B were up-regulated in eyes with AC, whereas IL-10 and the inhibitor of kappa B were down-regulated. In conclusion, the experimental findings in mice corroborate the epidemiological data showing that allergic inflammation influences the development of myopia.

Topics: Animals; Child; Cohort Studies; Conjunctivitis, Allergic; Cornea; Demography; Disease Progression; Epithelial Cells; Female; Humans; Incidence; Inflammation; Interleukin-6; Male; Models, Biological; Myopia; Ovalbumin; Proportional Hazards Models; Rats, Inbred Lew; Retina; Risk Factors; Tumor Necrosis Factor-alpha

Allergic conjunctivitis is mediated by eosinophilic infiltration and Th2 type immune responses. This study aims to elucidate the role of rapamycin, mTOR inhibitor, on OVA-induced experimental allergic conjunctivitis (EAC). Rapamycin administration intraperitoneally markedly reduced clinical signs, total and OVA-specific IgE and IgG1/G2a ratio in serum, and conjunctival eosinophilic infiltration. Infiltrations of CD11c

Topics: Animals; Conjunctiva; Conjunctivitis, Allergic; Female; Gene Expression; Immunoglobulin E; Immunoglobulin G; Immunosuppressive Agents; Mice, Inbred BALB C; Ovalbumin; Sirolimus; Th2 Cells

To evaluate galectin-3 (Gal-3), a β-galactoside binding protein, as a possible biomarker in ocular allergy and further investigated the role of endogenous Gal-3 in a murine model of ovalbumin (OVA)-induced allergic conjunctivitis (AC).. Conjunctival impression cytology specimens from control and patients with severe vernal keratoconjunctivitis, treated or untreated, were used to evaluate Gal-3 expression by immunocytochemistry. To investigate the mechanism of action of Gal-3, OVA-immunised BALB/c male wild-type (WT) and Gal-3 null (Gal-3. Patients with AC and OVA-sensitised WT mice exhibited increased levels of Gal-3 in the conjunctiva compared with control, an effect reverted by the action of Dex and TC therapy. Twenty-four hours after the final OVA challenge, total and anti-OVA IgE levels increased significantly in the blood of OVA-sensitised WT and Gal-3. Gal-3 contributes to the pathogenesis of ocular allergy and represents a relevant therapeutic target.

Topics: Adolescent; Adult; Animals; Anti-Allergic Agents; Biomarkers; Blood Proteins; Blotting, Western; Child; Conjunctivitis, Allergic; Disease Models, Animal; Drug Therapy, Combination; Female; Galectin 3; Galectins; Glucocorticoids; Humans; Immunoenzyme Techniques; Immunoglobulin E; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Tacrolimus

To determine the immunologic functions of TRPA1 or TRPV1 in allergic conjunctivitis (AC).. Mice were sensitized with ovalbumin (OVA), after which TRPA1 antagonist or TRPV1 antagonist was administered before topical OVA challenge. Expression of TRPV1 or TRPA1 in AC was examined by western blotting and multicolor immunofluorescence. Clinical signs, OVA-specific IgE, infiltration of inflammatory cells into conjunctivae (CJs), and Th2 cytokine in draining lymph nodes (LNs) were evaluated by microscopy, flow cytometry, and ELISA.. TRPV1 expression was increased in CJs and LNs from AC mice, but TRPA1 expression was only increased in LNs. TRPV1 antagonist but not TRPA1 antagonist attenuated the clinical signs of AC and OVA-specific IgE in sera. TRPV1 antagonist furthermore inhibited the infiltration of inflammatory cells into CJ and the production of Th2 cytokines in LNs.. TRPV1 antagonist but not TRPA1 antagonist may ameliorate AC by suppressing the Th2 response in LNs.

Topics: Acetanilides; Animals; Blotting, Western; Capsaicin; Conjunctivitis, Allergic; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eosinophils; Fluorescent Antibody Technique, Indirect; Immunoglobulin E; Male; Mast Cells; Mice; Mice, Inbred BALB C; Ovalbumin; Purines; TRPA1 Cation Channel; TRPV Cation Channels

Allergic conjunctivitis is an inflammatory eye disease mediated by Th2 type immune response. The role of extracellular superoxide dismutase 3 (SOD3) in immune response and allergic conjunctival inflammation was examined in a murine model for experimental allergic conjunctivitis (EAC). Allergic conjunctivitis was induced in mice by allergen challenge with ovalbumin in alum via the conjunctival sac. SOD3 was topically applied and allergy indicators were compared. Clinical signs associated with conjunctivitis, such as OVA-specific IgE production, IgG1/G2a ratio and eosinophil infiltration, were drastically reduced in mice treated with SOD3. They also had less dendritic cells and CD4

Topics: Allergens; Animals; Conjunctiva; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Eosinophils; Female; Immunoglobulin E; Immunoglobulin G; Inflammation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Superoxide Dismutase; Th2 Cells

Allergic conjunctivitis (AC) is an immunoglobulin E (IgE)-mediated and helper T cell 2 (Th2)--cell-mediated disease characterized by conjunctival eosinophilic infiltration. Previous study shows that IL-28A had anti-allergic activity in airway disease. In this study, we examined the effect of IL-28A on a mouse model of ovalbumin (OVA)-induced experimental allergic conjunctivitis (EAC).. Mouse EAC was induced by topical application of OVA after intraperitoneal (IP) sensitization with OVA in aluminum hydroxide (ALUM). Interleukin-28A was administered 1 hour before OVA challenge. Allergic conjunctivitis symptoms, eosinophil infiltration in the conjunctiva, antigen-specific IgE in the serum, and Th2 cytokine production by lymph node cells and splenocytes were subsequently analyzed.. Topical application of IL-28A to OVA-induced EAC reduced clinical symptoms, serum OVA-specific IgE, and the infiltration of eosinophils in the conjunctiva. In addition, topical administration of IL-28A suppressed the expression of IL-4, IL-5, and IL-13 (Th2-type cytokine) but promoted the expression of IFN-γ (Th1-type cytokine) by splenocytes and cervical lymph node cells in EAC mice. Immunofluorescence staining showed decrease expression of IL-4 and IL-5 in IL-28A-treated EAC conjunctiva.. Interleukin-28A shows therapeutic potential for allergic conjunctival inflammation.

Topics: Administration, Topical; Animals; Antibodies, Anti-Idiotypic; Cells, Cultured; Conjunctiva; Conjunctivitis, Allergic; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eosinophils; Female; Immunoglobulin E; Immunohistochemistry; Interleukins; Mice; Mice, Inbred BALB C; Ovalbumin; T-Lymphocytes

The aim of the current study was to investigate whether olopatadine and naphazoline hydrochloride reduce allergic conjunctivitis in mice through alterations in inflammation, NGF and VEGF. An allergic conjunctivitis mouse model was established using histamine or an antigen (ovalbumin), following which mice were treated with 1% olopatadine solution and/or 0.2 mg/ml of naphazoline hydrochloride. Histamine or antigen‑induced conjunctival vascular hyperpermeability was examined and the levels of inflammatory factors, cytokines, IgE, GMCSF and NGF were analyzed using ELISA in antigen‑induced conjunctival vascular hyperpermeability mice. In addition, VEGF protein expression was measured using western blotting in antigen‑induced mice. The results indicated that olopatadine and naphazoline hydrochloride significantly suppressed conjunctival dye leakage in mice with histamine or antigen‑induced conjunctival vascular hyperpermeability. In addition, treatment with olopatadine and naphazoline hydrochloride was able to reduce the levels of inflammatory factors (TNF‑α, IL‑1β and IL‑6), cytokines (IFN‑γ and IL‑4), IgE, GMCSF, and NGF in antigen‑induced conjunctival vascular hyperpermeability mice. The protein expression levels of VEGF in antigen‑induced conjunctival vascular hyperpermeability mice were reduced following treatment with olopatadine and naphazoline hydrochloride. These results suggest that treatment with olopatadine and naphazoline hydrochloride reduces conjunctivitis in mice via effects on inflammation, NGF and VEGF.

Topics: Animals; Blotting, Western; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Enzyme-Linked Immunosorbent Assay; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Histamine; Immunoglobulin E; Mice; Mice, Inbred BALB C; Naphazoline; Nerve Growth Factor; Olopatadine Hydrochloride; Ovalbumin; Vascular Endothelial Growth Factor A

Annexin A1 (ANXA1), a 37 kDa glucocorticoid-regulated protein, is a potent anti-inflammatory mediator effective in terminating acute inflammatory response, and its role in allergic settings has been poorly studied. The aim of this investigation was to evaluate the mechanism of action of ANXA1 in intraocular inflammation using a classical model of ovalbumin (OVA)-induced allergic conjunctivitis (AC). OVA-immunised Balb/c mice, wild-type (WT) and ANXA1-deficient (AnxA1(-/-)), were challenged with eye drops containing OVA on days 14-16 with a subset of WT animals pretreated intraperitoneally with the peptide Ac2-26 (N-terminal region of ANXA1) or dexamethasone (DEX). After 24 h of the last ocular challenge, WT mice treated with Ac2-26 and DEX had significantly reduced clinical signs of conjunctivitis (chemosis, conjunctival hyperaemia, lid oedema and tearing), plasma IgE levels, leukocyte (eosinophil and neutrophil) influx and mast cell degranulation in the conjunctiva compared to WT controls. These anti-inflammatory effects of DEX were associated with high endogenous levels of ANXA1 in the ocular tissues as detected by immunohistochemistry. Additionally, Ac2-26 administration was effective to reduce IL-2, IL-4, IL-10, IL-13, eotaxin and RANTES in the eye and lymph nodes compared to untreated WT animals. The lack of ANXA1 produced an exacerbated allergic response as detected by the density of the inflammatory cell influx to the conjunctiva and the cytokine/chemokine release. These different effects observed for Ac2-26 were correlated with diminished level of activated ERK at 24 h in the ocular tissues compared to untreated OVA group. Our findings demonstrate the protective effect of ANXA1 during the inflammatory allergic response suggesting this protein as a potential target for new ocular inflammation therapies.

