Antigen Provocation Research Articles

IgE, IgE Receptors and Anti-IgE Biologics: Protein Structures and Mechanisms of Action.

The evolution of IgE in mammals added an extra layer of immune protection at body surfaces to provide a rapid and local response against antigens from the environment. The IgE immune response employs potent expulsive and inflammatory forces against local antigen provocation, at the risk of damaging host tissues and causing allergic disease. Two well-known IgE receptors, the high-affinity FcεRI and low-affinity CD23, mediate the activities of IgE. Unlike other known antibody receptors, CD23 also regulates IgE expression, maintaining IgE homeostasis. This mechanism evolved by adapting the function of the complement receptor CD21. Recent insights into the dynamic character of IgE structure, its resultant capacity for allosteric modulation, and the potential for ligand-induced dissociation have revealed previously unappreciated mechanisms for regulation of IgE and IgE complexes. We describe recent research, highlighting structural studies of the IgE network of proteins to analyze the uniquely versatile activities of IgE and anti-IgE biologics.

Nasal Symptom Responses to Cat Dander Exposure are Comparable in the RMT Mobile and Fixed Naturalistic Exposure Chambers (NEC)

The RMT Mobile Naturalistic Exposure chamber (Mobile NEC) is a portable alternative to fixed chambers for controlled allergen exposure. It consists of a pop-up tent, in which milled cat hair is aerosolized using a modified robot-vacuum. We compared the allergic response to cat antigen provocation in the Mobile NEC to the previously validated Fixed NEC, where allergen shed from two resident cats is aerosolized.

Segmental Bronchial Allergen Challenge Elicits Distinct Metabolic Phenotypes in Allergic Asthma

Asthma is a complex syndrome associated with episodic decompensations provoked by aeroallergen exposures. The underlying pathophysiological states driving exacerbations are latent in the resting state and do not adequately inform biomarker-driven therapy. A better understanding of the pathophysiological pathways driving allergic exacerbations is needed. We hypothesized that disease-associated pathways could be identified in humans by unbiased metabolomics of bronchoalveolar fluid (BALF) during the peak inflammatory response provoked by a bronchial allergen challenge. We analyzed BALF metabolites in samples from 12 volunteers who underwent segmental bronchial antigen provocation (SBP-Ag). Metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by pathway analysis and correlation with airway inflammation. SBP-Ag induced statistically significant changes in 549 features that mapped to 72 uniquely identified metabolites. From these features, two distinct inducible metabolic phenotypes were identified by the principal component analysis, partitioning around medoids (PAM) and k-means clustering. Ten index metabolites were identified that informed the presence of asthma-relevant pathways, including unsaturated fatty acid production/metabolism, mitochondrial beta oxidation of unsaturated fatty acid, and bile acid metabolism. Pathways were validated using proteomics in eosinophils. A segmental bronchial allergen challenge induces distinct metabolic responses in humans, providing insight into pathogenic and protective endotypes in allergic asthma.

Inhibition of Angiogenic Factor Productions by Quercetin In Vitro and In Vivo.

Background: Angiogenesis is well known to be an important event in the tissue remodeling observed in allergic diseases. Although there is much evidence that quercetin, one of the most abundant dietary flavonoids, exerts anti-allergic effects in both human and experimental animal models of allergic diseases, the action of quercetin on angiogenesis has not been defined. Therefore, in this study, we first examined the action of quercetin on the secretion of angiogenic factors from murine mast cells in vitro. We also examined the action of quercetin on angiogenic factor secretion in the murine allergic rhinitis model in vivo. Methods: Mast cells (1 × 105 cells/mL) sensitized with ovalbumin (OVA)-specific murine IgE were stimulated with 10.0 ng/mL OVA in the presence or the absence of quercetin for 24 h. The concentrations of angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-α, IL-6 and IL-8 in the supernatants were examined by ELISA. BALB/c male mice immunized with OVA were challenged intranasally with OVA every other day, starting seven days after the final immunization. These mice were then orally administered quercetin once a day for five days, starting seven days after the final immunization. Clinical symptoms were assessed by counting the number of sneezes and nasal rubbing behaviors during the 10 min period just after OVA nasal provocation. The angiogenic factor concentrations in the nasal lavage fluids obtained 6 h after nasal antigenic provocation were examined by ELISA. Results: Quercetin significantly inhibited the production of angiogenetic factors induced by IgE-dependent mechanisms at 5.0 µM or more. Oral administration of 25.0 mg/kg quercetin into the mice also suppressed the appearance of angiogenetic factors in nasal lavage fluids, along with the attenuation of nasal symptoms. Conclusions: These results strongly suggest that the inhibitory action of quercetin on angiogenic factor secretion may be implicated in the therapeutic action of quercetin on allergic diseases, especially allergic rhinitis.

