Experimental Allergic Conjunctivitis Model Research Articles

Epinastine Cream: A Novel Once-Daily Therapeutic Agent for Allergic Conjunctivitis.

Purpose: To investigate the in vivo efficacy of epinastine cream in type I allergic models. Methods: The dose, timing, and antiallergic effect of epinastine cream on the conjunctiva were evaluated postapplication to the eyelid skin of guinea pigs with histamine- or ovalbumin-induced allergic conjunctivitis. Additionally, we assessed its antiallergic effects on the skin postapplication to the dorsal skin of guinea pigs with ovalbumin-induced passive cutaneous anaphylaxis. Efficacy was estimated by determining the amount of dye that leaked from conjunctival or dorsal skin tissue vessels as a measure of vascular permeability, scoring the severity of allergic symptoms, and observing the scratching behaviors using clinical parameters. Results: In the histamine-induced conjunctivitis model, epinastine cream strongly inhibited conjunctival vascular permeability in a dose-dependent manner. The inhibitory effect of 0.5% epinastine cream 24 h postapplication was significantly higher than that of 0.1% epinastine hydrochloride ophthalmic solution 8 h postadministration. Additionally, the 0.5% epinastine cream inhibited conjunctival vascular permeability 15 min postapplication, and the effect was sustained over 24 h. Furthermore, the 0.5% epinastine cream effectively suppressed clinical symptom scores and exhibited ameliorated scratching bouts in conjunctival allergic reactions in the experimental allergic conjunctivitis model. Additionally, it significantly inhibited vascular permeability in skin allergic reactions in the passive cutaneous anaphylaxis model. Conclusions: The results suggest that epinastine cream is a strong, long-lasting, and skin-penetrating inhibitor of type I allergic reactions. The 0.5% epinastine cream applied once daily could be a promising, potent, and long-acting therapeutic agent for allergic conjunctivitis.

Subconjunctival injection of human umbilical cord mesenchymal stem cells alleviates experimental allergic conjunctivitis via regulating T cell response

BackgroundT helper 2 (Th2) cells are thought to play critical roles in allergic conjunctivitis (AC). They release inflammatory cytokines to promote an allergic response in AC. Due to individual heterogeneity and long-term chronic management, current therapies do not always effectively control AC. Mesenchymal stem cells (MSCs) have been shown to be effective in treating allergy-related disorders, but it is unclear how exactly the Th2-mediated allergic response is attenuated. This study aims to elucidate the therapeutic effect and mechanism of the human umbilical cord MSCs (hUCMSCs) in a mouse model of experimental AC (EAC).MethodsA mouse EAC model was established by inoculating short ragweed (SRW) pollen. After the SRW pollen challenge, the mice received a single subconjunctival or tail vein injection of 2 × 106 hUCMSCs, or subconjunctival injection of hUCMSCs conditioned medium (hUCMSC-CM), and dexamethasone eye drops was used as positive control; subsequent scratching behavior and clinical symptoms were assessed. Immunostaining and flow cytometry were carried out to show allergic reactions and the activation of CD4 + T cell subsets in the conjunctiva and cervical lymph nodes (CLNs). Gene expression was determined by RNA-seq and further verified by qRT-PCR and Western blot. Co-culture assays were performed to explore the regulatory role of hUCMSCs in the differentiation of CD4 + naive T cells (Th0) into Th2 cells.ResultsSubconjunctival administration of hUCMSCs resulted in fewer instances of scratching and lower inflammation scores in EAC mice compared to the tail vein delivery, hUCMSC-CM and control groups. Subconjunctival administration of hUCMSCs reduced the number of activated mast cells and infiltrated eosinophils in the conjunctiva, as well as decreased the number of Th2 cells in CLNs. After pretreatment with EAC mouse serum in vitro to mimic the in vivo milieu, hUCMSCs were able to inhibit the differentiation of Th0 into Th2 cells. Further evidence demonstrated that repression of Th2 cell differentiation by hUCMSCs is mediated by CRISPLD2 through downregulation of STAT6 phosphorylation. Additionally, hUMCSCs were able to promote the differentiation of Th0 cells into regulatory T cells in CLNs of EAC mice.ConclusionsSubconjunctival injection of hUCMSCs suppressed the Th2-allergic response and alleviated clinical symptoms. This study provides not only a potential therapeutic target for the treatment of AC but also other T cell-mediated diseases.