Topics: Animals; Annexin A1; Blotting, Western; Conjunctivitis, Allergic; Cytokines; Dexamethasone; Disease Models, Animal; Eosinophils; Glucocorticoids; Immunoenzyme Techniques; Immunoglobulin E; Lymph Nodes; Male; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Peptides

Allergic conjunctivitis from an allergen-driven Th2 response is characterized by conjunctival eosinophilic infiltration. Although CCL20-CCR6 axis has been reported to play a proinflammatory role in several murine models of autoimmune diseases including allergic diseases, their underlying mechanism needs to be investigated. We here examined whether CCL20-CCR6 axis could play a role in the development of allergic conjunctival inflammation using murine experimental allergic conjunctivitis (EAC) model induced by ovalbumin (OVA) allergen. Mice were challenged with consecutive 10days of OVA via conjunctival sac after systemic challenge with OVA and cholera toxin in alum. Several indicators for allergy were comparatively evaluated in wild-type and CCR6 KO EAC mice. Wild-type mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. The absence of CCR6 suppressed IgE secretion and allergic conjunctival inflammation. Reduced allergic inflammation was ascribable to reduced cytokine responses from Th-2 type in draining lymph node although Th17, regulatory T cells and dendritic cell subsets are not affected by the absence of CCR6. In addition, neutralization of CCR6 ligand, CCL20 could repress allergic conjunctival inflammation. Our findings suggested that CCR6 might be crucial for optimal development of Th2 immune responses and further allergic conjunctival inflammation in EAC model.

Topics: Allergens; Alum Compounds; Animals; Chemokine CCL20; Chemokines, CC; Cholera Toxin; Conjunctivitis, Allergic; Cytokines; Dendritic Cells; Disease Models, Animal; Female; Immunoglobulin E; Inflammation; Lymph Nodes; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, CCR6; T-Lymphocytes, Regulatory; Th17 Cells; Th2 Cells

To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis.. Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure.. Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells.. Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.

Topics: Administration, Topical; Animals; Benzalkonium Compounds; Blotting, Western; Cell Line; Coculture Techniques; Conjunctiva; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Flow Cytometry; Hypersensitivity, Delayed; I-kappa B Proteins; Immune Tolerance; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Microscopy, Confocal; NF-kappa B; NF-KappaB Inhibitor alpha; Ovalbumin; Preservatives, Pharmaceutical; T-Lymphocytes

Acute allergic conjunctivitis is a constantly challenging condition that often requires steroids for effective management. Alternative treatment options are needed due to the potential side effects of steroids. Tacrolimus has been used for vernal/atopic conjunctivitis. The aim of our study was to investigate the therapeutic effect of topical administration of 0.03 % tacrolimus (eye drops or ointment) in comparison to 0.1 % dexamethasone in a mouse model of acute allergic conjunctivitis.. BALB/c mice were sensitized by an intraperitoneal injection of 10 μg/0.2 ml ovalbumin (OVA) absorbed on ALUM (2.0 mg) on days 1 and 8. They were challenged by topical instillation of 2 μl of 15 % OVA (absorbed in 10 % glycerol) twice daily, on days 15-21. Treatment was administered twice daily on days 17-21. Mice were randomly assigned topical treatment groups: Group 1, 0.1 % dexamethasone drops; Group 2, 0.03 % tacrolimus drops; Group 3, 0.03 % tacrolimus ointment; Group 4 PBS drops (control). On day 22 all mice underwent clinical evaluation, blood sampling for IgE levels, and conjunctivas were removed for eosinophil counting.. IgE and OVA-specific IgE levels were similar among all groups, demonstrating induction of allergic reaction in all mice. Significantly lower clinical scores were found among all treated groups as compared to controls (P < 0.001), while no significant difference was found among the three treatment groups (P > 0.05). Conjunctival eosinophil counts were significantly lower in Group 1 (P < 0.05) as compared to the other groups.. The clinical efficacy of topical 0.03 % tacrolimus was similar to 0.1 % dexamethasone for acute allergic conjunctivitis.

Topics: Acute Disease; Administration, Topical; Animals; Conjunctivitis, Allergic; Dexamethasone; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Glucocorticoids; Immunoglobulin E; Immunosuppressive Agents; Injections, Intraperitoneal; Mice; Ophthalmic Solutions; Ovalbumin; Tacrolimus; Treatment Outcome

To investigate the effect of Ag doses and administration time points on oral tolerance induction in experimental allergic conjunctivitis (EAC).. BALB/c mice were actively sensitised twice with ovalbumin (OVA) in alum, and then challenged twice with OVA in eye drops. Twenty minutes after the last challenge, the clinical appearance was evaluated. Twenty-four hours later, the conjunctivas, spleens and blood were collected for histological analyses, cytokine production assays, and measurement of serum Ig levels, respectively. The mice were fed with a high or low dose of OVA before sensitisation (prophylactic treatment). To assess the effect of therapeutic treatment, the high-dose Ag was administered after sensitisation. Control groups received phosphate-buffered saline without OVA. To determine the role of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in prophylactic low-dose oral tolerance, mice were injected intraperitoneally with neutralising antibodies during the entire experimental period. To assess the role of regulatory T cells, neonatally thymectomised mice were injected with depleting anti-CD25 monoclonal antibodies before the low-dose prophylactic treatment.. OVA-feeding was suppressive even when the mice were treated soon after Ag sensitisation. Both the low and high doses of oral OVA suppressed EAC. High-dose treatment significantly suppressed EAC-related cytokine production and serum Ig levels. IL-10 and TGF-β were less likely to be involved in prophylactic low-dose oral tolerance induction but regulatory T cells played a role.. Prophylactic and early therapeutic treatment of OVA feeding suppressed EAC. Both high and low doses of oral OVA induced oral tolerance but with different mechanisms.

Topics: Administration, Oral; Allergens; Animals; Antibodies, Neutralizing; Conjunctivitis, Allergic; Cytokines; Enzyme-Linked Immunosorbent Assay; Immune Tolerance; Immunoglobulin E; Immunoglobulin G; Injections, Intraperitoneal; Interleukin-2 Receptor alpha Subunit; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Ovalbumin; T-Lymphocytes, Regulatory; Thymectomy; Time Factors

Allergic conjunctivitis is characterized by itchy, watery and swollen eyes which occur in response to exposure to seasonal or environmental allergens. The early phase reaction of allergic conjunctivitis is primarily mediated by mast cell degranulation while the late phase reaction is driven by Th2 cells and eosinophils. Prostaglandin D(2) (PGD(2)), released from mast cells, is present in allergic conjunctival tears and may elicit classical allergic responses via interaction with the high-affinity DP2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells, CRTh2). Furthermore, antagonism of this receptor is well known to inhibit eosinophil chemotaxis, basophil activation and Th2 cytokine production. PGD(2), therefore, may be involved in both early and late phase reactions in response to allergen challenge.. Thus, we explored whether our novel and selective DP2 antagonist AM156 would be efficacious in animal models of allergic conjunctivitis. Furthermore, as respiratory syncytial virus (RSV) has been implicated in the pathogenesis of allergic conjunctivitis, we examined the effects of DP2 antagonism in a murine model of RSV ocular infection.. Utilizing a guinea pig ovalbumin model and a murine ragweed model we demonstrated that AM156 reduces redness, discharge and swelling in response to allergen challenge. These effects were equal to or greater than those of current clinical treatment options for allergic conjunctivitis including topical corticosteroids and a dual-mechanism antihistamine and decongestant. AM156 significantly reduced RSV-induced ocular inflammation and IL-4 production.. These results suggest that a topical DP2 antagonist such as AM156 may represent a novel therapeutic for allergic conjunctivitis.

Topics: Administration, Topical; Allergens; Ambrosia; Animals; Anti-Allergic Agents; Benzylamines; Conjunctivitis, Allergic; Conjunctivitis, Viral; Disease Models, Animal; Female; Guinea Pigs; Interleukin-4; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Receptors, Immunologic; Receptors, Prostaglandin; Respiratory Syncytial Virus Infections

CCR7 plays a key role in mobilizing tissue dendritic cells (DCs) to the lymphoid compartment for consequent elicitation of adaptive immunity. Interfering with CCR7 function therapeutically would therefore be anticipated to inhibit the progression of atopic conditions, for example, allergic conjunctivitis (AC). However, the CCR7-CCL19/CCL21 system in the ocular surface is poorly understood as is the precise role of DCs in AC immunopathogenesis. T cells from ovalbumin (OVA)-primed mice were adoptively transferred into wild-type (WT) hosts. Exogenous WT (eGFP(+)) versus CCR7(-/-) DCs were engrafted subconjunctivally (SCJ), and hosts were challenged with OVA (Texas-Red+) eye drops. AC immunopathogenesis was evaluated via clinical examinations, infiltration of mast cells and eosinophils, Th2 reactivity, and serum IgE levels. AC was also assessed in actively immunized mice challenged with OVA eye drops containing 1% anti-CCR7 antibody or isotype control. In eye-draining lymph nodes (LNs), OVA(+) SCJ engrafted WT DCs conferred upregulated CCR7 and caused augmentation of clinical signs. This result was corroborated by increased conjunctival infiltration, Th2 cytokines in LNs, and serum OVA-specific IgE. Strikingly, this was completely reversed with SCJ engrafted CCR7(-/-) DCs in all parameters tested. Furthermore, topical antibody blockade of CCR7 in actively immunized mice significantly inhibited AC. Ocular surface DCs via CCR7 expression contribute to the immunopathogenesis of AC, thereby allowing significant inhibition of this experimental condition via topical CCR7 antibody blockade.

Topics: Adoptive Transfer; Allergens; Animals; Antibodies, Monoclonal; Conjunctiva; Conjunctivitis, Allergic; Dendritic Cells; Immunoglobulin E; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Ophthalmic Solutions; Ovalbumin; Receptors, CCR7; T-Lymphocytes; Th2 Cells; Up-Regulation

Allergic conjunctivitis (AC) from an allergen-driven T helper 2 (Th2) response is characterized by conjunctival eosinophilic infiltration. Because curcumin has shown anti-allergic activity in an asthma and contact dermatitis laboratory models, we examined whether administration of curcumin could affect the severity of AC and modify the immune response to ovalbumin (OVA) allergen in an experimental AC model.. Mice were challenged with two doses of topical OVA via the conjunctival sac after systemic sensitization with OVA in aluminum hydroxide (ALUM). Curcumin was administered 1 h before OVA challenge. Several indicators for allergy such as serum immunoglubulin E (IgE) antibodies production, eosinophil infiltration into the conjunctiva and Th2 cytokine production were evaluated in mice with or without curcumin treatment.. Mice challenged with OVA via the conjunctival sac following systemic sensitization with OVA in ALUM had severe AC. Curcumin administration markedly suppressed IgE-mediated and eosinophil-dependent conjunctival inflammation. In addition, mice administered curcumin had less interleukin-4 (IL-4) and interleukin-5 (IL-5) (Th2 type cytokine) production in conjunctiva, spleen, and cervical lymph nodes than mice in the non-curcumin-administered group. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS), and curcumin treatment inhibited iNOS production in the conjunctiva.. We believe our findings are the first to demonstrate that curcumin treatment suppresses allergic conjunctival inflammation in an experimental AC model.