IgE repertoire and immunological memory: compartmental regulation and antibody function.

It is now generally recognized that bone marrow is the survival niche for antigen-specific plasma cells with long-term immunological memory. These cells release antibodies into the circulation, needed to prime effector cells in the secondary immune response. These antibodies participate in the surveillance for antigen and afford immune defence against pathogens and toxins previously encountered in the primary immune response. IgE antibodies function together with their effector cells, mast cells, to exert ‘immediate hypersensitivity’ in mucosal tissues at the front line of immune defence. The constant supply of IgE antibodies from bone marrow plasma cells allows the rapid ‘recall response’ by mast cells upon re-exposure to antigen even after periods of antigen absence. The speed and sensitivity of the IgE recall response and potency of the effector cell functions are advantageous in the early detection and elimination of pathogens and toxins at the sites of attack. Local antigen provocation also stimulates de novo synthesis of IgE or its precursors of other isotypes that undergo IgE switching in the mucosa. This process, however, introduces a delay before mast cells can be sensitized and resume activity; this is terminated shortly after the antigen is eliminated. Recent results from adaptive immune receptor repertoire sequencing of immunoglobulin genes suggest that the mucosal IgE+ plasmablasts, which have undergone affinity maturation in the course of their evolution in vivo, are a source of long-lived IgE+ plasma cells in the bone marrow that are already fully functional.

Platelet aggregation, secretion, and coagulation changes in children with asthma.

The chronic inflammation in asthma evolves by cells including eosinophils, mast cells and lymphocytes. Despite their principal function in hemostasis, platelets contribute to pathogenesis of asthma that activation of platelets occurs following antigen provocation and during asthma attack. Our aim was to evaluate the platelet functions and other hemostatic features of children with asthma, both during symptom-free period and asthma attack. We enrolled patients with asthma attack (n = 33), mild intermittent asthma (n = 18), mild persistent asthma (n = 15) and healthy children (n = 20). Demographic characteristics and disease-related features were noted. Platelet aggregation and secretion tests (expressed as ATP release) were performed by lumiaggregometer method by stimulation with collagen, epinephrine, ADP, thrombin, ristocetin and arachidonic acid. Plasma levels of D-dimer, factor VIII (FVIII) and von Willebrand factor (vWF) were assessed. There were no differences in platelet aggregation induced by agonists between study groups. ATP release from platelets of patients with asthma exacerbation induced by ADP was lower compared with mild intermittent asthma (P < 0.001). Epinephrine-stimulated ATP secretion was also lower in patients with asthma attack than mild intermittent (P = 0.039) and mild persistent asthma (P = 0.011) and controls (P = 0.018). vWF measurements were higher in children with asthma attack than other study groups (P = 0.001). However, FVIII was increased in patients with severe asthma attack. Asthma is a disease in which many immune cells play a role, one of which is the platelet. Distinctions in platelet secretion profiles and plasma levels of vWF and FVIII provide evidence that coagulation mechanisms might be critical for asthma pathogenesis.