The Effect of Thymoquinone on Experimental Model of Allergic Conjunctivitis

Amaç: Alerjik konjonktivit tedavisi güç bir hastalıktır; yeni tedavi seçeneklerine ihtiyaç vardır. Bu çalışmada anti-inflamatuar ve anti-alerjik özellikleri olan ve çörek otundan (Nigella Sativa) elde edilen timokinonun (TK) sıçan alerjik konjonktivit (AK) modeli üzerindeki etkisinin histolojik yöntemlerle araştırılması amaçlanmıştır. Yöntem: Çalışmaya 48 adet 4-6 aylık yetişkin Wistar Albino dişi sıçan dahil edildi. Sıçanlar; kontrol, AK, AK+Deksametazon, 10 mg/kg TK, AK+3 mg/kg TK ve AK+10 mg/kg TK şeklinde rastgele 6 gruba ayrıldı. AK geliştirmek için, 30 mg/ml adjuvan Al(OH)3 ve 0,3 mg Ovalbumin, serum fizyolojik içinde her sıçana 1 mL intraperitoneal (i.p.) olarak gün aşırı 7 kez uygulandı. Ardı sıra 15-21. günlerde doğrudan konjonktiva üzerine serum fizyolojik içinde %2'lik Ovalbumin 7 kez damlatıldı. Ait olduğu gruba göre TK ilgili dozlarda i.p. olarak uygulandı. Konjonktiva dokusu mast hücre sayıları (MHS) ile histopatolojik ve immünohistokimyasal MCT, TGFβ ve TNFα boyama sonuçları istatistiksel olarak değerlendirildi. Bulgular: Timokinonun tek başına sistemik uygulanması konjonktiva histolojisinde değişikliğe neden olmadı. Ovalbüminle duyarlı hale getirilen AK grubu kontrol grubuyla karşılaştırıldığında, konjonktiva epitelindeki MHS istatistiksel olarak anlamlı artış gösterdi (p<0,001). Deksametazon tedavi grubunda (p<0,001) olduğu gibi; TK tedavi gruplarında da MHS, AK grubuna göre daha düşük bulundu ancak bu fark istatistiksel olarak anlamlı değildi. TGF-β ile negatif boyanma, TNF-α ile zayıf fokal boyanma tedavi gruplarının ortak bulgusu olarak saptandı. Sonuç: Bu çalışmadaki doz ve sürelerle uygulanan ovalbümin, deneysel çalışmalar için uygun bir AK modeli oluşturmaktadır. Timokinonun uygulanan dozlarda yan etki oluşturmadığı ve AK gelişen durumlarda da kısmen anti-inflamatuar yönü ile tedaviye destek olabileceği, ancak istenen anti-alerjik etkiyi göstermediği düşünülmektedir.

Short ragweed pollen promotes M2 macrophage polarization via TSLP/TSLPR/OX40L signaling in allergic inflammation.