Topics: Allergens; Alum Compounds; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Movement; Conjunctiva; Conjunctivitis, Allergic; Curcumin; Eosinophils; Female; Hypersensitivity, Immediate; Immunoglobulin E; Injections, Intraperitoneal; Interleukin-4; Interleukin-5; Lymph Nodes; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase Type II; Ovalbumin; Spleen

Allergic conjunctivitis from an allergen-driven T helper type 2 (Th2) response is characterized by conjunctival eosinophilic infiltration. Association between signalling through Toll-like receptor 4 (TLR-4) and adaptive immune responses has been observed in allergic airway disease. We examined whether administration of bacterial lipopolysaccharide (LPS), a prototypic bacterial product that activates immune cells via TLR-4, could affect the development of allergic conjunctivitis and modify the immune response to ovalbumin (OVA) allergen in an experimental allergic conjunctivitis (EAC) model. Mice were challenged with two doses of OVA via conjunctival sac after systemic challenge with OVA in alum. Several indicators for allergy were evaluated in wild-type and TLR-4(-/-) mice with or without adding of different doses of LPS into OVA in alum. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, LPS administration markedly suppressed immunoglobulin (Ig)E-mediated and eosinophil-dependent conjunctival inflammation. In addition, mice sensitized with OVA plus LPS had less interleukin (IL)-4, IL-5 and eotaxin secretion than mice sensitized with OVA only. The suppression of allergic response by LPS administration was due to Th1 shift. In contrast, the presence of LPS during sensitization with OVA had no effect on severity of allergic conjunctivitis and Th2 responses in TLR4-4(-/-) mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses via the TLR-4-dependent pathway in the EAC model.

Topics: Administration, Topical; Allergens; Animals; Cells, Cultured; Chemokine CCL11; Conjunctivitis, Allergic; Disease Models, Animal; Female; Immunoglobulin E; Injections, Intraperitoneal; Interleukin-4; Interleukin-5; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Knockout; Models, Immunological; Ovalbumin; Specific Pathogen-Free Organisms; Th1 Cells; Th2 Cells; Toll-Like Receptor 4

Thymoquinone (TQ) is an active and potent compound in the oil of Nigella sativa, which has anti-inflammatory properties. The aim of this study was to evaluate the effects of TQ on ovalbumin (OVA)-induced allergic conjunctivitis (AC) in Balb/c.. The mice were divided into seven experimental groups; PBS Con, OVA Con, Conj, 0.05%TQ, 0.1%TQ, 0.5%TQ and Dex group. The mice were immunized and exposed to OVA, and eyes were treated with TQ and dexamethasone. Ocular symptoms were observed after last exposure to OVA. Eosinophils count in blood and Ophthalmic lavage fluid (OLF), recruitment of inflammatory cells in conjunctiva, serum IgE and OVA-specific IgE were evaluated by Giemsa and HE staining, and Enzyme-Linked Immunosorbent Assay (ELISA) respectively. High Performance Liquid Chromatography (HPLC) was used to determine the histamine level in OLF. The mRNA expression and protein level of cytokines were examined by real time RT-PCR and ELISA respectively.. Ocular symptoms of AC and other characteristics of allergic inflammation including IgE and OVA-specific immunoglobulin E (IgE) level, recruitment of eosinophils, histamine level, mRNA expressions and protein level of cytokines were remarkably increased in OVA-exposed mice compared with the control groups. Administration of TQ suppressed the ocular symptoms, inflammatory cell infiltration in conjunctiva, blood and OLF, increased level of serum IgE and OVA-specific IgE, and OLF histamine level in OVA-exposed mice. Furthermore, TQ abrogated the mRNA expression and serum level of interleukin including 1L-4, IL-5, IL-13 and transforming growth factor beta (TGF-β) in mice immunized and exposed to OVA.. Administration of TQ significantly reduced the ocular symptoms in AC by attenuating the recruitment of eosinophils, level of IgE, histamine and cytokines. Our findings suggest that TQ might be a useful intimation for the treatment and future research for AC.

Topics: Animals; Benzoquinones; Chromatography, High Pressure Liquid; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eosinophils; Histamine; Immunoglobulin E; Leukocyte Count; Mice; Mice, Inbred BALB C; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We evaluated the use of immunological biomarkers in transconjunctival immunotherapy by using cholera toxin B for treating experimental allergic conjunctivitis in a mouse model.. Balb/c mice were sensitized using intraperitoneal injections of ovalbumin (OVA) and were then divided into two groups. The first group was treated by topical instillation of OVA after the instillation of combined OVA and cholera toxin B (CTB) solution B group). The second group was treated by topical instillation of OVA alone (allergy group). The control group consisted of nonsensitized mice undergoing topical OVA instillation only. The numbers of eosinophils and CD4-positive lymphocytes in the conjunctiva were determined histologically, and the observation of immunoglobulin A (IgA)-positive cells in the conjunctiva was performed by immunohistochemistry. Cytokine concentration in the conjunctiva was determined by the protein-array methods. Messenger RNA expression of T-cell-specific markers, such as T-bet, GATA-3, and Foxp3, in the conjunctiva was detected by reversed transcriptase polymerase chain reaction.. The number of eosinophils and CD4-positive lymphocytes increased significantly in the allergy group compared with the control group (P < 0.001) but showed no difference between the CTB group and control group. Concentrations of interleukin 4 (IL-4) (P < 0.05), B-lymphocyte chemoattractant (P < 0.01), and thymus-expressed chemokine (P < 0.05) in the conjunctiva were significantly higher in the CTB group than in the other two groups. GATA-3 messenger RNA (mRNA) in the conjunctiva was expressed in the three groups, but T-bet and Foxp3 were not detected.. Transconjunctival immunotherapy using CTB can be evaluated by histological examination of eosinophils and CD4-positive T cells, and a mucosal immunity-associated chemokine and a helper T-cell-17-associated chemokine as biomarkers.

Topics: Animals; Biomarkers; CD4-Positive T-Lymphocytes; Cell Count; Cholera Toxin; Conjunctiva; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Eosinophils; Forkhead Transcription Factors; GATA3 Transcription Factor; Immunoglobulin A, Secretory; Immunotherapy; Leukocyte Count; Mice; Mice, Inbred BALB C; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Box Domain Proteins

Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action.. With in vitro studies apoptosis was evaluated in human eosinophils by flow cytometry and western blot of caspase-3 fragments. Eosinophil migration toward platelet-activating factor was evaluated by transwell assays. Interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and the chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation normal T cell expressed, and presumably secreted (RANTES) were measured using a high-throughput multiplex luminex technology. Annexin I and the chemochine receptor C-X-C chemokine receptor 4 (CXCR4) were detected by flow cytometry. With in vivo studies, allergic conjunctivitis was induced in guinea pigs sensitized to ovalbumin by an ocular allergen challenge and evaluated by a clinical score. Conjunctival eosinophils were determined by microscopy or eosinophil peroxidase assay.. In cultured human eosinophils, mapracorat showed the same potency as dexamethasone but displayed higher efficacy in increasing spontaneous apoptosis and in counteracting cytokine-sustained eosinophil survival. These effects were prevented by the glucocorticoid receptor antagonist mifepristone. Mapracorat inhibited eosinophil migration and IL-8 release from eosinophils or the release of IL-6, IL-8, CCL5/RANTES, and TNF-α from a human mast cell line with equal potency as dexamethasone, whereas it was clearly less potent than this glucocorticoid in inducing annexin I and CXCR4 expression on the human eosinophil surface; this was taken as a possible sign of glucocorticoid-dependent transactivation. In the guinea pig, mapracorat or dexamethasone eye drops induced an analogous reduction in clinical symptoms of allergic conjunctivitis and conjunctival eosinophil accumulation.. Mapracorat appears to be a promising candidate for the topical treatment of allergic eye disorders. It maintains an anti-allergic profile similar to that of dexamethasone but seems to have fewer transactivation effects in comparison to this classical glucocorticoid. Some of its cellular targets may contribute to eosinophil apoptosis and/or to preventing their recruitment and activation and to inhibiting the release of cytokines and chemokines.

Topics: Administration, Ophthalmic; Animals; Annexin A1; Anti-Allergic Agents; Apoptosis; Benzofurans; Blotting, Western; Caspase 3; Cells, Cultured; Conjunctiva; Conjunctivitis, Allergic; Cytokines; Dexamethasone; Eosinophils; Flow Cytometry; Guinea Pigs; Humans; Male; Mifepristone; Ophthalmic Solutions; Ovalbumin; Pentanols; Quinolines; Receptors, Glucocorticoid

To elucidate the ocular pharmacological properties of bepotastine besilate, a selective histamine H(1) receptor antagonist, when compared with other histamine H(1) receptor antagonists, using guinea pig allergic conjunctivitis models and in vitro models of eosinophil recruitment and mast cell membrane stabilization. Conjunctival vascular hyperpermeability was studied in guinea pigs passively sensitized with anti-ovalbumin antiserum or following subconjunctival injection of histamine. Modulation of eosinophil recruitment was evaluated for both platelet-activating factor (PAF)-induced eosinophil infiltration in guinea pigs and leukotriene B(4)-induced in vitro chemotaxis of guinea pig peritoneal eosinophils. Membrane-stabilizing effects of bepotastine also were studied with rat peritoneal mast cells stimulated with the ionophore A23187. Histamine H(1) receptor antagonists including bepotastine besilate were topically administered before ovalbumin, histamine or PAF challenges for in vivo experiments or were added directly to mast cell and eosinophil medium in vitro. Bepotastine besilate significantly inhibited conjunctival vascular hyperpermeability in a dose-dependent manner with maximal effect for bepotastine besilate 1.5%. In separate in vivo experiments, bepotastine besilate 1.0% was significantly more effective than levocabastine 0.025% in the passive sensitization model or olopatadine 0.1% in the histamine-induced hyperpermeability model. Bepotastine besilate 1.0% further suppressed PAF-induced eosinophil infiltration into conjunctival tissue more effectively than ketotifen 0.05%. Chemotaxis of guinea pig peritoneal eosinophils and histamine release from rat peritoneal mast cells in vitro were also inhibited by addition of bepotastine. Olopatadine had a weak effect as compared to that of bepotastine on eosinophil chemotaxis and no effect on mast cell histamine release in our study conditions. Bepotastine besilate was more potent than olopatadine, ketotifen, or levocabastine in reducing vascular hyperpermeability in various animal models of allergic conjunctivitis. Mast cell function and eosinophil chemotaxis were also inhibited in vitro with bepotastine, suggesting bepotastine acts as an inhibitor of allergic response through multiple mechanisms: histamine H(1) receptor antagonism, mast cell stabilization, and inhibition of eosinophil migration to ocular inflammatory sites.