Peroxisome proliferator-activated receptor-gamma expression in dendritic cells determines immune tolerance versus immune activation (HYP6P.256)

Abstract Dendritic cells (DCs) play a central role in regulating immune tolerance and activation but the molecular axis within DCs that dictates immune outcome is unknown. We investigated the role of Peroxisome proliferator-activated receptor-gamma (PPARγ) in DCs in establishment of immune tolerance in the airways using an experimental model. Using mice with selective deletion of PPARγ in CD11c+ cells (PPARγΔ) to ablate PPARγ expression in DCs, we show that DC-specific PPARγ expression exerts a dual role whose collective goal in response to inhaled antigen is to not only promote de novo Foxp3 expression in T cells but to actively dampen T effector cell development. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions and when tolerized PPARγΔ mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Absence of PPARγ promoted the expression of multiple pro-inflammatory cytokines such as IL-6 and IL-23 in DCs, limited Foxp3 induction but augmented IL-17 production in T cells with significant increase in the frequency of dual Foxp3+RORγt+ CD4+ T cells in PPARγΔ mice. In summary, our study identifies PPARγ as a central regulator in DC-mediated programming of the Foxp3/RORγt balance in CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.

The compatible solute ectoine reduces the exacerbating effect of environmental model particles on the immune response of the airways.

Exposure of humans to particulate air pollution has been correlated with the incidence and aggravation of allergic airway diseases. In predisposed individuals, inhalation of environmental particles can lead to an exacerbation of immune responses. Previous studies demonstrated a beneficial effect of the compatible solute ectoine on lung inflammation in rats exposed to carbon nanoparticles (CNP) as a model of environmental particle exposure. In the current study we investigated the effect of such a treatment on airway inflammation in a mouse allergy model. Ectoine in nonsensitized animals significantly reduced the neutrophilic lung inflammation after CNP exposure. This effect was accompanied by a reduction of inflammatory factors in the bronchoalveolar lavage. Reduced IL-6 levels in the serum also indicate the effects of ectoine on systemic inflammation. In sensitized animals, an aggravation of the immune response was observed when animals were exposed to CNP prior to antigen provocation. The coadministration of ectoine together with the particles significantly reduced this exacerbation. The data indicate the role of neutrophilic lung inflammation in the exacerbation of allergic airway responses. Moreover, the data suggest to use ectoine as a preventive treatment to avoid the exacerbation of allergic airway responses induced by environmental air pollution.

Hypersensitivity pneumonitis (extrinsic allergic alveolitis): a Canadian historical perspective.

Hypersensitivity pneumonitis (extrinsic allergic alveolitis): a Canadian historical perspective.

Peroxisome proliferator-activated receptor-gamma in CD11c+ lung dendritic cells programs peripheral CD4+ T cells for Foxp3 induction during airway tolerance. (P6001)

Abstract Peroxisome proliferator-activated receptor-gamma (PPARγ) is a widely expressed molecular regulator with anti-inflammatory functions. However, whether it has an anti-inflammatory role in dendritic cells (DCs) that influence the Treg/Teffector balance has not been experimentally addressed. In this study, we investigated the role of PPARγ in DCs regulating the delicate balance between tolerance versus inflammation in the periphery using a model of airway induced tolerance by inhaled allergen. Using mice with selective deletion of PPARγ in CD11c+ cells, we observed that PPARγ expression in CD11chi lung DCs is essential for de novo Foxp3 expression in CD4+ T cells. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions with significantly less expression of the enzyme in these cells when mice are immunized for airway inflammation. When tolerized CD11ccre/+ PPARγfl/fl mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Taken together, our study identifies PPARγ as a central regulator in DC-mediated programming of Foxp3 expression in naive CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.

New treatment strategies for allergic rhinitis utilizing the oral mucosa

SummaryThe oral mucosa is well known to be rich in dendritic cells (DCs), and the cervical lymph nodes (CLNs) are the draining lymph nodes for the oral mucosa. DCs play a crucial role in the induction of primary T cell‐dependent immune responses. Upon uptake of antigens on the oral mucosal surface, immature DCs in the oral mucosa are thought to migrate to the regional CLNs. The oral mucosa is a potential route for vaccine administration. We examined the efficacy and some candidate biomarkers of the clinical responses to sublingual immunotherapy (SLIT) with a cedar pollen extract against cedar pollinosis. We found that Lactobacillus KW3110 administered sublingually migrated to the CLNs, and was effective for decreasing the IgE production and nasal symptoms induced by antigen provocation. Through simultaneous stimulation with antigen, improved efficacy of SLIT may be achieved by an adjuvant with oral mucosal administration of Lactobacillus KW3110.