This study was to explore the role and mechanism of macrophages in pollen-triggered allergic inflammation. A murine model of short ragweed (SRW) pollen-induced experimental allergic conjunctivitis (EAC), and bone marrow (BM)-macrophages cultures were used. Typical allergic manifestations and TSLP-stimulated Th2 hyperresponse were observed in ocular surface of EAC model in wild-type (WT) mice induced by SRW. The M2 phenotype markers, Arg1, Ym1 and FIZZ1, were highly expressed by conjunctiva and draining cervical lymph nodes (CLNs) of WT-EAC mice when compared with controls, as evaluated by RT-qPCR and Immunofluorescent double staining with macrophage marker F4/80. The stimulated expression of TSLPR and OX40L by macrophage was detected in conjunctiva and CLNs by RT-qPCR, double staining, and flow cytometry. M2 macrophages were found to produce TARC and MDC. In contrast, EAC model with TSLPR-/- mice did not show allergic signs and any increase of Th2 cytokines (IL-4, IL-5 and IL-13) and M2 markers. In vitro cultures confirmed that SRW extract stimulates expression of TSLPR, OX40L, TARC, MDC, and three M2 markers by BM-macrophages from WT mice, but not from TSLPR-/- mice. These findings demonstrate that SRW pollen primes macrophage polarization toward to M2 phenotype via TSLP/TSLPR/OX40L signaling to amplify allergic inflammation.

Effect of Pycnogenol® on an experimental rat model of allergic conjunctivitis.

Allergic conjunctivitis (AC) is a frequent and challenging disease in ophthalmology practice. Cell protective effect of Pycnogenol® (PYC) depends on its antioxidant and anti-inflammatory properties. The aim of the study is to investigate the effect of PYC on an experimental AC model. Ovalbumin and Al(OH)3 were given seven times intraperitoneally (i.p.) every other day and ovalbumin installed everyday directly on conjunctiva to create an AC rat model. Then, PYC (3 or 10mg/kg i.p.) was applied in the study groups. Control rats were given adjuvant Al(OH)3 i.p. and topical saline on conjunctiva. A negative control group in which only PYC (10mg/kg/7days) was administered i.p. and an AC positive control group which have been given dexamethasone (1mg/kg/7days) was created. Mast cells were counted with a microscope; histological evaluation was performed with H-E and toluidine blue, mast cell tryptase, and TNF-α and TGF-β staining. Pycnogenol treatment alone did not show any detrimental effect. Mast cell count (MCC) decreased in both dexamethasone and 10mg/kg given PYC treatment groups compared to positive control group and these results were statistically significant (MCC 1.85± 0.69, p < 0.001; 2.42± 0.53, p = 0.003). Negative staining with TGF-β and weak focal staining with TNF-α were the common findings of dexamethasone and PYC treatment groups. The animal model of AC was successfully developed by using aforementioned way. PYC is a safe herbal product and it has alleviated the findings of ovalbumin-induced AC-similar to dexamethasone-histologically in this experimental model. These results are promising for the future of AC treatment.

Effects of Exposure to Ozone on the Ocular Surface in an Experimental Model of Allergic Conjunctivitis.

Based on previous findings that ozone can induce an inflammatory response in the ocular surface of an animal model and in cultured human conjunctival epithelial cells, we investigated whether exposure to ozone exacerbates symptoms of allergic conjunctivitis. We evaluated the effects of exposure to ozone on conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, production of inflammatory cytokines in tears, and aqueous tear production in a mouse model of allergic conjunctivitis. To validate our in vivo results, we used interleukin (IL)-1α-pretreated conjunctival epithelial cells as an in vitro substitute for the mouse model. We evaluated whether exposure to ozone increased the inflammatory response and altered oxidative status and mitochondrial function in IL-1α-pretreated conjunctival epithelial cells. In the in vivo study, ozone induced increases in conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, and production of inflammatory cytokines, accompanied by a decrease in tear volume. In the in vitro study, exposure to ozone led to additional increases in IL-6 and tumor necrosis factor-α mRNA levels, which were already induced by treatment with IL-1α. Ozone did not induce any changes in cell viability. Pretreatment with IL-1α increased the expression of manganese superoxide dismutase, and exposure to ozone led to additional increments in the expression of this antioxidant enzyme. Ozone did not induce any changes in mitochondrial activity or expression of mitochondrial enzymes and proteins related to mitochondrial function, with the exception of phosphor-mammalian target of rapamycin. Treatment with butylated hydroxyanisole, a free radical scavenger, attenuated the ozone-induced increases in IL-6 expression in IL-1α-pretreated conjunctival epithelial cells. Therefore, we conclude that exposure to ozone exacerbates the detrimental effects on the integrity of the ocular surface caused by conjunctival allergic reactions, and further increases the inflammatory response in IL-1α-pretreated conjunctival epithelial cells.