Topics: Animals; Capillary Permeability; Cells, Cultured; Chemotaxis, Leukocyte; Conjunctiva; Conjunctivitis, Allergic; Disease Models, Animal; Dose-Response Relationship, Drug; Eosinophils; Guinea Pigs; Histamine; Histamine H1 Antagonists; Histamine Release; Leukotriene B4; Male; Mast Cells; Ovalbumin; Peritoneal Cavity; Piperidines; Platelet Activating Factor; Pyridines; Rats; Rats, Wistar

Antigen (Ag)-presenting cells (APCs) participate in the development of allergic conjunctivitis (AC). However, the conjunctival APCs that take up Ag in AC have not been identified. We sought to clarify the phenotypes of conjunctival APCs that take up, process, and present Ag to T cells during the development of experimental AC.. Splenocytes from naïve ovalbumin (OVA)-specific T cell receptor transgenic (DO11.10) mice were stimulated with either OVA, gold, or OVA-gold to evaluate stimulation-induced proliferation. Naïve DO11.10 mice were subconjunctivally injected with PBS, gold, or OVA-gold. Twenty-four hours later, conjunctivas were harvested for immunohistochemistry and electron microscopy to identify cells that engulfed OVA-gold.. Stimulation of DO11.10 splenocytes with OVA-gold but not gold alone induced similar levels of proliferation as OVA alone. Subconjunctival injection of OVA-gold, but not gold alone, induced infiltration of eosinophils and CD4-positive T cells into the conjunctiva. The cells that took up OVA-gold into their cytoplasma expressed Cluster of Differentiation (CD) 11b, CD68, major histocompatibility complex (MHC) class II.. It appears that conjunctival macrophages are APCs in the development of experimental AC.

Topics: Animals; Antigen-Presenting Cells; Biomarkers; Conjunctiva; Conjunctivitis, Allergic; Cytoplasm; Inflammation; Macrophages; Mice; Ovalbumin; Phagocytosis; Phenotype; Spleen

We investigated the efficacy of cyclosporine A (CyA) eye drops on ocular symptoms in late phase and delayed-type reactions in guinea pig allergic conjunctivitis models. An emulsion of ovalbumin (OVA) and Freund's complete adjuvant (FCA) was intraperitoneally injected into guinea pigs, and 15% OVA solution was applied topically to the eyes to elicit late phase reactions. Following the early phase reaction, increased scores for hyperemia, swelling, edema, and discharge were detected 6 h after antigen challenge, and CyA eye drops significantly inhibited the increase in scores for edema and discharge, the increase in the number of infiltrating inflammatory cells, and the percentage of eosinophils among polymorphonuclear leukocytes in conjunctival tissue. To induce delayed-type reactions, guinea pigs were sensitized by injecting FCA into the footpad, followed by injections of purified protein derivative into palpebral conjunctivae 24 d later. Increased scores for hyperemia, swelling, and discharge were detected 6 h after the induction of delayed-type allergy, and CyA eye drops significantly inhibited the increase in scores for hyperemia and swelling. In contrast, betamethasone sodium phosphate eye drops showed a tendency to inhibit the symptoms in both late phase and delayed-type reactions, or inflammatory cell infiltration in the late phase reaction, but the inhibition was not significant. These results suggest that CyA eye drops are useful for suppressing ocular symptoms in both late phase and delayed-type reactions in allergic conjunctivitis models.

Topics: Animals; Conjunctiva; Conjunctivitis, Allergic; Cyclosporine; Disease Models, Animal; Eosinophils; Freund's Adjuvant; Guinea Pigs; Hypersensitivity, Delayed; Immunosuppressive Agents; Instillation, Drug; Leukocyte Count; Leukocytes, Mononuclear; Male; Ophthalmic Solutions; Ovalbumin; Time Factors

Allergic conjunctivitis is an inflammatory eye disease mediated by Th2-type cytokines and Staphylococcus aureus (S. aureus) colonization has been suggested as playing a role. This study used an experimental allergic conjunctivitis model to determine whether colonization by S. aureus affects the development of allergic conjunctivitis and modifies the immune response to OVA allergen. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, inoculation of S. aureus markedly accelerated the signs of allergic conjunctivitis and was associated with higher levels of IgE Ab in serum. In addition, mice inoculated with S. aureus had more IL-4, IL-5, IL-13 and eotaxin secretion than non-inoculated group. In contrast, inoculation of TLR2(-/-) mice with S. aureus had no effect on severity of allergic conjunctivitis. The findings suggest that activation of TLR2 signal by S. aureus induces Th2-type immune responses and accelerates experimental allergic conjunctivitis.

Topics: Animals; Conjunctivitis, Allergic; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Histocytochemistry; Immunoglobulin E; Lymphoid Tissue; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Specific Pathogen-Free Organisms; Staphylococcal Infections; Staphylococcus aureus; Th2 Cells; Toll-Like Receptor 2

Corneal allografts transplanted into hosts with allergic conjunctivitis experience an increased incidence and swifter tempo of immune rejection compared to corneal allografts transplanted to nonallergic hosts. Previous findings suggested that increased risk for rejection was not a local effect produced by an inflamed eye, but was due to perturbation of the systemic immune responses to alloantigens on the corneal allograft. We tested the hypothesis that another allergic disease, airway hyperreactivity (AHR), would also increase the risk for corneal allograft rejection. Induction of AHR with either ovalbumin (OVA) or short ragweed (SRW) extract prior to keratoplasty resulted in a steep increase in the speed and incidence of corneal allograft rejection. Delayed-type hypersensitivity (DTH) responses to corneal alloantigens were closely associated with corneal allograft rejection. However, the deleterious effect of AHR on corneal allograft survival was not reflected in a heightened magnitude of allospecific DTH, cytotoxic T lymphocyte and lymphoproliferative responses to the alloantigens on the corneal allograft. Unlike Th2-based immediate hypersensitivity, CD8+ T-cell-based contact hypersensitivity to oxazolone did not increase the risk for corneal allograft rejection. Thus, Th2-based allergic diseases significantly reduce the immune privilege of the corneal allograft and represent important risk factors for consideration in the atopic patient.

Topics: Animals; Asthma; Bronchial Hyperreactivity; CD8-Positive T-Lymphocytes; Conjunctivitis, Allergic; Corneal Transplantation; Disease Models, Animal; Female; Graft Rejection; Graft Survival; Isoantigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Risk Factors; T-Lymphocytes, Cytotoxic; Transplantation, Homologous

Topics: Allergens; Animals; Antigens; Conjunctiva; Conjunctivitis, Allergic; Fractals; Guinea Pigs; Hyperemia; Male; Ovalbumin; Photography

The effects of cyclosporine A eye drops on the early-phase reaction were investigated in a type-I allergic conjunctivitis model.. Mice were actively sensitized with ragweed (RW) absorbed on aluminium hydroxide gel and challenged with RW for 10 days (single challenge model) or 10-14 days (repetitive challenge model) after the first sensitization. For the evaluation of itching, ovalbumin was used as an antigen instead of RW. The effects of cyclosporine A eye drops on increased vascular permeability, mast cell degranulation, and itching were evaluated and compared with those of other anti-allergic eye drops.. In the single challenge model, cyclosporine A eye drops significantly inhibited the increase in vascular permeability and histological evaluations showed suppressed degranulation of mast cells. Disodium cromoglycate (DSCG) eye drops showed only a slight tendency to inhibit the increase in both pathophysiological parameters. Ketotifen or betamethasone eye drops significantly inhibited the increase in vascular permeability. The order of potency in the single challenge model was ketotifen > cyclosporine A > betamethasone. In the repetitive challenge model, cyclosporine A eye drops significantly inhibited the increase in vascular permeability and DSCG eye drops showed only slight inhibition. Ketotifen or betamethasone significantly inhibited the increase in vascular permeability. The order of potency in the repetitive challenge model was cyclosporine A > betamethasone > ketotifen. The effect of cyclosporine A eye drops on the itch-scratch response was studied. Cyclosporine A and DSCG significantly reduced the itch-scratch response in the single and repetitive challenge models; the effect of cyclosporine A in the repetitive challenge model was more potent than in the single challenge model.. Those results suggest that administration of cyclosporine A eye drops inhibit the early-phase reaction in type-I allergic conjunctivitis, which may be mediated by the suppression of mast cell degranulation. This action of cyclosporine A eye drops may be involved in the therapeutic effect of cyclosporine A on allergic conjunctivitis.

Topics: Administration, Topical; Ambrosia; Animals; Anti-Allergic Agents; Betamethasone; Capillary Permeability; Cell Degranulation; Conjunctivitis, Allergic; Cromolyn Sodium; Cyclosporine; Disease Models, Animal; Immunosuppressive Agents; Ketotifen; Male; Mast Cells; Mice; Mice, Inbred BALB C; Ophthalmic Solutions; Ovalbumin; Pruritus

The aim of this study was to examine the levels of platelet-activating factor (PAF) and lyso-PAF in tears from experimental animals developing allergic conjunctivitis (AC).. AC was induced in guinea pigs by application of ovalbumin in eye drops. Tear samples were collected from 5 actively sensitized animals and from 5 unsensitized control animals before the challenge, and 1, 2, 4, and 6 h postchallenge. C18:0-PAF, C18:0-lyso-PAF, C16:0-PAF, and C16:0-lyso-PAF levels in the tear samples were determined using liquid chromatography-tandem mass spectrometry.. The concentrations of C16:0-PAF, C16:0-lyso-PAF, and C18:0-lyso-PAF were measurable in both unsensitized and sensitized groups, whereas C18:0-PAF was undetectable in tear samples from either group. The levels of C16:0-PAF, C16:0-lyso-PAF, and C18:0-lyso-PAF in sensitized animals increased throughout the time course of the experiment, whereas there was no corresponding increase in the levels of these molecules in the unsensitized group. There were strong correlations between the concentrations of C16:0-PAF and C16:0-lyso-PAF, both in the sensitized and in the unsensitized group, and the concentrations of C16:0-lyso-PAF and C18:0-lyso-PAF within each group.. The data from this study demonstrated that the levels of PAF and lyso-PAF increase in tears in a guinea pig model of AC development and implicate a role for PAF for the development of AC.