Allergic Rhinitis and Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) was identified in 1980s as a protein that increases vascular permeability and induces endothelial cell-specific mitosis. VEGF plays an important role in angiogenesis during the embryonic stage and in angiogenesis and in increasing vascular permeability during postnatal life, both physiologically and pathologically. Great progress has been made in studies of VEGF, mainly in the field of oncology, and VEGF-targeted therapy has been successfully used to treat patients with cancer. In research related to chronic inflammation, several reports concerning rheumatoid arthritis or retinopathy and VEGF have been published. In the lower respiratory tract, increased levels of VEGF have been detected in biological samples from patients with asthma. However, VEGF has not been studied in detail in upper-airway diseases, such as rhinosinusitis. This review article focuses on VEGF and allergic rhinitis to advance studies of VEGF in chronic inflammation of the upper respiratory tract. VEGF levels in nasal secretions and nasal lavage fluid were higher in perennial allergic rhinitis than in nonallergic rhinosinusitis, after, rather than before, the antigen provocation test. The major VEGF isoforms were confirmed to be VEGF₁₂₁ and VEGF₁₆₅ in allergic rhinitis. Expression of VEGF mRNA was higher in serous versus mucous acini. In allergic rhinitis, serous acini produced significant quantities of VEGF, which was hypersecreted after antigen provocation. VEGF seems to play an important role in the pathophysiology of allergic rhinitis. Modulation of VEGF function seems to contribute to the successful treatment of conditions with airway inflammation such as allergic rhinitis.

Effect of vascular endothelial growth factor on nasal vascular permeability

One of the major functions of vascular endothelial growth factor (VEGF) is increasing vascular permeability. We previously reported that VEGF is present in nasal secretions in rhinosinusitis, particularly in allergic rhinitis, and is hyperproduced immediately after antigen provocation. However, its function in nasal mucosa has not yet been investigated. This study was designed to estimate the increased vascular permeability by VEGF in nasal mucosa and to discuss its possible role in allergic rhinitis. Prospective animal experiment. Hartley guinea pigs were used. Ethyl carbamate was injected into the abdomen for anesthesia, followed by intravenous injection of Evans blue into the femoral vein. Phosphate buffered saline, histamine, or VEGF was then applied to nasal cavities. Nasal mucosa was harvested after saline reflux from heart with phlebotomy by amputating the abdominal aorta. Tissue samples were soaked in formamide for 12 hours at 63 degrees C to extract dye exudates from nasal mucosa. To analyze vascular permeability, dye was quantified by colorimetry. VEGF increased vascular permeability in nasal mucosa, and was more than 1 x 10(6) times more potent than histamine on the basis of molar units. Although increased vascular permeability by VEGF was not inhibited by histamine 1 receptor antagonist, it was markedly inhibited by anti-VEGF receptor antibodies. VEGF increased vascular permeability to a greater degree than histamine in nasal mucosa via a different pathway. Regulating VEGF may be a new therapeutic option for persistent nasal symptoms in allergic rhinitis.

Reduced expression of IL‐3 mediates intestinal mast cell depletion in diabetic rats: role of insulin and glucocorticoid hormones

Rats turned diabetic by alloxan treatment are refractory to systemic anaphylactic shock, in a direct association with reduced intestinal haemorrhage and tissue response to antigen challenge. As diabetic rats show reduction in mast cell numbers in different body compartments, this study was undertaken to investigate the influence of alloxan diabetes on mast cell population as well as the expression of the mast cell growth factor interleukin (IL)-3 in the small intestine of rats. We also analysed the putative involvement of endogenous insulin and glucocorticoid hormones in this phenomenon. There was a significant decrease in the number of mast cells present in the small intestine (ileum segment) of diabetic rats. Likewise, the immunohistochemical analysis revealed that IL-3 labelling was markedly attenuated in diabetic rats, as compared with normal animals, a phenomenon which paralleled with a decreased mRNA expression as attested by Reverse transcriptase-polymerase chain reaction technique. Treatment with insulin and with the steroid receptor antagonist RU 486 restored basal mast cell numbers, normal levels of IL-3 labelling and mRNA expression for IL-3 in the ileum of diabetic rats. In conclusion, our findings show that there is a causative relationship between down-regulation of mast cell numbers and the expression of IL-3 associated with diabetic state. In addition, as both parameters were suppressed by administration of insulin and RU 486, it indicates that an imbalance between the systemic levels of insulin and glucocorticoid hormones seems to be implicated in the reduction in intestinal mast cell population and refractoriness to antigen provocation in alloxan diabetes.