The C-C chemokine receptor 6 (CCR6) is crucial for Th2-driven allergic conjunctivitis

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.

Comparison of the Effects of Topical Cyclosporine A 0.05%, Cyclosporine A 2%, Epinastine Hydrochloride 0.05%, and Prednisolone Acetate 1% on Allergic Inflammation in an Experimental Allergic Conjunctivitis Model

To investigate the effects of topical cyclosporine A 0.05%, cyclosporine A 2%, epinastine hydrochloride 0.05%, and prednisolone acetate 1% in an experimental allergic conjunctivitis model. Forty Wistar albino male rats were randomly divided into 5 groups. All the rats were immunized by an intraperitoneal injection of 100 μg of ovalbumin. After 2 weeks, topical treatment was administered: cyclosporine A 0.05% 4 times a day to group 1, cyclosporine A 2% 4 times a day to group 2, epinastine HCl 0.05% 2 times a day to group 3, and prednisolone acetate 1% 4 times a day to group 4 for the following 7 days. Group 5 was designated as the control group. Both eyes of the rats were enucleated on the 22nd day of the study. The excised sections were stained by toluidine blue for mast cell count and by hematoxylin-eosin for histopathological evaluation. The 2 solutions of cyclosporine A and epinastine were more effective than prednisolone acetate (P < 0.05) when compared with the control in histopathological scoring. There were statistically significant differences between the treatment groups and the control group (P < 0.05); however, no differences were observed between the treatment groups in the mast cell number. Topical 0.05% cyclosporine A is as effective as 2% cyclosporine A and epinastine in suppressing mast cell-mediated type I allergic conjunctivitis in an experimental allergic conjunctivitis model.

Rebamipide Suppresses PolyI:C-Stimulated Cytokine Production in Human Conjunctival Epithelial Cells

We previously documented that ocular surface epithelial cells could regulate ocular surface inflammation and suggested that, while Toll-like receptor 3 upregulates, EP3, one of the prostaglandin E2 receptors, downregulates ocular surface inflammation. Others reported that rebamipide, a gastroprotective drug, could not only increase the gastric mucus production, but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. The eyedrop form of rebamipide, approved in Japan for use in the treatment of dry eye diseases, upregulates mucin secretion and production, thereby suppressing superficial punctate keratopathy on the ocular surface of patients with this disease. In the current study, we investigated whether rebamipide has anti- inflammatory effects on the ocular surface. To examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells, we used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction assay. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model. Rebamipide could suppress polyI:C-induced cytokine production and the expression of mRNAs for CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. In our EAC model, the topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration. The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.

Interaction Between Conjunctival Epithelial Cells and Mast Cells Induces CCL2 Expression and Piecemeal Degranulation in Mast Cells