Topics: Animals; Conjunctivitis, Allergic; Eye; Guinea Pigs; Male; Ovalbumin; Platelet Activating Factor; Tears; Time Factors

To study the effects of ketotifen fumarate, olopatadine, and levocabastine on ocular active anaphylaxis in guinea pigs and on ocular immediate hypersensitivity in albino rats.. Clinical grading scores and Evans blue dye leakage to eyelids and to eyeballs were assessed in five treatment groups (n = 10): ketotifen fumarate 0.025%, olopatadine 0.1%, levocabastine 0.05%, negative control, and positive control.. At 20 minutes after challenge, edema scores for ketotifen-treated guinea pigs were statistically significantly lower than those for levocabastine or olopatadine. Active treatment significantly reduced vascular leakage in both models. Ketotifen significantly reduced vascular leakage in eyelids compared with the other drugs. In guinea pigs, vascular leakage in eyeballs was significantly reduced with ketotifen fumarate compared with olopatadine and levocabastine.. In the guinea pig model, ketotifen was more effective than olopatadine and levocabastine at reducing conjunctival edema and vascular permeability in eyelids and eyeballs. In the rat model, ketotifen was more effective at reducing vascular permeability in eyelids than olopatadine and levocabastine.

Topics: Anaphylaxis; Animals; Capillary Permeability; Conjunctivitis, Allergic; Dibenzoxepins; Disease Models, Animal; Edema; Eyelids; Guinea Pigs; Histamine H1 Antagonists; Ketotifen; Male; Olopatadine Hydrochloride; Ophthalmic Solutions; Ovalbumin; Piperidines; Rats

Antigen (Ag)-specific T cells are thought to play a key role in pathogenesis of chronic allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). In order to investigate the trafficking of Ag-specific T cells in experimental immune-mediated blepharoconjunctivitis (EC), we established a novel AC model in DO11.10 T cell receptor (TcR) transgenic (Tg) mice. DO11.10 TcR-Tg mice were challenged with eye drops of whole OVA protein, OVA peptide 1-15, 321-335, or 323-339. Their eyes were histologically examined. Conventional proliferation assay was performed against each Ag. Phenotypes of infiltrating cells and kinetics of Ag-specific T cells were investigated by immunohistochemistry. Adoptive transfer of CD4(+) Ag-specific T cells from DO11.10 TcR-Tg to WT mice was performed. The distribution of KJ1-26(+) cells was investigated in recipient mice. The challenge of OVA peptide 323-339 induced infiltration of inflammatory cells in conjunctivae in a dose dependent manner, accompanied by the proliferative responses of splenocytes. Immunohistochemical analysis revealed Agspecific/ non-Ag-specific T cells, macrophages, and eosinophils in conjunctivae. Infiltration of Ag-specific T cells increased 24 hr later. Transfer of CD4(+) cells from DO11.10 TcR-Tg to WT mice induced EC depending on the number of transferred cells. Ag-specific T cells were detected in the conjunctivae and spleens of recipient mice, though its numbers were significantly smaller compared to DO11.10 TcR-Tg mice. The challenge of OVA peptide 323-339 induced EC in DO11.10 TcR-Tg mice without prior sensitization. The response was mediated by CD4(+) Ag-specific T cells. The trafficking of Ag-specific T cells in EC was clearly visualized.

Topics: Adoptive Transfer; Animals; Biomarkers; CD11b Antigen; CD4-Positive T-Lymphocytes; Conjunctiva; Conjunctivitis, Allergic; Disease Models, Animal; Dose-Response Relationship, Immunologic; Mice; Mice, Transgenic; Ophthalmic Solutions; Ovalbumin; Peptide Fragments; Receptors, Antigen, T-Cell, alpha-beta; T-Cell Antigen Receptor Specificity

Platelet-activating factor (PAF) plays important roles in allergic reactions. In particular, there are many concerns about PAF, eosinophils, and the chronicity of allergic diseases. The purpose of the present studies is to elucidate the role of PAF in eosinophil activation at conjunctiva and to confirm the efficacy of Apafant (a potent PAF antagonist) ophthalmic solution in chronic experimental allergic conjunctivitis. Guinea pigs were actively immunized and allergic conjunctivitis was induced by repetitive instillation of 2.5% ovalbumin. PAF solution was topically applied and eosinophil activation was assessed by measuring the eosinophil peroxidase (EPO) activity in the tear fluid. Itch-scratching episodes and clinical symptoms scores were evaluated in the repetitive challenge conjunctivitis. From the instillation of PAF solution into guinea pig eyes, which were in a state of chronic allergic conjunctivitis, a significant increase in EPO activity was observed, and this increase was inhibited by pre-treatment with Apafant. In the repetitive challenge model, the animals treated with Apafant ophthalmic solution showed a significant reduction of clinical symptoms and the itch-scratch response in both the first and the second challenges. PAF has an activity, that induces mediator release from eosinophils in the conjunctival tissues and may be involved in the chronic phase of allergic conjunctivitis.

Topics: Administration, Topical; Animals; Azepines; Chronic Disease; Conjunctiva; Conjunctivitis, Allergic; Dose-Response Relationship, Drug; Eosinophil Peroxidase; Eosinophils; Guinea Pigs; Male; Ophthalmic Solutions; Ovalbumin; Platelet Activating Factor; Tears; Triazoles

In the present study, we investigated the participation of chemical mediators in the development of experimental allergic conjunctivitis in rats. Cetirizine (a histamine H1 receptor antagonist), ramatroban (a thromboxane A2 (TXA2) receptor antagonist) and zafirlukast (a cysteinyl leukotrienes (cys-LTs) receptor antagonist) were orally administered from day 14 to day 42 during repeated topical antigen challenge. An increase in reactivity to antigen- and histamine-induced eye scratching behavior was observed by topical sensitization in sensitized rats. Although increased reactivity to antigen was not influenced by cetirizine, ramatoroban and zafirlukast, increased reactivity to histamine was significantly inhibited by ramatroban. The development of conjunctival edema was also observed for topical sensitization. Cetirizine caused no inhibition of the development of conjunctival edema, but ramatroban and zafirlukast inhibited the development of conjunctival edema. In addition, the number of eosinophils in the conjunctiva was increased by topical sensitization. Cetirizine had no significant effect on the increase in the number of eosinophils. However, ramatroban and zafirlukast were effective in inhibiting an increase in the number of eosinophils induced by topical sensitization. These results indicate that TXA2 is involved in increased histamine reactivity, and TXA2 and cys-LTs are associated with not only the conjunctival edema but also eosinophil infiltration during the development of experimental allergic conjunctivitis in rats.

Topics: Administration, Oral; Animals; Carbazoles; Cetirizine; Conjunctivitis, Allergic; Disease Models, Animal; Edema; Eosinophilia; Histamine H2 Antagonists; Indoles; Leukotriene Antagonists; Male; Membrane Proteins; Ovalbumin; Phenylcarbamates; Pruritus; Rats; Rats, Wistar; Receptors, Histamine H2; Receptors, Leukotriene; Receptors, Thromboxane A2, Prostaglandin H2; Sulfonamides; Tosyl Compounds

How the early phase allergic reaction affects the late phase reaction remains unclear. We examined this issue with an experimental model of allergic conjunctivitis that permits the two reactions to be disconnected from each other.. Experimental immune-mediated blepharoconjunctivitis (EC) was initiated in Brown Norway rats by transferring ovalbumin (OVA)-specific T cells and then challenging with OVA-containing eye drops. To induce early phase reaction, a mast-cell activator, C48/80, was challenged together with or without OVA. Rats were evaluated clinically and eyes were harvested for histologic examination and for evaluation of chemokine expression by reverse-transcriptase PCR.. The rats challenged with OVA alone developed the T-cell-mediated late phase reaction histologically, but not clinically, in the absence of early phase reaction. While rats challenged with C48/80 with or without OVA exhibited clinical signs of the early phase reaction, the clinical late phase reaction was observed only in the OVA+C48/80 group. Eosinophilic infiltration into the conjunctiva during the late phase reaction of the OVA+C48/80 group markedly exceeded that of rats challenged with either OVA or C48/80 alone. RANTES (regulated on activation, normal T-cell expressed and secreted), an eosinophil attractant, was expressed both in the OVA+C48/80 and OVA groups, while eotaxin was expressed at equivalent levels in all three groups.. The mast-cell-mediated early phase reaction potentiates the T-cell-mediated late phase reaction, and RANTES is involved in eosinophilic infiltration induced by antigen-specific T cells. Other molecules induced by allergen-specific T cells activated in an as yet unknown manner by the mast cells may be responsible for the infiltration of eosinophils.

Topics: Animals; Blepharitis; Cell Movement; Chemokine CCL5; Conjunctivitis, Allergic; Enzyme-Linked Immunosorbent Assay; Eosinophilia; Eosinophils; Flow Cytometry; Hypersensitivity, Delayed; Lymphocyte Activation; Male; Mast Cells; Ovalbumin; p-Methoxy-N-methylphenethylamine; Rats; Rats, Inbred BN; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes

The effects of ketotifen and lodoxamide on eosinophil infiltration were assessed in a guinea pig model of allergic conjunctivitis. The two active treatments were coded in this masked study in which 30 male guinea pigs, sensitized to chicken egg albumin (ovalbumin), were randomly assigned to one of three groups: Group 1, instillation of 0.9% NaCl into the conjunctival sac of both eyes; Group 2, instillation of 0.025% ketotifen into the left eye and 0.9% NaCl into the right eye; Group 3, instillation of 0.1% lodoxamide into the left eye and 0.9% NaCl into the right eye. Ovalbumin was administered topically to each eye, except in Group 1 where it was only applied to the left eye. (111)In-oxine labeled eosinophils were injected into the jugular vein of each guinea pig; the animals were sacrificed 17 hours after ovalbumin had been applied. The level of radioactivity in the ketotifen- and lodoxamide-treated eyes was approximately 60% of that in the saline-treated eyes. Moreover, the mean level of radioactivity in the ketotifen- and lodoxamide-treated eyes was comparable with the mean level of radioactivity in the saline-treated eye of Group 1, which had not been exposed to allergen. These results indicate that the therapeutic effects of ketotifen and lodoxamide in allergic conjunctivitis may be partly mediated by an inhibitory effect on eosinophils.

