Th1/Th2 response profiles to the major allergens Cry j 1 and Cry j 2 of Japanese cedar pollen

Cry j 1 and Cry j 2 are known to be the major allergens of Japanese cedar pollen. A comparative study was carried out on the immune responses to stimulation with Cry j 1 and Cry j 2 in 24 symptomatic patients and six nonallergic subjects. In T-cell proliferation assays, mean stimulation indexes (SI) were 10.6 for Cry j 1 and 11.7 for Cry j 2 stimulation, respectively, in the allergic patients. Two of the nonallergic subjects showed strong T-cell proliferation to both allergens, while the remainder did not. All the allergic subjects (17/17) showed high titers of anti-Cry j 1 IgE antibody at a mean value of 165 U/ml, whereas only 64% responded to Cry j 2 with low titers at a mean value of 26 U/ml. Nonallergic subjects did not respond with IgE production. Allergic subjects were further examined for their cytokine production profiles. All allergic subjects tested (16/16) produced high levels of interferon-gamma (IFN-γ) in response to Cry j 1 with a mean value of 918 pg/ml, while only five subjects showed significant elevation of IFN-γ production in response to Cry j 2 with a mean value of 679 pg/ml. The remainder produced small amounts of IFN-γ. Cry j 1 induced higher levels of interleukin (1L)-10 gene expression than did Cry j 2 stimulation, while both allergens induced IL-4 expression at a similar level. The IL-12 p35 gene was constitutively expressed, whereas the IL-12 p40 gene expression in Cry j 1-stimulated cells was elevated eightfold over that of nonstimulated cells. Increased expression of the IL-12 p40 gene was negligible in Cry j 2-stimulated cells. Thus, Cry j 1 stimulated mixed features of Th1 and Th2-like responses, while Cry j 2 played a minor role in inducing IgE production and cytokine (IFN-γ, IL-10, and IL-12) production, except for IL-2 production and strong T-cell proliferative activity. Therefore, it was concluded that Cry j 1 is the more important allergen, and that T-cell proliferation assays do not necessarily reflect the level of allergenicity.     

Changes in quantity of Cry j1, a major cedar pollinosis allergen,in Cryptomeria japonica pollen during spring in Japan

The changes in quantity of Cry j1 in Cryptomeria japonica pollen were assessed during the course of spring in Japan. C. japonica pollen was collected from the same trees in Hiroshima and Yamanashi Prefectures, and Cry j1 was quantified by enzyme-linked immunosorbent assay. The quantities of Cry j1 were found to gradually decrease, and this trend was similar in both Hiroshima and Yamanashi Prefectures. In Yamanashi Prefecture, no significant differences in the quantities of Cry j1 between the upper and lower parts of the tree were observed in samples collected from March 1 to 14. Furthermore, the quantities of Cry j1 on March 1, 5, 10 and 14 were significantly lower than those on February 1. These results suggest that expression of C. japonica pollen levels in terms of particle numbers is not sufficient and that quantities of Cry j1 vary throughout the course of spring.     

Cry j 2, a major allergen of Japanese cedar pollen, shows polymethylgalacturonase activity

We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity to PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by β-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.     

Cry j 1 isoforms derived from Cryptomeria japonica trees have different binding properties to monoclonal antibodies

BACKGROUND: We identified five Cryptomeria japonica trees producing Cry j 1 isoforms that cannot be detected in a sandwich ELISA using two monoclonal antibodies, J1B01 and J1B07, suggesting that the binding affinity of these isoforms for both monoclonal antibodies is low. OBJECTIVES: The binding properties of the Cry j 1 isoforms produced by five trees to J1B07 and J1B01 were examined. The complementary DNA (cDNA) sequences of the Cry j 1 isoforms were also determined. METHODS: To clarify the binding properties of these Cry j 1 isoforms to J1B01 and J1B07, Cry j 1 was quantified in pollen samples collected from each of the five trees, by sandwich ELISAs using polyclonal antibodies and either J1B01 or J1B07. The cDNA sequences of isoforms with different binding properties were determined. To test the assumption that amino acid substitutions affect the binding affinities of Cry j 1 isoforms for monoclonal antibodies, cleaved amplified polymorphic sequences (CAPS) markers representing the putative polymorphisms were used to analyse additional trees. RESULTS: Four of the five trees produced Cry j 1 isoforms with extremely low binding affinity for J1B07, whereas the other tree produced two different isoforms with low binding affinity for either J1B01 or J1B07. Cry j 1-encoding cDNA sequences for one of the four trees and for the exceptional fifth tree indicate that amino acid substitutions at positions 55 and 352 in mature Cry j 1 affect its binding to J1B01 and J1B07, respectively. This was supported by the results of CAPS analysis. CONCLUSION: The existence of Cry j 1 isoforms with low binding affinity for either J1B01 or J1B07 was established. Furthermore, a single amino acid substitution is involved in this difference in binding affinity for each monoclonal antibody.     