Intraepithelial mast cells are observed in giant papillae tissue samples obtained from patients with atopic keratoconjunctivitis (AKC)/vernal keratoconjunctivitis (VKC). We examined the roles of interaction between the conjunctival epithelial cells and mast cells. The interaction between human mast cells and conjunctival epithelial cells (HCjE) was investigated using a coculture model. Protein array analysis, ELISA, and real-time PCR were performed to test the interaction. Tissue samples (n = 6) from giant papillae were resected for therapeutic purposes, and subjected to immunohistological analysis of CCL2 expression. Recombinant CCL2 (10 ng/mL) was reacted with the cultured human mast cells and ultrastructural analysis was performed. A ragweed (RW)-induced mouse experimental allergic conjunctivitis model was used to examine ccl2 mRNA expression and mast cell morphology. Protein array and real-time PCR analyses showed that CCL2 protein/mRNA expression was induced by mast cell-HCjE coculture. Upregulation of CCL2 mRNA was observed in mast cells, whereas in situ CCL2 expression was observed at the conjunctival epithelium of the giant papillae by immunohistochemistry. Ultrastructural analysis showed that recombinant CCL2 treatment induced piecemeal degranulation (PMD) in the mast cells. Ultrastructural analysis of tissues from the giant papillae showed PMD of mast cells within the conjunctival epithelial cells. The RW-induced experimental allergic conjunctivitis model showed increased ccl2 mRNA expression and PMD morphology in the conjunctivae. Mast cell-conjunctival epithelial cell interaction induces CCL2 expression and subsequent PMD.

Epistatic Interactions Associated with Stevens–Johnson Syndrome

Stevens-Johnson syndrome (SJS) is an acute inflammatory vesiculobullous reaction of the skin and mucosa, often including the ocular surface, and sometimes progresses to toxic epidermal necrolysis (TEN). SJS/TEN may be initiated by certain types of medication coupled with possible infection; however, few susceptibility genes have been identified as indicators for risk prediction, except for certain human leukocyte antigen alleles, although several candidate susceptibility genes were identified by candidate gene and genome-wide approaches. As an alternative genetic model, gene-gene interactions between candidate genes were investigated using high-dimensional variable selection methods such as iterative sure independence screening. Linkage disequilibria around the toll-like receptor (TLR)3 and prostaglandin E receptor (PTGER)3 genes were also investigated using additional single nucleotide polymorphisms in an extended additional sample set. A murine experimental allergic conjunctivitis (EAC) model was used to examine the functional interactions. Iterative sure independence screening analyses of Japanese SJS/TEN patients with ocular surface complications revealed 2 interactions that exerted SJS/TEN susceptibility effects, which were locus pairs of TLR3-PTGER3 and HLA-A-IL1α. Furthermore, functional interactions between TLR3 and EP3 (the protein of PTGER3) were demonstrated using the EAC model. Identification of functional interactions between TLR3 and EP3 supports an epistatic interaction conferring an increased risk for SJS with ocular complications.

Toll-like receptor 4 signalling attenuates experimental allergic conjunctivitis

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.

Staphylococcus aureus accelerates an experimental allergic conjunctivitis by Toll-like receptor 2-dependent manner

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.

Expression of β Defensins in Ocular Surface Tissue of Experimentally Developed Allergic Conjunctivitis Mouse Model

To investigate the expression of beta-defensin in the ocular surface of a mouse experimental allergic conjunctivitis (EAC) model. Mouse beta-defensin (mBD)-1 and -2 mRNA expressed in conjunctival tissue of both the EAC and the control group were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. mBD-1 and mBD-2 mRNA expressed in cornea, limbus, and conjunctival epithelium of the EAC and the control group were also analyzed using a laser-assisted microdissection technique and the RT-PCR method. There was no significant difference in the amount of mBD-1 between the EAC and the control group. However, the EAC group had significantly less mBD-2 (0.23 +/- 0.15 x 10(4) copy/microl) than the control group (3.61 +/- 4.87 x 10(4) copy/microl) (P < 0.01, Mann-Whitney U test). mBD-1 mRNA was positive for all examined tissue in both groups. The expression of mBD-2 mRNA in the EAC group was definitely positive for the corneal and conjunctival epithelium and negative for the limbus. mBD-2 mRNA was expressed significantly less in the EAC model than in the control. Analysis by site in the ocular surface tissue revealed that this reduction was caused by the diminished expression of mBD-2 in the limbus.