Topics: Administration, Topical; Animals; Anti-Allergic Agents; Cell Movement; Conjunctivitis, Allergic; Eosinophils; Guinea Pigs; Ketotifen; Male; Ovalbumin; Oxamic Acid

Genetic background determines the histological features of experimental immune-mediated blepharoconjunctivitis (EC) in rats, which is a model for human allergic conjunctivitis (AC). A great number of lymphocytes predominate in EC of Lewis rats, while less lymphocytes and more eosinophils are present in that of Brown Norway (BN) rats. Although this difference could be attributed to their systemic Th1/Th2 dominancy, it remains unclear whether some regulatory mechanisms may exist in the inflammatory site in the conjunctiva. Here, we aim to investigate this hypothesis by comparing the expression levels of inflammatory mediators in the conjunctiva between the two strains. EC was induced in Lewis and BN rats by transfer of ovalbumin (OVA)-specific CD4(+) T-cell lines followed by eye drops of OVA as antigen challenge, and then was clinically and histologically evaluated. Reverse-transcription (RT)-PCR was performed to compare the expressions of cytokines and cytokine receptors (Rs) in conjunctivas of both strains of rats either with or without EC. To confirm the biological significance of interferon (IFN)-gamma R expression, phosphorylation of signal transducers and activators of transcription (STAT)-1 was examined in the conjunctivas, followed by subconjunctival injection of IFN-gamma. BN T cells contained interleukin (IL)-4 and IFN-gamma, while Lewis T cells expressed no IL-4. Transfer of those cells induced more severe EC in Lewis rats. RTPCR using naive conjunctivas detected more IL-4, IFN-gamma, and IFN-gamma R beta-chain RNA expression in BN rats. After the EC induction, BN rats expressed significantly higher amounts of IFN-gamma R beta-chain, and upregulation of interferon regulatory factor (IRF)-1 was observed. Phosphorylation of STAT-1 was more remarkable in BN rats. The findings demonstrate differential expression of IFN-gamma R and signaling through IFN-gamma in the conjunctiva between the two strains. This may be due to differences in histopathological character between the two strains.

Topics: Animals; Blepharitis; CD4-Positive T-Lymphocytes; Cell Line; Conjunctiva; Conjunctivitis, Allergic; DNA-Binding Proteins; Humans; Interferon gamma Receptor; Interferon-gamma; Male; Ovalbumin; Phosphorylation; Rats; Rats, Inbred BN; Rats, Inbred Lew; Receptors, Interferon; Signal Transduction; Species Specificity; STAT1 Transcription Factor; Trans-Activators

The effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on experimental allergic conjunctivitis, induced by ocular challenge with antigen in actively sensitized guinea pigs, were investigated. NSAIDs reduced the increase in prostaglandin D2 (PGD2) and E2 (PGE2) in the ocular lavage fluid. The inhibition of NSAIDs to these increases was approximately 90%-95%. NSAIDs also lowered itch-scratch response (ISR) to approximately one-third to one-half of the vehicle-treated group. However, these drugs scarcely affected plasma exudation in the conjunctiva. Ketotifen, an H1 histamine receptor antagonist, inhibited both pathophysiological changes (inhibition: 70%-80%). However, this drug was less efficacious than NSAIDs in reducing PGD2 and PGE2 levels. Moreover, topical administration of histamine induced ISR and plasma exudation; in contrast, PGD2 induced ISR exclusively. These results suggest that a part of antigen-induced ISR may be attributable to PGs. However, PGs may not play a key role in plasma exudation; other mediators such as histamine may be involved.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens; Benzophenones; Benzopyrans; Bromobenzenes; Conjunctivitis, Allergic; Diclofenac; Dinoprostone; Disease Models, Animal; Evans Blue; Exudates and Transudates; Eye; Guinea Pigs; Histamine; Histamine Release; Immunization; Injections, Intraperitoneal; Injections, Subcutaneous; Ketotifen; Male; Ovalbumin; Propionates; Prostaglandin D2; Pruritus; Therapeutic Irrigation

Interferon gamma (IFN-gamma) knockout mice exhibit severe allergic conjunctivitis (AC), indicating that IFN-gamma regulates the development of AC. The authors examined whether this inhibitory effect of IFN-gamma is exerted during the induction or effector phase of experimental AC.. Experimental immune mediated blepharoconjunctivitis (EC) was induced in Brown Norway (BN) rats, using ovalbumin (OVA) as the antigen. To investigate the role of IFN-gamma in the induction phase, EC was induced by active immunisation and IFN-gamma (10 micro g/time, total 70 micro g), or phosphate buffered saline (PBS) as a control, was injected intraperitoneally every other day from the day of immunisation. The rats were challenged with OVA eye drops 13 days after immunisation, and 24 hours later, the eyes were harvested for histology. To examine the effects of IFN-gamma in the effector phase, OVA specific T cells were transferred into syngeneic rats and IFN-gamma (10 micro g/time, total 50 micro g) or PBS was injected each day after the transfer until induction of EC 4 days later with an OVA challenge. To investigate the role of endogenous IFN-gamma during the effector phase, an anti-IFN-gamma monoclonal antibody (3 mg/time) was injected on days 3 and 4.. Injection of IFN-gamma into actively immunised rats suppressed eosinophilic infiltration but not infiltration of mononuclear cells. In contrast, neither IFN-gamma nor anti-IFN-gamma affected EC in passively immunised rats.. IFN-gamma is a suppressive cytokine for the development of EC and exerts this suppressive effect during the induction phase.

Topics: Adoptive Transfer; Animals; Blepharitis; Cell Count; Cell Line; Conjunctiva; Conjunctivitis, Allergic; Cytokines; Immunity, Active; Immunity, Cellular; Immunoglobulin E; Interferon-gamma; Male; Mice; Ovalbumin; Rats; Rats, Inbred BN; T-Lymphocytes

To determine whether the number of filled conjunctival goblet cells and mucin gene expression are altered in a mouse model of allergic conjunctivitis.. A/J mice were sensitized intraperitoneally with cat dander or the peptide P3-1 from the protein Fel d1. Two weeks later, the mice were challenged for 7 consecutive days with eye drops containing the allergens. Conjunctival tissue was harvested at 0, 6, 24, or 48 hours after final antigen challenge. Control samples were naïve animals and mice sensitized with cat dander and challenged with OVA-peptide or PBS. The mean number of filled goblet cells per square millimeter in three forniceal fields for each group was determined in wholemounts of conjunctiva prepared using rhodamine-phalloidin labeling followed by confocal microscopy. RNA was isolated from conjunctiva of the contralateral eye and taken for relative quantitation of mRNA of the goblet cell mucin Muc5AC and the epithelial membrane-spanning mucin Muc4, by real-time RT-PCR.. The number of filled goblet cells was significantly decreased with both cat dander and P3-1, after final ocular challenge (P < 0.001). The most significant decrease over naïve mice was seen at 6 hours after final challenge with both allergens. The number of filled goblet cells was still decreased but was returning toward naïve levels at 24 hours (P < 0.05), and at 48 hours no significant difference was seen compared with naïve, PBS-treated, and OVA-peptide-treated control samples. For both cat dander and P3-1, Muc5AC and Muc4 mRNA was found to be decreased at the time of final ocular challenge. The level of Muc5AC mRNA from goblet cells rebounded from the decrease to show an increase over control by 24 hours after final challenge, and by 48 hours, the mRNA level had returned to naïve control range. In contrast, significant increases in Muc5AC mRNA were evident after final control challenge with PBS or OVA-peptide, indicating a potential irritant effect of drop application. The Muc4 mRNA level was significantly reduced at all time points except 24 hours after the last challenge. By comparison with allergen-challenged eyes, no change in Muc4 message levels was noted at any time point in OVA-peptide- or PBS-treated control eyes.. These findings demonstrate that, in the conjunctiva of mice, repetitive application of allergens induces a reduction in the number of filled goblet cells and a decrease in Muc5AC and Muc4 mRNAs. After a period of 24 to 48 hours, the goblet cell number return to naïve levels, and goblet cell mucin mRNA levels return to above or within normal range, indicating a rapid recovery in the mucus secretion system.

Topics: Allergens; Animals; Cell Count; Conjunctiva; Conjunctivitis, Allergic; Epithelium; Female; Glycoproteins; Goblet Cells; Mice; Mice, Inbred A; Microscopy, Confocal; Models, Animal; Mucin 5AC; Mucin-4; Mucins; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

FK506 has been used for treatment of cell-mediated immune disorders such as graft rejection in transplantation or Behçet disease. To evaluate the effectiveness of FK506 in another ocular disease model, we injected FK506 in rats with experimental allergic/immune-mediated blepharo conjunctivitis (EAC) the induction mechanism of which depends on cell-mediated immunity.. Lewis rats were immunized with ovalbumin (OVA) in emulsion of complete Freund's adjuvant (CFA). We injected 2 (n = 6), 20 (n = 6) or 200 (n = 5) microg of FK506 intramuscularly daily from the day of immunization (day 0) to day 6. Control rats were not treated with FK506 (n = 4). In addition, we injected 200 microg of FK506 from day 7 to day 13 (n = 12) to compare the timing of FK506 administration (day 0 to day 6, n = 12; control, n = 12). Twenty-one days after immunization, all rats were challenged with OVA by eye drops, and 24 h later they were killed after clinical evaluation and their eyes, blood and draining lymph nodes were harvested for histology, antibody titers and proliferation assay or flow cytometric analysis. In another set of experiments, rats that had received OVA-primed lymph node cells did (n = 9) or did not (n = 9) receive additional FK506 by injection daily for 4 days. Four days after transfer, these rats were challenged with OVA and evaluated as mentioned. To investigate possible suppression of disease by topical administration of FK506, both actively immunized and passively immunized rats received OVA together with 0.3% (weight/volume) of FK506 (n = 16) or vehicle (n = 10) by eye drops and 24 h after challenge, rats were evaluated as mentioned.. Development of disease, induced by either active or passive immunization, was inhibited in the group treated with 200 microg of FK506, regardless of timing of administration. Cellular proliferative responses to OVA were inhibited only in this group. Flow cytometry demonstrated a decrease of about 20% in the proportion of all cells made up by CD4-positive T cells. Topical administration of FK506 inhibited the development of EAC, though not significantly.. Systemic treatment with 200 microg of FK506 either in the induction or the effector phase inhibits the development of EAC in Lewis rats. Topical administration is not so effective as systemic administration.