Vascular Endothelial Growth Factor Produced in Nasal Glands of Perennial Allergic Rhinitis

Vascular endothelial growth factor (VEGF), a pleiotropic polypeptide that mediates endothelial cell-specific responses such as induction of angiogenesis and vascular leakage, is hyperproduced in a variety of inflammatory disorders. In asthma, VEGF hyperproduction promotes mucosal edema by enhancing vascular leakage. However, in allergic rhinitis, details of the pathophysiological importance remain unclear. This study was designed to investigate and discuss the pathophysiological significance of VEGF in nasal secretions from perennial allergic rhinitis sufferers. Seven allergic rhinitis patients sensitized with house-dust mites and 12 chronic rhinosinusitis patients were enrolled. Nasal secretion VEGF was quantified and compared between groups. In allergic rhinitis cases, nasal lavage VEGF was estimated before and after the antigen provocation. Nasal gland VEGF was immunohistochemically investigated. VEGF messenger RNA (mRNA) levels in serous and mucous acini were analyzed by laser microdissection and light cycler-polymerase chain reaction. VEGF levels in nasal secretions and nasal lavage from allergic rhinitis were higher than in nonallergic rhinosinusitis, after rather than before antigen provocation. VEGF mRNA expression was higher in serous versus mucous acini. These results are consistent with the immunohistochemistry results. In allergic rhinitis, there was significant VEGF production in serous acini, which was hypersecreted after antigen provocation. VEGF may play an important role in pathophysiology of allergic rhinitis.

Change in Prostate Specific Antigen Following Androgen Stimulation is an Independent Predictor of Prostate Cancer Diagnosis

We tested the hypothesis that a single exogenous androgen injection in men with low prostate specific antigen would provoke a differential prostate specific antigen response that would correlate with the presence and volume of cancer at biopsy. Following institutional review board approval 40 men with prostate specific antigen between 2.5 and 4.0 ng/ml were given 1 intramuscular injection of 400 mg testosterone cypionate at the start of the study. Prostate specific antigen and early morning serum testosterone were measured at baseline, 48 hours, and weeks 1, 2 and 4. All men underwent 12-core transrectal ultrasound guided biopsy at week 4. Of the 40 men 18 (45%) were diagnosed with prostate cancer. The mean change in prostate specific antigen from baseline to 4 weeks was 3.1 to 3.4 ng/ml (9.7%) in men found to have benign findings on biopsy compared to a mean increase of 2.9 to 3.8 ng/ml (29%) in those with prostate cancer (p = 0.006). The change in prostate specific antigen following androgen stimulation was significantly associated with the percent of tissue involved with cancer and it was an independent predictor of cancer diagnosis on univariate and multivariate analysis. An increase in prostate specific antigen following androgen stimulation in men with prostate specific antigen between 2.5 and 4.0 ng/ml was highly predictive of the subsequent diagnosis of prostate cancer and it correlated with disease volume. If these findings are corroborated, prostate specific antigen provocation may become an important strategy to identify men at risk for harboring prostate cancer and minimize the number undergoing unnecessary biopsies.

Effect of ageing on pulmonary inflammation, airway hyperresponsiveness and T and B cell responses in antigen‐sensitized and ‐challenged mice

The effect of ageing on several pathologic features of allergic asthma (pulmonary inflammation, eosinophilia, mucus hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized. To evaluate lung inflammation, mucus metaplasia and AHR in relationship with age in murine models of allergic asthma comparing young and older mice. Young (6 weeks) and older (6, 12, 18 months) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF), total inflammatory cell count and differential were measured. To evaluate mucus metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by quantitative PCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA. AHR developed in both aged and young OVA-sensitized/challenged mice (OVA mice), and was more significantly increased in young OVA mice than in aged OVA mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA mice than in young OVA mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA mice. All aged OVA mice had increased IL-5 and IFN-gamma mRNA expression in the lung and IL-5 and IFN-gamma protein levels from spleen cell cultures compared with young OVA mice. OVA-IgE was elevated to a greater extent in aged OVA mice. Although pulmonary inflammation and mucus metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-gamma and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in ageing animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.