Comparison of Topical Dexamethasone and Topical FK506 Treatment for the Experimental Allergic Conjunctivitis Model in Balb/c Mice

To evaluate the efficacy of topical dexamethasone and topical FK506 treatment on allergic inflammation in conjunctiva, we performed a comparative study using a mouse experimental allergic conjunctivitis model. The experimental allergic conjunctivitis model was developed by ovalbumin (OVA) instillation after OVA sensitization by intraperitoneal application of a mixed solution of OVA and aluminum hydroxide. The Balb/c mice of the experimental allergic conjunctivitis model were divided into three groups, depending on whether or not they received topical treatment and which type of treatment they received. The allergy group comprised six mice without topical treatment; the dexamethasone (Dx) group comprised six mice receiving topical 0.1% dexamethasone ointment treatment; and the FK506 (FK) group comprised six mice receiving topical 0.1% FK506 ointment treatment. The negative control group comprised six mice with neither sensitization nor topical treatment. For the histological examination, frozen sections of the eyelids, eyeballs, and lacrimal glands were stained with acid Giemsa stain or, in the immunohistochemical method, with alkaline phosphatase-antialkaline phosphatase (APAAP), for CD4. Densities of eosinophils and CD4-positive lymphocytes in the conjunctival tissue were counted. Productive C epsilon gene expression in the conjunctival tissue, including the lacrimal gland, was also evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). In the histological study, the cell density of eosinophils infiltrating into subconjunctival tissues was 66.3 +/- 34.7 cells/mm(2) (mean +/- SD) for the allergy group, 2.5 +/- 4.2 cells/mm(2) for the Dx group, 4.2 +/- 8.6 cells/mm(2) for the FK group, and 3.7 +/- 7.6 cells/mm(2) for the negative control group. The cell density of CD4-positive cells infiltrating into subconjuctival tissues was 145.9 +/- 66.4 cells/mm(2) for the allergy group, 10.0 +/- 12.9 cells/mm(2) for the Dx group, 10.1 +/- 15.5 cells/mm(2) for the FK group, and 21.7 +/- 17.8 cells/mm(2) for the negative control group. The allergy group showed significant differences in the density of eosinophils and CD4-positive cells compared with those of the Dx (P < 0.0001) and FK (P < 0.0001) groups. In the RT-PCR study, the expression of productive C epsilon gene was positive in the Dx group, but negative in the allergy group, the FK group, and the negative control group. Topical Dx and FK506 treatments were both efficacious in suppressing eosinophil and lymphocyte infiltration into subconjunctival tissue in the mouse experimental allergic conjunctivitis model. However, the influence of their IgE productive mechanisms seemed to differ, causing different efficacy of productive C epsilon gene expression in the conjunctival tissue.

Mast-cell activation augments the late phase reaction in experimental immune-mediated blepharoconjunctivitis.

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.

O-76 - An experimental model of allergic conjunctivitis by repeated sensitization/challenge with Japanese cedar pollen in guinea pigs

O-76 - An experimental model of allergic conjunctivitis by repeated sensitization/challenge with Japanese cedar pollen in guinea pigs

Interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis

Both nitric oxide and prostaglandins induce vasodilatation which is an important feature of local inflammation. The purpose of the study described here was to investigate a possible interaction between these two types of mediators in an experimental model of allergic conjunctivitis. A conjunctival allergic reaction was induced with antigen in sensitized guinea pigs. Conjunctival vascular permeability changes were evaluated with the prophylactic use of an inhibitor of nitric oxide synthase (L-NAME) and a cycloxygenase inhibitor (indomethacin). To study a possible interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis, the levels of nitrite and PGE 2 were determined in lavage fluid. The prophylactic use of L-NAME on the formation of conjunctival edema in response to topical PGD 2 administration was studied by measurement of albumin levels in lavage fluid. Both nitric oxide and PGE 2 are synthesized in response to antigen provocation and after histamine administration. Nitric oxide and PGE 2 are produced simultaneously in the conjunctiva and they showed identical synthesis profiles in response to antigen provocation. Pretreatment with L-NAME inhibited the synthesis of PGE 2 whereas exogenous administration of nitric oxide increased the level of PGE 2 in lavage fluid. Prophylactic treatment with L-NAME significantly inhibited the PGD 2 induced albumin extravasation. Nitric oxide seems to play an important role in the acute phase of allergic conjunctivitis it may stimulate PGE 2 production and acts as a secondary mediator in PGD 2 and histamine induced conjunctival edema.