Topics: Adoptive Transfer; Animals; B-Lymphocytes; Blepharitis; CD3 Complex; CD4-Positive T-Lymphocytes; Conjunctivitis, Allergic; Disease Models, Animal; Immunosuppressive Agents; Injections, Intramuscular; Leukocyte Common Antigens; Lymph Nodes; Lymphocyte Activation; Male; Ophthalmic Solutions; Ovalbumin; Rats; Rats, Inbred Lew; Tacrolimus; Vaccination

In allergic conjunctivitis, the early phase reaction has been studied extensively both in humans and animals. Although cellular infiltration is the main feature of the late phase reaction, the role of cellular immunity remains unclear. The purpose of this study was to elucidate the role of cellular immunity both in the induction and effector phases of experimental allergic blepharoconjunctivitis (EAC). To analyse the involvement of cellular immunity in the induction phase, 6-8-week-old male Lewis rats were immunized with ovalbumin (OVA) emulsified with complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), TiterMaxR (TM), aluminum hydroxide [Al(OH)3], or without any adjuvant. Three weeks after immunization, the rats were challenged with OVA by eye drops, and 24 hr later they were euthanized and their eyes, including the lids, blood, and lymph nodes were harvested for analysis of disease and immune responses. The results indicated that adjuvants were necessary to induce disease as well as both cellular and humoral immunity. Al(OH)3, CFA and TM induced stronger disease and cellular immunity than IFA. The intensity of disease correlated with that of cellular immunity. To further investigate the involvement of cellular immunity in EAC, lymph node cells collected from immunized rats were adoptively transferred into naive syngeneic recipients that were challenged 4 days later with OVA. EAC developed in the recipients of lymph node cells that were also stimulated in culture with OVA. These recipient rats developed cellular infiltration in the lid and conjunctiva, in a dose-dependent manner. These results suggest that cellular immunity played a major role in the development of EAC, both in the induction and effector phases.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Aluminum Hydroxide; Animals; Antibody Formation; Blepharitis; Conjunctivitis, Allergic; Freund's Adjuvant; Immunity, Cellular; Lymphocytes; Male; Ovalbumin; Poloxalene; Rats; Rats, Inbred Lew; Time Factors

The role of substance P in experimental allergic conjunctivitis induced by egg albumin was investigated with guinea pigs. Increase in vascular permeability of the conjunctiva induced by antigen was significantly inhibited after repeated application of capsaicin. Substance P contents in the conjunctiva of guinea pig were decreased by topical instillation of antigen to the eyes, suggesting that substance P was released from the conjunctiva due to antigen-antibody reaction. Moreover, subconjunctival injection of substance P resulted in a dose-related conjunctivitis, and vascular permeability in the conjunctiva was also increased by substance P. In substance P-induced conjunctivitis, a significant edema was observed in the bulbar and palpebral conjunctiva, but no hyperemia was noted in all instances. Histamine contents of the conjunctiva and tears were not influenced by subconjunctival injection of substance P. However, topical application of antigen and subconjunctival injection of compound 48/80 caused a significant decrease in histamine content, and content of tear was increased by both treatments. An increase in vascular permeability induced by antigen application was significantly inhibited by intravenous injection of FK-888, which is a specific and potent NK1 receptor antagonist. From these results, it is suggested that substance P is responsible for allergic conjunctivitis to some extent, and the conjunctival hyperpermeability induced by substance P occurs through NK1 receptor on the blood vessels, rather than by the direct action on the conjunctival mast cells during allergic conjunctival reactions.

Topics: Animals; Capillary Permeability; Capsaicin; Conjunctiva; Conjunctivitis, Allergic; Dipeptides; Disease Models, Animal; Guinea Pigs; Histamine; Indoles; Male; Mast Cells; Ovalbumin; p-Methoxy-N-methylphenethylamine; Serine Proteinase Inhibitors; Substance P; Tears

The effect of benzodiazepine (BZP) on experimental allergic conjunctivitis was studied. Male Hartley guinea pigs were sensitized with ovalbumin (OVA) and then homologous anti-OVA serum was injected intravenously into guinea pigs for passive sensitization. BZP was administered at a dose of 5 mg/kg by intraperitoneal injection (i.p.) according to the following schedule respectively; a single, repeated twice a day for 3 days, repeated twice a day for 3 days plus a single and repeated twice a day for 7 days. OVA challenge was performed to the conjunctiva, 20 minutes later, conjunctival edema during the early phase response (EPR) was observed and again at 6 hours later, both eosinophil infiltration into the conjunctiva and the platelet activating factor (PAF) serum level during the late phase response (LPR) were examined. BZP did not inhibit the development of conjunctival edema during the EPR. A single dose of BZP did not inhibit, but repeated doses of BZP for 3 or 7 days significantly suppressed eosinophil infiltration during the LPR. And after repeated doses of BZP for 7 days, all PAF serum levels during the LPR were under the lower detection limit of the assay. These results suggest that BZP has an inhibitory effect on antigen-induced eosinophil infiltration into the conjunctiva during the LPR.

Topics: Animals; Antigens; Benzodiazepines; Conjunctiva; Conjunctivitis, Allergic; Eosinophils; Guinea Pigs; Male; Ovalbumin; Platelet Activating Factor

The role of nitric oxide in allergic conjunctivitis was studied in a guinea pig model. The eyes of sensitized guinea pigs were challenged with ovalbumin (20 micrograms per eye) or histamine (20 micrograms per eye). Synthesis of nitric oxide (NO) was inhibited using L-NAME (200 micrograms per eye) or aminoguanidine (200 micrograms per eye). The formation of conjunctival edema was graded and levels of nitrite, a breakdown product of nitric oxide were measured in lavage fluid. Conjunctival vasopermeability was determined by measuring the albumin concentration in the fluid on the surface of the eye (lavage fluid). Animals were treated with sodium nitroprusside (SNP) or phenylephrine after which histamine induced conjunctival vasopermeability changes were measured. Drugs were administered topically with the other eye serving as a control. Both ovalbumin and histamine produced a marked inflammatory response including hyperaemia and edema. At the top of the inflammatory response occurring 30 min after challenge, increased levels of nitrite, a breakdown product of NO, were measured in lavage fluid. Prophylactic treatment with L-NAME or aminoguanidine resulted in a significant inhibition of the NO synthesis. Both L-NAME and aminoguanidine decreased conjunctival vascular permeability and edema formation significantly. Administration of SNP resulted in a marked dilatation of conjunctival blood vessels and produced a dose-dependent increase of vascular permeability. Addition of SNP to histamine significantly enhanced conjunctival edema and potentiated vascular permeability. These results indicate that NO is produced in the acute phase of allergic conjunctivitis and mediates vasodilatation after topical provocation with ovalbumin or histamine in sensitized guinea pigs. The resulting increase of the conjunctival blood flow subsequently increases the vascular permeability and enhances conjunctival edema formation. Inhibition of NO synthesis leads to a reduction of conjunctival hyperaemia and subsequently reduces the formation of edema.

Topics: Animals; Arginine; Capillary Permeability; Conjunctival Diseases; Conjunctivitis, Allergic; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Female; Guanidines; Guinea Pigs; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Ovalbumin; Phenylephrine

The effect of fenitrothion on experimental allergic conjunctivitis was studied using guinea pigs. Guinea pigs were passively sensitized with anti-sera against ovalbumin (OVA) or Japanese cedar pollen. Eight days after the sensitization, 6 x 10(-5) mg/kg to 6 mg/kg of fenitrothion were administered subcutaneously. Two days after the injection, OVA or Japanese cedar pollen was given in the conjunctival sac and the allergic conjunctivitis was examined. OVA or Japanese cedar pollen induced the allergic conjunctivitis, depending on the challenge dosage (OVA). However, no adverse effect of fenitrothion was observed on allergic conjunctivitis challenged with OVA or Japanese cedar pollen. An anti-allergic agent, ketotifen, suppressed the allergic intensity. These results indicate that fenitrothion has no adverse effect on the experimental allergic conjunctivitis.

Topics: Animals; Cholinesterase Inhibitors; Conjunctivitis, Allergic; Dose-Response Relationship, Drug; Fenitrothion; Guinea Pigs; Immunization, Passive; Ketotifen; Ovalbumin; Pollen; Trees

The purpose of this study was to assess the activity of some marketed products in ocular non-immune and immune type I hypersensitivity reactions, and during intra-ocular type III hypersensitivity. In order to compare these activities, we improved and validated three different models of ocular allergic reaction already known for their ability to reproduce allergic conjunctivitis or uveitis. Allergic conjunctivitis was induced by ocular immediate hypersensitivity after instillation of compound 48/80 in the rat, or an active anaphylaxis reaction with ovalbumin immunisation and challenge in the guinea pig. Uveitis was induced by a reverse passive anaphylaxis reaction using intra-vitreal rabbit anti-bovine IgG anti-serum sensitisation and intravenous bovine gamma-globulin challenge in the rabbit. Clinical scores and blood-tissue permeability indices were studied. Using the same schedule of ocular instillation, the effects of Livostin (levocabastine 0.05%), Almide (lodoxamide 0.1%), Opticrom (sodium cromoglycate 2%), Ocufen (flurbiprofen 0.03%), Acular (ketorolac 0.5%) and 0.3% chlorpheniramine maleate were compared to positive and negative controls. We demonstrated the potent activity of chlorpheniramine maleate 0.3% and Livostin in both allergic conjunctivitis models. Significant activity was also evidenced with Almide, which was only active in the non-immune allergy model, while Opticrom was definitely not active in these models. In the uveitis model, Acular and Ocufen are active and potent drugs, while Livostin and Almide were not active. These results are discussed with respect to the models used and the mediators involved.