Existence of exine-free airborne allergen particles of Japanese cedar (Cryptomeria japonica) pollen

We investigated whether exine-free pollen allergen particles exist together with the intact pollen grains of Japanese cedar (Cryptomeria japonica) in the air during the pollen season in Yamagata City. First, we separated the allergen particles in an Andersen multi-stage air-sampler according to their aerodynamic diameters. The amount of major allergen (Cry j I) on each stage of the sampler was determined by a sensitive fluorometric sandwich ELISA, and the pollen count of the same samples was done by light microscopy after Carberla staining. Cry j I was found in stages 1 to 6, whereas most of intact and ruptured pollen grains were microscopically observed only in stages 1 and 2. Second, we suctioned the air through a tandem membrane filter system (the first filter, Nuclepore filter with 5 microns-pores; and the second, Millipore filter with 0.3 micron-pores). None of the pollen grains was detectable on the 0.3 micron-pore filter with light microscopy. However, Cry j I was detectable in the aqueous extract from the second filter. From these results, we concluded that pollen-free Cry j I existed in the air of Yamagata City during the pollen season.     

Cry j I, a major allergen of Japanese cedar pollen, has pectate lyase enzyme activity

In the course of analyzing the partial amino acid sequences of Cry j I, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, we found a peptide fragment which has a significant homology to some pectate lyase isozymes secreted by plant pathogenic bacteria. Therefore, we investigated whether Cry j I has pectate lyase activity. Cry j I reacted with polygalacturonic acid, resulting in the release of unsaturated uronide products. The optimum temperature and pH for the reaction were 60–70°C and pH 10. The enzymatic reaction had an absolute Ca²⁺ ion requirement. These characteristics were very compatible with the character of the pectate lyase isozymes reported previously. These results clearly show that Cry j I has pectate lyase activity.     

Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent allergy in Japan. Recently, the Japanese cedar pollen allergen Cry j 3 was cloned as a homologue of Jun a 3, which is a major allergen from mountain cedar (Juniperus ashei) pollen. However, native Cry j 3 has not been isolated and there are no reports on its allergenic activity. The aims of this study were to isolate native Cry j 3 and assess its immunoglobulin E (IgE)-binding capacity in patients with Japanese cedar pollinosis. METHODS: Native Cry j 3 was purified from Japanese cedar pollen by multidimensional chromatography. We assessed the IgE-binding capacity using sera from patients allergic to Japanese cedar pollen by immunoblot analysis and ELISA. Moreover, we assayed the capacity of Cry j 3 to induce histamine release from the patients' leukocytes. We cloned cDNA corresponding to purified Cry j 3 from a cDNA library of Japanese cedar pollen. RESULTS: We isolated native Cry j 3 as a 27-kDa protein. The IgE-binding frequency of Cry j 3 from the sera of patients allergic to Japanese cedar pollen was estimated as 27% (27/100) by ELISA. Cry j 3 induced the release of histamine from leukocytes. We cloned the cDNA and named it Cry j 3.8. Cry j 3.8 cDNA encoded 225 amino acids and had significant homology with thaumatin-like proteins. CONCLUSIONS: Cry j 3 is a causative allergen in Japanese cedar pollinosis and may play crucial roles in the cross-reactivity with oral allergy syndrome.     