Efficacy of nedocromil sodium and cromolyn sodium in an experimental model of ocular allergy.

Because ocular allergic disorders are important in terms of frequency and severity, there is a constant search for new topical antiallergic drugs. Nedocromil sodium has recently been introduced as a potential substitute for the mast cell stabilizer cromolyn sodium. We compared the efficacy of topical 2% nedocromil sodium to that of 2% cromolyn sodium in treatment of the early-phase reaction of an experimental model of allergic conjunctivitis. Guinea pigs were challenged topically with egg albumin 14 days after systemic immunization. Fifteen minutes before, immediately prior to, and 15 minutes after topical challenge, the animals received either 2% nedocromil sodium or 2% cromolyn sodium topically in one eye and phosphate buffered saline (PBS) in the other eye. Antigen-induced increase in vascular permeability was measured by the extravasation of intravenously injected Evans blue dye. Animals that were immunized and topically challenged but did not receive Evans blue were used for histologic studies. Evans blue extravasation from ocular tissues significantly decreased in drug-treated eyes compared with PBS-treated eyes (P < .01). Further, the cellular infiltrate (mast cells, eosinophils, and neutrophils) in the substantia propria of the conjunctiva was markedly reduced in the drug-treated eyes. No significant difference was observed between the nedocromil sodium- or cromolyn sodium-treated eyes. Topical 2% nedocromil sodium reduces the early-phase reaction of the allergic response as effectively as 2% sodium cromoglycate in a guinea pig model of ocular anaphylaxis.

Cetirizine reduces inflammatory cell recruitment and ICAM-1 (or CD54) expression on conjunctival epithelium in both early- and late-phase reactions after allergen-specific challenge

Background: Allergen-specific conjunctival challenge (ASCC) is a safe and reproducible experimental model of allergic conjunctivitis and a useful tool in the evaluation of effectiveness and possible mechanisms of action of drugs commonly used in the treatment of allergic diseases. Objective: The protective effect of cetirizine on inflammatory changes after ASCC was assessed in 12 patients with rhinoconjunctivitis caused by Parietaria judaica in a double-blind study. Methods: After a screening ASCC was performed, patients were randomized into two treatment groups; each patient was given cetirizine (oral tablets) 10 mg twice daily, or matching placebo for 3½ days in off-pollen season. Clinical evaluation (itching, hyperemia, lacrimation, and swelling of eyelids) and cytologic assessment (number of inflammatory cells in conjunctival scraping and evaluation of intercellular adhesion molecule-1 (ICAM-1)/CD54 expression on epithelial cells) were performed at baseline, 30 minutes (i.e., early-phase reaction [EPR]), 6 hours, and 24 hours (i.e., late-phase reaction [LPR]) after ASCC, before and after treatment. Results: The EPR clinical events and the EPR total number of inflammatory cells were significantly reduced by cetirizine compared with placebo. The LPR clinical events and inflammatory cell recruitment were reduced by cetirizine in a similar manner. Both eosinophil and neutrophil numbers were decreased by active drug in EPR and LPR. Furthermore, ICAM-1/CD54 expression was significantly reduced by cetirizine in both the EPR and LPR compared with placebo. Conclusions: This study shows that cetirizine has a protective effect on clinical and cellular EPR and LPR events (including ICAM-1/CD54 expression on epithelium) induced by ASCC. (J ALLERGY CLIN IMMUNOL 1995;95:612-21.)