Aldose reductase inhibitor zopolrestat restores allergic hyporesponsiveness in alloxan-diabetic rats

This study was undertaken to investigate the role of the aldose reductase in the refractoriness of diabetic rats to allergic inflammation. Wistar rats were actively sensitized with a mixture of Al(OH) 3 plus ovalbumin and intrapleurally challenged with ovalbumin, 14 days later. Diabetes was induced by intravenous injection of alloxan into fasted rats, 7 days before sensitization, and the aldose reductase inhibitor zopolrestat was administered after 3 days of diabetes induction, once a day during 18 consecutive days. The treatment with zopolrestat restored antigen-induced protein extravazation and mast cell degranulation in the pleural cavity of diabetic sensitized rats. Zopolrestat also significantly reversed the suppression in the increase of total and specific levels of serum immunoglobulin E (IgE) noted in sensitized animals under conditions of diabetes. In addition, we noted that the drop in the pleural mast cell numbers as well as the increase in serum corticosterone levels in diabetic rats were inhibited by the drug. Our findings show that zopolrestat restored the hyporesponsiveness of diabetic rats to antigen provocation, in parallel with impairment of alloxan-induced mast cell depletion and hypercorticolism, indicating that polyol pathway activity seems to play an important role in these phenomena.

Th2-Cell-Mediated Chemokine Synthesis Is Involved in Allergic Airway Inflammation in Mice

Eosinophilic inflammation in the bronchial mucosa has been recognized as a prominent pathological feature of bronchial asthma. Th2 cells have been implicated in the local infiltration and activation of eosinophils. The migration of eosinophils as well as Th2 cells is controlled by chemokines, suggesting a crucial role of chemokines in the pathogenesis of bronchial asthma. To elucidate the mechanism by which Th2 cells induce eosinophilic inflammation, a Th2-cell-dependent murine model of asthma was employed in this study. Along with the infiltration of eosinophils and antigen-specific Th2 cells, CC chemokine receptor-3-active eotaxin, monocyte chemoattractant protein (MCP)-3 and RANTES, as well as CC chemokine receptor-3-inactive MCP-1 were produced in the lungs of Th2-cell-transferred mice after antigen provocation in vivo. On the other hand, differentiated antigen-specific Th2 cells produced MCP-3 and RANTES but not eotaxin or MCP-1 upon stimulation in vitro. Chemokines synthesized by Th2 cells and other cell types are involved in the development of eosinophilic inflammation in bronchial asthma.

Inhibition of the Antigen Provoked Nasal Reaction by Second-generation Antihistamines in Patients with Japanese Cedar Pollinosis

Epinastine hydrochloride and fexofenadine hydrochloride, the second-generation antihistamines, are largely used in the indication of allergic rhinitis in Japan. The purpose of this study was to compare the protective efficacy of epinastine hydrochloride or fexofenadine hydrochloride using a nasal provocation test with Japanese cedar pollen allergen. A single-dose, placebo-controlled, single-blind crossover clinical study was conducted in patients with Japanese cedar pollinosis. The pollen exposure was done by the antigen provocation by disc method and involved repeated provocation five times per day. Among the active agents studied-epinastine hydrochloride and fexofenadine hydrochloride-epinastine hydrochloride significantly decreased the number of sneezing attacks and the quantity of nasal discharge for 3 hours after drug administration compared with placebo, a finding supported by the quantity of nasal discharge in the nasal findings. In this study, fexofenadine hydrochloride showed no significant difference compared with placebo. This study demonstrates better protection with epinastine hydrochloride than with fexofenadine hydrochloride or placebo in a nasal provocation test with Japanese cedar pollen allergen.