Molecular cloning of a class IV chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen and competitive inhibition of its immunoglobulin E-binding capacity by latex C-serum

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified. OBJECTIVE: The aim of this study was to characterize a novel allergen with a high frequency of IgE binding. METHODS: The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA of C. japonica pollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition. RESULTS: The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients with C. japonica pollinosis and/or latex allergy to purified CJP-4. CONCLUSION: We identified CJP-4 as a novel and fourth C. japonica chitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile between C. japonica chitinase and latex C-serum indicated that C. japonica chitinase should be an important pan-allergen in C. japonica pollen.     

Immunodominance of seven regions of a major allergen, Cry j 2, of Japanese cedar pollen for T-cell immunity

The immunodominant regions of the Japanese cedar pollen allergen Cry j 2 for T-cell immunity were determined with whole peripheral blood lymphocytes (PBL) derived from seven allergic patients and three nonallergic subjects. Cry j 2–stimulated T-cell proliferation was inhibited by anti-HLA-DR, but not by anti-HLA-DQ antibody, indicating that the responding T cells recognized the allergen peptides associated with HLA-DR molecules. It was found that seven regions of Cry j 2. i.e., regions corresponding to amino acid numbers 1–26, 70–84, 151–167, 187–203, 252–279, 283–314, and 345–362, were immunodominant for T-cell proliferation. Thus, Cry j 2 bears a limited number of immunodominant regions despite polymorphic features of HLA-DR in the immune system. This suggests the possibility of molecularly designing Cry j 2 antagonists that could downregulate allergic reactions to Japanese cedar pollen.     

Identification and characterization of a group 2 conifer pollen allergen from Chamaecyparis obtusa, a homologue of Cry j 2 from Cryptomeria japonica

BACKGROUND: Not only Cryptomeria japonica (Japanese cedar) pollen but also that of Chamaecyparis obtusa (Japanese cypress) induces the allergic symptoms of Japanese cedar pollinosis. However, allergens from C. obtusa pollen have not been as well characterized as those from C. japonica pollen. OBJECTIVE: We sought to identify and characterize a homologue of the second major allergen of C. japonica pollen, Cry j 2, from the pollen of C. obtusa. METHODS: An allergen homologous to Cry j 2 was identified in C. obtusa pollen extract by immunoblot analysis, probed with anti-Cry j 2 monoclonal antibodies and purified by a series of column chromatographic steps. RESULTS: The allergen isolated from the extract showed a slightly diffuse band of 45 kDa and closely spaced double-bands of 42 and 45 kDa on SDS-PAGE, under reducing and non-reducing conditions, respectively; the bands were approximately 5-7 kDa larger than those of Cry j 2. In 24 of 30 residues, the N-terminal amino acid sequence of the allergen was identical with corresponding sequence in Cry j 2. Most patients with pollinosis who were IgE antibody-positive to Cry j 2 were shown to be IgE antibody-positive to this allergen, and the IgE antibody levels to both allergens were highly correlated. CONCLUSION: The results indicate that the allergen isolated from C. obtusa pollen in this study is a homologue of Cry j 2. The allergen was designated as Cha o 2 according to the WHO/IUIS Allergen Nomenclature Subcommittee recommendation.     

Sensitivity to two major allergens (Cry j I and Cry j II) in patients with Japanese cedar (Cryptomeria japonica) pollinosis

Background: Japanese cedar (Cryptmeria japonica: CJ) pollinosis is one of the most important allergic diseases in Japan. Recently, the second major allergen (Cry j II) was isolated from CJ pollen. There have been no prevalence studies of sensitivity to Cry j I and Cry j II among a large number of patients with pollinosis. Objective: This study was conducted to evaluate the prevalence of sensitivity to Cry j I and Cry j II. We measured specific IgE antibodies to these allergens in the sera of 145 patients. Furthermore, comparison of the sensitivity to Cry j I and Cry j II was examined by the hisiamine release assay. Methods: Specific IgE antibodies to Cry j I and Cry j II were assayed by a fluorometric ELISA. Allergen-specific histamine release was measured by a radioimmunoassay kit, Results: More than 90% of 145 patients had specific IgE antibodies to both allergens. the remainder had specific IgE to either one or the other. There were seasonal changes in the level of specific IgE. The changes in the levels of anti-Cry j II IgE antibodies were parallel to those of anti-Cry j I IgE. The histamine release assay with leucocytes from the patients demonstrated that the allergenic potency of the two allergens is almost the same. Conclusion: Cry j II is an as important a major allergen as Cry j I.     