Topics: Anaphylaxis; Animals; Blood Proteins; Capillary Permeability; Conjunctivitis, Allergic; Disease Models, Animal; Evans Blue; Guinea Pigs; Hypersensitivity, Immediate; Iris; Male; Ovalbumin; p-Methoxy-N-methylphenethylamine; Passive Cutaneous Anaphylaxis; Rabbits; Rats; Rats, Wistar; Uveitis

Immunological tolerance can be induced by the oral administration of antigen. We induced oral tolerance rats to experimental allergic conjunctivitis by ovalbumin and investigated the suppression of inflammation. In groups which had started eating antigen both before and after the immunization, the serum anti-ovalbumin IgE level measured by passive cutaneous anaphylaxis reaction was significantly lower than in a group that had not been fed antigen. The intensity of experimental allergic conjunctivitis in the group which had started eating antigen before immunization, was significantly suppressed in regard to the leakage of Evans Blue from conjunctival vessels 30 minutes after the challenge and the neutrophil infiltration 6 hours after. The dye leakage of the group which had started feeding after immunization was also significantly suppressed. There was a positive correlation between the serum IgE level and the leakage of Evans Blue (r = 0.90, p < 0.001). These results suggest that the suppression of antigen-specific IgE antibody production caused by oral tolerance affected the decrease of local inflammation on the conjunctiva.

Topics: Administration, Oral; Allergens; Animals; Conjunctivitis, Allergic; Immune Tolerance; Male; Ovalbumin; Rats; Rats, Wistar

Emedastine [1-(2-ethoxyethyl)-2-(4-methyl-1-homopiperazinyl)- benzimidazole difumarate] was evaluated for topical ocular anti-histaminic activity in histamine and antigen stimulated conjunctivitis models. Concentration-dependent inhibition of histamine induced vascular permeability changes occurring in the conjunctiva was observed when the time interval between topical ocular administration and histamine challenge ranged from 1 min to 8 hr. The calculated ED50 values obtained using intervals of 1 min, 30 min, 2, 4 and 8 hr were 0.0002%, 0.000035%, 0.0029%, 0.019% and 0.19%, w/v, respectively. Comparisons of relative potency 30 min post dosing between emedastine and other anti-histamines demonstrated that emedastine is equipotent to ketotifen, and 7, 7, 10, 10, 100, 357, 3333, and 5813 times more potent than brompheniramine, chlorpheniramine, clemastine, pyrilamine, levocabastine, pheniramine, diphenhydramine, and antazoline, respectively. Emedastine (0.1%) failed to significantly attenuate either serotonin or platelet-activating-factor induced vascular permeability changes indicating high selectivity for the histamine H1 receptor. In a passive conjunctival anaphylaxis model in guinea pigs, significant inhibition of the allergic response was observed following topical ocular administration of emedastine 5 min or 30 min prior to antigen challenge (ED50s 0.0046% and 0.00022%, respectively). These data clearly indicate that emedastine has potential as a topical ocular anti-histamine for treating allergic conjunctivitis.

Topics: Administration, Topical; Anaphylaxis; Animals; Benzimidazoles; Capillary Permeability; Conjunctiva; Conjunctivitis, Allergic; Drug Evaluation, Preclinical; Guinea Pigs; Histamine; Histamine H1 Antagonists; Male; Ophthalmic Solutions; Ovalbumin; Platelet Activating Factor; Rats; Rats, Sprague-Dawley; Serotonin

The aim of this study has been to determine whether the presence of lymphocytic infiltrates observed in the histology of ocular allergic conditions in humans or in the late phase of ocular anaphylactic reactions in experimental animals is a non-specific event dependent only on the degranulation of mast cells, or is conditioned by a specific response to antigen. With this in mind, responses to antigen and to a non-immunological mast cell degranulator (compound 48/80) were compared in an experimental model of allergic conjunctivitis. Rats were sensitised to ovalbumin and challenged topically in the left conjunctival sac either with ovalbumin or compound 48/80. The presence of T cells and activated T cells in the infiltrate was studied by immunohistochemical staining on conjunctival tissue obtained at 4, 24, and 48 hours after challenge. Ovalbumin sensitised and challenged rats showed increased numbers of T cells in the conjunctival infiltrate, statistically significant when compared with compound 48/80 challenged rats at 48 hours and with controls at 4, 24, and 48 hours. The number of T cells was significantly higher in compound 48/80 challenged rats only at 48 hours when compared with controls. As for the number of activated T cells, only ovalbumin sensitised and challenged rats showed significantly increased levels of these cells compared with both sensitised animals challenged with compound 48/80 and controls at 4 and 24 hours after challenge. These results suggest that the infiltration of the conjunctiva by activated T lymphocytes is, at least in part, dependent on a specific response to antigen.

Topics: Animals; Antibodies, Monoclonal; Conjunctiva; Conjunctivitis, Allergic; Disease Models, Animal; Immunoenzyme Techniques; Immunoglobulin E; Lymphocyte Activation; Male; Ovalbumin; p-Methoxy-N-methylphenethylamine; Rats; Rats, Wistar; T-Lymphocytes

The relationship between the release of histamine, a major mast cell mediator of conjunctival type I reactions, and the production of a prostanoid, prostacyclin (prostaglandin I2, PGI2), was examined in a guinea pig model of allergic conjunctivitis. Guinea pigs were sensitized topically and challenged by repeated conjunctival instillation of fluoresceinyl ovalbumin. Histamine and 6-keto-PGF1 alpha, the stable product of the spontaneous degradation of PGI2, were measured in tears by radioimmunoassays. Clinical type I reactions and tear histamine appeared by 8 days and increased up to 22 days during the initial sensitization, with notable variations between animals. The kinetics of histamine and 6-keto-PGF1 alpha release in tears were examined over a 24-hr period after the antigen challenge. Histamine release was maximal during the first 10 min and returned to baseline values by 1 hr in all instances. The 6-keto-PGF1 alpha release also peaked during the first 10 min but continued for an extended period. The ratio of tear 6-keto-PGF1 alpha to histamine increased more than 16-fold over the 2 hr after antigen challenge. Late-phase reactions with second peaks of histamine or 6-keto-PGF1 alpha in the tears were observed in two different guinea pigs 4-8 hr after antigen challenge. Histamine applied to the eyes of naive guinea pigs also induced the release of 6-keto-PGF1 alpha in tears. Histamine appeared to act as a primary mediator, stimulating the secondary production and release of PGI2 by constitutive (eg, vascular) and possibly infiltrating inflammatory cells during an allergic conjunctival reaction.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Conjunctivitis, Allergic; Female; Guinea Pigs; Histamine Release; Ovalbumin; Radioimmunoassay; Tears

Evans blue (EB) dye extravasation has been used as a reliable and objective parameter of the increased vascular permeability of an allergic conjunctivitis experimental rat model that closely mimics human ocular allergy. Five male Wistar rats, previously immunized (Group 1), had DL-dithiothreitol (DTT) applied topically to one eye 15 min prior to topical challenge with egg albumin (EA). The fellow eye (control) received phosphate buffered saline (PBS) 15 min prior to receiving EA. Immediately prior to challenge, the rats were injected intravenously with EB. After 30 min, the animals were killed and the dye extracted from the eyes. The intensity of EB extravasation was determined by spectrophotometry at 620 nm. EB extravasation was significantly higher in the eyes that received DTT than in those that received PBS. Groups 2, 3 and 4 of nonimmunized rats served as additional controls: Group 2 for DTT toxicity, Group 3 as a proof of the reaginic antibody mediation and Group 4 as a control of EB extravasation under normal conditions. Five additional groups of five rats each were immunized and both eyes of each rat received DTT 15 min before EA challenge. One eye of each rat received 0.1% dexamethasone sodium phosphate topically (Group 5), 0.1% pyrilamine maleate (Groups 6 and 7), and 2% disodium cromoglycate (DSCG) (Groups 8 and 9). The fellow eye received the solvent of each drug topically (control). In the eyes treated with antiallergic drugs, EB extravasation decreased 40% for dexamethasone, 44.1% and 10.4% for pyrilamine, and 51.4% and 51.2% for DSCG.

Topics: Animals; Capillary Permeability; Conjunctiva; Conjunctivitis, Allergic; Cromolyn Sodium; Dexamethasone; Disease Models, Animal; Dithiothreitol; Evans Blue; Male; Ovalbumin; Pyrilamine; Rats; Rats, Inbred Strains

Acute and recurrent allergic conjunctival reactions were induced in guinea pigs by repeated conjunctival applications of fluoresceinyl ovalbumin (FL-OA) for up to 30 months. Early type I conjunctival reactions developed 11 to 25 days after the initial conjunctival exposure to FL-OA. Continuous topical challenges during a six- to 30-month period caused a variety of reactions, including papillary changes and massive hyperplasia of the conjunctival-associated lymphoid tissues. Hyperplasia of lymphoid tissues was induced during a shorter period (two to five months) with a mixture of FL-OA and phorbol ester. Culture fluid from hyperplastic conjunctival lymphoid tissue showed a ratio of IgG1/IgG2 antibody production of up to 15. A low level of recurrence of type I reactivity, after an initial desensitization phenomenon due to a loss of reactive mast cells, correlated with prominent follicular hyperplasia of the conjunctival-associated lymphoid tissue.

Topics: Animals; Antibody Formation; Cells, Cultured; Conjunctiva; Conjunctivitis, Allergic; Enzyme-Linked Immunosorbent Assay; Female; Guinea Pigs; Hyperplasia; Immunoglobulin G; Lymphoid Tissue; Organ Culture Techniques; Ovalbumin

Topics: Allergens; Animals; Capillary Permeability; Conjunctivitis, Allergic; Guinea Pigs; Leukotriene B4; Ovalbumin

Inbred guinea pigs selected for high (IMM/S) respectively low (IMM/R) responsiveness to ovalbumin (OA) as measured by induced respiratory anaphylaxis, were investigated for atopic immune responses of their conjunctival mucosa. IMM/S animals sensitized either by inhalation of OA, or by instillation of antigen into the conjunctival sac, developed regularly an acute ocular inflammatory response to topical (conjunctival) challenge with the allergen. A minimum of 1 microgram OA dropped repeatedly into the conjunctival sac was enough for both ocular and systemic sensitization of the animals, but the minimal dose of effective challenge was considerably higher. In IMM/R animals, ocular hypersensitivity was not achieved by inhalation of OA, but after topical administration of the antigen some of the IMM/R strains could be challenged to ocular anaphylactic responses of the same intensity as observed in IMM/S animals. The conjunctivae of both IMM/S and IMM/R animals could be sensitized passively by intraperitoneal injection of guinea pig sera containing homocytotropic antibodies to OA, but topical administration of such sera had no effect.

Topics: Administration, Inhalation; Administration, Topical; Anaphylaxis; Animals; Antibodies; Bronchi; Bronchial Provocation Tests; Conjunctiva; Conjunctivitis, Allergic; Dose-Response Relationship, Immunologic; Guinea Pigs; Immunization; Ovalbumin; Respiratory Hypersensitivity