A new method of counting airborne Japanese cedar (Cryptomeria japonica) pollen allergens by immunoblotting

We have devised a new method of counting pollen allergen particles, modified from the fluorescent immunoblotting technique of Schumacher et al. Airborne Japanese cedar pollen allergens collected on Burkard's sampling tape were transferred onto a nitrocellulose membrane. The membrane was then treated with antiallergen mouse monoclonal antibody conjugated with alkaline phosphatase. Pollen allergens were detected as purple spots on the nitrocellulose membrane after phosphate substrate staining was performed. Pollen allergen particles were visible under a stereoscopic microscope or to the naked eye and could thus be counted easily. This new counting method takes less time than previous methods and requires no special skill.     

Analysis of sequential immunoglobulin E-binding epitope of Japanese cedar pollen allergen (Cry j 2) in humans, monkeys and mice

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported to occur naturally in Japanese monkeys (Macaca fuscata) as well as in humans. Most human patients and monkeys with pollinosis have specific IgE for Cry j 2, a major allergen of CJ pollen. OBJECTIVE: The main purpose of this study was to identify IgE B cell epitopes of Cry j 2 using a synthetic peptide in humans, monkeys and mice. METHODS: We synthesized 38 overlapping peptides that span the entire length of Cry j 2. We examined the B cell epitopes of Cry j 2 that are recognized by IgE in the sera of human patients and monkeys with pollinosis and immunized mice using synthetic peptides of Cry j 2. We also examined the reaction of Cry j 2-specific mouse monoclonal IgG antibodies to the peptides. Furthermore, we conducted a histamine release assay with leucocytes from a pollinosis patient using human serum albumin (HSA) conjugated with the peptides as a B cell epitope. RESULTS: We found that 16 of the 20 pollinosis patients who had specific IgE to Cry j 2 also exhibited IgE reaction with some Cry j 2 peptides. Of these 16 patients, 10 exhibited IgE reaction with Cry j 2 peptide no. 13 (121GQCKWVNGREICNDRDRPTA140). Five of the seven monkeys with CJ pollinosis exhibited a reaction with peptide no. 13. Furthermore, IgE in mice immunized with Cry j 2 and two mouse monoclonal IgG antibodies reacted with peptide no. 13. Peptide no. 13-conjugated HSA showed the release of histamine from basophils. Furthermore, to determine the minimum epitope in peptide no. 13, we conducted an enzyme-linked immunosorbent assay inhibition test. The core of the epitope in humans, monkeys and mice was 124KWVNGREI131. CONCLUSION: We found that 124KWVNGREI131 is an important B cell epitope recognized by IgE in humans, monkeys and mice.     

Assessment of cross-reactivity between Japanese cedar (Cryptomeria japonica) pollen and tomato fruit extracts by RAST inhibition and immunoblot inhibition

BACKGROUND: An association between pollinosis and sensitivity to fruits and vegetables has been reported. Although Japanese cedar (Cryptomeria japonica) pollinosis is one of the most widespread diseases in Japan, there have been no reports demonstrating cross-reactivity between Japanese cedar pollen and other plant food. OBJECTIVE: The aim of this study was to demonstrate cross-reactivity between Japanese cedar pollen and tomato fruit (Lycopersicon esculentum) using RAST inhibition and immunoblot inhibition. METHODS: The RAST and immunoblot inhibition were performed using sera from patients with oral allergy syndrome (OAS) after ingesting fresh tomatoes. We identified some proteins that took part in cross-reactive IgE by the determination of N-terminal amino acid sequences and a homology search through the SWISS-PROT database. RESULTS: In the RAST inhibition, the bindings of IgE from the sera from four out of five (4/5) subjects to Japanese cedar pollen discs were inhibited by more than 50% by preincubation of the serum with tomato fruit extracts. Likewise, the IgE bindings to tomato fruit discs were inhibited more than 50% by Japanese cedar pollen extracts in 3/5 sera. In immunoblot inhibition, IgE binding activities of some protein bands on both membranes were decreased by heterologous inhibitors. However, the combinations of these protein bands involved in cross-reactivity were different between patients. CONCLUSION: We have demonstrated cross-reactivity between Japanese cedar pollen and tomato fruit using RAST inhibition and immunoblot inhibition.     

Identification of the second major allergen of Japanese cedar pollen

We isolated and characterized the second major allergen (Cry j II) from Japanese cedar pollen. We found that most patients with this pollinosis had IgE antibody to this protein in addition to IgE antibody to Cry j I; however, some sera reacted only with Cry j I or Cry j II. IgE-ELISA inhibition studies revealed that Cry j I and Cry j II had no cross-allergenicity. Cry j II did not react with anti-Cry j I monoclonal antibodies. In SDS-PAGE under a non-reducing condition, Cry j II showed a band at the 37 kDa position, compared with the 45-50 kDa bands of Cry j I. N-terminal amino acid sequence of Cry j II was completely different from that of Cry j I.     

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.     

Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross-allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level

BACKGROUND: Pollens from species of Cupressaceae family are one of the most important causes of respiratory allergies worldwide. In Japan, many patients with pollinosis have specific IgE to both pollens of Japanese cypress (Chamaecyparis obtusa) and Japanese cedar (Cryptomeria japonica). The sequences between Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar pollens, respectively, are 80% identical. OBJECTIVE: This study was undertaken to identify T cell epitopes in Cha o 1, and to elucidate the mechanism of cross-allergenicity between Cha o 1 and Cry j 1, at the T cell level. METHODS: T cell epitopes in Cha o 1 were identified by the reactivity of T cell lines, generated from 19 patients, to stimulation with overlapping peptides. The subsets of T cell clones specific to rCha o 1 were determined according to their ability to produce IL-4 and IFN-gamma. Peptide specificities of two T cell clones were determined by stimulation with the peptides from Cha o 1 and Cry j 1. RESULTS: Four dominant and six subdominant T cell epitopes were identified in Cha o 1. While four T cell epitopes, p11-30, p211-230, p251-270 and p331-350, were common to Cha o 1 and Cry j 1, 4 T cell epitopes, p61-80, p71-90, p311-330 and p321-340, were considered to be unique to Cha o 1. The subsets of T cell clones were predominantly of T helper2-type. One T cell clone recognized p16-30, which is common to Cha o 1 and Cry j 1, but another recognized p321-330, which is unique to Cha o 1. CONCLUSION: Presence of both T cells reactive to T cell epitopes common to Cha o 1 and Cry j 1 and T cells specific to T cell epitopes unique to Cha o 1 in patients with pollinosis contributes to prolonged symptoms after the cedar pollen season in March and the following cypress pollen season in April.     

Identification of a sequential B-cell epitope on major allergen (Cry j 1) of Japanese cedar (Cryptomeria japonica) pollen in mice

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides. The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. METHODS: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. RESULTS AND CONCLUSION: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 ((141)GVEPVHPQDGDALTLRTATN(160)), though those antibodies did not react with other peptides. IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be (145)VHPQDGDA(152).     

Pilot study of Japanese cedar pollen exposure using a novel artificial exposure chamber (OHIO Chamber)

For basic and clinical studies of Japanese cedar (JC) pollinosis and its treatment, experimental facilities for exposure to pollen under stable environmental conditions are becoming increasingly desirable. We developed an artificial exposure chamber (OHIO Chamber) that allows the dispersal of fixed concentrations of JC pollen in stable environments with a unique pollen supply system, air flow system for fixing the concentration of JC pollen, system for monitoring the number of pollen grains, and automated pure water washing and drying system. In the chamber, temperature and relative humidity (RH) could be successfully maintained at 22±1.1 °C and 45±5%, respectively. The spatial distribution of pollen concentrations in the chamber was within 10% of target, including when subjects were present. Only a few or no pollen grains were detected in the chamber after automatic washing and drying. We conducted a pilot tolerability and safety study in 15 JC pollinosis patients who were exposed to 15 000 pollen grains/m³ for 2 h. Symptoms manifested on average 33 min after start of exposure. The subjects experienced no serious side-effects, and pollen exposure at 15 000 grains/m³ was confirmed safe. After exposure, the number of intranasal and intraocular pollen grains was 469 and 602, respectively. The lower number of pollen grains in the nose than in the eyes was considered due to sneezing and nasal discharge. Further studies are needed to clarify the number of pollen grains required for the occurrence of symptoms in the OHIO Chamber.